Itofs, A.Sc[)in
H. HtrmM
Vol. 12
w
H.
sell
KARGER
Concepts m 8acteHal Virulence
Contributions to
Microbiology
Vol. 1 Z
Series Editors
/Lve/ Schmidt iVuppenal
Heiko Hef-wald Lund
KAKGER
Concepts in Bacterial
Virulence
Volume Editors
.TT,::
Wayne Russell Lund
Heiko Herwald Lund
27 figures, I in color, and 7 tabies> 2005
K AKG E IV
Basel ■ Freiburg ■ Paris ■ London ■ New York ■
Bangalore ^ Bangkok ■ Singapore ■ 1 okyo ■ Sydney
Contributions to Microbtofogy
formerly 'Concepts in Immunopathology' and
'Contribudons to Microbiology and immunology'
Wayne Russeff, PhD Heiko Herwald, PhD
Lnnd University Lund University
Dept. of CelJ and Molecular Biology DepL of Cell and Molecular Biology
Section for Clinical and Experimeutal Section for Clinical and Experimental
Infectious Medicine Infectioiiii Medicine
PUmBM PlanB]4
Tomavageii 10 Tomavagen 10
S-221 84 Lund (Sweden) S-221 84 Lund (Sweden)
Library of Congress Catalogme-in-PublkaHon Daia
Concepts in bacterial viriilcnce / volume editors, Wayne Russell,
Heiko HenvalfL
p. ; cm. -(Conlribulioni to mkrobtology^ ISSN 1420-9519 ;
V. 12>
Irtcludcx bibHogi-iiphical references and index.
ISBNf 3.fi05J^77a6'9 fhard cover: alk. p^sper)
I. Virulence (Microbiology) 2. Molecular microbiology. 3. Endo
lo>:ins, I. Russell, W^yr^. \\. jlerwald, Heiko. IIL Series.
IDNLM^ I. Virulence Fatlori'-physiology. 2, Bacicria-patho-
gcnscity. 3, Communicable DisiMscs-microbiology
Wl <J077SUE V.I2 2004 /QW 730 C744 2004]
QR\75.Cf>b2WA
6l5,9'52y3^c22
20040 1 S270
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All Tighis reserved. No part of thss publication may be jransJated into other language^ repnoduced or
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Prinlcd in Switzerland on acid-free paper by Reinhardl Druck, Basel
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ISBN 3^055-7786-9
v.4--.-----.-7A+^ + R + -+«
Contents
VII Foreword
titfS5elUW,;Herwafd, K £Lund)
Tccons
Bishop^ R.E. fTurur^ttF)
ZS Bacterial Ex-otvixins
PopofF,M.R_ {Paris)
55 Capsular Patysaccharides andThcJr Role inViriflcnce
Taylor, C-M-, itol^-E3. I-S. (Mancliestfir)
Adhesin
&
67 FifnbrJa=e+ Pilj| FI^Lgetla and Bacterial Virulence
90 Gram-PoiltfveAdh^iljii
Tnfny, S.R. [Braunschwuig)
114 Microbiil Pathogenesh -ind Dconim Development
Rckncr^ A. (Lyngby)" Hdiby, N. (CopniFragcn)' Tulkcr-Niclscn^T.; Molin, S. (Lyngbyl
Enz/mc&
112 B^ctenal Peptidn^es
Putcrtipa, J. (Krakow/Athjens, G^-); Pike, R-N. fCEaj^l4mJ
V
131 Bacteria] Invasins: Molecular Systems Dedicate to the Invasion of
Host Tissues
Cainbrormep E.U; Sciineewind, O. (Chicago, IIJ,)
Signaling and Gene R^gulatton
no Bacterial Iron Transport Related to Virulence
Bsaim, V (Tubingfsn)
234 Pathogenicity Islands andTheir Role in Bacterial Virulence and Survival
Hoclihgt. B.; Dobrindt, U; Hacker, J. (Wiiraburg)
255 Horizontal and Vertk&l Gene Transfer: The Life History of Pathogens
La;vrence, J.G. (Pittsburgh. Pa,)
272 Subject Index
Coniems
VI
Foreword
With rtie current volume of the Karger book sefies Comnbutions to
Microbiology, we attempt to summarize some of the most important virulence
mechiuiisms in bacterial ijifectious diseases. In many cases the disease pathway
begins with the invasfon of the host and ends with the outbreak of physiologi-
cal responses that may lead to severe complications and ultimately death. Over
the years it has been shown that the interplay between pathogenic bacteria and
the host is complex and finely balanced. The ability of successful pathogens to
survive in an imiinmologically hostile environment is provided by a large
armamentarium of virulence mechanisms, which includes bacterial factors thai
evade, neutralise or counter the host defense systems, but also manipulate hosi
homeostasis and normal cell fijnctions. In order to give a comprehensive
update, we were able to recruit some of the most eminent scientists in ijifectious
diseases to give an overview of the most important recent findings in their
fields. We hope that this volume provides a thought-provoking update on these
important medical issues.
Limd, May 2004 Wayne Russell
Heiko Her^'aJd
vii
Toxins
Russell W, Herwald H (eds): Concepts in Bacterial Virulence!
Contrib Microbiol. Basel, Karger, 2005, vol 12, pp 1-27
Fundamentals of Endotoxin
Structure and Function
Russell E. Bishop
Departments of Laboratory Medicine and Pathobiology, and Biochemistry,
University of Toronto, Toronto, Canada
In 1892, flichard Pfeiffer first defined endotoxin as a heat-stable toxic sub-
stance that was released upon disruption of microbial envelopes [1]. The toxic-
ity is now known to be a consequence of the host inflammatory response, which
appears to be optimally adapted for the clearance of most local infections.
However, when severe infections become distributed systemically, the inflam-
matory response can lead to septic shock and death. Most of the early efforts to
determine the signal transduction events that occur between the presentation of
endotoxin to the myeloid cells of the immune system and the production of
inflammatory cytokines have utilized lipopolysaccharide (LPS) from gram-
negative bacteria [2], The bioactive lipid A component of LPS is arguably the
most potent of the substances that fit Pfeiffer's endotoxin definition, and lipid A
has become synonymous with endotoxin. However, many other inflammatory
mediators derived from bacteria can also be regarded as endotoxins, including
peptidoglycan, the diacylglycerylcysteine moiety of bacterial lipoproteins, and
bacterial nucleic acid signatures, to name only a few. The recent discovery that
Toll-like receptor 4 (TLR4) is the lipid A inflammatory signal transducer has
been folJowed by the identification of signal transducers for different Inflam-
matory mediators [3, 4]. Coincident with these developments in endotoxin
signaling has been the revelation that pathogenic gram-negative bacteria can
modulate the structure of lipid A in order to evade detection by the host immune
system. This article summarizes the recently elucidated pathways for the
biosynthesis of lipid A in enteric bacteria, which provide a framework for
understanding lipid A structure and fijnction in all gram-negative bacteria.
Readers are referred to the recent review of Raetz and Whitfield [5] for a more
complete treatment of LPS structure and function that accounts for its diversity
in more divergent organisms.
Overview of the Gram-Negative Cell Envelope
The cell envelope of gram-negative bacteria (fig. 1) consists of the inner
membrane (IM), the peptidoglycan (murein) and the outer membrane (OM) [5].
The IM is a phospholipid bilayer, much like the plasma membrane of eukaryotic
cells, and is permeable to lipophilic compounds. Numerous integral trans-
membrane a-helical proteins and peripheral membrane proteins are primarily
responsible for transport, cell signaling and metabolic fijnctions [6]. The IM pro-
vides a topologically closed environment for the vectorial translocation of ions to
generate a transmembrane electrochemical potential or proton-motive force that
governs cellular energetics. Proteins synthesized with a cleavable amino -terminal
signal peptide can be targeted for export across the TM [7]. The periplasm is the
gelatinous material between the OM and the IM. It contains enzymes for nutrient
breakdown as well as binding proteins to facilitate the transfer of nutrients across
the IM. Additionally, the murein sacculus in the periplasmic space is composed
of alternating N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid
(MurNAc) sugars that are cross-linked by short peptide bridges [8]. The highly
reticulated murein layer plays a crucial role in maintaining the cell's characteris-
tic shape and in countering the effects of osmotic pressure. The murein is bridged
to the OM by the abundant covalently bound murein lipoprotein, while numerous
low-abundance non-covalently-bound lipoproteins are anchored to the inner
leaflet of the OM and a few are anchored to the outer leaflet of the IM.
The OM is unique to gram-negative bacteria, and its role is to serve as a
protective structure. Tlie lipid arrangements of the OM are highly asymmetric.
While phospholipids [70-80% phosphatidylethanolamine (PtdEtn), 20-30%
phosphatidylglycerol (PtdGro) and cardiolipin] occupy the inner leaflet, LPS
molecules pack against one another in a tight architecture in the outer leaflet of
the OM [9]. Due to the low fluidity of lipid A hydrocarbon chains and the strong
lateral interactions between LPS molecules, the OM bilayer is impermeable to
lipophilic compounds and, thus, serves as an important permeability barrier for
gram-negative bacteria [10]. To allow uptake of essential nutrients, the OM is
studded with trimeric |3-barrel proteins, known as porins, which allow diffusion
of solutes with a molecular weight below approximately 600 daltons. Additional
P-barrel proteins in the OM are adapted for the uptake of particular nutrients that
carmot gain access through porins, and a few OM |3-barrel proteins function as
enzymes [11]. One consequence of porins is that the OM is believed to lack any
transmembrane electrochemical potential.
LPS is composed of three parts: the proximal, hydrophobic lipid A region,
which anchors LPS to the outer leaflet of the OM, the distal, hydrophilic
0-antigen repeats, which extend into the aqueous medium, and the mterconnect-
ing core oligosaccharide (fig. 2). The 0-antigen and core sugars are not essential
Bishop
LPS
GPL
Transmembrane
p-barrel protein
/
/
Lipoprotein
Murein
Prolon
motive
force
Transmembrane a-hellcal protein
Outer
membrane
Periplasmic
space
Inner
membrane
Fig, L Molecular organization of the gram-negative cell envelope. The OM is an
asymmetric bilayer with an outer leaflet of LPS and an mner leaflet of glycerophospholipids
(GPL). The integral OM proteins are exclusively transmembrane (3-barrels. Lipoproteins
anchored to the OM inner leaflet can link the OM to the murein exoskeleton. The energy-
transducing IM is a phospholipid bilayer that supports the proton motive force and contains
transmembrane ct-helical proteins. The periplasmic space is the region between the IM and
OM and contains numerous globular proteins.
for survival, but they provide bacterial resistance against various antimicrobial
agents including detergents and the membrane attack complex of serum comple-
ment [12], Wild-type cells that produce 0-antigen are termed 'smooth' due to
their glossy colony morphology, while those that lack 0-antigen are termed
'rough'. The term LPS formally applies only to the molecule that contains the
Endotoxin Structure and Function
3
Hex
Hex
Hex
O-antigen
Outer core
EtN-P-P-/ Hep
EtN-P'
Kdo
/
Kdo
P^GIcN) ( GlcNKP
Inner core
Lipid A
Bishop
0-antigen polysaccharide, while molecules that lack 0-antigen, as in the case of
Neisseria, are more appropriately termed lipooligosaccharide or LOS, Lipid A is
a target for the development of antibiotics and anti-inflammatory agents because
it is both essential for survival and a potent inflammatory mediator
TLR Signaling
When LPS is shed from the bacterial surface durmg infection, lipid A
recognition in mammalian cells is mediated by the TLR4 signal transduction
pathway [13, 14]. LPS is first recognized by the circulating acute phase LPS-
binding protein (LBP), which then interacts with the glycosylphosphatidylinositol-
anchored CD 14 on the surface of myeloid cells. Subsequent interaction with
TLR4 and its associated factor MD2 initiates a cascade of signahng pathways
that, in turn, elicit the production of cationic antimicrobial peptides (CAMPs),
a variety of cytokine and chemokine molecules, and the costimulatory mole-
cules that are expressed on the surface of antigen-presenting cells and further
signal the presence of an infection to the cells of the adaptive immune system
[15]. Upon activation, TLR4 recruits to its intracellular Toll-interieukin recep-
tor homology region (TIR), the adapter protein MyD88, which associates by a
homotypic protein-protein interaction with its own TJR domain (fig. 3). Another
homotypic protein-protein interaction between the death domains of MyD88
and the mterieukin-l receptor-associated kinase lRAK-1 initiates the autophos-
phorylation of lRAK-1, which then associates with a signal transduction way
station known as tumor necrosis factor-a (TNF-ct) receptor-associated factor-6
(TRAF-6). An ubiquitin-conjugating enzyme complex is bound to TRAF-6
along with the TAK-1 kinase complex, which is anchored by the TAB adapter
proteins [3]. The pathway impinges on the master regulator of inflammation
known as nuclear factor kB (NFkB), which activates transcription of inflam-
matory response genes. However, NFkB is normally sequestered in the cyto-
plasm in complex with its inhibitory subunit IkB. Proteolytic degradation of
IkB enables NFkB to migrate inlo the nucleus and activate inllammatory gene
Fig. Z Structural organization of LPS, The most highly conserved region of the LPS
molecule is the lipid A domain, which is an acylated and phosphorylated disaccharide of
glucosamine. Assembly of lipid A is contingent upon the addition of the two 8-carbon Kdo
sugars, which are the only essential components of the inner core. The inner core normally
includes three 7-carbon Hep sugars and can be modified by the addition of phosphate and
pEtN substituents. Outer core sugars provide the acceptor for 0-antigen ligation, but tend to
be composed of hexose sugars that differ between species. The 0-antigens represent the most
highly species-variable component of the LPS molecule.
Endotoxin Structure and Function
TLR4
LPS/LBP
MD2jp»
MyD88
IRAK-1
TRAF-6
Ubc13 _4.TAK-1
IKK y
i
fi
a
Degradation
t
NFkB
TNF-a; IL-13; costimulatory molecules
Cationic antimicrobial peptides
'M
NFkB
Fig. 3. TLR4 signal transduction pathway. LPS released from the surface of gram-
negative bacteria is bound to the circulating LPS-binding protein (LBP) and delivered to the
glycosylphosphatidyhnositol-anchored CD 14 on the surface of myeloid cells. The leucine-
rich repeats (LRR) of CD14 are also shared with the extracellular domain of TLR4, which,
in association with MD2, can transduce a signal to its intracelJular 77/?. TIR-TIR interactions
with the adapter protein MyD88 promote mteractions between the death domains (DD) of
MyD88 and the interleukin-1 receptor-associated kinase IRAK-l. Autophosphorylation of
lRAK-1 promotes an association with the TNF-a receptor-associated factor TRAF-6, which
anchors both the kinase TAK-1, by its TAB adapter proteins, and the dimeric ubiquitin-
conjugating enzyme complex composed of UevlA and Ubcl3. Subsequent phosphorylation
events activate the trimeric IkB kinase complex IKK, which phosphorylates the NFkB
inhibitory subunit IkB and targets it for proteolytic degradation. The master regulator of
inflammatory response gene expression NFkB is then released and migrates into the nucleus
where inflammatory response genes are transcriptionally activated.
expression. IkB is targeted for proteolysis upon phosphorylation catalyzed by
the IkB kinase complex (IKK), which is itself phosphorylated by the TAK-1
kinase in a manner that depends on the ubiquitin-conjugating enzyme complex
in association with TRAF-6. TAK-1 also phosphorylates mitogen-activated
Bishop
protein kinases that impinge on the AP-I transcription family members Jun and
Fos, leading to further immune activation.
The response to LPS includes locaJ inflammation, which is highly beneficial
in providing antibacterial defenses. If infection persists, however, the subse-
quent systemic responses, including the overwhelming production ofTNF-a and
interleukin-lp by the host immune system, can lead to septic shock [16]. Efforts
to understand the lipid A signal transduction pathway were largely motivated
by a desire to develop endotoxin antagonists for the treatment of septic shock.
The discovery that some bacteria can evade host immune defenses by modifying
the structure of lipid A suggested that naturally occurring lipid A structures
may function as potent endotoxin antagonists. The microbial pathways for the
biosynthesis of lipid A and its derivatives have been elucidated recently and pro-
vide powerful tools for the investigation of endotoxin signaling, in addition to
illustrating the pathogenic mechanisms utilized by gram-negative bacteria.
Re Endotoxin Biosynthesis
The recent completion of the Raetz pathway for lipid A biosynthesis [5]
hinged on the serendipitous discovery of lipids X and Y in a conditional
PtdGro-deficient pg.?^ mutant o^ Escherichia coli in 1979 [17]. Lipid X was
subsequently shown to be a diacylglucosamine i-phosphate bearing R-3-
hydroxymyristoyl (3-OH-14:0) groups at positions 2 and 3, while lipid Y only
differed from lipid X by the presence of a palmitoyl (16:0) group in acyloxy-
acyl linkage at position 2 [18, 19]. Around the same time, the determination of
the correct chemical structure of lipid A [20] revealed possible biosynthetic
routes for the production and utilization of Mpids X and Y. The accumulation of
these glucosamine-based phospholipids in the PtdGro-deficient mutant proved
to be a consequence of a second unlinked conditional mutation in the gene
pgsB(lpxB) [21, 22], which was later shown to encode the lipid A disaccharide
synthase. LpxB generates the P-1 ',6-glycosidic bond that is a characteristic fea-
ture of lipid A [23]. Both lipids X and Y couJd activate macrophages in a simi-
lar manner as lipid A [24], but only lipid X proved to be a substrate for LpxB
[23], raising doubts about the physiological significance of lipid Y. Lipid A
biosynthesis is now known to occur in four separate cellular compartments,
namely, the cytoplasm, the cytoplasmic face of the IM, the periplasmic face of
the IM, and ui the OM (where the origin of lipid Y was recently found).
Conceptually, it is helpful to recognize that lipid A and the core oligosaccharide
are assembled together as a single unit starting in the cytoplasm and moving to
the cytoplasmic face of the IM, but the subsequent lipid A modifications and
en bloc ligation of O-antigen occur in the extracellular compartments.
Endotoxin Structure and Function 7
OH
^fi
O
.OH
Q=i UDP
!pxA
UDP-GlcNAc
o
Nl
O
^
UDP
Acetate
In
2+
IpxC (envA)
UDP
3-OH-C1.-ACP
OH
0=^ nhI
HOl< O
HO
(2X)
IpxD {firA)
HOli'^OH
o
HO
HO
9ho \
^^'^^
o
"03PO
o^ nh|
HO
/pxM /pxL
(msbS) {htrB)
Ci4-ACPCt2-ACP
KdOj-lipid A
(Re endotoxin)
O
o=<. nhI
HOK 0=^ OPO3'
HO I
UDP
OH
o=<, nh]
0< 0P03=
HO
UMP
HOI
/pxH
fpxB
UDP
Lipid X
OH
o< o^ nhI
HO I
"O3PO
/(dtA (waa>A)
;2x)CMP-Kdo
ATP
0=^ nhI
HO< ^^ OP^S^
HOI
Lipid IV^
F/^. 4, The Raetz pathway for synthesis of Kd02-iipid A, LpxA catalyzes the addition
of 3-OH-14:0 to position 3 of UDP-GlcNAc. LpxC then removes the acetamido group at
position 2, which allows LpxD to add a second 3-OH-]4:0 group. LpxH cleaves the
nucleotide to generate lipid X, which is condensed with UDP-diacyl-GlcN to generate the
disaccharide 1 -phosphate. The 4'-kiiiase LpxK then generates lipid IV^, which is converted
into Kdo2-lipid TV^ by a bifiinctional Kdo transferase KdtA. Kdo2-lipid IV^ is a substrate for
the LpxL and LpxM acyltransferases, which generate the acyloxyacyl linkages at positions
2' and 3', respectively.
The moJecular genetics and enzymology of the conserved steps of Jipid A
biosynthesis are best characterized in E. coli, as shown in figure 4. The Raetz
pathway begins with the key precursor molecule UDP-GJcNAc, which is also the
first substrate for peptidoglycan biosynthesis. The first enzyme in lipid A
biosynthesis is a cytoplasmic acyltransferase LpxA, which selectively transfers
thiol ester-activated 3-OH-14:0 from acyl carrier protein (ACP) to the 3-OH of
UDP-GlcNAc [25]. The crystal structure of LpxA revealed a homotrimeric
Bishop
8
molecule that self-associates by a distinctive left-handed parallel p-helix
motif [26]. E, coli LpxA is extraordinarily selective for 3-OH-14:0-ACP as
the acyl donor substrate while the Pseudomonas aeruginosa LpxA prefers
3-OH-10:0-ACP, However, the specificity could be modulated by mutating cer-
tain key residues lining the active site cleft [27]. For example, the specificity for
the G173M mutant oi E. coli LpxA was shifted to 3-OH-10:0-ACP. In contrast^
the specificity of P. aeruginosa LpxA could be extended to accommodate
3-OH-l4;0-ACP in the corresponding M169G mutant. These findings demon-
strated the existence of precise hydrocarbon rulers in LpxAs, which can explain
variations in lipid A acylation that are observed between different organisms.
The acylation of UDP-GlcNAc by LpxA is thermodynamically unfavorable
[25], and the first committed step in lipid A biosynthesis is the subsequent
deacetylation catalyzed by LpxC (EnvA) [28, 29]. LpxC is a Zn^^-dependent
enzyme that is an established target for antibiotic development [30, 31]. The
recent crystal and NMR structures of Aquifex LpxC revealed tu^o slightly differ-
ent models for the mechanism of catalysis [32, 33], but both include a critical
role for Zn^^. Most LpxC inhibitors are hydroxamate compounds that interact
with the catalytic Zn^^ ion. Current challenges are aimed at the development of
inhibitors with the ability to evade efflux pumps that provide resistance, particu-
larly in pseudomonads [34, 35].
Following deacetylation, an N-linked 3-OH-14:0 moiety is incorporated
from ACP by LpxD (FirA) to generate UDP-2,3-diacylglucosamine [36]. A
highly selective pyrophosphatase LpxH then cleaves UDP-2,3-diacylglucosamme
to form lipid X [37, 38]. Next the disaccharide synthase, LpxB, condenses
UDP-2,3-diacylglucosamine and lipid X to generate the |3-T,6-linkage found
in all lipid A molecules [23]. The membrane-bound 4' kinase LpxK then phos-
phorylates the disaccharide 1 -phosphate to produce lipid IV^ [39, 40], which is
an important pharmacological agent because it functions as an endotoxin anta-
gonist in human cell lines [41, 42]. Next, two 3-deoxy-Z)-manno-2-octulosonic
acid (Kdo) sugars are incorporated by a Kdo transferase, which is encoded by
the kdtA (waaA) gene, using the labile nucleotide CMP-Kdo as the Kdo donor
[43]. The final lipid A biosynthetic steps that occur on the cytoplasmic side
of the IM depend on the prior addition of the Kdo sugars and involve the trans-
fer of lauroyl (12:0) and myristoyl (14:0) groups from ACP to the distal glucos-
amine unit to produce acyloxyacyl Linkages; the reactions are catalyzed at the
2'-position by LpxL (HtrB) and at the 3'-position by LpxM (MsbB), respect-
ively [44-46]. Under conditions of cold growth at 12''C, LpxL is replaced by
LpxP, which has a preference for palmitoleate (16:lcisA^) in ACP [47, 48]. The
incorporation of an unsaturated acyl chain into lipid A likely increases mem-
brane fluidity under cold growth conditions. Viable mutants that lack acyloxy-
acyl linkages in lipid A are attenuated for virulence and reveal the importance
Endotoxin Structure and Function
of the lipid A acylation pattern in inflammation [49, 50]. All other enzymatic
steps of the Raetz pathway, and those for the biosynthesis of CMP-Kdo, are
essential for cell viability and, thus, provide potential targets for antibiotic
development
Assembly of LPS
Kd02-lipid A, also known as Re endotoxin, can be regarded as the simplest
chemotypeof LPS [5], Completion of thecore-Kdo2-lipid A molecule involves the
subsequent addition of core sugars to the nascent Kdo2-lipid A anchored on the
cytoplasmic side of the IM [51]. The two essential 8-carbon Kdo sugars are
regarded as pait of the inner core, which is normally extended to include three
7-carbon Z.-glycero-Z)-manno-heptose (Hep) sugars (fig. 2). Core oligosaccharide
synthesis is contingent upon modification with phosphate at position 4 of the first
Hep, which can be followed by the addition of phosphoethanolamine (pEtN) at the
same position. Phosphate also normally occurs at position 4 of the second Hep,
and pEtN modification at position 7 of the second Kdo can occur under Ca^^-rich
growth conditions [52]. The so-called 'deep-rough' mutants have defects in the
inner core heptose sugars and are sensitive to detergents and hydrophobic anti-
biotics [53], The outer core sugars are predominantly hexoses and exhibit a greater
degree of structural diversity than is seen in the inner core and lipid A regions [54].
The outer core sugars provide the acceptor residue for 0-antigen ligation.
The 0-antigen is synthesized and anchored to a carrier lipid, undecaprenyl
phosphate, in the IM. The remarkable diversity in 0-antigen structures reflects the
multitude of glycosyl transferases that utilize various sugars and create diverse
glycosidic linkages, combined with the occasional presence of substoichiometric
sugar modifications [5, 55]. However, biosynthesis of all 0-antigens is initiated
by the formation of a common diphosphate linkage between the first sugar and
undecaprenyl phosphate. 0-antigen units are then completed in the cytoplasm
and transported to the periplasmic face of the IM by one of three distinct pathways
termed Wzy-dependent, ATP binding cassette (ABC) transporter-dependent, and
synthase-dependent. The most common of these is the Wzy-dependent pathway,
which is characteristic of £". coli and is followed by polymerization of 0-antigen
units on the periplasmic face of the IM. Recent studies have implicated an
essential ABC transporter MsbA in translocating the core-Kdo2-lipid A mole-
cule to the periplasmic side of the IM [56-60]. Core-Kdo2-lipid A and poly-
merized 0-antigens from the various pathways are then linked together by a
common ligation mechanism at the periplasmic surface of the IM.
The completed LPS is transported for assembly in the OM by a poorly under-
stood process. Interestingly, certain integral membrane proteins can passively
Bishop
10
promote the translocation of phospholipids across the IM [61], but MsbA is
required for the transport of both LPS and phospholipids to the OM [56, 57],
Jt has been known for more than 25 years that phospholipids freely exchange
between the IM and OM, while LPS transport appears to be unidirectional [62,
63], The mechanism by which LPS is translocated to the outer leaflet of the OM
is unknown, but it may depend on the highly conserved OM protein OMP85
[64], which is also implicated in the assembly of OM proteins [65].
The OM Permeability Barrier
LPS contains phosphate and acidic sugars and is therefore negatively
charged. In order to reduce the electrostatic repulsion between LPS molecules at
the cell surface, the bacterial OM sequesters divalent cations, mainly Mg^^ [66,
67], which neutralize the negative charges and maintain the integrity of the OM.
The presence of hydrogen-bond donors and acceptors in the lipid A molecule
allows for additional lateral interactions that cannot occur between phospholipid
molecules [67], Moreover, the six or seven saturated acyl chaijis of lipid A serve
to reduce the fluidity of the OM bilayer compared with the IM. The tight lateral
interactions between LPS, combined with low membrane fluidity, provides a
permeability barrier in the OM to lipophilic solutes and detergents [10].
Mechanism of Action of CAMPs
The requirement for Mg^"^ ions to bridge LPS molecules at the cell surface
is an Achilles' heel for the OM. Numerous CAMPs are produced in nature, but
the main types produced by the immune system are the small a-helical proteolytic
digestion products that are released from precursors known as the cathelicidins,
and the disulfide-bonded p-sheet peptides known as the defensins. CAMPs can
navigate through the OM by a nonporin pathway termed the 'self-promoted
uptake pathway' [68]. They are ijiitially unstiojctured in aqueous medium, and
their initial electrostatic interactions with the bacterial surface serves to displace
some Mg^"^ ions. The reduced dielectric constant at the membrane interface
induces dehydration of peptide bonds that become hydrogen-bonded in ct-helical
or p-sheet structures. The induced structure reveals the amphipathic nature of
CAMPs, which may promote changes in phase and/or motion in the OM bilayer
and, in turn, facilitates their translocation through the hydrocarbon layer, TTiese
peptides are then thought to target the IM bilayer and to produce a detergent-like
disruption of permeability. Some possible consequences of IM permeation
include the fatal depolarization of the transmembrane potential across the IM,
Endotoxin Structure and Function 1 1
leakage of cytoplasmic contents, cell lysis and cell death. The actions of CAIVfPs
are thought to selectively target bacterial membranes [69]. The outer leaflet of the
bacterial IM is negatively charged because it contains anionic phospholipids,
whereas eukaryotes tend to sequester anionic lipids internally. Moreover, choles-
terol molecules, which are embedded only in the eukaryotic plasma membrane,
could stabilize the lipid bilayer and, thus, reduce the activity of CAMPs.
Lipid A Modifications
Considering the importance of Mg^^ in maintaining the OM permeability
barrier, it is not surprising that Mg^"^ limitation can regulate the covalent structure
of lipid A. Mg^^ limitation is also believed to signal the presence of an intra-
cellular environment [70]. For example, in the phagocytic vacuoles of macro-
phages, the natural resistance-associated macrophage protein 1 (Nrampl) serves
to pump divalent cations into the cytosol, thereby withholding Mg-^^ required for
bacterial growth [71], Figure 5 outlines several covalentmodificationsof lipid A
found under Mg-^^- limited growth conditions that have been characterized in
E. coli and Salmonella enterica [72 75]. Three enzymes function to modify the
acylation pattern of lipid A. LpxO is a hydroxylase that generates 5'-2-hydroxy-
myristate (2-OH-14:0) at position 3' [76]. PagP is a transacylase that incorpo-
rates a palmitate chain at position 2 [77], while PagL is a deacylase that removes
the 0-linked 3-OH-14:0 chain at position 3 [78]. Moreover, the phosphate
groups at positions 1 and 4' of the lipid A disaccharide backbone can be modi-
fied with 4-amino-4-deoxy-L-arabinose (L-Ara4N) and/or pEtN, which serve to
reduce the overall negative charge of lipid A [79, 80].
Roles in Counteracting CAMPs
Lipid A modifications provide a dual protective mechanism against
CAMPs. First, substituting the phosphate groups on lipid A with L-Ara4N and
pEtN could effectively weaken the electrostatic attraction between the nega-
tively charged cell surface and CAMPs. In fact, the resultant neutralization
of the negatively charged bacterial surface is associated with resistance to
polymyxin B, a lipid A-binding cationic cyclic peptide antibiotic, in E. coli and
S. enterica [81, 82]. Moreover, lipid A acylation may block the hydrophobic
interaction between CAMPs and the membrane bilayer. Lipid A pahnitoylation
by PagP has been shown to provide bacterial resistance against CAMPs [83].
Possibly, the resultant hepta-acylated lipid A could further reduce OM fluidity
and, thus, prevent CAMP insertion. The pattern of lipid A acylation is also
Bishop
12
L-Ara4N
(pmrE/
pmrHFIJKL)
S-2-0H
(IpxO)
pEtN
(pmrC)
G-O-Deacylation
PalmitatefpagPj
Fig. 5. Regulated covalent lipid A modifications. The conserved lipid A nucleus can be
modified by the addition of L-Ara4N and pEtN to the phosphate groups, by the 5'-2-hydroxy-
lation of the secondary myristoyl group at position 3\ by the removal of the 3-OH- 14:0 group
at position 3, and by the addition of a pahnitate chain at position 2. Modifications to the acy-
lation of Hpid A are under the direct control ofPhoP/PhoQ, while the phosphate modifications
are controlled indirectly by PhoP/PhoQ through the downstream effectors PmrA/PmrB.
known to be critical in mediating its endotoxic activity through interactions
with the TLR4 signal transduction pathway [50, 84], Hepta-acylated lipid A
bearing a palmitate chain can function as an endotoxin antagonist, which blocks
the inflammatory effects of the hexa-acylated lipid A in human cell lines [85,
86]. Consequently, modifications to the acylation pattern of lipid A may^
remarkably, block both direct interactions between CAMPs and the bacterial
cell, and the induction of CAMP synthesis in the eukaryotic host. The enzymes
responsible for S-2-hydroxylation and 3-0-deacylation are absent from E. coli
and then- roles are less clear, but they may serve to stabilize lateraJ LPS inter-
actions by introducing new hydrogen-bond donors [67].
The PhoP/PhoQ and PmrA/PmrB Two-Component
Regulatory Systenns
Gram-negative bacteria use the PhoP/PhoQ two-component signal transduc-
tion pathway to respond to Mg^"^-limited environments that can be encountered
during infection [87]. PhoQ is a membrane-bound sensor kinase that detects
Endotoxin Structure and Function
13
Mg^"^ and can phosphorylate and activate the transcriptional regulatory protein
PhoP [88]. Mutants altered in the PhoP/PhoQ system display greatly reduced
virulence. PhoP controls the expression of over 40 different genes, many of
which are involved in Mg^"^ transport and in lipid A modification. For example,
transcription of pagP, pagL and IpxO, which are involved in the modification
of lipid A acyl chains, are under the direct influence of PhoP/PhoQ [76-78].
The PmrA/PmrB two-component regulatory system is one of the down-
stream effectors of the PhoP/PhoQ system, and is required for the modification
of lipid A with pEtN and L-Ara4N [80], PmrA is the transcriptional response
regulator and PmrB is the membrane-bound sensor kinase. While PmrA can be
activated by PhoP/PhoQ via a mediating protein PmrD [89], the PmrA-induced
genes can also be activated independently of PhoP/PhoQ by exposure of PmrB
to Fe^"^ or mild acidic conditions [90]. PmrA/PmrB activation has also been
shown to repress PmrD expression [91], which thereby creates a negative feed-
back loop. Interestingly, CAMPs themselves have been reported to activate
PhoP/PhoQ in Salmonella and PmrA/PmrB in Pseudomonas [92, 93],
L-Ara4N Cluster
PmrA/PmrB is onJy one of several clusters of pmr genes that were origi-
nally identified in polymyxin-resistant mutants of £^. coli [94, 80]. The pmrF
(pbgP) locus encodes an operon of 7 open reading frames pmrHFIJKLM, of
which the first 6, together with the unlinked pmrE (ugd), are required for
L-Ara4N synthesis. The proposed biosynthesis and attachment of L-Ara4N to
lipid A is shown in figure 6. The first step involves the conversion of UDP-
glucose into UDP-glucuronic acid catalyzed by a dehydrogenase encoded by
pmrE. Complex regulation of dehydrogenase gene expression reflects the fact
that UDP-glucuronic acid is a precursor for both colanic acid-containing cap-
sules and L-Ara4N [95]. Next, Pmrl (ArnA) catalyzes the oxidative decarboxy-
lation of UDP-glucuronic acid to generate a novel UDP-4-keto-pyranose
iiitennediate [96]. PmrH (AniB) then catalyzes a transamination reaction using
glutamate as the amine donor to generate UDP-L-Ara4N [97]. The crystal
structure of PmrH has verified that a pyridoxal phosphate cofactor contributes
to the catalytic mechanism [98]. Interestingly, Pmrl contains a second domain
that formylates the 4-amine of UDP-L-Ara4N. The resultant UDP-L-Ara4-
formyl-N is transferred by PmrF (AmC) to the membrane-anchored unde-
caprenyl phosphate, forming undecaprenyl phosphate-Z.-Ara4-formyl-N [97].
The formylation step may drive forward the equilibrium of the transamination
step, which is thermodynamically unfavorable. Formylation may also facilitate
translocation across the IM by neutralizing positive charge. It is speculated that
Bishop
14
UDP-glucose
.OH
UDP-/.-Ara4N
o
.T^o 2NAD+ Ho^V^'^v NAD'" CO. ^^
UDP UCP
Colanic acid capsule
Q Glutamate
NH, N-10-formyl ^-^
OHi
™^^
UDP
a-Ketoglutarate
arnB (pmrH)
THF HO
UCP
am^ (pmr/J
'^
UDP
P-O
f^),>'
arnC (pmrF)
UDP
NH
J/
o
HO-
PO
arnT (pmrK)
o-p-o- Lipid A core ^'>
>'^ OH
Transport?
n
\^
P-of-^)
Deformylation?
1 1
Fig. 6. Pathway for attachment of L-Ara4N to lipid A. The Ugd dehydrogenase con-
verts UDP-glucose into UDP-glucuronic acid, which is a precursor for both colanic acid cap-
sular polysaccharides and L-Ara4N. The first committed step of L-Ara4N biosynthesis is the
AmA-catalyzed oxidative decarboxylation, which generates a novel UDP-4-keto-pyranose
mtermediate.Transainination catalyzed by ArnB is followed by formylation due to a second
catalytic domaiji in AmA. Transfer of the fonnylated monosaccharide to undecaprenyl phos-
phate by ArnC is presumably followed by translocation to the periplasmic side of the IM for
deformylation. Undecaprenyl phosphate Z.-Ara4N is the substrate for ArnT, which transfers
L-Ara4N to the lipid A acceptor.
a putative transporter may be specific for the formylated compound and that
deformylation may then occur at the periplasmic surface [97], These steps
would ensure the vectorial translocation of the lipid across the IM and avoid
futile cycling. The necessity of the deformylation step is dictated by the fact that
undecaprenyl phosphate-L-Ara4N is the substrate for PmrK (ArnT), which cata-
lyzes the final transfer of L-Ara4N to lipid A at the peripJasmic surface of the
IM [99, 100]. Roles for the remaining /?mr genes in the transport and periplas-
mic deformylation reactions are suspected, but remain to be established.
EptA
The putative pEtN adding enzyme EptA has recently been cloned from
E, coll [101], and a homologous gene from Neisseria has been associated with
the addition of pEtN to lipid A [1 02], The EptA-encoding gene is the upstream
Endotoxin Structure and Function
15
-OCcte
OH
'■lO^O
o-
o
eplA (pmrC)
,OC<He
o o-
NH3
iNH.
'1 10.^0
0=
o
Diacylglycerol
V
OH
.OCcre
pagP
-\-
PtdEtn
sn-1-lyso-RdEln
Fig. 7. Modification of lipid A with pEtN and palmitate. EptA at the periplasmic side
of the IM uses PtdEtn as the pEtN donor to generate diacylglycerol and pEtN-modified lipid
A, PagP also uses PtdEtn (or another glycerophospholipid) as the palmitoyl donor in the OM
to generate sn-1-lyso-PtdEtn and hpid A modified by the addition of a pahnitoyi group.
open reading frame that is part of the pmrAB operon, and is also known as
pmrC (pagB) [103, 104]. PtdEtn is the reported pEtN donor (fig. 7) and several
EptA homoJogues are likely responsible for pEtN addition to other cell enve-
lope components including the inner core sugars of LPS. It is noteworthy that
roughly one third of E, coli lipid A carries a diphosphate moiety instead of the
monophosphate at position 1 [56], and that the putative phosphorylating
enzyme shares with EptA the ability to generate a phosphodiester bond at the
same position in lipid A.
PagP
PagP is encoded by a PhoP/PhoQ-activated gene and functions to transfer
a palmitate chain from a phospholipid to the hydroxyl group of the N-linked
3-OH-14:0 chain on the proximal glucosamine unit of lipid A [77, 83]. PagP
was the first enzyme of lipid A biosynthesis shown to be localized in the OM
Bishop
16
[77]. Since thiolester-containing substrates are not available in the extracellular
compartments, PagP uses a phospholipid as the palmitoyl donor instead (fig. 7).
PagP appears to be responsible for the production of lipid Y as a side reaction
in IpxB mutants. It was first identified in the salmonellae due to its role in pro-
viding resistance to CAMPs [83], and was subsequently purified from E. coll
[77]. In addition to these enteric pathogens, PagP homologues are present in the
respiratory pathogens Legionella pneumophila and Bordetella bronchlseptlca,
where PagP has been shown to be necessary for disease causation in animal
models of infection [105, 106]. In B. bronchlseptlca, PagP is controlled by a
different two-component virulence signal transduction pathway known as
BvgA/BvgS, and palmitoylation occurs at the 0-linked 3-OH-14:0 chain on the
distal glucosamine sugar [106]. PagP homologues are also found in Yersinia,
Photorhahdus and Erwlnia species, which adopt pathogenic lifestyles in ani-
mals, insects, and plants, respectively. Current efforts to understand the struc-
ture and function of PagP are aimed at developing a treatment for infections
caused by this important group of pathogens. The structure and dynamics of
PagP in detergent micelles have been determined by both NMR spectroscopy
[107] and X-ray crystallography [Bishop and Prive, unpubl. data].
PagP is an 8-stranded antiparallel (3-barrel preceded by an N-terminal
amphipathic a-helix. The ^-barrel is well defined in the structure while the
extracellular loops are not. Unlike other |3-barrel membrane proteins, proline
residues at two sites between (3-strands disrupt the continuity of hydrogen
bonding in the outer leaflet half of the PagP p-barrel. These non-hydrogen-
bonded regions are located between strands p-1 and p-2, generating a (3-bulge,
and between strands (3-6 and (3-7. The (3-bulge is largely responsible for the
highly dynamic nature of the extracellular loop L1 [107]. Additional features
not seen in any other (3-barrel membrane protein include a tilting of the PagP
barrel axis by 30^ with respect to the membrane normal and the presence of an
interior hydrophobic pocket in the upper half of the |3-barrel [Bishop and Prive,
unpubl. data]. The hydrophobic pocket harbors a single detergent molecule and
functions as a hydrocarbon ruler that allows the enzyme to distinguish pahni-
tate from otlier acyl chains present in phospholipids. Mutation of Gly 88 lining
the bottom of the hydrophobic pocket can modulate the acyl chain length speci-
ficity of PagP [Bishop and Prive, unpubl. data]. Internalization of phospholipid
palmitoyl groups within the barrel interior likely occurs by lateral diffusion
through the non-hydrogen-bonded regions between the |3-strands in the upper
half of the molecule.
Three putative catalytic residues were identified by site-directed mutagene-
sis and mapped to the extracellular loops Li and L2, indicating that the active
site is localized at the cell surface in the most dynamic region of the molecule
[1 07]. The putative catalytic residues project their side chains toward the barrel
Endotoxin Structure and Function 17
interior and are positioned above the hydrocarbon ruler [Bishop and Prive,
unpubl. data]. The requirement of invariant His 33, Asp 76, and Ser 77 for cataly-
sis might suggest that PagP utilizes an acyl-enzyme mechanism characteristic
of known serine esterases. However, the putative active site residues are not
organized into a catalytic triad that could enhance the nucleophiJic character
of Ser 77 [107]. The presence of two non-hydrogen-bonded regions that could
provide shnultaneous access for both substrates to the |3-barrel interior raises
the distinct possibility that PagP catalysis proceeds through the formation of a
ternary complex. Such a mechanism could promote the direct transfer of the
palmitoyi group from the phospholipid donor to the lipid A acceptor without the
formation of an acyl-enzyme intermediate, but the detailed mechanism of PagP
catalysis remains to be elucidated.
The clear alignment of the PagP active site with the OM outer leaflet cre-
ates an important topological problem for the enzyme. How does PagP access
phospholipids if OM lipid asymmetry is maintained? Chelating agents such as
EDTA can strip a fraction of LPS from the bacterial surface [108]. A large body
of evidence indicates that EDTA promotes the migration of phospholipids into
the OM outer leaflet [10]. Indeed, brief treatment of cells with EDTA rapidly
induces lipid A palmitoylation through a process that is independent of both
pagP gene regulation and de novo protein synthesis [Bishop, unpubl. data].
Lipid A palmitoylation induced by EDTA in vivo also requires functional MsbA
[Bishop, unpubl. data], which is presumably needed to replenish phospholipids
lost from the OM inner leaflet. These findings suggest that PagP may function
to maintain the OM permeability barrier under Mg^"^-limited growth condi-
tions, in addition to providing CAMP resistance and converting lipid A into an
endotoxin antagonist.
LpxO
An Fe^"^/a-ketoglutarate-dependent dioxygenase homologue in Salmonella
has recently been shown to catalyze the hydroxylation of lipid A and is
expressed in a PhoP/PhoQ-dependent manner [76]. Under aerobic conditions,
LpxO uses molecular oxygen to hydroxylate the 3' secondary acyl chain to gen-
erate 2-OH-14:0-modified lipid A (fig. 8). Homologues are found in other
gram-negative bacteria that similarly incorporate S-2-0H groups into their lipid A.
The function of S-2-hydroxylation is unknown, but the authors speculate that
the action of leukocyte acyloxyacyl hydrolase, an enzyme that releases sec-
ondary acyl chains from the lipid A of phagocytosed bacteria, would release
2-OH-14:0, which is possibly converted into 2-OH-14:0-CoA, a known
inhibitor of protein N-myristoylation needed for cell signaling functions.
Bishop
18
.0-Cofo
O2 +
a-keloglutarate
IpxO Jte2+
^^ CO2 +
succinate
HoO^
.OCOTfa
pagL
Fig. 8. S-2-hydroxylation and 3-0-deacylation of lipid A, LpxO is an LM Fe^"^/a-
ketoglutarate-dependent dioxygenase homologue that uses molecular oxygen to incorporate
a hydroxyl group into the secondary myristoyl group at position 3', PagL is an OM lipase
that removes the 3-OH-l^:0 group at position 3,
S-2-hydroxylation may also function to provide an additional hydrogen-bond
donor that could stabilize the lateral interactions between LPS molecules in
the OM [67]. Given that S-2-hydroxylation is contingent upon lipid A acylation
by LpxM, the LpxO reaction could occur on either side of the IM without inter-
fering with the sequential steps of the Raetz pathway. However^ LpxO is pre-
dicted to be anchored on the periplasmic face of the IM.
PagL and Rhizobium Lipid A
Lipid A 3-0-deacylase activity was observed in Salmonella during investi-
gations of PagP in membranes from a PhoP-constitutive mutant [77]- The
responsible enzyme was subsequently identified as the PagL gene product,
which proved to be the second enzyme of lipid A metabolism that is located
in the OM [78]. PagL functions to deacylate the 0-linked 3-OH-14:0 chain at
the proximal glucosamine unit of lipid A (fig. 8). By exposing the 3-OH group
in lipid A, PagL may provide a new hydrogen-bond donor to stabilize the lat-
eral interactions between LPS molecules in the OM [67]. Although a similar
Endotoxin Structure and Function
19
reaction had been described in Rhizobium legiiminosarum membranes [109],
PagL homologues are only found in the various serovars of Salmonella.
Lipid A recovered from Rhizobium species is structurally quite different from
£". coli lipid A, a fact that may reflect the symbiotic relationship between nitrogen-
fixing rhizobia and leguminous plants, which normally mount an innate immune
response to endotoxin. Rhizobium lipid A biosynthesis proceeds according to the
Raetz pathway, but the molecule is subsequently remodeled by numerous modify-
ing enzymes. Besides the absence of phosphate groups at positions I and 4' [I 10],
due to the presence of specific phosphatases [11 1, 112], the distal glucosamine
sugar exhibits a 27-OH-28:0 acyl chain as part of a characteristic acyloxyacyl moi-
ety at position 2' and a galacturonic acid residue at position 4' [113, 1 14]. LpxQ
is the third OM enzyme found to be involved in lipid A modification [115, 1 16],
and catalyzes the oxidation of the proximal 1-dephospho sugar to generate an
acylated 2-ammogluconate moiety.
Rhizobium lipid A serves to illustrate a fundamental point that is supported
by functional genomics; namely^ that the essential enzymes of the Raetz pathway
are highly conserved in gram-negative bacteria and that the observed variations
in lipid A structure are a consequence of the presence of additional modifying
enzymes. Aside fi^om variations in lipid A structure due to cytoplasmic ACP-
dependent acyl transferases [1 17-1 19] and Kdo transferases [ 1 20, 121] with dis-
tinct substrate specificities, it appears that most modifying enzymes act on the
lipid A nucleus in the extracytoplasmic compartments. These observations may
reflect a need to avoid futile cycling and to maintain a sequential order of Raetz
pathway reactions. These principles should faithfully guide future discoveries of
new enzymes that are employed to generate novel lipid A structures in diverse
organisms.
Perspectives
LPS structure and function are unique to gram-negative bacteria, but some
intriguing parallels are seen with the cholesterol and glycosphingolipid-rich
lipid rafts, and N-luiked protein glycosylation pathways of eukaryotic cells.
Both lipid A and eukaryotic glycolipids differ from phospholipids by the pres-
ence of hydrogen-bonded lateral interactions that tend to exclude phospholipids
leading to the formation of detergent resistant lipid domains [67, 122].
Additionally, the undecaprenyl phosphate-dependent pathways for the synthe-
sis and mcorporation of 0-antigens into the core-Kdo2-lipid A molecule at the
IM mirrors the dolichol phosphate-dependent pathway m the endoplasmic
reticulum, where Glc3-Man9-GlcNAc2 is incorporated into targeted protein Asn
residues [1 23]. Finally, it now appears that many of the Raetz pathway enzymes
Bishop
20
are conserved in the genomes of plants, perhaps reflecting the presence of lipid
A-like molecules in plastids [5].
Lipid A and its regulated covalent modifications exhibit profound effects on
bacterial and human physiology. Novel endotoxin antagonists and immune adju-
vants have already been developed from modified lipid A structures [124, 125],
By revealing the biochemical details of lipid A structure and function we hope to
understand its role in bacterial pathogenesis and to intervene with novel treat-
ments for infection. However, we must remind ourselves that multiple molecular
subtypes of lipid A are acting in concert in the bacterial cell. The need to unravel
the interactions between individual lipid A modifications will provide fertile
ground for future research.
Acknowledgments
Work in the author's laboratory was supported by the Canadian [nstitutes of Health
Research. Eileen K Lo is acknowledged for her assistance with the initial drafts of this
manuscript.
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32 Coggins BE, Li X, McClerren AL, Hindsgaul O, Raetz CR, Zhou P: Structure of the LpxC
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Bishop
22
35 Pirrung MC, Tumey LN, McClerren AL, Raetz CR: High-throughput catch-and-release synthesis
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Endotoxin Structure and Function 23
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95 Mouslim C, Groisman EA: Control of the Salmonella ugd gene by three two-component regula-
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96 Breazeale SD, Ribeiro AA, Raetz CR: Oxidative decarboxylation of UDP-glucuronic acid in
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97 Breazeale SD, Ribeiro AA, Raetz CR: Origin of lipid A species modified with 4-amino-4-deoxy-
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Endotoxin Structure and Function 25
99 Trent MS, Ribeiro AA, Lin S, Cotter RJ, Raetz CR: An inner membrane enzyme in Salmonella
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106 Preston A, Maxim E, Toland E, Pishko EJ, Hai'vill ET, Caroff M, Maskell DJ: Bordetella
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1 14 Que NL, Ribeiro AA, Raetz CR: Two-dimensional NMR spectroscopy and structures of six lipid
A species from Rhizobium etli CE3. Detection of an acyloxyacyl residue in each component and
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26
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Russell E. Bishop
6213 Medical Sciences Building, I King's College Circle
Toronto, Ont. M5S I A8 (Canada)
Tel. +1 416 946 7103, Fax +1 416 978 5959, E-Mail russell.bishop@utoronto.ca
Endotoxin Structure and Function 27
Toxins
Russell W, Herwald H (eds); Concepts in Bacterial Virulence,
Contrib Microbiol. Basel, Kaiger, 2005, vol 12, pp 28 54
Bacterial Exotoxins
Michel R. Popoff
Unite dcs Bactcrics anacrobics ctToxincs, Fnstitut Pasteur, Paris, France
Amongst the various mechanisms developed by pathogenic bacteria to
cause disease, toxins play an important role, since they are responsible for the
majority of symptoms and lesions during infection. Exotoxins act at a distance
from the infectious site and can diffuse through the organism. While some
cytotoxins can cause disruption of cells permitting the pathogens access to nutri-
ents, other toxins are only active on specific cells, for example intestinal cells,
neuronal cells, or leukocytes. This is achieved by the recognition of specific cell
surface receptors. When bound to the receptor, toxins can unleash their toxic
program at the cell membrane by interfering with signal transduction pathways,
pore formation, or enzymatic activities towards membrane compounds. In con-
trast, other toxins enter the cytosol, and recognize and modify specific intracel-
lular targets. According to the nature of the target and the type of modification,
intracellular active toxins cause a dramatic alteration of cellular functions such
as protein synthesis, cell homeostasis, cell cycle progression, vesicular traffic,
and actin cytoskeletal rearrangements. Alternatively, invasive bacteria can
directly inject toxins or virulence factors into target cells. This chapter is a com-
parative overview of the molecular mechanisms of the main bacterial exotoxins.
Toxins Active at the Cell Surface
Toxins Modulating Signal Transduction Pathways
Some enterotoxigenic Escherichia coli and other gram-negative entero-
pathogens (Yersinia enterocolitica, Vibrio cholerae) secrete heat-stable entero-
toxins (STs) that can cause acute diarrhea in humans and animals. These toxins
are small peptides which fall into two subgroups: methanol-soluble (STa or ST-I)
and methanol-insoluble (STb or ST-II) toxins. Analysis of STs shows they possess
a similar structure, containing 3 segments joined by 3 disulfide bridges. Alal3 in
Hormone-like
toxins
CI", H2O
Adenylcyclase
Pore-forming
toxins
Fig. L Toxins that alter cell homeostasis. Some of the mechanisms used by bacteria to
modify cell homeostasis are depicted. E. coli heat-stable enterotoxin (STa) binds to the extra-
cellular domain of transmembrane guanylate cyclase, resulting in an increase in cyclic GMP, and
secretion of CI" and H2O, PFT inserted into the membrane cause leakage of ions and HjO, CT
and E. colt heat-labile toxins enter the cell cytosol and ADP-ribosylate the Gsu: subunit of het-
erotrimericG proteins, leading to a permanent active molecule by inhibition of its GTPase activ-
ity and subsequent stimulation of adenylcyclase. The resulting increase in cyclic AMP induces
the secretion of Cl~ and HjO. PT inactivates the inhibitory heterotrimeric G protein Gia, lead-
ing to a upregulation of adenylcyclase activity. Bacterial adenylcyclases, such as EF from
anthrax toxin and Bordetella adenylcyclase (Cya), can also modulate cAMP levels in the celJs.
the flexible central segment plays a key role in the toxin's activity. This residue
is probably involved in the interaction of the toxin with its receptor. In the case
of STa, the secreted protein encompasses 18-19 amino acids, including 6 cys-
teines, and is capable of forming 3 disulfide bridges to create a highly stable
molecule. The carboxy-terminal segment of STs shares similarities with ionu-
phores and is therefore expected to interact with metal ions. Enteroaggregative
E. coli (EAggEC) strains also produce a heat-stable enterotoxin related to STa
with similar pathological effects,
STa induces watei^ diarrhea without causing obvious histological morpho-
logical damage. The toxin binds to the extracellular domain of guanylate cyclase
(GC-C) localized on the apical membrane of enterocytes. GC-C consists of
4 domains: an extracellular domain, a transmembrane segment, a kinase-like
domain and an enzymatic domam, which catalyzes the formation of cyclic GMP
(cGMP) (fig. I), The kinase-like domain has an inhibitory effect on the catalytic
Bacterial Exotoxins
29
activity. Binding of STa to the extracellular domain of GC-C has been suggested
to induce a conformational change in the protein kinase-liJce domain resulting in
an uncontrolled increase of GC-C activity. Elevation of intracellular cGMP
activates protein kinase II (cGKIl), which in turn stimulates the cystic fibrosis
transmembrane conductance regulator (CFTR) C\~ channels. This results in a
net fluid secretion through activation of apical CI" channels in parallel with the
inhibition of coupled NaCl transporters. Hiese findings have been confirmed in
GC-C knockout mice, which have a lower intestinal GC-C activity and do not
exhibit a secretory response to STa treatment [reviewed in 1].
STa was the first ligand found to bind GC-C and later studies demonstrated
that the hormones guanylin and uroguanylin are the natural ligands for this
receptor. These hormones have been shown to be involved in the regulation of
fluid and electrolyte transport in many tissues. Guanylin and uroguanylin con-
sist of 15 amino acids and are highly homologous to STa.
Toxins with Enzymatic Activity at the Cell Surface
That Alters Cell Signaling
Phospholipases
The first toxin that was recognized to possess an enzymatic activity was
the Clostridium perfringens a-toxin. This protein is a zinc-dependent phospho-
lipase C, which degrades phosphatidylcholine and sphingomyelin. Both
in vitro and in vivo studies have shown that it has cytolytic, dermonecrotic, and
hemolytic activities, and is lethal to animals at low doses. The toxin causes
membrane damage to a variety of different human and animal cell types includ-
ing platelets, leukocytes, and fibroblasts, as well as erythrocytes. It is the major
toxin involved in gangrene, which is characterized by extensive local tissue
destruction and necrosis progressing to profound shock and death. The secreted
protein consists of 370 amino acids (43 kD), and contains 2 domains, an
a-helical amino-terminal domain (residues 1-246) harboring the active site,
and a p-sandwich carboxy-terminal domain (residues 256-370), which medi-
ates membrane binding. The carboxy-terminal domain is structurally similar to
eukaryoLic calcium-binding C2 domains, which are involved in Ca^"*" -dependent
phospholipid binding. a-Toxin preferentially binds to phospholipids in the
intact membrane, opening the active site of the toxin and resulting in cleavage
of phospholipids [2]. In the activated state, the active site contains two tightly
bound zinc ions and one loosely bound zinc ion and is accessible for substrate
binding, whereas in the closed or inactive conformation, the active site is
occluded and one zinc ion binding site is lost [2-A].
In addition to its lytic activity, a-toxin is also involved in intracellular sig-
naling and the activation of endogenous metabolism cascades. Diacylglycerol and
ceramide generated from limited hydrolysis of phospholipids and sphingomyelin,
Popoff
30
respectively, activate endogenous phospholipases A2, C and D, and protein
kinase C. This in turn stimulates membrane phospholipases and initiates the
arachidonic acid pathway leading to the production of proinflammatory molecules
(prostaglandins, thromboxanes, and leukotrienes responsible for vasodilatation,
bronchostriction), and platelet aggregation [4].
Other bacterial phospholipases include phospholipase C from Pseudomonas,
Listeria, and various Clostridium species, phospholipase A from Helicobacter
pylori, phosphatidyl inositol phospholipase C from Bacillus, Clostridium, and
phospholipase D from Corynebacterium.
Bacteroides fragilis Enterotox in
B. fragilis enterotoxin (BFT) induces morphological changes in cultured
intestinal and renal cells, including cell rounding, increase in volume, and efface-
ment of microvilli and apical junctional complexes. BFT has zinc-dependent
protease activity, which has been shown to cleave the extracellular domain of
E-cadherin, the primary protein of the zonula adherens. Experimental studies
have led to the proposed two-step hypothesis, whereby the extracellular domain
of E-cadherin is cleaved by BFT, followed by intracellular degradation by as yet
unidentified protease(s). As a consequence, nuclear signaling and actin rearrange-
ment occur, which leads to the production of proinflammatory cytokines,
diminished epithelial barrier function, and activation of apical membrane ion
transporters. These cytotoxic effects are reversible, since 2-3 days after toxin
treatment cells appear normal [reviewed in 5].
Pore- Forming Toxins
So far more than 80 toxins have been identified that act by forming a trans-
membrane pore in the target cell. The general mechanism of pore-forming
toxins (PFT) is to bind to cell surface receptors where they then oligomerize.
The insertion of the oligomer into the cell membrane results in the formation of
a channel, which impairs the osmotic balance of the cell and causes cytolysis.
Most of the PFTs are cytolytic and/or hemolytic and they have been classified
into several families [for review see 6-8].
RTX toxins (repeats in toxin) are synthesized by many gram-negative
pathogens (Escherichia, Proteus, Pasteurella) . Members of the RTX toxin fam-
ily, including cytolytic toxins, meta'lloproteases and lipases, share a common
gene organization and distinctive structural features. They are secreted by
the type I secretion system which is mediated by the Sec machinery. At the
carboxy-terminal end, RTX contains 10^0 repeats of glycine- and aspartate-rich
nonapeptide domains. Most RTX toxins are posttranslationally activated by
acylation. The prototype of this family is the a-hemolysin (1 lOkD) of £. coli
and its target receptors on leukocytes have been identified as members of the |32
Bacterial ExotoxJns
31
integrin family. Insertion of a-hemolysin into the membrane, probably mediated
by four predicted hydrophobic a-hehces in the amino-terminal region, leads to
the formation of a hydrophilic- and cation-selective pore of at least 1 nm in
diameter [9], A related family of hemolysins consists of streptolysin S and
streptolysin S-like cytolysins expressed in streptococci.
Cholesterol-binding cytolysins are produced by a wide variety of bacterial
species including Streptococcus, Bacillus^ Clostridium^ and Listeria. Perfringo-
lysin O (PFO) is one of the best-studied toxins from this family. PFOs are
secreted as water-soluble monomers, which contain 4 domains rich in [3-strands.
A short hydrophobic loop in domain 4 is involved in the binding to cholesterol
[10], After cholesterol binding, PFO undergoes a conformational change result-
ing in the unfolding of domain 3 a-helices and the formation of two amphipathic
p-hairpins in each monomer. This leads to an association of neighboring
monomers and the subsequent formation of a large p-barrel, which then inserts
into the membrane forming the pore. In general, cholesterol-bindmg cytolysins
form large pores (300 A) containing about 50 monomers [1 1].
Staphylococcus aureus a-hemolysin, aerolysin and the binary staphylo-
coccal leukocidins, such as LukF, are also synthesized as monomers consisting
of a very hydrophilic sequence essentially arranged in ^-sheets. Binding of
monomers to an as yet unidentified cell receptor triggers the heptamerization of
the toxin, which adopts a mushroom shape with cap, rim and stem domains. The
amino-terminus detaches from the core monomer unmasking a small hydro-
phobic surface and assembles with the corresponding domains of the neighboring
monomers to form the cap. In contrast to PFO, only one antiparallel [S-hairpin
loop of each monomer unfolds and contributes to the stem formation, which
consists of l4-stranded p-barrels and results in pores with a small diameter
(15^5 A) [11, 12]. Aerolysin is secreted as an inactive precursor, which binds
to aglycosylphosphatidylinositol (GPI)-anchored protein. The toxin is activated
by cleavage of a carboxy-terminal peptide (40 amino acids) by soluble proteases
(trypsin or chymotrypsin) or furin. The localization of the aerolysin receptor on
lipid rafts probably facilitates toxin oligomerization [13]. Clostridium septicum
a-toxin, which is responsible for gangrene, shares a similar mode of activation
and pore formation with aerolysin [14].
The multicomponent leukocidins and ^-hemolysin from S. aureus also
assemble in hexamers (1:1 stoichiometry), which form transmembrane pores
[7]. One component (class S) is involved in the recognition of a cell suiface
receptor and allows the binding of the other component (class F). The p-toxin
from C perfiingens, which is involved hi necrotic enteritis, is related to S. aureus
a- and 7-hemolysin, and triggers pore formation [15].
C perfringens enterotoxin is a toxin that causes food poisoning via the
specific binding of the enterotoxin to receptor(s) from the claudin family,
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32
present on enterocytes. This complex is then able to associate with additional
membrane proteins, including occludin, to form larger complexes. It has been
suggested that these complexes form pores in the plasma membrane, which
alters the permeability of the plasma membrane for small molecules and
ultimately causes cell death by lysis or metabolic shut-down [16],
Superantigens
A particular class of bacterial toxins referred to as superantigens (entero-
toxins, toxic shock syndrome toxms from Staphylococcus and Streptococcus)
are characterized by their ability to bind both MHC class 11 molecules and T cell
receptors. UnJike conventional antigens that are presented to the T cell receptor
in complex with the MHC class II molecule, superantigens bind to the T cell
receptors and MHC class 11 molecules outside the classical antigen-binding
groove. This results in a massive antigen-independent proliferation of the targeted
T lymphocytes, leading to the release of various cytokines and inflammatory
factors [6],
Intracellularly Active Toxins
Inhibition of Protein Synthesis
Diphtheria Toxin - Inactivation of Elongation Factor 2
Corynebacterium diphtheriae is a human pathogen that normally colonizes
the throat The bacterium secretes a potent toxin, also known as diphtheria toxin
(DT), which is one of the most extensively studied and well-understood bacterial
toxins. Once DT has entered the bloodstream it can affect various organs, caus-
ing serious complications such as nephritis and cardiac dysfunction associated
with high mortality rates. DT is a single-chain protein of 58kD encompassing
three structural and functional domains: a carboxy-terminal domain rich in
|3-sheets (domain R), which binds to cell surface receptors, a central transloca-
tion domain containing 9 a-helices (domain T), and the amino-terminal catalytic
domain consisting of a mixLure of a- and (3-sLrucLures wilh a cleft forming the
active site (domain C). The toxin is activated by proteolysis at a furin cleavage
site located in an exposed loop between Cysl86 and Cys201. The amino-
terminal fragment corresponds to the catalytic domain and remains Imked by a
disulfide bridge to the rest of the molecule.
The receptor for DT has been identified as heparin-bhiding epidermal
growth factor- 1 ike growth factor precursor which forms complexes with other
membrane components, including CD9, heparin sulfate proteoglycans and inte-
grins. Epidermal growth factors are synthesized as transmembrane proteins,
which are subsequently cleaved close to the transmembrane segment to release
Bacterial ExotoxJns
33
DT
ER
Golgi
ADP-ribosylation
Pore-forming toxin
CPE
ST, VT
Hydrolysis of
N-glycosidic bond
in ribosomal RNA
Leakage of
nucleotides and
amino acids
Fig, 2. Toxins that inhibit proLein synthesis, DT enters the cytosol via the early endosomes
(EE) and inactivates EF2 by ADP-ribosylation, which results in impaired protein synthesis.
Pseudomonas exotoxin A (ExoA), Shiga toxm (ST) and E. coli verotoxin (VT) enter cells via
the Golgi apparatus and ER. While ExoA inactivates EF2, ST and VT impair ribosomal RNA
function by cleaving an N-glycosidic bond in the 60S subunit. PFT such as C perfhngens
enterotoxin (CPE) inhibit protein synthesis by inducing leakage of nucleotides, amino acids,
and other small molecules.
the active growth factor Once bound to the receptor complex, DT is proteo-
lytically cleaved by furin and internalized into cells by receptor-mediated endo-
cytosis via clathrin-coated vesicles. DT is then transported to late endosomes
and lysozomes where further degradation occurs. An acidification of the early
endocytic vesicles (pH less than 6) triggers a conformational change in the
T domain, to form a molten globule structure exposing hydrophobic sites (in
particular TH5-7 and TH8-9) that insert into the membrane forming cation-
selective channels. The ammo-terminal fragment of DT is then translocated
in an unfolded state from the endosomes into the cytosol where it inhibits pro-
Lein synthesis by ADP-ribosylaLion of elongation factor 2 (EF2) [see details in
17-21].
The DT catalytic domain belongs to a family of mono-ADP ribosyltrans-
ferases, which bind to NAD and transfer the ADP-ribose group to a specific
residue on the target protein. The active site is conserved among the bacterial
ADP-ribosylating toxins. It consists of an a-helix bent over a p-strand, which
forms the NAD-binding cavity that is flanked by two residues (His and Glu)
that have a major role in catalytic activity. The ADP-ribosylation of diphtamide
715 by DT prevents the binding of EF2 to tRNA, resulting in the mhibition of
protein synthesis (fig. 2).
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34
Pseudomonas Exotoxin A - Inactivation of EF2
Pseiidomonas exotoxin A (ExoA) is a 66-kD single-chain protein, which
shares the same mechanism of action as DT. ExoA is the major virulence factor
of the opportunistic pathogen Pseudomonas aeruginosa, which often infects
immunocompromised patients. The toxin is synthesized as a precursor, con-
taining an amino-terminal signal peptide that directs the polypeptide into the
type II secretion pathway. The crystal structure reveals three distinct domains:
an amino-terminal domain consisting of 17 antiparallel (^-strands that recog-
nizes the cell surface receptor, a central domain composed of 6 oc-helices form-
ing the translocation domain, and a carboxy-terminal domain containing the
catalytic site. ExoA binds to Jipoprotein-receptor-related protein (LRP), which
is a multifunctional scavenger receptor that is expressed by many cell types.
Upon binding to LRP, ExoA is internalized into the cell by receptor-mediated
endocytosis. Inside the endosome, the toxin is cleaved by furin, which results in
two fragments. The enzymatic domain is transported from the Golgi to the
endoplasmic reticulum (ER), where it is then translocated to the cytosol. In the
cytosol the enzymatic domain of ExoA catalyzes the ADP-ribosylation of EF2,
resulting in an inhibition of protein synthesis and ultimately leading to cellular
death [22, 23] (fig. 2).
Shiga Toxin - Inactivation of Ribosomal RNA
Another family of toxins consists of Shiga toxin, Shiga-like toxins, vero-
toxins, and verocytotoxins which are expressed by several enteric pathogens,
including Shigella dysenteriae and enterohemorrhagic E, coli. This group of
toxins plays an important role in the disease pathogenesis of a number of severe
complications, such as hemorrhagic colitis and the hemolytic uremic syndrome.
Shiga toxins are composed of a catalytically active subunit (A subunit) and
a receptor recognition subunit (B subunit). The B subunit that recognizes the
cell surface receptor globotriosyl ceramide Gb3 consists of 5 B fragments that
form a symmetrical ring-like structure in solution. The catalytic domain is
located in the A subunit, which is activated by proteolytic cleavage leading to
two fragments (Al and A2) that are linked together by a disulfide bridge.
Several studies have previously shown that Shiga toxin enters the cell by the
clathrin-dependent pathway and is then transported directly from early/recycling
endosomes to the Golgi apparatus and then to the ER [24], However, a clathrin-
independent mechanism has also been described involving lipid rafts [25].
Activation of the catalytic domain probably occurs in the trans-Golgi
network and/or in endosomes by the action of furin^ and to a lesser extent by
other cellular proteases. The Al fragment is released into the cytosol and inacti-
vates the 60S subunit of host cell ribosomes by cleaving the N-glycosidic bond
of adenosine 4324 of the 28S ribosomal RNA of the 60S subunit. This induces
Bacterial Exotoxins
35
a dramatic inhibition of cellular protein synthesis (fig. 2). It has been reported that
Shiga toxin and verotoxins also cause apoptosis characterized by DNA degrada-
tion and subsequent cell lysis by an independent mitochondrial pathway [26].
Alteration of Cell Homeostasis
Alteration of Heterotrimeric G Protein Signaling
Cholera Toxin. Cholera is a serious epidemic disease characterized by
severe diarrhea and dehydratation, caused principally by the cholera toxin (CT).
Other members of the CT family are the E. coli heat-labile enterotoxins LT-1
and LT-1 1. The CT gene is localized to filamentous bacteriophage DNA and can
be chromosomally integrated or replicated as a plasmid [27]. Similarly, the
heat-labile enterotoxin genes are located on plasmids (LT-l) or are integrated
into the chromosome (LT-II) [28]. CT and lethal toxin (LT) subunits are
exported across the bacterial membrane by Sec proteins and assemble in the
periplasm. In V. cholerae, CT is actively secreted through the outer membrane,
while the release of LT-I depends on cell lysis [for a review, see 29].
Like Shiga toxin, CT and LTs consist of an A subunit (28 kD) and 5 B
subunits (1 1 kD each) assembled in a pentamer (AB5 structure). The A subunit
is proteolytically activated by a ff cholerae endopeptidase into two com-
ponents Al (approximately 22kJI)) and A2 (approximately 5.5 kD) which
remain linked by a disulfide bridge. The carboxy-terminal part of A2 extends
through the central pore of the B pentamer and is linked noncovalently to the
B subunits.
CT is internalized into noncoated vesicles after binding of the B subunits
to ganglioside GMl, which is located at the epithelial cell surface. GMl directs
the toxin into lipid rafts from where it enters the Golgi via early and late endo-
somes in a Rab9-dependent pathway [30]. In the perinuclear region of the
Golgi, the A subunit dissociates from the B subunits and enters the ER via
coatomer 1-coated vesicles. The carboxy-terminal sequence of the A2 fragment
contains an ER retention sequence (KDEL), which recognizes the receptor
Erd2p and directs the Golgi-ER trafficking of CT [31]. B subunits lacking an
ER retention signal are also LransporLed to the ER, via an unknown niechanism,
and translocate into the cytosol via the Sec61 complex [32]. The Al fragment
is responsible for the enzymatic activities of the toxin in the presence of the
membrane factor Arf. This activity includes NAD hydrolysis of ADP-ribose and
nicotinamide, and transfer of ADP-ribose to Argl87 of the ot-subunit of stimu-
latory protein (Gsct), leading to stimulation of adenylcyclase and elevated
intracellular cAMP. The increased cAMP levels lead to an activation of protein
kinase A, which subsequently phophorylates numerous substrates in the
cell [33]. This results in an increase of Cl~ secretion by intestinal crypt cells
(fig. I) and a decrease of NaCl-coupled absorption by villus cells.
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36
Pertussis Toxin. Pertussis toxin (PT) is an important virulence factor of
Bordetella pertussis, the causative agent of whooping cough in humans. PT is
a hexameric protein consisting of an enzymatic A domain (subunit SI) and five
binding B domains (subunits S2-S5). Whereas the 5 B subunits of CT are iden-
tical and arranged in a regular pentamer, the correspondmg PT subunits are
distinct (n-26kD) and organized as an oligomer (S5-S2-S4-S3-S4). This
structure forms a disc-like base upon which the pyramid-shaped enzymatic A
subunit (SI) rests. The different B subunits form a pentameric domain in the
center of the B oligomer, consisting of a ring of 30 antiparallel p-strands, which
is surrounded by a barrel of five a-helices. The pore of the barrel is partially
penetrated by the carboxy-terminus of SI.
Glycoproteins and glycolipids found on many types of eukaryotic cells
have been shown to act as a receptor for the B oligomer of PT, seemingly via
carbohydrate-recognizing domains on subunits S2 and S3. The interaction of
the toxin with cells of the immune system leads to the induction of lympho-
cytosis, inhibition of macrophage migration, adjuvant activity, and T cell mito-
genicity. The T cell mitogenic effect is mediated by the B oligomer and is
thought to be independent of the S 1 subunit of the toxin. PT possibly undergoes
a retrograde transport to the ER to deliver SI into the cytosol, although SI does
not contain an ER retention sequence [34].
Internalization of PT is mediated by endocytosis through coated pits, and
seems to be routed to the late endosome and to the Golgi apparatus.
The SI subunit of PT, which shares high homology with the enzymatic
domains of CT and LT, catalyzes the ADP-ribosylation of the inhibitory
a-subunits of the heterotrimeric GTP-binding proteins (G proteins) involved in
a variety of signaling pathways. This results in the prevention of the a-subunit
coupling with the corresponding (3/7-subunits, an increase of adenylcyclase
activity, which is no longer negatively regulated, and the impairment of several
second-messenger pathways including an increase in cAMP (fig. 1).
Adenylcyclase Activity
Bordelella Adenylcyclase
The adenylate cyclase toxin (Cya) of 5. pertussis, the whooping cough
agent, is a major virulence factor required for the early phases of lung coloni-
zation. Cya is a single-chain 177-kD protein consisting of two domains. The
toxin is activated after posttranslational palmitoylation of the protein at Lys856
and Lys963. The enzymatic activity of Cya is located within the proximal 400
amino acids at the amino-terminus. The carboxy-terminal part, also referred
to as the hemolysin domain, contains several glycine and aspartate-rich non-
apeptide repeats that are related to those found in RTX toxins and represent the
main Ca^"^-binding site of the protein. In addition to its intrinsic hemolytic
Bacterial Exotoxins
37
activity, this domain mediates the binding to and internalization of the toxin into
eukaryotic cells. CyaA can penetrate a wide range of cell types, including erythro-
cytes and immune cells. In macrophages, neutrophils and dendritic cells, CyaA
has been demonstrated to bind specifically a^^2 integrin (CDUb/CDl 8) [35].
After internaJization, possibly directly through the plasma membrane, Cya is
cleaved and the catalytic domain is released into the cytosol, where it increases
the cAMP levels in a cahnodulin-dependent fashion (fig. 1). The toxin allows
the pathogen to escape the host immune response by intoxicating neutrophils
and macrophages, causing phagocyte impotence, and inducing apoptosis [36].
Anthrax Edema Toxin
Anthrax toxin is a tripartite toxin consisting of the protective antigen (PA),
edema factor (EF), and lethal factor (LP). PA is the binding component, which
permits the entry of either EF or LF into the cell. The combination PA and EF
is termed anthrax edema toxin, while PA and LF is termed lethal toxin (a fur-
ther description of LT can be found in the section Apoptosis below). The genes
of the three components are localized on a large plasmid (pXOI) present in
virulent Bacillus anthracis strains. The proteins are secreted by means of a
signal peptide [reviewed in 37].
PA is secreted as an inactive protein (83 kD), which is activated after
removal of a 20-kD amino-terminal peptide. The cleavage site contains the
RKKR motif, which is sensitive to proteases such as trypsin or furin. The active
protein (PA63) has four domains [38]: an amino-terminal domain (domain 1)
that is relatively hydrophobic and which is involved in the binding of EF orLF,
a heptamerization domain (domain 2) containing a large amphipathic flexible
loop implicated in membrane insertion, a small domain of unknown function
(domain 3), and a carboxy-terminal receptor-bindmg domain which is rich in
P-strands (domain 4) [39, 40]. The cell surface receptor for PA has been iden-
tified as a membrane protein containing a von Willebrand factor A domain that
is located in lipid rafts [41, 42]. Receptor-bound PA is activated at the cell sur-
face and clusters in lipid rafts, resulting in the formation of PA63 heptamers
that bind EF or LF. The complex is endocyLosed and transported to endosonial
compartments where the low pH induces a conformational change in the PA63
heptamers, leading to its insertion into the membrane and the formation of
water-filled channels. The translocation of EF and LF into the cell occurs by
different strategies. While LF is ftilly translocated into the cell cytoplasm, EF
remains membrane bound, exposing its catalytic domains to the cytosoiic com-
partment [43]. EF is an adenylcyclase, which is only active when associated
with calmodulin (fig. 1). Ca^^-bound calmodulin is much more efficient at acti-
vating EF than the Ca^"^-free form. The catalytic domain of EF is homologous
with B. pertussis adenylcyclase, and contains the consensus ATP binding motif
Popoff
38
(GxxxxGKS). The conversion of ATP by EF leads to an increase in intracellular
cAMP levels. These effects are reversible and transient, since EF is instable in
the cytosol. In human monocytes, EF enhances lL-6 production and inhibits
LPS-dependent tumor necrosis factor (TNF) synthesis. It has been speculated
that the main role of anthrax edema toxin is to impair the function of phago-
cytosing cells such as polymorphonuclear cells and macrophages, which may
facilitate the early stages of bacterial infection [37].
Arrest of Cell Cycle
Cytolethal distending toxins (CDTs) belong to a recently discovered family
of toxins, which cause irreversible cell cycle arrest and ultimately death of the
target cells. CDT was first described in 1987 when certain strains of E. coli
were found to cause cytopathic effects that were distinct from those induced by
E. coli toxins such as LT, ST, verotoxin, and hemolysm. Cells that are sensitive
to CDT first increase in size (3- to 5-fold), followed by a slowly developing cell
distention, that finally leads to cell death. Apart from E. coli, CDTs are produced
by a wide variety of gram-negative bacteria including Shigella, Hemophilus
ducreyi, Actinobacillus actinomycetemcomitans, H. pylori, and Campylobacter
[44]. Jn E. coli, it has been shown that CDT is encoded by three adjacent or
slightly overlapping genes, cdtA, cdtB, and cdtC, all of which are required for
the activity of the toxin. While CdtB contains the enzymatic activity, CdtA and
CdtC are required for the translocation of CdtB into the target cell. Internali-
zation of CDT from H. ducreyi occurs via endocytosis mediated by clathrin-
coated pits. The toxin has been shown to traffic through the Golgi apparatus
into the cytosol and the nucleus. The proposed mechanisms of action of CDTs
are not yet fully elucidated; however, it has been reported that the toxin blocks
cells in the G2 phase of the cell cycle by preventing dephosphorylation of the
inactive form of cdc2. In addition CdtBs possess DNase I activity that causes
double-strand DNA breaks (fig. 3) [45].
Apoptosis
Vacuolating Cy to toxin
The vacuolating cytotoxin (VacA) is one of the most important virulence
factors produced by H. pylori, a causative agent of severe gastric diseases such
as ulcers and cancer. VacA has been shown to induce large cytoplasmic vacuoles
in cultured cells and apoptosis in gastric epithelial and parietal cells. Cleavage
of the secreted VacA protein (95 kD) results in an amino-terminal 34- to 37-kD
(p37) and a carboxy-terminal 58-kD (p58) fragment that remain associated with
each other. The p58 fragment mediates VacA monomer binding to the target cell
via a GPI-anchored protein, which leads to VacA oligomerization in the membrane
and the formation of anion-selective channels that release bicarbonate, chloride
Bacterial Exotoxins
39
ADP-
ribosylation
C3
EDIN
Glucosylation Deamidation Proteolysis
Transglutamination
^3® CNF
clostridial ^^^ YopT
toxins
±
r
^
Rho. Rac, Cdc42
GDP
bound
Bacterial GAPs
SptP
YopE
ExoS
ADP-ribosylation
Clostridial binary toxins
SpvB
\
Bacterial GEF
SopE
GTP
bound
Glucosylation
Actin
filaments
Actin
monomers
/
RhoK
LT
Ras
Raf
Proteolysis
LF
J.
MAPKK
CDT
DNase
Nuclear factors
Proliferation, differentiation
— ^ Cell cycle arrest
Phosphatase
Cdc25C
> Cdc2-P
Fig. J< Bacterial toxins that modify intracellular signaling, actin cytoskeleton
rearrangement and cell cycle progression. Clostridial binary toxins and other toxins injected
by the type tit secretion system (SpvB) depolymerize actin filaments by ADP-ribosylation
of actin monomers. While the large clostridial toxins and C3 inactivate Rho-GTPases and
YopT impairs the translocation of Rho-GTPases to the membrane, CNF and DNT induce an
activation of Rho-GTPases. In contrast, SopE and YopE activate Rho-GTPases via a GEF
activity, or inactivate these molecules through a GAP activity, respectively. These factors are
involved in the coordinated remodeling of the actin cytoskeleton permitting the bacterial
invasion and the subsequent restitution of the normal cell architecture after bacterial entry,
C. sordelUi LT and anthrax LT (LF) downregulate the Ras signaling pathway by glucosylation
of Ras molecules (LT) or proteolysis of MAPK kinase, whose subsequent molecular mecha-
nisms and cell effects are still unclear. CDT interfere with the cell cycle through DNase
activity, which induces DNA damage and subsequent cell cycle arrest. CDT probably also
acts on the regulation of cyclin-dependent kinase (Cdc2) by converting this molecule to its
phosphorylated inactive form.
Popoff
40
and urea from the cell cytosol [46, 47]. VacA toxin channels are then internal-
ized and transported to the late endosomal compartments where they change the
anion permeability, leading to an enhancement of the vacuolar ATPase proton
pump activity [40, 41]. It has also been reported that the p34 fragment of VacA
targets mitochondria leading to the release of cytochrome c, activation of
caspase 3 and cell apoptosis [48].
Anthrax Lethal Toxin
B. anthracis LT is a zinc metaJ loprotease that causes hyperinflammatory
conditions in macrophages, the release of reactive oxygen intermediates, and
secretion of proinflammatory cytokines, such asTNF-a and Lnterleukin- 1 p [49].
LF (90 kD) is composed of 4 domains. As discussed for EF, domain 1 (amino
acids 1-254) consisting of a 12-helix bundle, is involved in the interaction
with PA. Interestingly, the structure of domain 2 is similar to that of the
catalytic domain o^ Bacillus cereus VIP2 (vegetative insecticidal protein) and
C. perfringens iota toxui (see below). However, LF is devoid of ADP-ribosylating
activity. Domain 3 forms a small helical bundle, which is required for the substrate
recognition and domain 4 (residues 552-776), consisting of a nine-helix bundle
packed against a four-stranded (S-sheet, contains the metal loprotease active site
(HExxH). Analysis of the ci7Stal stinjctui^e revealed that domains 2, 3 and 4 form
a long deep groove that holds the 16-residue amino-terminal tail of mitogen-
activated protein kinase kinase 2 (MAPKK-2) [50].
Subsequent studies have shown that MAPKK-2 is not the only target for LF,
since MAPKK-1 to 7 (except MAPKK-5) are also cleaved and inactivated by
this enzyme [51, 52]. In macrophages, LF also uihibits the extracellular signal-
regulated kinase (ERK), c-Jun N-terminal kinase (INK), and p38 MAPKs
pathways (fig. 3). While high concentrations of LF cause cell necrosis, low con-
centrations (200ng/ml) induce apoptosis m macrophages. However, in order to
trigger apoptosis, cells have to be activated, for mstance by LPS or other inflam-
matory mediators. Apoptosis of activated macrophages was found to be dependent
on p38 inactivation, however, the mechanism is not fully elucidated [53].
Alteration of Vesicular Traffic, Blockade of Neuroexocytosis,
Clostridial Neurotoxins
The mode of action of botulinum (BoNT) and tetanus (TeTx) neurotoxins
consists of four steps: binding, internalization, translocation and intracellular
activation [see also reviews 54—58], BoNT and TeTx recognize specific recep-
tors on unmyelinated areas of the presynaptic membrane. The precise identity
of neurotoxm receptors has still to be determined; however, gangliosides from
the Gib series and synaptic vesicle-associated proteins known as synaptotagmins
(a family of membrane-trafficking proteins) seem to be involved [59].
Bacterial ExotoxJns
BoNT/B
TeTx
Proteolysis
>
Inhibition of
exocytosis
BoNT/D, F, G
SNAP25
VAMP
Synaptic
iicle
.** .... B0NT/CI
: / \ \
_* ■ X
SynlaxJn
BoNT/E Bo NT/A
Assembly of
SNARE complex
Fig. 4. Toxins that interfere with vesicular traffic. BoNTs and TeTx are zinc-dependent
proteases, which cleave SNARE proteins (VAMP, SNAP25 and syntaxin) and result in
SNARE complexes with a reduced stability. This prevents synaptic vesicles from frjsing with
the presynaptic membrane.
Neurotoxin bound to its receptor is internalized by receptor-mediated
endocytosis. An essential difference between BoNTs and TeTxs is that the BoNTs
are directly endocytosed in clathrin-coated vesicles, resulting in a translocation
of the light chain into the cytosol. In the peripheral nervous system, the BoNT
light chain blocks the release of acetylcholine at the neuromuscular junctions,
leading to a flaccid paralysis. In contrast, TeTx is sorted to the fast axonal retro-
grade transport route, and delivered to the motoneurons, which are located in
the spinal cord. TeTx enters inhibitory interneurons probably via coated vesi-
cles, permitting the delivery of light chain into the cytosol where it inhibits the
release of glycine and GABA.
The light chains of clostridial neurotoxins contain a conserved zinc-
dependent proteolytic site (His-Glu-x-x-His) with endopeptidase activity [60,
61 ], It has been shown that the different neurotoxins preferentially target proteins
belonging to the SNARE (soluble N-ethylmaleirrude-sensitive flision protein
attachment protein receptors) family, comprising the three membrane-associated
proteins VAMP/synaptobrevin, SNAP-25, and syntaxin. While TeTx, BoNT/B, D,
F and G cleave VAMP/synaptobrevin and BoNT/A and E cleave SNAP25,
BoNT/Cl utilizes both SNAP25 and syntaxm as substrates (fig. 4). Each neuro-
toxm recognizes its substrate at specific bindmg sites termed SNARE motifs (two
in VAMP and syntaxin, and four in SNAP25), resulting in a cleavage pattern
which is characteristic for each toxin. It should be noted that TeTx and BoNT/B
cleave VAMP at the same site. While SNARE proteins are unstructured in solu-
tion, when they lie parallel to the membrane surface, they assemble in a ternary
complex (SNARE complex) consisting of four tightly packed a-helices.
Popoff
42
The SNARE complex is able to recruit a number of soluble cytosolic proteins
such as NSF (N-ethymaleimide-sensitive factor) and SNAPs (soluble NSF
accessory proteins). The resulting 20S SNARE complex has been recognized as
essential in vesicle targeting and fusion. It has been shown that this complex is
rapidly disassembled by NSF-dependent hydrolysis of ATR Assembly and disas-
sembly of SNARE proteins within the complex are thought to be essential in the
exocytosis process. Importantly, clostridial neurotoxins can only cleave SNARE
proteins when they are disassembled. The cleavage of SNARE proteins by
clostridial neurotoxins results in a reduction of SNARE complex stability and
impaired neurotransmitter release. Even though VAMP, SNAP25 and syntaxin
have different physiological properties at neuromuscular junctions, all clostridial
neurotoxins cause similar symptoms. However, the intensity and duration of
neurotransmission inhibition vary depending on the neurotoxin [56^ 57].
Alteration ofActin Cytoskeleton and Small G Protein Signaling
Toxins Active on Actin
Actin ADP-Ribosylating Toxins. Actin ADP-ribosylating toxins are binary
toxins which share a common structure, composed of two individual pro-
teins, a binding/translocation component and an enzyme component, which are
norJinked and assemble on the target cell. So far three families have been iden-
tified. The iota family, which encompasses iota toxin, produced by C, perfringens
type E, Clostridium spiroforme toxin and an ADP-ribosyltransferase synthe-
sized by some strains of Clostridium difficile. The second family (C2 family)
contains the C2 toxins expressed by Clostridium botulimim type C and D, which
have been shown to cause necrotizing enteritis and diarrhea. The third family
concerns the insecticidal binary toxins or VIP produced by B. cereus and Bacillus
thuringiensis [62].
The binding component binds to the surface of the target cell and is essen-
tial for the import of the toxin into the cell. For this, the binding component
has to be activated by protease cleavage. In solution, the binding components
of iota and C2 toxins (lb and C2-1I, respectively) can be processed by trypsin
or a-chymotrypsLii. However, unprocessed lb and C2-II can also bind to the
cell surface receptor, but do not mediate the entry of the enzymatic compo-
nent. The processed binding component recognizes specific cell membrane
receptors, heptamerizes and forms small ion-permeable channels that trap the
enzymatic component into endocytic vesicles. The enzymatic component is
subsequently translocated into the cytosol [63-66],
The enzymatic component catalyzes the ADP-ribosylation of actin
monomers at Argl77 but not of polymerized F-actin, since Argl77 is located in
the actin-actin binding site. The cumbersome ADP-ribose at the actin-binding
site prevents the nucleation and polymerization of ADP-ribosylated actin
Bacterial ExotoxJns
43
monomers. Moreover, ADP-ribosylated actin acts as a capping protein, it binds to
the barbed end of the actin filament and inhibits the further addition of unmodi-
fied actin monomers. Actin filaments depolymerize at the pointed end and the
released actin monomers are immediately ADP-ribosylated (fig. 3), In addition,
ADP-ribosylation inhibits the intrinsic ATPase activity of actin. Cell micro-
injection of ADP-ribosylated actin monomers induces the same effect as C2 or iota
toxin. This results in a complete disassembly of the actin filament and accumu-
lation of actin monomers [67, 68]. While the microtubules are unaffected, the
intermediate filaments are disorganized. As a consequence cells become rounded,
detach from the surface, and die [reviewed in 68^ 69]. Studies with epithelial and
endotheJial cells have shown that clostridial ADP-ribosylating toxins alter the
tight and adherens junctions resulting in a loss of cell barrier function [70, 71].
While toxins of the iota family modify aJl actm isoforms^ including cellular and
muscular isoforms, C2 toxins only interact with cytoplasmic and smooth muscle
7-actin. Substrates for VIP have not yet been reported.
Type III Secretion System-Dependent A DP-Ribosylating Toxins, Nontyphoid
Salmonella strains that are commonly associated with severe systemic infections
carry a large plasmid harboring spv genes, which are required for bacterial growth
in macrophages and monocytes. Among the four-gene operon (spvABCD), it has
been demonstrated that the spvB gene, encoding a 65,6-kD protein, is essential
for the virulence phenotype. Based on database searches it has been proposed that
SpvB has two iunctional domains, an amino-terminal domain related to the insecti-
cidal toxin Teal from Photorhabdus himinescens with an as yet unknown mecha-
nism of action, and a carboxy-terminal domain that shares homology with the
ADP-ribosylating part of iota, C2 and VIP Recombinant SpvB ADP-ribosylates
nonmuscle actin and microinjection of SpvB into CHO cells causes a breakdov/n
of actin filaments (fig. 3). In vivo studies have shown that SpvB is crucial for the
vLnjlence in mice while a mutant strain lacking the spvB gene shows marked
attenuation of virulence [72]. Evidence has been provided demonstrating that
SpvB is injected into host cells by a type HI secretion system. Once bacteria have
entered epithelial cells and macrophages, SpvB is expressed after 6h, and in
infected macrophages SpvB-dependent cytotoxicity is evident after 10-12h. Like
SptP, SpvB reverses the actin cytoskeleton reorganization mediating bacterial
entry, and permits the infected cells to regain their normal architecture after inva-
sion. Another ADP-ribosyJtransferase toxin that also targets actin and which is
secreted into the target cell by a type III system has been found in Aeromonas
salmonicida (AexT) [73].
Toxins Activating Small G Proteins
Enzymatic Modification of the GTPase Site, Some E, coli strains have been
shown to produce cytotoxic necrotizing factors (CNFs). To date, two variants
Popoff
44
termed CNFl and CNF2 have been characterized. CNFl is synthesized by
strains mainly isolated from human urinary infections and neonatal meningitis,
whereas CNF2 is produced by strains that infect animals [74].
Both factors are highly homologous at the amino acid level (86% identity)
and are produced as single-chain proteins with a molecular weight of about
llOkD. CNFs are related to the dermonecrotic factor (DNT) from Bordetella,
and homologous sequences to the cnfJ gene have been found in the genomes of
Yersinia pestis and Yersinia pseudotuberculosis. CNF toxins consist of three
functional domains: an amino-terminal domain (amino acids 1-299), which is
involved in the recognition of a cell surface receptor, a central domain (amino
acids 299-720) containing two hydrophobic regions which have been proposed
to translocate the toxin across the cell membrane, and a carboxy-terminal
(720-1,014) catalytic domain. TTie carboxy-terminal domain of CNFl has a
novel protein fold as determined by crystal structure analysis. This unusual
compact domain is formed by a central p-sandwich, that is composed of two
mixed |3-sheets, and surrounded by helices and extensive loop regions [75].
CNFl catalyzes the deamidation of Ghi63 in Rho and Gln61 in Rac and
Cdc42 to glutamic acid. Gln63/Gln61 are located in the switch II region of the
Rho protein. This region has an important function in the turn-off mechanism
of RhoGTPases and is essential for GTP hydrolysis by this family of proteins
[76, 77]. Thereby, CNFl blocks the RhoGTPases in their active form linked to
GTP. Studies with fibroblasts (Vero cells) have shown that CNFl causes dense
actin stress fibers and focal contact point formations, whereas in epithelial cells
(Hep2) the formation of lamellipodia and filopodia predominates. In both cell
types, CNFl leads to cell spreading resulting from the increase in actin filament
formation at the leading edge and anchorage of actomyosin filaments to focal
contact points. This is followed by contraction of these filaments in a similar
way to that seen in actin-based motility. These findings suggest that in epithe-
lial cells CNFl first activates Cdc42 and Rac followed by the activation of Rho,
whereas in fibroblasts activation of Rho is predominant [78].
Activation of RhoGTPases by CNFs is only transient and it has been shown
that deactivation of Rac correlates with an increase in the susceptibility of
its deamidated form to ubiquitin/proteasome-mediated degradation. During
the first phase of CNF intoxication, which corresponds to the activation of
RhoGTPases, uroepithelial cells begin spreading followed by intense membrane
ruffling. In the next phase of intoxication, lamellipodia are replaced by filopodia,
cells become highly motile, and there is an alteration in cellularjunction dynam-
ics. This probably favors bacterial internalization, which requires coordinated
RhoGTPase activation and inactivation for a maximal efficiency [79].
Type III Toxin-Activating RhoGTPases by Guanine Nucleotide Exchange
Factor Activity. Salmonella enters the cell by a trigger mechanism that induces the
Bacterial Exotoxins
45
formation of large membrane ruffles, which engulf the bacteria. The subsequent
rearrangements of the actin cytoskeleton and the plasma membrane are reminis-
cent of lameJIipodia and filopodia responses stimulated by various agonists such
as growth factors, hormones, or activated oncogenes. It has been demonstrated
that Cdc42 and to a lesser extent Rac are involved in the Salmon el la-dependent
cytoskeletal rearrangements. These effects are mediated by SopE, which is
delivered into the cell by a type III secretion system. Like guanine nucleotide
exchange factors (GEFs), SopE activates RacI, Rac2, Cdc42, RhoG, and also
to a lesser extent RhoA by catalyzing the exchange of GDP for GTP [80].
Interestingly, SopE2, an isoform of SopE, interacts with Cdc42 but not with
Racl [81].
SopE binds to the switch 1 and switch II regions of Cdc42 and promotes
guanine nucleotide release. This mechanism is similar to that used by the eukaryo-
tic Dbl-like exchange factor Tiaml in complex with Racl (fig. 3). However, the
catalytic domain of SopE has a different structure to that of Tiaml and interacts
with the switch regions via a GAGA motif [82]. SopE also acts as a GEF for Rab5
and mediates the recruitment of Rab5 in its GTP form to phagosomes containing
Salmonella. This promotes the fiision of these phagosomes with early endosomes,
preventing their transport to lysozomes and subsequent destruction [83]. In addi-
tion, activation of Cdc42 and Rac by SopE leads to stimulation of p2 1 -activated
kinase (PAK) and subsequent activation of JNK, the N4AP kinase pathway and a
number of transcriptional factors [80].
Toxins Inactivating Small G Proteins
ADP-Ribosylating C3 Exoenzyme. C. botulinum C3 exoenzyme belongs
to the family of Rho-ADP-ribosylating toxins. Other C3-like ADP-
ribosyltransferases have been identified in S. aureus and B. cereus and are
termed EDIN (epithelial differentiation inhibitor) and B. cereus exoenzyme,
respectively. It should be noted that genes encoding EDIN have a higher preva-
lence in S. aureus strains isolated from infection sites than in strains isolated
from nasal carriers [84].
The C3-like exoenzymes ADP-ribosylaLe Asn41, which is located within
the (3-strand, align next to the switch 1 region of the Rho-GTPases [85]. However,
the Asn41 residue is not accessible when Rho is associated with GDI (guanine
nucleotide dissociation inhibitor), resulting in a protein that is resistant to C3
exoenzyme ADP-ribosylation. Studies have shown that ADP-ribosylation of
Rho-Asn41 by C3 exoenzyme does not affect the activity of the protein [86, 87],
but prevents Rho translocation to the membrane that is required for its activation
and subsequent interaction with effector molecules [83]. While C3 exoenzyme
recognizes RhoA, B and C, but not RhoE, EDIN ribosylates all four proteins.
This results in the disassembly of actin filaments (fig. 3).
Popoff
46
Glucosylating Toxins. Glucosylating toxins, also referred to as large
clostridial toxins, are proteins with a molecular weight of approximately
250-300 kD. The family consists of C. difficile toxin A and B (ToxA, ToxB),
Clostridium sordellii LT and hemorrhagic toxin, and Clostridium novyi a toxin
(a-novyi). In Clostridium isolates that cause intestinal disease and myonecro-
sis, the toxins are considered to be the main virulence factors.
The glucosylating toxins are single chain proteins containing thi^ee func-
tional domains. In ToxA and ToxB, the carboxy-terminal domains contain mul-
tiple repeated sequences and are involved in cell surface receptor recognition.
A trisaccharide (Gal-al 3Gal-pl^GlcNac) has been found to be the motif
recognized by ToxA. The central domain contains hydrophobic sequences that
are thought to mediate the translocation of the toxin across the membrane and
the enzymatic and cytotoxic activity (DxD motif) of the toxins is found at the
amino-terminus. Sequence analysis has revealed that ToxB and LT are highly
homologous (76% amino acid sequence identity) and are more distantly related
to ToxA and a-novyi (48-60% identity) [88].
The large clostridial toxins enter cells by receptor-mediated endocytosis.
The cytotoxic effects are blocked by endosomal and lysosomal acidification
inhibitors (monensin, bafilomycin Al, ammonium chloride) and the inhibiting
effects can be bypassed by an extracellular acidic pulse. This indicates that the
large clostridial toxins translocate from early endosomes upon an acidification
step. At low pH, ToxB and LT induce channel formation in cell membranes and
artificial lipid bi layers, and show an increase in hydrophobicity [89, 90]. This
is thought to involve a conformational change and insertion of the toxin into the
membrane mediated by the hydrophobic segment of the central domain.
Large clostridial toxins catalyze the glucosylation of 21-kD G proteins
using UDP-glucose as the sugar donor (with the exception of a-novyi that prefer-
entially uses UDP-N-acetylglucosamine) (fig. 3). The toxins transfer the glucose
or N-acetylglucosamine moiety to the acceptor amino acid Thr37 of Rho or
Thr35 of Rac, Cdc42 and Ras proteins [91, 92]. Rho complexed to GDI is not a
substrate for glucosylation, and modified Rho does not bind to GDI [93].
The conserved glucosylated Tin* (Thr37/35) is located in switch I of
Rho/Ras GTPases. Thr37/35 is involved in the coordination of Mg^"^ and sub-
sequently to the binding of the (3 and -y phosphates of GTR The hydroxy! group
of Thr37/35 is exposed at the surface of the molecule in its GDP-bound form,
which is the only accessible substrate for glucosylating toxins. Crystal structure
analysis of Ras modified by LT shows that glucosylation prevents the forma-
tion of the GTP conformation in the effector loop of Ras, which is required for
the interaction with the effector Raf [94]. Similar results were found when
RhoA glucosylation by ToxB was studied [91]. It has been shown that glucosyl-
ation of GTPase by the toxins reduces the intrinsic GTPase activity, completely
Bacterial Exotoxins
47
inhibits GTPase-activating protein (GAP)-stimulated GTP hydrolysis, and leads
to accumulation of the GTP-bound form of Rho at the membrane [93, 95].
The modification of Rho proteins by the large clostridial toxins induces
cell rounding, the loss of actin stress fibers, reorganization of cortical actin, and
disruption of the intercellular junctions. ToxB and ToxA have been reported to
trigger apoptosis as a consequence of Rho glucosylation. In addition to the
effects on the cytoskeleton, the inactivation of Rho proteins impairs other
cellular functions such as endocytosis, exocytosis, NADPH oxidase regulation,
and transcriptional activation mediated by JNK and/or p38 [88].
Proteolytic Toxins. YopT is one of the six Yop effector proteins which are
injected into host cells by the Yersinia type HI secretion system. This protein
inactivates Rho-GTPases leading to the disruption of actin filaments and the
accumulation of inactive RhoA in the cytosol (fig. 3). Recently, it has been
reported that YopT is a cysteine protease that cleaves prenylated Rho-GTPases
near their carboxy-termini and results in the release of these proteins from the
membrane [96].
Rho-GTPases Inactivating Toxins by GAP Activity. As discussed earlier,
Salmonella enters nonphagocytic cells by delivering effector proteins, such as
SopE, into the host cell cytosol by the type ill secretion system that directly
modulates host actin dynamics to facilitate bacterial uptake. Importantly, the
infected cells quickly recover from the above-mentioned cytoskeletal rearrange-
ments. It has been shown that the reversal of actin cytoskeleton rearrangements
is promoted by SptP, another type Ill-secreted protein, which acts as a GAP for
Cdc42 and Rac.
SptP is a modular molecule that consists of an amino-terminal domain
that shares sequence similarity with YopE of Yersinia spp. and ExoS of
P. aeruginosa and binds to Rac and Cdc42 but not Rho in the GTP-bound
form. The carboxy-terminal domain is related to YopH and several eukaryotic
tyrosine phosphatases [97]. Crystal structure analysis revealed that SptP binds
Racl exclusively through an amino-terminal four-helix bundle domain that
targets the nucleotide and both the switch I and switch II regions of the
GTPase.
Interestingly, eukaryotic GAPs show a larger surface of interaction with
Rho-GTPases than SptP. This outlines the minimal structure involved in the
GAP activity and argues for a convergent evolution of eukaryotic and bacterial
GAPs. SptP binding to Rac does not change the conformation of the carboxy-
terminal domain (tyrosine phosphatase domain). It is possible that the GAP
domain targets the tyrosine phosphatase to its relevant substrate(s) [98, 99] and
it has been speculated that the tyrosine phosphatase activity of SptP is involved
in the downregulation of the subsequent nuclear response to Cdc42 and Rac
stimulation [ 1 00]. While SopE is rapidly degraded by the proteasome pathway, the
Popoff
48
degradation kinetics of SptP is much slower, permitting the transient reorgani-
zation of the actin cytoskeleton involved during bacterial invasion [101].
YopE from Yersinia, E\oS and ExoT from F aei^ginosa are secreted into
macrophages by the type III secretion system and display a GAP activity towards
Rho-GTPases. Despite the fact that the amino acid sequences are not highly
conserved among these proteins, the GAP domains of YopE and ExoS show a
similar structure to that of SptP with a conserved Arg finger that is essential for
activity [102]. These factors induce actin cytoskeleton disorganization and cell
rounding, and support the antiphagocytic activity permitting the survival of the
bacterium [103, 104]. In addition, ExoS exerts an ADP-ribosyltransferase activity
towards several proteins including Ras [105],
Concluding Remarks
Whilst most of the bacterial toxjns form pores that act on cell membranes,
many of them have the ability to enter host cells and enzymatically modify
intracellular targets. As discussed in the present review, while some toxins con-
tain specific translocation domains that attach to the cell membrane forming
small pores, others lack such domains and are directly injected into cells by a
type III secretion system.
Over the last years, evidence has accumulated showing that many bacter-
ial toxins interfere with physiological processes by modulating host effector
systems. In contrast to the host, bacteria, however, are not able to regulate these
cascades, since toxins often act in an uncontrolled manner. This may trigger a
noxious amplification of the signal and lead to severe systemic complications
from the infection. Among the numerous potential cellular targets, bacterial
toxins have only selected some key physiological pathways, such as the inacti-
vation of EF and ribosomal RNA, leading to the inhibition of protein synthesis,
as well as interfering with cell homeostasis by stimulating the overproduction
of secondary messengers. It is also interesting to note that even though the regu-
lation of acLin polymerization requires a large number of proteins, bacterial
toxins only act on two essential targets, namely monomeric actin and Rho-
GTPases.
Many toxins target the same host effector systems; however, the physio-
logical effects can differ from species to species. For instance, Clostridium
secretes actin-modifying exotoxins, which act at a distance from the bacterium
and disrupt cell barriers and tissues, permitting massive bacterial colonization
of necrotic tissues. In contrast, some bacteria use specific toxins at the site of
infection which interfere with the cytoskeleton, facilitatmg the invasion into
target cells or preventing phagocytosis.
Bacterial Exotoxins
49
Finally, the specificity of bacterial toxins makes these molecules highly
attractive as potential therapeutic agents (for instance, botulinum neurotoxins
and ijnmunotoxinsX valuable tools in cell biology and the vectorization of
molecules into cells.
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Michel R. Popoff
Unite des Bactdries ana6robies et Toxines, Institut Pasteur
28 rue du Dr Roux, FR-75724 Paris Cedex 15 (France)
Tel. +33 1 456838307, Fax +33 I 40613123, E-Mail mpopoff@pasteur.fr
Popoff
54
Toxins
Russell W, Herwald H (eds): Concepts in Bacterial Virulence!
Contrib Microbiol. Basel, Karger, 2005, vol 12, pp 55-66
Capsular Polysaccharides and
Their Role in Virulence
Clare M. Taylor, Ian S. Roberts
School of Biological Sciences, University of Manchester, Manchester, UK
Bacterial pathogens exhibit a number of virulence factors that enable them
to invade and colonize the tissues of host organisms. A number of these viru-
lence factors are displayed on the cell surface and include adhesins that medi-
ate attachment to host cells, toxins that may be secreted resulting in host tissue
damage, and the possession of molecules that render them resistant to host
antimicrobial defences. Capsular polysaccharide (CPS) has long been recognized
as an important virulence determinant in isolates capable of causing infection
in humans and animals [1]. CPS is found on the outermost surface of a wide
range of the bacteria [2] and may be linked to the cell surface via covalent
attachments to phospholipid or lipid A molecules [3]. In contrast, extracellular
polysaccharide (EPS) molecules appear to be released onto the cell surface with
no visible means of attachment. Such EPS can be loosely associated with the
cell surface and easily sloughed off as slime.
CPS molecules are highly hydrated and typically constitute more than 95%
water [4]. They are composed of repeating single monosaccharide units that are
joined by glycosidic linkages. CPS may be homo- or heteropolymers and can be
substituted with both organic molecules such as acetyl groups, and inorganic mole-
cules such as phosphate. In addition, two monosaccharides may be joined m a
number of configurations due to the presence of multiple hydroxyl groups within
each monosaccharide that may be involved in the glycosidic linkage. Thus, CPS
are a diverse range of molecules that can differ not only in their constituent mono-
saccharides but also in the manner in which they are joined. This diversity is illus-
trated in bacterial species such as Escherichia coli where over 80 distinct capsular
serotypes have been described while in Streptococcus pneumoniae , there are over
90 capsular serotypes. The introduction of branches and substitution with organic
or inorganic molecules to polysaccharide chains adds a further layer of structural
complexity. However, chemically identical CPS may also be synthesised by
different bacterial species. The group B capsule of Neisseria meningitidis, a homo-
polymer of ct2, 8-1 inked N-acety I neuraminic acid (NeuNAc), is identical to the Kl
antigen oiE. coli [5], while the CPS of Pas teu re I la muitocida type D is identical
to the E. coli K5 capsule which comprises repeating disaccharides of glucuronic
acid linked to N-acetylglucosamine [6]. The apparent conservation of particular
CPS structures between taxonomically diverse genera of bacterial species raises
intriguing questions regarding the evolution of capsule diversity and the acquisi-
tion of capsule biosynthesis genes.
Functions of Bacterial Capsules
As the polysaccharide capsule represents the outermost layer of the bacte-
rial cell, it is not surprising that the capsule mediates interactions between the
bacterium and its immediate environment. Accordingly, a number of functions
has been ascribed to bacterial capsules. Each of these functions (resistance to
desiccation, adherence, resistance to nonspecific host immunity, resistance to
specific host immunity) is directly relevant to pathogenicity and as such con-
tributes to the role of CPS as a virulence factor.
Resistance to Desiccation
As CPS are highly hydrated molecules that surround the cell surface, they
may protect bacteria from the harmful effects of desiccation [7]. This property is
probably most relevant in the transmission and survival of encapsulated bacteria
in the environment demonstrated in the cases of isolates off. coli, Acinetobacter
calcoaceticus and Erwlnia stewartii, which have been shown to be more resis-
tant to desiccation than their isogenic acapsular mutants [8]. Furthermore, the
capsule probably provides protection during transmission from host to host. In
the case of E. coli, genes encoding enzymes for the biosynthesis of capsular
colanic acid have been shown to be upregulated m response to desiccation [8].
While the mechanism of regulation is unclear, it is thought that external osmo-
larity is altered during desiccation, and it has been shown tliat expression of
alginate EPS of Pseudomonas aeruginosa as well as expression of the Vi CPS
of Salmonella typhi, which is essential for virulence, are increased in response
to high osmoJarity [9, 10].
Adherence
CPS may mediate adhesion of bacteria to surfaces (both biotic and abiotic)
and to each other. Adhesion to abiotic surfaces may result in the establishment of
biofihns and EPS-mediated interspecies co-aggregation within biofilms can
enhance colonization of various ecological niches [11]. In addition, growth of
Taylor/Roberts
56
bacteria as a biofilm may offer some protection from phagocytic protozoa and pre-
sent nutritional advantages, while it is thought that the presence of EPS acts as a
permeabiMty barrier against antimicrobial agents [12]. While adhesion to host tis-
sues is undoubtedly a multifactorial process involving an array of bacterial surface
components, CPS has been implicated in the adhesion of a number of human
pathogens to host tissues. Streptococcus pyogenes or group A Streptococcus
(GAS) is responsible for a range of clinical infections including skin infections,
acute rheumatic fever, streptococcal pharyngitis, streptococcal toxic shock syn-
drome and necrotizing fasciitis [13, 14]. In the development of pharyngitis, colo-
nization of the pharynx by streptococci not only represents a vital stage in the life
cycle of GAS, but it is also likely that that the pharynx serves as a reservoir for
infection from which GAS may be disseminated to other hosts as well as causing
invasive infections such as necrotizing fasciitis. It has been demonstrated that
the hyaluronic acid capsule of GAS binds to CD44 molecules on the surface of
human keratinocytes, the predominant cell type in skin and the pharyngeal
epithelium [15]. Once bound, bacterial contact with the epithelial surface
induces lamellipodia formation on the surface of keratinocytes, which is not
observed in an isogenic acapsular mutant [16]. Gram-negative pathogens such
as Salmonella and Shigella spp. also induce lamellipodia formation following
binding to host epithelial cells; however subsequent fusion of the lamellipodia
entraps the bacteria, resulting in their internalization. GAS are inefficiently
internalized as a consequence of the possession of their hyaluronic acid cap-
sule. Furthermore, the binding of GAS to CD44 induces marked cytoskeletal
rearrangements and cell signalling events leading to the opening of intercellular
junctions, which is thought to promote tissue penetration by GAS [16]. Clearly
this is not the case for all encapsulated pathogens, as the case of GAS involves
molecular mimicry, with the CPS being identical to host hyaluronic acid. In
other pathogens, initial attachment to host cells has been shown to be inhibited
by encapsulation, as is the case for binding oi Klebsiella pneumoniae to epithelial
cell lines in vitro [17]. Paradoxically, encapsulated isolates of the same straui
adhered better to a mucus-producing cell line than an acapsular mutant. These data
suggest tliat in some cases the CPS may promote initial colonization of the mucus
layer, while subsequent interaction with the underiying epithelial layer is reduced
by the presence of a capsule, presumably due to the masking of bacterial
components required for specific interaction with the epithelial surface. These
observations support the notion that there is some form of co-ordinate regulation
of capsule expression during the early stages of infection.
Resistance to Non-Specific Host Immunity
During invasive infections of humans and animals by encapsulated
pathogens, interactions between the bacterial CPS and immune system of the
Capsules and Virulence 57
host play a critical role in determining the fate of the infection [18]. During
an innate host response, the bacterial capsule may confer some resistance to
complement-mediated killing. The main function of the complement system is
the binding of host peptides to foreign organisms. Once bound, these are rec-
ognized by specific complement receptors on host phagocytes that facilitate
opsonization and subsequent destruction. Thus, activation of the complement
cascade involves an array of serum and cell surface proteins and three pathways
of activation are recognized. In the classical pathway, an antibody response is
generated, while the alternative pathway can be activated in the absence of spe-
cific antigen-antibody recognition. The mannan-binding lectin pathway recog-
nizes surface polysaccharides and then activates the complement cascade [19].
These pathways generate C3 convertases that cleave C3 (the major complement
component) to C3b, which can then bind to the cell surface. Factor C3b and its
degradation product iC3b are the primary complement opsonins [20]. In the
absence of specific antibody, CPS is thought to activate the alternative pathway
in which C3b binds non-specifically to the bacterial surface. Bound C3b is then
activated by interaction with factor B and forms the C3 convertase C3bBb,
which binds to the bacterial surface along with further C3b. This complex
termed C3b2Bb acts as the C5 convertase and promotes formation of the mem-
brane attack complex (MAC), which can form pores in certain bacteria, causing
their destruction.
CPS that contain NeuNAc are known to be poor activators of the alternative
pathway [21 , 22] and it is thought that this is because NeuNAc binds directly to
factor H [21]. Bound factor H promotes the binding of factor 1 to C3b, forming
iC3b, which breaks the amplification loop of the cascade, which in turn prevents
formation of the MAC [23]. In such cases, the bacterial capsule usually acts in
concert with other surface structures such as the 0-antigen of lipopoly saccharide
to confer resistance to complement-mediated killing [24]. Thus, a particular
combination of surface structures can confer a high degree of resistance to the
innate immune response. In the case of other encapsulated pathogens, it is thought
that the presence of a CPS may actually provide a barrier to complement compo-
nents by pJiysically maskhig underlying surface stiiictures that would normally be
potent activators of the alternative pathway [24].
Finally, CPS may confer resistance to complement-mediated opsonophago-
cytosis. In the case of Staphylococcus aureus^ the presence of a thick capsule has
been shown to be antiphagocytic, as it interfered with recognition of cell-bound
C3b and iC3b by phagocytic receptors [25]. Shnilar observations have been
made in the case of yS*. pneumoniae where CPS also appears to block cell-bound
C3b [26]. Furthermore, many CPS are highly negatively charged molecules and
may also confer resistance to phagocytosis [1, 27, 28]. In addition to these direct
interactions between CPS and components of the complement system, certain
Taylor/Roberts
58
CPS may modulate the host's immune system by stimulating the release of
certain cytokines resulting in the disruption of the cell-mediated immune response
[29]. One such example is the CPS of K. pneumoniae, which was shown to
induce high levels of interleukin-lO (IL-10) in experimentally infected mice, in
contrast to an acapsular mutant [30]. High levels of lL-10 inhibit gamma
interferon-induced activation of macrophages, and therefore cell-mediated
reactions such as delayed-type hypersensitivity, which are normally visible
24—48 h after infection.
Resistance to Specific Host Immunity
Although many CPS elicit a specific (antibody-mediated) immune response
in the host, a certain small set of CPS are able to confer some resistance.
Capsules such as those that contain NeuNAc, e.g. E. coli K 1 and N. meningitidis
serogroup B [31] in addition to the E. coli K5 polysaccharide which is identical
to N-acetyl heparosan (precursor in heparin/heparan sulfate biosynthesis) [32],
are poorly immunogenic. Infected individuals only mount a poor immune
response to these antigens as a consequence of the structural similarities of these
capsules to host polysaccharides encountered abundantly in the extracellular
matrices [18, 33]. As a result, the expression of these capsules that mimic host
structures provides protection against the specific arm of the host's immune
response.
Polysaccharide Capsules of Pathogenic E. coll
A large number of capsule gene clusters, representing various capsular
serotypes, have been identified and cloned from a number of gram-negative
pathogens. In all cases, the capsule genes are clustered at a single locus allow-
ing for the co-ordinate regulation of capsule gene expression. Each of the cap-
sule serotypes appears to be represented within E. coli, and to date, E. coli
capsules are amongst those most intensively studied. Thus capsule gene clus-
ters of E. coli are regarded as a paradigm for capsule gene clusters in gram-
negative bacteria.
As previously mentioned, over 80 different serologically and chemically
distinct types of polysaccharide capsule have been described in E. coli [34].
Termed K antigens, these have been classified into four functional groups
(table 1) based on a number of biochemical and genetic criteria [35]. Most
pathogenic extra-intestinal E. coli express group 2 K antigens [2]. Group 2
CPS represent a heterogeneous group concerning composition, while in
terms of structure and cell surface assembly they resemble the capsules of
other gram-negative pathogens, N. meningitidis and Haemophilus influenzae.
Capsules and Virulence 59
Table L Classification of £. coli capsules [adapted from 34]
Characteristics
Group
1
2
3
4
Foriner K antigen
lA
11
1/n or 111
IB (0-antigen
group
capsules)
Co-expressed with
Limited range
Many
Many
Often 08, 09 but
serogroups
(08,09,020,0101)
sometimes none
Co-expressed with
No
Yes
Yes
Yes
colanic acid
J hermostabiJity
Yes
No
No
Yes
lerminal lipid moiety
Lipid A core
OL-
ot-
_.ipid A core in
in Klps; unhiown
G ycerophosphate
Glycerophosphate
Klps; unknown
for K antigen
for K antigen
Direction of chain
Reducin:^ terminus
Non-reducing
Non-reducing
Reducing
growth
terminus
terminus?
terminus
Polymerization system
Wzy-dependent
Processive
Processive?
Wzy-dependent
Transplasma
Wzx (PST)
ABC-2 exporter
ABC-2 exporter?
Wzx (PST)
membrane export
E evated levels o:^
No
Yes
No
No
CMP-Kdo synthetase
Genetic locus
cps near his and rfb
kps near serA
kps near serA
rfb near his
Thermoreeulated (not
No
Yes
No
No
expressed below 20°C)
Positively regulated
Yes
No
No
No
by Res system
Model system
K30
K1,1C5
KIO, K54
K40, 0111
Sijnilar to
Klebsiella^ Erwinia
Neisseria,
Haemophilus
Neisseria,
Haemophilus
Many genera
A model for assembly and attachment of group 2 capsules to the cell surface
is shown in figure I .
Genetic Organization and Regulation of £. co/i Group 2
Capsule Gene Clusters
A number of group 2 capsule gene clusters have been cloned, and analysis
has revealed that they have a conserved modular genetic organization, consisting
Taylor/Roberts
60
Biosynthesis of
Phosphatidyl-Kdo (PA-Kdo)
Ligation of PA-Kdo
^
Polymerization
initiation on an
acceptor
OM
o
Phosphatidic acid
2-Keto-deoxymanno-octonic
acid (Kdo)
Repeat monosaccharide
Fig. L A model for the assembly of group 2 capsules. Polysaccharide is poJymerized at
the non-reducing terminus and subsequently ligated to phosphatidyl-Kdo (PA-Kdo) at the reduc-
ing end prior to export across the cytoplasmic membrane. The presence of PA-Kdo nnay act as
a motiF for the export proteins as structurally diverse group 2 polysaccharides are all exported
via the same conserved export proteins. LM = Inner membrane; OM = outer membratie.
of three functiona] regions (fig. 2). Furthermore it appears that this modular
organization is applicable to capsule gene clusters of other bacteria [2]. Gene
expression is achieved following transcription from two convergent promoters
PI and P3 which flank regions I and 3, respectively. Regions I and 3 are con-
served amongst group 2 gene clusters and encode proteins necessary for the
transport of the polysaccharide from its site of synthesis to the cell surface.
Region 2 is serotype specific and encodes the enzymes responsible for biosyn-
thesis (where necessary) and polymerization of the individual monosaccharides
that comprise the particular polysaccharide. The size of this region is variable;
however, size is thought to reflect the complexity of the polysaccharide, as
region 2 in isolates that produce CPS with complex structures often encodes a
larger number of open reading frames [36].
Region 1 comprises 6 genes kpsFEDUCS organized in a single transcrip-
tional unit (fig. 2) that encode proteins involved in transport of the polysaccharide.
A single E. coli of^^ promoter (PI) has been mapped 225 bp upstream of /g^^Fand
transcription from PI generates an 8.0-kb polycistronic transcript that is subse-
quently processed to generate a stable 13-kb A??55'-specific transcript [37]. This
may facilitate the differential expression of KpsS, which may mfluence the
attachment of phosphatidyl-Kdo (2-keto-3-deoxymanno-octonic acid) to nascent
Capsules and Virulence
61
H-NS/BipA
-4-
> "♦■
kps F E D U C
T M
kfi D C
B
A
Fig. 2. Genetic organization and regiilarion of £". coli group 2 capsule gene clusters. In
this example, the gene cluster of £'. coli K5 is shown. The numbers at the top refer to the three
functional regions; the serotype-specific region, region 2, is shaded. PI and P3 represent the
region 1 and 3 promoters, respecrively, and the straight arrows denote the major transcripts.
polysaccharide and regulate its entry into the export machinery. An intragenic
Rho-dependent transcriptional terminator has also been identified within fqysF,
This may play a role in regulating transcription by preventing synthesis of
untranslated region 1 transcripts under conditions of physiological stress [38].
Region 3 of the gene cluster contains two genes kps M and /^^ 7" organized
in a single transcriptional unit [2, 39]. The promoter (P3), which has a typical
E. coli a70 -10 consensus sequence but no -35 motif, has been mapped 741 bp
upstream of the initiation codon of I<psM, No consensus binding sites for other
alternative a-factors or other DNA-binding proteins have been identified [40].
However, region 3 is subject to control by an antitermination process, conferred
by RfaH and ops elements. A cis-act'mg regulatory sequence termed ops, which
is essential for the function of RfaH has been identified 33 bp upstream of the
initiation codon of kpsM [40]. The ops element^ with the sequence GGCGGTAC,
is contained within a larger regulalory element of 39 bp termed JHLTMPsLart (just
upstream from many polysaccharide-associated gene starts) [41]. RfaH is known
to regulate a number of gene operons in E. coli including the hemolysin operon
and the gene clusters for LPS core and 0-antigen biosynthesis [42, 43]. In addi-
tion, RfaH is a homolog of NusG, an essential transcription elongation factor
that is necessary for Rho-dependent transcription termination and bacteriophage
\-N-mediated antitermination. RfaH is thought to act as a transcriptional elon-
gation factor that allows transcription to proceed over long distances. As such,
mutations in rfaH gwQ rise to increased transcription polarity throughout RfaH-
regulated operons without disrupting initiation from operon promoters [42].
Taylor/Roberts
62
To act, ops elements must be located on the nascent mRNA transcript, where they
recruit RfaH, and perhaps other proteins, promoting transcription elongation. It
is thought that the JUMPstart sequence on the mRNA molecule may permit the
formation of stem-loop structures at the 5' end, which mediate the interaction
with RfaH [41]. A mutation in rfaH or deletion of the JL'MPstart sequence has
been shown to abolish capsule production in E, coli Kl and K5 [40] and serves
to confirm the role of RfaH in the regulation of group 2 capsule gene clusters.
The genetic organization of region 2 is serotype specific and differs among
group 2 K antigens. In the case of K5, region 2 comprises 4 genes kfiABCD
[44] while Kl comprises 6 genes neiiDBACES [39], In each case, transcription
of region 2 proceeds in the same direction as that of region 3, which is impor-
tant in the RfaH-mediated regulation of region 2 expression [40].
A feature of group 2 capsule gene expression pertinent to pathogenicity is
that transcription from both PI and P3 is temperature regulated, enabling capsule
expression at 37°C but not at 18°C [37, 45]. Temperature regulation is in part
achieved via the action of the global regulatory protein H-NS (histone-like
nucleoid-associated protein), since hns mutants show comparable levels of tran-
scription from PI at both 18 and 37°C^ albeit lower than those usually seen in a
wild-type strain at 37''C, indicating that H-NS is required for maximal transcrip-
tion at 37°C as well as repression at 1 8°C [46]. This situation is analogous to the
H-NS-mediated thermoregulation of the virB promoter in Shigella flexneri. In
this system however, activation of the virB promoter has an absolute requirement
for the AraC-like protein VirF [47]. It is not yet clear whether an AraC-like tran-
scriptional activator is involved in modulating transcription from PI and P3.
Mutations in bipA also result in increased transcription at 18°C and reduced
transcription at 37°C [44]. Although this phenomenon mirrors the effect of muta-
tions in hns^ the phenotype of a bipA mutant cannot be explained by a loss of
H-NS function as this is unaffected in a bipA mutant. BipA was first described as
a tyrosine-phosphorylating protein in enteropathogenic E, coli (EPEC) [48].
EPEC bipA mutants are unable to trigger cytoskeletal rearrangements in host cells,
are hypersensitive to BPI (bactericidal/permeability-increasing protein) protein
and demonstiate increased flagella expression and motility [48], Fuithermore,
BipA is a GTPase with similarity to the TetO resistance protein and elongation
factor G (EF-G), both of which interact with ribosomes. These data have led to
the suggestion that BipA may represent a novel class of regulators that interact
directly with the ribosome by regulating translation elongation [48]. It is therefore
likely that BipA does not regulate PI and P3 directly, but that regulation is
achieved via interaction with other proteins that do modulate transcription of PI
and P3. This hypothesis is currently under investigation in our laboratory.
At 37°C, the mechanism of temperature regulation is further complicated
by the interaction of integration host factor (IHF) with PI . IHF is required for
Capsules and Virulence
63
optimal capsule gene expression and IHF binding sites have been identified that
flank P I [40], IHF usually acts to facilitate the activity of other regulatory proteins
[49] and as such it is likely that LHP also acts in concert with an as yet unidenti-
fied regulatory protein or proteins that act to control transcription from regions
1 and 3 at 37°C. However, the lack of IHF binding sites in the region 3 promoter
[40] demonstrates that the requirement for IHF is not absolute,
Ln summary, the regulation ofE. coli group 2 capsules is complex, hivolving
a number of overlapping regulatory circuits. However, of relevance to patho-
genicity and virulence, there are still many unanswered questions. How are
changes in temperature, such as those concomitant with entry into a susceptible
host, sensed and transduced to induce changes in gene expression? How is cap-
sule gene expression regulated in response to attachment and interaction with
host cells? While it is known that encapsulation is an important virulence fac-
tor, understanding the regulation of capsule expression during the stages of
infection still represents an interesting challenge. One fiirther important area
for fijture research is the understanding of the export of CPS onto the bacterial cell
surface. Such understanding will lend itself to the design of chemotherapeutic
agents targeted to selectively disrupt capsule export and therefore combat infec-
tions caused by encapsulated bacteria.
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17 Favre-Bonte S, Joly B, Forestier C: Consequences of reduction oi Klebsiella pneumoniae capsule
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21 Michaiek M, Mold C, Bremer E: Inhibition of the alternative pathway of human complement by
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22 Stevens P, Huang SNH, Welch WD, Young LS: Restricted complement activation by Escherichia
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23 Frank M, Joiner K, Hammer C: The function of antibody and complement in the lysis of bacteria.
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24 Howard CJ, Glynn AA: The virulence for mice of strains of Escherichia coli related to the effects
of IC antigens on their resistance to phagocytosis and killing by complement. Immunology
1971;20:767-777.
25 Cunnion M, Lee JC, Frank MM: Capsule production and growth phase influence binding of com-
plement to Staphylococcus aureus. Infect Immun 2001;69:6796-6803,
26 AbeytaM, Hardy GG, YotherJ: Genetic alteration ofcapsule type but not PspA type affects acces-
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27 Brown EJ, Joiner KA, Garther TA, Hammer CH, Frank MM: The interaction of C3b bound to
pneumococci with factor H (beta IH globulin), factor I (C3b/C4b inactivator), and properdin
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28 Hor\vilz MA, Silvei'stein SC: Influence of the Escherichia coli capsule on complement fixation
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29 Cross A: The biological significance of bacterial encapsulation. Curr Top Microbiol Immunol
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30 Yoshida K, MatsumotoT,TatedaIC, Uchida K,Tsujimoto S,Yamaguchi K: Induction of interleukin-
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31 Bhanacharjee A, Jennings H, Kenny C, Martin A, Smith I: Structural determination of the sialic
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32 Vann WF, Schmidt M, Jann B, Jann K: The struchjre of the capsular polysaccharide (K5 antigen)
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33 Wyle F, Artensteiji M, Brandt BL, Tramont EC, Kasper DL: [mmunological response of man to
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35 Whitfield C, Roberts IS: Structure, assembly and regulation of expression of capsules in Escherichia
coli. Mol Microbiol 1999;31:1307-1319.
36 Boulnois G, Drake R, Pearce R^ Roberts 1: Genon^e diversity at the serA-linked capsule locus in
Escherichia coli. FEMS Microbiol Lett 1992;100:121-124.
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of region 1 of the Escherichia coli K5 capsule gene cluster. J Bacteriol 1996;178:6466-6474.
38 Richardson JP: Preventing the synthesis of unused transcripts by Rho factor. Cell I99l;64:
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Escherichia coli Kl. Mol Microbiol I996;2I :22I-23L
40 Stevens MP, Clarke BR, Roberts IS: Regulation of the Escherichia coli K5 capsule geae cluster
by transcription antiterminalion. Mol Microbiol 1997;24:1001-1012.
41 Hobbs M, Reeves PR: The JUMPstart sequence: A 39 bp element common to several poly-
saccharide gene clusters, Mol Microbiol 1994;12:855-856,
42 Bailey MJ, Hughes C, Koronakis V: RfaH and the ops element, components of a novel system
controlling bacterial transcription elongation. Mol Microbiol 1997;26:845-851.
43 Marolda CL, VaJvano MA: Promoter region of the Escherichia coli 07-specific I ipopoly saccharide
gene cluster: Structural and functional characterization of an upstream untranslated mRNA
sequence. J Bacteriol 1998;180:3070-3079.
44 Petit C, Rigg GP, Pazzani C, Smith A, Sieberth V, Boulnois G, Jann K, Roberts IS: Region 2 of
the Escherichia coli K5 capsule gene cluster encoding proteins for the biosynthesis of the K5
polysacchai'ide. Mol Microbiol 1995;17:611-620,
45 Cieslewicz M, Vimr E: Thermoregulation of kpsF, the first region 1 gene in the kps locus for
polysialic acid biosynthesis in Escherichia coli KL J Bacteriol 1996;178:3212-3220,
46 Rowe S, Hodson N, Griffiths G, Roberts IS: Regulation of the Escherichia coli K5 capsule gene
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Microbiol 1998;28:265-279.
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Clare M. Taylor
School of Biological Sciences, 1.800 Stopford Building, University of Manchester
Oxford Road, Manchester, M 13 9PT (UK)
Tel. +44 161 2755601, Fax +44 161 2755656, E-Mail clare.taylor@man.ac.uk
Taylor/Roberts
66
Adhesins
Russell W, Herwald H (eds): Concepts in Bacterial Virulence.!
Contrib Microbiol. Basel, Karger, 2005, vol 12, pp 67 89
Fimbriae, Pili, Flagella and
Bacterial Virulence
Ann-Beth Jonson, Stajfan Normark, Mikael Rhen
Microbiology and Tumor Biology Center, Karolinska Institute,
Stockholm, Sweden
Filamentous surface structures have been detailed on gram-negative entero-
bacteria since the introduction of electron microscopy [1]. The bacteria
appeared to express two types of extruding appendages: wavy flagella exceed-
ing the length of the bacterium itself, and more rigid but somewhat thinner
'fimbriae' [2] or 'pili' [3]. Soon after the description of fimbriae and pili, it was
realized that their expression correlated with the ability of the bacteria to bind
to cells from potential host organisms. Fimbriated and piliated bacteria agglu-
tinated erythrocytes in a fashion resembling classical hemagglutination and
adhered to host epithelial cells [2, 4-6]. Moreover, for some strains bacteria-
induced hemagglutination was inhibited by the addition of the monosaccharide
mannose. This suggested that mannose is used as a receptor for adherence and
that the free mannose functions as a hapten. For other bacteria-erythrocyte
reactions hemagglutination was not inhibited by mannose implying another
receptor selectivity in the binding reaction [7-9].
Since the initial notion that fimbriae or pili function as specific adhesive
organelles that aid bacterial colonization of mucosal surfaces, a myriad of
bacterial adhesive factors have been described, many of which have turned out
to act as virulence factors and to have a fimbriaJ morphology. Thematically,
therefore, the unique but often separate binding specificity expressed by the vari-
ous fimbriae participates in determining host and mucosal tropism (fig. 1) [10,
1 1]. While such notions remain rather unchallenged, many recent observations
imply additional functions for fimbriae. Distinct fimbriae are known to bind
plasma proteins and to initiate intrinsic proteolytic cascades [12], whereas others
are capable of activating Ca^+ influx and signal transduction cascades in host
target cells [13]. In addition, fimbriae have been shown to act as invasion and
motility factors, whereas bacterial flagella that typically mediate bacterial
p
+
p
a
Fig. h a Transmission electron nnicroscopy of type IV piliated (P^) and nonpiliated
(P") N. gonorrhoeae, b The adherence of fluoreseently labelled P^ and P" bacteria to
human cervical tissue sections is shown. The presence of type IV pili enables the bacteria
to attach.
motility have also been ascribed functions in terms of bacterial adherence [14]
and in the initiation of proinflammalory responses [15]. One purpose of this
review is to highlight the more recently defmed virulence-associated functions of
fimbriae, pili (see The Role of Fimbriae in Pathogenesis of Mammalian Hosts as
Illustrated through a Few Examples) and flagella (see Flagella as Virulence
Factors) using a few illustrating examples, and to argue that these organelles have
a role in the infection pathogenesis beyond the first step of surface colonization.
Classification and Biosynthesis of Fimbria!
Adhesive Factors
The early notion of variation among fimbrial adhesive virulence factors
brought the need for classification schemes [16]. However, the multitude by
Jonson/Normark/RJien
68
which various bacterial organelles with different binding specificities started to
emerge soon implied that typing approaches could become difficult based on a
single characteristic, such as antigenicity or receptor specificity. Even for a
single fimbrial type, the antigenic variation could be significant [17], and for
many fimbriae no defined receptor structure was identified. Furthermore, not
all adhesive factors appeared typically 'fimbria!' in morphology although they
showed receptor-specific binding abilities [18, 19], Finally, in selected cases
even flagellaare known to function as adhesive organelles [14].
While fimbriae and flagella can be defined as distinct structures, they
share the need to create a polymer architecturally outside the ordinary bacterial
anabolic machinery. This is also reflected in the complex organization of genes
that are needed for either fimbrial or flagellar biosynthesis. The elucidation of
the precise events involved in fimbrial biosynthesis by several laboratories has
clearly formulated distinct fnnbrial 'families' and assembly pathways that
actually can define groups of fimbrial types. Thus, a given assembly pathway
can be used as a gross classification criterion [20], within which fimbriae can
be defined based on receptor specificity or antigenic variation.
Fimbriae Produced through the 'Chaperone/Usher' Pathway
The classical 'common' type-1 fimbriae that mediate mannose-sensitive
hemagglutination, and the P-blood-group-antigen-binding P-fimbriae, or Pap piU,
are produced through the so-called 'chaperone/usher' pathway [21]. The biogene-
sis and the basis for receptor recognition have been extensively studied for these
two types of fimbriae. Therefore, the defined biogenesis and function of type I
and Pap pili have functioned as guidelines when dissecting the molecular biology
of fimbriae not only belonging to this class, and as a 'reference system' when
dissecting other types of bacterial adhesins.
Yet, fimbriae that belong to this 'chaperone-usher' family come in several
different variants, and are not onJy defined to Escherichia coli. Gene clusters
that provide the fimbrial subunits, protein chaperones and outer membrane
anchors for the fimbrial shaft, as well as specific fimbrial regulatory genes
code for these fimbriae. Altogether nine 'biosynthelic' genes and two iiitrijisic
funbrial regulatory genes are included in the E. coli pap gene cluster responsi-
ble for the expression of P-fimbriae [21, 22]. The fimbrial constituents are
translocated to the periplasm through the housekeeping Sec system, and are met
by the fimbrial chaperones once translocated. Principally, the chaperones
translocate fimbrial subunits to the usher which then initiates translocation and
polymerization of the funbrial subunits across the outer membrane. Thus, the
f unbria elongates from the proxunal end of its shaft.
Initially, these fimbriae were considered genuine homopolymers of the
fimbrial protein subunit, the fimbrillin [23]. Furthermore, isolated fimbriae that
Fimbriae, Pili, Flagella and Bacterial Virulence 69
appeared as one major protein species in Coomassie-blue-stained SDS
polyacrylamide gels couJd agglutinate cells, implying that receptor recognition
was closely associated with fimbrial subunits [24]. However, during the dissection
of P-fimbrial biosynthesis it became evident that the formation of fimbria and
the ability of fimbriae to mediate adhesion or hemagglutination (receptor
recognition) could be separated [25, 26]. This showed that the P-fimbriae actually
were composite fibers. TTie fimbriaJ fibers include at least two distinct func-
tions: the constitution of a filament and the recognition of the receptor; these
functions were separable [27]. indeed, in addition to the major fimbrial subunit
PapA, P-fimbrial filaments were found to contain minor subunits, including the
PapE, PapF, PapK and PapG proteins located at the distal end of the fiber [21,
27]. The ability to bind the receptor resided in the PapG subunit, whereas other
tip-located Pap proteins functioned as initiators of fimbrial polymerization and
for adapting PapG to the fimbrial shaft. However, PapA alone forms the
micrometer long shaft and hence substantially dominates preparations of
isolated fimbriae [26]. This may explain why isolated fimbriae perform receptor
recognition but require sensitive staining techniques to reveal minor components
in gel analyses.
Not surprisingly, type 1 fimbriae are also composite fibers [22, 28-30],
and may even include minor components scattered throughout the filament
[31]. This could be due to a need to enhance fimbrial polymerization, and/or
due to a need to include receptor-binding entities along the fimbrial shaft [20,
31]. That is to say, the addition of minor nucleator or lectin components along
the shaft could increase the efficiency of polymerization, or the avidity of the
receptor-recognizing potential of the fimbrial filament. Other fimbriae belonging
to this class include the E. coli S-fimbriae recognizing sialyl galactosides and
type IC fimbriae [21, 22, 32].
Crystallographic studies have demonstrated that the periplasmic chap-
erone not only fulfils a transporting function for the respective pilus subunit
proteins in the periplasmic space as initially thought [33]. The pilus subunit
proteins have an incomplete immunoglobulin fold, due to the lack of the
seventh p-strand creating a large hydrophobic groove in the pilus subunit
protein. In pilus biogenesis this groove is transiently occupied by the Gl strand
of the chaperone [34]. At the site of the usher, the chaperone Gl strand is
replaced by the amino-terminal extension of the next subunit protein to become
incorporated via a donor strand exchange mechanism [35]. During donor strand
exchange, the subunit undergoes a topological transition that triggers the
closure of the groove and seals the amino-terminal extension of the incoming
subunit in place [36]. These findings help explain the ordered assembly of pili
heteropolymers. A contributing factor to the ordered assembly is the different
affinities that chaperone-subunit complexes have for the outer membrane usher
Jonson/Normark/RJien 70
protein [37]. Outer-membrane PapC molecular ushers discriminately recognize
peripJasmic chaperone-pilus subunit complexes. That the initiating step in pilus
assembly is an interaction between the adhesin, in complex with the chaperone
and the outer membrane usher, explains why the adhesin ends up at the pilus tip
[38]. Evidently, comparable complex strategies of assembly are aJso applied by
other fimbria and fimbrial types, and reflected in the multitude of participating
gene functions [20, 39].
The atomic structures of three minor fimbrial lectin subunits or lectin
domains associated with the cognate receptor have been determined [34, 40, 41],
While these three lectin proteins do not share obvious sequence identity, they share
a remarkably similar structural outline. The three protems in question that, respec-
tively, recognize mannosides (FimH of the type 1 fimbriae), Galal -^ 4Galp
(PapG of the P-fimbriae) and terminal N-acetyl-Z)-glucosamine (GafD or F17-
G of the F17 fimbriae) share an immunoglobulin-like folding pattern forming an
ellipsoid structure [41]. The receptor-binding pockets, however, seem to be
somewhat differently positioned in relation to the superimposed ijnaginary core
structure [41]. Thus, although all these fimbrial lectin proteins share the ability
to bind a small carbohydrate epitope and to become integrated into the fimbrial
filament, the lectin proteins apparently have not evolved just through modifi-
cations in one existing carbohydrate-binding pocket.
Fimbrial lectins are interesting candidate antigens for vaccine development.
Due to the incomplete structural nature of the adhesin, vaccine trials have been
conducted with adhesin-chaperone dimeric complexes. The FimH/FimC complex
provided protection against uropathogenic E, coli in both a murine and a primate
cystitis model [42^ 43].
The CSl Fimbrial Family
Fimbriae belonging to the class of the CSl fimbrial family are assembled
in a manner that phenotypically resembles the 'chaperone-usher' pathway [20].
The CSl fimbria forms the prototype of this class that includes several antigenic
variants, including the classical CFA/1 fimbriae of enterotoxigenic E, coli
(ETEC), and the type II pili o^ Burkholderia cepacia [44, 45]. Tlie CSl fimbrial
subunit CooA is translocated to the periplasm through a Sec-dependent path-
way, and then assisted by a protein CooB with chaperone-like function [20, 46,
47]. CooA is then fed to a larger transmembrane protein CooC concomitant
with fimbrial polymerization. However, polymerization needs the presence of a
minor fimbrial subunit protein CooD, which functions both as an initiator and
the lectin subunit [45, 48].
The constituents of the transport and assembly machinery do not show
apparent amino acid sequence homology to the P-fimbrial chaperone or usher
components. In addition, the number of CSl -specific genes that participate in
Fimbriae, Pili, Flagella and Bacterial Virulence 71
funbrial biogenesis as well as the number of specific fimbrial components
tend to be more restricted within the CSl family. Still, the number of
genes involved may not be a definitive characteristic of a fimbrial class;
N-acetyj-D-glucosamine-binding F17 fimbriae also need only four genes for
their expression in E. coll K12 and yet show many characteristics of the
P-fimbrial family [48, 49].
T^pe IV Pili
Type IV pili are multifunctional adhesive structures expressed by a num-
ber of diverse microorganisms, including Neisseria meningitidis. Neisseria
gonorrhoeae, Pseudomonas aeruginosa, Dichelobacter nodosus and Moraxella
bovis [50]. Related structures have also been identified in Vibrio cholerae
(toxin-coregulated pili, Tcp) and enteropathogenic E. coli (bundle-forming pili,
Bfp) [51, 52]. Type IV pili are typically 5-7 nm in diameter and can extend
several micrometers in length (fig. 1). They share an unusual (amino-termmal)
N-methyl phenylalanine, a high conservation of the amino-termLnal 32 amino
acids, and a proposed immunogenic carboxy-terminal disulfide-bound region.
As with other types of fimbriae, type IV pili are composed primarily of a smgle
protein subunit, termed pilin, which are arranged in a helical conformation with
5 subunits per turn. In addition, and somewhat unorthodoxal for prokaryotic
structural proteins, type IV pili can be glycosylated and/or phosphorylated
depending on the bacterial species [53-57]. Type IV pilus assembly is hypothe-
sized to occur within the cytoplasmic membrane or periplasm. The assembly of
pili requires a nucleotide-binding protein, a polytopic inner membrane protein,
the prepilin peptidase, and a multimeric outer membrane protem that forms a
pore in the outer membrane for pilus protrusion [58].
One most astonishing aspect of type IV pili is their ability to intimate their
initial contact through pilus retraction. A core set of mechanisms, fiber assem-
bly and extension, fiber adhesion, fiber disassembly and retraction, account for
these functions. Genetic analysis has revealed multiple clusters of genes, scat-
tered through the microbial genome coding for type IV pilus biogenesis genes,
as well as for major pilin and minor pilin-like proteins. The fact that ahnost 40
genes have been identified in P. aeruginosa as essential for biogenesis and func-
tionality of type IV pili evidently reflects the complexity both in pilus assem-
bly and function [59, 60]. While bacterial fimbriae belonging to chaperone/usher
or CSl family may have evolved through a divergent evolutionary need to pro-
duce sticky, surface-located adhesive organelles [20], type IV pili may share
evolutionary origins with filamentous bacteriophages [61], and with genes
required for bacterial type II protein export and DNA uptake systems [62].
Type IV pili bind to a variety of surfaces, including both 'inert' nonbiological
surfaces, to other bacteria, as well as to eukaryotic cells. In the case of type IV
Jonson/Normark/RJien 72
pili, the tip of the pilus binds to specific receptors on mammalian epithelial
cells as an initial engaging event. Pili attached to cells are always observed
anchored to surfaces at their distal end, and broken pili also only attach via an
end [63]. In P. aeruginosa, the above-mentioned carboxy-terminal disulfide-
bonded region is exposed at the tip of the pilus and binds the carbohydrate
moiety of the asialo-GMl and asialo-GM2 glycosphingolipids on epithelial
cells [64, 65].
Consequently, type TV pili of Neisseria are composed of a major pilus
subunit PilE and several other pilus-associated proteins, which have different
functions in pilus assembly and adhesion [66, 67]. One of these proteins is PilC,
which is associated with the tip and the shaft of the pili [68] and the basal part
in the outer membrane [69]. Adhesion o^ Neisseria to cells requires PilC, which
appears to function as a tip adhesin, although it is also found in the cell membrane.
The pili of Neisseria recognize and interact with the cell surface receptor
complement regulator CD46 [70].
Fimbriae Produced through the Extracellular
Nucha tor Pathway: Curli Organelles
Many enterobacteria are capable of expressing elongated surface
organelles, called AgfA fimbriae, with an 'aggregative' and chemically robust
character [71, 72]. AgfA fibers appear not as straight but rather as twisted,
curly structures and hence are referred to as 'curli' fimbriae [73]. Curli fibers
of £". coli and Salmonella enterica sv Tyhpimurium are coded for by the cfg
gene cluster. The cluster consists of two divergently transcribed units that
include the csgABC diud csgDEFG genes, respectively. Although curli fibers are
coded for genetic elements comparable in size to the P-fimbrial pap operon [74,
75], the curli fiber polymerization process is apparently different. Interestingly,
curli fibers show all the typical characteristics of amyloid fibers, such as the
binding to the dye Congo red. However, unlike amyloid formation in human
neurodegenerative disorders such as Alzheimer's disease, curli amyloids require
a specific assembly machinery [76]. Thus, the CsgA and CsgB fimbrial subunits
appear to be secreted out from the bacteria [72, 74], where after an interactioji
between the subunits in the extracellular compartment then leads to polymer-
ization. The CsgA subunit occurs in excess in the isolated filament, whereas
in vitro both the CsgB subunit [72] and the isolated CsgA subunit [76, 77] are
capable of self-polymerization. Thus, as in analogy with type 1, P- and CSI
funbriae the assembly of curli organelles also involves a nucleator component
(CsgB), proteins with apparent chaperone functions (CsgE) [76], or a nucleator
center (CsgG) [78]. As with type IV pili, curli fibers have a rather diverse
spectrum of receptor targets. Curli fibers are reported to mediate binding to
mouse small intestinal epithelial cells [73], in addition to various plasma and
Fimbriae, Pili, Flagella and Bacterial Virulence 73
extracellular matrix proteins [12, 71, 79, 80]. One reason for this promiscuity
might reside in the participation of curii in the formation of biofilms [81, 82].
A more flexible binding specificity might be more efficient in collecting
various organic molecules into the biofilm as compared to an organelle with a
highly specific, but concomitantly more narrow receptor repertoire. Since the
CsgD transcriptional regulator also affects bacterial production of cellulose an
important role of curli might be to interact with cellulose fibrils in an extra-
cellular matrix [83].
The Role of Fimbriae in Pathogenesis of Mannnnalian
Hosts as Illustrated through a Few Examples
Chape roneAJsher Fimbriae and Urinary Tract Infection
Adhesion
E. coli is by far the most common causative agent of urinary tract infections
(UTI) [84]. Consequently, the role of £". coli fimbriae in the infection patho-
genesis of UTi has been given much attention, and has been used as a template
for the analysis of other fimbria! structures [21, 85].
The ability to express certain types and sets of fimbriae seems overrepre-
sented among urinary tract isolates of £. coli. The expression of type 1 fimbriae
appears to be both an miportant colonization factor and a factor contributing to
the persistence in the bladder epithelium [85]. However, the pattern of mannose
binding by the protein FimH is somewhat different among commensal and UTI
E. coli; UTI isolates seem capable of binding Z)-mannose whereas commensals
seem to prefer trimannoside structures [86]. This difference in specificity
resides in minute differences m the FimH fimbrial lectin molecule as coded for
by separate alleles of JimH. Uroplakins, or rather mannosides contained on
uroplakin, are believed to be the actual epithelial receptor in the urinary tract
[84, 87]. Thus, it appears that the type 1 fimbria can be equipped with differ-
ent variants of FimH, and that the receptor preferences expressed by FimH
in tuni steer the mucosal tropism of the bacterial even within a single host
organism.
The P-fimbriae is another group of bacterial adhesins often expressed by UTI
isolates of £". coli, in particular among strains causing upper UTI and urosepsis
[84, 88]. P-fimbriae recognize the core Gala I -^ 4Gal|3 entity contained in
blood group antigen-carrying globoseries glycolipids [78]. Thereby, as the
receptor is present on cells linmg the human urinary tract, it provides an adhe-
sion target for P-fimbriated bacteria ascending from the bladder to the ureter
and further up into the kidney [84]. As with the FimH protein of type 1 pili,
PapG possesses allelic polymorphism: the class 1, 11 and 111 adhesins. Of these,
Jonson/Normark/RJien 74
the class II G adhesin recognizes most members of Galal — > 4Gal|3-containing
globoseries glycolipids and has been considered important for kidney infection
in persons with a nonobstiucted urinary tract [84, 89-91].
Beyond Adherence
Besides mediating adherence to the urinary tract epithehum, type 1 and
P-fimbriae have been implicated in the later phases of infection, and in the
generation of innate proinflammatory responses in the infected urinary tract
epithelium. First, although cystitis-associated E. coLi have generally been
regarded as noninvasive bacteria, type I fimbriated E. coli have been observed
to enter human bladder epithelial cell lines in vitro in a FimH-dependent manner
[87]. Invasion could be mimicked by applying FimH-coated beads, and invasion
was associated with host protein tyrosine phoshorylation and host actin
cytoskeleton rearrangement [92]. This suggests that FimH alone, in analogy
with Yersinia invasion factor Inv [93], can activate host signal transduction
events that subsequently trigger actin cytoskeletal rearrangements in the host
leading to bacterial uptake. Later it was observed in a mouse cystitis model that
the bacteria were internalized into bladder epithelial cells and subsequently
formed a biofilm-like mass [94]. Apart from type 1 fimbriated bacteria, uroplakin
was also found in the biofihn. Thus, type 1 fimbriae appear multifunctional in
the pathogenesis of UTI; they mediate initial adherence, invasion and seem to
participate in the formation of an intracellular biofihn.
Many types of fimbriae, including type 1, type IC and P-fimbriae have all
been associated with the induction of proinflammatory responses in epithelial
cells [95-97]. Type 1 fimbriated E. coli induce cytokine expression from both
A498 kidney epithelial cells as well as in bladder cell lines [96, 98]. However,
in bladder epithelial cells the majority of the IL-6 response seems to derive
from lipopolysaccharide (LPS) signalling through the CD14-TLR4 pathway
[98]. Still, type \lfimH^ fimbriae appear to be somewhat more potent inducers
of IL-6 as compared to type \lfimH~ bacteria in LPS-hyporesponsive A498
cells. Likewise^ type IC fimbriae, also associated with cystitis, augment bacte-
rial lL-8 release from A498 cells [95]. It is thus possible that bacterial attach-
ment, the prerequisite for the infection in the first place, is also one cause foq
the symptoms of cystitis.
The mechanism by which P-fimbriae induces signal transduction
casacades in kidney A498 cells appears complex, and differs from those mecha-
nisms used by type 1 fimbriae [96, 99]. Binding of P-fimbriated bacteria causes
a release of ceramide in the target cells concomitant with an activation of
cytokine release [96]. Cytokines, such as TNF-a, also cause the release
of ceramide from sphingomyelin, which eventually results in the activation of
transcription factor NF-kB [100]. It has thus been suggested that ceramide
Fimbriae, Pili, Flagella and Bacterial Virulence 75
Initial adherence
CD46
Tight adherence
Cellular responses
Pilus retraction
CD44
ICAM-1
.2 +
Ca signalling
Trigger lysosome exocytosis/LAMP-1 to cell surface
Fig. 2. Initial adherence of type FV piliated Neisseria involves initial contact with ceJJ
surface receptors followed by sophisticated cell signalling leading to tight adherence and
invasion of host cells. Failure in the pilus retraction events and/or host cell signalling leads
to lost or changed adherence patterns, and a loss of ability to enter and invade host cells.
release caused through attachment of P-fimbriated E. colt could induce nuclear
responses as a result of ceramide release. Furthermore, the LPS-recognizing
Toll-like receptor TLR4 has been implicated in P-fimbria-induced host responses
[99]. Possibly^ P-fhnbriae can adapt both cermJde- and TLR4-mediated signals
to induce NF-kB nuclear translocation. Binding of P-fimbriated bacteria to A498
cells also caused an upregulation in the expression of TLR4mRNA suggesting
that one function of P-fimbria-mediated host cell responses might be to modify
the surface of the host cell to better accommodate or promote the infection.
Type IV Pili in Sequential Attachment and Invasion
of Pathogenic Neisseria
Adhesion
The important initial interaction between pili of Neisseria and its host cell
occurs through the receptor molecule CD46;, a human cell surface protein
involved in the regulation of complement activation. In cultured epithelial cells,
binding of pili to CD46 is followed by release of Ca^"^ from intracellular stores
[13, 101], This Ca^"^ transient is sufficient to mediate exocytosis of a pool of
the lysosomal/late endosomal vacuoles resultmg in the increase of surface
lysosomal components such as h-Lamp-1, and possibly other factors that could
contribute to a tighter adherence of bacteria. During initial contact between bacte-
ria and cells, pilus retraction exerts tensile forces upon the plasma membrane
(fig. 2) [102]. The mechanical forces applied to the plasma membrane trigger actm
polymerization accompanied by accumulation of phosphotyrosine-containing
Jonson/Normark/RJien
76
proteins, which leads to the formation of compact microcolonies and so-called
pilus-associated cortical plaques on the host cell [103, 1 04]. The cortical plaque
structures are characterized by the accumulation of actin and actin-associated
proteins, and trigger recruitment of transmembrane proteins such as CD44,
ICAM-1, EGFR, and components of the cortical cytoskeleton, i.e. ezrin and
cortactin, and contain tyrosine-phosphorylated host cell proteins beneath the
microcolony [104, 105].
At later times after infection, bacteria disperse from the microcolonies, pili
disappear, and individual diplococci become intimately associated with the host
plasma membrane. Pilus loss, bacterial dispersal, and intimate adhesion are all
blocked in ap/'/r mutant [106, 107]. The full set of rearrangements requires the
expression of both type IV pili and PilT. Obviously, pilus retraction could account
for elongation of microvilli towards the bacterial microcolony and bring the
host cell and bacterial membrane into close contact [108]. For example pilT
mutants of N. gonorrhoeae are unable to make intimate contact with or form
attaching effacing lesions on epithelial cells [95]. P. aeruginosa pilT mutants
are not infective in corneal tissue and exhibit reduced cytotoxicity to epithelial
cells in culture [109 1 1 !].
To summarize, type IV pili do not only simply anchor the bacteria at the cell
surface, they initiate a multistep adhesion cascade, which starts with a loose
adherence and ends with the intimate attachment of bacteria [1 12]. Establishment
of intimate attachment appears to require an intensive host-pathogen cross talk,
and a complex sequence of bacteria-host cell interactions. Type IV pili also assist
in the formation of biofilms [1 12] that may support further tissue colonization
and protect the bacteria against antibodies and antibiotics.
Beyond Adhesion
In an experimental model system oi Neisseria infection, using transgenic
mice expressing human CD46, the crossing of the blood-brain barrier by
bacteria occurred in CD46 mice but not in nontransgenic mice, indicating an
important role for CD46 in meningococcal meningitis [113]. Intranasal
infection of CD46 mice required piliated bacteria for the development of disease,
supportmg that CD46 facilitates pilus-dependent mteractions at the epithelial
mucosa.
Binding ofFimbrial Structures to Extracellular Components
Although a primary role of fimbriae indeed might be to mediate adhesion
and subsequent events through binding to specific structures on host (epithe-
lial) cells, it has recently become evident that fimbriae can also bind various
connective tissue proteins, as well as plasma and serum proteins. Moreover,
Fimbriae, Pili, Flagella and Bacterial Virulence 77
binding to selected plasma components can induce subsequent intrinsic cascades
leading to the activation of zymogen proteases and the release of biologically
active host peptides [12, 80], Such observations illustrate that fimbriae may
contribute to the infection pathogenesis even after they have assisted adhesion
and invasion.
The F17 fimbriae occur characteristically in E. coli isolates causing
diarrhea and septicemia in newborn calves. F17 fimbriae mediate binding to the
calf intestinal epithelium, which suggests a role for F17 fimbria in the intesti-
nal colonization. In addition, the F17 fimbria is capable of binding plasmino-
gen [114] and the extracellular matrix protein laminin [1 15]. Binding to laminin
is inhibited by the receptor analogue N-acetyl-Z)-glucosamine, indicating that
carbohydrate receptors on the extracellular matrix protein are recognized by the
minor fimbrial lectin protein GafD [115]. The binding to plasminogen is not
inhibited by the receptor analogue, but instead the binding leads to conversion
of plasminogen to proteolytically active plasmin. Binding and activation of
plasmin is not unique to F17 fimbriae. For instance, it has been shown that
meningitis-associated S-fLmbriae and S. enterica sv Typhimurium type 1 fim-
briae as well as curii fibers are both plasminogen binders and activators [79,
1 14, 1 16, 1 17]. Such observations suggest that fimbriae may assist bacteria dur-
ing tissue dissemination by directing them to extracellular matrix proteins, and
by coating them with proteolytically active proteins that enable the bacteria to
penetrate through the tissue. Indeed, enterobacteria capable of binding and acti-
vating plasminogen have been shown to degrade extracellular matrix proteins,
and to penetrate reconstituted basement membranes in vitro [117].
Yet another aspect of binding to plasma proteins is illustrated by the ability
of bacterial curIi fimbriae to activate the contact phase pathway of the coagu-
lation system, and thereby to induce proinflammatory reactions [12, 80]. Factor
XI, factor XII, prokallikrein and H-kJninogen are absorbed to curilated E. coli
and S. enterica sv Typhimurium, but not to isogenic noncurliated mutants.
Binding of the contact phase proteins by the curiiated bacteria lead to a rapid
release of vasodilatory bradykinin from kininogens and to prolonged clotting
times of the infected plasma. While it is difficult to ascertain the biological
fijnction of such reactions, possibly they reflect an aspect of the innate line of
defenses, such observations imply that a more massive encounter with curiiated
bacteria may contribute to the symptoms of septic shock [80].
Pili and Motility
The term twitching motility was used by Lautrop [118] in 1961 to describe
flagella-independent spasmodic movements of bacteria. Twitching motility
occurs in a wide range of bacteria, and has been well studied in N. gonorrhoeae
and P. aeruginosa. It occurs on solid, wet surfaces and is mediated by type IV
Jonson/Normark/RJien 78
pili. Twitching motility occurs by extension, tethering, and then retraction of type
TV pili, which operate in a manner similar to a grapping hook, which has been
shown by elegant studies in A', gononhoeae [1 02], Myxococcus xanthiis [119], and
P. aeruginosa [1 11],
Type IV pili serve as an initial bridge between bacteria and cells, and twitch-
ing motility allows bacteria to spread in the infected tissue. P. aeruginosa is
an important pathogen, being the major cause of lung damage in patients
suffering from cystic fibrosis as well as of opportunistic infections in immuno-
compromised individuals, such as burn victims or patients undergoing chemo-
therapy. Twitching motility has been shown to be important for infection by
P. aeruginosa as well as for biofilm formation, which appears to be involved in
chronic infection [110, 112].
Type JV pili generate considerable force by retraction [102, 120]. For
some pilus-dependent functions, the amount offeree is critical, e.g. in host-cell
responses and movement of bacteria through viscous mucous layers. Although
pilT mutants adhere and colonize surfaces, pilT mutants are aviruient in many
experimental model systems. PilT mutants are unable to retract their pili, lead-
ing to hyperpiliation and loss of twitching motility. It could be speculated that
a signal could pass from the tip of the fiber to its base, by the propagation of a
helix dislocation or a mechanical force such as tension, compression or flexion.
The dislocation signal that reaches the base of the pilus could induce a beneficial
movement response to the cell.
PilT, an ATPase associated with various cellular activities (AAA), seems
to act as a molecular motor [121, 122]. Pilus retraction is thought to occur by
filament disassembly mediated by PilT, a process that has been estimated to
occur at around 1 ,000 pilin subunits per second. Genetic studies and structural
data support the following molecular model. The cytoplasmic membrane has a
reservoir of the prepilin subunits that are cleaved by PilD, the prepilin pepti-
dase, and then polymerized into pili. In the model PilT is actively involved in
the dissociation of a pilus. PilT is a member of the GspE family of hexameric
AAAs, and one PilT unit could hydrolyze several (up to six) ATP molecules in
the process of dissociating one pilin subunit. It is possible that epithelial cells
sense the amount of force generated by pilus retraction and respond in a similar
manner.
Phase Variation of Pilus Structures
As mentioned above, fimbriae and pili of the same type can be expressed as
antigenic variants. For example, separate strains of UTI E. coli can express sepa-
rate antigenic variants of the major fimbrial subunit protein [123], and a single
strain can contain more than one P-fimbrial gene cluster [124]. Furthermore, as
different P-fimbrial gene clusters may contain separate /?£j!/?G alleles [89], and
Fimbriae, Pili, Flagella and Bacterial Virulence 79
as P-fimbriae are subject to phase variation [125], the set-up provides E. coli
with flexibihty in terms of varying antigenicity and ftinction of P-fimbriae.
Still, for a given strain the repertoire is restricted to the number of fimbria! gene
clusters contained, and hence usually narrow.
One extraordinary characteristic of the pathogenic Neisseria species is
their enormous capability to vary their surface pili [17]. In this context, the
changing in the antigenic structures of surface proteins is certainly an impor-
tant immune escape mechanism [ 1 26]. Furthermore, the variation also modifies
the function of these adhesions [127-130]. Small alterations on the primary
structures of neisserial pilins cause changes in immunoreactivity, post-
translational modification, adhesive function, and ability to form bundles of pili.
As a consequence, the pathogens can selectively interact with certain cell types
and thus occupy special niches in their host. Many pilin variants that promote
strong adhesion to host cells also aggregate into laminar bundles, whereas vari-
ants that promote weaker adhesion tend to exist as single filaments [131, 132]. It
is unclear how bundling promotes adhesion. Bundles could promote bacterial
aggregation, increase receptor avidity by oligomerizing binding sites, or increase
pilus stiffness. Bundles might also promote twitching motility by promoting
coordinated fiber extensions and retraction processes that would be unfeasible
with less-ordered structures.
Flagella as Virulence Factors
Like fimbriae, flagella are protein polymers, each flagellum consisting of
thousands of flagell in monomers [1 4]. These filaments are connected to the cell
surface through the 'hook' structure, and the basal structure that forms the rota-
tion device and that traverses the bacterial cell wall. Consequently, flagella are
complex structures and coded for by a large set of genes. While the primordial
role of flagella is to ensure motility, either as swimming movement in liquid
medium or as swarming on solid surfaces, these traits are also applied in bacte-
rial virulence [14]. For example, flagel la-mediated moLilily acts as a virulence
function for V. cholerae [133], Helicobacter pylori [134] and for Proteus
mirabilis [135]. The former two pathogens are noninvasive colonizers of the
digestive tract. Evidently, these bacteria apply motility to gain contact with the
intestinal or gastric mucosal cells, respectively, and thus to establish the infection.
P. mirabilis, on the other hand, is believed to apply motility for ascending from
the ureter to the bladder, and further up to kidney structures. For V. cholerae and
//. pylori, the role of flagella as virulence factors is also supported through
transcriptome analyses, which show an upregulation of motility genes in de facto
infecting bacteria [136-138]. For P. mirabilis, the swarming state involves a
Jonson/Normark/RJien
80
transition to a hyperflagellated state and an upreguiation, the expression of
selected virulence functions [135].
Besides mediating motility, flagella are in many instances known to adapt
functions typically ascribed to fimbriae. The flagellar FliC and FliD proteins of the
gram-positive anaerobe Clostridium difficile, a causative agent of pseudo-
membranous colitis, have been shown to bind both to mouse catcall mucous
and cultured cells [139]. Similarly, a nonflagellated P. aeruginosa mutant was
shown to be attenuated in a mouse pneumonia infection model. In parallel
flagellin was been shown to bind GMl, asialoGMl and GDI glycolipids
in vitro [140]. For S. enterica sv Enteritidis, nonflagellated mutants are abro-
gated for their ability to adhere to gut epithelium and epithelial cells, and for
their ability to invade host cells [141].
Whereas some bacteria, like S. enterica sv Typhimurium, can phase-variate
between the expression of two alternative flagellar subunit proteins, others,
like Vibrio parahaemolyticus and Aeromonas spp., apply two separate sets of
flagella: polar and lateral sets [14]. The different flagellar sets expressed by
Aeromonas primarily associate with a shift in motility, the lateral set being used
for swarming. However, there is also evidence for different adhesive characters
disposed by polar and lateral Aeromonas flagella [142]. Therefore, as for R
mirabilis, the switch to a swarming phenotype reflects a more fiindamental
alteration in the expression of the bacterial virulence potential.
As with fimbriae, flagella also activate host cell signal transduction cas-
cades and inflammatory responses. At least in part, this response originates
from the fact that flagella, like bacterial LPS, act as pattern molecules that
are recognized by the host innate responses. While LPS is recognized by
TLR4, flagellin from both gram-positive and gram-negative bacteria is rec-
ognized by TLR5 [15]. The interaction between flagellin and TLR5 signals
via Myd88 to cause activation of inflammatory responses [143]. Both LPS
and flagellin can cause tolerance in host cells, the cells becoming non- or
hyporesponsive after prior exposure to the ligand. What is interesting in this
context is that LPS and flagellin can cause cross-tolerance, at least in cell
lines [144].
The flagellar assembly pathway is related to the contact-dependent, so-called
type 111 protein secretion pathway that is applied by many pathogens, like
S. enterica sv Typhimurium and Yersiniae^ for the translocation of bacterial viru-
lence protein into host cells [145]. In selected cases it has been observed that
the flagellar basal body and hook structures in >S'. enterica sv Typhimurium can
substitute for the transport function of virulence proteins [146, 147]. While this
was observed against a background with the ordinary secretion machinery inac-
tivated, it suggests that the flagellar protein secretion potential, normally reserved
for flagellar components, also could be applied for more sinister purposes.
Fimbriae, Pill, Flagella and Bacterial Viruience 81
Concluding Rennarks
The ability to express surface structures related to adhesiveness and motility
appears to be a widespread ability among prokaryotes, reflecting the necessity of
corresponding traits for microorganisms. In many cases these organelles medi-
ate colonization and adhesion of the bacteria to their growth niche: the plant root
or a vertebrate epithelium. However, the further investigations proceed from
describing adhesion to resolving the biogenesis and host responses, the more
complex the functions of the ad priori adhesive and motility organelles appear.
Indeed, type IV pili are known not merely to function as passive adhesive
fibers, but in addition as dynamic machines that participate in a surprising num-
ber of functions: adhesion to host cell surfaces, modulation target cell specificity,
twitching motility, DNA transformation, and bacterial autoagglutination.
Furthermore, fimbrial receptor recognition can actually represent the prelude to
a much more elaborated host-parasite cross talk. This is illustrated by type 1
fimbria-mediated activation of host signal transduction cascades that result in
concomitant bacterial internalization, or by P-fimbria that activates TLR4-
mediated proinflammatory and causes increase in TLR4 expression to further
amplify the process. Considering the impact of adhesion and motility in viru-
lence, it is interesting to note that, at least in selected cases, the same protein-
aceous extensions are being applied both for adhesion and motility. Perhaps it
is the extendedness of the structure that makes it suitable for such purposes. It
is important to mention that the host has evolved systems that recognize bacte-
rial flagellar It remains to be evaluated whether there are specific innate
specific recognition systems for fimbriae or whether the ability of fimbriae to
initiate proinflammatory responses in fact reflects attempts to eradicate bacte-
ria] colonization.
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Mikael Rhen
Microbiology and Tumor Biology Center, Karolinska Institute
Nobels vag 16, SE-171 77 Stockholm (Sweden)
Fax +46 8 301797, E-Mail mikael.rhen@mtc.ki.se
Fimbriae, Pili, Flagella and Bacterial Virulence 89
Adhesins
Russell W, Herwald H (eds); Concepts in Bacterial Virulence,
Contrib Microbiol. Basel, Kai'ger, 2005, vol 12, pp 90^1 13
Gram-Positive Adhesins
Susanne R. Talay
GBF-Gcrman Research Centre for Biotechnology, Braunschweig, Germany
In the process of bacterial infection, adhesion to host tissues represents an
initial and essential step. Adhesion allows the pathogen to attach to and colonize
specific sites of the body, thereby withstanding eradication through cleansing
mechanisms such as excretion and peristalsis. Once attached to the target tissue,
bacteria may either remain extracellular, multiply, and eventually spread into
deeper tissue, or trigger their own uptake by host cells, resulting in an intra-
cellular location that may allow the pathogen to persist or further spread within
the cellular or subcellular compartment.
Bacterial surface components that mediate adherence are called adhesins.
Among gram-positive pathogens, surface proteins represent the largest group of
adhesins, although other factors such as polysaccharides and lipids may also
display adhesive functions. Targets for these microbial adhesins are host mole-
cules found on mucosal surfaces, skin, and wounds. Depending on the strength
of this interaction, adhesins allow the pathogen to loosely associate with or
intimately bind to specific cells or tissues. Most gram-positive pathogens express
multiple adhesins that may bind to either the same or distinct target molecules.
Multiple adhesins of one pathogen are likely to be involved in different stages
of an infection, expressed under different environmentally determined condi-
tions, and may display a redundant function. Li the present article, adhesins
of pathogenic gram-positive bacteria belonging to the genus Streptococcus,
Staphylococcus and Listeria, as well as the most important host molecules targeted
by these adhesins are reviewed.
The Extracellular Matrix -A Major Target for Pathogens
Many adhesins function by specifically recognizing and binding to various
components found in the extracellular matrix (ECM) of the host. The ECM
forms the major structural support for cells and tissues and is responsible for
maintaining the strength and elasticity of the body. Thus, it is ubiquitously present
and frequently exposed in cases such as trauma and injury, a situation that renders
its constituents ideal targets for many adhesins. The following section gives a
short overview on the major ECM components, their structure and their basic
function.
Collagens
Collagens are the most abundant protehis in the mammalian body and it is
well recognized that collagens fulfill an important structural role in the ECM in
a number of tissues. More than 25 distinct collagen types have been identified,
in which identical or distinct a chains form a triple helix. Collagens can be
divided into fibril-forming interstitial collagens (e.g. types I, II, III, V, and XI)
and non-fibril-forming collagens such as type IV, VI, and X [1]. Type I collagen
is found bi tendons and muscle, while type II collagen is the major constituent of
cartilage. The nonfibrillar type IV collagen is the major constituent of basement
membranes, forming a network with laminins, nidogen, and sulfated
proteoglycans. Collagen IV is composed of six chains (al-a6) that form three
basic sets of triple helical molecules. Collagens may interact with a variety of
factors, Lncludmg other matrix components such as fibronectin and laminin, as
well as matrix metal loproteinases. The binding of collagen to cells is mediated
by integrins, which constitute another group of receptors for collagens. Currently
four collagen-binding integrins are known, a|(3|, cd2Ph otioPi ^^id ctnPi, that
mediate cellular binding and signalling. Bacterial binding to collagens, such as
cartilage collagen and basement membrane collagen, represent important adhesion
mechanisms among pathogens.
Fibronectin
Fibronectin, which exists both as a soluble protein in plasma and as a
fibrillar polymer in the ECM, is a large glycoprotein involved in cell adhesion,
migration, and differentiation. Fibronectin exists as a dimer composed of two
250-kD subunits which are carboxy-terminally linked via a pan- of disulfide
bonds [2]. Each subunit contains three distinct types of modules, the type I, II
and III modules (fig. 1). Fibronectin efficiently binds to cell surfaces via
numerous integrins, including the classic fibronectin-binding integrin, agp,
integrin. Integrin bindmg is mediated by an RGD sequence and also involves
secondary sites on the fibronectin molecule. In addition to the interaction with
integrms, fibronectm associates with heparin, collagen/gelatin, and fibrin.
Heparin binding is governed by three domains that interact with heparan sulfate
proteoglycans. Bmding to collagen is mediated by type I repeats 6—9 and the
two type II repeats. The two fibrin-binding sites are located at the carboxy- and
Gram-Positive Adhesins
91
a
COOH
HOOC
5>3-NH;
50 Residues
NHj-
T ^
PRR
1 I 2 1 _3 I 4 1 5"! [W| M H- COOH
Spacer
FnBR
LPATG
Fig, L a Modular structure of fibronectin (one subunit). Module types 1, 2 and 3 are
symbolized by pentagons, hexagons, and circles, respectively. The amino-terminal domain
('"^Fl), the collagen-binding domain (CBD), alternatively spliced sites (curved labels) and
the major integrin-binding site (RGD) are labelled. bl\\t extended tandem |3-zipper model
of Sfbl binding to '"^Fl. A model of the amino-terrmnal domain is shown on top. Short con-
secutive segments of a fibronectin-binding repeat form antiparallel p-strands on triple-
stranded (3-sheets ofall five homologous Fl modules, c Molecular organization of Sfbl from
S, pyogenes. S = Signal peptide; A = nonhomologous region; PRR = proline-rich repeats;
spacer — upstream fibronectin-binding region; 1-5 — Fn-binding repeats; W — cell wall
spanning sequence; LPATG = cell wall anchor; M = membrane spanning region. (The
tandem zipper model was kindly provided by Dr. Ulrich Schwarz-Linek, University of
Oxford, UK,)
ammo-teniiinal part of the molecule, the major site being formed by type I
modules 4 and 5 located at the amino-terminal domain. Fibrin-binding and
cross-linking to fibronectin via factor XJlla is important in the generation of
fibrin clots that form a provisional ECM network in the wound healing process.
Fibronectin is an ideal target for many pathogens due to its wide presence in
exudates, blood, wounds, as well as on the surface of cells.
Laminin
Laminin is a 900-kD glycoprotein and is a major component of the basement
membrane. Its macromolecular structure is formed by assembly of tbree distinct
Talay
92
polypeptide chains, a, p, and 7 [3]. Laminin functionally interacts with other
components of basement membranes such as collagen IV and a variety of proteo-
glycans and ECM molecules. More than 10 different isoforms of laminin are
known to be involved in cell proliferation and attachment, as well as in chemotaxis
and angiogenesis. In the case of epithelial and endothelial injury, basement
membrane components such as lammin are likely to be exposed and may serve
as target structures for bacterial colonization of damaged tissue,
Elaslin
Elastin is the major ECM protein of lung, skin and large arteries such as
the aorta, imparting characteristics of extensibility and elastic recoil [4]. Elastin
is formed by polymerization and cross-linking of its precursor tropoelastin.
This process of ordered self-aggregation is called coacervation. Once deposited
in tissues, polymeric elastin is not subject to turnover, but is able to sustain its
mechanical resilience through millions of cycles of extension and recoil.
Elastin consists of approximately 36 domains with alternating hydrophobic and
cross-linking characteristics. The rubber-like mechanical properties result from
the repetitive hydrophobic domains of tropoelastin that display an unstructured
organization with higher entropy in the relaxed state, and a structured organi-
zation with lower entropy in the extended state. The major binding partners for
tropoelastm are fibrillins, the main components of microfibrils which themselves
may be attached to cells, Elastin serves as a target for pathogenic staphylococci,
which use this molecule for adhesion to host tissue.
Viironectin
Vitronectin is a multiftinctional 75-kD glycoprotein present in blood and
the ECM [5]. It binds collagen, plasminogen and the urokinase receptor, and
stabilizes the inhibitory conformation of plasminogen activation inhibitor-1,
thereby regulating the proteolytic degradation of the ECM. It further interacts
with glycosaminoglycans via its carboxy-terminal part, and integrins of the ay
family via an ROD sequence located at the most ammo-terminal part Binding
of the RGD sequence to integrins induces signalling cascades, and mediates
attachment and spreading of cells on the matrix. Through its localization in the
ECM and its binding to plasminogen activation inhibitor-1, vitronectin can
potentially regulate the proteolytic degradation of this matrix. In addition,
vitronectin binds to complement factors, heparin and thrombin-antithrombin III
complexes, and therefore participates in the regulation of clot formation. The
biological functions of vitronectin can be modulated by proteolytic enzymes,
and exo- and ecto-protein kinases which are present in blood. Like fibronectin,
vitronectin is an ideal target for adhesins of pathogens due to its presence in the
ECM, in blood, and at sites of tissue injury.
Gram-Positive Adhesins
93
Fibrinogen
Fibrinogen is a 340-kD plasma glycoprotein composed of six polypeptide
chains, two Aa, two B[3, and two -y chains that form a dimer. In the vascular
system, fibrinogen mediates platelet adherence and aggregation at sites of
trauma and injury, thereby acting as an important clotting factor [6]. Upon
interaction with thrombin, subsequent stabilization of the fibrin clot is achieved
by transglutaminase/factor XJIla-mediated cross-linkage of the 7 and a chains
of fibrinogen. Binding to platelets is mediated through the interaction of
fibrinogen with integrin a]jb(33 on the platelet surface. In addition to its function
in the coagulation system, fibrinogen also participates in inflammatory
responses. Fibrinogen mediates leukocyte attachment to the vessel wall and
transmigration through the endothelium. Fibrinogen binds to aMp2 integrin on
leukocytes and to ci^\^2 ifitegrin on macrophages, thereby regulating phagocytic
clearance of fibrin clots during wound healing. Interaction with integrins is
governed by two RGD sequences and other defined epitopes on the fibrinogen
molecule. In addition, fibrinogen has the ability to bind a variety of factors such
as fibronectin, collagen, and components of the fibrinolytic system, implicat-
ing this protein as a key factor in matrix organization, remodelling and wound
repair. Many gram-positive pathogens have evolved distinct factors that speci-
fically bind fibrinogen, evoking bacterial adhesion, aggregation, and evasion of
phagocytosis.
G lye OS am in oglycan s
Glycosaminoglycans are polysaccharide chains covalently linked to a protein
core to form proteoglycans. Being composed of distinct repeating disaccharide
units, these molecules can be divided into different classes such as heparan
sulfate, dermatan sulfate, and chondroitin sulfate. Glycosaminoglycans are pre-
sent in the ECM of connective tissue but are also expressed on the surface of
eukaryotic cells. Heparan sulfate [7] and dermatan sulfate [8] are ubiquitously
found on the surface of cells and in the ECM and skin. Glycosaminoglycans
fimction as stabilizers, cofactors and coreceptors of cytokines and chemokines,
regulators of enzymatic activity, and signalling molecules in response lo injury
or infection. Glycosaminoglycans may mediate adherence and entry of pathogens
including bacteria, viruses and parasites [8].
Streptococcal Adhesins
Streptococcus pyogenes
S. pyogenes, the group A Streptococcus, is an important human pathogen
that causes localized infections of the respiratory tract and the skin, but also in
Talay
94
severe invasive diseases, such as sepsis and toxic shock-like syndrome. Severe
nonsuppurative sequelae such as acute rheumatic fever and glomerulonephritis
may follow primary group A streptococcal infection. S. pyogenes initiates infec-
tion by interacting specifically with host molecules present on mucosal surfaces
or skin. A variety of different adhesins that either bind to identical or distinct tar-
get molecules are expressed by lS". pyogenes (table 1). Among the large number
of bacterial factors that bind to host molecules, onJy those for which adhesive
properties were clearly demonstrated are herein termed adhesins.
S. pyogenes possesses at least nine distinct fibronectin-binding adhesins.
Some of these occur in a large number of serotypes, such as Sfbl protein or
FBP54, whereas others such as Ml or M3 protein are exclusively expressed by
Ml or M3 serotypes, respectively. Among all fibronectin-binding adhesins of
S. pyogenes, Sfbl protein and its allelic variant Fl are the most extensively stud-
ied. Identified iii 1992, Sfbl/Fl was shown to act as an adhesin on epithelial cells
[9^ 10]. Sfbl protein has a modular architecture [11], and binds to fibronectin via
two distinct domains [12, 13]. The carboxy-terminal repeat region and the adja-
cent nonrepetitive domain termed spacer 2 or UR synergistically bind to two dis-
tinct regions on the fibronectin molecule: the amino-terminal fibrin-binding
fragment (harboring fibronectin Fl modules 1-5) and the gelatine/collagen-
binding fragment (harboring Fl modules 6-9 and the two F2 modules) [14]. The
carboxy-terminal repeat region of Sfbl was demonstrated to be sufficient to
mediate adherence to epithelial cells [14]. However, besides this activity, Sfbl
acts as a potent invasin that triggers internalization into eukaryotic cells [14-18].
Sfbl mediates attachment to epithelial cells of the oral mucosa and the lung, but
also to endothelial cells [1 8]. Binding to human cells was shown to be dependent
on the presence of fibronectin-binding integrins [17], leading to the concept that
fibronectin acts as a bridging molecule between bacteria and host cell mtegrins.
Besides its potential to bind to cell surfaces, Sfbl has the ability to recruit collagen
via prebound fibronectin, a mechanism that enables the bacteria to form
aggregates and renders the organism capable of colonizing collagen matrix [19],
The overall pathogenic potential of this protein is underlined by vaccination
studies using recombinant Sfbl that protected mice fi'om lethal 5*. pyogenes
infection [20]. Recently, the first three-dimensional stmcture for a bacterial
fibronectin-binding peptide, the B3T peptide derived from the Streptococcus
dysgalactiae FnBP, in complex with the ^FHFl module of fibronectin was
obtained. Based on this structural information, a compelling model for the inter-
action of the fibronectin-binding repeats of Sfbl with the amino-terminal
domain of fibronectin was developed, termed the tandem (3-zipper model [21].
Short motifs within each of the carboxy-terminal repeats of Sfbl w^ere predicted
to form antiparallel (5 strands along the five Fl modules in the amino-terminal
domain of fibronectin (fig. 1)^ leading to binding affinities in the nanomolar
Gram-Posilive Adhesins
95
Table L Streptococcal adhesins
Adhesin
Ligand niolecu e
Target cells, tissue
Reference no.
S. pyogenes
Sfbl/Fl
fibronectin, collagen
pharynx and lung
epithelial cells,
endothelial cells,
CO agen matrix
9 19,21
F2/PFBP
fibronectin
n.c.
22,23
FBP54
fibronectin
buccal epithelial cells
24
hba
fibronectin
epithelial cells
26
hbaB
fibronectin
epithe ial cells
27
Protein H
fibronectin, M proteins
epithelial cells
28,29
Ml protein
fibronectin
epithelial cells
30 32
ITA
fibronectin, macrophage
epithe ial cells.
33,34
scavenger receptor
macrophages
M3 protein
type 1 and IV collagen,
fibronectin
collagen matrix
35,36
Cpa
type ] CO lagen
n.c.
37
HA capsule
type ] and IV collagen,
collagen matrix.
35,38
Cl)44
keratinocytes
M6 protein
CD46
keratinocytes
39,40
M proteins
g ucosaminoglycans,
epithelial cells,
29,41,42
M proteins
Tbroblast ce Is
Lbp
aniinin
epithe ial ce Is
43
SpeB
aJiLinin, glycoproteins
n.d.
44
R?8
n.d.
cervical epithe ial cells
45
Scl A/Scl 1
n.d.
pharyngeal cells
46,47
Sc B/ScJ2
n.d.
fibrob ast ce s
48,49
S, agalactiae
ScpB
fibronectin
pharynx and ung
50,51
epithelial cells
^mb
aminin
n.c.
52
A pha C protein
n.d.
cervica epithelial ce s
53
S. pneumoniae
SpsA/CbpA/PspC
SC, slgA, factor H
plgR-expressing cells
56-61
Phosphoiy choline
PAF receptor
endothelia and
epithelial eel s
62
PavA
fibronectin
n.d.
63,64
n.d. ->]ot determined.
Talay
96
range. This is extremely important since high affinity binding is a prerequisite
for bacterial attachment, a mechanism that has to withstand shear forces occur-
ring on the mucosal surfaces or during the mternalization process.
Protein F2 or PFBP are homologous but distinct fibronectin-binding
proteins, found in most isolates of S. pyogenes lacking the sfbllprtFl gene
[22, 23]. Like Sfbl, protein F2 possesses two binding domains that interact with
fibronectin.
Among the genes encoding fibronectin-binding proteins, the gene for
FBP54 is the most abundant and found in all S. pyogenes isolates [24]. Although
lacking the classical membrane anchor motif of gram-positive surface proteins,
it appears to be localized on the streptococcal surface by a distmct mechanism
[25], thereby acting as an adhesin for buccal epithelial cells but not for HEp2
cells [24]. These data also indicate that distinct fibronectin-binding factors may
target different cell types and have a substantial effect on cell tropism.
Two other recently discovered fibronectin-binding proteins are Fba and
FbaB [26, 27]. Thtfba gene was found m 5 serotypes of S. pyogenes including
M types 1 and 49. An Fba mutant showed diminished adhesion to HEp2 cells,
suggesting that this protein has adhesive properties [26]. However, it should
be noted that the FbaB protein was only found in serotype M3/M 1 8 S. pyogenes
isolates and appears to be genetically most closely related to protein F2 [27].
Protein H, a member of the M protein family, binds to fibronectin in a unique
manner [28]. Unlike the proteins described so far that mainly interact with the
type 1 or type 11 module containing domains of fibronectin, protein H binds to the
type III modules. In addition, protein H was shown to mediate streptococcal
aggregation through a so-called AHP sequence that also promoted adhesion to
epithelial cells [29].
Ml protein, another member of the M protein family, was demonstrated to
bind fibronectin [30], and Ml -specific antibodies efficiently blocked adherence
to HeLa cells. Moreover, an Ml -deficient mutant showed reduced adherence
and invasion, indicating that Ml protein acts as an adhesin and invasin m serotype
Ml S. pyogenes strains [31]. Importantly, as in the case of Sfbl protein, agPi
integruis are the termmal receptor proteins on the cellular surface [32].
Lipoteichoic acid (LTA) was suggested to interact with fibronectin or
hydrophobic residues on the cellular surface. It was defined as a first step
adhesin, mediating low affinity and reversible binding to the ligand, whereas
protein adhesins with high affinity binding to the ligand were termed second
step adhesins [reviewed in 33]. At least one other cellular receptor exists for
LTA: the type I macrophage scavenger receptor which exhibits a broad range of
binding specificity [34].
Recent findings identified M3 protein as an important adhesin that binds
to soluble type 1 and type IV collagen as well as to the native collagen matrix
Gram-Positive Adhesins
97
Fig. Z Serotype M3 S. pyogenes adhering to collagen type I fibers via the M3 protein,
(The scanning electron-microscopic image was kindly provided by Dr. Manfred Rohde, GBF,
Braunschweig, Germany.)
[35] (fig- 2). The amino- terminal variable but M3-specific region of M3 protein
is essential for collagen binding, explaining why other M proteins lack this
function. Besides attaching bacteria directly to collagen matrix, aggregation
of soluble collagen on the bacterial surface leads to formation of large bacte-
rial aggregates that facilitate the colonization process [35]. The only other
collagen-binding protein of 5". pyogenes described so far is Cpa, which was
identified in the M49 serotype and was suggested to mediate attachment to
immobilized type 1 collagen [37],
In highly encapsulated MI8 streptococci, collagen-binding activity and
adhesive properties are mediated by the hyaluronic acid (HA) capsule. The
assumption that M3 protein and streptococcal HA could indeed mediate adherence
to the collagenous matrix was demonstrated ex vivo on native collagen fibers
and in vivo by using a skin infection mouse model [35]. Apart from binding to
collagen, HA interacts with human CD44 on the surface of keratinocytes, acting
as an adliesin for the major cell type of the human pharyngeal epithelium and
external skin [38]. This finding was of particular importance since former studies
suggested an inhibitory role of HA in streptococcal cell attachment. The current
concept, however, is that HA may act as an adhesin itself but may also mask
binding interactions of other streptococcal surface molecules, depending on the
type of the M serotype or tissue [38].
Another target receptor present on the surface of keratinocytes is
CD46, the membrane cofactor protem which is bound by M6 protein [39]. The
carboxy-terminal region of M6 protein as well as the short consensus domains
3 and 4 of CD46 were shown to be crucial for M6/CD46-mediated keratinocyte
Talay
98
attachment [40]. Although structurally closely related, M proteins represent a
heterogeneous group of adhesins with respect to their ligands or target cells
[41]. In contrast to the binding properties displayed by individual M proteins,
such as the fibronectin-binding activity of Ml protein or the collagen-binding
of M3 protein, homophilic interactions of M protein [29] and interactions with
glycosaminoglycans [42] represent common adherence mechanisms among
several types of M proteins. This is underlined by the finding that interactions
with several types of glycosaminoglycans such as dermatan sulfate and heparan
sulfate are predominantly, although not exclusively, mediated via the conserved
carboxy-terminal part of the M proteins [42].
Laminin, another constituent of the ECM, also represents a target for
S. pyogenes. Two lam in in- binding proteins are known, Lbp that has adhesive
properties for epithelial cells [43], and SpeB, the secreted cysteine protease
which also displays glycoprotein-binding activity [44]. Whether SpeB indeed
mediates adherence to host cells remains to be determined.
Within S. pyogenes, three adhesins have been identified of which the ligand
molecules are still unknown. R28, a highly repetitive surface protein related to
the Streptococcus agalactiae surface proteins Rib and a, binds to cervical
epithelial cells [45], and two distinct collagen-like proteins termed SclA/Scll
and SclB/Scl2 were shown to bind to pharyngeal and fibroblast cells, respec-
tively [46-^9]. Since the set genes appear to be prevalent in all S. pyogenes
serotypes, are differentially regulated, and display adhesive function, precise
functional analysis of these potentially important factors will be helpful to
understand their role in the infection process.
During recent years a large number of S. pyogenes adhesins have been
identified and considerable progress has been made by analyzing the molecular
mechanisms underlying the process of bacterial attachment to host cells and tissue.
Future challenges will be to elucidate the three-dimensional stnjcture of receptor/
ligand complexes that will lead to a better understanding of the molecular
nature of these interactions, and the development and use of appropriate in vivo
and ex vivo models for studying the role of the adhesins in the infection process.
The emerging number of available biockout cell lines and mice will serve as
helpful tools, defining a promising interdisciplinary cutting edge between mouse
genomics and infection biology.
S. agalactiae
S. agalactiae, the group B streptococcus, is a gram-positive commensal of
the human vagina, but also the major cause of neonatal sepsis and meningitis.
vS". agalactiae may also cause serious infections in immunocompromised adults.
Compared to S. pyogenes, the number of adhesins identified so far is relatively
small (table I). The host molecules known to be targeted by S. agalactiae are
Gram-Positive Adhesins
99
fibronectin [50, 51], laminin [52, 53], and cytokeratin 8 [54]. The only known
fibronectin-binding factor of group B streptococci is C5a peptidase (ScpB), a
large serine protease that is secreted but also attached to the streptococcal sur-
face. Purified recombinant ScpB was demonstrated to bind to immobilized
fibronectin [51], as well as to HEp2 and A549 cells [50],
Lmb, a surface-associated lipoprotein belonging to the Lral family of pro-
teins, was shown to mediate attachment of group B streptococci to laminin [52].
Whether Lmb indeed acts as an adhesin remains to be determined. Other data
suggest a direct role for the alpha C protein in adherence to cervical epithelial
cell [53]. The alpha C protein is the prototype for a family of long tandem repeat-
containing surface proteins that also include R28 of S. pyogenes and Esp of
Enterococcus faecalis . The cellular receptor for alpha C protein is, as in the case
of R28, still unknown. The molecular nature of another streptococcal adhesin
that binds to cytokeratin 8 [54], a molecule potentially important for coloniza-
tion of keratinized epithelium or damaged cells, also remains to be identified.
Streptococcus pneumoniae
*S. pneumoniae, the pneumococcus, is a natural colonizer of the nasopharyn-
geal epithelium and has the ability to penetrate the epithelial barrier, to translocate
into deeper tissue, where it can cause severe infections such as pneumonia,
meningitis and sepsis. Although binding Xo laminin, type IV collagen, and
vitronectin was described over a decade ago [55], onJy three adhesins that bind
to other target molecules have been identified in this streptococcal species.
To date, the best-studied adhesin of S. pneumoniae is SpsA, also named
CbpA or PspC [56-58]. SpsA binds to human secretory IgA [56, 59], mediates
adherence to activated human cells [57], and uses the human polymeric
immunogJobulin receptor as a terminal receptor on the surface of host cells for
adherence and translocation [60]. In addirion to these properties, SpsA is a pro-
tective antigen that also binds to factor H [58, 61], suggesting a multifunctional
role for this adhesin.
Attachment of pneumococci to activated cells was also shown to be mediated
Lhrough phosphory] choline on the bacterial surface, employing the platelet-
activating factor (PAF) receptor as a target molecule on the cellular surface
[62]. PAF receptor-mediated adherence was found to be coupled to invasion of
epithelial and endothelial cells, suggesting a direct role for this interaction in
subcellular spreading of the pathogen [62].
Among the various ECM molecules, fibronectin is one of the target mole-
cules used by pneumococci for attachment [63]. The binding site of pneumo-
cocci was suggested to be located within the carboxy-terminal portion of
fibronectin. Immobilized rather than soluble fibronectin was shown to be bound
by this bacterial species, discriminating this binding factor from most of the
Talay
100
fibronectm-bindiiig proteins found in S, pyogenes or Staphylococcus aureus,
which efficiently bind to soluble fibronectin as well. PavA, a surface-associated
pneumococcal protein, was identified as receptor for immobilized fibronectin
[64], It displays high similarity to FBP54, its orthologue found in S, pyogenes.
Evident data demonstrate that PavA is essential for virulence [64]; however, its
precise role in mediating cell or tissue adherence remains to be defined.
Staphylococcal Adhesins
S, aureus is an important opportunistic pathogen of humans and animals. The
spectrum of diseases ranges from superficial skin infection to serious infections
such as endocarditis, septic arthritis, and community-acquired and nosocomial
sepsis. Besides this, S. aureus is a major cause of infections originating from
catheters and implanted synthetic medical devices.
Many S. aureus isolates have the ability to bind fibronectin. Most strains
express FnbpA and FnbpB (table 2), two related fibronectin-binding proteins
encoded by closely linked genes [65-67]. These two proteins were shown to bind
soluble and immobilized fibronectin via their carboxy-terminal repeat region^
whereas FnbpA was also shown to bind fibrinogen via its amino-terminal A
domain [68], In vitro infection experiments employing distinct cell types as well
as isogenic S, aureus strains either expressing or lacking one or both Fnbps
revealed that fibronectin-coated devices, human epithelial cells, endothelial
cells, and T lymphocytes are targets for Fnbp-mediated adhesion [69-73]. As in
the case for the S, pyogenes fibronectin-binding proteins Sfbl/Fl and Ml, the
underlying mechanism for this interaction was shown to be the use of fibronectin
as a bridging molecule between the bacteria and host cell integrins such as
a5pi integrin [71, 74, 75]. Consequently, S. aureus Fnbps may act as invasins
governing the uptake of staphylococci by human epithelial and endothelial cells
[74—78]. Analogous to the fibronectin-binding repeat region of Sfbl from S. pyo-
genes^ the domain in fibronectin which is recognized by the Fnbp repeat region
is located at the amino-tenninus of the molecuJe, being composed of five Fl
modules [79-82]. The interacting Fnbp repeat region was suggested to be
unfolded, undergoing a conformational shift upon interaction with the Fl mod-
ules of fibronectin [83, 84]. Based on recent NMR-based structural data, FnbpA
contains 1 1 fibronectin-binding repeat segments, each of which can potentially
bind sequential Fl modules, most likely through the tandem p-zipper mecha-
nism that has also been suggested for Sfbl protein [21] (fig. 1). Altogether, these
findings provide substantial insight into the molecular mechanisms of fibronectin-
mediated adherence of pathogenic cocci. Whether Fnbps of S. aureus are also
able to recruit collagen via prebound fibronectin remains to be determined.
Gram-Positive Adhesins 1
Table Z S. aureus adhesins
Adhesm
^igand mo ecule
Target ce Is, tissue
Reference no.
FnbpA
fibronectin, fibrinogen
epithelial cells, endothelial
cells, mammary g ands,
'1' lymphocytes
65 75
FnbpB
fibronectin
epithelial cells, endothelia
cells, mammary glands
67, 69 75
Ebh
fibronectin
9
85
Cna
CO agen
cartilage
86 94
ClfA
fibrinogen
thrombi, implanted
biomateria
95 100
ClfB
fibrinogen, cytokeratin
thrombi, implanted
biomaterial, keratinocytes,
nasal epithe ia ce s
101 102
SasG
9
nasa epithe ia cells
103, 105
Pis
9
nasal epithelial cells
104, 105
Bbp
bone sia oprotein
bone tissue
106
Spa
vWF
damaged endothe ium
108
vWbp
vWF
7
109
Map/F.ap
fibronectin, fibrinogen,
epithelial cells, fibroblast
110-116
vitronectin, bone sialo-
ce Is
protein, thrombospondin,
collagen, osteopontin
lCAM-1
E!mp
fibronectin, fibrinogen,
vitronectin, collagen
9
t
117
EbpS
elastin
?
118 120
PI A
9
bio film formation,
ce l-cell adhesion
121
Capsule
9
■
epithe ial cells,
endothelial cells
122
Another fibronectin-binding protein of 5. aureus is Ebh, a large Kl-
megadalton surface-associated protein that has been shown to bind soluble and
irrmnobilized fibronectin [85]. The role of Ebh in cell adherence is, however,
still undefined.
Cna, the collagen-bindmg factor of S. aureus is an important adhesin which
mediates attachment to collagen substrates and collagenous tissues [86, 87].
In addition to this, Cna is abJe to mediate adherence to cartilage, a poten-
tially important mechanism during septic arthritis [88, 89] and/or osteomyelitis
[90]. The ligand-binding domain of Cna was identified to be located on a
1 68-amino-acid-long segment within the amino-terminal A domain of the protein
Talay
102
[91]. A synthetic peptide mimicking a subdomain of this segment inhibited
collagen binding to the bacteria and identified the critical residues for collagen
binding [92]. Structural resolution of the binding domain revealed a trench-
shaped organization of the binding module that was predicted to accommodate
the collagen triple helix [93]. interestingly, collagen binding to S. aureus cells
is inhibited by capsule expression, suggesting a masking role for the surface
polysaccharide [94]. This is in contrast to the collagen-binding characteristics
observed in S. pyogenes where HA capsule expression does not inhibit but
enhances collagen binding of 6". pyogenes by directly binding to collagen [35].
S. aureus expresses two adhesins that mediate binding to fibrinogen, ClfA
and ClfB. ClfA enables S. aureus to adhere to fibrinogen-containing substrates
such as plasma clots and to clump in the presence of fibrinogen, giving this
protein its name: clumping factor [95]. ClfA is a potentially important viru-
lence factor since ClfA negative mutant staphylococci showed reduced viru-
lence in a rat endocarditis model [96]. The ligand binding domain of ClfA was
mapped to a 329-amino acid segment within the amino-terminal A domain [97].
ClfA recognizes the carboxy-terminus of the y chain of fibrinogen, a region
also recognized by the oiu]^^^ integrin on platelets, and thus inhibits platelet
aggregation [98]. Analogous to the integrin/fibrinogen interaction, ClfA-mediated
fibrinogen binding is affected by Ca^"^ [99], The structural basis for this inter-
action was found by analyzing the crystal structure of the fibrinogen-binding
domain. A variant of the immunoglobulin (IgG) fold, a structure found in IgG,
was defined to mediate adhesion, placing ClfA into the IgG fold group of
adhesins [100].
ClfB, the second fibrinogen-binding clumping factor and adhesin of
S. aureus, has an overall organization similar to ClfA [101]. However, in contrast
to ClfA, ClfB binds to the a and p chains of fibrinogen. Another characteristic
of ClfB is its abiUty to bind cytokeratin 10 via the amino-terminal A domain [102].
It was shown to promote adherence to human keratinocytes and desquamated
nasal epithelial cells, suggesting that this adhesin plays an important role in
nasal colonization [102].
SasG, a recently identified surface protein of S. aureus [103], and Pis, a
surface protein of methicillin-resistant 5". aureus [104], also promote adherence
to desquamated nasal epithelial cells [105]; their receptor on the cellular surface
is, however, still unknown.
Bone sialoprotein (BSP) is bound by Bbp, a surface protein of S. aureus
[106]. BSP is present in high concentrations in newly formed bone tissue, the
osteoid, and thus suspected to be of relevance in osteomyelitis, an infection
mostly affecting the osteoid. Bbp, like ClfA and ClfB, belongs to the Sdr family
of surface proteins, characterized by the presence of carboxy-terminal serine-
apartic acid dipeptide repeats [107].
Gram-Positive Adhesins
03
S. aureus has the ability to adhere to von Willebrand factor (vWF), a
multimeric glycoprotein present at damaged endothelial sites. Two proteins
have been identified that mediate binding ofS. aureus to human vWF: staphylo-
coccal protein A (Spa) and vWbp [108, 109]. Binding to soluble or immobilized
vWF may not onJy be responsible for 5'. aureus endovascular adherence but also
increase the risk of disturbed hemostasis and vascular thrombosis, both symptoms
observed during severe ^S". aureus infection.
A surface-associated protein with broad matrix protein binding specificity
was identified in 1993 [110], and subsequently characterized as Map or Eap
protein [111, II 2]. Map/Eap was shown to bind fibrinogen, fibronectin, thrombo-
spondin, vitronectin, bone sialoprotein, osteopontin and collagen, and occurs as
a secreted but also surface-associated protein [110-112]. Map/Eap was demon-
strated to mediate adherence to cultured epithelial and endothelial cells [113,
1 14], and appears to enhance staphylococcal internalization into eukaryotic cells
[115]. Furthermore, due to its binding ability towards ICAM-1 and the result-
ing impairment of leukocyte recruitment, Map/Eap plays a role as anti-inflam-
matory immune modulator [116].
Emp, another surface-associated protein of 5. aureus, binds to fibronectin,
fibrinogen, vitronectin and collagen [1 17]. Like Map/Eap, Emp lacks the carboxy-
terminally located LPXTG membrane anchor motif present in several gram-
positive adhesins, but is found on the surface of ^. aureus cells where it may
display adhesive function.
The ECM component elastin is a target for EbpS, an elastin-binding protein
[118]. The elastin-binding domain was localized within the amino-terminal por-
tion of the transmembrane molecule, encompassing 2 1 amino acid residues shown
to be exposed on the surface of intact S. aureus cells [119, 1 20]. As for Ebh, vWbp,
and Emp, its role in mediating cell adherence remains to be investigated.
In addition to the various protein adhesins, S. aureus expresses poly-
saccharides that display an adhesive function: PIA, the polysaccharide inter-
cellular adhesin, is required for biofihn formation and cell-to-cell adhesion
[121]. Capsular polysaccharide of serotype 5 or 8, most frequently found to be
expressed by S. aureus isolated from human infections, bmds to monocytes as
well as to epithelial and endothelial cells, demonstrating adhesive properties for
the S. aureus capsule [122].
Other Gram-Positive Adhesins
Listeria monocytogenes is a gram-positive food-borne human pathogen
that causes listeriosis, a severe invasive infection during which bacteria are
disseminated to the fetoplacental unit and the central nervous system. Although
Talay
104
the overall number of cases of listeriosis is low, the severity of infection is high
and the factors responsible for host cell interaction and spreading are well
studied. L. monocytogenes expresses two important invasins, internalin A and
B (InlA, InlB), that also mediate adhesion to host cells. The cellular receptor for
InJA was shown to be human E-cadherin [123], a cell surface adhesion
molecule contributing to cell cohesion via homophilic dimerization and formation
of adherens junctions. Interestingly, the species specificity of listeriosis arises
from a single amino acid variation in E-cadherins of distinct species: the presence
of a proline residue at position 16 in human E-cadherin was demonstrated to be
crucial for cell interaction, explaining the finding that mouse and rat E-cadherin
harboring a glutamic acid residue at that position was not susceptible for
listeriosis [124, 125]. Different mammalian cell lines have varying susceptibilities
to InJA and InlB. The human intestinal epithelial cell line Caco-2 and the
hepatocyte HepG2 cells are targets for InlA and InlB. Interaction with
monkey kidney Vero cells, mouse hepatocytes, and human endothelial cells is
mediated via InlB [125]. Three receptor molecules have been identified for
InlB [reviewed in 126]. InlB binds to HGF-R or Met, a receptor tyrosine
kinase that acts as a receptor for hepatocyte growth factor [127], to gClq-R
or p32, a receptor of the complement component Clq [128], and to
proteoglycans [129].
Recent work has demonstrated that autolysLns of gram-positive pathogens
may also display adhesive properties. The first autolysin shown to act as an
adhesin was AtlE o^ Staphylococcus epidermidis, a commensal of the skin and
an opportunistic pathogen [1 30]. AtlE was suggested to play a role in the attach-
ment to polystyrene surfaces and to vitronectin, thereby contributing to biofilm
formation of S. epidermidis on implanted polymers. Aas, an orthologous
autolysin of Staphylococcus saprophyticus, mediates adhesion and binds to
fibronectin [131]. The third autolysin found to mediate bacterial attachment
was Ami of L monocytogenes [132]. Adhesive properties were localized within
the noncatalytic carboxy-terminal cell wall-anchoring domain, composed of
so-called GW modules, short dipeptide repeats containing the amino acid
residues glycine and tryptophane [133]. Linkage of GW modules to LTA, as
well as to glycosaminoglycans, anchor GW module-containing proteins to the
surface of gram-positive bacteria [134], GW modules are found within all
adhesive autolysins described herein, but also in eight other listerial proteins
including InlB [134]. Thus, to define the adhesive properties of the yet unchar-
acterized GW module-containing proteins will be a future goal. Interestingly,
Cwp66 of Clostridium difficile, the first identified adhesin of this gram-positive
spore-forming pathogen belonging to the genus Clostridia, exhibits homology to
the catalytic domain of CwlB, the autolysin of Bacillus subtilis [135]. In contrast
to the above-described adhesive autolysins, Cwp66 lacks repetitive GW modules
Gram-Positive Adhesins 105
but may be linked to the gram-positive cell wall via an alternative mechanism,
explaining its surface localization.
It is important to mention that a variety of adhesins, colonization and
cross-linking factors have been identified and characterized in commensal
gram-positive bacteria such as oral streptococci, enterococi, and staphylococci.
Since these adhesins were not the subject of this chapter, the reader should be
referred to these reviews [136-140] summarizing the adhesive mechanisms of
commensal organisms that may also play an important role as opportunistic
human pathogens in the susceptible host.
Concluding Remarks
Among bacterial virulence factors adhesins represent an important group.
Many gram-positive pathogens express adhesins with a broad specificity, as
well as adhesins that recognize particular target molecules such as collagen or
fibronectin. These proteins have evolved in distinct gram-positive and gram-
negative pathogens via convergent mechanisms. Adhesins very often function
synergistically and are highly specific factors that are a prerequisite for infec-
tion which subsequently governs the interplay between the microbe and the
host. In particular cases, they may even have a direct impact on the phenotype
of a disease such as septic arthritis in case of the S. aureus collagen-binding
adhesin Cna [88, 89] or in autoimmune reaction based on the M3 protein of
S. pyogenes [35].
Further characterization of the concerted fianction of multiple adhesins is
a hallmark in understanding the initiation and progress of infection caused by a
particular pathogen. Defining the target molecules in the adhesion process will
help to understand individual host susceptibilities, and will link recent data on
molecular interactions with epidemiological data collected over a whole century.
The growing knowledge in the field of molecular mechanisms of pathogen
adhesion will open up new perspectives in prevention and treatment strategies.
Rational drug design based on the availability of structural data on receptor/1 igand
complexes, fine-tuned vaccination approaches based on minimal functional
domains, and identification of new vaccine candidates are the challenging
perspectives of future research in this field.
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Gram-Positive Adhesins 109
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Gram-Positive Adhesins
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Susaniie R. Talay
Department of Microbial Pathogenesis and Vaccine Research
GBF-German Research Centre for Biotechnology
Mascheroder Weg U DE-38124 Braunschweig (Germany)
Tel. +49 5346 912110, Fax +49 531 6181 708, E-Mail sta@gbf.de
Gram-Positive Adhesins 1 1 3
Adhesins
Russell W, Herwald H (eds); Concepts in Bacterial Virulence,
Contrib Microbiol. Basel, Kai'ger, 2005, vol 12, pp 1 14-13!
Microbial Pathogenesis and
Biofilm Development
Andreas Reisner^, Niels H0iby^, Tim Tolker-Nielsen^,
S0ren Molirv"
^BioCentrum-DTU, Technical University of Denmark, Lyngby, and
''DepartmenL of Clinical Microbiology, Rigshospitalet, Copenhagen, Denmark
Microbial infections constitute a major cause of premature death in large
parts of the world, and for several years we have seen an alarming tendency
towards increasing problems of controlling such infections by antibiotic treat-
ments. It is hoped that an improved understanding of the infectious cycles of
different microorganisms will eventually lead to improved treatments. Several
bacteria have evolved specific strategies for virulent colonization of humans in
addition to their otherwise harmJess establishment as environmental inhabitants,
Jn many such cases biofilm development seems to play a highly significant role
in connection with chronic infections [1].
Bacterial growth on surfaces depends on several factors [2], In nature, surfaces
are probably often conditioned with a thin film of organic molecules, which may
serve as attractants for bacterial chemotactic systems and which subsequently
permit bacterial growth to occur. In laboratory model systems the growth of the
surface-associated bacteria is supported by the nutrient supply in the moving or
standing liquid, A benchnriark of biofilm formation by several organisms in vitro
is the development of three-dimensional structures that have been termed 'mat-
uration', which is thought to be mediated by a differentiation process. Maturation
into late stages of biofilm development resulting in stable and robust structures
may require the formation of a matrix of extracellular polymeric substances (EPS),
which are most often assumed to consist of polysaccharides. A recent striking
finding is that DNA released fi'om biofilm cells may be important as an initial
matrix former [3]. At later times other EPS molecules may add to the shape and
quality of the mature biofilm structure. Figure 1 summarizes the principle steps
involved in the development of microbial biofilms.
1) Reversible attachment 2) Irreversible attachment 3) Cell proliferation
"v.
c:)
■=^>
<?
^
^=>
d>
^^M
.-^^^■.^qaF^^v.^^^^
4) Biofilm maturation
5) Dissolution
Fig, L The biofilm development cycle. Biofilm development is depicted as a geiieraJ
scheme involving attachment to the surface, formation of a tight association between bacteriaT
cells and a surface, growth and intercellular adhesion allowing microcolony formation, mat-
uration including EPS matrix development, and local dissolution leading to release of bacteria,
which may eventually restart the cycle.
How do bacteria know that they are located in a biofilm? There is no doubt
that cell density is an important factor that distinguishes the usually dilute
suspensions of planktonic cells in water from the very cell-dense surface com-
munities found where organic matter is abundant. One answer to the question
therefore is: very high cell density. Another characteristic of biofilms and other
types of surface-associated communities is the prevalence of Lnternally hetero-
geneous environments and microenvironments, often generated and maintained
by the presence of EPS, For the biofilm-associated bacteria this scenario is
recognized as gradients of nutrients and stress factors. For planktonic cells such
gradients rarely play a role. It is often argued that attachment to surfaces is the
most important feature, and that surface-induced gene expression is therefore
one of the key determmants of biofilm development. It should be remembered,
however, that cellular contact with the substratum in a biofilm is a transient
phenomenon (but niosL likely important for early gene activation), which is
quickly converted to a state where essentially all bacterial cells are located far
above the surface in microcolonies or in EPS-embedded 'mushrooms'. In these
entities it is difficult to imagine any bacterial sensing of the surface association
as a physical signal.
Thus, it seems that biofilm-associated bacteria must respond to the (1) very
high cell density and (2) to the various positive and negative gradients. If it is
assumed that bacterial evolution is mainly connected to the dominant life form
of these organisms, and that bacteria in natural environments almost exclusively
live an active proliferating life associated with surfaces (in biofilms), it is to be
Biofilms and Pathogenesis
115
expected that evolution has provided bacteria with properties that allow adaptation
to life under high cell density conditions in environments with nutrient and
antagonist gradients.
This leaves the following issues as the major comumon themes for biofilm
investigations related to the microbial capacity to develop mature, heterogeneously
structured surface-associated communities: How are the specific structural fea-
tures in a biofihn created and maintained? Which functions are involved in the
adaptation to high cell densiries and nutrient gradients? How do biofilm bacteria
evolve, and what are the major selective forces?
In the following we will present an overview of the current understanding
of microbial biofilm development and its clinical relevance in relation to
two examples of gram-negative pathogens, Escherichia coli and Pseudomonas
aeruginosa, for which the biofilm lifestyle seems to be relevant during the
course of infection.
E co/j
As the dominant facultative anaerobe of the normal human intestinal flora,
E, coli remains harmlessly confined to the intestinaJ lumen. However, highly
adapted clones have evolved the ability to cause a broad spectrum of diseases
ranging from urinary tract infection (UTI) and diarrhea to sepsis and meningitis
[4]. Many of these infections are initiated by bacterial colonization of mucosal
surfaces of the genitourinary, gastrointestinal or respiratory tracts. Successful
establishment in the host depends on the ability to overcome host defenses and
shear forces present at most of these surfaces. Since biofilm formation has also
been suggested to be an ancient bacterial survival strategy [5], it seems possible
that at least a fraction of pathogenic E, coli clones have conserved or evolved
the ability to enter a sessile lifestyle in multicellular biofilm communities in the
host environment. Through investigations in recent years we now begin to realize
that bacterial cell-cell interactions among £'. coli cells on biotic and abiotic sur-
faces play a more significant role in pathogenicity than previously anticipated.
It has therefore been of significant interest to clarify the mechanism(s) by
which this organism colonizes surfaces and develops into substantial and robust
biofilms.
In vitro Biofilm. Development
Since E, coli K-12 has been the workhorse bacterium for molecular bio-
logists for neariy 50 years, standard laboratory strains became model organisms
used in an approach to assign a developmental program to E. coli biofilms
formed in vitro. A simple genetic screen was implemented utilizing 96-well
Reisner/H0iby/Tolker-Nielsen/Molin 1 16
microliter dishes as abiotic substrates for biofilm development in vitro, allowing
large-scale isolation of mutants attenuated in biofilm formation under static
conditions.
Underlined by microscopic observations, the results of these initial studies
were integrated in a developmental model for E, coli biofilm formation [6],
According to this model, E. coli K-12 utilizes flagel la-mediated motility and
type 1 pili to initiate early attachment processes. The major phase-variable outer
membrane protein Ag43 was implicated in further development of microcolonies,
and in agreement with the classical role ascribed to exopolysaccharides in
stabilization of mature biofilms, the production of colanic acid was found to be
required for the development of normal biofilm architecture in vitro.
In subsequent similar approaches, additional factors have been found to
affect biofilm formation of £. coli on abiotic surfaces in conventional growth
media; however, only the effects of a few of them have been studied in detail
[7]. The intracellular localization of most of the proposed effector proteins such
as the disulfide bond formation catalyzing DsbA or the acetate kinase AckA
suggests an indirect influence, possibly by altering expression, assembly or
function of already implicated surface appendages and outer membrane pro-
teins. The importance of others such as the stress-response sigma factor RpoS
or the stringent response proteins RelA and SpoT might simply indicate the
requirement for metabolic pathways and stress responses within the hetero-
geneous biofilms that are less important during exponential growth in suspension.
Interestingly, the growth of E. coli K-12 biofilms in continuous hydro-
dynamic culture leads to the identification of biofilm-promoting factors,
reflecting the reduced biofilm-forming capability of K-12 lab strains under these
conditions. An E. coli ompR234 mutant was isolated from the glass surface of
a long-term continuous culture that was found to constitutively overexpress
curii fimbriae [8]. The significantly improved biofilm formation phenotype was
independent of flagella [9]. In 2001, Ghigo [10] discovered that conjugative
plasmids enhance biofibn formation on submerged Pyrex slides under continu-
ous flow when the expression of conjugative pili is derepressed. Mutant analysis
demonstrated that at least for plasmid F, fujictionai conjugative pili are indeed
necessary to obtain the observed induction. In a subsequent study, evidence was
provided that the promotion of biofilm formation in the presence of the con-
jugative transfer genes of plasmid F is mdependent of flagella, type I pili or
Ag43 synthesis [11].
As the biofilm lifestyle is thought to be fundamentally different from
bacterial life m mixed suspension, major differences in gene expression were
expected to be encountered upon switching from planktonic to biofilm growth.
This view was confirmed by an experimental approach that used random chromo-
somal insertions of a promoterless lacZ reporter gene [12], A large fraction
Biofilms and Pathogenesis 1 17
(38%) of 885 fusions was differentially expressed in a curli-promoted static
E. coli K-12 biofilm when compared to planktonic cells. However, a recent
microarray analysis of a biofilm formed by a wild-type K-12 strain under
continuous flow indicated a more modest impact on global gene expression
[13], The transcript level of only 5.4 and 13.6% of the 4,290 protein-encoding
genes was found to be significantly different as compared to expression in
either exponential or stationary planktonic culture, respectively. It is unclear
whether these drastically different results in terms of changes in global gene
expression can be ascribed to the different strain background and/or the exper-
imental setup.
Due to the exclusive focus on K-12 strains in the vast majority of genetic
studies, the relevance of the implicated factors for biofilm formation of non-
domesticated E. coli isolates remains uncertain. For example, whereas the role of
type I and curli fimbriae in the adherence of Shiga toxin-producing E. coli has
been confirmed [14], a recent study suggests that the expression of colanic acid
blocks adhesion of uropathogenic E. coli (UPEC) to inert abiotic surfaces [15].
Given the significantly elevated genome size of pathogenic E. coli as compared
to K-12, determination of the diversity of molecular mechanisms used by the
species E. coli in bacterial cell-cell interactions will necessitate the application
of the already established molecular approaches at least to prototypic clinical
E. coli isolates.
Gastrointestinal Biofilms
As a minority member of the normal flora of the large intestine in verte-
brates, E. coli has to compete for nutrients with approximately 500 other
indigenous species. In principle, successful coexistence can only be achieved
by a growth rate that is at least equivalent to the washout rate from the intestine
or by adherence to the intestinal epithelial cells [16]. Indeed, E. coli is capable
of growing rapidly in intestinal mucus both in vivo and in vitro, whereas growth
in luminal contents seems to be poor [17]. In addition, in situ hybridization
experiments detected only separated single cells of commensal E. coli strains
within the niucus layer but no bacterial cells associated with the epithelium [17,
18]. Thus, benign E. coli cells do not seem to be able to overcome the innate
barriers that impede colonization in a healthy host and the natural lifestyle of
these strains appears to be to reside and grow within the mucus layer almost
exclusively as single cells.
In contrast, each highly adapted E. coli clone causing diarrheal disease
has evolved efficient ways to penetrate the mucus layer and stably adhere to
the underlying epithelial cells even at intestinal sites normally not colonized by
E. coli, such as the small bowel mucosa [19]. As for other mucosal pathogens,
surface colonization by diarrheagenic E. coli is a prerequisite to initiate disease.
Reisner/Hsiby/Tolker-Nielsen/Molin 1 18
Not surprisingly therefore, the most useful phenotypic assay for the diagnosis
and differentiation of diarrheagenic E. coli pathotypes is an adherence assay
using monolayers of epithelial HEp-2 cells. Strikingly, the adherence pattern of
members of two major pathotypes of diarrheagenic E. coli, enteropathogenic
(EPEC) and enteroaggregative (EAEC) E. coli involves - in addition to binding
to eukaryotic cells - apparent strong interactions between bacterial cells leading
to three-dimensional structures typically obsei"ved in bacterial biofilms. EPEC
develop a characteristic localized adherence pattern appearing as microcolonies
on the surface, whereas EAEC appear to aggregate both on the surface as well
as more distantly from the epithelium in a characteristic stacked-brick config-
uration [19]. Most importantly, similar biofilm-like adherence patterns have
also been observed for both EPEC and EAEC in vivo.
While the adherence to epithelial cells has been extensively studied, little
information is currently available about the factors that trigger bacterial cell-cell
adherence or the relevance of the size of these cell aggregates for pathogenicity
[20]. Although the plasmid-encoded bundle-forming pili (BFP) of EPEC have
been suggested to mediate interbacterial interactions allowing formation of
three-dimensional microcolonies on the surface of epithelia [21 ], BFP-expressing
EPECs were found to bind to epithelial cells rather than to already formed
microcolonies. Interestingly, BFP are subject to morphological changes from
thin to thick pili as infection proceeds, resulting in loosening and dispersal of
the aggregates [20]. A bfpF mutant that was found unable to undergo this morpho-
logical change was significantly attenuated in virulence, indicating that formation
and dispersal of microcolonies are both important for virulence.
Likewise, plasmid-encoded thin aggregative adherence fimbriae were found
to mediate the adherence and aggregation pattern of EAEC strains in vivo and
in vitro [19]. Interestingly, the aggregative adherence pattern also requires expres-
sion of a secreted coat protein designated Aap (antiaggregation protein), which
appears to promote dispersal of EAEC on the intestinal mucosa by forming a protein
capsule on the bacterial surface. Mutations in aap lead to increased aggregation
and significantly reduced mucus penetration in vitro, indicating that bacterial
cell-ceil adherence has to be tightly controlled in order to be advantageous iji tlie
intestinal environment [22].
Nevertheless, a large fraction of EPECs and EAECs lack BFP and aggrega-
tive adherence fimbriae, respectively [19, 23]. Thus, E. coli clones seem to have
evolved various divergent pathways to solve the same problem.
Inti'acellular Biofilm-Like Pods in UTI
The human urinary tract is usually a sterile system protected from the
intestinal microflora by nonspecific resistance mechanisms that include phago-
cytosis, endotoxin-induced shedding of bladder epithelial cells, and the flushing
Biofilms and Pathogenesis 1 19
effect of urine flow. However, UTIs are considered to be the most common
bacterial infections [24], with UPEC remaining the predominantly isolated
species [25]. Generally, UPECs are thought to migrate from the gastrointestinal
tract to the periurethral area where they eventually enter the bladder via the
urethra [26]. Further transport into the kidneys may even enable an invasion
into the bloodstream.
Since intestuial E. coli clones are not equally able to survive within and col-
onize the urinary tract, UPECs are thought to be equipped with a variety of vir-
ulence factors including various adheshis of fimbrial nature such as curli, type 1
pili, P, S, and FlC fimbriae [27]. These surface appendages bind to specific
host cell receptor molecules and facilitate attachment of bacteria to specific
epithelial cells they encounter during their transit [28]. However, despite the
clear importance of cell-surface interactions dui'ing the course of infection^ bac-
terial cell aggregates typical for biofilm formation have not been demonstrated
on epithelial cells in vivo.
Recent evidence suggests a novel role for biofilm-like cell-cell interactions
during recurrent UTI. After artificial UTI infection of mice, Anderson et al.
[29] observed large pod-like bacterial cell aggregates within superficial cells of
dissected bladders whereas uninfected bladders appeared smooth. Bacteria
within the pods had a uniform coccoid morphology, were interconnected by
fibers and encased in a polysaccharide matrix. Although the presence of per-
sistent E. coli in the bladder following acute UTI has been shown before, these
large biofilm-like pods are observed after only 24 h of infection and represent
a previously unrecognized intracellular microbial community and might play a
role in the frequent recurrence of uncomplicated UTI (cystitis). However, the
occurrence of these bacterial cell communities in human UTI has not yet been
demonstrated.
Colonization of Indwelling Devices
For every artificial appliance placed in humans there is a corresponding
microbial infection [30]. Tlie crucial importance of biofilms associated with
conLaminaLion of medical ijnplanL devices has been well established. Although
E. coli has been found to adhere to implanted endotracheal tubes and contact
lenses [6, 3 1], it is predominantly isolated from the surface of urinary catheters.
Catheter-associated UTIs are indeed the most common among nosocomial
infections. For example, 1 0-50% of patients experiencing short-term (<7 days)
urinary catheterization [32], and virtually all patients undergoing long-term
(> I month) catheterization became infected [33].
During early stages of infection, E. coli is assumed to be present as a single
species, whereas longer catheterization periods commonly lead to the forma-
tion of mixed communities of mainly gram-negative opportunistic pathogens,
Reisner/Hsiby/Tolker-Nielsen/Molin 120
including /? aei^ginosa, Proteus mirabitis, and Klebsiella pneumoniae [34].
Such E. C(? //-dominated biofilms formed on the luminal surfaces can reach
more than 400 (Jim in height, are usually embedded in a polysaccharide matrix
[35], and can contain minerals such as hydroxyapatite and struvite that crystallize
at the biofilm-urine interface as a result of the elevated pH achieved by bacterial
urease activity. Although symptoms are seldom associated with the infection
initially, ultimate blockage of the inner lumen of the catheter and/or ascent of
bacteria to the bladder and kidney manifest severe consequences for the patient
if left untreated.
Further support for a biofilm mode of growth after catheter colonization is
derived from studies indicating that bacteria in these biofilms survive the urinary
concentrations of antibiotics generated by standard treatment [36]. As a conse-
quence, removal of the colonized device is the only efficient way to clear the
infection. Given these complications generated by biofihns, several attempts
have been made to prevent infection and bacterial colonization of catheters by
incorporating conventional antibiotics or biocides such as silver oxide into the
catheter material [34, 36]. Unfortunately, although the onset of bacteriuria
could be delayed for several days with some catheter materials and treatments^
most of these strategies were ineffective in preventing colonization [31].
A better insight into biofilm formation and ecology on catheters therefore
appears to be required in order to identify more suitable and specific drug targets
or to design more resistant catheters. It needs to be addressed whether initial
colonization by E. colt supports a later establishment of other pathogens.
Subsequent colonizers could attach to initial E. coli biofilms or benefit from
provision of more suitable conditions in the local microenvironment such as
changes of pH and nutrient supply. Interactions between different species during
biofihn formation such as coaggregation might play an important role, as such
phenomena have already been observed between Jactobacilli and UPEC [37].
However, since standardized in vitro and in vivo models are crucial for
obtaining any relevant information about virulence mechanisms, the lack of a
nondestructive, longitudinal monitoring system is a major problem faced in
indwelling-device-related biofilm research. A recently described mouse model
of chronic biofihn infection that relies on biophotonic imaging of biolumines-
cent reporter bacteria constitutes an appealing approach to overcome this
bottleneck [38].
P. aeruginosa
R aeruginosa is an environmental microorganism found especially in fresh-
water and soil. In humans, R aeruginosa may cause a wide range of infections.
Biofilms and Pathogenesis 121
The most prevalent and severe chronic lung infection in cystic fibrosis (CF)
patients is caused by mucoid, biofilm-forming F aeruginosa, which has become
endemic in CF patients [1]. CF is the most common congenital, inherited disease
among Caucasian populations with an incidence rate of 1:2,500-1:4,500. The
pathology of the lung infection, however, is similar in severe chronic obstructive
pulmonary disease, where the number of patients is much higher.
fn vitiv Biofilm Development
In contrast to the biofilm development for E, coli, which appears to be a case
of relatively simple self-assembly processes in concert with surface association,
R aeruginosa is considered an example of a more elaborate biofilm develop-
mental pathway involving several distinct steps of early and late maturation.
Most of the work clarifying this developmental cycle has been performed with
reference strains - PAOl, R aeruginosa 14 and PAK - and so far it appears that
at least these strains share the major features of the biofihn developmental
cycle. In particular, the highly structured R aeruginosa biofilms (comprising
'mushrooms', 'towers', voids and water channels) observed under some condi-
tions have been a challenge to molecular geneticists, and below we will briefly
summarize the current understanding of how the development progresses and is
controlled.
It is first of all important to stress that structural biofilm development by
R aeruginosa appears to be conditional. The immediate environment is a key
determinant of the eventual biofilm structure, illustrated by the finding that in
flow chambers supplied with a citrate minimal medium R aeruginosa forms a
flat biofilm, while in flow chambers supplied with glucose minhnal medium it
forms a heterogeneous biofilm with mushroom-shaped multicellular structures
[39]. In a series of investigations, it was shown that the formation of the flat
R aeruginosa biofilm occurs via initial growth of sessile bacteria forming
microcolonies at the substratum, followed by expansive migration of the bacteria
on the substratum, resulting in the formation of a flat biofilm [39]. Since biofilm
formation by a /? aeruginosa pilA mutant (which is deficient in biogenesis of
type IV pili) occurred without the expansive phase that resulLs in discrete
protruding microcolonies, it was suggested that the expansive migration of the
bacteria on the substratum is type IV pili-driven, and that the shift may be
induced by some sort of limitation arising in the initial microcolonies.
TTie formation of the mushroom-shaped structures in the heterogeneous
glucose-grown R aeruginosa biofihn was shown to occur in a sequential process
involving a nonmotile bacterial subpopulation^ which formed the initial micro-
colonies by growth in certain foci of the biofilm, and a migrating bacterial subpopu-
lation, which initially formed a monolayer on the substratum, and subsequently
formed the mushroom caps by climbing the microcolonies [40].
Reisner/H0iby/Tolker-Nielsen/Molin 122
TTie nature of bacterial cell agglutinating factor(s) in very dynamic
P. aeruginosa biofilms is not known at present. A role of alginate as acell-to-cell
interconnectLng substance has been proposed previously [41], but recently it
was concluded that alginate is not expressed at any significant level in such
in vitro biofilms and therefore cannot be a key structural determinant under the
defined conditions [42]. As we will see later, this situation is completely reversed
in biofilms developing in some clinical cases, where alginate production appears
to be essential for robust biofilm development. Some bacterial cell populations
are apparently kept in the biofilm by substances that allow type IV pili-driven
migration. Since twitching motility is powered by a mechanism involving
extension, grip, and retraction of type IV pili [43], it is possible that type IV pili
can play a role as cell-to-cell and cell-to-substratum interconnecting compounds.
It has been reported that extracellular DNA may play a role as a cell-to-cell
interconnecting substance in P. aeruginosa biofilms [3, 44], and interest-
ingly there is evidence that type IV pili bind to DNA [45]. Yet, other bacterial
cell -to- sub stratum and cell-to-cell connections keep the pilA mutant bacteria
substratum-associated and agglutinated in the biofihns. Evidence is emerging
that a novel type of fimbriae may function as adhesin in P. aeruginosa biofilms
[46], and that certain exopolysaccharides may function as cell-to-cell intercon-
necting substances [Friedmann and Kolter, pers. commun.]. Such compounds
could likely interconnect nonmigrating P. aeruginosa populations.
The apparent complexity of the biofilm developmental cycle of/? aeruginosa
has stimulated the search for genetic regulatory activities, and the findings of
Davies et al. [47] that quorum-sensing control seems to be essential for normal
biofilm formation was in accord with the characteristics of the process. In light
of the current knowledge about the above-described steps of biofilm development
for this organism it is, however, important to emphasize that so far no specific
target for quorum-sensing control has been identified as relevant for these
particular processes. It therefore remains to be seen whether quorum sensing is
regulating any of the described process features such as bacterial cell-cell
adherence, colony climbing or population differentiation.
Chronic Lung Infections in CF
CF patients are intermittently colonized with nonmucoid P. aeruginosa
strains for an average of 12 months before the infections become chronic, and the
presence of mucoid strains and an antibody response is a sign of chronicity [48^
49]. The chronic P. aeruginosa lung infections in CF patients is responsible for
most of the morbidity and mortality of these patients [50], and this state of the
infection constitutes a lung-associated biofilm [51, 52]. The biofilm is charac-
terized by the mucoid phenotype of/? aeruginosa producing an abundance of
alginate [53]. In the conductive zone of the lungs the majority of the bacteria
Biofilms and Pathogenesis 123
stay inside the mucus and grow under anaerobic conditions using nitrate as
electron acceptor [54]. Most of the bacteria are not located on the epithelial
cells, but they induce an endobronchitis and endobronchiolitis without spreading
to the blood or to other organs [54, 55], In the respiratory zone of the airways,
however, the environment is aerobic [56]. Foci of pneumonia in the aJveolar
tissue with extensive infiltration of polymorphonuclear leukocytes (PMNs)
surround localized biofilms of/? aeruginosa which are situated within the alve-
oles and alveolar ducts [55, 57]. The location and organization of the bacteria
in these biofilms are similar to those observed in mucoid colonies and Ln spu-
tum fi^om CF patients with microcolonies of mucoid P aeruginosa [58]. High
levels of antibodies are produced against alginate and other P. aeruginosa anti-
gens, but elimination of the infections is not accomplished [59], and the result-
mg persistent bnmune-compl ex-mediated inflammation is the major cause of the
lung tissue damage [59]. The biofilm mode of growth is resistant to the patients'
defense mechanisms and to antibiotic treatment [59] and is the major reason for
the persistence of the infection lasting for more than 30 years in some patients.
Adaptation of P. aerugmosa to CF Lungs
The CF lung is a stressfijl environment for P. aeruginosa, and, therefore,
they have developed a range of survival strategies. When particles of >5 [xm
containing bacteria are inhaled, they are deposited in connection with the gel
phase of the mucus on the airway surfaces in the relatively small conducting
zone of the central airways, which are covered by ciliated epithelial cells and
coordinated movements of these cilia beating in the sol phase (=epithelial lin-
ing fluid) remove the gel phase of the mucus towards the trachea [56]. The gel
phase of the mucus is produced by submucosal glands and goblet cells. In normal
persons the effect of the cilia's beating (also named the mucociliary escalator)
removes the mucus towards the trachea in this way rapidly (60 )xm/s) clearing
the bacteria within 6h [54, 60]. This clearance mechanism is the most impor-
tant part of the noninflammatory defense mechanism of the respiratory tract. In
CF patients, however, the basic defect of the CFTR protein leads to a reduced
volume of the epithelial lining fluid [60], and the mucociliary clearance of the
bacteria is therefore greatly reduced, leading to robust bacterial growth [54] and
recruitment of the inflammatory defense mechanisms (PMNs) [59]. When par-
ticles of 2-5 |JLm containing bacteria are inhaled, they are deposited in the much
larger peripheral respiratory zone of the lungs without mucus or cilia, and the
major defense mechanism are the alveolar macrophages, which belong to the
inflammatory defense mechanisms [56]. In accordance, bronchoalveolar lavage
studies on CF infants have shown that recruitment of the inflammatory defense
mechanisms (dominated by the phagocytic cells, PMNs and macrophages)
takes place when aspirated microorganisms are colonizing the lower respiratory
Reisner/Hsiby/Tolker-Nielsen/Molin 124
tract [61]. When PMNs and macrophages engulf bacteria there is a metabohc
burst in the phagosomes leading to a release of reactive oxygen species^ some
of which are leaked to the environment [62]. These oxygen radicals induce
killing, DNA damage and mutations in the bacteria [62, 63].
Oxygen radicals produced by the inflammatory response (PMNs) induce
mutations in e.g. the mucA gene leading to the alginate production, which is
characteristic for F aeruginosa biofilm infections in CF [64], Alginate, on the
other hand, is an oxygen radical scavenger [65] and provides mucoid R aeruginosa
with protection against further DNA damage compared to nonmucoid strains
[66]. Alginate can also make the bacteria resistant to phagocytosis by PMNs
and macrophages [67], Alginate production of P. aeruginosa biofilms in CF lungs,
therefore, seems to be the major mechanism of adaptation permitting mucoid
strains to persist in the hostile environment of oxygen radicals originating from
the phagocytic cells of the inflammatory defense mechanisms.
The lungs consist of the central conducting zone and the peripheral respi-
ratory zone. When /? aeruginosa grow in the peripheral respiratory zone (niche)^
the growth condition is comparable to growth in an aerobic or microaerophilic
incubation chamber (5-20% oxygen). The respiratory zone is the area of the
lungs where the venous blood becomes oxygenated in the dense capillary net-
work of the alveoles, thus providing continuous culture conditions with nutrient
and oxygen from the blood [56]. The central conductive zone of the respiratory
tract (the bronchi), on the other hand, where P aeruginosa is located in sputum,
is a completely different niche^ since no oxygen is present in sputum [54].
Sputum consists mainly of dead PMNs and an abundance of released DNA [68]
and leukocyte proteases [69] originating from PMNs in addition to mucus. In
sputum the environment is anaerobic and the growth condition for P aerugi-
nosa is comparable to a batch culture in the stationary phase. There is not so
much blood supply of the conducting zone compared with the respiratory zone
[56] and the bacteria are located inside sputum and not at the epithelial surface
[54]. Under these conditions P aeruginosa may rely on anaerobic growth with
N03~ as the electron acceptor [54].
In cases of infection with mucoid P aeruginosa cells, wliich dommates
chronic infections, a pronounced antibody response against the bacteria is
observed in connection with deteriorating lung function and poor prognosis. In
contrast, the few CF patients colonized only with nonmucoid P aemginosa
have a low antibody response, and they maintain their lung fiinction at the same
nearly normal level similar to that of CF patients without chronic infection [70],
The persistent PVJN inflammation around P aeruginosa infection areas in the
respiratory zone destroys the lung tissue of the infected foci of the lungs of
the CF patients [71], The alveolar macrophages in this zone [61], which migrate
to the lymph nodes [56], are antigen-presenting cells, which are important for
Biofilms and Pathogenesis 125
initiating the antibody production of the B lymphocytes. Colonization of the
conducting zone of the lungs, on the other hand, primarily leads to obstruction
due to the abundance of mucus, and antibody production and lung tissue damage
of the respiratory zone are normally not severe [54]. These observations sug-
gest that severe respiratory failure in CF patients is caused by infection of the
respiratory zone with mucoid P. aeruginosa located in biofilms [55, 57], Pieces
of these biofilms are visible in gram-stained smears of sputum from CF patients
[58]. Although the mucoid phenotype of P. aeruginosa is characteristic for
colonization of the respiratory zones in CF patients, nonmucoid variants of the
same genotype are regularly present simultaneously in sputum [66]. The reason
for this diversity has so far been obscure [58], but indications from in vitro
investigations of stratified bacterial populations may be relevant for a better
understanding of the phenotypical diversity of infectious P. aeruginosa popula-
tions in CF lungs [57, 72-74]. In a population of lung-associated mucoid
P. aeruginosa, isogenic nonmucoid variants could represent a subpopulation of
the original infecting cells (most likely not mucoid) occupying a niche in which
mucoidy is not selectively favorable. Alternatively, the nonmucoid variants may
be phenotypic revertants arising either as 'cheaters', benefiting from the algi-
nate production of other bacteria within the biofihn, or as niche specialists in
the anaerobic conductance zone. The fact that these variants seem to appear as
individual bacteria outside the mucoid biofilm areas in sputum may indicate
that they predominantly derive from the anaerobic zone.
Antibiotic Therapy
Bacteria growing in biofilms are often much more resistant to antibiotics
than planktonic cells of the same isolate. Minimal inhibitory concentration and
minLmal bactericidal concentration may be increased 100- to 1,000-fold in old
biofilms, whereas young biofilms are less resistant [75]. In contrast, planktonic
bacteria released from such resistant biofilms are most often found to be as
sensitive to antibiotics as the original planktonic cells [75]. Biofilm-induced
resistance to antibiotics can be caused by several factors, such as slow growth,
reduced oxygen concentrations at the base of the biofilm, penetration barriers
e.g. binding of positive charges on the antibiotic molecules to the negatively
charged alginate polymers, the presence of 13-lactamase from the bacteria which
cleaves and/or traps (3-lactam antibiotics and overexpression of efflux pumps
[53, 76]. The increased resistance of biofilm bacteria usually results in the
failure of antibacterial therapy with respect to eradication of the bacteria, but
the antibiotic treatment regularly leads to temporary clinical improvement of
the patient [53].
The development of traditional mechanisms of resistance to antibiotics
occurs frequently in CF due to the intensive selective pressure provided by the
Reisner/Hsiby/Tolker-Nielsen/Molin 126
large amount of antibiotics used in these patients [53]. Mucoid and nonmucoid
variants of the same strain are frequently simultaneously present in sputum but
the nonmucoid variants are more resistant to antibiotics, possibly reflecting a
higher antibiotic selection pressure outside the alginate biofilm [66]. The num-
ber of/? aeruginosa in sputum may be as high as lO^-lO'^CFU/ml. The high
number of bacteria implies that mutations do occur in sputum. In addition, high
frequencies (>30%) of hypermutable F aemginosa variants have been found in
CF lung infection [77, 78], and the mutator strains (hypermutable strains) show-
ing >20-fold higher mutation frequency than control strains [78] were also mul-
tiply resistant. The observations fr"om R aeruginosa strains from CF patients
showed the occurrence of a high frequency of hypermutable P. aeruginosa, a
high level of resistance to many antibiotics and, in the case of ciprofloxacin,
several different mutations which increased over time [79]. Jn addition, muta-
tions can be induced by means of oxygen radicals from PMNs, which in vitro
leads to alginate production due to mutations in the mitcA gene [64]. Furthermore,
there is an antioxidant imbalance in the CF lung, which leads to oxygen radical
damage [80]. Taken together, all these observations have led us to suggest that
it is the chronic inflammation dominated by PMNs which induces a high level
of mutations in R aeruginosa in the CF lungs and that the resistant mutants are
then selected by the heavy use of antibiotics. These conventional resistance
mechanisms are then added to the physiological resistance caused by the biofilm
mode of growth in the CF lung.
Perspectives
There is an increasing documentation concernmgthe importance of biofibns
in connection with microbial infections - in particular in relation to persistent
infections of opportunistic pathogens. The detailed investigation of several micro-
bial biofilms has produced interesting information indicating that the multi-
cellular life of bacteria may have its own genetic background that is controlled
by bacterial interactions, which in some cases may resemble complex eukaryotic
tissue development. One important question in relation to pathogenic bacteria
is whether it is possible to extrapolate from these detailed in vitro observations
and mechanisms to the conditions in the infected host. A word of caution is
probably warranted: it is important to keep in mind that there is no indication
of a consensus developmental program, and we therefore must resolve the indi-
vidual biofilm pathways case by case. We also have strong ijidications that the
in vitro biofilm conditions applied in the laboratory cannot be compared to those
prevailing in the host, and it is therefore important to develop better model
systems, if not performing the investigations in vivo. The genomic diversity of
Biofilms and Pathogenesis 127
bacteria is an additional complication; different isolates of the same species
often behave quite differently from each other or when compared with reference
strains or laboratory strains. We also have to keep in mind that simple molecu-
lar identification and characterization of various bacterial cell-cell interaction
mechanisms only constitute the first step in an approach to interfere with cell-
cell interactions necessary for virulence. Since the overall physical strength and
resistance of biofilms to shear force presumably play a critical role in vivo, a
better understanding of the binding forces exhibited by the individual implied
molecular factors is required to identify realistic drug targets.
We now have some fundamental knowledge about the principles of bacterial
life forms which seem to be important for a range of pathogens causing severe
therapeutic problems in the clinic, and the technological and conceptual advances
that have been made during the last 10 years of biofilm research should be applied
with increased intensity in the investigations of infectious diseases. In particular,
it will be important to establish the boundaries for our extrapolations from
in vitro biofilm studies to the conditions prevaihng in clinical cases, just as we must
expand our investigation scenarios to encompass conditions which much better
reflect what goes on in cases of suspected biofilm infections.
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Soren Molin
Molecular Microbial Ecology Group, BioCentrum-DTU
Building 301, DTU
DK-2800 Lyngby (Denmark)
Tel. +45 45252513, Fax +45 45887328, E-Mail sm@biocentrum.dtu,dk
Biofilms and Pathogenesis 131
Enzymes
Russell W, Herwald H (eds); Concepts in Bacterial Virulence,
Contrib Microbiol. Basel, Kai'ger, 2005, vol 12, pp 132-180
Bacterial Peptidases
Jan Potempa^, Robert N. Pike^
^Department of Microbiology, Faculty of Biotechnology, Jagiellonian University,
Krakow, Poland, and Department of Biochemistry and Molecular Biology,
University of Georgia, Athens, Ga., USA;
''Department of Biochemistry and Molecular Biology, Victorian Center for Oral Health
Sciences and CRC for Oral Health Sciences, Monash University,
Clayton, Australia
Enzymes that catalyze the hydrolysis of peptide bonds are referred
to as proteases or peptidases. They are widely distributed in nature, where a
variety of biological functions and processes depend on their activity.
Regardless of the complexity of the organism, peptidases in general are
essential at every stage m the life of every individual cell, since all protein
molecules produced must be proteolytically processed and eventually
degraded. Therefore, it is not surprising that throughout cellular life forms,
genes encoding proteases occur at a relatively high frequency, ranging from
1.15% {Pirellula sp.) to 6.06% (Buchnera aphidicola) of the total gene count,
with the average being about 3%). Among bacterial species which are patho-
genic for humans, the number of peptidases known and putatively functional
ranges from 9-15 in small genomes, such as those of the Mycoplasma
spp. (1.45-2.07%) of the total gene count) to 98 (2.64%) and 121 (2.85%) in
genomes stich as Pseudomonas aeruginosa and Escherichia coli, respectively.
Fortunately, only a small fraction of the expressed peptidases in any pathogen
impose a direct or indirect deleterious effect on their human host and may
therefore be considered a virulence factor. With respect to the number of pro-
tease genes, the record in the microbial world goes to Bacillus cereus [179
potentially functional peptidase genes out of a total of 5,243 genes (3.99%)].
In comparison, onJy three times more functional protease genes have been
identified in Homo sapiens (489 + 143 out of 23,531, 2.7% of the total gene
count).
Classification of Peptidases
Three major criteria are currently used to classify peptidases: (I) the reaction
catalyzed, (2) the chemical nature of the catalytic site, and (3) the evolutionary
relationship to other proteases, as revealed by the primary and/or tertiary structure
of the protein.
Based on the reaction they catalyze, peptidases are divided into two classes,
comprising the exopeptidases and endopeptidases. The exopeptidases act only
near the ends of polypeptide chains. Those acting at a free amino-terminus to
liberate a single amino acid residue, a dipeptide or a tripeptide are referred to as
aminopeptidases, dipeptidyl-peptidases, and tripeptidyl-peptidases, respectively.
On the other hand, exopeptidases that cleave a single residue or dipeptide from a
free carboxy-terminus are called carboxypeptidases and dipeptidyl-dipeptidases,
respectively. Other exopeptidases are specific for dipeptides (dipeptidases), or
the removal of terminal residues, either carboxy- or amino-terminal, that are
substituted, cyclized, or linked by isopeptide bonds. Isopeptide bonds are peptide
linkages other than those joining an ot-carboxyl to an a-amino group. This last
group is collectively referred to as the omega peptidases and is of particular
importance for prokaryotic organisms producing nascent proteins that start with
N-formylmethionine at the beginning of their sequence, which needs to be
removed.
In contrast to the exopeptidases, endopeptidases preferentially hydrolyze
peptide bonds in the inner regions of peptide chains, away from the termini.
Typically, the presence of fi^ee a-amino or a-carboxyl groups has a negative
effect on the activity of these enzymes, but it must be kept in mind that it is not
unusual for an endopeptidase to have both exo- and endopeptidase activity.
A subset of the endopeptidases, with activity limited to oligopeptides or fairly
short polypeptide chains, are referred to oligopeptidases.
According to the nature of their catalytic site, peptidases are divided
into 6 types differing in their catalytic mechanism. The aspartic peptidases,
sometimes incorrectly referred to as carboxypeptidases, have two aspartic acid
residues involved in the catalytic process. The cysteine-type peptidases (incor-
rectly called thiol peptidases) have a cysteine residue in their active center. The
metallopeptidases use a metal ion (commonly zinc) in their catalytic mecha-
nism. The activity of the serine-type peptidases depends on an active serine
residue, while threonine-type peptidases utilize a catalytic threonine. The last
group constitutes a number of peptidases that cannot yet be assigned to any par-
ticular catalytic type. Among prokaryotic organisms, including pathogenic bac-
teria, peptidases of all 6 catalytic types are common, although the frequency of
their appearance is often strongly disproportionate (see following sections).
Bacterial Peptidases
33
A third way to classify peptidases is based on the evolutionary and struc-
tural relationship among enzymes, inferred from the comparison of amino acid
sequences and/or tertiary structures. This method, introduced by Barrett et al.
[2003], and currently implemented in the MEROPS database server (www.
merops.ac.uk) [Rawlings et al., 2004], is a powerful tool, aJlowing the logical
classification of all peptidases, since the structural similarities within a family
of peptidases commonly reflect important similarities in catalytic mechanism
and other properties. However, in some cases, the classification is not fully con-
sistent with three-dimensional structural data, as observed for the structurally
distinct astacins and adamolysins, englobed in the same family Ml 2, or ser-
ralysins and matrixins, grouped into family MIO. This classification may even
extend to assigning the biological flinction of an enzyme for which only the
encoding DNA sequence is known. Therefore, the classification system briefly
described below will be used here to discuss bacterial peptidases.
The term 'family' is used to describe a group of peptidases in which each
member shows an evolutionary relationship to at least one other, either through-
out the whole sequence or at least in the part of the sequence responsible for cat-
alytic activity. Each family is identified by an upper-case letter representing the
catalytic type (A for aspartic type, C for cysteine type, M for metal lo-type, S for
serine type, T for threonine type, and U for unknown type), followed by a unique
number. A family that contains deeply divergent groups is sometimes divided
into subfamilies, identified by upper-case letters. Families are further clustered
into clans. A clan contains all the present peptidases that have evolved from a sin-
gle origin. It represents one or more families that show evidence of their evolu-
tionary relationship, judged by similar tertiary structures, or when structures are
not available, by the order of catalytic-site residues in the polypeptide chain and
often by common sequence motifs around the catalytic residues. Each clan is
identified by two letters, the first representing the catalytic type of the families
included in the clan (with the letter 'P' being used for a clan containing families
of more than one of the catalytic types: serine, threonine or cysteine).
For the purpose of this review it is worth introducing a fourth classification
of bacterial peptidases accordhig to their role in pathogenicity. Pathogenicity,
which is a term synonymous with virulence, is generally delineated as the abil-
ity of a bacterium to cause infection. Virulence factors represent either bacterial
products or a strategy that contributes to virulence, which entails the pathogen to
colonize the host, evade host defense mechanisms, facilitate dissemination, and
cause host damage [Jsenberg, 1988; Mekalanos, 1992]. In many respects, prote-
olytic enzymes produced by several pathogenic bacterial species fit mto the cat-
egory of virulence factors since they are directly involved in one or more of the
processes listed above. Taking into account the numbers of peptidases produced
by bacteria, relatively few can be considered sensu stricto as virulence factors. In
Potempa/Pike
134
this chapter we refer to peptidases, which preferentially target host proteins as
'primary virulence factors'. Many other peptidases are indirectly involved in
pathogenicity, since they are indispensable for the expression of virulence factors
per se. Such proteinases we call 'auxiliary virulence factors'. Finally, many other
peptidases have well defined housekeeping functions. They do not harm the host
either directly or indirectly, but are needed to withstand the stress of living in a
hostile environment. We name them 'bystander virulence factors'.
Aspartic Peptidases
The MEROPS database currently (March 24, 2004) contams a total of
19,682 peptidase-related sequences and aspartic peptidases represent 6.3% of all
peptidases, compared with 19.8% for cysteine, 30.2% for metal lo-, 35.0% for
serine, and 4.1% for threonine peptidases. The aspartic peptidases are subdi-
vided into six clans. Two clans (clans AC and AF) contain enzymes present only
in the major domain of living organisms made up by bacteria. Bacterial pepti-
dases also constitute a separate family within clan AD. They are represented by
three archetypal enzymes: lipoprotein signal peptidase (LspA) often referred to
as signaJ peptidase II (SPase II), a type IV prepilin peptidase and omptin.
SPase II participates in prolipoprotein translocation through the cytoplas-
mic membrane of both gram-negative and gram-positive bacteria. With the
exception of only three bacterial species, including Mycoplasma penetrans.
Mycoplasma gallisepticum and onion yellows phytoplasma, the gene encoding
a potentially functional protein has been found in all other species for which
there is a completely sequenced genome (total 94). SPase II is a good example
of a nonessential housekeeping enzyme, which, in the case of some pathogens,
can contribute to their virulence. Apparently in Listeria monocytogenes,
a gram-positive facultative intracellular human pathogen, temporally regulated
expression of surface lipoproteins is critical for efficient phagosomal escape of
L. monocytogenes. Mutants deficient in SPase II activity stayed entrapped
inside the phagosomes of infected macrophages and have severely attenuated
virulence [Reglier-Poupet et al., 2003].
The gene encoding a potentially functional homologue of the type IV
prepilin peptidase is strongly conserved amongst bacteria (clan AD, subfamily
24 A), although not to the same degree as SPase II. The enzyme cleaves, among
other substrates, the leader sequence from type 4 prepilins or prepilin-like
proteins secreted by a wide range of bacterial species. Its activity is required
for a variety of functions, including type 4 pilus formation, secretion of tox-
ins and other enzymes through the type II protein secretion system in gram-
negative bacteria, gene transfer and biofilm formation. In many regards,
Bacterial Peptidases 135
prepilin peptidase can be considered a housekeeping enzyme, but it contributes
to the expression of well-defined virulence factors in several pathogenic species.
In enteropathogenic E. coii, assembly of the type IV fimbriae known as the bun-
dle-forming pilus (BFP) is dependent on the activity of the prepilin peptidase
encoded by the bfpP gene [Anantha et al., 2000]. Biogenesis of BFP is required
for autoaggregation and localized adherence to host cells and enteropathogenic
E. coli mutants deficient in these surface appendages are nonvirulent in orally
challenged human volunteers. Similarly, a knockout of the prepilin peptidase
gene (pilD) in Legionella pneumophila greatly impaired the ability of the bac-
terium to grow within amoebae and human macrophage-like U937 cells [Liles
et al., 1999]. The mutant showed strongly attenuated virulence in animal models
due to the malfunction of the prepilin peptidase-dependent type 11 secretion
system operating inside the phagocytes [Rossier et al., 2004]. Jn the case of
Vibrio cholerae, functionmg of the extracellular protein secretion apparatus
encoded by the eps gene is strongly dependent on prepilin peptidase activity.
Deletion of the peptidase gene resulted in a dramatic decrease in cholera toxin
secretion and abolished surface expression of the type 4 pilus responsible for
mannose-sensitive hemagglutination [Marsh and Taylor, 1998].
In contrast to SPase 11 and the prepilin peptidase, which are good exam-
ples of auxiliary virulence factors, the plasminogen activating surface pepti-
dase, Pla, of the plague bacterium Yersinia pestis is a paradigm for the primary
virulence factor. The Pla surface peptidase resembles mammalian plasminogen
activators in function and converts plasminogen to plasmin by limited proteol-
ysis. At the same time, the Pla peptidase inactivates a2-antiplasmin, a potent
inhibitor of plasmin [Kukkonen et al., 2001], facilitating unrestrained activity
of this broad -spectrum peptidase that in turn degrades fibrin and noncollage-
nous proteins of the extracellular matrix and activates latent procollagenases.
This causes local damage of the connective tissue and enables the highly
efficient spread of Y. pestis from a subcutaneous site, where the pathogen is
introduced by a vector bite, into the cb-culation [Sodeinde et al., 1992]. In addi-
tion, independent of proteolytic activity, the Pla peptidase mediates Y. pestis
adhesion to basement membrane and invasion into human endothelial cells,
which may also contribute to dissemination of the bacterium in the host
[Lahteenmaki et al., 2001].
The Pla peptidase shares significant amino acid sequence identity (about
50%) with the E. coli integral outer membrane peptidases, OmpT and OmpR,
referred to as omptins. Since some serine protease inhibitors weakly affect OmpT
activity and site-directed mutagenesis studies appeared to implicate Ser99 and
His212 as the active site residues [Kj-amer et al., 2000], the omptins have been
classified as novel serine proteases (family S18) [Rawlings and Barrett, 1994].
However, the crystal structure of OmpT [Vandeputte-Rutten et al., 2001 ] followed
Potempa/Pike
136
by structure-guided site-directed mutagenesis [Kramer et al., 2001] proved that
OmpT activity depends on the Asp83-Asp85 and Asp2 1 0-His2 1 2 residues. These
residues are strictly conserved in all OmpT homologues described to date, includ-
ing PgtE of the Salmonella sp., peptidase SpoA of Shigella flexneri, putative
peptidases ofRhizobium loti, a new species of legume root nodule bacteria, plant
pathogens of the Ei-winia sp. and Agrobacterium tumefaciem, and of course
OmpP and the Pla peptidase. It is assumed that these peptidases have a consei^ved
fold, consisting of a lO-stranded antiparallel |3-barrel that protrudes far from the
lipid bi layer into the extracellular space with the catalytic site located in a groove
at the extracellular top of the vase-shaped p-barrel. Interestingly, activity of
omptins is critically dependent on a specific interaction with lipid A of the LPS
molecule [Kukkonen et al., 2004].
Omptins other than the Pla peptidase are typical housekeeping enzymes
with their function/s not yet entirely tmderstood. Nevertheless, they also seem
to be implicated directly or indirectly in bacterial pathogenicity [Stathopoulos,
1998]. The presence of the ompT gene in clinical isolates of E. coli has been
associated with complicated urinary tract disease [Webb and Lundigran, 1996],
a notion supported by the observation that OmpT cleaves protamine, a highly
basic antimicrobial peptide that is excreted by epithelial cells of the urinary
tract [Stumpe et al., 1998]. Similarly, PtgE expression by Salmonella enterica
may promote resistance to innate immunity by proteolytic inactivation of
a-helical cationic antimicrobial peptides. On the other hand, SopA from
S. flexneri, the causative agent of bacillary dysentery, cleaves the endogenous
autotransporter IcsA, which has an essential role in the formation of actin tails
in host cells, and therefore SopA might be indirectly involved in the actin-based
motility inside infected cells [Egile et al., 1997; Shere et al., 1997].
Among omptins only the Pla peptidase is a potent plasminogen activator
Interestingly, however, OmpT can be easily converted into the plasminogen
activator by subtle mutations at surface-exposed loops. Such conversion may
represent an interesting example of the evolution of a potent virulence factor
from a housekeeping protein [Kukkonen et al., 2001]. In the case of PgtE
from^S. enterica, the 0-antigen of LPS sterically prevents recognition of large-
molecular-weight substrates, rendering plasminogen activator activity cryptic
in this enteropathogen. The 0-antigen repeats also prevent plasminogen activa-
tion by the Pla peptidase and, in this context, it is now clear why Y. pestis lost
the genetic locus involved in 0-antigen synthesis [Kukkonen et al, 2004].
Collectively, it is apparent that the proteolytic activity of omptins con-
tributes to virulence in a variety of ways. Their contribution ranges from bacte-
rial defense and plasmin-mediated tissue infiltration to motility inside infected
cells. Fortunately, they are produced by only a limited number of gram-negative
bacteria which are pathogenic for plants and animals.
Bacterial Peptidases 137
Cysteine Peptidases
The MEROPS database contains 3,897 cysteine-peptidase-related
sequences (19.8% of the total sequences), which are divided into five phylo-
genetically related clans of proteins (CA, CD, CE, CF, and CH) and several
families which are provisionally without a clan assignment. Bacterial pepti-
dases are scattered among all of the clans except clan CH. It is a paradox, how-
ever, that although the bacterial ly derived cysteine peptidases, streptopain
(SpeB) of Streptococcus pyogenes and clostripain from Clostridium perfrin-
gens were among the first proteolytic enzymes ever characterized, cysteine
peptidases are underrepresented in prokaryotic organisms and show limited
variation. Just one family (family C40) encompasses more than one third of
the total cysteine peptidase count in prokaryotes (about 640 sequences). These
enzymes are exemplified by dipeptidyl-peptidase VI from Bacillus sphaericus
and murein endopeptidases (LytE and LytF) from Bacillus subtiUs and repre-
sent typical housekeeping peptidases. Biochemically characterized enzymes
have N-acetylmuramoyl-L-aJanine amidase activity [Kuroda and Seikiguchi
et aL, 1991; Moriyama et aj., 1996; Yamamoto et al., 2003] and are involved
in a peptidoglycan turnover. They are widespread among both gram-positive
and gram-negative bacteria and genes encoding from 1 to 6 functional homol-
ogous are present in at least 70 bacterial species with completely sequenced
genomes (out of 94). No association with virulence has been reported for this
group of peptidases.
Sortases (Family C60)
Peptidases comprising the C60 family constitute a functionally and struc-
turally related group of proteins expressed by all gram-positive species of
bacteria. The prototypical enzyme, referred to as sortase A (SrtA), was first
described in Staphylococcus aureus as an enzyme that is anchored in the plasma
membrane and is responsible for covalent tethering of protein A to the cell wall
[Mazmanian et al., 1999]. It is now known that SrtA attaches a range of impor-
tant surface proteins to the peptidoglycan component of S. aureus and many
other gram-positive bacteria, including virulence-related microbial surface com-
ponents recognizing adhesive matrix molecules (MSCRAMs). Substrates for
SrtA are easily recognized by a carboxy-terminally located sorting signal made
up by an LPXTG amino acid sequential motif, where X is any amino acid, fol-
lowed by a hydrophobic domain composed of about 20 amino acid residues and
a tail of positively charged residues. The hydrophobic domain and charged
residues hinder polypeptide chain translocation through the plasma membrane,
facilitating recognition of the LPXTG motif by SrtA. In a two-step transpepti-
dation reaction, sortase cleaves the LPXTG motif between the threonine and
Potempa/Pike
138
glycine residues and covalently attaches a polypeptide chain, via the carboxy-
terminal threonine, to the amino group of the pentaglycine crossbridge, thus
tethering the protein to the cell wall. Although the structure of peptidoglycan
crossbridging shows large variability in gram-positive bacteria, the mechanism
of surface protem attachment is strictly conserved.
A comparative genome analysis indicated that gram-positive bacteria fre-
quently encode more than one sortase (up to 7 paralogues) and an even larger
number of potential substrates (up to 40 per genome) with their characteristic
LPXTG-type cell wall sorting motif or derivatives thereof [Comfort and Clubb,
2004]. In contrast, a single gene coding for a sortase and only one potential sub-
strate have been identified thus far in only five gram-negative bacterial species.
The sortases can be partitioned into 6 distinct subfamilies (5 in gram-positive
and 1 in gram-negative bacteria) based on amino acid sequence. Members of
each subfamily are suggested to recognize a discrete variation of the sorting
motif [Comfort and Clubb, 2004]. In the bacterial species with more than one
sortase, usually the SrtA-like molecule is responsible for tethering of most cell
wall proteins in an organism, while additional sortase(s) have more specialized
functions. For example, in the case of S. aureus, sortase B (SrtB) recognizes
and anchors a protein known as IsdD, which is involved in heme iron transport
[Mazmanian et al., 2002, 2003]. This protein contains the NPQTN motif
instead of the classical LPXTG sorting sequence exploited by SrtA, but other-
wise the catalyzed reaction is identical. Also a protein, referred to as SvpA,
which is anchored to peptidoglycan by SrtB of L. monocytogenes has the sort-
ing motif, NAKNT, which is divergent from the one used by SrtA [Bierne et al.,
2004]. As in S. aureus, the genes encoding SrtB and its target, SvpA, are part
of the same locus. In S. aureus, isd genes are regulated by iron and encode fac-
tors for hemoglobin binding and the passage of iron, in the form of a heme
group, to the cytoplasm [Mazmanian et al., 2002].
Some of the six sortase genes encoded in the genome of Corynebacierium
diphtheriae are required for biogenesis of the pilus. Assembly of the fimbriae
involves the cleavage of pilin precursors at the classical sorting signal
(LPLTG), or at an LAFTG motif, by two different sortases, which then further
catalyze amide bond cross-linking of adjacent subunits or tethering to peptido-
glycan [Ton-That and Schneewind, 2003]. This covalent attachment of adjacent
pilin subunits has probably evolved m many gram-positive bacteria, since sor-
tase genes in close association with pilin subunit genes with sorting signals
were found in enterococci, streptococci, Actinomyces spp., and C. perfringens.
The NMR structure in solution of SrtA [llangovan etal., 2001] and the crys-
tal structure of SrtB [Zong et al., 2004] from S. aureus are available, revealing an
eight-stranded (3-barrel core structure with a helical subdomain at the amino-
terminal end, which is unique among peptidases. The topology of the p-barrel is
Bacterial Peptidases 139
identical in both enzymes with the critical cysteine residue (Cysl 84 and Cys223
in SrtB and SrtA, respectively) located at the tip of the (37 strand. Initially, it was
predicted that Cysl 84 and His 1 20 of SrtA form a thiolate-imidazolium ion pair
for catalysis [Ton-That et al., 2002] as in the papain cysteine peptidases.
However, pKa measurements for SrtB Cysl 84 and His 120 residues refuted the
involvement of the His residue in the transpeptidation reaction [Connolly et al.,
2003]. From the crystal structure of SrtB and conservation of the Arg233
(Argl 97 in SrtA) residue it is apparent that a unique Cys-Arg catalytic dyad con-
stitutes the foundation of the catalytic machinery of sortases.
By exposing anchored proteins and polymeric structures such as fimbriae,
the cell wall envelope of gram-positive bacteria can be considered to be a surface
organelle maintaining contact between the microbe and its environment. It is now
apparent that the assembly of these surface appendages is dependent on sortases.
In this regard, sortases can be considered to be house-keeping enzymes. However,
they are responsible for surface expression of acknowledged virulence factors,
which mediate adherence to host tissues, host cell invasion, iron acquisition, and
provide protection from assault by the formidable forces of the innate and
acquired immune system. Therefore, sortases can be considered to be the classi-
cal example of an auxiliary virulence factor. Indeed, it was shown that sortase
knockouts in various pathogenic bacteria, including S. aureus, Sweptococcus
/nutans, L. monocytogenes. Streptococcus gorcfonii, and Streptococcus pneumo-
niae, have significantly attenuated virulence when tested in several different ani-
mal models. In this way sortase(s) are a very good target for the development of
therapeutic inhibitors to fight gram-positive infections.
Family C66: IdeS Peptidase (MAC Protein)
A streptococcal protein (Mac) has been identified as a group A Streptococcus
(GAS)-secreted protein of 35 kD with homology to the a-subunit of Mac-1, a
leukocyte ^2 integrin. Mac binds to CDl 6 (Fc^ROIB) on the surface of human
polymorphonuclear leukocytes and inhibits opsonophagocytosis and production
of reactive oxygen species, which resulted in significantly decreased pathogen
killing [Lei et al., 2001]. Later, the MAC protein was shown to be identical to the
IdeS peptidase (IgG-degrading enzyme of 5*. pyogenes) [von Pawel-Rammingen
et al., 2002a, b], a previously unrecognized cysteine peptidase of 5". pyogenes. The
IdeS peptidase is an extremely specific enzyme, which exclusively cleaves the
heavy chain of IgG at the Gly237 residue in the hinge region. The enzyme is active
in human plasma and its ability to interfere with Fc-mediated phagocytic killing
has been demonstrated in a variety of bactericidal assays. These data collectively
show that the IdeS protease contributes to evasion of the adaptive immune system
by GAS by cleaving opsonizing IgG antibodies at the bacterial surface [von
Pawel-Rammingen and Bjorck, 2003]. There is, however, a debate as to whether
Potempa/Pike
140
the proteolytic activity of IdeS (MAC protein) is absolutely necessary for inter-
ference with phagocytosis, which may only be dependent on molecular mimicry
and the presence of the Arg-Gly-Asp amino acid motif in IdeS, which is involved
in the interaction of the enzyme with the human integrins, a^^j ^nd a^^^ [Lei
et al., 2002; von Pawel-Rammingen and Bjorck, 2003],
The occurrence of orthologues of the IdeS peptidase is limited to a very
small subset of the streptococci. In GAS, the enzyme occurs in two allelic
variants among GAS serotypes, where the amino acid sequences of the variants
differ from each other by about 15%. The only three homologues of the IdeS
peptidase identified thus far are in the genome of Streptococcus equi (two
genes) and m Streptococcus suis. One enzyme from S. equi was expressed and
the recombinant protein was shown to possess the same activity as the IdeS
peptidase [Lei et al., 2003]. A distant homologue was also identified in the
genome of Treponema denticola. The recombinant protein was expressed in
E. coli and shown to have a nonspecific, general peptidase activity [Potempa,
unpubl. data].
The activity of IdeS depends on a thiolate-imidazolium ion pair formed by
Cys94 and His262, which act as the active-site residues as in the papain-like
peptidases. These residues are conserved not only in the enzymes from S. equi
and S. suis, but also in the T. denticola homologue. However, the amino acid
sequence is unique and the crystal structure of the IdeS peptidase needs to be
solved to delineate the relationship of the enzyme to other cysteine peptidases.
Based on the present cumulative knowledge, it is apparent that the IdeS
peptidase evolved to a primary virulence factor. It is also a good example of the
possibility that bacteria may contain more peptidases than predicted from
sequence alignments.
Clan CA
All clan CA peptidases have a common fold motif, consisting of an amino-
terminal domain that is mostly a-helical and a carboxy-terminal domain fea-
turing an antiparallel |3-sheet, with the Cys and His catalytic residues forming
a thiolate-imidazolium dyad. However, it is also the most divergent and popu-
lous clan of the cysteine peptidases. The clan is divided into 12 families, of
which bacterial peptidases are found only in 6. Two of these families encom-
pass exclusively bacterial enzymes that have apparently evolved as important
virulence factors.
Family CI: The Papain Family
It is an evolutionary paradox that this major family of cysteine peptidases,
exemplified by papain and manmialian cysteine cathepsins and encompassing
more than 720 sequences, has only few representatives in bacteria. All together,
Bacterial Peptidases
41
only 47 homologues of papain have been identified, including 22 bacterial
species with a completely sequenced bacterial genome. In this context, it is inter-
esting to note that two Mycoplasma species, M. gallisepticum and M. penetrans,
carry three and two copies of a gene encoding a potentially active papain homo-
logue, respectively. However, among the genus Mycoplasma, these two species
are the richest with regard to their peptidase gene count.
Papain homologues occur predominantly in gram-positive species, the major
representative being aminopeptidase C. This enzyme from Lactococci spp. has
been thoroughly characterized [Vesanto et al., 1994; Fenster et al., 1997], and is
also present in pathogens, but there are no reports that this peptidase or its homo-
logues are involved in any aspect of bacterial pathogenicity.
Family C2: The Calpain Family
The protein fold of the peptidase unit for members of this family resem-
bles that of papain. In mammals they are represented by calcium-regulated
ubiquitous enzymes, but thus far only five highly diverged homologues have
been identified in prokaryotes. The recombinant enzyme from Porphyromonas
gingivalis, Tpr peptidase, was characterized as a general endopeptidase which
also cleaves the bacterial collagenase peptide substrate. However, the enzyme
has no collagenolytic activity [Bourgeau et al., 1992] and there is no indi-
cation that the Tpr peptidase is associated with the virulence of this major
periodontopathogen.
Family CIO: The Streptopain (SpeB) Family
The streptococcal cysteine peptidase was isolated and characterized in
1945 and was the second proteolytic enzyme after clostripain to be isolated
fi"om a prokaryote [Elliott, 1945]. For some time the identity of the peptidase
was mistaken for the streptococcal pyrogenic toxin termed SpeB (streptococcus
pyrogenic exotoxin B). The confusion ended when the entire genomes of
several strains of GAS were sequenced, showing that SpeB and streptopain are
the same protein. For historical reasons, however, streptopain is still very often
referred to as SpeB. The enzyme occurs in two variants, which differ only
in a single amino acid residue, glycine or serine, at position 164 from the amino-
terminus of the mature enzyme. Most strains of 5. pyogenes that are associated
with severe invasive diseases express a Gly variant and therefore present an
integrin-binding Arg-Gly-Asp motif at the surface-exposed loop. It was suggested
that the ability of streptopain to bind integrins may be linked to the pathogenicity
of these strains [Stockbauer et al., 1999].
Despite a lack of significant sequence similarity, the crystal structure
clearly indicates that streptopain belongs to the papain clan (superfamily) of
cysteine peptidases. The mature peptidase portion has the two-domain fold
Potempa/Pike
142
characteristic of other papain-like enzymes, with an amino- terminal domain
composed largely of a-helices and a carboxy-terminal domain based on a four-
stranded antiparallel (3-sheet, with the catalytic dyad in the same topological
orientation as in actinidtn, a close relative to papain. In contrast to the peptidase
domain, the profragment of streptopain has a unique fold. While an extended
strand of the prosegment runs the full length of the active site cleft in a direc-
tion opposite to that of a natural substrate, thus blocking the major specificity
pocket in the papain-like peptidase, in prostreptopain the inactivation mecha-
nism relies on displacement of the catalytically essential histidine residue by a
loop inserted into the active site [Kagawa et a!., 2000].
For more than 50 years, streptopain was recognized as a unique cysteine
peptidase unrelated to papain or any other known peptidase. The first homo-
logue of streptopain was identified in P. gingivalis^ a bacterium involved in the
pathogenesis of human periodontal disease [Madden et al., 1995], then another
one from the same microorganism was purified and characterized [Nelson et al.,
1999]. This peptidase, referred to as periodontain, shows a strong preference
for the degradation of unfolded polypeptide chains, with the human plasma
proteinase inhibitor, apantitrypsin, being an important exception. This major
inhibitor of human neutrophil elastase is very efficiently inactivated by cleavage
in the reactive site loop [Nelson et al., 1 998]. Locally, this may lead to a loss of
control of neutrophil peptidases and contribute to connective tissue damage. On
the other hand, any direct role of periodontain in P. gingivalis pathogenicity is
obscure. The enzyme, together with its homologue, is probably involved in gen-
erating nutrients in the form of short peptides which are an indispensable source
of carbon and energy for this asaccharolytic microorganism.
The MEROPS database lists only three streptopain homologues, two in
P. gingivalis and one in the genome of Baclewides thetaiotaomicron. However,
closer analysis of partially finished bacterial genome sequences revealed that
genes encoding potentially active streptopain-like peptidases are more widely
spread. Three different homologues were found in the genome of Prevotella
intermedia, two in Prevotella ruminicola, and one in each of Tannerella
forsythensis and Bacteroides fragilis. These genes encode either secreted or
intracellular proteins. Significantly, the potentially secreted enzymes carry
profragments with significant similarity to the proregion of streptopain. in the
context of streptopain, which is very likely to be a virulence factor, it would be
very interesting to elucidate the role of these streptopain homologues from other
bacterial species.
Streptopain is an outstanding example of a primary virulence factor with a
very broad spectrum of activity. The list of pathogenetically relevant, biologi-
cally important proteins processed, activated, or otherwise altered by the enzyme
is impressive. In vitro, streptopain cleaves the human interleukin-l|3 (IL-I|3)
Bacterial Peptidases
43
precursor to form bioactive IL-1 (3 [Kapur et al., 1 993a], processes the monocytic
cell urokinase receptor [Wolf et al., 1994] and degrades human fibronectin and
vitronectin, two abundant extracellular matrix proteins engaged in maintaining
host tissue integrity [Kapui" et al., 1993b]. In addition, streptopain activates latent
human matrix metal) opeptidases (MMPs), a process hypothesized to participate
in the extensive soft tissue destruction observed in some patients with invasive
streptococcal disease [Bums et al., 1996].
Streptopain is able to cleave IgG molecules at the hinge region of the
7-chain, generating two Fab fragments and one Fc fragment [Collin and Olsen,
2000]. Interestingly, although streptopain can also cleave antigen-bound IgG, it
does not affect antibodies bound to the bacterial surface through the Fc region
[Eriksson and Norgren, 2003]. In this way, streptopain's ability to cleave off the
Fc part of antigen-bound IgG contributes to the ability of GAS strains to escape
opsonophagocytosis, while not interfering with the formation of a host-like coat
of IgG immobilized on the bacterial surface through the Fc portion. This mech-
anism may significantly remforce the defenses of 5". pyogenes against attack by
the adaptive immune response. In addition to streptopain, this deterrence system
consists of (1) eel I- wall-anchored surface proteins of the so-called M protein
family, which binds IgG 'upside down' through the Fc fragment [Berge et al.,
1997]; (2) a secreted, highly specific endoglycosidase (EndoS) that targets con-
served N-linked oligosaccharides on IgG [Collin and Olsen, 2000], and (3) the
uniquely IgG-specific endopeptidase, IdeS (see family C66). Taken together, this
system is very effective hi protectmg S. pyogenes against opsonin-dependent
uptake and killmg by professional phagocytes [Collin et al., 2002].
Streptopain also seems to play a key role in shielding S. pyogenes from the
innate immune system. The enzyme induces release of dermatan sulfate from
the extracellular matrix resulting in the inactivation of antibacterial peptides
[Schmidtchen et al., 2001] or directly eliminates the bactericidal potential of
these peptides by degrading them [Schmidtchen et al., 2002], Finally, and pos-
sibly the most important role of streptopain in the pathogenicity ofS. pyogenes
is the ability of streptopain to directly release the potent peptide hormone,
bradykinin, from high-molecular- weight kininogen. This release is not under
the control of the host system [Hei'wald et al., 1996]. Bradykinin released
by bacterial pathogens has been shown to contribute to the dissemination of
infection [Sakata et al., 1996] and symptoms of sepsis and septic shock
[Herwald et al., 1 998, 2003; Tapper and Herwald, 2000]. Studies conducted with
animal models confumed the significant pathogenic potential of streptopain.
The purified enzyme is lethal to mice [Geriach et al., 1983] and can cause
myocardial necrosis when injected into rabbits, apparently due to its fibrinolytic
activity [Kellner and Robertson, 1954]. Moreover, active immunization of mice
with the purified streptopain elicits a protective response in a model of invasive
Potempa/Pike
144
disease, while mice injected with lethal doses of S. pyogenes were cured
by a single injection of streptopain-specific inhibitor [Bjorck et al., 1989].
Furthermore, experiments using a rat model of lung infection show that strep-
topain acts synergistically with either the streptococcal cell wall antigen or
streptolysin O to augment lung injury [Shanley et al., 1996], This observation
is especially intriguing in the context of the recent discovery that streptolysin O
is the functional equivalent of the type III secretion system in gram-positive
bacteria [Madden et al., 2001] and invites specuJation that in some circum-
stances streptopain may enter the host cell and act as an intracellular viiulence
factor.
Taking into account the results of in vitro and ex vivo experiments, it is
somewhat perplexing that the importance of streptopain as an indispensable vir-
ulence factor in vivo is still questioned. In one study, the importance of strep-
topain for the virulence of S. pyogenes has been demonstrated in a mouse
model using isogenic strains with the streptopain gene inactivated by genetic
manipulation [Lukomski et al., 1997]. In the follow-up in vivo investigation, it
was shown that streptopain helps S. pyogenes to resist phagocytosis [Lukomski
et al., 1998], contributes to soft tissue pathology, including necrosis, and is
required for efficient systemic dissemination of the organism from the initial
site of skin inoculation [Lukomski et al., 1999]. In stark contrast, in a well-
designed and executed study, Ashbaugh and Wessels [2001] proved that genetic
inactivation of the streptopain gene did not significantly attenuate murine inva-
sive infection, either after intraperitoneal or subcutaneous challenge. Also, in a
model of necrotizing fasciitis, a streptopain mutant organism was found to be
as effective in causing tissue damage, as the wild-type control strain [Ashbaugh
et al., J 998]. These results are in keeping with the clinical observation of an
inverse correlation between disease severity and streptopain production in vitro
by genetically related MlTl GAS isolates associated with invasive infection
[Kansal et al., 2000]. This paradox may be explained, at least partially, by the
ability of streptopain to proteolytically remodel S. pyogenes surface proteins.
Although this process is considered advantageous for bacteria [Rasmussen
and Bjorck, 2002], two studies have suggested that the overexpression of strep-
topain results in nonspecific degradation of the antiphagocytic protein M and
solubilizingofthe C5a peptidase [Bergeand Bjorck, 1995; Raederetal., 1998].
Together with degradation of secreted key virulence factors, such as superanti-
gens (streptococcal pyrogenic exotoxins) [Kansal et al., 2003], excessive pro-
duction of streptopain may therefore decrease the pathogenicity ofS. pyogenes.
This hypothesis is further corroborated by the observation that streptopain-
negative isolates have a survival advantage in vivo [Reader et al., 2000] and the
recent discovery that invasive MlTl GAS undergoes a stable phase shift to a
phenotype expressing no streptopain, but instead a full repertoire of secreted
Bacterial Peptidases 145
proteins, which are apparently degraded by active streptopain [Aziz et al.,
2004]. This phenotypic phase shift may be related to the marked resurgence of
severe, invasive and potentially fatal GAS infection, including the necrotizing
fasciitis and streptococcal toxic syndrome observed during the last 20 years.
The role of streptopain in GAS virulence confirms the ancient maxim that
even for a bacterial pathogen too much of a 'good thing' can be bad. Indeed,
S. pyogenes has developed its own system to regulate proteolytic activity and
protect its surface-associated array of key virulence factors. Firstly, expression
of streptopain is regulated at the transcriptional level [Heath et a!., 1999];
secondly, streptopain is produced as an inactive zymogen, which undergoes an
autocatalytic, multistep activation process assisted by the bacterial surface [Liu
and Elliott, 1965a, b; Collin and Olsen, 2000; Chen et al., 2003], and thirdly,
in vivo, the pathogen can coat its surface with the broad spectrum peptidase
inhibitor, a2"r"^croglobulin (a2M) immobilized through interaction with the
peptidoglycan-anchored protein, G-related a,2M-binding protein (GRAB).
Bound to GRAB, a2M protects protein M, and possibly other surface proteins,
from being cleaved by streptopain [Rasmussen et al., 1999]. In this context, it
is very interesting to note that S. pyogenes retains some of the streptopain
enzyme displays associated with the bacterial cell surface, where the enzyme
displays laminin-binding activity [Hytonen et al., 2001], Taking into account
the mechanism of peptidase inhibition by a2M, it is tempting to speculate that
the immobilized form of streptopain preserves proteolytic activity even in the
presence of a high concentration of this inhibitor. Such a feature may be par-
ticularly useful in soft tissue infections where the experimental and epidemio-
logical evidence strongly implies that streptopain plays a critical role in
promoting infection [Svensson et al., 2000].
Family C47: The Staphopain Family
At present, this family is limited to the Staphylococcus genus. Staphopain
occurs in two variants, apparently reflecting the duplication of an ancestral
gene. S. aureus expresses both variants, referred to as staphopain A and
staphopain B, which share about 47% identity at the amino acid sequence level
of the mature enzymes. The single staphopain o^ Staphylococcus epidermidis is
related to staphopain A (75% identity) [Dubin et al., 2001; Oleksy et al., 2004].
On the other hand, a gene encoding a close relative of staphopain B has been
cloned from Staphylococcus warneri, while a cysteine peptidase similar to the
staphopains was purified fi^om the growth medium o? Staphylococcus simulans
biowsLT s tap hylo lytic us [Donham et al., 1988; Neumann et al., 1993].
Both staphopains are processed from large precursors, but so far only
the crystal structure of the mature staphopains is available [Hofmann et al.,
1 993; Filipek et al., 2003]. Remarkably, despite the low sequence similarity to
Potempa/Pike
146
papain-like peptidases, the tertiary structure of the staphopains resembles the
overall fold of papain.
The reciprocal relationship present between the staphopains apparent at the
amino acid sequence level is also mirrored at the genetic level. The staphopain
A gene (scpA) occurs in a bicistronic operon (scpA), in which it is followed by a
gene (scpB) encoding a staphopain A-specific inhibitor. On the other hand, the
staphopain B gene (sspB) is part of the tricistronic operon sspABC, where sspA
and sspC encode the V8 protease and an inhibitor specific for staphopain
B, respectively [Rzychon et al., 2003a, b]. The staphopain inhibitors, ScpB
and SspC, termed staphostatins, have similar folds and apparently the same mech-
anism of target peptidase inhibition although they share less than 20% sequence
identity [Rzychon et al., 2003a, b, Dubin et al., 2003]. Nevertheless, they are
uniquely specific; ScpB affects only staphopain A activity, while SspC exclusively
inhibits staphopain B, without any cross-reactivity. In some cases, the reactivity of
the inhibitor does not extend to the orthologous enzyme from other staphylococcal
species [Dubin et al., 2004]. Apparently, evolution has hand-tailored these
inhibitors to control the activity of the coexpressed enzyme. Interestingly,
staphopains are secreted, while staphostatins are intracellular proteins, suggesting
that they function as so-called threshold inhibitors protecting cytoplasmic proteins
from any prematurely folded peptidases [Rzychon et al., 2003a, b]. The genetic
assembly of peptidase and inhibitor genes in cotranscribed, cotranslated units
provides the means for very efficient elimination of active staphopain fi^om the
cytoplasm.
The extracellular activity of S. aureus is also the subject of multilevel
control. All secreted peptidases, including both staphopains are coordinately
regulated at the transcriptional level by an accessory gene regulator operon
(agr) in a cell density-dependent manner [Janzon et al., 1989]. This regulation
is fine tuned by direct, strong repression of the transcription of the stpAB and
sspABC operons by SarA, the product of the staphylococcal accessory regula-
tor (sar) locus [Chan and Foster, 1998; Lindsay and Foster, 1999; Ziebandt
et al., 2001]. Additionally, this regulatory system is indirectly affected by the
alternative sigma factor c^ [Ziebandt et al., 2001] and probably by several
SarA-like transcriptional factors. Collectively, this highly complex network of
gene regulation assures the precisely coordinated synthesis of extracellular
proteins, including staphopains and other peptidases.
In the case of the proteinases, the regulation of their activity does not stop at
the transcriptional level. Aureolysin, the V8 peptidase (glutamylendopeptidase I)
and the staphopams are secreted as proenzyme forms and activated in a cascade-
like manner. It is well documented that aureolysin activates the zymogen of the
V8 peptidase, which in turn cleaves pro-staphopain B [Drapeau, 1978; Rice at al.,
2001]. Indeed, pro-staphopain B can be expressed in the zymogen form in E. coU
Bacterial Peptidases 147
and activated in vitro by the V8 peptidase (J. Potempa, unpubl. data). In contrast,
the means by which pro-staphopain A processing/activation occurs is obscure and
nothing is known as to whether this pro-enzyme is inactive or which proteinase
is responsible for its processing.
Tight regulation of staphopain expression, together with that of other
acknowledged virulence factors, including toxins and adhesins, may be con-
sidered as indirect evidence of their importance for the sui'vival of S. aureus
in vivo. This association has revitalized interest in staphylococcal extracellular
peptidases as markers of pathogenicity, a subject which has been neglected for
many years. Unfortunately, the results of recent investigations using animal
models of staphylococcal infection are contradictory and confusing. Firstly, it
was shown that a mutant strain deficient in the V8 peptidase was severely atten-
uated in virulence in mouse abscess, bacteremia and wound infection models
[Coulter etal., 1998]. However, the reduced virulence ofthis mutant was appar-
ently due to a polar effect on the expression of the sspB gene encoding
staphopain B, located downstream of the V8 peptidase gene (sspA) in the same
operon [Rice et al., 2001]. Indeed, this assumption was confirmed using a
S. aureus strain with the staphopain B gene eliminated by means of genetic
manipulation [Shaw et al., 2004]. In this study it was shown that only the sspB
gene knockout strain, but not the metalloproteinase (aureolysin) and staphopain
A-deficienr mutants were attenuated in the skin abscess model. However, these
results were not confirmed in a model of septic arthritis in mice. The inactiva-
tion of any of the peptidase genes did not affect the frequency or severity of
joint disease, indicating that, at least in this model, staphopain B does not act
as virulence factor [Calander et al., 2004].
Taken together, the role of staphopains in the physiology and virulence of
staphylococci is obscure, but stringent conservation of the stpA and sspB genes
among S. aureus strains, as well as preservation of the stpA-Wke gene among
coagulase-negative staphylococcal species, implies that their function is impor-
tant for staphylococcus survival in vivo. Amongst the bacterial proteinases,
staphopains are unique with regard to their secretion as zymogens and activa-
tion by Imiited proteolysis. In this respect they resemble sti'eptopain from
5*. pyogenes. In addition, for an as yet not understood reason they are tightly
regulated both at the transcriptional and posttranslational levels. At the protein
level their activity is released in a cascade pathway unique among bacterial
species and then is further controlled by highly specific inhibitors.
Family C39: Bacteriocin-Processing Peptidase
Bacteriocins are antimicrobial peptides produced by microorganisms
belonging to different bacterial taxonomic branches and used by microorganisms
for biological warfare and communications [Eijsink et al., 2002]. One type of
Potempa/Pike
148
these peptides is posttranslationaily modified (class I lantibiotics), while a sec-
ond type does not contain modified amino acids (class 11 nonlantibiotic bacterio-
cins). Both classes are ribosomally synthesized in the precursor form. In most
nonlantibiotic peptides and some lantibiotic peptides, the amino-terminal exten-
sions are composed of a very characteristic leader sequence termed the double-
glycine-type leader, which is cleaved after the second glycine, concomitant with
export carried out by members of a specific family of dedicated ATP-binding
cassette (ABC) transporters. The amino-terminal domain of these transporters,
absent in other ABC transporters, contains conserved cysteine and histidine
residues operating as the catalytic dyad. Also, other residues, including the glu-
tamate and aspartate residues which participate in peptide bond hydrolysis by
papain-like peptidases, are strictly conserved in this portion of the molecule,
which apparently has a canonical fold characteristic of papain [Havarstein et al.^
1995]. The peptidase domain, together with a central hydrophobic integral mem-
brane domain and a carboxy-terminal cytoplasmic ATP-binding domain, consti-
tutes the dedicated transport machinery which recognizes substrates and removes
leader peptides while translocating them across the cytoplasmic membrane. In
addition to bacteriocins, the ABC transporters are used to translocate peptide
pheromones [N4ichiels et al., 2001].
Bacteriocin-processing peptidases are widespread amongst both gram-
positive and gram-negative bacteria and constitute the second most numerous
family of cysteine peptidases in prokaryotes (after family C40). None has been
implicated as a virulence factor. On the contrary, as peptidases which are indis-
pensable for the maturation of bacteriocins, they can be utilized in expanding
applications using bacteriocins as natural food preservatives [Riley and Wertz,
2002].
Family C51: D-Alanyl-Glycyl Endopeptidase
Representatives of this family have thus far only been found in the three bac-
terial species, S. aureus, S. epidermidis, and S. pyogenes. The enzymes are phage-
derived and can degrade the cell wall envelope. Autolysins LytN and LytA from
S. aureus possess a D-alanyl-glycyl endopeptidase as well as N-acetyhnuramyl-
L-alanyl amidase activity, which is contained within the amino-terminal portion
of the polypeptide chain [Navarre et al., 1 999]. None of these autolysins has been
implicated in virulence. Conversely, it has been suggested that they may be used
to counter antibiotic-resistant staphylococcal infections [Fischetti, 2003].
Family C58: The YopT Peptidase Family
Bacterial pathogens share common strategies to infect and colonize animal
and plant host [Staskawicz et al., 2001]. One system, widespread among gram-
negative pathogens, referred to as the type 111 secretion system [Cheng and
Bacterial Peptidases 149
Schneewind, 2000; Cornells and Van Gijsegem, 2000] directly delivers different
classes of proteins to the host. These proteins, now collectively termed type IIJ
effectors, mimic, suppress, interfere^, or modulate host defense signaling path-
ways. Their sole function is to enhance pathogen survival, proliferation and dis-
semination and therefore may be considered to be primary virulence factors.
The structural scaffold to dispense type 111 effectors is conserved but 'delivered
goods' are custom designed to serve the particular needs of a given pathogen.
This is exemplified by the YopT peptidase [Cornelis, 2002] and its homologues
from Yersinia spp. and plant pathogens, including Pseudomonas syringae
[Axtell et al., 2003], which, despite sharing the same fold and catalytic mecha-
nism, target a different set of substrates inside host cells. In addition to the YopT
peptidase onthologues, an overlapping set of pathogens has adopted a cysteine
peptidase with a different fold and evolutionai^y origui (clan CE) [Orth^ 2002]
as the type III effectors.
The YopT peptidase is one of six proteins called Yop effectors (YopH,
YopE, YopJ/YopP, YopO/YpkA, YopM, and YopT) injected into the host cell by
the Yersinia type III secretion system [Juris et al., 2002]. They function in con-
cert to thwart the host immune system. YopT itself exerts a cytotoxic effect in
mammalian cells when delivered by the type 111 secretion system [Iriarte and
Cornelis, 1 998]. This effect is due to proteolytic cleavage of posttranslationally
modified Rho GTPases by the YopT peptidase [Shao et al., 2002]. Apparently
the YopT peptidase specifically recognizes prenylated Rho GTPases and exe-
cutes a proteolytic cleavage near their carboxy-termini [Shao et al., 2003b].
This leads to the loss of the carboxy-terminal lipid modification on these
GTPases, resulting in their release from the membrane and irreversible inacti-
vation. Globally, this causes a disruption of the actin cytoskeleton, exerting a
powerfiil antiphagocytic effect and thus protectuig the pathogen from being
killed by phagocytes.
AvrPphB is an avirulence (Avr) protein from the plant pathogen P. syringae
that can trigger a disease resistance response in a number of host plants. The
crystal structure revealed that the topology of the catalytic triad (Cys-His-Asp),
together with other structural features, resembles that for papain-like pepti-
dases, particularly staphopain [Zhu et al., 2004]. AvrPphB has a very stringent
substrate specificity and apparently exerts only a single proteolytic cleavage in
the Arabidopsis serine/threonine kinase PBSl [Shao et al., 2003a]. It is sug-
gested that the cleavage product is recognized by RPS5, a member of the class
of R proteins that have a predicted nucleotide-binding site and leucine-rich
repeats. In a resistant host these molecular events induce a hypersensitive
response.
The avr genes of the YopT family are common amongst plant pathogens as
well as symbiotic plant bacteria and multiple Avr proteins are found in a single
Potempa/Pike
150
Pseudomonas strain. They all function as specific peptidases targeting different
substrates in the plant host or possibly cleaving the same substrates at different
positions, generating signals detected by distinct R proteins. It is speculated that
the large number of YopT-like proteins found in plant pathogens may reflect
coevolutionary pressures in which the evolution of a new R protein in the host
that detects the cleavage products of a given peptidase selects for a pathogen
with new protease variants [Axtell and Staskawicz, 2003; Zhu et al., 2004].
Clan CD
This clan was recognized based on a conserved sequential motive His-Gly-
spacer-Ala-Cys encompassing the catalytic His-Cys dyad present in caspases,
peptidases involved in apoptosis and cytokine activation (family 14), gingipains
(family 25), plant and animal legumains, processing proteinases (family 13),
bacterial clostripain (family 1 1), and separase, a proteinase required for sister
chromatid separation during anaphase (family 50) [Chenetal., 1998]. The addi-
tional common feature of all these enzymes is a substrate specificity dominated
by a specific PI residue recognition, which is asparagine (legumain), lysine
(Kgp), arginine (Rgp, clostripain, and separase), or aspartic acid (caspases).
Although crystal structures are only available for caspases and one gingipain, it
is expected that representatives of other families in the clan will aJso have a
similar fold. The hallmark of this fold is a six-stranded parallel (i-sheet in the
middle of the molecule sandwiched by three a-helices on each side [Eichinger
et al., 1999]. Out of the five CD clan families known so far, three are found in
bacteria.
Family CI I: The Clostripain Family
Clostripain was identified and partially purified in 1937 from the culture
filtrate of Clostridium histolyticum. The enzyme was then characterized as a
cysteine peptidase that is strictly specific for Arg-Xaa (Xaa stands for any
amino acid) peptidyl bonds. The mature, active clostripain is a noncovalent het-
erodimer derived from an inactive precursor through the autocatalytic removal
of a 9-residue linker peptide [Witle el al., 1996, 1994]. Al least 16 closLripaiii
onthologues homologues were identified in microbial genomes, most of them
in Clostridium spp. [Labrou and Rigden, 2004]. None of them was ever impli-
cated as a virulence factor in clostridial infections. On the contrary, clostripain
is a very useful enzyme in technology, both in sequence analysis and in enzy-
matic peptide synthesis [Gunther et al., 2000].
Family CIS: The Legumain Family
Mammalian asparaginyl endopeptidase (AEP) or legumain is a recently
identified lysosomal cysteine peptidase belonging to clan CD. To date it has been
Bacterial Peptidases
51
shown to be involved in antigen presentation within main-histocompatibility-
complex (MHC) class 11-positive cells and in proprotein processing [Shirahama-
Noda et al., 2003; Manoury et al., 1998; Sarandeses et al., 2003]. Genes
encoding potentially active legumain homologues have thus far only been found
in a few bacterial species, including Caulobacler crescentus, P. aeruginosa,
Pseudomonas putida, P. syringae, Xanthomonas axonopodis, and Xanthomonas
campestris. Their function awaits elucidation.
Family CI 4: The Caspase Family
Caspases are important players in the programmed cell death of multi-
cellular organisms ranging from humans to sponges [Wiens et a!., 2003].
Comparative genomic studies have provided evidence which indicates that the
eukaryotic apoptotic system emerged by acquisition of several central apop-
totic effectors, including caspases, from a-protobacteria as a consequence of
mitochondrial endosymbiosis [Koonin and Aravind, 2002]. Therefore, it is not
surprising that homologues of caspases, referred to as paracaspases and meta-
caspases [Aravind and Koonin, 2002], are abundant in diverse bacteria, par-
ticularly those with complex development, such as Streptomyces, Anabaena,
Mesorhizobium, Myxococcus, and a-protobacteria. The role of these ancient
enzymes in bacterial physiology is obscure.
Family C25: The Gingipain Family
So far gingipains have only been found in P. gingivalis, the major pathogen
of adult onset periodontal disease. They are represented by the products of three
genetic loci conserved amongst clinical and laboratoi7 strains of/? gingivalis,
one (kgp) encoding a lysine-Xaa peptide bond-specific endopeptidase (gingi-
pain K, Kgp) and two others, rgpA and rgpB, which are arginine-Xaa-specific
enzymes (Arg-gingipains, Rgps) [Curtis et al., 1999; Potempa et al., 1995]. The
nascent translation products of gingipain genes undergo complex proteolytic
processing and posttranslational modifications [Veith etal., 2002]. In the case of
Kgp and RgpA, initial polypeptide chain fragmentation is necessary for assem-
bly of a noncovaJenl complex composed of the catalytic, hemoglobin-binding
and hemagglutkiation/adhesin domains [Potempa et al., 2003]. This complex is
either anchored to the outer membrane through a glucan moiety attached to the
carboxy-terminus of the domain derived from the carboxy-terminal portion of
the nascent product, or released into the growth media in the nonglycated form.
RgpB lacks the additional hemoglobin-binding and adhesin domains, but still
undergoes complex modification consisting of the autoproteo lytic removal of
the profragment and either truncation at the carboxy-terminus (the secreted form
of the enzyme) [Mikolajczyk et al., 2003] or glycosylation at the carboxy-
terminus, the latter allowing RgpB to form an association with the cell envelope
Potempa/Pike
152
[Veith etal., 2002]. Collectively, gingipain activity constitutes at least 85% of the
general proteolytic activity produced by /? gingivalis [Potempa et al., 1997].
In every respect, gingipains can be considered to be primary virulence
factors for P. gmg/vfl//5-dependent initiation and/or progression of periodontal
disease. As peptidases, tJiey target a large set of disease-relevant substrates
which can be directly associated with the clinical hallmarks of the disease
[Potempa et aJ., 2000]. Due to the large number of substrates it targets, gingi-
pain activity is also thought to provide this asaccharolytic organism with nutri-
ents. However, gingipains are certainly broad spectrum peptidases. Actually, in
many cases they act with the precision and sophistication of the tailored host
peptidases, mimicking their function. The best example of how P. gingivalis can
manipulate the host is the use of the gingipains to affect the major proteolytic
cascades of coagulation, complement activation, fibrinolysis and kinin genera-
tion [Imamura et al., 2003].
The coagulation cascade is targeted at several levels by Rgps, which
convert factor X, factor IX, protein C and prothrombin to active peptidases by
limited proteolysis, thus mimicking the action of host enzymes [Imamura et al.,
1997, 2001a, b; Hosotaki et al., 1999]. In the case of factor X activation, this
functional mimicry additionally involves enhancement of the Rgp-converting
activity in the presence of phospholipids and Ca^"^, two critical cofactors of
the normal coagulation cascade [Imamura et al., 1997]. The factor X activation
is very efficient, with the catalytic potency in some cases matching that of
natural activators. In this context it is worth emphasizing that gingipains are not
controlled by host inhibitors, in stark contrast to the clotting factors. In vivo, at
periodontal disease sites, the procoagulant activity of Rgps is apparently
negated by the fibrinogen degradation carried out by Kgp [Scott et al., 1993;
Imamura et al., 1995a, b], which contributes to a bleeding tendency, a hallmark
of the disease, which correlates positively with the presence of P. gingivalis at
discrete periodontal pockets. Collectively, the interaction of gingipains with the
coagulation cascade leads to local, uncontrolled release of thrombin, an enzyme
with a multitude of diverse biological activities, including the stimulation of
prostaglandin, IL-1 and pi ate let- activating factor release by endothelial cells
and macrophages. These mediators are considered predominant factors in the
tissue destruction process in periodontal disease.
Another trademark of periodontitis is the increased flow of gingival fluid
from periodontal pockets. This symptom can be directly associated with the
unrivalled (compared to other bacterial proteases) ability of gingipains to
release bradykinin. Physiologically, this potent mediator is released from high-
molecular-weight kininogen by plasma kallikrein, which in turn is generated
from prokallikrein by activated Hageman factor (factor Xlla). Rgps shortcut
this cascade by activation of plasma prekaMikrein, with kinetics, which are
Bacterial Peptidases
53
better than those observed in prekalHkrein activation by factor Xlla [Imamura
et al., 1994]. In addition, Rgps working in concert with Kgp, can release
bradykinjn directly from high-molecular-weight kininogen [Imamura et al.,
1995a, b]. Bradykinin exerts powerful biological activities and is responsible
for pain and local extravasation at the site of infection/inflammation leading to
edema, which underlies the mechanism of generation of gingival crevicular
fluid.
The main targets for gingipains amongst factors of the complement
cascade seem to be the proteins C3 and C5, but the mode of action on these
factors is different. While C3 is destroyed, thus disabling the bactericidal and
opsonizing ability of activated complement, the functional chemoattractant,
C5a, is released from C5 by the action of the gingipains [Wingrove et al., 1992;
Discipio et al., 1996]. In addition, gingipains can enhance the chemotactic
activity of IL-8 [Mikolajczyk-Pawlinska etal., 1998]. Cumulatively, this gingi-
pain-mediated generation of potent chemoattractants may lead to excessive
neutrophil accumulation at periodontal sites, another clinical sign of active
disease.
A large set of cell surface proteins and receptors, including the LPS recep-
tor (CD14) [Sugawara et al., 2000; Tada et al., 2002], the C5a receptor (CD58)
[Jagels et al., 1 996], the IL-6 receptor (TL-6R) [Oleksy et al., 2002], and ICAM-1
[Tada et al., 2003] are targeted by the gingipains. Although the cleavage of
these proteins may significantly contribute to P. gingivalis-'mdiXxcQd. pathologi-
cal changes in the periodontium, activation of protease-activated receptors
(PARs) desei'ves special emphasis. PARs mediate cellular responses to a vari-
ety of extracellular serine peptidases [Ossovskaya and Bunnett, 2004]. The four
known PARs constitute a subgroup of the family of seven-transmembrane
domain G protein-coupled receptors and activate intracellular signaling path-
ways typical for this family of receptors. Activation of PARs involves prote-
olytic cleavage of the extracellular domain, resulting in formation of a new
amuio-terminus, which acts as a tethered ligand. PAR-1 , PAR-3, and PAR-4 are
relatively selective for activation by thrombin whereas PAR-2 is activated by a
variety of proteases, including trypsin and tryptase [Gabazza et al., 2004^. Rgps
specifically activate intracellular signaling pathways through cleavage of PAR-2
on neutrophils [Lourbakos et al., 1998], PAR-1 and PAR-4 on platelets
[Lourbakos et al., 2001 b], and PAR-1 and PAR-2 on human oral epithelial cells
[Lourbakos et al., 2001a] with efficiency matching that for the endogenous
agonists. Collectively, hijacking of the PAR-dependent signaling pathways
illustrates the ability of the gingipains to carry out functional mimicry, which
contributes to potentiation of local inflammatory responses and can be directly
linked to bone resorption, the most profound clinical sign of advanced peri-
odontal disease.
Potempa/Pike
154
The list of proteins cleaved by gingipains discussed above is far from com-
plete. A more complete set includes P. gingivalis extracellular proteins, as well
as many other host proteins, such as hemoglobin and heme/iron-binding pro-
teins, cytokines, bactericidal peptides, host peptidase inhibitors, proteins of the
extracellular matrix, latent matrix metalloproteinases, and epithelial junctional
proteins. The significance of these protein cleavages for periodontal disease
pathogenicity is often speculative, but there is no doubt that gingipains can^
out an extremely diverse set of interactions with the host. Consistently, strains
with the gingipain genes disabled by genetic manipulation have severely
decreased virulence [O'Brien-Simpson et al., 2001] and the pathogeneicity of
P. gingivalis can be supressed in vivo by gingipain-specific inhibitors [Curtis
et al., 2002]. Finally, immunization with the gingipains as antigens has protec-
tive effects, as observed in animal models of P. gingivalis infection [Gibson and
Genco, 2001; Gibson et al., 2004; Rajapakse et aJ., 2002].
Clan CE
This clan contains five families recognized thus far, three are found exclu-
sively in viruses, one is unique for bacteria (family C55) and one is widespread
among cellular organisms, except the archae (family C48). The archetypal
enzyme of clan CE is the cysteine peptidase from adenovirus, adenain. Although
adenain has a unique scaffold not seen in cysteine peptidases outside clan CE,
the active site contains a Cys-His-Glu triplet and an oxyanion hole in an
arrangement similar to that in papain [McGrath et al., 2003; Ding et al., 1996].
In this respect, the CE clan peptidases represent a powerful example of conver-
gent evolution at the molecular level.
Family C48: The Ulpl Endopeptidase Family
In eukaryotic cells, the modification of proteins by a small ubiquitin-like
modifier (SUMO) plays an important role in the function, compartmentaliza-
tion, and stability of target proteins, contributing to the regulation of diverse
processes [Muller et al., 2004; Melchior et al., 2003]. The covalent modifica-
tion of proteins by SUMO-l is reversible and is mediated by SUMO-specific
proteases. These proteases are ubiquitous in eukaryota and are thought to have
a dual function. They are responsible firstly for the initial processing of SUMO- 1
by cleavage of the precursor peptide at the carboxyl-terminus of the protein, and
secondly for the subsequent processing and cleavage of high molecular weight
SUMO-1 conjugates, releasing SUMO-1 and reducing the conjugation status of
the target proteins. Homologues of these peptidases have thus far only been
found in a few gram-negative bacteria, including Bradyrhizobium japonicum,
Chlamydia muridarum. Chlamydia trachomatis, Mesorhizobium loti, P. syringae
and X. campestiis. In the genomes of these organisms, representing animal and
Bacterial Peptidases 155
plant pathogens and plant symbionts, up to 3 genes encoding potentially func-
tional SUMO-specific peptidases are present, but their role in symbiosis or vir-
ulence has not been established. However, taking into account the importance
of SUMO conjugation for the functioning of eukaryotic cells [Yeh et al., 2000],
it is tempting to speculate that bacterial homologues of SUMO-specific pepti-
dases are also active inside the host cell, subverting its function to benefit the
pathogen, as in the case of the YopJ peptidases described below.
Family C55: The YopJ Peptidase Family
It is fascinating to note that amongst the type 111 secretion effectors, human
and plant pathogens, as well as plant symbionts, have evolved two conserved
families of cysteine peptidases with completely different folds. Both families
mimic the proteolytic activity of eukaryotic proteins that are essential for the
normal maintenance of host signaling. Members of the YopT family discussed
earlier have a typical papain-like fold which has been crafted by pathogen evo-
lution to yield a new, specific role in bacterial pathogenicity. The YopJ family
described here apparently evolved using the scaffold of SUMO-specific pepti-
dases (see above). Regardless of their differences in structure and specificity,
both groups of enzymes target a limited number of intracellular substrates,
specific cleavage of which subdues the host reaction to benefit the invading
pathogen.
YopJ, one of the effector molecules injected into the host cell by Y. pestis
was the first protein m this family recognized as a peptidase, based on a com-
parison of the predicted secondary structure of YopJ to that of the known struc-
ture of the adenovirus cysteine peptidase, which revealed significant similarity
between these two proteins [Orth et al., 2000]. Indeed, the intact catalytic dyad
of Cys-His is absolutely necessary for YopJ to exert biological activity in
the host eukaryotic cell. Also, the ability of the YopJ homologue, AvrBsT (the
effector molecule secreted via the type HI pathway by X. campestris pathovar
campestris), to trigger the hypersensitive response in plants, was shown to be
dependent on the proteolytic activity of AvrBsT. In the case of YopJ, the activ-
ity was exerted by cleaving SUMO- 1 -conjugated proteins. Now, it has become
clear that plant homologues of YopJ are also cysteine peptidases with SUMO sub-
strate specificity, since it was shown that XopD, an X. campestris pathovar vesi-
catoria type III effector injected into plant cells, translocated to subnuclear foci
and hydrolyzed SUMO-conjugated proteins in vivo [Hotson et al., 2003]. This
indicates that SUMO protein deconjugation is a common strategy utilized by
animal and plant pathogens to alter signal transduction. The SUMO-dependant
pathway of intracellular signaling is very ancient and evolutionarily conserved
in eukaryotic cells. So is its sensitivity to proteolytic interference by YopJ,
which cleaves SUMO-conjugated proteins in yeast, resulting in a blockage of
Potempa/Pike
156
the mitogen-activated protein kinase (MAPK) kinase-dependent pathway of
signaling [Yoon et al., 2003]. The cleavage of SUMO conjugates in mammahan
cells by Yersinia YopJ peptidase also blocks MAPK kinase [Collier-Hyams
et al., 2002] paralyzing both the innate and adaptive immune responses. There
are, however, some differences between the function of different YopJ pepti-
dases, which apparently reflects adaptation to the specific lifestyle of a given
pathogen. An AvrA protein from common, mild enteropathogen of humans,
S. enterica serovar typhimurium, although 86% similar in amino acid sequence
to YopJ, only inhibits NF-kB signaling and augments apoptosis in human
epithelial cells, giving rise to speculation that AvrA may limit virulence in
vertebrates in a manner analogous to the avirulence factors in plant [Collier-
Hyams et al., 2002]. The lack of an avrA allele in strains of Salmonella typhi
and Salmonella paratyphi [Prager et al., 2000], which evade epithelial defenses
and results in severe systemic diseases seems to support this hypothesis.
In summary, in the case of animal pathogens, SUMO protein deconjugation
interferes with the innate Lmmune response by blocking cytokine production and
inducing apoptosis in the infected cells. The infected host cell cannot respond
to invaders because YopJ-like peptidases disrupt an essential posttranslational
modification that is required for activation of mammalian MAPK and NF-kB
pathways [Orth, 2002].
Clan CF
The crystal structures of two peptidases from this clan are known and they
are clearly unique. As yet, only one family was distinguished (family CI 5).
Family CI 5: The Pyroglutamy I- Peptidase I Family
Pyroglutamyl-peptidases remove the amino terminal pyroglutamate (pGlu)
residue from specific pyroglutamyl substrates [Cummins and O'Connor, 1998].
To date, three distinct forms of this enzyme have been identified, but only type
1 pyroglutamyl-peptidase is a cysteme peptidase with a unique fold. The active
enzyme is apparently a homotetramer [Odagaki et al., 1999]. Both in mammals
aiid prokaryotes, it is located in the cytoplasm and displays a broad pyroglu-
tamyl substrate specificity. Genes encoding pyroglutamyl-peptidase I occur in
several, mostly gram-positive bacterial species, but there are no reports that this
enzyme activity may be related to virulence.
Metallopeptidases
Metallopeptidases are hydrolases in which the nucleophilic attack on a
peptide bond is carried out by a water molecule activated by a divalent metal
Bacterial Peptidases 157
cation, which is usually zinc, but examples where cobalt, manganese or nickel
are used have been reported. The metal ion is usually immobilized by three
amino acid ligands, His, Glu, or Asp. In addition to the metal ligands, at least
one other residue is involved in catalytic hydrolysis of the peptide bond exercis-
ing the functions of a general base in catalytic solvent polarization. In many
cases this residue is a glutamate.
At present the MEROPS database allocates metallopeptidases to 15 clans
recognized by the type and number of metal ions required for catalysis and,
within these broad groups, by the sequential arrangement of the metal ligands
and the catalytic residue. Within clans, separate families are distinguished based
on structural similarities. The most divergent and densely populated clan is MA
featuring the zincins, in which the water nucleophile is bound by a single zinc
ion ligated to two His residues in a sequential motif of His-Glu-Xaa-Xaa-His^ in
which Glu is the general base and Xaa stands for any amino acid. Depending on
the third Zn ligand, which is either a Glu or His/Asp located downstream of the
Zn-binding motif, clan MA is divided into two subclans, MA(E) and MA(M)
[Gomis-Ruth, 2003], respectively. These subclans putatively represent separate
evolutionary lines of metallopeptidases after a very ancient divergence within
clan MA. Also, peptidases grouped into clan MM utilize the His-Glu-Xaa-Xaa-
His motif and use an Asp residue to ligate zinc, but they are structurally unre-
lated to clan MA enzymes. The other well-defined and characteristic sequential
motifs involved in zinc chelation include His-Xaa-Xaa-Glu and His (clan MC),
His-Xaa-Xaa-Glu-His and Glu (clan ME), His-Xaa-Glu-Xaa-His with the third
ligand unidentified (clan MK), His-Xaa-Xaa-Xaa-Asp and His-Xaa-His (clan
MO) and His-Ser-His-Pro-(Xaa9)-Asp (clan MP).
In contrast to the limited occurrence of aspartic and cysteine peptidases
amongst bacteria, metallopeptidases are widespread and they have representa-
tives in 50 out of the 52 distinguished families of this class of enzymes. Even
more interestingly, three metallopeptidases, including the FtsH protease [clan
MA(E), family M41], methionyl aminopeptidase (clan MG, family M24), and
homologues of sialoglycoprotease from Mannheimia (Pasteurella) haemolytica
(Clan MK, family M22) are the only peptidases of any catalytic class which are
absolutely conserved among bacterial species. Apparently, this trio features
essential house-keeping enzymes and, therefore, a perfect target for the devel-
opment of inhibitors, which, by blocking the activity of these peptidases, should
arrest or kill most bacteria. Methionyl aminopeptidase I is an especially attrac-
tive target since the reaction it catalyzes, i.e. removal of the formylated amino-
terminal methionine residue fi^om newly synthesized polypeptide chains, is
unique to bacteria. TTierefore, one would expect that specific inhibitor of the
methionyl aminopeptidase should exert no side effects on eukaryotic organisms,
thus resembling the action of classical antibiotics. Unfortunately, however, the
Potempa/Pike
158
mammalian homologues of methionyl aminopeptidase are also susceptible to
bacterial enzyme inhibitors. Collectively, the promise of effective new drugs and
the obstacles with regard to cross-reactivity has fuelled intense interest in the
detailed investigation of this family of peptidases, which are of known tertiary
structure, have a characterized mechanism of catalysis and are subject to inhi-
bition by an array of different compounds [Bradshaw et al., 1998; Bazan et al.,
1994; Douangamath et al., 2004; Oefner et al., 2003; Hu et al., 2004; Towbin
et al., 2003; Copik et al., 2003; Klein et al., 2003; Li et al., 2004].
Using the FtsH protease as a target to fight bacterial infection seems to
be an even more challenging task than targeting the methionyl aminopeptidase
I. FtsH is a member of the AAA superfamily (ATPases associated with diverse
cellular activities), which includes proteins involved in a variety of cellular
processes characterized by conserved regions which include an ATP-binding site
and a metal lopeptidase domain. These ATP-dependent proteases mediate the
degradation of membrane proteins in bacteria, mitochondria and chloroplasts.
They combine proteolytic and chaperone-like activities and thus form a mem-
brane-integrated quality control system [Langer, 2000]. In bacteria, the FtsH
peptidase is anchored to the cytoplasmic membrane with the catalytic domains
exposed to the cytoplasm. In addition to being involved in quality control of inte-
gral membrane proteins, FtsH peptidase is involved in the posttranslational con-
trol of the activity of a variety of important transcription factors [Schumann,
1999]. In this way, FtsH peptidase is involved in the regulation of the stress
response together with other chaperones with proteolytic activity, including
serine peptidases such as ClpXP, ClpAP, HslUV and Lon [Hengge and Bukau,
2003; Wong and Houry, 2004]. However, unlike the serine peptidase chaperones,
FtsH has never been implicated as an agent contributing to pathogenic fitness of
a pathogen until recently, when it was shown that a S. aureus ftsH mutant was
attenuated in a murine skin lesion model of pathogenicity [Lithgow et al., 2004].
The biological function of the sialoglycopeptidase in M. (Pasteurella)
haemolytica has been investigated in some detail. The 35-kD enzyme isolated
from the culture supernatant of this bacterium is active at neutral pH and
is remarkably specific for 0-siaJoglycoproteins. It cleaves human eiythrocyte
glycophorin A, which is 0-glycosylated at several positions, with a major
site of cleavage at Arg31-Asp32, but does not cleave N-glycosylated proteins
or nonglycosylated proteins [Abdullah et al., 1992]. The importance of the
enzyme in the pathogenicity of bovine pneumonic pasteurellosis (shipment
fever) caused by M. (Pasteurella) haemolytica is not clear, although the enzyme
may interfere with cell-cell adhesion or with cytokine receptor binding through
the cleavage of the cell surface 0-sialoglycoproteins [Sutherland et al., 1992]
during the development of the host immune response in the cattle lung. Also,
the sialoglycopeptidase-mediated enhanced adhesion to bovine platelets may
Bacterial Peptidases 159
initiate platelet aggregation and fibrin formation in alveolar tissue in pneu-
monic pasteurellosis [Nyarko et al., 1998].
Genes encoding potentially active homologues of the sialoglycopeptidase
are conserved across all cellular forms of life, but their biological function is
still a puzzle. The essentiality nature of this gene for some bacteria indicates
that the enzyme has a very important biological function, but either we do not
know its physiological substrate(s) or the protein carries out a function unre-
lated to proteolytic activity. At least in the case Schizosacchawmyces pombe the
sialoglycopeptidase homologue has been shown to be involved in pro-protein
processing [Ladds and Davey, 2000].
The large number of bacterial metallopeptidases excludes the possibility of
a systematic description of each family of these peptidases in the context of
their involvement in pathogenicity. Jt is interesting to note that a relatively large
number of peptidase families in clans MA(E) (7 out of 16) and MA(M) (6 out
of 12) have no counterparts in any other cellular form of life outside the
(archae) bacterial kingdom. In addition to peptidases, which are strongly impli-
cated as virulence factors, only members of families specific for bacteria are
discussed below in more detail.
Family M4: Thermolysin Family
Thermolysin, an extracellular metallopeptidase isolated from Bacillus
thermoproteolyticus, constitutes an archetype, not only of this family, but
also for bacterial metallopeptidases in general. Enzymes homologous to ther-
molysin are expressed by several pathogens, including L. monocytogenes,
S. epidermidis, S. aureus, Enterococcus faecalis, C. perfringens, Helicobacter
pylori, P. aeruginosa and V cholerae. Their involvement in pathogenicity is
generally related to the broad substrate specificity of these peptidases, which
can attack several physiologically important host proteins. A significant
amount of data has been generated regarding the destructive function of
pseudolysin from P. aeruginosa, an enzyme known for its strong elastinolytic
activity [Wretlind and Wadstrom, 1977; Galloway, 1991]. This peptidase, also
referred to as P. aeruginosa eiastase, exerts its destructive action by direct
degradation of several connective tissue proteins [Kessler et al., 1977; Heck
et al., 1986; Galloway, 1991] and, indirectly, by inactivation of host proteinase
inhibitors, including a (-antitrypsin [Morihara et al., 1979]. Through its fib-
rinogenolytic and fibrinolytic activities, the eiastase may disturb homeostasis
and induce changes in the structure of the vascular wall, causing leakage of the
plasma component, including cells into the extravascular tissue. This activity
can potentially induce a hemorrhagic tendency and damage of infected tissue
[Komori et al., 2001]. In lungs, the enzyme may degrade surfactant proteins
SP-A and SP-D, which have important roles in the innate immune response.
Potempa/Pike
160
This mechanism significantly contributes to the virulence mechanism in the
pathogenesis of chronic P. aeruginosa infection [Mariencheck et al., 2003].
This data correlate well with the observation suggesting that the P. aeruginosa
elastase is a potent inflammatory factor in a mouse model of diffuse panbron-
chiolitis [Yanagihara et al., 2003] and that the control of elastase release by
P. aeruginosa may be beneficial for patients with diffuse panbronchiolitis. Also,
pseudolysin seems to play an essential role in the initiation and/or maintenance
of a corneal infection [Hobden, 2002].
The role of pseudolysin orthologues in other pathogenic bacteria is less well
understood and requires further investigation. Nevertheless, aureolysin from
S. aureus has been shown to contribute to connective tissue degradation by host
peptidases through inactivation of host proteinase inhibitors [Potempa et al.,
1986, 1991]. It may also assist m S. aureus dissemination by degradation of bac-
terial adhesins [McAleese et al., 2001]. A similar function is suggested for the
hemagglutinin/peptidase of V cholerae, which may be responsible for the detach-
ment of these bacteria from cells through digestion of several putative adhesion
receptors [Finkelstein et al., 1992]. On the other hand, the L. pneumophila Msp
protease can significantly suppress antibacterial human phagocyte responses and
contribute to the pathogenesis of Legionnaire's disease [Sahney et al., 2001]. A
totally different mechanism seems to be utilized by the gelatinase (GelE) secreted
by E.faecalis. This enzyme, which is also termed coccolysin, is implicated as a
virulence factor by both epidemiological data and animal model studies and can
apparently contribute to the dissemination of E. faecalis by fibrin degradation
[Waters et al., 2003]. It is also possible that some of the manifestations of inflam-
matory conditions in the presence of E. faecalis are related to coccolysin-
catalyzed inactivation of endothelin [Makinen and Makinen, 1994].
Family M6: Immune Inhibitor A Family
The name of this family, also known as thuringilysm family and belonging
to the metzincin clan (MA(M)) [Gomis-Ruth, 2003], refers to the ability of pro-
teins initially isolated from Bacillus thuringiensis to inactivate the antibacterial
activity of insect hemolymph [Ediund et al., 1976]. It is now known that this
protein is a metallopeptidase, exerting its insecticidal activity by proteolytic
degradation of attacins and cecropins, two classes of antibacterial proteins in
insects, and thus kills insect larvae [Dalhammar and Sterner, 1984; Lovgren et
al., 1990]. This unique property contributes to the use of 5. thuringiensis in bio-
logical pest control. Fortunately, this kind of peptidase, which is very effective
in disabling the most important weapon of the host innate defense, is limited to
insect pathogens. Nevertheless, several bacterial peptidases of different catalytic
classes have been described to be able to inactivate human antibacterial peptides,
once again indicating the importance of this activity in bacterial pathogenesis.
Bacterial Peptidases
61
Family M9: Microbial Collagenase
By virtue of being able to degrade collagen, one of the major proteinaceous
constituents of the connective tissue and extracellular matrix, bacterial peptidases
with this activity are by default recognized as virulence factors [Harrington, 1996].
The members of this family are common among Clostridium spp., Bacillus spp.,
and Vibrio spp. Despite the potential ability to inflict extensive tissue damage
and facilitate spreading of infection, the precise role of microbial collagenases m
pathogenicity remains unclear.
Family MI
This family is divided into two subfamilies in MEROPS, though according
to somewhat dubious criteria. Both belong to the metzincin clan [Gomis-Riith,
2003], as well as those of the -equally cryptically subdivided- family 12. Subfamily
lOA encompasses predominantly eukaryotic MMPs. Probable orthologues have
been identified in the genomes of archaebacteria {Methanosarcina acetivorans,
Methanosarcina mazei Gol, Methanosarcina bar/ceri), uncultured crenarchaeote,
and bacteria (Bacillus anthracis, Listeria innocua, L. monocytogenes, Leptospira
intenvgans, and S. pneumoniae). In the latter cases, function as putative virulence
factors or housekeeping enzymes remains to be assessed. According to MEROPS,
subfamily lOA would fiarther encompass a secreted 20-kD metallopeptidase toxin,
B.Jragilis toxin (BFT). The toxin also known as fragilysin is considered an impor-
tant factor in the pathogenicity of infections with enterotoxigenic B. fragilis
(ETBF), a recently identified enteric pathogen of children and adults. Fragilysin
can directly damage human colonic mucosa [Riegler et al., 1999]. This effect is
apparently dependent on cleavage of E-cadherin, the primary protein of the zonula
adherens, leading to modification of epithelial cell morphology in vitro and result-
ing in increased fluid secretion into the intestine, which is clinically manifested
as diarrhea [Wu et al., 1998; Sears, 2001]. AJso, fragilysin contributes to mtesti-
nal mucosal inflammation by stimulation of the expression of the neutrophil
chemoattractant cytokine, IL-8 [Sanfilippo et al., 2000]. According to another
classification, fragilysin, together with three paralogues and an orthologue m the
photosynthetic cyanobacterium Nostoc punctiforme, would constitute an indepen-
dent family within the metzincins, though structurally probably related to MlVTPs
[Gomis-Ruth, 2003].
Only bacterial peptidases are grouped in subfamily lOB, which are exem-
plified by the major metalloproteinase secreted by Serratia marcescens, termed
serralysin. The other members of the subfamily include aeruginolysm, an alka-
line protease from P. aeruginosa, mirabilysin (ZapA protease) from Proteus
mirabilis, and several peptidases from Erwinia spp. Aeruginolysin seems to play
a major role in the pathogenesis of eye infections by enhancing P. aeruginosa
attachment to corneal epithelium [Pillar et al., 2000] and is a target for vaccine
Potempa/Pike
162
development, and chemotherapy for bacterial eye infections. On the other hand,
mirabilysin is considered to be an important virulence factor because it degrades
host immunoglobulins, contributing to immune evasion during urinary tract
infection [Walker et al., 1999; Almogren et al., 2003].
Family M26: IgA 1 -Specific Peptidase
Many of the important mucosal bacterial pathogens, including Haemophilus
influenzae, Neisseria gonorrhoeae, Neisseria meningitides, S. pneumoniae and
successful members of the human resident flora, such as Streptococcus mitis,
Streptococcus oralis, and Streptococcus sanguinis, have developed peptidases
exclusively specific for cleavage at the hinge region of IgAl. These peptidases
apparently belong to three catalytic classes, but only enzymes belonging to
the serine (family S6) and metal 1 ©peptidase (family M26) classes have been
thoroughly characterized. The IgAl-metallopeptidases are produced by
Streptococcus spp., with a significant exception being GAS (S. pyogenes), while
Haemophilus and Neisseria spp. produce serine-type IgA peptidases. Taken
together, these peptidases are a striking example of convergent evolution to the
same function by bacterial vinilence factors [KJlian et al., 1996]. All these
enzymes cleave peptide bonds at a PI proline residue within the hinge region of
IgAl, separating the antigen-binding Fab fragment from the Fc fragment. This
mode of cleavage, which removes the Fc effector domain of the IgAl molecule,
not only eliminates the protective effect of the immunoglobulins, but can also
serve to camouflage the bacteria with Fab fragments, which mask the epitopes
recognized by intact, functional antibodies. Despite this narrow specificity, which
is precisely aimed to not only disable the effector molecules of host immune sys-
tem and to take advantage of them, the exact role of these enzymes in bacterial
pathogenesis is still unclear. This is due to the lack of an appropriate animal
model to test the contribution of these enzymes to pathogenicity, since they only
cleave human, gorilla or chimpanzee IgAl molecules [Reinholdt and Kilian,
1991].
In the context of convergent evolution it is worth mentioning the IgA spe-
cific metal lopeptidase produced by Clostridium ramosum here (family M64)
[Kosowska et al., 2002]. This enzyme has specificity for cleavage of both IgAl
and IgA2 molecules, which is a clear adaptation to the commensal lifestyle in
the human gut, where both IgA isotypes are abundant.
Family M27: Tentoxilysin
Neurotoxins produced by several serotypes of Clostridium botulinum
(BoNT type A-G) and Clostridium tetanum (TeNT) are the most potent natural
toxins known to date. The toxins exert their biological effects at subfemtomolar
Bacterial Peptidases
63
concentrations and they are released into the environment upon bacterial lysis
as a single polypeptide chain of 150kD. Proteolytic cleavage executed by host
peptidases generates a two-chain, mature, active neurotoxin composed of a
heavy chain ( 1 00 kD) and a light chain (50 kD) held together by a single disul-
fide bridge. The heavy chain is responsible for the specific binding of the toxin
to presynaptic membranes and the translocation of the light chain into the
neuron. The light chain is a very specific metal lopeptidase with activity limited
to a small subset of proteins, including VAIVTP/synaptobrevin, SNAP-25 and
syntaxin, which play key roles in synaptic signal transduction [Schiavo et al.,
1992a, b; Montecucco and Schiavo, 1994]. Cleavage of these proteins directly
leads to the clinical manifestations of tetanus and botulism.
Cumulatively, tentoxilysins represent a very interesting example of the
development of extremely specific and potent virulence factors. Fortunately,
their occurrence is limited to a few Clostridium spp.
Family M34: Anthrax Lethal Factor
The anthrax toxin is one of the most lethal natural toxins. It is produced by
Bacillus anthracis and spores of these bacteria are the active component of the
most deadly bioweapon developed by mankind. The toxin is composed of three
proteins, includmg protective antigen (PA), edema factor (EF) and lethal factor
(LF). PA binds to specific cell surface receptors and, upon proteolytic activa-
tion by cell membrane-associated furm-like host peptidases, forms a membrane
channel through which EF and LF enter the cell. LF is a unique multidomain
metal lopeptidase with a very narrow specificity to cleave the amino-terminus
of mitogen-activated kinase kinases 1 and 2 (MMPKKl and MMPKK2). The
cleavage inactivates the signal transduction pathway dependent on these
kinases. This signaling pathway plays a fiindamental role in the overall intra-
cellular signaling network, thus the overall signaling in the cell is compromised.
Family M 56: BlaRJ Peptidase (S. aureus)
The BlaRl peptidase from S. aureus is a metallopeptidase which cleaves a
repressor (Blal) of the synthesis of the (3-lactamase enzyme BlaZ by this bac-
terium [Hackbarth and Chambers, 1993]. Thus, this peptidase controls antibiotic
resistance by controlling the production of the |3-lactamase. The BlaRl pepti-
dase orthologue, Mec Rl, only found in methicillin resistant 6". aureus (MRSA),
controls the formation of the penicillin-binding protein 2a (PBP 2a) and thereby
controls the resistance of the bacterium to methicillin [Hackbarth and Chambers,
1993; Brakstad and Maeland, 1997]. The BlaRl molecule consists of two
domains, an extracellular penicillin-binding domain and an integral-membrane
zinc metallopeptidase domain [Zhang et al., 2001]. Upon penicillin binding,
the BlaRl peptidase autoactivates, then cleaves the repressor of p-lactamase
Potempa/Pike
164
synthesis, providing an interesting 'signal transduction' system which mediates
this antibiotic resistance in the highly pathogenic staphylococcus species.
Family M66: StcE Protease
The StcE metallopeptidase, member of the cholorerilysins within the
metzincin clan MA(M) [Gomis-Riith, 2003] is produced by the enterohemor-
rhagic 0157:H7 strain of £". coll, which causes diarrhea, hemorrhagic colitis,
and the hemolytic uremic syndrome, specifically cleaves CI inhibitor (also
known as CI esterase inhibitor). The peptidase is quite specific for CI inhibitor
and does not appear to cleave other proteins, although it has been shown to
cause aggregation of cultured T cells, the significance of which is not com-
pletely understood [Lathem et al., 2002]. CI inhibitor is known to control
potent proinflammatory and procoagulant enzymes, and thus its inactivation by
the bacterial peptidase is likely to cause proinflammatory effects which may be
consistent with the disease outcomes caused by this strain of E. coli. Further
experiments will be required to elucidate how critical this enzyme is to patho-
genesis by this strain of the bacterium.
Family M73: Camelysin
Camelysin (casein-cleaving metal loprotease) is found on the surface of
B. cereijs, whose genome encodes a total of four paralogues. Possible ortho-
logues have been identified in the genomes of Oceanohacillus iheyensis (five
sequences) and B. anthracis (two sequences). Single sequences are further found
in B. thuringiensis, B. subtilis, and Bacillus halodurans (Gomis-Ruth; personal
communication). This bacterium is known to cause food poisoning and nosoco-
mial diseases. Camelysins do not have a sequence consistent with metallopro-
teases, but the enzyme is active against a broad range of proteinaceous substrates,
and mass spectrometry analyses strongly indicate the association of a zinc ion
with each enzyme molecule. Disruption of the gene for the enzyme causes a
marked loss in the proteolytic activity of membranes from the bacterium and it|
is possible that the enzymatic activity plays a role in the pathogenic activity of
the organism, although this remains to be fimily established [Grass et al., 2004].'
Serine Peptidases
Peptidases which utilize a serine residue as the main catalytic residue are
the biggest group of peptidases, making up 35% of the total peptidases listed in
MEROPS. The serine peptidases are widespread across all organisms and are
divided into 10 clans on the MEROPS database [SB, SC, SE, SF, SH, SJ, SK,
SP, SR and S- (the last contains currently unassigned peptidases)]. Bacterial
Bacterial Peptidases 165
proteases are present in all of these clans, except SH, SP and SR, which will
therefore not be considered any further here.
By definition, this catalytic class contains a serine residue acting as the
nucleophile during catalysis. Usually (as applies to enzymes in clans SB, SC and
SK) the catalytic Ser residue combines with His and Asp residues to form the
classical catalytic triad exemplified by chymotrypsin, the archetypal enzyme of
the serine protease class. Variations on this do exist, for instance enzymes in
clans SE and SJ use a Ser/Lys dyad to accomplish catalysis, while those in SF
use either a Ser/Lys or a Ser/His dyad.
There are over 60 families represented within the serine-type catalytic
class and many of these are subdivided into subfamilies. The sheer number of
proteases in this catalytic class which are found in bacteria defies their being
mentioned in any representative manner here. Thus the most interesting or well-
characterized examples with direct relevance in pathogenicity were selected for
presentation here.
Family SIB
The glutamyl endopeptidase I, better known as endoproteinase GluC or the
V8 protease from S. aureus, is a member of the SIB family. The roles of this
enzyme are somewhat related to pathogenicity (see section Family C47: The
Staphopain Family above), but this enzyme is better known for its widespread
biotechnological use as a specific protease in sequencing applications. Its struc-
ture has recently been solved [Prasad et al., 2004]. This family also contains the
Spl peptidases, which have recently been identified as a new operon which is
positively controlled by the Agr virulence regulator, indicating a possible role
in pathogenesis by S. aureus [Reed et al., 2001].
Family SIC
An interesting group of peptidases is formed by members of the SIC family,
which is required for growth at high temperatures by a number of organisms, such
as E. coli. Some of these enzymes, generically termed protease Do (also referred
Lo as DegP or HtrA), have been characterized as being associated with the viru-
lence of 5. enterica serovar typhimurlum. Yersinia enterocoUtica and S. pyogenes.
DegP from E. coli has a fascinating dual function of acting as a chaperone and a
peptidase, depending on the temperature of the environment. In the chaperone
phase, a hydrophobic patch of amino acids plays the presumptive role of binding
unfolded proteins and mediating their refolding. During chaperone operation, the
active site for the peptidase is 'walled off', preventing substrate binding and catal-
ysis. A change in the environmental conditions triggers the opening of the active
site to substrates and allows catalysis. This fascinating mechanism allows the pep-
tidase to process many different proteins needed for pathogenesis by the bacteria.
Potempa/Pike
166
Family SID
The family is entirely composed of the endoproteinase lysC and endopro-
teinase Arg-C, which have applications in the sequencing of proteins due to
their high specificity for lysine and arginine amino acids at the cleavage point,
respectively. An endoproteinase Arg-C orthologue from P. aeruginosa is
thought to act as a virulence factor in cornea] infections by this bacterium
[Engel etal., 1998].
Family S6
The IgA I -specific serine endopeptidases which are found in Neisseria
spp. and some Haemophilus spp. are typical members of the S6 family. In
A^. gonorrhoeae, the enzyme has been postulated to play a role in evading the
host immune response by specifically cleaving IgAl [Vitovski et al., 1999].
It has been suggested that the enzyme plays a role in bacterial invasion of host
cells [Lin et al., 1997]. However, whether the IgAl -specific serine endopep-
tidase is a crucial virulence factor has yet to be determined [Johannsen et al.,
1999].
Family S8A
This group of serine proteases contain enzymes generally referred to as
subtilisin-like enzymes, named after the archetypal enzyme of the group. The
family contains a large number of enzymes, most likely second only to family
SIA which contains the mammalian chymotrypsin-like enzymes. The subtil-
isins and chymotrypsin-like enzymes are examples of convergent evolution,
arriving at the same function and catalytic groups, but grafted onto very differ-
ent scaffolds.
Perhaps the best-characterized virulence factor of this family is the C5a pep-
tidase from group A and group B Streptococci, exemplified by the enzyme from
S. pyogenes. As the name suggests, this enzyme cleaves the C5a component of
complement, destroying its ability to act as a chemotaxin for polymorphonuclear
leukocytes [Hill et al., 1 988]. Recent studies suggest that this enzyme is also able
to bind to fibronectin, which may be important in the binding and invasion of
host cells by group B streptococci [Beckmann et al., 2002; Cheng et al., 2002b].
Recently, much effort has been invested into the development of C5a peptidase-
based vaccines for the treatment of group A and B streptococcal infections [Shet
et al., 2003; Cheng et al., 2002a].
Family S9B
Members of the family S9B are generally dipeptidyl peptidases, which
cleave two amino acids at a time from the terminii of proteins. The bacterial
peptidases in this subfamily are exemplified by the dipeptidyl aminopeptidase
Bacterial Peptidases 167
IV, from organisms such as R gingivalis [Banbula et al., 2000; Kumagai et al.,
2000]. The enzyme is apparently important for the virulence of P. gingivalis,
since bacteria lacking the protease or with a mutation in the catalytic domain
have attenuated virulence [Kumagai et al., 2003].
Family SI 4 and SI 6
The S14 family is primarily composed of the endopeptidase Clp enzymes,
originally discovered and characterized m E. coli. Endopeptidase Clp enzymes
are rather similar to Lon proteases (SI 6 family) in that their activity as a pepti-
dase is linked to the hydrolysis of ATP. The enzymes contain an ATP binding
and catalysis domain and a distinct peptidase domain [Wang et al., 1997]. Some
studies suggest that these enzymes are the functional equivalents of the protea-
some complex found in all mammalian cells, which is crucial for the control of
protein turnover in these cells. Interesting support for this hypothesis is pro-
vided by a recent study which suggests that the Clp enzyme is important for
survival of bacteria which are in the stationary phase [Weichart et al., 2003].
The catalytic dyad of Lon proteases consists of Ser and Lys. The enzyme
is normally induced under stress conditions [Botos et al., 2004], and animal
studies suggest it is highly unportant S. enterica serovar typhimurium virulence
[Takayaetal., 2003].
Conclusions
As is evidenced by the above review, which is by necessity not absolutely
comprehensive, there is a wealth of information about bacterial peptidases. In
many instances, however, knowledge is just starting to be accumulated about
specific families or enzymes within families. Bacterial peptidases span a
tremendous range of mechanisms, and frequently have surprising associations
with additional domains which carry out separate functions. This adds a fasci-
nating range to the potential activities of these enzymes. In many cases, the
potential for inhibitors of the enzymes to be used as antibacterial agents will
continue to drive the active and thriving research in this important field.
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Jan Potempa
Department of Microbiology, Faculty of Biotechnology
Jagiellonian University
ul. Gronostajowa 7
PL-30-387 Krak6w (Poland)
Tel. +48 12 664 6343, Fax +48 12 664 6902, E-Mail poiempa@arches.uga.edu
Potempa/Pike
180
Enzymes
Russell W, Herwald H (eds): Concepts in Bacterial Virulence.!
Contrib Microbiol. Basel, Karger, 2005, vol 12, pp 181 209
Bacterial Invasins: Molecular Systems
Dedicated to the Invasion
of Host Tissues
Eric D. Cambronne, Olaf Schneewind
Committee on Microbiology, University of Chicago, Chicago, 111., USA
Bacterial pathogens have devised several strategies for their survival in
the tissues of vertebrate hosts. Some of these strategies are common to a wide
distribution of bacterial species, while others are quite speciaHzed and unique
to a particular pathogen. The term invasin has been traditionally assigned to
virulence factors that specifically promote Lntemalization of a bacterium by a
host cell. This designation may also be collectively assigned to general viru-
lence strategies required for host colonization. A typical pathogen must use
one or a combination of mechanisms to colonize the host. Factors that pro-
mote colonization can be functionally quite diverse, from the release of a
toxin into its surrounding environment, to the display of an individual surface
ligand promoting receptor binding on a host cell. Many pathogens have
evolved specialized macromolecular structures dedicated to the delivery of
effector molecules directly into the cytosol of target cells. These injection sys-
tems bypass the requirement for association of a toxin with a target through
diffusion, and therefore appear to be highly efficient pathogenic strategies.
Collectively, these meclianisms allow the pathogen Lo maniptilate molecular
processes of host cells to promote adhesion, cytotoxicity, in some cases
phagocytosis, and often general subversion of both the innate and adaptive
immune systems. The end result is the establishment of an environmental
niche in host tissues that will allow for the perpetuation of the bacterium. The
secretion of polypeptides from the bacterial cytosol to targets in or beyond the
cell wall envelope is a requirement common amongst most pathogenic strate-
gies. Generalized secretion pathways are utilized or modified to accommo-
date these virulence strategies, resulting in specialized systems dedicated to
the invasion of host tissues.
The Bacterial Cell Wall
The bacterial cell wall envelope provides the molecular scaffolding for the
display of virulence factors and also provides the framework for the assembly
of dedicated secretion systems. With few exceptions, bacteria may be catego-
rized based on the composition and morphology of their cell wall. All bacteria
contain an inner cytosolic compartment that is surrounded by a phospholipid
membrane. Morphological distinctions become apparent beyond this primary
barrier. In gram-positive bacteria, the inner membrane is surrounded by
an elaborate peptidoglycan cell wall consisting of polymerized subunits of
N-acetylmuramic acid-(pl-4)-N-acety]glucosamine. These glycan polymers
are cross-linked via transpeptidation of murein peptides that are covalently
attached to the D-lactyl groups on N-acetylmuramic acid. In general, the
framework of the gram-positive cell wall is further supported by the inclusion
of techoic acid, lipotechoic acid, or lipoglycan polymers, which may be cova-
lently linked to the wall peptidoglycan or anchored to the outer leaflet of the
inner membrane through lipid modification [I ]. The gram-negative cell wall
consists of a thin layer of peptidoglycan beyond the inner membrane. Outside
of the peptidoglycan wall, a second phospholipid outer membrane is assem-
bled. The outer leaflet of the outer membrane is composed of lipopolysaccharide
(LPS or endotoxin), consisting of lipid A, an oligosaccharide core, and a
distal 0-antigenic polysaccharide [2]. The 0-antigen is a key virulence deter-
minant, often leading to the promotion of inflammation at the infection site.
LPS can be described as the molecular signature of a particular pathogen, and
several pathogens have devised schemes to alter their LPS structure, promot-
ing serum resistance [3]. The outer membrane is also the destination of surface
proteins required for adherence and for pore-forming protein complexes [4].
The double membrane arrangement of the gram-negative bacterium provides
a compartment distinct from the cytoplasm called the periplasm. This com-
partment is typically rich in enzymatic factors required for adaptation to the
extracellular environment, and proteins that influence the proper folding of
secretion substrates [5]. Proteins destined for display on the surface of a gram-
negative bacterium must therefore contain information that will provide for
navigation through the inner membrane, the periplasmic space, and for inser-
tion into the outer membrane. This contrasts with the navigation of a surface-
displayed protein in a gram-positive bacterium, which need only contain
information for secretion beyond the inner membrane. Gram-positive bacteria
have devised a strategy for the display of proteins on their surface involving
the covalent linkage between the polypeptide and the peptidoglycan itself,
while the mechanisms for the secretion of factors beyond the cell wall are
poorly understood.
Cambronne/Schneewind 182
Generalized Secretion Strategies
Proteins that are destined for localization in compartments outside of the
bacterial cytosol must be translocated across the inner membrane. The Sec
pathway is often required for this translocation process. This involves the deliv-
ery of a nascent signal-bearing precursor polypeptide to a specialized secretion
apparatus (Sec translocase) in the inner membrane [6]. This process may be
accomplished using either a posttranslational or cotranslational mechanism.
The posttranslational secretion mechanism is thought to be utilized primarily
for soluble proteins synthesized in the cytosol [7]. In the gram-negative
bacterium Escherichia coli, newly translated polypeptide harboring an amino-
terminal secretion signal is bound by a cytosolic chaperone SecB. This secre-
tion signal consists of 18-30 hydrophobic residues that are preceded by a
charged domain and succeeded by a signal peptidase cleavage site [6]. SecB
delivers the signal-bearing precursor to the Sec translocase complex, composed
of SecD, SecE, SecF, SecG, SecY, and YajC [7]. SecB is predicted to maintain
the signal-bearing polypeptide in a secretion-competent conformation [8].
SecB associates with another factor SecA, which is a soluble translocation
ATPase. SecA associated with signal-bearing precursor polypeptide binds the
SecY translocase component, and the hydrolysis of ATP to ADP by SecA
promotes delivery of short segments of the polypeptide into the transloca-
tion channel, consisting of SecE, SecG, and SecY [9]. The signal sequence is
retained by the translocase until a signal peptidase cleaves the signal peptide at
a particular site, allowing for the release of the mature polypeptide. Signal peptid-
ases have been identified for the general secretion of soluble proteins (signal
peptidase 1), lipoproteins (signal peptidase 11), and for virulence-associated
prepilins (prepilin peptidase) for the assembly of type IV pili [10-12].
Cotranslational secretion of signal-bearing precursor polypeptides is in
general thought to be associated with the proteins destined for insertion in
the inner membrane after translocation [7]. The secretion mechanism involves
the stalling of translation initiated thi'ough the binding of the bacterial signal
recognition particle (SRP) to the signal sequence. SRP is a ribonucleoprotein
complex consisting of the factor Ffh (P48) and a 4.5S ribonucleic acid [13].
Translation resumes when SRP binds to the inner membrane receptor FtsY and
is displaced. The ribosome proceeds with translation and provides the force for
the translocation of the nascent polypeptide through the Sec translocase [14],
Proteins destined for insertion into the inner membrane may be retained by two
mechanisms durmg Sec -mediated translocation. Type I membrane proteins con-
tain a noncleavable signal/anchor sequence that mserts into the membrane,
whereas type C membrane proteins contain a downstream stop transfer/membrane
anchor sequence that retains the mature polypeptide after cleavage [15, 16].
Bacterial Invasins
83
Outer membrane proteins often assume (3-barrel structures prior to insertion.
Proteins that are destined for insertion into the outer membrane or secreted
beyond the outer membrane are often folded in the periplasm. DsbA and DsbC
catalyze disulfide bond formation, and are both important in several of the
gram-negative secretion systems [5]. In general, the Sec pathway is thought to be
conserved among the gram-positive bacteria. Bacillus subtillis carries homologs
of all of the Sec genes except SecB and SecG. It is assumed that gram-positive
bacteria may utilize functional homologs of SecB for the chaperone-mediated
delivery of secretion substrates to the Sec translocase [17].
A second general secretion system has been described recently. This sys-
tem is called the twin-arginine translocation system or TAT pathway, named for
the secretion signals consei"ved in the amino-termini of the substrates. The con-
sensus amino-terminal secretion signal consists of a positively charged segment
bordered by two consecutive arginine residues, a nonspecific amino acid, and
two hydrophobic residues [18, 19]. This translocation system is likely devoted
to the secretion of prefolded substrates and enzyme complexes in the cytoplasm
that are required for general physiology and destined for localization in the
periplasm [20]. Recent observations suggest that the TAT pathway may be asso-
ciated with the secretion of virulence factors however, and examples of this
requirement have been demonstrated in E. coli and Pseudomonas aeruginosa
[21, 22]. In E. coli, the TAT translocation system is composed of TatA, TatB,
TatC, and TatE, all integral membrane proteins. TatA, TatB, and TatC have been
purified in complexes and are believed to be the structural components of the
translocase [23, 24]. This relatively simple complex has been implicated in
the secretion of Shiga toxin in enterohemorrhagic E, coli and was found to be
required for toxin-mediated cytotoxicity of cultured cells [22]. The TAT system
appears to be conserved among most bacteria, including gram-positive species,
and future investigation may show this mechanism to be an important virulence
determinant [25]. The general strategies required for protein secretion in bacte-
ria are represented in figure I .
Invasive Strategies of Gram-Positive Pathogens
Typical gram-positive pathogens have only a single barrier separating then"
cytoplasmic membrane from the extracellular environment. Secretion beyond or
localization to the cell wall represent two general mechanisms employed to
establish infections [1]. The secretion of toxins is a common feature, but the
mechanistic process is poorly understood beyond the scope of Sec-mediated
translocation. The display of virulence factors on the surface of the pathogen is
a second common feature of the gram-positive pathogen. This mechanism
Cambronne/Schneewind 184
Fig. L General strategies involved in the secretion of bacterial proteins, a Gram-positive
bacteria translocate signal-bearing precursor proteins through the inner membrane (IM) via the
Sec pathway. Extracellular soluble proteins may passage through the peptidoglycan to diffuse
in the extracellular space (1). Proteins destined for display on the surface of the bacterium
(2) are often covalently linked to the cell wall peptidoglycan (CW). b Gram-negative bacteria
transport signal-bearing soluble proteins to the periplasm via the Sec translocase. Proteins may
be transported to the extracellular space by mechanisms requiring specialized secretion sys-
tems (1), or fold and insert into the outer membrane (OM) (2), Proteins destmed for insertion
m the mner membrane are bound by bacterial SRP at which time ribosomal synthesis is stalled,
SRP binding to the membrane-boiuid receptor (R) promotes release of SRP from the precursor
at the Sec translocase, where translation will resume, promoting insertion of the membrane
protein (3). The Tat system is employed for the transport of prefolded substrates and enzyme
complexes that localize in the periplasm (4).
involves the covalent linkage of secreted polypeptides to the peptidoglycan itself
This process is accomplished by a specialized membrane bound transpeptidase
called sortase [26, 27], The sortase mechanism was first characterized in
Staphlyococcus aureus, a ubiquitous pathogen that causes a variety of human
infections. Sortases have subsequently been identified in numerous gram-positive
pathogens, including Listeria monocytogenes. Streptococcus spp.. Bacillus
anthracis, and others [28]. Staphylococcal protein A (Spa) is a surface protein
that binds serum immunoglobulins to protect the bacterium from complement-
mediated destiuction. Spa is synthesized as a signal-bearing precursor that will
promote its Sec-mediated translocation. Spa also contains a consensus carboxy-
terminal sorting signal that consists of an LPXTG sequence motif followed by a
hydrophobic stretch of 15-19 amino acids and a distal positively charged tail of
5-10 residues [29]. The hydrophobic/charged domam m the carboxy-terminus
functions to retain Spa in the membrane after signal peptidase cleavage (P2
Bacterial Invasins
185
NK
NH.
NH,
NK
NH,
-GN-MN-
I
L-Ala
I
I
Gtyg-L-Lys
D-AJa
t
-GN-MN-
I
L-Ala
GN-MN
L-Ala
Gly^-L-Lys
D-Afa
LPXTG
+
Fig. 2, The sorting reaction in S. aureus. PI precursor protein substrates that harbor an
amino-termina] signal peptide and a carboxy-terminal sorting signal are exported from the
cytoplasm through the Sec translocase (1). The amino-terminal secretion signal is cleaved by
signal peptidase generating a P2 precursor, which is retained in the plasma membrane by the
carboxy-terminal sortijig signal (2), Sortase (SrtA) catalyzes a cleavage reaction between the
threonine and glycine residues of the LPXTG motif, generating a thioester enzyme interme-
diate (3). The acyl-enzyme intermediate is resolved through nucleophilic attack by a free
amine group on lipid n, resuhing in amide linkage of the sortase substrate to the pentaglycine
cross-bridge (4). The mature surface protein is incorporated into the cell wall through a trans-
glycosylation reaction (5), IM = Inner membrane.
precursor). Sortase catalyzes a transpeptidation reaction between the threonine
and glycine residues of the LPXTG motif, where a proteolytic cleavage event
links the thieonyl carboxyl group to an active site cysteine, generating an acyl-
enzyme intermediate through thioester linkage [30], The carboxyl group of thre-
onine is then amide linked to the amino group of the pentaglycine cross-bridge
in the murein tetrapeptide segment of the lipid II cell wall precursor [31]. The
reaction product is incorporated into new peptidoglycan polymers through
transglycosylation, resulting in the mature cell wall-anchored Spa polypeptide.
The general staphylococcal sorting reaction is represented in figure 2.
The identification of cell wall-anchored surface protems based on carboxy-
terminal sequence analysis has revealed the potential for numerous virulence-
associated factors, including C5 peptidase in Sti^eptococcus pyogenes, internal in A
Cambronne/Schneewind
186
in L. monocytogenes, and neuraminidase in Streptococcus pneumoniae [28].
Variations on the consensus sorting reaction have also emerged, which include
multiple sortase enzymes encoded by the same organism that recognize alternate
sorting signals, such as the recognition of the NPQTN consensus sorting signal
by SrtB in S. aureus [32]. Further, evidence suggests that the expression of sortase
enzymes is environmentally regulated, promoting the display of a particular set
of surface proteins under specific conditions.
L monocytogenes is a food- and water-borne pathogen that causes infec-
tions ranging from gastroenteritis to septicemja. It is an intracellular pathogen
that employs a particularly interesting mechanism for migration through host
tissue. The bacterium requires at least two surface factors for entry into cultured
cells. Intemalin A (InlA) and internalin B (InlB) each contain Sec-mediated
amino-terminal signal sequences but are recruited to the bacterial surface
by two different mechanisms [33]. L. monocytogenes harbors two sortase
genes, sriA and srtB. SrtA is required for the cell wall anchoring of InlA, which
contains a consensus LPXTG sorting signal. Deletion of the srtA locus results
in a defect in invasiveness sunilar to that of an inlA mutant [34]. InJB con-
tains carboxy-terminal repeat regions that promote a noncovalent interaction
with lipoteichoic acids in the cell wall peptidoglycan [35]. InJA binds to the
E-cadherin receptor on epithelial cells, while InlB interacts with the comple-
ment receptor gClqR, glycosaminoglycans, and the tyrosine kinase receptor Met
[36—38]. Activation of signal transduction cascades promotes phagocytosis and
the bacterium is enveloped in a phagocytic vesicle. To combat the acidification
of the phagocytic vacuole, the bacterium expresses the enzymes listeriolysin O
(LLO) and phosphatidylinositol phospholipase C (PlcA), which are secreted
and promote degradation of the vacuolar membrane [39, 40]. This event allows for
escape from the phagocytic vacuole and promotes bacterial multiplication in the
host cell cytoplasm. Listeria expresses another factor ActA, a membrane pro-
tein exposed on the bacterial surface. ActA recruits the host Arp 2/3 complex
resulting in nucleation of actin filaments at the surface of the bacterium [41,
42]. ActA acts as a molecular mimic, functioning in a similar fashion to the
WASP family of proteins. The WASP proteins are activated tlirough binding of
cellular GTPases, and conformational changes promote Arp 2/3 complex
recruitment [43]. The assembly of an actin tail propels the bacterium through
the cytoplasm, generating pseudopod-like extensions that promote phago-
cytosis by neighboring cells, resulting in the formation of a double membrane
vacuole in the neighbor cell. In addition to the secretion of LLO and PlcA,
phosphatidylcholine phospholipase C (PIcB) has been implemented in the
escape of the bacterium from this specialized vacuole [44]. The intracellular
growth cycle of the bacterium has been shown to result in localized tissue
destruction with minimal exposure to components of the immune system.
Bacterial Invasins
187
Invasive Strategies of Gram-Negative Pathogens
Gram-negative pathogens have devised an array of mechanisms to pro-
mote colonization. Most of these strategies incorporate the modification of a
generalized secretion pathway to either promote the display of a surface molecule
for colonization, or deliver eifector molecules beyond the bacterial envelope.
Specialized secretion systems may be generally divided into two categories, those
that promote the release of diffusible protein factors to the surrounding envi-
ronment and systems that promote the delivery of effector proteins directly
into the cytosol of target cells. There are currently five specialized secretion
systems described for gram-negative bacteria (type 1-V) that appear dedicated
to virulence. Type 1 secretion incorporates a Sec-independent process to
deliver toxin to the extracellular space without a periplasmic intermediate.
Type 11 secretion, the main terminal branch of the general secretory pathway
(GSP), represents a two-step translocation mechanism where factors secreted
by the Sec pathway are transported by a protein complex that contains a char-
acteristic outer membrane secretin. The type III secretion mechanism is a Sec-
independent translocation process that involves the direct delivery of effector
molecules from the bacterial cytoplasm to the cytosol of a target cell through
a specialized channel or needJe complex. Type IV secretion systems are simi-
lar to bacterial conjugational systems and harbor the ability to transfer proteins
and/or nucleic acids into a target cell using either a one- or two-step translo-
cation process. Type V secretion represents an alternate terminal branch of the
GSR Often referred to as the autotransporter mechanism, type V substrates are
secreted by the Sec pathway and contain information in their carboxy-termini
that promotes incorporation in the outer membrane and delivery of the amino-
terminal domain outside of the cell. Each of these systems are discussed in detail
below and figure 3 is a representation of the basic features associated with these
secretion mechanisms.
Surface Proteins
The expression of a molecule on the suiface of the bacterium, not unlike
the display of surface proteins in gram-positive pathogens, represents a mecha-
nism for colonization in some gram-negative bacteria. A prototypical example
of this mechanism is the display of the factor invasin in Yersinia pseudotuber-
culosis and Yersinia enterocolitica. The expression of invA m a noninvasive
strain off. coli results in the phagocytosis of the bacterium by cultured mam-
malian cells [45]. Invasin is a modular protein that harbors an amino-terminal
outer membrane localization domain, as well as an extracellular carboxy-terminal
domain that consists of repeats of an IgG-like fold, and an adhesive tip [46, 47].
It has been determined that invasin binds to pi integrin receptors localized on
Cambronne/Schneewind 1 88
OM
irr
cwc
IM
1*r
Fig. 3. Basic features of the five classes of gram-negative protein secretion systems,
(I) The type I mechanism involves a single, Sec-independent translocation event that incorpo-
rates an inner membrane (IM) ABC transporter for energy generation, (II) The type II mecha-
nism represents a two-step translocation process, where signal-bearing precursor proteins are
traiisported to the periplasm via the Sec pathway. Mature substrates are transported through an
outer membrane (OM) secretin. (DQThe type III mechanism incorporates a single translocation
step to transport substrates from the bacterial cytoplasm into the cytosol of eukaryotic cells. The
substrate is transported through a basal body complex, an outer membrane secretin, and a nee-
dle complex that penetrates the target cell membrane. (fV, left) The type IV secretion system is
employed to transfer substrates into host cells. This process requires the assembly of a pilus
structure at the outer membrane, a core assembly in the periplasm, and inner membrane-
associated ATPases. (FV, right) Pathogens may also employ the type FV mechanism for secretion
of diffusible toxins to the environment in a Sec-dependent manner. (V) Autotransporters are
secreted by the type V pathway A typical substrate is traiislocated to the periplasm by the Sec
translocase. Insertion into the outer membrane promotes the secretion of the amino-terminal
passenger domain, Autoproteolysis releases the diffusible mature protein. Localization of energy
generatmg enzymes are indicated by *. CW = Cell wall peptidoglycan.
the apical surface of M cells, located amongst the folHcle-associated epithelia
and lymphoid follicles of the small mtestine, commonly referred to as Peyer's
patches [48, 49]. M cells sample contents of the intestinal lumen and transport
particles contained in vesicles to the basolateral surface^ which is rich in immune
cells such as macrophages and polymorphonuclear leukocytes. The bmding of
invasin to M cells may therefore represent an early mechanism mvolved in the
Yersinia infection process, as the bacteria have a specific tropism for lymphoid
tissues. Numerous examples of adhesion factors have been identified, often
associated with the protein subunits localized in the tip of pili or fimbriae.
Bacterial Invasins
189
Examples mclude the PapG adhesin of type I pili in E, coli, and the major pilin
subunit PilA on the type IV pili ofP aeruginosa [50, 51].
Type I Secretion
The type I secretion mechanism involves the one-step translocation of a
secretion substrate in a Sec- independent manner [52], This mechanism is
employed for the secretion of diffusible toxins into the extracellular space. The
type I mechanism has been demonstrated for the secretion of a-hemolysin (HlyA)
in E. coli, as well as for the secretion of Bordetella pertussis adenylate cyclase
and P aeruginosa protease [53]. In each instance, the toxin is recruited to a
translocation complex that assembles upon association with the substrate. The
type I secretion system is relatively simple in architecture, consisting of only three
factors, each required for transport of the substrate, A characteristic feature of the
system is the presence of an ATP-binding cassette (ABC) protein transporter [52,
54], ABC transporters are inner membrane proteins that are found in a wide range
of organisms, including gram-positive bacteria, lower eukaryotic, and mammalian
cells, and are normally associated with the transport of small molecules. The
secretion of HlyA has been extensively studied and the synthesis of the pro-
hemolysin precursor protein (proA) requires a lipid modification for activation to
mature HlyA [55], The cytosolic factor HlyC as well as an acyl-carrier protein
(ACP) are required for the myristoylation or palmitoylation of two lysine residues
[56]. HlyC acts as an acyl-transferase for this process. Although this lipid modi-
fication step is requu"ed for the hemolytic activity of HlyA, this event is not
required for the type I-dependent secretion of HlyA [57]. After modification,
HlyA binds to the ABC transporter HlyB at the inner membrane [58], The
sequence information required for secretion of HlyA is contained in the poly-
peptides carboxy-terminal 48 amino acids, and unlike signal sequences in Sec-
mediated substrates, the signal sequence of type 1 substrates is not cleaved after
translocation [59, 60]. The H lyB transporter associates with a second factor HlyD
independent of substrate binding. The HlyD protein spans both the inner and
outer membrane and trimerization of HlyD in the presence of HlyB bound to
HlyA results in the recruitment of the outer membrane protein TolC [58], Each
subunit of the trimeric TolC contains an amino-terminal p-sheet domain that
inserts into the outer membrane. A second carboxy-terminal a-helical domain
extends deep into the periplasmic space and forms a barrier between the
periplasm and the amino-terminal pore-like structure [61], The binding of ATP to
HlyB in the presence of HlyA may result in the specific recruitment of TolC by
HJyD, The HlyB/HlyD/TolC complex then supports the HJyB mediated trans-
location of HlyA through successive rounds of ATP hydrolysis [62], resultmg in
the delivery of HlyA to the extracellular space. Eleven tandem glycine-rich
repeats (LXGGXGND) contained in the carboxy-terminus of HlyA are required
Cambronne/Schneewind 1 90
for calcium binding. Calcium-bound HlyA is competent for insertion in the host
cell membrane, and results in pore-mediated leakage of the target cell [63].
Type II Secretion
The GSP represents the primary route for translocation of polypeptides to
the extracellular space among gram-negative bacteria. The type EI secretion
mechanism represents the archetype for protein secretion in the GSP, and has
been designated the main terminal branch. The type II pathway is associated
with the secretion of virulence factors in several bacterial pathogens. Alternate
GSP branches include the secretion of autotransporters (type V secretion), the
chaperone/usher-mediated assembly of P or type I pili in E. coli, the assembly
of type IV pili in P. aeruginosa and Neisseria gonorrhea, and the assembly of
curli in E. coli [64]. The factors required for extrusion of filamentous bacterio-
phage from the bacterial envelope also share conserved components with the GSP
[65]. One common feature associated with all of these strategies is the requirement
for the Sec-mediated translocation of secretion substrates to the periplasm. The
type II secretion pathway therefore represents a two-step translocation process,
incorporating distinct secretion reactions for translocation across the inner and
outer membranes.
The type Il-dependent secretion of pullulanase in Klebsiella oxytoca is a well-
studied example of this secretion mechanism. I\illulanase (PuIA) is a lipoprotem
of the a-amylase family that enzymatically degrades the complex carbohydrate
pullulan to maltotriose subunits, a substrate that may be transported into the bac-
terium [66]. The secretion of PulA requires the products of at least 25 genes, 14
of which are specifically involved in the translocation of PulA beyond the outer
membrane [67]. After secretion to the periplasm through the Sec pathway, The
PulA precursor is subjected to diacyl glyceride modification and cleaved by
signal peptidase [68]. The lipid-modified PulA is retained in the outer leaflet of
the inner membrane by an aspartyl residue located at the amino-terminus of the
mature polypeptide. Factors required for the type ll-dependent translocation
step are localized within several compartments. A cytoplasmic ATPase (GspE)
associates willi the inner membrane through interaction with a second factor
(GspL), an inner membrane protem that harbors a carboxy-terminal cytoplasmic
domain [69, 70]. This interaction, coupled with ATP hydrolysis by GspE, may
provide the energy required for PulA transport. Four additional integral mem-
brane proteins GspC, GspF, GspM, and GspN are thought to assemble into a basal
body complex, since the factors harbor carboxy-terminal domains that extend
into the periplasm [71]. A characteristic feature of the type II apparatus is the
requirement for periplasmic pseudopilin proteins. These factors, all harboring
prepilin signal sequences, are secreted by the Sec pathway and processed by the
inner membrane-associated prepilin peptidase GspO, which will also N-methlyate
Bacterial Invasins
91
the pseudopilin subunits [72]. Five pseudopilin factors, GspG, GspH, Gspl, GspJ,
and GspK, are processed in this manner, and assemble into a channel-hke pilus
structure Ihiking components of the inner and outer membranes [73], GspD is an
outer membrane secretin required for the export of type II substrates. GspD is
inserted into the outer membrane and assembles into a dodecameric channel-
forming structure^ a process that requires the outer membrane chaperone GspS
[74], Functional homologs of the GspD secretin are conserved amongst most of
the alternate branches of the GSP and the GspD secretin is also conserved
among the type 111 secretion systems (see below). The type ll-mediated export
of PulA may occur through its association with the basal body complex, with
signal recognition most likely residing in the secondary or tertiary structure of
the secretion substrate after folding in the periplasm. PulA is transported to the
outer membrane secretin GspD and exported to the extracellular space. Similar
type II secretion mechanisms have been identified for the release of exotoxin A,
elastase, and phospholipase C in /? aeruginosa [71].
The secretion of AB-type holotoxins is also mediated by a type Il-dependent
process. This class of toxins includes the cholera toxin of Vibrio cholerae, E. coli
enterotoxin, and the Shiga-like toxins of £". coli and Shigella dysenteriae [75].
Cholera toxin is composed of two separate polypeptides CtxA and CtxB. Sec-
dependent secretion of the subunits to the periplasm results in proteolytic cleavage
of signal peptides and the formation of an intramolecular disulfide bond in CtxA
prior to cleavage. The CtxB subunits assemble into a pentameric ring structure
and bind the carboxy-terminal domain of the CtxA subunit, generating the
CtxA|-CtxB5 holotoxin [76]. Secretion of the holotoxin requires components of
the eps gene cluster, which encodes several factors homologous to the type II
secretion system in Klebsiella [77]. Export of the holotoxin will result in the
binding of the CtxBg subunits to a G^vii ganglioside on the surface of intestinal
epithelial cells [78], Reduction of the disulfide in CtxA by host cytosolic
thioredoxin promotes the release of the mature CtxA toxin from the CtxBs
pentameric ring, where it will function to activate host cell adenlyate cyclase,
resulting in the massive cellular fluid loss associated with the diarrhea in
cholera disease [79].
Type II J Secretion
The delivery of polypeptides from the bacterial cytoplasm directly into the
cytosol of target host cells without the generation of an extracellular interme-
diate is the halknark feature of the type III secretion system [80]. Effector pro-
terns that are translocated into host cells harbor enzymatic activities that
manipulate cellular processes of the eukaryotic host, resulting in a variety of
processes that culminate in perpetuation of the bacterium at the infection site
[81]. The type III secretion mechanism was first characterized in pathogenic
Cambronne/Schneewind 1 92
Yersinia species, but has subsequently been identified and extensively studied
in various pathogens including enteropathogenic E. colt (EPEC), P. aeruginosa,
Salmonella enterica and Shigella flexneri [82]. Analysis of genetic infornnation
has revealed the potential for type III systems in several other gram-negative
bacteria, and thus may represent a highly conserved pathogenic strategy [83].
Even though the process of injection of virulence factors by the type III
pathway is a recently described phenomenon, the type III secretion apparatus
appears both structurally and fiinctionally similar to the basal body of the fla-
gellar secretion system in gram-negative bacteria. In fact, the flagellar secretion
system is now considered a type III pathway and recent observations suggest
that the flagellar export system may also support the secretion of virulence factors
[84]. Type III secretion systems most likely evolved from the flagellar machinery
to support colonization in new nutrient-rich environments such as those found
in higher eukaryotes. Yersinia species employ the type III pathway to maintain
an extracellular lifestyle in the lymphoid tissues of their mammalian hosts and
cause a variety of diseases ranging from bubonic plague in Yersinia pestis to
acute enteritis in Y. enterocolitica. This is accomplished through the type III
injection of effector proteins called Yops {Yersinia outer proteins) into host
macrophages, resulting in prevention of phagocytosis and eventual apoptotic
death of the host cell [85].
With the exception of the assembly of the secretion apparatus, the transloca-
tion of type in secretion substrates represents a Sec-independent process. The type
III secretion system consists of three principle components, an inner membrane-
associated basal body, an outer membrane secretin, and an extracellular needle
complex. Assembly of a jflinctional Yersinia type III apparatus requires the prod-
ucts of at least 21 ysc {Yersinia secretion) genes [86-88]. Eleven of these genes are
conserved amongst other type IJI systems, including nine that are conserved with
the flagellar basal body [89]. In general, the Yersinia type III secretion apparatus
must be assembled in a similar fashion to the flagellar secretion system, where
assembly of the basal body complex precedes any substrate dehvery. The initiation
of the assembly of the basal body complex in Yersinia likely begins with mem-
brane inseition of the FliF homolog YscJ, after Sec-mediated translocation [86, 90,
9 1 ]. This event will allow the association of inner membrane proteins YscD, YscR,
YscU, YscV and accessory factors to form the basal body complex. YscN is
homologous to the Flil ATPase in flagellar secretion and contains the Walker boxes
A and B, which are characteristic conserved ATP-binding domains [92]. YscN is
predicted to provide the energy for the transport of type 111 secretion substrates and
is required for Yop secretion. YscC is homologous to the GspD secretin involved
in the transport of molecules in the type II padiway, and requires the outer
membrane lipoprotein YscW for its localization and for the formation of the char-
acteristic dodecameric rings in the outer membrane, resulting in outer membrane
Bacterial Invasins
93
channel formation [93, 94]. Accessory factor association between the basal body
complex and the YscC secretin provides a conduit between the two components of
type III secretion system, an assembly step that is not conserved with flagellar
secretion. Secretion of the factors YscF,YscO,YscP, and YscX promote the assem-
bly of the needle complex [95-97],
Although the type III needle complex remains to be isolated from Yersinia,
needle complexes have been purified from Salmonella, Shigella, and E. coll
[98-100], The YscF protein has been determined to be the main component of
the needle complex, where the protein multimerizes in a right-handed helical
fashion. The YscF homolog MxiH of Shigella displays 5.6 subunits per turn,
and is polymerized from the distal tip into a conduit as long as 50 nm with a
width of Vnmi and a central tube of 2-3 nm [101, 102]. YscO and YscP are
secreted by the basal complex. YscP has recently been suggested to participate
in substrate recognition, as yscP mutant strains secrete an increased amount of
the needle component YscF but fail to secrete Yop proteins in vitro. Mutations
in the amino-terminus of YscU suppress the yscP mutant phenotype^ reducing
the amount of secreted YscF to wild-type levels and restoring the secretion of
Yop proteins [95]. These results suggest that YscO, YscP^ and YscU may control
type ill secretion at the level of substrate specificity, allowing for a switch
between the secretion of structural components and the delivery of Yop sub-
strates, similar to the switch between hook and filament proteins in the flagel-
lar apparatus [89], Assembly of the YscF needle complex would then represent
the final step of assembly and provide a switch for the recognition of type 111
secretion substrates and the delivery of effector Yop proteins.
The hydrophobic nature of the YscF polymer has been predicted to provide
a mechanism for the piercing of the host cell cytoplasm [96]. An alternate
hypothesis suggests that three secretion substrates, YopB, YopD, and LcrV, each
required for the translocation of Yop proteins^ form a translocation pore in the
host cell membrane allowing for subsequent delivery of effector proteins [80,
103-106],
X enterocolitica secretes 14 polypeptides via the type III pathway. One
cui'ious feature of each of these proteins is tliat they do not contain any amino
acid sequences that would suggest the presence of a conserved type 111 secre-
tion signal. Experiments performed usuig reporter proteins have revealed the
presence of minimal secretion information contained in the amino-terminal
8-15 residues [107-110]. The nature of the minimal secretion information
remains controversial. Scanning mutagenesis studies employed to determine
the residues required for secretion of YopE revealed that no specific residues
were necessary. Further, introduction of frameshift mutations in the minunal
signal did not affect the secretion of reporter fusion constructs [107]. These
results prompted the hypothesis that the minimal signal information is actually
Cambronne/Schneewind 1 94
contained in the mRNA rather than the protein. Th^ yop mRNA might therefore
recruit a translational complex to the type Til apparatus promoting a cotransla-
tiojial secretion mechanism.
The nature of the 5' mRNA/amino-terminal signal hypothesis has been
highJy contested however, and independent studies suggested that a mutant that
generated multiple mutations in the mRNA sequence without affecting the amino
acid sequence of the protein was indeed secreted [109], This implied that the
amino acid sequence, rather than the mRNA, contained the information required
for type 111 secretion. Construction of a synthetic amphipathic amino -terminal
signal that contained alternating serine and isoleucine residues between positions
2 and 9 of YopE also supported secretion [109], Recent observations in the mini-
mum secretion signals of YopE and YopQ have again resurrected the mRNA
secretion signal argument, as it was discovered that minimum signals, such as the
1-10 positions of YopQ, do not tolerate frameshifts unless a downstream sup-
pressor region of mRNA is included that contains codons 1 1-13 [1 10]. Further,
single substitutions in codons 2 and 10 caused a defect in the secretion reporter
fusions in the context of 10 but not 15 codons. Finally, multiple mutations in the
wobble positions of yopQ 1-10 did not support the secretion of the reporter, again
suggesting that mRNA rather than protein sequences initiate the transport of sub-
strates via the type III pathway [1 1 0],
Beyond the context of the amino- terminal minimal secretion signal, experi-
mental evidence suggests that type III substrates may require a second signal for
their injection into the cytosol of host cells, and the presence of Syc (specific Yop
chaperone) proteins may be required for the injection process [105, 1 11]. In gen-
eral, Syc proteins are small acidic proteins that form dimers in the bacterial cyto-
plasm. Each chaperone appears to specifically bind a partner effector Yop protein
in the cytoplasm and structures have been determined for secretion substrate/
chaperone complexes [112]. Studies that examined the role of both the amino-
terminal and chaperone-mediated secretion signals demonstrated that a defective
secretion signal, when linked in context to the full length YopE protein^ v^^s
secreted in an SycE-dependent manner, suggesting that the chaperone mediated
secretion signal does not requii'e the presence of a functional amino-terniinal
signal [113], This prompted the hypothesis that Yop proteins harbor two indepen-
dent secretion signals, the first required for initiation of the substrate into the type
III pathway^ and the second for injection into host cells.
K enterocolitica transports a class of at least six factors into the cytosol of
the host cell, each of which harbors an enzymatic function. All of these factors,
which include YopE, YopH, YopM, YopO, YopP, and YopT, share sequence
homology to proteins of eukaryotic origin, suggesting the pathogen evolved
these strategies through intimate interaction with the host over time. Although
all of the type III pathogens share a conserved mechanism for the delivery of these
Bacterial Invasins
195
effector proteins, the effector proteins themselves may or may not be conserved
between pathogens. YopE is only cytotoxic to HeLa cells when injected via the
type 111 pathway. Jt is a characteristic GTPase-activating protein (GAP) that acts
upon the Rho family ofeukaryotic GTPases. YopE inactivates RhoA, Racl, and
CDC42 by accelerating the conversion of GTP to GDP in these factors [114,
115]. This mechanism results in an inhibition of actin polymerization at the site
of bacterial contact, YopH is a protein tyrosine phosphatase (PTPase) that is
involved in the dephosphorylation of focal adhesions [1 16]. The amino-terminal
domain of YopH appears to be a targeting domain that binds to pi 30^^ and
focal adhesion kinase (FAK) [1 17]. The carboxy-terminal domain harbors the
phosphatase domain and acts specifically to dephosphorylate these substrates,
resulting in an interruption of stress fiber formation [118], YopM is required for
virulence in mice and has been suggested to target to the nucleus and may influ-
ence transcription in the host cell, inhibiting inflammatory cytokine production
[119, 120]. YopO is similar in sequence to RhoA kinase. YopO functions as an
autophosphorylating serine threonine kinase that is activated in vitro through bind-
ing to actin [121]. The protein is believed to phosphorylate the Rho family of
GTPases and enhance the inhibition of actin polymerization [122]. YopP acts as
an inhibitor of IkB in the NF-kB pathway and also inhibits the MAP kinase path-
way [123-125]. It has been reported that YopP is a cysteine protease that may
function through a protein degradation pathway [126]. The cumulative effects
of disrupting the NF-kB and MAP kinase pathways result in inhibition of the
proinflammatory response, thus preventing the production of the cytokines
TNF-a and lL-8 [123, 125]. YopP also induces apoptosis in macrophages,
which is likely to be a cumulative result of the failure to activate the NF-kB sig-
naling pathway and through the cleavage of Bid, a proapoptotic member of the
Bcl-2 family [127]. YopT is also a cysteine protease that has been demonstrated
to cleave RhoA^ Racl, and CDC42 at their carboxy-termini, sites that are prenyl-
ated for membrane anchoring [128]. Cleavage releases the factors from the
membrane resulting in a defect in actin polymerization at the site of bacterial
contact.
EPEC, a food- and water-borne pathogen that is a causative agent of
human infantile diarrhea, uses the type III pathway to establish attaching and
effacing lesions on intestinal epithelium [129, 130], These bacteria inject a pro-
tein, translocated intimin receptor (Tir), which is subsequently displayed on the
surface of the gastric epithelial cell [131]. The bacterial cell displays a ligand
for this receptor on its outer membrane, called intimin. interaction between the
two factors results in tight binding between the bacterium and host cell. This
event coupled with the cumulative effects of the type III injection of other factors
will promote actin pedestal formation at the site of contact, allowing extra-
cellular colonization and destruction of surrounding tissue.
Cambronne/Schneewind 1 96
S. enterica serovar spp. cause a variety of diseases in humans and animals,
ranging from acute food poisoning and gastrointestinal inflammation to typhoid
fever and septicemia. In general pathogenic Salmonella species are food-
and water-borne pathogens that have a tropism for the intestinal epithelium.
S. enterica serovar spp. harbor the genes encoding two separate type 111 secre-
tion systems on their chromosome. The first system, designated Salmonella
pathogenicity island 1 (SPI-I), is employed to invade nonphagocytic epithelial
cells [132]. Salmonella uses the SPI-1 type 111 pathway to inject several effec-
tor molecules that leads to a massive reorganization of actin filaments promot-
ing the formation of membrane ruffles and eventual phagocytosis. Similar to
effector proteins in Yersinia, many Salmonella effectors target the signaling
processes governing actin polymerization. Sip A stabilizes F-actin through
binding to the T-plastin protein [133]. SopE and SopE2 function as guanine
nucleotide exchange factors (GEF) to activate Rac-1 and CDC42 [134, 135]. In
order to promote recovery of the cytoskeleton, SptP is injected. SptP is a multi-
functional enzyme that contains an amino-terminal YopE-like GAP domain and
a carboxy-terminal YopH-like tyrosine phosphatase domain. SptP counteracts
the enzymatic effects of SopE and SopE2 by downregulating Rac-1 and CDC42
[136, 137]. The second type HI system located at SPI-2 appears to manipulate
vesicular trafficking, allowing for perpetuation of the bacterium in a special-
ized vacuole, and is required for systemic infections [138].
Pathogenic Shigella species are typically water-borne pathogens that are a
causative agent of bacillary dysentery, an infection of the colon. Shigella species
utilize a type III secretion for the invasion of epithelial cells. Shigella are
believed to enter epithelial cells from the basolateral surface. After engulfment
by intestinal M cells and presentation to lymphoid macrophages, Shigella
secretes an apoptotic factor IpaB which allows the bacterium to spread to adja-
cent cells [139]. Shigella also employs the type III pathway to promote phago-
cytosis by the epithelial cell through the cumulative effects of IpaA, IpaB, IpaC,
and IpaD [140]. Unlike Salmonella^ Shigella escape from acidified vesicles and
reside in the host cell cytoplasm. An outer membrane protein IcsA nucleates
actin polymerization tlii'ough the bindmg to N-WASP and the fomiation of die
Arp2/3 complex [141, 142]. Production of cytoskeletal filaments at the pole of
the bacterium propels the organism into neighboring cells, similar to the process
described fori, monocytogenes.
Type IV Secretion
The type IV secretion mechanism is employed for a wide range of func-
tions in gram-negative bacteria. Several species utilize the type IV mechanism
for interbacterial conjugative transfer of mobilized genetic elements. Pathogenic
Agi'obacterium tumefaciens employs the type IV system for the transfer of
Bacterial Invasins
197
tumorigenic DNA and protein into host plant cells, and several vertebrate
pathogens such as Brucella spp,, B, pertussis, Helicobacter pylori, and Legionella
pneumophila use a modified type IV secretion system for the secretion of
toxins or the delivery of effector proteins into the host cell [143, 144], There is
evidence to suggest that the secretion of substrates through the type IV appara-
tus requires a Sec-dependent translocation step, such as in the secretion of
pertussis toxin; however, specialized systems such as those for H, pylori and
L pneumophila may bypass this requirement [ 1 45], All type IV systems represent
a modification of the conjugative transfer system found in strains of £". coli.
In general, the mechanism involves the assembly of a secretion apparatus with
a pilus-like projection that will provide intimate contact between the donor and
recipient cell [146].
The transfer of DNA from bacterium to host in the pathogen A. tumefaciens
represents the archetype for the type IV secretion pathway. Substrate translocation
requires the products of the VirB-encoded system: VirBl-1 1 and VirD4 [147].
The mechanisms of VirB-mediated type IV transport have been extensively
studied, and the system is currently employed as the general model for the type
IV mechanism in animal pathogens [144]. Evidence suggests that the type IV
apparatus is assembled to extract the major pilin subunit VirB2, a cyclic
polypeptide, through the outer membrane [148]. The secretion and processing
of the pilin subunits is a Sec-dependent process. VirB2 forms a pilus through
multimerization and contains a second minor pilin subunit VirB5. Pilin subunits
interact with an outer membrane lipoprotein VirB7 and are thought to assemble
at the outer membrane [149]. VirB6 is an inner membrane protein that may
provide the connection between components of the inner and outer membrane,
as well as guide assembly of the periplasmic core [150]. VirB? also interacts
with VirB9, an outer membrane component, and VirB8, a muramidase localized
in the periplasm that may provide for organization of the complex through
wall peptidoglycan [149, 151]. Energy for the transport of type FV substrates
is provided by the activities of three separate ATPases, The VirB4 dimer is
localized in the inner membrane. A second inner membrane ATPase, hexa-
meric VLi'Bl I assembles into a ring stmcture and may provide a route for trans-
location of type IV substrates [152^ 153]. VirB 11 also interacts with the
periplasmic core component VirB 10. The third ATPase VirD4, also called
'coupling protein', is localized in the bacterial cytoplasm and is involved in
substrate recognition [144].
The ptl system in B. pertussis represents an interesting link between the
type II and type IV secretion pathways. 5. pertussis is the causative agent of
whooping cough, where pertussis toxin, an AB-type holotoxin, is the primai^
virulence determinant. Pertussis toxin is exported by the Ptl type IV secretion
apparatus. The Ptl system appears functionally distinct from other type IV
Cambronne/Schneewind 1 98
secretion systems. Rather than supporting injection into host cells, the Ptl system
exports pertussis toxin to the extracellular space, where the toxin is available to
associate with the target cell membrane [145]. The mature enzyme acts as an
ADP-ribosylating factor of G proteins in the host cell. Unlike the T-DNA
translocation process, the PtxA and PtxB subunits are translocated to the
periplasm, where they are processed and assembled into the PtxA|-PtxB5 holo-
toxin [154]. This protein complex then becomes a substrate for type IV-mediated
export. Nine structural components of the Ptl system are homologous to the
VirB system, where PtxA represents the major pilin subunit. The system also
contains two membrane-associated ATPases, PtIC and PtlH, which are homo-
logous to VirB4 and VirBll, respectively.
Recent discoveries have provided evidence that type IV secretion systems
are competent in the delivery of effector proteins directly into the cytosol of
host cells. H. pylori is a causative agent of several gastrointestinal syndromes
ranging from peptic ulcers to MALT lymphoma and adenocarcinoma.
Pathogenic strains of H, pylori harbor the CAG pathogenicity island which
encodes a VirB-like type IV secretion system [155]. CagA is translocated
by the type IV pathway into the host cell cytosol where it becomes tyrosine-
phosphorylated and proteolytically processed to a carboxy-terminal phosphoryl-
ated fragment. The injection of CagA results in a change in the
phosphorylation state of associated host cell factors, and is required for
virulence [156], L. pneumophila and Brucella species require type IV secre-
tion systems for survival in intracellular vacuoles [157]. L, pneumophila is
the causative agent of Legionnaire's disease, a severe respiratory pneumonia.
L. pneumophila targets alveolar macrophages where it employs the Dot/Icm
type IV secretion system for intracellular survival. The Dot/Icm transporter is
more distantly related to the VirB system, but is homologous to the IncI con-
jugation system in 5. flexneri, and is competent for conjugational transfer of
DNA [158]. L. pneumophila bypasses destruction mediated by the endocytic
pathway by creating an endoplasmic reticulum-like vacuole, presumably
through the injection of effector molecules into the host cytosol [157], RalF
was the first factor identified to be an effector substrate. RalF is a guanine
nucleotide exchange factor that functions to activate the ADP ribosylation
factor (ARf) family of GTPases [159]. The factor has been localized on the
surface of the Legionella-conx&mxng vacuole in a Dot/Icm-dependent manner^
and is required for the early recruitment of ARFl, but is not required for
intracellular survival of the bacterium. A second factor LidA has recently
been identified to be exported in a Dot/Icm-dependent manner and localizes to
the phagosomal surface [160]. It has been hypothesized that this factor may
function as a gatekeeper for the premature release of other factors. It is not yet
clear how L. pneumophila modulates the type IV pathway to support the export
Bacterial Invasins
199
of nucleic acid/protein hybrids or effector protein substrates under different
environmental stimuli. The requirement for at least 24 genetic loci to promote
intracellular survival suggests that the Dot/Icm transporter may be far more
sophisticated than the VirB-like type IV systems [157].
Type V Secretion (Autotrunsporters)
The autotransporter pathway represents an alternate branch of the GSP that
serves as a simplified mechanism for the translocation of substrates out of the
cell. Analysis of various genomes suggests that the autotransporter mechanism
is widely conserved across gram-negative bacteria [83]. Rather than a require-
ment for factors in the periplasm or outer membrane translocases, the auto-
transporter secretion substrate harbors information in its carboxy-terminus for
insertion into the outer membrane, and for translocation of the amino-terminal
domain out of the bacterium. Pathogenic species of Neisseria secrete Igal pro-
tease using the type V mechanism to promote sui'vival in interstitial fluids. The
activated enzyme, once exported will function to degrade secretory antibodies
[161]. Igal protease is synthesized as a preproenzyme that harbors an amino-
termmal signal sequence to initiate its translocation across the inner membrane
through the Sec pathway. The signal sequence is cleaved by signal peptidase, and
the carboxy-terminaJ domain folds into a (5-barrel stiuctui'e promoting insertion
of the proenzyme in the outer membrane [162]. Insertion in the outer membrane
generates a porin-like channel for the export of the amino-terminal passenger
domain. Transport of the amino-terrmnal domain through the channel promotes
an autoproteolysis event, cleaving the proenzyme between the N- and C-terminal
domains at a proline residue [163], This proteolytic event will result in the
release of a diffusible active enzyme.
Examples of autotransport have also been described for the Hap adhesin of
Haemophilus influenzae, the IcsA protein ofS./lexneri, and the adhesin YadA of
X enlerocolitica. The IcsA autotransporter represents a modification of the type
V pathway, where an outer membrane serine protease SopA is required for the
cleavage of the IcsA passenger domain, which promotes proper actin cytoskele-
Lal nuclealion [164]. YadA in X enterocoliiica may represent another modifi-
cation of the type V pathway, where the amhio-terminal passenger domain is
delivered to the extracellular space, but is not cleaved from the carboxy-terminal
p-barrel [165], Further, YadA assembles into trimers in the outer membrane and
the amino-terminal heads assume a lollipop-like structure extending from the
narrow stalk domain. YadA is involved in the resistance to complement mediated
lysis.
A second variation of the type V pathway involves a two-component
secretory system, where the synthesis and secretion of an enzyme substrate
requires a single outer membrane transporter for delivery to the extracellular
Cambronne/Schneewind 200
space. The ShlA hemolysin of Serratia marcescens is synthesized as a pro-
enzyme that contains an amino-terminal signal sequence^ promoting its Sec-
dependent translocation to the periplasm [166]. The ShlB polypeptide also
contains an amino-terminal signal sequence and is exported through the
Sec pathway [167]. Both ShlA and ShlB are processed by a signal peptidase
and fold into mature species. The ShlB protein folds into a p-barrel structure
that inserts into the outer membrane. This event is required for the trans-
location of the enzymatic substrate ShlA. Other examples of this modified
type V pathway include the secretion of filamentous hemagglutinin (FHA)
by B, pertussis^ and the secretion of the HpmA hemolysin by Proteus
mirabiiis [168],
Concluding Remarks
Molecular mechanisms that promote bacterial colonization are seemingly
countless, however the accumulation of an ever-increasing body of information
has allowed for the detection of common themes in pathogenesis. Strategies
employed for the translocation of protein from the bacterial cytoplasm to targets
in or beyond the cell wall envelope represent prime examples of this common-
ality. Not only are the mechanisms for protein secretion conserved across
species, many seemingly distinct secretion mechanisms share common compo-
nents. Although protein secretion mechanisms represent only a fraction of the
virulence strategies employed by bacteria, several of these processes represent
primary virulence determinants. It appears that several pathogens use a combi-
nation of secretion mechanisms to establish infection, and the identification of
mechanisms by analogy has allowed for rapid progress in the classification of
a particular pathogen arsenal. This of course provides the potential for rapid
biochemical characterization of secretion systems and theu" protein substrates,
as well as development and application of therapeutic targets to cover a wide
range of bacterial species.
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Eric D, Cambronne
Section of Microbial Pathogenesis, Yale University School of Medicine
295 Congress Avenue, New Haven, CT 06536 (USA)
Tel. + 1 203 737 2404, E-Mail cambronne@yale,edu
Bacterial Invasins
209
Signaling and Gene Regulation
Russell W, Herwald H (eds); Concepts in Bacterial Virulence,
Contrib Microbiol. Basel, Kai'ger, 2005, vol 12, pp 210-233
Bacterial Iron Transport Related
to Virulence
Volkmar Braiin
Mikrobiologie/Membranphysiologie, Universitat Tiibmgen,
Tiibingen, Germany
The Problem of Iron Supply
Under oxic conditions, iron occurs in the Fe^"^ valence state and forms
insoluble polymeric hydroxyl-aquo complexes. Therefore, all aerobically livmg
organisms that contain iron in many cytosolic and membrane-bound redox pro-
teins, in particular in respiratory chains, have developed means to solubilize Fe^^.
Bacteria and fiingi synthesize iron-complexing compounds, designated sidero-
phores, which are secreted, bind extracellular Fe^^, and are transported as Fe^^
complexes via specific transport systems into the cells, where Fe^^ is released
from the complexes, usually by reduction to Fe^^, and then incorporated into
heme, iron-sulfur proteins, and other forms of protein reaction centers.
Higher organisms synthesize heme, which is the most abundant form of
iron-containing compounds. OnJy a small percentage of the heme occurs in free
form; most of it is incorporated into hemoglobin and bound to hemopexin.
Important extracellular iron-binding proteins in higher organisms are
transferrin and lactoferrin and intracellular ferritin. Transferrin is the predomi-
nant iron carrier that delivers iron to cells. The di-iron complex is taken up by
transferrin receptors, and the iron is released in endosomes and then further
metabolized. Lactoferrin is the predominant iron-binding protein in secretory
fluids. Transferrin and lactoferrin bind Fe^"^ so tightly that the free Fe-^"^ con-
centration in equilibrium with these proteins is in the order of 1 ion per liter.
The extreme Jack of iron inhibits growth of microorganisms. However, some
bacteria synthesize transferrin and lactoferrin receptor proteins exposed at the
bacterial cell surfaces, which remove the iron from transferrin and lactoferrin
and transport iron across the outer membrane.
This short overview focuses on some prominent examples of iron supply
systems formed by human pathogenic bacteria. The reader is referred to more
comprehensive reviews on specific aspects [1 24].
Overview of Bacterial Iron Transport Systems
Transport across the Cytoplasmic Membrane
The design of Fe^"*" transport systems across the cytoplasmic membrane is
the same for gram-negative and gram-positive bacteria. The systems belong to
the ATP-binding cassette (ABC) transporters, which consist of a binding protein,
a permease, and an ATPase (fig. 1). The binding proteins of gram-negative bac-
teria are located in the periplasm. In gram-positive bacteria, the binding proteins
are linked by a lipid of the murein-lipoprotein type (triacyl-glyceryl cysteine) to
the outer surface of the cytoplasmic membrane. The permease consists of one or
two proteins that are incorporated into the cytoplasmic membrane and trans-
locate Fe^"*", Fe^"'"-siderophores, or heme across the cytoplasmic membrane. The
ATPase provides the energy derived from ATP binding and subsequent ATP
hydrolysis [25].
Crystal structures have been determined for two Fe-'^-binding proteins,
FbpA of Neisseria gonorrhoeae and hFbpA of Haemophilus influenzae [26],
and for the ferrichxome-binding protein FhuD, which binds structurally related
siderophores of the hydroxamate type and the antibiotic albomycin [16, 27].
The crystal structures of FbpA and hFbpA are similar, but differ from that
of FhuD. The three proteins are composed of two globular domains; in FbpA
and hFbpA, these domains are connected by a hinge region that permits closure
of the globular domains upon binding of Fe^"^ (liice a Venus fly trap). In con-
trast, the two globular domains of FhuD are connected by a rigid, kinked
a-helix that allows onJy a slight movement of the globular domains. The crys-
tal structure of an entire ABC transporter, the vitamin B12 transporter of
Escherichia coll, has recently been unraveled. The ABC transporter consists of
the BtuC permease and associated BtuD ATPase [28], and the BtuF-binding
protein [29]. Since the BtuF structure is similar to FhuD and the transmem-
brane topology of BtuC is comparable to that of FhuB [15] which transports
ferrichrome across the cytoplasmic membrane [30], it is predicted that the
structure of the vitamin B12 transport system is representative for the ferric
siderophore and heme transport systems. BtuF can be positioned via salt
bridges on top of the BtuC permease. BtuCD forms a translocation channel that
is large enough to accommodate vitamin B|2. In the crystal, the channel is open
to the periplasmic side and closed to the cytoplasmic side. BtuD controls open-
ing of the BtuC channel. The two BtuD subunits located at the inner side of the
Iron Transport Related to Virulence 21 1
n
Q
X
LU
CD
i2
c
a
fPhuD
CQ
FhuC
FhuC
ATP ADP + P| ATP ADP + Pj
m
lij
Q
.2
OM
PP
CM
C
Albomycin
Rifamycin CGP4832
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212
H
I
C-
O
H2N-' I
H
o
H,C-C^.^
H^C^ o
N
I
O
Fe
O"
H
0"-N
\CH,), y
.r.0
3*
o=c
v./
I
N-
/
H
CHo-O-C-CHn-C-0,
CH3O
I
H ^C
NH
I
I
■N' "0
H
HOOC-CH I
ho-(!:h ^°
.s
O"
r
"A
N
(
V_
_y
CH
Albomycin
Rifamycin CGP4832
/
Fig. L Crystal structure of the FhuA outer membrane (OM) transport protein off", coli
with bound anribiotics albomycin (a) and rifamycin (b) CGP 4832, which are transported by
FhuA. The structures of the antibiotics derived from the crystal structures (c, d) and the chem-
ical formula {e,f) are shown, a, A The model illustrates the subcellular location of the proteins
TonB, ExbB, and ExbD, which form the energy-transducing complex between the cytoplas-
iTiic membrane and the outer membrane, the transport proteins across the cytoplasmic mein-
brane, and the interactions of the proteins. This protein arrangement is typical for all transport
systems of gram-negative bacteria that transport Fe^"^, Fe^"^-siderophores, and heme. For
further ijiformadon, see the text. PP - Periplasm; CM — cylopJasmic membrane.
cytoplasmic membrane are in close contact to the two BtuC subunits. Binding
of ATP moves the two BtuD subunits closer together. This might rearrange the
two BtuC subunits such that the channel opens to the cytoplasmic side, BtuF
loaded with vitamin B|2 is bound to BtuC, delivers vitamin B12 to BtuC, and
triggers ATP hydrolysis. The BtuD molecules move apart, which in tum closes
the BtuC channel to the cytoplasmic side and opens it to the periplasmic side
for the next round of vitamin B12 transport.
Transport across the Outer Membrane
Gram-negative bacteria contain an outer membrane that forms a perme-
ability barrier for hydrophilic substrates above a certain molar mass, which in
E. coli is 600 daltons [31]. The inner diameter of the porins through which the
substrates diffuse across the outer membrane determines the substrate size. The
Fe^"^ siderophores usually have a molecular weight greater than 600 and cannot
Iron Transport Related to Virulence
213
difFuse with a sufficient rate through porins. In addition, their concentration is
too low for diffusion to satisfy the growth requirement - in the order of 10^
iron ions per cell per generation. The siderophores, heme, and the iron-binding
proteins adsorb to outer membrane proteins, which not only serve as receptors
but also function as transporters across the outer membrane. The iron com-
pounds are thereby concentrated at the bacterial cell surface and are sub-
sequently actively transported by an energy-consuming process across the
outer membrane into the periplasm. There is no energy source in the outer
membrane to drive active transport. Energy is provided by the cytoplasmic
membrane through the proton motive force [32]. TonB, ExbB, and ExbD are
the three known proteins that relay the energy from the cytoplasmic membrane
into the outer membrane [33, 34], These proteins are located in the cyto-
plasmic membrane and interact with each other, and TonB interacts with the
outer membrane transport proteins. It is thought that these three proteins
respond to the proton motive force of the cytoplasmic membrane (e.g.^ the proton
gradient)^ react with a conformational change, and store the energy as poten-
tial energy. Upon interaction of energized TonB with the outer membrane
transporters^ the bound iron compounds are released from their binding sites
and a channel is opened through which the iron compounds diffuse into the
periplasm.
The crystal stnictiires of three outer membrane iron transporters FhuA [35,
36], FepA [37], and FecA [38, 39], and the vitamin B|2 transporter BtuB [40] pro-
vide a conceptual framework of how these transporters might function. The struc-
tures reveal a p-barrel composed of 22 antiparallel p-strands that form a channel.
The channel is cjosed by a globular domain, which is designated as the cork, plug,
or hatch. Binding of the substrates to the transporters occurs at a site well above
the cell surface. Very strong bmding occurs through approximately ten-amino acid
side chains with a binding constant in the nanomolar range. Energy input is
required to release the substrates from their binding sites and to move the cork so
that a channel is formed through which the substrates gain access to the periplasm.
The theory is that TonB transfers potential energy to the transporters, which alter
their confomiation to open a channel, TonB is deenergized, and tlie ti'ansportei's
close the channels after the iron compounds have passed through by diflflision. The
genetically and biochemically identified sites of interaction between TonB and the
transporters are located in the TonB box of the transporters and a region around
residue 160 of TonB [41, 42]. The crystal structures and electron spin resonance
determinations of nitroxide-substituted TonB box residues of BtuB demonstrate
that the TonB box is exposed to the periplasm and moves upon binding of the sub-
strates to the transporters [43]. The TonB box and the substrate-binding sites are
far apart, which implies long-range structural transitions throughout the entire
transporter Transport across the outer membrane is mechanistically not coupled
Braun
214
to transport across the cytoplasmic membrane. The two membrane transport
processes occur independently of each other.
Iron Transport Associated with Virulence
Iron-Controlled Bacterial Functions
Since iron is an essential element, but available only in growth-Jimiting
concentrations, those bacteria that multiply in the human body express potent
iron transport systems. The relationship of iron transport to virulence is usually
not easy to establish since bacteria normally express several iron transport sys-
tems. Knocking out one system by mutation might not result in conversion of a
pathogenic strain to a nonpathogenic strain since other iron transport systems
take over the iron supply For example, a pathogenic £'. coli strain may transport
Fe^^ by the siderophores aerobactin, enterobactin, salmochelin, citrate, ferri-
chrome, and heme, and Fe^"^ via the/eo-encoded transport system. tonB, exbB,
and exbD are the only genes involved in all energy-coupled outer membrane
iron transport systems of gram-negative bacteria, lonB mutants are impaired in
virulence in various animal infection systems [44, 45], However, some bacteria
contain up to three tonB and exbB, exbD genes, which might participate in dif-
ferent iron uptake systems (see, for example. Iron Transport of Vibrio cholerae
Related to Virulence), In addition, it is usually not known which iron transport
system is important for proliferation at a specific infection site. Moreover, the
iron limitation usually encountered in the human body could serve as an envi-
ronmental signal that tells a bacterial strain its location in the human body. This
could induce expression of genes required for multiplication, but might not be
directly related to the iron supply Therefore, different approaches are required
to elucidate a relationship between iron transport and virulence. Such studies
have involved knocking out a particular iron transport system and a genome-
wide search for the expression of genes in vivo compared to the expression of
genes in synthetic media under iron-deplete and iron-replete conditions. Such
large-scale expression profiles usually reveal genes related to the iron supply.
These genes encode proteins for siderophore biosynthesis and transport, heme
transport, hemolysins^ and toxins. The most prominent toxin is the diphtheria
toxin, which is synthesized under iron-limiting conditions. Other iron-regulated
toxins are the Shiga toxin of Shigella and E, coli strains, the hemolysins/
cytolysins of Serratia marcescens and certain E, coli strains, exotoxin A of
Pseudomonas aeniginosa, and the tetanus toxin oi Clostridium tetani. By dam-
aging cells, the toxins can mobilize intracellular iron sources and make them
available to bacteria. S. marcescens, for example, colonizes the intestine of
Caenorhabditis elegans and kills the nematode, S, marcescens mutants are
Iron Transport Relaied to Virulence 215
impaired in virulence when they carry a transposon in the hemolysin gene or in
a siderophore biosynthesis gene [46].
Stress by Iron Surplus
Not only iron shortage, but also iron surplus can affect the outcome of a
bacterial infection. Aerobic metabolism constantly creates hydrogen peroxide
and superoxide radicals. If too much H2O2 is formed, it might not be completely
destroyed by catalase and peroxidase. In the Haber- Weiss reaction, the oxygen
radical reacts with H2O2 to form the highly reactive hydroxyl radical and
hydroxyl anion. In the Fenton reaction, Fe^"^ converts H2O2 to the hydroxyl
radical and hydroxide anion. Fe-^^ oxidizes the oxygen radical to oxygen. H2O2,
the oxygen radicals, and the hydroxyl radicals damage DNA, lipids in mem-
branes, and proteins. The lack of regulation of iron metabolism could, therefore,
be deleterious to cells [47]. This has been demonstrated for E. coli, in which
a mutation in the/wr (iron uptake regulator) gene renders cells sensitive to
oxygen. An additional mutation in the recA gene, which is involved in DNA
repair, kills cells when they are cultivated under oxic conditions [48]. The sur-
plus of reactive intracellular free iron might result from an uncontrolled import
and the lack of intracellular iron storage proteins. Iron uptake is controlled by
the/wr gene in most gram-negative bacteria and certain gram-positive bacteria
with a low GC content and by the dtxR gene in most (GC-rich) gram-positive
bacteria. When the intracellular iron concentration reaches a certain level, the
Fur and DtxR proteins are loaded with Fe^"^ and repress transcription of genes
encoding iron transport proteins and enzymes that synthesize siderophores [7].
Two types of iron storage proteins contribute to intracellular iron homeostasis
in bacteria [22]. Ferritins are also found in eukaryotes, and heme-containing
bacterioferritins are only found in bacteria. Both types are composed of 24 identi-
cal subunits that form an almost spherical shell into which more than 2,000 Fe^"^
ions can be deposited. The FtnA ferritin of E. coli accumulates iron in the post-
exponential growth phase in the presence of excess iron in the medium and sup-
ports subsequent growth under non-deficient conditions. Helicobacter pylori and
Campylobacter Jejuni express a similar protein that stores iron and protects cells
against oxygen damage. No physiological role has been ascribed to the Bfr bacte-
rioferritin of E. coli, but a bfr mutant of/? aeruginosa is sensitive to peroxides.
Dps is another iron-binding protein that forms a shell, but with 12 sub-
units. Dps is probably less important for iron storage than for protecting DNA
against the combined action of iron and H2O2.
Iron Transport ofE. coli <3«(i Shigella Related to Virulence
Pathogenic E. coli strains express ten outer membrane proteins that transport
ferric siderophores and heme (table I). All the ferric hydroxamates (aerobactin,
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216
Table L Iron transport systems o^ E. coli
Substrate
Outer membrane
Peri plasn lie
Cytoplasiiiic membrane
protein
protein
proteins
Enterobactin
FepA
FepB
FepD", FepG^ FepC'
Salmochelin
IroN
FepB
FepDS FepGS FepC''
Catecholates
Cir
FepB
FepD", FepG^ FepC'
Catecho ates
Fiu
FepB
FepD^ FepG^ FepC
Ferrichrome
FhuA
FhuD
FhuB^ FhuC''
Aerobactin
lutA
FhuD
FhuB% FhuC
Coprogen
FhuE
FhuD
FhuB^ FhuC'
Citrate
FecA
FecB
Fece, FecD", FecE''
Heme
ChuA
ChuP
ChuU^■^ ChuV''^'^
Yersiniabactin''
FyuA
NI
YbtP, YbtP
Fe^^-
FeoB
^Transmembrane transport proteins in the cytoplasmic membrane,
^ATPase.
"Designations adapted from S. dysenteriae which is justified by the highly homologous E. coli
and Shigella genomes. In E. coll K-12 ChuA alone is sufficient to support heme-dependent
growth but the transport system in the cytoplasmic membrane may increase sensitivity to
heme and rate of heme uptake.
^The transport system of yersiniabactin is encoded on pathogenicity islands which occiu"
in various Enterobacteriaceae.
The nomenclature of reference 58 was used. For further details, see text and references 8
and 49-5 L NJ = Not identified.
ferrichrome, coprogen) for which specific transporters are found in the outer
membrane are transported by the same transport system across the cytoplasmic
membrane. The same holds true for the ferric catecholates, including ferric entero-
bactin and presumably ferric salmochelin^ which are transported across the cyto-
plasmic membrane by the same system. It is not clear whether or to what extent
the entile FepBCD transport system is involved in the ferric salmochelin tians-
port. The heme transport system has been characterized in Shigella dysenteriae
and its phylogenetic distribution in enteric bacteria has been determined [52]. The
assignment of the heme genes to functions is based on the fh'st functionally char-
acterized heme transport system of Yersinia enterocolitica [53]. Heme and aero-
bactin transport^ as well asTonB are required for vinjience of the uropathogenic
E. coli strain CFT073 in a mouse model of urinary tract uifection [54]. In addition,
E. coli strains isolated from patients with an intra-abdominal infection have been
shown to secrete a protease, Hbp, that degrades hemoglobin. Hbp binds the released
heme [55] and promotes the growth of Bactero ides fingi lis, which is frequently
Iron Transport Related to Virulence
217
associated with E. coli in intra-abdominal infections. In a mouse infection model,
Hbp contributes to the pathogenic synergy of these two organisms in abscess
development. Heme transport systems are widely distributed among gram-positive
and gram-negative bacteria [10, 11].
The Fe-^'^-yersiniabactm transport system is frequently encoded on a 'high
pathogenicity island', which occurs in several Enterobacteriaceae [56], but is
also present in strains with less pathogenic potential [57]. The transport system
of Fe^"^-yersiniabactin across the cytoplasmic membrane is interesting since the
two permease protems YbtP and YbtQ are each fused with the ATPase [58], as
is found with human ABC export proteins. Subcutaneous infection by a ybtP
mutant fails to cause disease \n mice, a route that mimics Yersinia pestis trans-
mission by fleas causing bubonic plague.
To date there has been no association reported between virulence and the
ferric citrate transport system, in which FecB (binding protein), FecCD (perme-
ase), and FecE (ATPase) catalyze transport across the cytoplasmic membrane. A
nearly identical transport system is located on a pathogenicity island of Shigella
flexneri [59]. Coliform isolates oiE. coli and Klebsiella pneumoniae from bovine
inflammatory infections (mastitis) contain FecA, as evidenced by anti-FecA anti-
bodies [60], and FecA is being considered as a vaccine component for the treat-
ment of mastitis. A study of the regulation of the ferric citrate transport proteins
uncovered a new type of transcription regulation. The inducer of the transcription
of the transport genes binds to the FecA outer membrane protein and elicits a sig-
nal that is transmitted by FecA across the outer membrane to the FecR protein,
which transmits the signal across the cytoplasmic membrane. In the cytoplasm,
the Feci sigma factor is activated and directs the RNA polymerase specifically to
the promoter of the^ec transport genes upstream offecA [61, 62].
Siderophores like ferrichrome and coprogen, which are not synthesized by
E. coli or any other bacteria, but which are transported by many bacteria, includ-
ing E. coli, might be used during coinfection with fungi that synthesize the
siderophores or during bacterial growth outside the human body. The large vari-
ety of transport systems for ferric siderophores and heme found in E. coli and
Shigella are typical for pathogenic bacteria. The systems are distiibuted among
bacteria by horizontal gene transfer. For example, the aerobactin synthesis genes
are found on plasmids in E. coli and Salmonella, on pathogenicity islands in
S. flexneri and Shigella sonnei^ and on the chromosome of Shigella boydil and
certain E. coli strains [8, 63]. Another example is the recently discovered iroN
gene, which was originally identified in Salmonella enterica and then shown to
contribute to the uropathogenicity of £■. coli isolates [64, 65]. iroN is encoded on
a pathogenicity island on the chromosome [64] and on a transmissible plasmid
[65]. In a mouse model of ascending urinary tract infection, IroN contributes to
colonization of the bladder, kidneys, and urine [64].
Braun
218
In addition to the Fe^"*" transport systems, E. coli also contains an Fe^"*"
transport system, which is encoded by the feoAB genes [23], This transport sys-
tem functions Linder anoxic conditions, as found in the colon and in biofihns.
Iron Transport o/ Salmonella Related to Virulence
S. enterica serovar Typhimurium has h-on transport systems similar to those
of E. coli and Shigella, but so far no heme or ferric citrate transport system
has been described. However, a heme transport gene operon similar to that in
>S'. dysenteriae is encoded on the Salmonella typhimurium genome. The known
systems include those related to the outer membrane transporters FhuA, FepA,
FoxA, Cir, and IroN. An additional transport system presumably transports iron,
as was first demonstrated for the sfuABC iron transport system of 5'. marcescens
[66] and then for the fbpABC system of A^. gonorrhoeae, hJbpABC (hit ABC) of
H. influenzae [18], and yfuABC of Y. pestis [67]. sitA encodes a putative peri-
plasmic permease, sitB an ATPase, and sitCD a permease [68]. However, sitABC
is not homologous to the sfuABC-ty^iQ transport systems, but is homologous to
yfeABC o^Y. pestis and it transports Mn'^"'" with a much higher affinity than Fe^"*".
The Sit system is widely distributed in all S. enterica serovars and is required
for full virulence of *S. typhimurium [69]; the Yfe system is essential for virulence
of Y. pestis [70]. Iron transport systems are redundant, depending on the test sys-
tem, since depleting one system may have no effect on bacterial virulence. The
S. enterica genome also carries the feoAB genes, which encode an Fe^"^ transport
system. Single mutations of sitA,feoB, or iucD (Fe^^-aerobactin transport) in
S.flexneri do not impair the growth of these bacteria on a Henle cell monolayer;
however, triple mutants do not form plaques [71].
A novel siderophore, designated salmochelin, was discovered only recently
in S. enterica serovar Typhimurium LT2. The iroB gene product, encoded in the
iron-regulated gene cluster iroNEDCB, shows sequence similarity to glycosyl
transferases. This fmding prompted a search for the fionction of IroB. Indeed, IroB
was shown to encode an enzyme that glucosylates enterobactin at the 5' position
of the benzoyl ring, forming a C-C bond [106]. The published tentative structure
carries the two glucosyl moieties inserted between two 2,3-dihydroxybenzoylser-
ine residues [49]. In a Salmonella culture, sahnochelin is more abundant and is
more soluble than enterobactin. Therefore, it might be less able to elicit antibod-
ies than enterobactin, which serves, bound to serum albumin, as a hapten.
Transport of Fe-'^-salmochelin across the outer membrane is mediated by IroN
and to a lesser extent by the FepA and Cir transporters.
Iron Transport of?, aeruginosa Related to Virulence
Pyoverdin and pyochelin are two well-studied siderophores that supply
iron to P. aeruginosa. A number of indications show a relationship between
Iron Transport Related to Virulence 2 1 9
iron supply and virulence of P. aemginosa in animal infection models:
derepression of siderophore synthesis genes, synthesis of the siderophores
pyoverdin and pyochelin and the related transport proteins, release of iron
from the host iron-binding proteins transferrin and lactoferrin, and reduction
of virulence of mutants deficient in synthesis of siderophores or Fe^"^-
siderophore transport proteins. In addition, exotoxin A synthesis is controlled
by the iron supply via the Fur repressor. A tonB mutant devoid of Fe-'^ uptake
via pyoverdin, pyochelin, and heme grows in the muscles and lungs of
immunosuppressed mice, but does not kill the animals [72]. Pyoverdin- and
pyochelin-negative double mutants multiply, but do not kill the mice; however,
intranasal inoculation of wild-type bacteria results in multiplication and killing
[73]. PvdS (see below) is an ECF sigma factor synthesized in chronic lung
infections affiliated with cystic fibrosis and contributes to the synthesis of
exotoxin A [74].
Complex regulatory devices underlie iron-mediated control of gene expres-
sion in P. aeruginosa. For example, iron-loaded Fur does not bind to the pro-
moter of the toxA gene of exotoxin A, but acts via the pvf:/,^ gene product, which
regulates 26 iron-repressible genes. pvdS encodes an ECF sigma factor of the
Feci type (see Iron Transport of £". coli and Shigella Related to Virulence), and
its synthesis is repressed by binding of Fe^"^-Fur to the pvdS promoter [75]. The
activity of PvdS is controlled by pyoverdin secreted in the growth medium;
pyoverdin (probably Fe-'"'" -pyoverdin) binds to the FpvA protein in the outer
membrane. FpvA displays several functions: it acts as a signal receiver and as a
signal transmitter across the outer membrane, and it transports Fe-'^ -pyoverdin
across the outer membrane. The signal is transmitted by the FpvR protein across
the cytoplasmic membrane into the cytoplasm, where PvdS is converted into an
active sigma factor. Since PvdS is active in mutants lacking FpvR and over-
expression of FpvR inactivates PvdS, FpvR probably fijnctions as an anti-sigma
factor of PvdS [75]. PvdS directs the RNA polymerase to the promoter of the
kon-repressible genes, uicluding the pyoverdm synthesis genes. fpvR transcrip-
tion is repressed by Fe^"^-Fur, as is transcription of a second ECF sigma factor
gene,^v/. Fpvl synthesis is regulated like PvdS synthesis via Fe^"^ -pyoverdin,
FpvA, and FpvR, and controls synthesis of FpvA.
Heme uptake by P. aeruginosa is mediated by two systems, one of which
is encoded by the phuRSTUVW genes (fig. 2) [76]. This system is very similar
to the heme transport system of Y. enterocolitica. Heme is bound to the PfuR
outer membrane protein that transports heme across the outer membrane.
Further transport into the cytoplasm is achieved by an ABC transporter. The
other heme transport system is sunilar to the heme transport system of
S. marcescens and involves a hemophore that is secreted, releases heme from
hemoglobin, and delivers it to the outer membrane transport protein (fig. 2).
Braun
220
hemP
R
S
T
U
V
Yersinia enterocolitica
Yersinia pestis
hmuX Y P R
phuR
S
u
V
w
Pseudomonas aeruginosa
1=11
t>
shuS
A
Shigella dysen tehee
T
w
X Y u y
Vibn'o cholerae
huLA tonBI exbBI hutB
exbD1
D
hxuC
Haemophilus Influenzae % i i
hxuB
hxuA
hgpA
hemO
hmbR
Neisseria meningitidis
hpuA
hpuB
has! S
R
D
B
Serratia marcescens
hasR
Yersinia pestis
Bordetella pertussis
rhul R
bhuR
(ECF*) hasR
Pseudomonas aeruginosa r[^ — [N n~
F
A
D
^=t>
Outer membranG receptor; perlplasmic binding protein; integrai membrane protein; ATPase subunit;
i=t>
Hemophore; hemophore secretion system; protein for heme utiiization
Fig, 2. Heme transport systems of gram -negative bacteria. The upper panel shows the
transport genes and some promoters (P). In the lower panel, genes for hemophore synthesis,
secretion, and regulation, and not the actual heme transport genes are shown for S. marcescens,
Y. pestis, and F aeruginosa. The HasA hemophores are secreted by the type 1 secretion mecha-
nism catalyzed by the proteins HasD, HasE, and HasF. HasB is structurally and functionalJy a
TonB-like protein, hasi and hasS, and rhuJ and rhuR encode a transcription-signaling device of
the FeclR type in which the ) proteins represent extracytoplasmic membrane (ECF) sigma fac-
tors that receive signals from outside the cytoplasm and the R or S protein transfers the signals
across the cytoplasmic membrane. In Bordetella pertussis, rhuIR regulates transcription of the
bhuRSTUV \\^m^ transport genes [for fijrther mformation, see 10, 62, 77].
Iron Transport Related to Virulence
221
In S. marcescens, regulation of heme transport gene transcription is mediated
by a signaling device of the FecIRA type [77]. Heme-loaded hemophore binds
to the HasR heme transporter and induces transcription of the hasR gene
via HasI, which functions as an ECF sigma factor, and HasS, which acts as an
anti-sigma factor. Since P. aeruginosa contains genes homologous to those in
S. marcescens and arranged similarly, it is likely that the two Has regulatory
systems function similarly.
Analysis of the genome of P. aeruginosa predicts nine additional regula-
tory devices of the FecIRA, HasISR, FpvA/Fpvl, FpvR, and PvdS type. These
systems usually have the same gene arrangement asfecIRA, and the outer mem-
brane proteins contain an extended amino-terminus, which in FecA interacts
with FecR [12-14].
In addition to surface signaling elicited by the iron substrates, P. aeitiginosa
controls iron usage by a number of additional regulatory mechanisms. For
example, pyochelin synthesis and uptake is repressed by Fe^"^-Fur, which binds
to promoters of the synthesis and uptake genes. The regulatory protein PchR
acts as a repressor in the absence of pyochelin and as an activator in the pres-
ence of pyochelin [78]. Regulation of ferric enterobactin usage is mediated by
a two-component system consisting of the PfeS signal receiver and the PfeR
response regulator. Ferric enterobactin in the periplasm binds to the PfeS sensor
kinase, which is aiitophosphorylated and transfers the phosphate group to the
receiver domain of PfeR. Phosphorylated PfeR functions as a transcription
activator of the pfe A gene, which encodes the high-affinity PfeA outer mem-
brane transporter [79]. In this iron transport system and in all the other iron
transport systems studied in P aeruginosa, the transported substrate induces
synthesis of the cognate transport system. This is achieved by various mecha-
nisms, but always results in the economic adaptation of the cells to the available
iron source. If only iron depletion of the Fur protein would derepress gene tran-
scription, many of the approximately 13 iron transport systems would be
synthesized, even though only the one for the available iron soui'ce would be
required.
Iron Transport o/Vibrio cholerae Related to Virulence
Three heme transport systems have been identified in V cholerae^ repre-
sented by the outer membrane transporters HutA, HutR, and HasR [80]. A huiA
hutR double mutant is impaired, but not completely unable to use hemin as an
iron source. The triple mutant hulA hutR hasR is completely devoid of heme
utilization. V. cholerae HasR is similar to the HasR proteins of S. marcescens
and P. aeruginosa^ which receive heme from the hemophore that releases heme
from hemoglobin. In addition to the use of heme via transporters across the
outer and cytoplasmic membranes, V. cholerae can use the iron complexes of
Braun
222
the siderophores vibriobactin, enterobactin, and ferrichrome [81], The transporters
are preferentially coupled to one of the twoTonB proteins present in V choleme
[82], HasR, VctA, and IrgA, the latter two transport Fe^^-enterobactin [83], are
only coupled to TonB2, whereas HutA, HutR, ViuA (Fe^^-vibriobactin trans-
porter) and FhuA (ferrichrome transporter) can use TonBl and TonB2 [80]. \n
an infant mouse model, the triple mutant competes with the wild-type strain,
which indicated additional iron sources in vivo [80]. Analysis of gene tran-
scription in the rabbit ileal loop model have revealed enhanced transcription of
heme and Fe^^ transport genes and of they^o^^ genes, which encode an Fe^^-
transport system [84] that may have supplied the necessary iron.
Functions of Iron in Neisseria Related to Viimlence
A tonB mutant of Neisseria meningitidis does not actively transport iron
and is unable to replicate within epithelial cells [85], N, gonorrhoeae and
N. meningitidis transport iron across the cytoplasmic membrane by an ABC
transporter encoded by the JbpABC genes [18], which are similar to the sfuABC
genes of *S. marcescens, the hfbpABC (hitABC) of//, influenzae, and the yf ABC
genes of Y. pestis (see Iron Transport of Salmonella Related to Virulence).
No siderophore seems to be involved in iron transport. In A^. gonorrhoeae and
//. influenzae, the iron might be delivered by the host transferrin and lactoferrin,
which bind to highly specific outer membrane receptor proteins composed of two
polypeptides: TbpA and TbpB for the transferrin receptor, and LbpA and LbpB
for the lactoferrin receptor The B components are lipoproteins and discriminate
between iron-loaded and iron-unloaded transferrins and lactoferrins. The
A components are similar to TonB-coupled ferric siderophore and heme
transporters. TonB is not only required for the transport of iron across the outer
membrane, but also for the release of Fe^"^ from transferrin and lactoferrin [21],
The A and B components act in concert and interact with each other Proteolytic
degradation of TbpB is strongly influenced by coupling of TbpA to TonB.
A^. gonorrhoeae mutants that lack the transferrin receptor do not elicit symptoms
of urethritis in human male volunteers [86].
Two hemoglobin receptors have been identified in A^. meningitidis: a tu^o-
component receptor designated HpuAB and a one-component receptor designated
HpmR. No siderophores have been identified in Neisseria. However, Neisseria can
utilize Fe-^^-enterobactin taken up via a TonB-coupled transporter across the outer
membrane and an ABC transporter across the cytoplasmic membrane [5, 87].
Iron Transport cj/ Staphylococcus aureus Related to Virulence
In S. aureus^ several iron transport systems seem to operate. Ferrichrome is
actively transported [88], and recently heme transport has been correlated with
proteins (Isd) on the cell surface that are anchored to the murein by Iavo sortases
Iron Transport Related to Virulence 223
[89]. S. aureus binds transferrin [90] and haptoglobin-hemoglobin [91]. In certain
strains, slime production is enhanced by iron limitation [92]. Iron homoeostasis
is regulated by the Fur repressor, whose synthesis is repressed by a homologous
protein, PerR, which also regulates synthesis of the iron storage proteins ferritin
and MrgA, a Dps homolog. PerR is required for full virulence of >S. aureus in a
murine skin abscess model [93]. The cell wall of 5". aureus and Staphylococcus
epldermidis contains the Tpn transferrin-binding protein, which is synthesized
under iron-limiting growth conditions and elicits antibody formation in human
serum and peritoneum upon staphylococcal infections [94]. The Tpn protein is the
cell wall glyceraldehyde-3-phosphate dehydrogenase, which also binds plasmin
[95], It is assumed that the released iron is taken up into the cytoplasm by ABC
transporters. Two such ABC transporters, encoded by the sir ABC and sstABCD
genes, have been partially characterized [96].
Fe3"^-Siderophores as Antibiotic Carriers
Multidrug resistance against currently used antibiotics forms an increasing
problem in the treatment of bacterial diseases. One way out of the resistance
dilemma is the development of new antibiotics. Since most antibiotics have been
discovered during the decades of large-scale random screening, new strategies
will have to be exploited. One possibility is the use of transport systems to trans-
port antibiotics into cells. There are examples in which active transport, as
opposed to diflfiision, decreases the minimal inhibitory concentration (MIC) of an
antibiotic more than 100-fold [97].
Antibiotics with F^^-Hydroxamate Carriers
Most antibiotics diffuse into bacteria, and their rate of diffusion and their
activity at the target sites determine their efficiency, as measured by the MIC. In
gram-negative bacteria, the outer membrane forms an additional permeability
barrier in addition to the cytoplasmic membrane, and renders gram-negative
bacteria less sensitive to many antibiotics than gram-positive bacteria. However,
if antibiotics are actively transported across the outer membrane, their MIC
could be lower in gram-negative than in gram-positive bacteria because the
antibiotics are accumulated ui the periplasm and form a steep concentration gra-
dient into the cytoplasm, thereby enhancmg the diffusion rate, or the antibiotic
might even be actively transported across the cytoplasmic membrane.
There are naturally occurring antibiotics that consist of an antibiotically
active moiety and a siderophore carrier The best-studied example is albomycin,
which is composed of a trihydroxamate that binds Fe^"^, a peptide linker, and a
thioribosyl pyrimidine moiety that inhibits tRNA^*^"" synthetase [98]. Albomycin is
Braun
224
highly active toward gram-positive and gram-negative bacteria. The MIC against
an E. coli strain is 200 times lower (0,05 |JLg/mJ) than of ampicillin (12.5 jjug/mJ).
The high specific activity comes from the active transport across the outer
membrane and the cytoplasmic membrane into bacteria via the transport system
of the structural analogue ferrichrome. The ferrichrome analogue serves as carrier
of the antibiotically active thioribosyl pyrimidine group. After transport into the
cytoplasm, iron is released from albomycin, and the thioribosyl pyrimidine
group has to be cleaved from the carrier to be inhibitory. Tn E. coli, this is
mainly achieved by peptidase N [1, 3, 97], Mutants devoid of peptidase N acti-
vity are resistant to albomycin, and albomycin then serves as an iron carrier.
Most of the thioribosyl pyrimidine moiety remains inside the cell, whereas the
carrier is released into the culture medium. Albomycin is one of the very few
antibiotics for which transport^ intracellular activation^ and target have all been
characterized.
Albomycin has been cocrystallized with FhuA to determine whether it
binds to the ferrichrome binding site of FhuA and where the bulky side chain is
located in FhuA (fig. 1). The crystal structure reveals that the Fe-^^-hydroxamate
portion of albomycin occupies the same site on FhuA and is bound by the same
amino acid side chains as ferrichrome [99], The thioribosyl pyrimidine moiety
binds in the external pocket via five residues that are not involved in ferricKrome
binding. The crystal structure also reveals the hitherto unknown conformation of
albomycin and the conformation in the transport-competent form. Unexpectedly,
albomycin assumes two conformations in the crystal - an extended and a com-
pact conformation. Both conformations fit into the external cavity of FhuA and
occupy seven different amino acid ligands. The solvent-exposed external cavity
of FhuA is sufficiently large to accommodate the voluminous side chain of
albomycin.
After transport across the outer membrane by FhuA^ albomycin binds to
FhuD in the periplasm. FhuD subsequently delivers albomycin to the permease
in the cytoplasmic membrane. Cocrystals of FhuD with bound albomycin have
been obtained in sufficient quality to determine the structure [100]. In contrast
to FhuA, where albomycin sits inside the molecule, in FhuD albomycm is
exposed to the surface of the protein. The thioribosyl moiety is not even seen in
the crystal since it is not fixed to the protein and is thereby flexible. The fixa-
tion of albomycin at the surface of FhuD explains the broader substrate speci-
ficity of FhuD in contrast to FhuA since space is less restricted at the protein
surface than within a protein.
Results of studies with albomycin demonstrate that the proteins involved
in transport across the outer membrane and the cytoplasmic membrane tolerate
substantial modifications of the substrate. The modular design of albomycin can
be synthetically mimicked. Antibiotics that are ineffective because of poor entry
Iron Transport Relaied to Virulence 225
into the cells can be chemically linked to ferrichrome and then transported into
cells as ferrichrome derivatives.
CGP 4832 is a semisynthetic rifamycin derivative with an activity against
many gram-negative bacteria 200-fold higher than that of unmodified rifamycin
[101], The reason for the increased activity of CGP 4832 is its energy-coupled
transport by FhuA across the outer membrane ofE. coli [101]. The use of FhuA
as transporter is surprising since CGP 4832 does not contain iron and has no
structural resemblance to ferrichrome or any other hydroxamate. To obtain
insights into how CGP 4832 is transported by FhuA, the crystal structure of
FhuA loaded with CGP 4832 was determined [102], CGP 4832 occupies in
FhuA largely the same site as ferrichrome (fig. 1 ). Nine residues that bind CGP
4832 also bind ferrichrome. Of 16 amino acid residues that bind CGP 4832,
5 residues recognize those side chains of CGP 4832 in which it differs from
unmodified rifamycin. Two additional amino acid residues specifically bind the
unique CGP 4832 side chains, whereas the other residues bind to sites that CGP
4832 shares with rifamycin. The crystal structure reveals the conformation of
CGP 4832, which demonstrates a completely different structure than that of ferri-
chrome. Unlike albomycin, CGP 4832 is not transported via FhuBCD across
the cytoplasmic membrane [101]. Rather, its active transport across the outer
membrane results in an elevated concentration in the periplasm, which facilitates
diffusion across the cytoplasmic membrane. It is the active transport across the
outer membrane that reduces the MIC 200-fold.
Salmycins have been isolated from SU^eptomyces violaceiis 37290 (DSM
8286) and are highly active against staphylococci and streptococci (MIC
10 |JLg/ml). Salmycins consist of an Fe-^'^-siderophore with a ferrioxamine group
and an antibiotically active aminodisaccharide, which in salmycin B consists of
a 2-ketoglucose linked to the 2-position of a 6-methylaminoheptopyranose
[103]. It is assumed that the aminodisaccharide is released from the carrier by
cleavage of the ester bond. Salmycins seem to inhibit protein synthesis by a yet
unknown mechanism.
Ferrimycins are among the first sideromycins discovered [97]. The action
of ferrimycins is antagonized by ferroxamine B, which competes for ferri-
mycin uptake. Ferrimycin inhibits incorporation of amino acids into proteins
of ^. aureus SG5I I. Ferrimycin is difficult to isolate and for this reason has
recently been studied less than aJbomycin and salmycin.
Antibiotics with Fe^^-Catecholate Carriers
Enterobactin is the most prominent catecholate siderophore with an
extremely high Fe-^"^ stability constant. It consists of three dihydroxy benzoyl
serine residues linked to a cyclic trimer by ester bonds. No natural Fe-^"^-
catecholates with antibiotic activity are known. However, chemically synthesized
Braun
226
catechol-substituted cephalosporins display MIC values below 1 |jLg/ml [104,
105], particularly against gram-negative bacteria, including R aeruginosa. Their
antimicrobial activities can exceed the activity of the unsubstituted
cephalosporins more than 100-fold, Their high activity is related to their active
transport into the periplasm^ where the target, the murein transpeptidase, is
located. They are transported across the outer membrane by the Fe-^"^-catecholate
transport proteins Fiu and Cir [26]. Iron limitation increases the susceptibility of
E. coll strains since low iron derepresses Fiu and Cir synthesis.
Resistance to Fe^^-Siderophore Antibiotics
Resistant bacteria emerge on every nutrient agar plate containing anti-
biotics that are carried into the bacteria by active Fe-^"^-siderophore transport
systems. The higher the number of genes involved in a particular transport system,
the higher the frequency of resistance. However, when two transport systems
are used by an antibiotic, for example Cir and Fiu for the cephalosporin cate-
cholates, the frequency of resistant mutants is low. Although the high resistance
frequency seems to prevent development of such antibiotics as antibacterial
drugs, the in vivo situation might be quite different In cases where an iron
transport system is important for the proliferation of the pathogenic bacteria^
loss of the iron transport system is detrimental. Even when several iron trans-
port systems exist and only one is inactivated by resistance to a particular
antibiotic, the inactivated system might be the one that is essential for the bac-
teria to survive and multiply at the site of infection in the human host. Under
these circumstances, it does not matter whether the number of bacteria is reduced
by the antibiotic or by loss of the iron supply since under both conditions the
immune defense system gains time to cope with the infection.
Concluding Remarks
Iron deficiency was also designated nutritional immunity which meant that
growth inhibition by lack of iron prevents bacterial multiplication. Lack of
growth or growth retardation gives the natural and the adaptive immunity sys-
tem the chance to cope with an infection. Iron is the only nutrient for which an
essential role in growth of many bacterial pathogens causing various diseases
in humans and animals has been demonstrated. There are certainly many more
nutrients which play a decisive role in extra- and intracellular multiplication of
bacteria. However, it is difficult to identify these nutrients. Large-scale expres-
sion profiles of metabolic genes in bacteria isolated from human patients with-
out further cuJturing and from animal models may indicate metabolic pathways
from which the nutrients may be derived. From a purely scientific point of view
Iron Transport Relaied to Virulence 227
the iron supply systems are of great interest with regard to the various ways
insoluble Fe^"^ is complexed by siderophores, heme, transferrin, and lactoferrin
and transported into the bacterial cells by distinct and very sophisticated mech-
anisms. For the avoidance of iron shortage and iron surplus the transport systems
are regulated by various means, iron-dependent repression, downregulation by
small RNAs, transcription enhancement by two-component systems, and tran-
scription initiation by surface signaling. In the fliture, a detailed knowledge of
iron uptake and intracellular iron metabolism may be applied to interfere with
bacterial growth as a means to control bacterial diseases, and siderophore
antibiotics (sideromycins) may be used when treatment with other antibiotics
fails because of resistance.
Acknowledgments
I would like to thank Klaus Hantke for preparation of figure 2, Michael Braun for
preparation of figure 1, and Karen A, Brune for critically reading the manuscript. The
author's work was supported by the Deutsche Forschungsgemeinschaft (Forschergruppe
^Bakterielle ZeUJiiiUe: Synthese, Funktion und Wijkort', Br 330/14-2) and the Fonds der
Chemischen Industrie,
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Prof. Volkmar Braun
Mikrobiologie/Membranphysiologie, Universitat Tubingen
Auf der Morgenstelle 28, DE-72076 Tubingen (Germany)
Tel. +49 7071 2972096, Fax +49 7071 2975843
E-Mail volkmar.braun@mikrobio.uni-tuebingen.de
Iron Transport Related to Virulence 233
Signaling and Gene Regulation
Russell W, Herwald H (eds); Concepts in Bacterial Virulence,
Contrib Microbiol. Basel, Kai'ger, 2005, vol 12, pp 234-254
Pathogenicity Islands and Their Role
in Bacterial Virulence and Survival
Bianca Hochhiit, Ulrich Dobrindt, Jorg Hacker
Institut fur molelaiJare Infektionsbiologie, Universitat Wiirzburg,
Wiirzburg, Germany
Infections caused by bacterial pathogens are still a significant problem in
modern medicine. Therefore, the identification of the factors that are related to
the infections and the understanding of the processes involved in the evolution
of pathogenic bacteria from their nonpathogenic progenitors is an important
subject of research. It has long been known that acquisition of virulence deter-
minants by horizontal gene transfer is one of the major driving forces in the
emergence and evolution of new pathogens [reviewed in 1^]. Furthermore, our
knowledge of the organization of the bacterial genome has greatly increased
within the last few years due to the availability of more than 120 completely
sequenced eubacterial genomes, including those of almost all pathogenic
bacteria, which has introduced a new area of pathogen research. It has become
evident that the typical bacterial genome consists of a conserved 'core gene
poor comprising genes that encode essential structural features and fundamental
metabolic pathways, and a 'flexible gene pool' that is more variable and encodes
functions only advantageous under specific growth conditions. Core genes are
characterized by a relatively homogenous G + C content and tliey are normally
encoded in stable regions of the chromosome that are conserved in their orga-
nization in closely related species. In contrast, the flexible gene pool comprises
variable regions of the chromosome and various mobile genetic elements such
as plasmids, bacteriophages, IS elements and transposons, conjugative trans-
posons, integrons and superintegrons that are transferred between different
organisms by the means of natural transformation, transduction or conjugation.
Many of the genes encoding toxins, adhesins, secretion systems, invasins or
other virulence-associated factors have been found to be encoded by mobile
genetic elements [overviews in 5^ 6]. Furthermore, the analysis of the genomes
of closely related species has revealed that the conserved chromosomal
backbone is interspersed with large regions that exhibit features of former
mobile genetic elements that have been termed genomic islands (GEls) [7, 8].
GEIs are broadly distributed and seem to be a common theme in most bacterial
genomes. Originally, such elements were identified in uropathogenic
Escherichia coii strains and were designated 'pathogenicity islands' (PAIs),
because they encoded key virulence factors of these bacteria [9], However,
when regions with similar features were also found in nonpathogenic bacteria
where they encoded other accessory functions, it was recognized that these
elements are not limited to bacterial pathogens, but are present in most bacteria
that have been analyzed. In this chapter, the role of GEIs in bacterial virulence
and survival will be discussed.
The Concept of GEIs
Features of GEIs
A comparative analysis of microbial genome sequences has revealed that
bacterial genomes can harbor variable and frequently significant amounts of
foreign DNA [3]. The genome size of different variants of the same species or
closely related species can vary by more than one megabase, which can be
accounted for by the acquisition of large blocks of DNA such as plasmids,
bacteriophages and GEIs, as well as by the acquisition of smaller pieces of foreign
DNA that have been described as 'islets'. Generally, GEIs represent distinct
pieces of DNA that have most of the following features in common suggesting
that they originate from events of lateral gene transfer [10].
(1) GEIs are present in the genomes of many bacteria but absent from the
genomes of closely related strains or species. (2) GEIs occupy relatively large
regions of the chromosome and can cover between 10 and more than 100 kb,
which may reflect the introduction of large pieces of DNA into a new host by
horizontal gene transfer Some strains also carry smaller pieces of DNA (1-1 Okb)
that have been termed 'genomic islets' in contrast to the larger islands. (3) GEIs
differ in their G + C content and their codon usage from that of the conserved
regions of the chromosome. (4) GEIs are often flanked by direct repeats that
may have been generated during integration of GEl-specific regions into the
host chromosome via site-specific recombination. (5) GEIs are frequently asso-
ciated with tRNA loci. The 3' end of tRNA genes have been recognized as pre-
ferred target sites for the integration of foreign DNA [reviewed in i 1]. (6) GEIs
often possess functional or cryptic genes coding for factors that are involved in
genetic mobility such as integrases, transposases, phage genes and origins of repli-
cation. Furthermore, GEIs normally do not represent homogenous elements but
Pathogenicity Islands 235
rather are generated by multistep processes including DNA rearrangements via
IS elements which is reflected by mosaic-like structures, (7) Some GEls tend to
be unstable DNA regions due to recombination between the flanking direct
repeats, between IS elements or between other regions of homologous sequences.
Generally, little is known about the mechanisms that have led to the acquisition
of GEIs and there are only few examples of inter- or intracellular mobilization
of GEls [12-16],
GEls are prevalently found in organisms that show frequent gene transfer
by bacteriophages and plasmids which are regarded as possible precursors of
GEls [8]. However, GEls have also been described in bacteria that exhibit
natural competence such as Helicobacter pylori. Neisseria gonorrhoeae and
Streptococcus pneumoniae, and that tend to introduce smaller pieces rather than
large regions of foreign DNA into their genome [17-19].
GEIs Contribute to Bacterial Fitness
Besides selfish genes such as genes involved in recombination and trans-
fer or modification of DNA, GEls often carry determinants that are beneficial
for their host bacterium in certain environments thereby increasing bacterial fit-
ness and consequently survival. GEls were divided into different subtypes
reflecting their contribution to the respective microbial lifestyle [8] (table 1),
GEls that encode virulence traits were defined as 'pathogenicity islands' (PAls).
The original definition of GEls was based on the characteristics of PAls in patho-
genic E. coll, but mtensive studies of the genome structure of bacterial pathogens
resulted in the identification of similar structures in many phylogenetically
unrelated organisms includmg gram-negative as well as gram-positive bacteria
(tables 2-3). Typical virulence factors encoded on PAls include toxins, adhesins
and fimbriae, factors involved in host cell entry, capsules, secretion systems
and iron uptake systems. Based on the broad distribution of PAls, it can be con-
cluded that they have contributed significantly to the evolution of virulent vari-
ants. However, the still growing number of genome sequences has made it clear
that GEIs are not restricted to pathogenic species. GEIs contributing to the
adaptation to specific growth conditions or the interaction with a eukaryotic
host organism have been described in environmental, commensal or symbiotic
bacteria and have been designated 'symbiosis islands', 'ecological islands' or
'resistance islands', according to the respective encoded functions. Relatively
well-studied examples of GEIs include the symbiosis island of Mesorhizobium
melioti that carries genes required for nitrogen fixation, whereas GEIs such as
the mec region enhance survival of staphylococci in hospitals where they have
to face antimicrobial substances. Other islands encode enzymes involved in the
degradation of phenolic compounds or for uptake and metabolism of certain
carbohydrates (table I ). Finally, a recently described island in Magnetospirillum
Hochhut/Dobrindt/Hacker 236
Table L Examples of GEIs
Subtype
of Designation
Organism
encoded
functions
Reference
PAJ
PAI [J536
Escherichia coli
P fimbriae,
a-hemolysin
26
PA]
HPl
Yersinia spp.
Iron uptake
21
ECl
HPI
Fecal Escherichia coli,
Salmonella enierica
subgroups 111 + IV
Iron uptake
23,24
FCl
Clwscr94
Salmonella
senfienberg
Sucrose uptake anc
metabolism
3
ECJ
etc e ement
Pseudomonas
putida
Degradation of
pheiiolic compounds
16
ECl
Vlagnetosome
Magnetospir ilium
Foimation of
20
is and
gryph is waldense
magnetosomes
REI
STIl
Salmonella enierica
DTI 04
Antibiotic resistance
76
\-\
mec ocus
Staphylococcus
aureus
Antibiotic resistance
77
SYI
Mesorhizobium
melioti
Nitrogen fixation
78
SYl
Sinorhizobiiim
fredii
Type 111 secretion
system
79
ECJ =
- Ego ogical is and;
REl - resistance is and; SYI - symbiosis is and.
gryphiswaldense is required for the formation of magnetosomes and the char-
acteristic magnetotactic phenotype of these bacteria [20]. Interestingly, some
GEJs have been assigned to different subtypes depending on the habitat and
genetic background of the respective bacterium. An example is the so-called
'high pathogenicity island' (HPI) that was originally found in derivatives of
Yersinia spp. exhibiting increased virulence in mice [21]. As this island and the
associated iron uptake system have been found in many pathogenic and non-
pathogenic enterobacteria [22-24], HPI can be considered as a 'broad host
range GEI'. Whereas it contributes to virulence in pathogenic variants and has
therefore been defined as a PAI^ it enhances the capability of fecal E. coli,
Klebsiella spp. and nonpathogenic Salmonella enierica spp. to grow under iron-
limiting conditions and has therefore been defined as an 'ecological island' in
these strains.
Pathogenicity Islands
237
Table 2, PAIs of pathogenic Enterobacteriaceae
Organism
Designation Encoded traits
Size, kb Junction
Integrase Insertion site Reference
o
:x
c
O
o
cr
'-I
5"
a:
&>
o
Escherichia colt 536
(UPEC)
Escherichia coli 536
(UPEC)
Escherichia coli 536
(UPEC)
Escherichia coli 536
(UPEC)
Escherichia coli J96
(UPEC)
Escherichia coll J96
(UPEC)
Escherichia coli
CFT073 (UPEC)
Escherichia coli
CFT073 (UPEC)
Escherichia coli
AL862
Escherichia coli
Ec222 (APEC)
Escherichia coli C5
PAII
536
PAIH
536
PAIIII
536
PAIV
536
PAII
J96
PAin
J96
PAH
CFT073
PA I IIcn'07;
PAI
ALS62
VAT^PAl
PAII
C5
a-Hemolysin, put,
adhesions
a-Hemolysio, put,
P fimbriae (Prf),
adhesion
S fimbriae (Sfal), iro
siderophore system,
hemoglobin protease
K15 capsule
ot-Hemolysia,
P fimbriae (Pap)
a-HemolySLQ,
P fimbriae (Prs),
cytotoxic necrotizing
factor L (CNFl)
a-Hemolysin,
P fimbriae (Pap)
P fimbriae (Pap),
iron acquisition
afa% adhesin
Vat autotransporter
ct-Hemolysin, P fimbriae
(Prs), cytotoxic
necrotizing factor 1
(CNFl), heat-resistant
hemagglutinin
75.8
102
76.8
>75
>170
110
58
71
61
22
100
DR 16 bp
DR 18 bp
DR 46 bp
DR 23 bp
DR9bp
NoDR
DR 14 bp
or DR 136 bp
(imperfect)
No
DR 18 bp
CP4-like selC
(cryptic?)
P4-liJce leiiX
Sfx-like
P4-Iike
7
DR 135 bp P4-lLke
P4-like
P4-like
P4-like
P4-like
thrW
pheV
pheV
pheU
pheV
pheU
pheU
pheV
Sfil-like ihrW/yagV
(truncated)
? leuX
26
26
26
Unpublished
32
32
29
29
80
81
82
Escherichia coli
E2348/69 (EPEC)
FspC-PAI
Autotransporter/
eaterotoxin
Escherichia coli
LEE
^ ype in secretion,
F2348/69 (EPEC)
invasion
o
JO
Escherichia coli
EDL933 (EHEC)
LEE
lype III secretion,
invasion
3
Escherichia coli
RW 1374 (SI EC)
LEE
Type III secretion,
invasion, parts of the
she PAI (S. /Jexneri 2a)
CI.
Escherichia coli
LEE
Type HI secretion,
RDEC-1 (REPEC)
invasion, put. adhesion
Escherichia coli
83/39 (RFPEC)
LEE
Type 111 secretion,
invasion, put. adhesin,
enterotoxin
Escherichia coli
LEE
lype 111 secretion,
84/110-1 (REPEC)
invasion
Escherichia coli
FPEC
Diffuse adherence
135/12
Afa-PAI
adhesin
Escherichia coli
(FHFC)
LPA
Serine protease (Espl),
vitamin R|2 1'^ceptor
(BtuB), adhesion
Escherichia coli
IPAl'l
Invasion
10407 (ETEC)
Pathogenic
HPI
Yersiniabactin
Escherichia coli,
nonpathogenic
Salmonella
(PAi rv53o)
synthesis, transport
Yersinia enterocolytica
HPI
Yersiniabactin
to
Ye8081
synthesis, transport
^o
Yersinia
HPI
Yersiniabactin
pseudotuberculosis
synthesis, transport
Yersinia peslis
HPI (pgm
locus)
Yersiniabactin synthesis,
transport, herriin uptake
15.2
35
43
>80
59.5
85
>11
33
46
31^3
45
36
102
NoDR
NoDR
NoDR
9
NoDR
No DR
DR 23 bp
(imperfect)
9
No DR
DR 25 bp
NoDR
NoDR
DR 17 bp
ISlOO
DR 17 bp
No
No
CP4-like
No
P4-like
P4-like
P4-like
P4-like
CP4-l)ke
Yes
P4-like
P4-)ike
P4-like
P4-)ike
ssrA
selC
selC
pheV
pheU
pheU
pheV
pheV
selC
selC
asnT
asnT
asnT, U, W
asnT
83
84
27
85
37
61
61
86
87
88
23,24
21
12
89
Table 2. (continued)
Organism
Designation Encoded traits
Size, kb Junction
Integrase Insertion site Reference
Sh i gel la jlexn eri
SH[-1
{she)
Enterotoxin (Set),
protease (Pic)
X
o
o
nr
Shigella flexneri
SHl-2
Aerobactin synthesis,
CO icin V uniiiunity
c
o
a.
Shigella flexneri
SRL
Ferric diciLiate
transport, antibiotic
resistances
£0
Shigella flexneri
Shi-0
Genes involved in
O
serotype conversion
^^
Salmonella enterica
SPl-1
Type III secretion,
sv yphimuriiim
invasion into epithe ial
ce s, apoptosis
Salmonella enterica
SPI-2
"ype III secretion,
invasion into
monocytes
Salmonella enterica
SPl-3
Invasion, survival
in macrophages
Salmonella enterica
SPl-4
Invasion, siu'viva
in monocytes
Salmonella enterica
SPI-5
SPI-1 effector
protein (SopB)
Salmonella enterica
sv iyphi
SPl-7
Vi exopolysaccharide
production
Erwinia amylovora
Ea321
hrp PAl
Type III secretion,
effectors
46.6
23-30
66
11
40
40
17
25
7
134
60
DR 22 bp
(imperfect)
DR 14 bp
NoDR
NoDR
NoDR
NoDR
NoDR
NoDR
DR 55 bp
P4-like
CP4-]ike
Yes
Yes
No
No
No
No
No
Yes
Yes
pheV
selC
serX
ihrW
Between
JhlAlmuiS
valV
selC
Putative
tRNA gene
serT
pheU
pheV
68
90,91
92
93
41
43
44
45
46,94
47
56
DR ^ Direct repeat; APEC ~ avian pathogenic £. coli\ REPEC = rabbit enteropathogenic E. coli\ ETEC = enterotoxigenic E. coli\
LPA = locus of proteolysis activity; SRL = Shigella resistance locus; put, = putative; STEC = Shiga toxin-producing E. cod.
Table 3. Examples of PAIs of gram-positive bacteria
o
Organism
Designation
Encoded traits
Size, kb Junction Integrase
Insertion site Reference
Staphylococcus
aureus RM4282
Staphylococcus
aureus RN3984
Staphylococcus
aureus COL
Staphylococcus
aureus RF122
Staphylococcus
aureus T^ 1 1 4
SaPU
SaPI2
SaPr3
SaPIbov
etd PAI
Enterococcus faecaiis Enterococcus
faecalis PAI
Toxic shock syndrome
toxin-] (TSST-1)
Toxic shock syndrome
toxin- 1 (TSST-1)
Enterotoxin
serotypes B, K, Q
Toxic shock syndrome toxin- 1
(TSST-1), enterotoxin C
Exfoliative toxin D,
glutamyl endopeptidase
Cytolysin, surface proteiji
(Esp), aggregation
substance
Clostridium difficile
Pathogenicity
Enterotoxin (TcdA),
locus (PaLoc)
cytotoxin (TcdB)
Streptococcus
PPII
Iron uptake system
pneumoniae
Pathogenic Listeria
LIPI-1
PrfA-dependent virulence
gene cluster (phospholipases,
listerio ysin, ActA)
Listeria ivanovii
LIPI-2
Internalins,
sphingomye inase C
15
16
16
15
19
27
22
DR 1 7 bp Yes
DR 1 7 bp Yes
DR 74 bp ?
DR 5 bp No
150 DR 10 bp Yes
NoDR
NoDR
NoDR
No
No
No
Near tyrB
Near trp
gene cluster
■7
LQtergenic
Intergenic
No DR Recombinase ye/A
14
14
95
96
Intergenic 97
60
Intergenic 58
18
Lntergenic 57
Intergenic 57
PAIS Contribute to Virulence of Bacterial Pathogens
PAIs of Enterobacterial Pathogens
Most of the characterized GEIs so far have been found m members of the
Enterobacteriaceae (table 2), which may in part be explained by the fact that
this family has been intensively studied, but also mdicates that PAJs have played
a pivotal role in the evolution of enterobacterial pathogens. E. coli normally
lives as a harmless commensal in the bowels of humans or animaJs, but some
variants have the potential to cause gastrointestinal as well as extraintestinal
infections [25]. Pathogenic E. coli can be linked to a variety of quite diverse
symptoms that include enteric diseases that range from cholera-like diarrhea to
severe dysentery and hemorrhagic colitis, cystitis or pyelonephritis, septicemia
and meningitis. Based on their mode of pathogenesis, virulent E. coli have been
classified into different pathotypes such as uropathogenic E. coli (UPEC),
enteropathogenic E. coli (EPEC), enterohemorrhagic E. coli (EHEC), entero-
toxigenic E. coli or enteroinvasive E. coli and the pathogenetically related Shigella
species. They are characterized by the expression of specific virulence factors
that enable them to exploit new niches in their host and to disrupt the normal
host physiology. In pathogenic E. coli and Shigella spp., many of these key
virulence factors are encoded on PAJs, which underlines their importance in the
formation of the various pathotypes. The diversity of diseases that are associated
with E. coli infections is also reflected by the structural and functional varieties
in PAIs (see table 2, fig. 1). Whereas some PAIs are widely distributed among
different enterobacterial species [e.g. HPI and the Jocus of enterocyte efface-
ment (LEE)], others are closely related to a specific pathotype. Furthermore,
most strains carry multiple PAIs that can cover more than 5% of the genome.
For example, at least five PAIs (PAI \^2t to PAI V535) have been identified in the
chromosome of the uropathogenic isolate E. coli 536 (table 2) [26].
Besides PAIs that have been identified on the basis of functional studies, a
still increasing number of putative GEIs have been detected in the completely
sequenced genomes of pathogenic E. coli and Shigella flexneri. However, it has
yet to be investigated whether the encoded factors contribute to virulence or fit-
ness of the respective pathogen [27-29]. Most PAIs of pathogenic E. coli exhibit
a mosaic-like modular structure and although some PAIs show similarities in
respect to the presence and linkage of certain virulence determinants, there is
also a great variability in regard to size, organization and chromosomal local-
ization even among strains of the same patho- or serotype [26, 30]. Interestingly,
some tRNA genes seem to represent hot spots for the integration of foreign
DNA including PAIs. The majority of PAIs in enterobacteria is linked to either
selC, the gene for a selenocysteine-specific tRNA, or one of two genes for a
phenylalanine-tRNA, p/?eP^ or p/?ei7. Whereas the associated integrase genes
Hochhut/Dobrindt/Hacker 242
^
\A^
seIC int
-►
DR
» I I
F17-like fimbriae?
CS12-like fimbriae? a-Hemolysin
m
my
PAN
536
DR
m^mm^mrm
(\
:i
sefC int Secreted Intimin Tir Type III secretion system
Prophage 933L Proteins
^
se/C int
^
selC int
sefC
■_^»<hHOH
Protease B12receptor
Adhesin
Aerobactin synthesis
■-/
Survival in macrophages
EDL933
LPA
SHI-2
SPI-3
Fig. i. Examples of jvWC-associated PAls of Enterobacteriaceae. The orgardzarion of
^^/C-associated PAls is shown. Known or putative virulence genes are shown as gray arrows
and their (predicted) function is given, ORFs with similarity to transposases are indicated by
hatched arrows. Also shown are genes for a CP4-Hke integrase (int). With the exception of
SPI-3 from S, enterica, highly similar genes are present in all islands. Finally, direct repeat
sequences in PAI ]^26 ^nd the prophage sequence present m EPEC EDL933 are also shown.
are well conserved and seem to be specific for the linked tRNA gene, the further
structural organization and nucleotide sequence of islands that are integrated
into identical sites in their respective bacterial host are not necessarily closely
related but rather encode functions that determine the lifestyle and pathotype of
the bacterium (fig. 1).
The best-studied PAls of £. coli belong to the LEE island and PAls of
uropathogenic strains. One important trait of UPEC isolates is the presence of
adhesins that enables them to adhere to uroepithelial cells [31], Besides type I
fimbriae, PAls often carry genes that are specific for P fimbriae that bind to the
Gal a(l-4)Gal moieties of glycoproteins and S-fimbrial adhesions.
Furthermore, UPEC produce the pore-forming toxin oc-hemolysin, several iron
uptake systems, as well as capsules that function as a protection against the host
Pathogenicity Islands
243
defense. All these traits are encoded by PAJs that are similar to each other, but
not identical in the different isolates [26, 30, 32]. Interestingly, adhesin and
toxin gene clusters are often linked with each other, suggesting a coevolution of
these factors [33].
The LEE island encodes the outer membrane adhesion protein intimin, a
type III secretion system and several secreted effector proteins. One of these
secreted proteins is Tir (translocated intimin receptor) that is inserted into the
eukaryotic host cell membrane where it serves as a receptor for intimin to mediate
binding of the bacterium to the host cell [34]. Strains carrying the LEE locus
cause characteristic attaching and effacing lesions. When LEE is transferred to
E. coli K-12, it exhibits the same phenotype which indicates the potential of
LEE to transform a nonpathogenic strain into a more virulent variant [35]. The
LEE island has been identified in E. coli isolates of humans and many animals
as well as in Cilrobacter wdenthim [36]. Sunilar to PAI Igj^ of UPEC strain
536, the LEE locus is located next to selC in some EHEC and EPEC strains
(fig. 1), but can also be associated w<f'\X\\ pheV or pheil in other isolates. When
LEE sequences of EHEC, EPEC and the rabbit isolate RDEC-1 were compared,
it became evident that the esc genes encoding the secretion apparatus were
highly conserved, whereas the other genes were less similar than it would have
been expected from clonal lineages. This may reflect the differences in interactions
with the specific host but also suggests that the LEE locus has been acquired
more than once during the evolution of £'. coli [36, 37].
Similar to pathogenic E. coli, PAls have played a fundamental role in the
evolution of the genus Salmonella. Five PAJs (SPI-1 to SPL5) have been iden-
tified in a range of serovars of 5'. enterica and were characterized in more detail.
Furthermore, additional chromosomal regions that exhibit features of GEls
have been found in the genomic sequences of serovars Typhimurium and Typhi
[38, 39]. SPI-1 is regarded as a very ancient island which was already intro-
duced into the genome of a common ancestor of S. enterica and Salmonella
bongon [40]. Consequently, it has become a stable part of the chromosome and
lacks most of the typical traits of PAls. Similar to the LEE locus, SPl-1 encodes
a type III secretion system including the components of the secretion appara-
tus, effector proteins, specific chaperones, and virulence gene regulators [41].
SPl-1 mediates invasion of host cells and induction of macrophage apoptosis
[reviewed in 42]. SPI-2 (located next to valV) encodes a second type III system
that is required for systemic infections and replication within macrophages
[43], Similarly, SPI-3 and SPl-4 have also been shown to be involved in intra-
macrophage survival [44, 45]. As for PAI I535 and LEE, SPI-3 is associated with
selC, but seems to have lost the corresponding integrase gene (fig. 1). The
mglBC operon of SPI-3 is not only required for replication m macrophages, but
also for in vitro growth under low Mg^"^ conditions. SPI-5 encodes an effector
Hochhut/Dobrindt/Hacker 244
protein (SopB) that acts as a substrate for the SPI-1 -encoded secretion
apparatus. This is an example of a tight connection between different PATs
of one strain [46]. Finally, the so-called SPI-7 is only present in a subset of
S. enterica isolates including S. enterica serovar Typhi CT18 that produce the
Vi capsular polysaccharide. The corresponding genes reside on a 134-kb island
that seems to have evolved from several independent insertion events and
carries a region with similarity to the pilus genes of the conjugative plasmid
R46 [47] .
PA Is of Other Gram-Negative Pathogens
Besides in Enterobacteriaceae, PAJs are also present in the genomes of
several other gram-negative bacterial pathogens and can contribute significantly
to the viiulence potential of their host bacterium. In the case of Vibrio cholerae,
two PAIs have been described [48, 49]. The first Vibrio pathogenicity island
VPI-1 is present in all epidemic and pandemic strains, but absent in most
nonpathogenic strains. The 39.5-kb island encodes a type IV pilus, the toxin-
coregulated pilus (TCP) that functions as an essential intestinal colonization
factor in humans and animal models [50]. Besides its role as an adhesion factor,
TCP also functions as the receptor for the cholera toxin encoding filamentous
phage CTXcp [51]. Therefore, acquisition of VPT-1 seems to be a prerequisite
for the emergence of highly pathogenic V. cholerae variants. Furthermore, VPI- 1
is linked to cholera toxin production because toxT, the gene for a transcriptional
activator of the AraC family, also resides on the island. ToxT is involved in both
activation of the top gene cluster and the toxin genes. A second pathogenicity
island, VPI-2, has recently been found to be present in the majority of toxigenic
(CTXcl)-positive) strains, but absent from nontoxigenic isolates [49]. VPI-2
encodes a neuramidase and a putative metabolic pathway for amino sugars. The
role of these determinants for either virulence or fitness of V. cholerae has yet
to be elucidated. Similarly, the impact of a putative pathogenicity island in the
genome of Legionella pneumophila serogroup 1 Philadelphia- 1 (LpPI-I) that
carries genes for a type IV secretion system is still unclear [52]. In contrast, the
role of the cag island in the virulence of//, pylori has been intensively studied.
This island is only present in H. pylori strains that are associated with severe
forms of gastroduodenal disease (type 1 strains) suggesting that acquisition of
this region has been an important event in the evolution of more virulent forms
of//, pylori [19]. Like LpPI-1, the cag island encodes a type IV secretion sys-
tem that resembles other toxin secretion systems as well as transport systems
that are required for transfer of DNA. It has been shown that Cag A is delivered
by the island-encoded secretion apparatus into host cells where it induces
cellular growth changes that are specific for infections with type I strains of
//. pylori [53].
Pathogenicity Islands 245
Finally, PAJs have also been identified in animal and plant pathogens;
however, they have not been as extensively studied as in human pathogens. Tn
Dichelobacter nodosus, the causative agent of foot rot in sheep, two chromo-
somal regions with PAI-typical features have been described [54, 55]. However,
their role in virulence is still unclear.
Similar to many enterobacterial pathogens of humans and animals, several
gram-negative plant pathogens also use type IIJ secretion systems to inject
effector proteins into plant cells that induce a plant tissue defense line includ-
ing programmed cell death. The corresponding genes have been designated hrp
(hypersensitivity response) or hrc (hypersensitivity response and conserved)
and form PAJ-like regions that can be located either on the chromosome or on
plasmids [reviewed in 56]. This reveals common themes in virulence of human,
animal or plant pathogens.
PA Is in Gram-Positive Pathogens
Chromosomal regions with the typical features of PAIs in gram-negative
bacteria are less frequently found in gram-positive pathogens. However, a few
regions that exhibit some of the characteristics of PAIs have also been identi-
fied in gram-positive bacteria (table 3). For example, virulence gene clusters in
Listeria spp. or Clostridium difficile are not flanked by direct repeats or linked
to mobility genes, but have been described as PAI-like elements [57, 58]. In
S. pneumoniae, the characterization of an iron uptake system that is required for
full vu-ulence revealed that the corresponding genes for an ABC transporter are
linked to a recombinase gene in a 27-kb region designated as PPll [18].
Furthermore, GEls seem to be crucial elements for genetic exchange in staphylo-
cocci. Besides the above-mentioned methicillin resistance islands, staphylo-
coccal pathogenicity islands (SaPIs) contribute to horizontal transmission of
resistance and toxin genes in Staphylococciis aureus [reviewed in 59]. PAIs of
S. aureus share many of the criteria of PAIs of enterobacteria and are mobiliz-
able by phage transducdon (see also below). SaPIs of human as well as animal
isolates have been distinguished by the different toxin types they encode such
as superanligen LoxJns (e.g. LoxJc shock syndrome toxin), exotoxins and enlero-
toxins. Finally, a large chromosomal island has recently been identified in the
genome of an Enterococcus faecalis isolate that caused an infectious outbreak
[60]. This island encompasses more than 150kb, is flanked by direct repeat
sequences and exhibits a lower G + C content than the rest of the genome. It
encodes several putative virulence factors including a cytolysin and a surface
protein that contributes to the colonization of the bladder. A closer investigation
o^E. faecalis isolates revealed that structural variations of the island occur with
relatively high frequencies thereby enabling strains harboring this island to
modulate their virulence potential.
Hochhut/Dobrindt/Hacker 246
PAIS and Genome Plasticity
The relatively high genetic flexibility of a bacterium is thought to facilitate
the access to new ecological niches and may represent an advantage over organ-
isms with less flexible genomes. As already discussed, PAIs have contributed to
the long-term evolution of many bacterial pathogens, but beyond that, they may
also be involved in relatively recent changes within the genetic information of
an organism thereby modulating the virulence potential of a strain.
Intact or rudimentary mobility genes on PAIs give evidence that they
have been acquired by means of horizontal gene transfer such as transduction or
conjugation; however, so far only little is known about the actual mechanisms
that have been involved. It has been assumed that integration into the recipient's
chromosome, at least in some cases, was mediated by site-specific recombina-
tion similar to the integration mechanism of several bacteriophages. This is
supported by the findings that some island-encoded integrases still have the
potential to carry out these reactions [61, 62]. Most PAIs have undergone modi-
fications such as deletions and mutations within the direct repeat sequences or
mobility genes (fig, 2), Often, these processes have resulted in a relatively sta-
ble integration of PAIs in the bacterial chromosome which has been designated
as 'homing'. Examples of PAIs that have become locked in the chromosome are
some of the islands specific to S. enterica (also see above). This may at least
partly reflect the fact that the encoded traits have become indispensable for the
host bacterium and are becoming part of the core chromosome.
In contrast to such presumably very ancient elements, other islands are still
mobilizable and can be transferred from one bacterium to another, at least in
laboratory settings. VPl-l has been transmitted among V choleme strains by a
transducing vibriophage, CP-Tl [15] and there is also one report that VPl-1
itself may correspond to a functional prophage [63]. Furthermore, almost iden-
tical regions to VPI have been found in the chromosome of some Vibrio mimicus
isolates, which suggests a relatively recent gene transfer between these two
species [64], General transduction also plays a role for transfer of SaPlI and
related islands in S. aureus. Even though these PAIs are not self-transferable,
they can be propagated by staphylococcal phages such as c(>80a and c|>13 [14]
in a mechanism reminiscent of the relationship between the defective coli-
phage P4 and its helper phage P2 [65]. Besides mobilization by transduction,
conjugation may have played a role for the acquisition of PAIs and it is not
unlikely that PAIs may have been derived from conjugative plasmids. In the
last few years a number of elements termed 'integrating conjugative elements
(ICEs)' or 'conjugative transposons' have been reported to be normally
integrated in the chromosome, but can excise in a precise manner to be subse-
quently transferred by close cell-to-cell contact [66, 67]. Similar to PAIs, these
Pathogenicity Islands 247
E
virC
Horizontal gene transfer
tRNAgene
I I ! I I rT~r
DR ini
virA virB virC
e
I I— 1 1 I
IS
Acquisition of new PAIs
S E
PAH
PAI2
Increased virulence potential
Chronnosome
Recombination, integration
Chromosome H- pre-PAl
mob DR
Deletions, point mutations,
DNA rearrangements
Chromosome + PAI
Amob
Deletion of PAIs/
parts of PAIs
E
Decreased virulence potential
Fig. 2. PAJs and genome plasticity. Evolution of PAIs is based on acquisition of novel
DNA by horizontal gene transfer followed by point mutations, recombination and deletion
events that can render the PAl inunobile. The viruJence potential of a bacterium can subse-
quently be either increased by introduction of new PAIs or decreased by partly or complete
deletions of PAIs. int ^ Integrase gene; wr ^ virulence-associated gene; mob ^ mobility
genes; Amo6 = truncated mobility genes; DR = direct repeat sequences.
elements encode site-specific recombinases and lack the ability to replicate
autonomously.
Finally, several islands seem no longer mobilizable, but have a tendency to
delete from the chromosome either as a complete unit or in parts [12, 19, 61,
68, 69]. Precise deletion from the chromosome has been observed for PAIs of
the UPEC isolate 536 and requires functional integrase genes [70] (our unpubl.
results). Furthermore^ an increase of deletion incidences was observed under
certain environmental conditions [71]. It has been speculated that loss of virulence
determinants may play a crucial role during the transition fi^om an acute state of
disease to chronic infections [8, 72]; therefore it v^ill be interesting to further
Hochhut/Dobrindt/Hacker
248
investigate how the content of virulence genes can be modulated by environ-
mental, bacterial or eukaryotic host factors.
Similarly, rearrangements or deletions within islands are often mediated
by coresiding transposons or IS elements (fig. 2). This is especially true for
H. pylori where an ongoing adaptation between bacterium and host based on
1^605 mediated DNA rearrangements within the cag island (see also PAIs of
Other Gram-Negative Pathogens) has been described [reviewed in 73]. Full
virulence of//, pylori depends on an intact cag island, whereas deletions within
the island render the bacterium less pathogenic. The interaction of//, pylori
with epithelial cells results in an elevated production of cytokines such as
interleukin-8 (IL-8). This induction of IL-8 production correlates with the
presence of a complete cag island, whereas //. pylori strains carrying only parts
of the island induce lL-8 at significantly lower levels [74].
Conclusions
GEIs contribute to virulence and survival of pathogens in several ways.
First, the acquisition of GEIs has been described as 'evolution in quantum
leaps', because they often carry more than one virulence or fitness determinant
[75]. These GEI-encoded factors enable the bacterium to colonize novel niches
in the eukaryotic host and facilitate the adaptation to the respective environ-
mental conditions. This increase of fitness gives an advantage over coresiding
bacteria. Furthermore, the genome of many bacterial pathogens contains more
than one GEI that encode important virulence factors, thereby determining the
capability to cause disease. In addition, to ensure coordinated expression of
virulence or virulence-related genes that are located on GEIs, a tight connection
to regulatory networks of the bacterium has evolved, as well as a link of island-
encoded regulators to genes encoded elsewhere in the genome. Finally, an
ongoing mobilization and transfer of GEIs as well as reorganization, partial or
complete deletion of existing GEIs affect long-term (macro-) as well as short-term
(micro-) evolution of pathogenic bacteria.
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Jorg Hacker
Institut fijr molekulare Infektionsbiologie
Rontgenring 1 1, DE-97070 Wiirzburg (Germany)
Tel. +49 931 312575, Fax +49 931 312578, E-Mail j\hacker@maiLuni-wuerzburg.de
Hochhut/Dobrindt/Hacker 254
Signaling and Gene Regulation
Russell W, Herwald H (eds): Concepts in Bacterial Virulence.!
Contrib Microbiol. Basel, Karger, 2005, vol 12, pp 255-271
Horizontal and Vertical Gene Transfer
The Life History of Pathogens
Jejfrey G. Lawrence
Pittsburgh Bacteriophage Institute and Department of Biological Sciences,
University of Pittsburgh, Pittsburgh, Pa., USA
Viewpoints regarding the evolution of pathogenic bacteria have themselves
evolved over the past two decades. Although it is perhaps extreme to suggest
different teleological camps have been established, it is fair to say that opinions
regarding the evolution of pathogens are varied, and the strength of different
points of view have waxed and waned. Initially, many viewed pathogenic bac-
teria as being specialized, highly derived bacteria, which evolved complex and
intimate associations with their hosts. In this way, special evolutionary mecha-
nisms were perhaps responsible for the origin or persistence of pathogens.
Gradually, a viewpoint that every microorganism was adapted to a particular
niche was widely accepted, and pathogenicity represented just another bacterial
lifestyle; therefore, no special evolutionary forces were at play. The evolution
of well-studied pathogens could even be used as models for how other bacteria
adapted to their environment.
Somewhat surprisingly, perhaps, data collected in the 'genomic era' have
brought opinion back to the view that the evolution of pathogens indeed may
encompass evolutionary paths typically not experienced by nonpathogenic bac-
teria. That is, the association of pathogens with particular hosts results in
smaller effective population sizes, low genetic diversity, infrequent recombuia-
tion and other factors influencing their evolution as dictated by their population
genetics. As a result, pathogens would not serve as good models for the evolution
of nonpathogenic bacteria that do not share these population genetic constramts.
As discussed below, both viewpoints are perhaps true, when applied to the differ-
ent stages of pathogen evolution. At the heart of the difference between the stages
of pathogen evolution are the relative roles of gene acquisition via horizontal
gene exchange versus gene loss (genome degradation). Rather than representing
different paths of pathogen creation or modification, these modes of genomic
evolution likely represent a continuum or pathway along which a single lineage
may travel.
Early Examples of Horizontal Gene Transfer
Horizontal gene transfer (HGT) is defined as the transfer of genetic material
between bacterial cells uncoupled with cell division [1-3]. In contrast, vertical
inheritance is the transmission of genetic material from mother cell to daughter
cell during cell division. Most often, HGT refers to gene transfer across large
phylogenetic distances (that is, between otherwise unrelated organisms),
whereby genes are integrated into a replicon by illegitimate means. On occasion,
HGT is used to denote allelic exchange among closely related bacterial strains
where integration occurs via homologous recombination; herein that process
will be referred to as ' recombination \
Some of the eariiest examples of HGT involved the transfer of antibiotic
resistance genes [4], many times among pathogens, which were often facilitated
by the localization of these genes on plasmids. Here, virulent strains of bacteria
could acquire resistance to antibiotics at alarmmgly high rates, ones inconsistent
with the evolution of such a complex trait from preexisting genetic material via
random point mutation. Further investigation revealed that the bacteria had
obtained a gene conferring antibiotic resistance from another bacterium. This
observation reinforced the idea that the strong selection imposed by the adoption
of a pathogenic lifestyle allowed investigators to see otherwise rare evolutionary
events, like horizontal gene exchange. As a result^ this process of gene exchange
was not considered to be a potent evolutionary force. A cogent model of bacterial
evolution relied on the systematic periodic selection of random mutations arising
in the population [5], The exchange of genes among bacterial strains was not
considered to be important until many years later [6-8], and the potential high
rates of occurrence were not appreciated until rather recently [9, 10].
Pathogenicity Islands
The special role of HGT in pathogen evolution was reinforced as the|
sequences of bacterial genes becanue abundant [11], Early analyses suggested
that the genes encoding virulence fionctions in many pathogens were somehow
different from other genes in the chromosome; differences often included changes
in overall nucleotide composition (%GC), codon usage bias, association with
mobile genetic elements, and association with tRNA genes (frequent sites of
Lawrence
256
bacteriophage integration). The term 'pathogenicity island' was coined to denote
the distinct evolutionary histories reflected by these bits of genetic material, histo-
ries that were not shared with the remaining genes in the chromosomes [12-17].
Almost uniformly, genes encoding virulence factors mapped to pathogenicity
islands, thus unplicating HGT in virtually every step of pathogen evolution.
Functions encoded by pathogenicity islands included those required for adhesion
and invasion [12, 13], type III secretion systems for altering host cell metabolism
[18-20], toxin production [21-23] and a host of metabolic capabilities including
the acquisition of phosphate and iron at low concentrations [24].
ing Pathogens by Gene Transfer
While the role of HGT in pathogen evolution was compelling, it is not the
only route to pathogen creation. In some cases, pathogens are merely bacteria
found in the wrong place at the wrong time. For example, Legionella persists in
macrophages using mechanisms that evolved to allow it to passage through its
more common Entamoeba host [25, 26]; this strategy may be common among
pathogens [27, 28]. Clostridium tetanus is just a soil anaerobe delivered unexpect-
edly hito the human body via a puncture wound [29]; certainly some of the more
unexpected results of tetanus infection [e.g. autism-like symptoms, 30] are not
considered traits resulting from strong selection for particular vuTjIence functions.
Even the well -characterized pathogen Salmonella enterica has been implicated in
causing disease in nematodes, which may represent their primary host [3 I].
Yet other times, pathogens inhabit a somewhat different ecological niche
than do their nonpathogenic ancestors. For example, the ancestor of 5. enterica
was likely an intestinal-dwelling bacterium which never invaded epithelial cells.
Here, new physiological capabilities are required for the pathogen to succeed in
its new environment, and acquisition of fully functional genes from other
pathogens is an effective strategy in making this transition [32-35]. Analyses of
many genomes have demonstrated that similar genes are found in diverse
organisms, and that their evolutionary histories reflect frequent travel among
genomes [1, 36-38]. As a whole, one may view pathogen evolution as the gain
of genes via HGT coupled with the loss of genes (necessary from a population
genetic point of view, as discussed below), which changes the ecological capa-
bilities of a bacterial ta^on (fig. 1).
The power of HGT ui creating pathogens from nonpathogens is strikingly
demonstrated in the examination of the complete genome sequences of four
strains of Escherichia coli, including one benign laboratory straui [39], two
pathogenic strains of £". coli [23, 40] and the phylogenetically very closely related
strain Shigella flexneri [41] (despite being placed in a different genus, gene
Horizontal and Vertical Gene Transfer 257
oidEFG
oi<i)(m
o
y
2
Gene loss
newKLM
Ancestral DNA retained in
derived lineage
QldRST
oldABC
newNOP
newHJK
Chromosome of
ancestral lineage
Introduced
DNA
DNA not
maintained
newUVW
Chromosome of
derived lineage
Fig. L The role of HGT in changing a bacterial species. Here, an ancestral taxon gains
(black genes and arrows) and loses (gray genes and arrows) both chromosomal and episomal
genes. Both classes of events alter the phenotypic capabilities of the bacterium, and both
classes of events may increase the pathogenicity of the bacterium (see text).
EDL933(0157:H7)
enterohemorrhagic
Fig, 2. Genetic differences between three completely sequenced isolates off, coli. the
nonpathogenic strain MG1655 (hold line), the uropathogenic strain CFT073 (gray line), and the
enterohemorrhagic strain EDL933 (thin line). The number of genes shared among genomes, or
unique to a genome, is shown in the appropriate location in the Venn diagram [after 40].
sequences group Shigella within the E. coli complex [42, 43]). Even though high
sequence identity among genes shared among the four strains place them all in
the same species [43], Jess than 40% of the collective gene pooJ among the three
named strains of £. coli is shared (fig. 2), and nearly 47% of the genes are unique
to one of the three taxa [40]. Each strain has numerous genes found only in that
genome, the lion's share found in the two disparately pathogenic E. coli strains
Lawrence
258
(fig. 2). In contrast, the Shigella genome has many pseudogenes and prophages,
but fewer unique genes, implicating large-scale gene transfer as a factor in its
evolution to a lesser degree [41], Indeed the role of gene loss (the cadA and ompT
genes) in maintaining virulence has been noted in Shigella [44, 45]. Taken
together, the variation among these strains shows that gene transfer can act
quickly to introduce genes that allow for dramatic changes in lifestyle, but, as
discussed further below, it is not the only route.
Generalized Lifestyle Alteration
Examination of variation among natural isolates of £. coli shows that
huge dynamics in gene content are evident even among these nonpathogenic
isolates [46^8]. Therefore, the lessons imparted by pathogenicity islands are
extensible to the examination of other, nonpathogenic bacteria. If pathogenicity
islands can allow for rapid adoption of the pathogenic lifestyle, one could posit
that the introduction of other genes would allow for similarly effective
invasion of nonpathogenic niches [32-34]. Examination of the genomes of
numerous bacteria shows that nonpathogenic bacteria have a great deal of DNA
that is 'atypical' with respect to the majority of genes in the genome and could
have been introduced recently by HGT [49]. As seen in figure 3, genomes of
both pathogenic and nonpathogenic bacteria show abundant signs of recent
gene acquisition.
The methods employed to generate figure 3 rely on a simple premise: genes
introduced into a genome from a donor chromosome all share one characteristic;
they did not evolve for long periods of time in their current genomic context.
Each organism experiences a unique set of 'directional' mutation pressures
[50-52] which impart signature patterns of nucleotide composition [53-55],
codon usage bias [56], nucleotide strand bias [57-59], dinucleotide signatures
[60—62] and patterns detected by Markov chain models [63]. In effect, genes
evolving in the same genome Mook alike' due to the mutational proclivities of the
DNA polymerase, the composition of the nucleotide pools during replication, the
nature and efficiencies of the DNA mismatch-repair systems, abundance of tRNA
species and other factors. As a result, 'atypical' genes are often interpreted as
having evolved in a different genomic context, their unusual features reflecting
the different mutational pressures of their parental donor genome. Initially, these
genes are readily detected as having unusual compositional pattems, but over
time^ these patterns are erased as genes evolve in their new genomic context
[54, 55].
Alternatively, genes introduced by HGT can be detected since their relation-
ship to homologues in other bacterial genomes will not be congruent with the
Horizontal and Vertical Gene Transfer 259
Pseudomonas aeruginosa
Escherichia cofi
Mycobacterium tuberculosis
Bacillus halodurans
Vibrio choleras
Bacillus subtilis
Syr^echocystis PCC6803
Deinococcus radiodurans
Xylella fastidiosa
Pasteurefia multocfda
Lactococcus lactis
Archaeoglobus fulgidus
Neisseria meningitidis Z2491
Neisseria meningitidis MC58
Halobactenum NRC-1
Thermotoga maritima
Mycobacterium leprae
Pyrococcus abyssi
Pyrococcus horikoshii
Methanobacterium Ihermoautotrophicum
Aeropyrum pernix
Campylobacter jejuni
Haemophilus inlluenzae
Helicobacter pyfoh 26695
Aquifex aeollcus
Thermoplasma acidophilum
Methanococcus jannaschii
Treponema pallidum
Borrefia burgdorferi
Rickettsia prowazekii
Mycoplasma pneumoniae
Ureaplasma urealyiicum
Buchnera aphidicola
Mycoplasma genitalium
Typical genes
Atypical genes, with atypical
phylogeny
T
2
T
3
T
4
T
5
Megabases of protein-coding DNA
relationships of other genes in the same genome. These 'phylogenetic' methods
can include the direct comparison ofdendrograms to identify genes with uimsual
branching patterns [64], or examine relationships en masse to detect genes with
discordant relationships [65]. Each method has the abiHty to detect different sets
of genes, since each relies upon different sets of criteria for the identification of
potentially transferred genes [66—68]. A comparison of these 'parametric' methods
of identifying genes potentially mtroduced by HGT with phylogenetic methods
that identify genes unique to a lineage show that the two approaches are, for the
most part, congruent in their predictions [68]. Therefore, it is fair to say that some
lessons learned from the evolution of pathogens - that is, rapid adaptation can
occur via HGT - are extensible to the evolution of nonpathogens.
6
Fig, 3. The amount of recently acquired DNA in 34 bacterial and archaeal genomes,
as inferred from the identification of genes with atypical sequence features, including aberrant
nucleotide composition, dinucleotide signatures and codon usage bias patterns; these atypi-
cal genes were confirmed as being horizontally transferred by performing a phylogenetic
concordance test. (That is, the strongest matches to the gene in the database differed signifi-
cantly from the set of strongest matches shown by other genes in the chromosome.)
Lawrence
260
Pathogens with Little Foreign DNA
Examination of figure 3 shows that many pathogens have little recently
acquired DNA, This observation would seem to conflict with the conclusion
that gene acquisition plays such a strong ro]e in the evolution of pathogens. Yet
there is good theoretical and empirical evidence that HGT would be of lesser
importance in the evolution of virulent or host-restricted pathogens, or special-
ized bacterial symbionts, like those with small genomes (fig. 3). That is, one
may consider pathogen evolution to be a two-step process. Fu:st, HGT allows
the introduction of genes which allow adoption of the pathogenic lifestyle.
Metabolic and physiological capabilities may be augmented, and pathogenicity
islands will be detected in the genomes. However, as the pathogen adapts to its
new role, HGT becomes both less important and less feasible, and further evolu-
tionary change is accommodated by alteration of existing genes. As existing
foreign genes ameliorate to their new genomic context [54], few genes will be
detected as 'foreign' using the methods employed for generating figure 3;
phylogenetic methods would still detect ancient transfer events, as evidenced by
the facile detection of the transfer of the phenylalanine tRNA synthase mto the
ancestor of spirochetes from an archaeal donor [64],
Factors Reducing Rate of HGT in Pathogens
Three primary influences lead to the reduction in the rate of HGT into
pathogen genomes. First, many symbionts and pathogens have a reduced expo-
sure to the agents facilitating gene transfer: conjugation, transduction and trans-
formation [69]. With a lower opportunity for exposure to foreign DNA, fewer
foreign genes would be detected in the genomes of these relatively sheltered
organisms. Second, fewer genes may be of utility to organisms that have adapted
to a specialized environment. Here, the pathogen may have no use for the major-
ity of foreign genes that are introduced into its genome, since few would offer
functions of utility [70, 71], Lastly, tlie changes m population stmcture coincident
with specialization - lower population size and rare rates of recombination - raise
the threshold for an effectively neutral mutation [70]- As a result, fewer genes,
even those offering a potential benefit, would be retained; the benefits they con-
fer would differ significantly from selective neutrality (that is, small benefits
are unable to allow a gene to persist in the face of stochastic changes in gene
frequency - termed random genetic drift - that dominate the fates of variant
alleles at low population sizes; this effect whereby genes which would confer a
benefit in a larger population cannot do so m a small population has been termed
'effective neutrality' [72, 73]), and the genes would be lost.
Horizontal and Vertical Gene Transfer 261
It is this same loss of population size that leads to genome decay in many
pathogens, like Mycobacterium leprae [74] or Rickettsia prowazekii [75-77]. In
these cases, the populations are insufficiently large to retain the genes present in
the ancestral organism, and potentially deleterious mutations - those that elimi-
nate gene function by producing pseudogenes - accumulate. That is, although
the genes so mutated may have provided a serviceable function, the losses of the
genes were uisufficiently detrimental to prevent pseudogene formation; as a
result, the mutations were effectively neutral, given the population size, structure
and rate of recombmation. The effects of such population bottle necks are evident
in many pathogens just beginning this process of genome decay, including
Salmonella typhi [78, 79] and Mycobacterium tuberculosis [80-83].
Correlated Genome Changes
In addition to pseudogene accumulation and the failure to retain genes
introduced by HGT, the genomes of pathogens may experience other phenomena
at abnormally high rates. Again, these events reflect a decreased ability for cells
containing such deleterious rearrangements to be eliminated from the population,
not an increased rate of their initial occurrence. For example, inversions that do
not contain the origin or terminus of replication are rare [84]. However, such
chromosome rearrangements are common in many bacterial genomes, including
Bordatella pertussis, Rickettsia, and Salmonella typhi [85-87].
The increased numbers of inversions in B. pertussis (and, to a lesser extent,
in Bordatella parapertussis) are thought to have resulted from an accumulation
of transposable elements [88]. The IS elements provide sites of DNA identity in
inverted orientation at which homologous recombination may act, thus creating
an inversion [89]. Similar recombination between IS elements in direct orientation
may lead to potentially large chromosome deletions, a phenomenon deduced to
have occurred in the Buchnera aphidicola genome. A similar accumulation of
IS insertions, especially of IS/, is seen in Shigella [41, 90]. In both cases, IS
elements are not more prone to transpose in these genomes; rather, the strains
carrying large numbers of IS elements are not removed from the population
since the insertions are insufficiently detrimental.
Gene Loss during Pathogen Evolution
As noted above, several gene losses at the ompT [45] and cadA [44] loci
were critical for the evolution of pathogenicity in Shigella [91]. Similarly, loss
of genes - especially those involved in the producrion of surface antigens - was
Lawrence
262
important in the evolution of the highly virulent strains B. pertussis and
B. parapertussis from the relatively broad host-range pathogen Bordatella
bronchiseptica [88]. Common genes losses were detected [92] when comparing
the smaller genomes ofM. leprae and Mycobacterium bovis [92] to the larger
genome of M. tuberculosis, suggesting that adaptation occurred via loss of
function and not gain of functions by way of horizontally transferred genes;
indeed no genes are found to be unique to M. bovis [92], unlike the situation
with pathogenic E. coli [40].
These changes reflect more than just the inevitable loss of genes that are no
longer under selection for function [93]. Rather, gene loss can be beneficial if the
gene product interferes with the functions of the newly evolving pathogen, either
by diverting metabolic flux along an unproductive pathway or by actively creat-
ing substances that attenuate its vinalence. Alternatively, chromosomal deletions
may be beneficial if the loss of DNA removes potentially problematic DNA
sequences, like genetic parasites [93], or inverted DNA (as discussed above) that
may interfere with chromosome replication and segregation [58, 94-97].
Gene Modification during Pathogen Evolution
Although gene gain and gene loss are effective means by which the char-
acter of a bacterial species may change, we have so far overlooked perhaps the
most fundamental mode of bacterial evolution: gene alteration by mutational
processes. Mutation has played a critical role in the origin or maintenance
of pathogenicity in many organisms. For example, increased virulence of
B. pertussis is due in part to an increased level of expression of the ptxA gene,
facilitated by mutations which increased the strength of its promoter sites and
binding sites for the BvgA regulatory protein [88]. Here, it was not gene gain
that led to toxin production but an increased level of expression of a preexisting
toxin gene.
A different kind of mutational processes, replication slippage, plays an
LinporlanL role in regulating the expression of antigenic loci in a stochastic fash-
ion in both Haemophilus influenzae and Neisseria species [98-100]. Here, genes
can be turned on or off at random via the addition of microsatellite repeats
embedded within protein coding genes (allowing in-frame translation in only 1
of 3 slippage states), or genes may be attenuated in expression by the addition or
subtraction of bases in its promoter region. Lastly, single point mutations can
bring about enormous changes in virulence. Yersinia pestis, the causative agent
of bubonic plague, is virtually indistinguishable from its parent strain Yersinia
pseudotuberculosis [101]. TTiis 'instant species' apparently has recently emerged
by virtue of only a handful of genomic modifications [102, 103].
Horizontal and Vertical Gene Transfer 263
a
Recipients of these
donor fragments
are counterselected
Recipients of these
donor fragments
are counterselected
X
Gene flow between
ancestral and derived
taxa is reduced in the
neighborhood of
acquired genes
A'
B'
a
>*^"^^<3
E'
F'
G'
These fragments are
poor or nonfunctional donors
b
A
B
^X^y^AKi
l_ack of recombination leads to an accumulation of
differences in the neighbortiood of the acquired genes
A'
B^
C
>^"^^<^
K^
F
G'
Fig. 4, The localized decrease in homologous recombination as catalyzed by an HGT
event that leads to ecologically different lineages of bacteria, a After genes (depicted in
black) are acquired by lateral transfer, homologous recombination in flanking genes
decreases, both because recombination events leading to the addition or removal of the
acquired genes are counterselected, or because fragments with ends within the acquired
genes serve as poor donors, b Lower rates of recombination lead to increased sequence diver-
gence (denoted by gray shading) at flanking genes due to their inability to participate in local
periodic selection events [figure after 35; 1 10].
Interplay between HGT, Mutation and Recombination
Though seemingly distinct processes, genes introduced by HGT or modified
in an adaptive way by mutational processes affect the process of intraspecific
recombination (that is, gene exchange among closely related microorganisms)
in a very particular way. While mutations and HGT events introduce potentially
important genetic variation into a population, recombination among strains dis-
seminates this genetic information among closely related strains. Among strams
of a bacterial 'species' - defined as those which exchange genes at high fre-
quency by homologous recombination [6] - strains can show dramatic differ-
ences in the environments they inhabit. This phenomenon has been shown for
Lawrence
264
Free-living, benign ancestor
Gain of pathogenicity islands
Broad host range pathogen
Host specialization
Virulent, narrow host range pathogen
am
i
Escherichia coli
Escherichia coli 0157:H7
Salmonella typhimurium
Bordatella brochiseptica
Salmonella typhi
Population crasli, loss
of recombination
I
Virulent, host-dependent pathogen
Massive gene loss,
pseudogene accumulation,
genome decay
Obligate, host-dependent pathogen
I
Mycobacterium tuberculosis
Bordatella pertussis
Mycobacterium leprae
Ricf^ettsia
Mycoplasma
Fig. 5. The pathway to pathogenicity (see text). Steps in the evolution of pathogens are
depicted on the left, and examples of bacteria exhibiting these properties are depicted on the
right.
natural isolates of E. coli and other enteric bacteria [104-106], and is also
evident in the strong genotypic and phenotypic differences between pathogenic
and nonpathogenic strains of E. coli [40]. Therefore, differences that are adap-
tive for one strain may not be adaptive for other strains; as a result, recombina-
tion events which introduce genes into a nonadaptive strain background, or
remove important genes that were not present in the donor taxa, will be coun-
terselected [35], In effect, the events which cause phenotypic differentiation
among strains lead to genetic isolation of these strains (fig. 4); therefore^
the processes of pathogenicity island acquisition may contribute to the lack
of recombination among strains, therefore catalyzing subsequent genome
reduction.
Genome Evolution and the Progression of Pathogenicity
As detailed above, pathogens may evolve through several distinct phases,
each of which is characterized by different evolutionary mechanisms acting to
shape the content and composition of their genomes. This process is outlined in
figure 5. At the start, a presumably benign, free-living ancestor adopts a patho-
genic lifestyle after acquiring virulence factors by HGT Here, HGT acts as it
does for many bacterial Imeages in providing genetic modules for rapid and
effective exploitation of a new environmental niche. At this point, populations
of broad host-range pathogens may be similar to their nonpathogenic sisters in
Horizontal and Vertical Gene Transfer
265
terms of population size and structure. This stage may be typified by pathogenic
strains of £. coli, for example.
However, host specialization (as seen in S. typhi, for example) leads to
lower population sizes, lower rates of recombination, and eventual gene loss.
B. pertussis shows an intermediate phenotype, whereby many genes have been
lost, IS elements are accumulating, and pseudogenes are evident. M. leprae rep-
resents a genome in massive decay, wherein the pseudogenes abnost outnumber
functional genes. Eventually, this period of genome instability passes; pathogens
with extremely small genomes (e.g., Rickettsia or Mycoplasma) remain as the
result. In endosymbionts which experience similar processes of genome decay
[107, 108] - this period of stability can last for millions of years [109].
Conclusions
While the introduction of pathogenicity islands by HGT is considered a hall-
mark in the evolution of pathogenic bacteria, this process represents only one step
in a multifaceted and complex evolutionary process. Some of the principles of
pathogen evolution are widely applicable to the evolution of nonpathogen organ-
isms (e.g., adaptation via the acquisition of foreign gene), while others are not.
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270
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105 Gordon DM: Geographical structure and host specificity in bacteria and the implications for tracing
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Jeffrey G, Lawrence
Pittsburgh Bacteriophage Institute and Department of Biological Sciences
University of Pittsburgh, Pittsburgh, PA 15260, USA
Tel. +1 4 12 624 4204, Fax +1 412 624 4759, E-Mail jlawrenc@pitt,edu
Horizontal and Vertical Gene Transfer 271
Subject Index
ActAJnvasion, role 187
Actin-ADP-ribosylating toxins
families 43
mechanism of action 43, 44
species distribution 43
AdbesijQS
extracellular matrix binding
collagen 91
elastin 93
fibrinogen 94
fibronectin 91, 92
glycosaminoglycans 94
laminin 92, 93
overview 90, 91
vitronectiji 93
Listeria monocytogenes 104, 105
miscellaneous gram-positive bacteria
adhesins 104, 105
Staphylococcus aureus 1 1 - 1 04
Streptococcus agalactiae 96, 99
Streptococcus pneumoniae 96, 100, 101
Streptococcus pyogenes 94—99
Agrobacterium tumefaciens, type IV
secretion 197, 198
Z)-Alanyl-g]ycyl endopeptidase, functions
149
Albomycin, iioa siderophore carriers 224,
225
Anthrax toxin
edema toxin
components 38
mechanism of action 38,39
lethal factor metallopeptidase 164
lethal toxin 41
Antibiotics, iron siderophore carriers
iron catecholate carriers 226, 227
iron hydroxamate carriers 224-226
resistance 227
L-Ara4N, synthesis and lipid A
modification 14, 15
Arg-C, functions 167
Bacillus anthraciSy see Anthrax toxin
Bacteriocin-processing peptidases,
functions 148, 149
Bacteroides fragilis, enterotoxin 3 1
Bbp, adhesin functions J 03
Biofilm
development cycle 114, 115
Escherichia coll
gastrointestinal biofilms 118, 119
indwelling device colonization 120,
121
in vitro iiludit;s \ I6-I i8
pathogenesis, role 116
urinary tract infection 119, 120
prospects for study 127,128
Pseudomonas aeruginosa
antibiotic resistance 126,127
cystic fibrosis chronic lung infections
123-126
in vitro studies 122, 123
signaling 1 15, 1 16
BlaRl, functions 164, 165
272
BoNT, see Botulinum toxin
Bordetella pertussis, see also Cya; Pertussis
toxin
type rv secretion 198, 199
Botulinum toxin (BoNT), mechanism of
action 41,42
Btu proteins, structures 211,213
C3 exoenzyme, mechanism of action 46
C5a peptidase, functions 167
Calpain, bacterial functions 142
Camelysin, functions 165
CAMPs
hpid A modifications, protection 12, 13
mechanism of action 1 1, 12
signaHng pathway activation 14
Capsular polysaccharide (CP)
attachment 55
Escherichia coli
classification of capsules 59,60
genetic orgaruzation/regulation, group
2 capsule gene clusters 60-64
fujictions
adherence 56, 57
desiccation resistance 56
host immune resistance 57-59
structures 55, 56
Caspases, bacterial functions 152
Cathelicidins, see CAMPs
Cell wall, components 1 82
CGP 4832, iron siderophore carriers 226
Cholera toxin (CT)
mechanism of action 36
secretion 192
structure 36
trafficking 36
ClfA, adhesui functions 103
ClfB, adhesin functions 103
Clostridium
botulinum toxin, ^ee Botulinum toxin
C3 exoenzyme 46
Clostridium perfringes a- toxin
mechanisms 30, 31
phospho I ipase C activity 30
structure 30
enterotoxin 32, 33
glucosylating toxins 47, 48
neurotoxin mechanisms 42, 43
Clostripain, bacterial functions 151
Clp, functions 168
Cna, adhesin functions 1 02, 1 03, 1 06
Collagen, features and adhesin binding
91
Collagenase, bacterial functions 162
Corynebacteriiim diphlheriae, see also
Diphtheria toxin sortases 139
Curli organelles, assembly 73,74
Cya
adenylate cyclase activity 37
cell internalization 38
structure 37
Cytolethal distending toxins (CDTs)
mechanism of action 39
species distribution 39
Cytoplasmic membrane, iron transport
211,213
Cytotoxic necrotizing factors (CNFs)
mechanism of action 45
structure 45
types 44,45
Defensins, see CAMPs
DegP, functions 166
Dichelobacter nodosus, pathogenicity
islands 246
Wpeptidyl aminopeptidase, bacterial
functions 167, 168
Diphtheria toxin (DT)
elongation factor 2 inhibition 34
receptor 33, 34
structure 33
transport 34
Ebh, adhesin functions 102
EbpS, adhesin fiinctions 104
Elastin, features and adhesin binding 93
Elongation factor 2 (EF2), inliibition by
exotoxins 34, 35
Emp, adhesin functions 104
Endotoxin, see also Lipopolysaccharide
definition 1
history of study 1
Enterobactin, iron siderophore carriers
226, 227
Subject Index
273
En lerococcus faecal is ^ pathogenicity
islands 246
EptA, pEtn modification of lipid A 15, 16
Escherichia coli
biofilms
gastrointestinal biofilms 1 18, 1 19
indwelling device colonization 120,
121
in vitro studies 116-118
pathogenesis, role 116
urinary tract infection 119, 120
capsular polysaccharide
classification of capsules 59,60
genetic organization/regulation, group
2 capsule gene clusters 60-64
fimbriae, urinary tract infection 74-76
horizontal gene transfer 257-259
iron transport related to virulence
216-219
pathogenicity islands 242-244
Exotoxin
Cell-surface-active toxins
Bacteroides fragilis enterotoxin 31
phospholipases 30, 31
pore-forming toxins 31-33
signal transduction pathway
modulation 28-30
supcrantigens 33
definition 28
intracellularly active toxins
actin alterations 43-49
apoptosis induction 39^1
cell cycle arrest 39
celJ homeostasis alterations 36-39
protein synthesis inhibition 33-36
trafficking alterations 41^3
overview of mechanisms 28,49
therapeutic application prospects 50
Extracellular polysaccharide, sloughing 55
Fba, adhesin functions 97
FBP54, adhesin fixnctions 97
FbpA, structure 21 1
FecA, structure 214
FepA, structure 214
Ferrimycins, iron siderophore carriers 226
FhuA
antibiotic carrier 225
structure 214
FhuD
antibiotic carrier 225
structure 21 1
Fibrinogen, features and adhesin binding
94
Fibronectin, features and adhesin binding
91,92
Fimbriae
adhesive functions 67
biosynthesis
chaperone/usher pathway 69-71
extracellular nucleator pathway 73
classification 68-74
CSl fimbria! family 71,72
pathogenesis, roles
Escherichia coli urinary tract infection
74-76
extracellular component binding 77,
78
Flagella
functions 67, 68
virulence factors 80,81
FnbpA, adhesin functions 101
FnbpB, adhesin functions 101
F2/PFBP, adhesin ftjnctions 97
FtsH, therapeutic targeting 158,159
Genome evolution, see also Horizonta
gene transfer; Pathogenicity islands
gene loss during pathogen evolution
262, 263
gene modification during pathogen
evolution 263
horizontal gene transfer interplay with
mutation and recombination
264, 265
pathogenicity progression 265,266
Genomic islands (GEIs), see also
Pathogenicity islands
bacterial fitness, role 236,237
definition 235
features 235, 236
genome plasticity, role 247-249
high-pathogenicity island 237
Gingipains, bacterial functions 1 52-1 55
Subject Index
274
Glycosaminoglycans, features and adhesin
binding 94
Gsp proteins, functions 191, 192
Guanylate cyclase, STa binding 29, 30
Helicobacter pylori, see also Vac A
pathogenicity islands 245
type IV secretion 199
Hly transporters, functions 190, 191
Horizontal gene transfer (HGT)
correlated genome changes 262
definition 256
examples 256
gene detection and abundance 259,
260
interplay with mutation and
recombination 264, 265
pathogen creation from nonpathogens
257-259
pathogenicity islands 256, 257, 259
pathogenicity progression and genome
evolution 265, 266
pathogens with little foreign DNA 261
rate reduction factors in pathogens 261,
262
IdeS peptidase, functions 140, 141
Immune inhibitor A, bacterial functions
161
Lmmunoglobulin-A] -specific peptidases
metallopeptidases 163
serine peptidases 1 67
Liner membrane (IM)
CAMP permeability 11 J2
structure, gram-negative bacteria 2, 3
Lnvasins
defuiiiions 1 81
gram-negative pathogen invasive
strategies 188-201
gram-positive pathogen invasive
strategies 184-187
Iron
bacterial function, virulence 215,
216
binding proteijis 210,211,216
siderophores as antibiotic carriers
iron catecholate carriers 226, 227
iron hydroxamate carriers 224-226
resistance 227
surplus and stress 216
transport related to virulence
Escherichia coli 2 1 6—2 1 9
Neisseria 223
Pseudomonas aeruginosa 2 1 9, 220,
222
Salmonella 219
Shigella 218
Staphylococcus aureus 223, 224
Vibrio cholerae 222, 223
transport systems
cytoplasmic membrane 211,213
outer membrane 213-215
prospects for study 227, 228
Kdo transferase, lipid A biosynthesis 9
Klebsiella oxytoca, type II secretion
pathway 191, 192
Laminin^ features and adhesin binding 92,
93
Lbp, adhesm functions 99
Legionella pneumophila, type IV secretion
199,200
Legumain, bacterial functions 151,
152
Lethal toxin (LT)
metallopeptidase 1 64
structure 36
Lipid A
biosynthesis 7-1 0, 20, 2 1
modifications
l-Ara4N synthesis and modification
14, 15
CAMP counteraction, role 1 2, 13
EptA in pEtn modification 15, 16
LpxO in hydroxylation 18, 19
magnesium effects on covalent
structure 12
PagL deacylation 19, 20
PagP in palmitoylation 1 6
prospects for study 21
Rhizobium features 19, 20
TolJ-like receptor 4 binding 1
Lipid X, structure 7
Subject Index
275
Lipid Y, structure 7
Lipopolysaccharide (LPS), see also Lipid
A; 0-antigen
assembly 10, 1 1
cation binding 1 1
components 2, 3
definition 3, 5
structure 4
transport 10, 1 1
Lipoteichoic acid (LTA), adhesin functions
97
Listeria monocytogenes
adhesins 104, 105
invasion strategies 187
Lmb, adhesin functions 100
LpxA, lipid A biosynthesis 8,9
LpxD, lipid A biosynthesis 9
LpxO, lipid A hydroxylation 18, 19
LysC, functions 167
Magnesium
lipid A structure modulation 12
lipopolysaccharide permeability, role
12
PhoP/PhoQ signaling, limited
environments 13, 14
Map/Eap, adhesin functions 104
M proteins, adhesin functions 97-99,
106
Murein, gram-negative bacteria 2
MyD88, Toll-like receptor signaling 5
Neisseria, iron transport related to
virulence 223
Nuclear factor-KB (NF-kB), Toll-like
receptor signaling 5,6
0-antigen
assembly 10
rough versus smooth 3
OmpT, functions ] 37
Omptins, functions 137
Outer membrane (OM)
CAMP permeability 1 1
iron transport 213-215
lipopolysaccharide transport 10,11
structure, gram-negative bacteria 2, 3
11
PagL, lipid A deacylation 19, 20
PagP
catalytic mechanism 17, 18
lipid A palmitoylation 16
outer membrane localization 16, 18
palmitoyl donors 17
species distribution 17
structure 17
Papain, bacterial functions 141,142
Pathogenicity islands (PAls)
enterobacterial pathogens 242-245
examples (table) 238-241
genes 257
genome plasticity, role 247-249, 256,
257, 259
gram-negative pathogens 245, 246
gram-positive pathogens 246
high-pathogenicity island 237
PavA, adhesin functions 101
Peptidases
aspartic peptidases 1 35-1 37
classification 1 33-1 35
cysteine peptidases
D-alanyl-glycyl endopeptidase 149
bacteriocin-processing peptidase 148,
149
calpain family 142
caspase family 152
clan CA peptidases 141
clan CD peptidases 1 51
clan CE peptidases 1 55
clan CF peptidases 157
clostripain family 151
gingipain family 152-155
IdeS peptidase 140, 141
legumain family 151, 152
papain faniily 141, 142
pyroglutamyl-peptidase I family 157
sortases 138-140
staphopain family 146-148
streptopain family 142-146
Ulpl endopeptidase family 155, 156
YopJ pepidase family 156,157
YopT peptidase family 149-151
gene abundance, bacteria 1 32
metallopeptidases
anthrax lethal factor 164
Subject Index
276
BlaRl 164, 165
camelysin 165
collagenase 162
immune inhibitor A family 161
immunoglobulin-Al -specific
peptidases 163
MIO family 162, 163
sequence conservation 158
StcE 165
tentoxilysin 163, )64
therapeutic targeting 158, 159
thermolysin family 160, 161
serine peptidase families, functions
165-168
Pertussis toxin
mechanism of action 37
structure 37
PhoP/PhoQ
CAMP activation 14
signaling, magnesium-limited
environments 1 3, 14
Phospholipases, exotoxins 30,31
PI A, adhesin functions 104
Pili
adhesive functions 67
Neisseria pathogenesis, role 76, 77
phase variation of structures 79, 80
twitching motility, role 78,79
type IV pili 72,73,76,77
Pla peptidase, functions 136, 137
PmrA/PmrB
CAiMP activation 14
signaling, lipid A modification 14
Pore-forming toxins (PFTs)
cholesterol-binding cytolysins 32
classification 31
Clostridium perfringens cutcrotoxiu
32,33
RTX toxins 31,32
Staphylococcus aureus 32
Prepelin peptidase, functions 135,
136
Proteases, see Peptidases
Protein H, adhesin functions 97
Pseudomonas aeruginosa
biofilms
antibiotic resistance 126,127
cystic fibrosis chronic lung infections
123-126
in vitro studies 122,123
iron transport related to virulence 219,
220, 222
Pseudomonas exotoxin A, elongation factor
2 inhibition 35
Pyroglutamyl-peptidase I family, functions
157
Raetz pathway, lipid A biosynthesis 7-10,
20,21
Re endotoxin, assembly 1
Rhizobiiim, lipid A features 19, 20
Rho GT Pases
activators
cytotoxic necrotizing factors 45
SopE 46
glucosylation 47, 48
toxins, GAP activity 48
YopT proteolysis 48
Salmonella
iron transport related to vhulence 219
pathogenicity islands 244, 245
Salmycins, iron siderophore carriers 226
SasG, adhesin functions 103
ScpB, adhesin functions 100
Secretion pathways
Sec pathway 183, 184
TAT pathway 1 84
type I 188-191
type II 188, 189, 191, 192
type 11 [ 188, 189, 192-197
typelV 188, 189, 197-200
typeV 188, 189,200,201
Sllil, adhesijj Functions 95
Shiga toxin
ribosomal RMA inactivation 35, 36
species distribution 35
structure 35
Shigella
horizontal gene transfer 257-259
iron transport related to virulence 218
pathogenicity islands 242
type III secretion 197
Shi proteins, functions 201
Subject Index
277
Siderophores, antibiotic carriers
iron catecholate carriers 226, 227
iron hydroxamate carriers 224—226
resistance 227
Signal peptidase II (SpPase II), functions
135
Signal recognition particle (SRP), functions
183
SNARE, neurotoxin targeting 42, 43
SopE, mechanism of action 46
Sortase A (SrtA)
functions 138, 139, 187
structure 139, 140
Sortase B (SrtB)
functions 139, 187
structure 139, 140
SpeB, adhesin functions 99
SpsA, adhesin functions 100
SptP, mechanism of action 48
STa
guanylate cyclase binding 29, 30
structure 28, 29
Staphopain, bacterial functions 146-148
StaphylococcaJ protein A (Spa), functions
104, 185, 186
Staphylococcus aureus
adhesins 101-104
iron transport related to virulence 223,
224
pathogenicity islands 246
pore-forming toxins 32
sortases 138, 139
STb
mechanism of action 28,29
structure 28
StcE, functions 165
Streptococcus ugulucliuc, adhesms 96, 99
Streptococcus pneumoniae, 2idhQs\ns 96,
100, 101
Streptococcus pyogenes y adhesins 94-99
Streptopain, bacterial functions 142-146
Superantigcns, immune response 33
TAT system, overview 1 84
Tentoxilysin, functions 163,164
Tetanus toxin (TeTx), mechanism of action
41,42
TTiermolysin, bacterial functions 160, 161
Toll-like receptor 4 (TLR4)
fimbria interactions 82
lipid A binding 1
signaling 5-7
TonB, iron transport, role 214
TRAF-6, Toll-like receptor signaling 5, 6
Type III secretion system dependent ADP-
ribosylating toxins
mechanism of action 44
species distribution 44
Ulpl endopeptidase family, functions 155,
156
VacA
mechanism of action 39,41
structure 39
Vibrio cholerae
iron transport related to vimlence 222,
223
pathogenicity islands 245
Vitronectin, features and adhesin binding
93
V8 protease, functions 1 66
vWbp, adhesin functions 1 04
Yersinia, invasion strategies
surface proteins 188-190
type III secretion 193-196
YopE
ftmctions 195, 196
mechanism of action 49
YopH, functions 195, 196
YopJ, funciions 156, 157
YopM, functions 195, 196
YopO, functions 195, 196
YopP, functions 195, 196
YopQ, functions 195
YopT
funcrions 1 49-151, 195, 196
mechanism of action 48
Ysc proteins, functions 193,194
Subject Index
278
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