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Bacterial Invasion
of Host Cells

Edited by
Richard |. Lament

CI.A' * *••

CAMBRIDG

www.cambr idgeorgft /ao-j 21 009542

Bacterial Invasion of Host Cells

This book concerns the intimate association between bacteria and host cells.
Many bacterial pathogens are able to invade and survive within cells at mu-
cosal membranes. Remarkably, the bacteria themselves orchestrate this pro-
cess through the exploitation of host cellular signal transduction pathways.
Intracellular invasion can lead to disruption of host tissue integrity and per-
turbation of the immune system. An understanding of the molecular basis
of bacterial invasion and of host cell adaptation to intracellular bacteria will
provide fundamental insights into the pathophysiology of bacteria and the
cell biology of the host. The book details specific examples of bacteria that are
masters of manipulation of eukaryotic cell signaling and relates these events
to the broader context of host-pathogen interaction. Written by experts in
the field, this book will be of interest to researchers and graduate students in
microbiology, immunology, and biochemistry, as well as molecular medicine
and dentistry.

richard j. lam o nt is Professor of Oral Biology in the College of Dentistry,
University of Florida, and he works on the adhesion and invasion of oral
pathogens, biofilm formation, and intercellular communication.

Published titles

1. Bacterial Adhesion to Host Tissues. Edited by Michael Wilson 0521801079

2. Bacterial Evasion of Host Immune Responses. Edited by Brian Henderson and
Petra Oyston 0521801737

3. Dormancy and Low-Growth States in Microbial Disease. Edited by Anthony
R.M. Coates 0521809401

4. Susceptibility to Infectious Diseases. Edited by Richard Bellamy 0521815258

Forthcoming titles in the series

Mammalian Host Defence Peptides. Edited by Deirdre Devine and Robert

Hancock 0521822203
The Dynamic Bacterial Genome. Edited by Peter Mullany 0521821576
Bacterial Protein Toxins. Edited by Alistair Lax 052 18209 IX
The Influence of Bacterial Communities on Host Biology. Edited by Margaret

McFall Ngai, Brian Henderson, and Edward Ruby 0521834651
The Yeast Cell Cycle. Edited by Jeremy Hyams 0521835569
Salmonella Infections. Edited by Pietro Mastroeni and Duncan Maskell

0521835046

Over the past decade, the rapid development of an array of techniques in
the fields of cellular and molecular biology has transformed whole areas of
research across the biological sciences. Microbiology has perhaps been influ-
enced most of all. Our understanding of microbial diversity and evolutionary
biology and of how pathogenic bacteria and viruses interact with their animal
and plant hosts at the molecular level, for example, have been revolutionized.
Perhaps the most exciting recent advance in microbiology has been the de-
velopment of the interface discipline of Cellular Microbiology, a fusion of
classic microbiology, microbial molecular biology, and eukaryotic cellular
and molecular biology. Cellular Microbiology is revealing how pathogenic
bacteria interact with host cells in what is turning out to be a complex evo-
lutionary battle of competing gene products. Molecular and cellular biology
>- are no longer discrete subject areas but vital tools and an integrated part
O of current microbiological research. As part of this revolution in molecular
O biology, the genomes of a growing number of pathogenic and model bac-
CQ teria have been fully sequenced, with immense implications for our future
rv understanding of microorganisms at the molecular level.
— Advances in Molecular and Cellular Microbiology is a series edited by re-

^~ searchers active in these exciting and rapidly expanding fields. Each volume
-< will focus on a particular aspect of cellular or molecular microbiology and
13 will provide an overview of the area; it will also examine current research.
— i This series will enable graduate students and researchers to keep up with the
LJ rapidly diversifying literature in current microbiological research.

AM CM

Q Series Editors

"; Professor Brian Henderson

•^ University College London

<^j Professor Michael Wilson

— i University College London
O

^ Professor Sir Anthony Coates

= St. George's Hospital Medical School, London

{j Professor Michael Curtis

Z St. Bartholomew's and Royal London Hospital, London

Advances in Molecular and Cellular Microbiology 5

Bacterial Invasion
of Host Cells

EDITED BY

Richard J. Lamont

University of Florida

"BTff

CAMBRIDGE

UNIVERSITY PRESS

CAMBRIDGE UNIVERSITY PRESS

Cambridge, New York, Melbourne, Madrid, Cape Town, Singapore, Sao Paulo

Cambridge University Press

The Edinburgh Building, Cambridge CB2 2RU, UK

Published in the United States of America by Cambridge University Press, New York

www.cambridge.org

Information on this title: www.cambridge.org/9780521809542

This publication is in copyright. Subject to statutory exception and to the provision of
relevant collective licensing agreements, no reproduction of any part may take place
without the written permission of Cambridge University Press.

First published in print format

iSBN-13 978-0-511-18514-4 eBook (NetLibrary)
isbn-io 0-511-18514-6 eBook (NetLibrary)

ISBN-13 978-0-521-80954-2 hardback
isbn-io 0-521-80954-1 hardback

Cambridge University Press has no responsibility for the persistence or accuracy of urls
for external or third-party internet websites referred to in this publication, and does not
guarantee that any content on such websites is, or will remain, accurate or appropriate.

Contents

Contributors ix

Preface xiii

1 Invasion mechanisms of Salmonella 1

Beth A. McCormick

2 Shigella invasion 25

Chihiro Sasakawa

3 How Yersinia escapes the host: To Yop or not to Yop 59

Geertrui Denecker and Guy R. Cornells

4 Stealth warfare: The interactions of EPEC and EH EC with

host cells 87

Emma Allen-Vercoe and Rebekah DeVinney

5 Molecular ecology and cell biology of Legionella

pneum op hi la 1 2 3

Maelle Molmeret, Dina M. Bitar, and YousefAhu Kwaik

6 Listeria monocytogenes invasion and intracellular growth 161

Kendy K.Y. Wong and Nancy E. Freitag

7 N. gonorrhoeae: The varying mechanism of pathogenesis in

males and females 203

Jennifer L. Edwards, Hillery A. Harvey, and Michael A. Apicella

8 Group A streptococcal invasion of host cells 239

Harry S. Courtney and Andreas Podhielski

W
H

O
U

9 Invasion of oral epithelial cells by Actinobacillus
actinomycetemcomitans 275

Diane Hutchins Meyer, Joan E. Lippmann, and Paula Fives-Taylor

10 I n vasi o n by Porphyron) onas gingiva I is 29 5

Ozlem Yilmaz and Richard J. Lamont

Index 3 1 5

Contributors

Emma Allen- Vercoe

Department of Microbiology and Infectious Diseases

University of Calgary

Health Sciences Centre

Calgary, Alberta

Canada

Michael A. Apicella

Department of Microbiology
The University of Iowa
Iowa City, Iowa 52242
USA

Dina M. Bitar

Department of Microbiology and Department of Medical Microbiology

and Immunology
Faculty of Medicine
Al-Quds University
Jerusalem, 19356
Israel

Guy R. Cornells

Biozentrum

70 Klingelbergstrasse

CH 4056 Basel

Switzerland

Harry S. Courtney

Research Service (151)
Veterans Affairs Medical Center
Memphis, Tennessee 38104
USA

Geertrui Denecker

Biozentrum

70 Klingelbergstrasse

CH 4056 Basel

Switzerland

Rebekah DeVinney

Department of Microbiology and Infectious Diseases

g University of Calgary

5 Health Sciences Centre

g Calgary, Alberta

o Canada

Jennifer L. Edwards

Department of Microbiology
The University of Iowa
Iowa City, Iowa 52242
USA

Paula Fives-Taylor

Department of Microbiology and Molecular Genetics
University of Vermont
Burlington, Vermont 05405
USA

Nancy E. Freitag

Seattle Biomedical Research Institute and the Department of Pathobiology
University of Washington
Seattle, Washington 98109
USA

Hillery A. Harvey

Department of Microbiology
The University of Iowa
Iowa City, Iowa 52242
USA

Yousef Abu Kwaik

Department of Microbiology and Immunology
University of Kentucky Chandler Medical Center
Lexington, Kentucky 40536-0084
USA

Richard J. Lamont

Department of Oral Biology
University of Florida
Gainesville, Florida 32610
USA

Joan E. Lippmann

Department of Microbiology and Molecular Genetics
University of Vermont

o
Z

Burlington, Vermont 05405 2

C
H
O

USA

Beth McCormick

Department of Pediatric Gastroenterology and Nutrition

Mucosal Immunology Laboratory

Massachusetts General Hospital

Charles town, Massachusetts 02129-4404

USA

Diane Hutchins Meyer

Department of Microbiology and Molecular Genetics
University of Vermont
Burlington, Vermont 05405
USA

Maelle Molmeret

Department of Microbiology and Immunology
University of Kentucky College of Medicine
Lexington, Kentucky 40536
USA

Andreas Podbielski

Department of Medical Microbiology & Hospital Hygiene
University Hospital Rostock
D-18057 Rostock
Germany

Chihiro Sasakawa

Institute of Medical Science, University of Tokyo, 4-6-1, Shirokanedai

Minato-ku

Tokyo 108-8639

Japan

Kendy K.Y. Wong

Seattle Biomedical Research Institute and the Department of Pathobiology
University of Washington
Seattle, Washington 98109
USA

Ozlem Yilmaz

Department of Pathobiology
3 University of Washington

£ Seattle, Washington 98195

3 USA

Preface

Few microbiologists are likely to forget the moment when the sheer scale
and diversity of the microbial world became apparent to them. Similarly, it
is a sobering thought that, in (or more accurately on) the human body, bac-
teria outnumber human cells by at least 10 to 1. Fortunately, most of these
bacteria behave as good guests should, and they are content to remain on the
other side of the physical barriers that separate us from the outside world.
It is inevitable that at such a large gathering, some guests, the pathogens,
will misbehave, and worse, start a fight. For many years it was thought that
the battle between host and pathogen at the mucosal membranes was fought
at arm's length. The bacteria lobbed toxins and other noxious agents at the
host and the host returned the favor with antibodies. Bacteria that ventured
too close were rapidly dispatched by the professional killing machines, the
phagocytic cells. Some bacteria, such as Mycobacterium tuberculosis, however,
proved inconveniently recalcitrant to intracellular killing and could become
permanent guests within macrophages. Nonetheless, despite the apprecia-
tion that mitochondria originate from intracellular bacteria, the notion that
mucosal pathogens could be intimately involved with nonprofessional phago-
cytes is relatively new.

We now know that a wide variety of organisms are capable of direct-
ing their own entry into epithelial cells and other host nonphagocytic cells.
These bacteria engage in a remarkably sophisticated molecular dialogue with
host cells in order to manipulate signal transduction pathways and effectuate
bacterial entry. In such an immunologically protected, nutrient-rich envi-
ronment, bacteria can thrive. At first glance the burden of an intracellular
bacterial load would appear to bode ill for the host cell. However, a long
evolutionary relationship has resulted, in many cases, in a more balanced
encounter between invasive organism and host, whereby the bacteria have

adapted to minimize the degree of damage to the host. In other words, both
bacteria and host try to make the best of their enforced cohabitation.

The long-term consequences of such an arrangement can only be specu-
lated on. Does the presence of intracellular bacteria help the host by providing
a continuous low level stimulation of the immune system? Could intracellular
bacteria aid in normal development of the epithelium as has been suggested
for Bacteroides species in the intestine? Conversely, is it possible that sup-
pression of apoptotic cell death by some organisms could be a cofactor in
cancer development? While indulging speculation, it is interesting that, in
what is called the Penrose- Hameroff orchestrated objective reduction model,
microtubule stability can perform a cognitive role through quantum compu-
tations within the neurons of the brain. If bacteria that can impinge upon
microtubule stability were to gain access to the brain, the results could be
g startling!

In the following chapters the molecular bases of invasion, and the out-
comes for host and bacterium, are discussed for variety of Gram-negative
and Gram-positive species. Also included are organisms that subvert host
cell signaling but do not appear to locate intracellularly in large numbers,
or indeed use these pathways to prevent uptake into phagocytic cells. In a
volume of this size, obviously we cannot be comprehensive; nonetheless,
although some specific invasive species are not included, the themes that
are developed are broadly applicable. In that vein, each of the authors has
addressed the topic according to his or her own perspective, so individual
chapters are uniquely informative.

w
ft

Bacterial Invasion of Host Cells

CHAPTER 1

Invasion mechanisms of Salmonella

Beth A. McCormick

Salmonella enterica serovar Typhimurium is a facultative intracellular pa-
thogen that causes gastroenteritis in humans and a systemic disease similar
to typhoid fever in mice. Following oral ingestion, bacteria colonize the in-
testinal tract and then penetrate the lymphatic and blood circulation systems.
Passage of eukaryotic organisms through the intestinal epithelium is thought
to be initiated by bacterial invasion into M cells and enterocytes. The process
of epithelial cell invasion can be studied experimentally because S. enterica
serovar Typhimurium invades cultured epithelial cells in vitro. Many of the
genes required for epithelial invasion have been found within eukaryotic
pathogenicity island 1 (SPI-1), which is a contiguous 40-kb region at centro-
some 63 of the chromosome. SPI-1 genes encode a bacterial type III secretion
apparatus and several effectors, which contribute to pathogenesis through an
interaction with eukaryotic proteins. The type III secretion apparatus is a mul-
tiprotein complex that is thought to build a contiguous channel across both
the bacterial and epithelial cell membranes, resulting in efficient translo-
cation of bacterial effectors directly into the cytosol of epithelial cells. The
secreted effectors are thought to interact with eukaryotic proteins to activate
signal transduction pathways and rearrange the actin cytoskeleton, leading to
membrane ruffling and engulfment of the bacterium. This chapter discusses
the mechanism by which S. typhimurium enter into host cells.

CLINICAL DESCRIPTION

S. enterica, gram-negative bacteria of the family Enterobacteriaceae, cause
a variety of diseases in humans and other animal hosts. Salmonella serovars
fall into two general categories: those that cause enteric (typhoid) fever in
humans (S. typhi and S. paratyphi), and those that do not (S. enteriditis and

S. typhimurium) . Enteric fever is a systemic illness characterized by a high,
sustained fever, abdominal pain, and weakness. Millions of cases of enteric
fever are reported annually throughout the world, and, without antimicro-
bial therapy, the mortality rate is 15% (Hook, 1990). Nontyphoidal serovars
of S. typhimurium produce an acute gastroenteritis, characterized by intesti-
nal pain and usually nonbloody diarrhea, which is a serious public health
problem in developing countries (Hook, 1990). Approximately 40,000 cases
of nontyphoidal Salmonella infection are reported annually in the United
States, and the nontyphoidal Salmonella are responsible for more deaths in
the United States than any other foodborne pathogen, with a mortality rate
of approximately 1% (Hohmann, 2001). Because S. typhimurium is usually
self-limiting in healthy adults, it is often not reported to public health offi-
cials, and the total number of annual cases in the United States has been
g estimated to be 1-3 million. However, in infants, in the elderly, in immuno-

s compromised individuals, or in response to certain serovars, nontyphoidal

8 Salmonella, such as S. choleraesuis, infection can also result in bacteremia and

^ establishment of secondary focal infections (i.e., meningitis, septic arthritis,

x or pneumonia). Antibiotic treatment of nontyphoidal salmonellosis is not

£ recommended, because it may prolong the illness; this is most likely due to

the killing of the normal gut microbiota, which exert a protective effect on the
intestine (Hohmann, 2001). Bacteremia and its associated secondary infec-
tions can be treated with antibiotics, such as ampicillin or cephalosporins,
but this treatment has recently become a serious problem as a result of the
rapidly growing number of Salmonella isolates that are multidrug resistant.
Most infections with Salmonella (typhoidal and nontyphoidal) are con-
tracted through contaminated food or water, and, although it is very rare,
direct person-to-person transmission can occur. Studies with healthy vol-
unteers have demonstrated that 10 6 -10 9 organisms are necessary to cause
symptomatic illness (Hook, 1990). Most organisms are killed by the low pH of
the stomach, but those that persist target the colon and small intestine (distal
ileum) as their portal of entry into the host. The surviving bacteria then di-
rect their internalization by the epithelial cells (both enterocytes and M cells)
lining the intestine. S. typhimurium pass through the intestinal epithelial bar-
rier to gain access to the lymphoid follicles, from which point the bacteria
are eventually transported to the bloodstream and more distant sites of in-
fection, such as the liver and spleen (Hook, 1990). Nontyphoidal Salmonella
remain primarily at the level of the intestinal epithelium and submucosa (ex-
cept during bacteremia) , where they elicit an acute inflammatory response
that manifests itself as fever, diarrhea, and abdominal cramping (Hohmann,
2001). Although in healthy adults the symptoms usually abate within a few

days, it can take at least 1 month to fully clear all Salmonella from the gastroin-
testinal tract, reflecting the fitness of these bacteria for their gastrointestinal
niche (Hohmann, 2001).

SALMONELLA ENTRY: SALMONELLA PATHOGENICITY ISLAND-1

One of the first noteworthy in vitro activities observed regarding
Salmonella-host cell interactions was the ability of this organism to induce
its own uptake into epithelial cells, which are not normally phagocytic. This
unusual phenotype, termed invasion, allowed for the identification and char-
acterization of invasion genes associated with SPI-1. Galan and Curtiss (1989)
were the first to characterize the S. typhimurium invasion locus, inv, which was
identified by complementation of a noninvasive mutant of S. typhimurium.
In particular, using an in vitro system of cultured epithelial cells, these in- 5

vestigators discovered that highly virulent S. typhimurium strains carrying &

inv mutations were defective for entry into but not attachment to Henle-407 *

cells. Moreover, when administered perorally to Balb/c mice, the inv mutants w

of S. typhimurium had higher 50% lethal doses than their wild-type parents >

(Galan and Curtiss, 1989). £

Since this original observation, significant progress has been made to- £

ward understanding the molecular mechanisms that lead to Salmonella entry c*

into cells. As subsequently discussed in detail, contributions by a variety of Eg

o
laboratories have established that the key ingredient of the machinery used g

by Salmonella to gain access into nonphagocytic cells is the type III secretion £

system encoded at centrosome 63 of its chromosome. Type III secretion sys-
tems are widely distributed among plant and animal pathogenic bacteria that
share the property of engaging host cells in an intimate manner. Composed
of more than 20 proteins, these systems are regarded as one of the most com-
plex protein secretion systems discovered. Such complexity is largely due to
their specialized function, which is not only to secrete proteins from the bac-
terial cytoplasm but also to deliver them to the inside of the eukaryotic host
cell. Adding to this level of complexity is the temporal and spatial restrictions
that govern their activity.

The regulation of Salmonella pathogenicity islands

Salmonella have evolved spatially and temporally regulated systems,
which secrete proteins that allow for the microorganism to invade the intesti-
nal epithelium. In Salmonella, these delivery systems are encoded on regions
of the bacterial chromosome termed pathogenicity islands (Darwin and Miller,

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1999a; Hansen-Wester and Hensel, 2001). Many of the genes required for
epithelial invasion have been found within SPI-1, which is a contiguous 40-kb
region at centrosome 63 of the chromosome (Mills et al., 1995). SPI-1 genes
encode a bacterial type III secretion apparatus and several effectors, which
contribute to pathogenesis through an interaction with eukaryotic proteins
(Fig. 1.1 A) . Salmonella invasion gene expression is known to be modulated by
multiple environmental signals, including osmolarity, oxygen tension, pH,
and stage of growth (Lee and Falkow, 1990).

Specifically, the expression of SPI-1 genes appears to be regulated at
several stages in a complex manner by regulators within SPI-1, including
HilA and InvF, and those outside SPI-1, such as the two-component reg-
ulators, the flagella associated genes, and the small DNA binding proteins
(Fig. 1.1B). Salmonella does not constitutively express the virulence pheno-
types associated with the SPI-1 type III secretion system (TTSS-1). In vitro 5

inducing conditions that result in optimal expression of TTSS-1 include high &

osmolarity, low oxygen tension, slightly basic pH, and the growth rate of the z

bacteria (Lee and Falkow, 1990). The primary mechanism for controlling the w

production of TTSS-1 factors in response to environmental and physiological >

cues is by transcriptional regulation. The expression of genes encoding the K

TTSS-1 apparatus and most of the effectors requires HilA, a transcription £

factor encoded on SPI-1. HilA, a member of the OmpR/ToxR family, directly c*

binds to and activates the promoter of SPI-1 operons and functions as a cen- Eg

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tral regulator of invasion gene expression (Lostroh et al., 2000). Osmolarity, g

oxygen, and pH coordinately affect the transcription of hilA, and changes in £

the level of HilA mediate the regulation of the SPI-1 TTSS-1 by the same

Figure 1.1. (facing page). Type III secretion genes of SPI-1 and their regulation. (A) An
overview of the type III secretion system encoded on SPI-1 includes subunits of a type III
secretion apparatus, effectors secreted by the apparatus, factors required for their efficient
translocation, and transcriptional regulators. The part of the island encoding a
high-affinity iron transporter (sitBCDA) is not depicted. (B) Sequential upregulation by
factors encoded on SPI-1 leads to expression of the type III secretion system. When in
vitro environmental conditions are favorable for invasion gene expression (low pH, low
oxygen tension, and high osmolarity), HilD derepresses the hilA promoter. The straight,
solid arrows show that HilA protein directly activates the expression of structural genes
such as the prgs and another regulatory gene, invF. HilA transcription initiated at Pi nv p
results in a long mRNA that continues through sic A. Although not illustrated, InvF, as a
complex with SicA, directly activates the expression of effectors such as SipB, SopE, and
SopB/SigD. HilD also makes a small direct contribution to invF expression by slightly
upregulating the activity of a promoter far upstream of the start of invF translation.
(Adapted from P.C. Lostroh and C.A. Lee, Microh. Infect. 3: 1281-1291, 2001.)

environmental conditions. Notably, no environmental condition has ever
been documented that affects HilA-dependent TTSS-1 genes without also
affecting hilA expression.

SPI-1 encodes four additional transcriptional regulators besides HilA;
these include InvF, HilD, SprB, and SprA/SirC. To date, genetic evidence
implicates a cascade of transcriptional activation in which HilD, HilA, and
InvF act in sequence to stimulate TTSS-1 gene expression in vitro (Fig. 1.1B).
Initially, HilD, an AraC/XylS family member, binds directly to several sites
within PhM and derepresses hilA expression. HilA then binds to conserved
sequences located between — 54 and — 37 relative to both invF and prgH start
sites. Activation of P prg H a ^d Pi nv p results in the transcription of prgHIJK-
orgAB and InvFGEABCspaMNOPQRS. Therefore, HilA directly activates the
expression of the structural type III secretion genes as well as the transcription
g factor InvF. InvF, an AraC-like transcriptional regulator, promotes expres-

s sion of HilA-activated effector genes by inducing their transcription from a

8 second HilA-independent promoter (Darwin and Miller, 1999b). That is, InvF

^ activates a promoter upstream of sicA, causing additional expression of sicA-

X sip BCD A. Furthermore, it has been demonstrated that two transcriptional

£ regulators of SPI-1, HilC and HilD, allow the expression of hilA by coun-

teracting the action of an unknown repressor (Lucas and Lee, 2001). These
complex regulations appear to ensure that invasion genes are appropriately
expressed when Salmonella infects the host.

SPI-2 also encodes for a protein secretion system similar to that encoded
by SPI-1. Unlike SPI-1, which is found in all Salmonella lineages, SPI-2 is
found only in S. enterica serovars and its acquisition most likely led to the
divergence of S. enterica and S. bongori. SPI-2 is located at minute 30 of the
S. typhimurium chromosome and has been implicated in the systemic phase
of infection (Hensel et al., 1998; Shea et al., 1999). Expression of SPI-2 is
induced within macrophages (Cirillo et al., 1998), and it appears to mediate
bacterial survival within macrophages through evasion of NADPH oxidase-
dependent killing (Vasquez-Torres et al., 2000) , and interference with cellular
trafficking of Salmonella containing vacuoles (Uchiya et al., 1999).

The type III secretion complex

A TTSS is a complex organelle composed of more than 20 proteins (Fig.
1.2). Subsets of these proteins are organized into a supramolecular structure,
termed the needle complex, which spans the bacterial envelope (Kubori et al.,
1998). This structure resembles the basal body of flagella, suggesting an
evolutionary relationship between these two organelles. Indeed, components

InvE

SpaM/InvI

SpaO

OrgO

OrgB

Outer membrane

Periplasm
Inner membrane

PrgI

PrgJ

(needle)

InvG InvH
'(secretin pore)

InvA, SpaPQRS
(export apparatus)

PrgK (top inner membrane ring)
PrgH (bottom inner membrane ring)

InvC/SpaL
(ATPase)

SpaN/InvJ
(specificity regulator)

Figure 1.2. Schematic representation of the SPI-1 type III secretion system. The secretion
machinery is made up of approximately 20 proteins that span the inner and outer
membranes and direct the secretion of proteins without the classical sec-dependent signal
sequence.

of the TTSS share amino acid similarity to flagellar proteins. The needle
complex of the SPI-1 encoded TTSS has been characterized in some detail
(Kubori et al., 2000; Zhou and Galan, 2001). It consists of a mul tiring base
composed of the SPI-1 encoded proteins PrgHIJK. PrgH alone multimerizes
into a tetrameric structure, but when complexed with PrgK it oligomerizes
into ring-shaped structures that resemble the base of the needle complex
of flagella. InvG has also been reported to form part of the base, and this is
consistent with the observation that InvG forms a ring in the outer membrane
in the presence of a helper lipoprotein, InvH. PrgI and PrgJ appear to form
part of the bore of the needle. The core components, which comprise the
needle-like complex, are highly conserved among different gram-negative
pathogens, suggesting a common mode of operation. However, regulation
of the secretion event is not well understood except that it requires energy in
the form of ATP (Eichelberg et al., 1994). Further, unlike the sec-dependent
pathway of bacterial protein secretion, type III secretion does not require a
signal sequence on the protein to be exported, and in this respect it shares
similarities with the bacterial flagellar export system.

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The needle complex is constructed in an orderly manner (Kubori et al.,
1998, 2000; Zhou and Galan, 2001) . The proteins that make up the base of the
complex are secreted through the inner membrane by the sec-mediated path-
way. Once in the periplasm, the proteins form a complex that associates with
a set of inner membrane proteins that share extensive sequence similarity
to the components of the flagellar export apparatus. The resulting complex
is restricted to the export of only the proteins that are necessary to make the
needle structure. Once this foundation is made, the type III secretion appa-
ratus becomes competent for the export of other type III secreted proteins,
including those that are targeted to the inside of host cells.

Although much progress has been made in characterizing the secretion
apparatus itself, little is known about how the effector proteins are subse-
quently translocated across the eukaryotic cell membrane. To date, three
g proteins, SipB, SipC, and SipD, are required for the translocation of effector

s proteins into the host cell, although the mechanisms by which SipB, SipC,

8 and SipD exert their functions is not understood. Although these three pro-

^ teins have been shown to be required for translocating effector proteins into

x the cytoplasm of the host cells, SipB, SipC, and SipD are not essential for

£ the secretion process (Collazo and Galan, 1997). At least 13 proteins that

are delivered by the SPI-1 TTSS have been identified: AvrA, SipA, SipB,
SipC, SipD, SlrP, SopA, SopB, SopD, SopE/E2, SptP, and SspHl (see ref-
erences in Zhou and Galan, 2001). During the infection process, these pro-
teins are presumably translocated into the cytosol of the host cell, where
they engage host cell components to induce host cellular responses and pro-
mote bacterial uptake. Although some of these effector proteins are encoded
within SPI-1, several effector proteins are encoded outside this pathogenicity
island.

INTERNALIZATION OF SALMONELLA BY THE HOST EPITHELIUM

Animal models of Salmonella infection

A key feature of Salmonella pathogenesis is the ability of these bacteria
to induce their own internalization by the normally nonphagocytic epithelial
cells that line the intestine. Interactions between Salmonella and the intesti-
nal epithelium were first described by Takeuchi, who orally infected guinea
pigs with S. typhimurium (Takeuchi, 1967). From this early work it was de-
termined that bacteria that closely contact the epithelial cells lining the intes-
tine, primarily the ileum, elicit the local degeneration of filamentous actin in
apical microvilli and the underlying terminal web. The morphology of other

areas of the apical surface, either on the same cell or adjacent enterocytes,
remains unaffeced. Subsequently, extruded membrane (described as mem-
brane ruffles) surrounds the bacteria, resulting in their internalization into
membrane-bound vacuoles. Once the bacteria are internalized, the overlying
apical membrane regains its microvillar morphology, and despite these dras-
tic changes to the apical cytoarchitecture and the presence of intracellular
S. typhimurium, infected enterocytes remain healthy. Interestingly, although
some bacteria become internalized by enterocytes, the majority remain in
the intestinal lumen (Watson et al., 1995). Similar observations have been
reported in other animals, including calves, pigs, and primates, all of which
present with a diarrheal gastroenteritis in response to S. typhimurium and
other related Salmonella strains (Bolton et al., 1999; Rout et al., 1974; Wallis
and Galyov, 2000).

<
>

Cell culture models of Salmonella infection S

As a way to investigate in more detail the changes to the host intesti- w

nal epithelium during early Salmonella-host cell interactions, a number of >

cell culture models have been developed. Initial studies were performed with K

epithelial cell lines that, when cultured on porous filter supports, establish £

electrically resistant epithelial monolayers with full apical-basolateral po- c*

larity. Two polarized cell lines used in these early studies were the Madin §

o
Darby canine kidney cell line (M DCK) , derived from dog kidney distal tubule g

cells, and the Caco-2 cell line, derived from human colonic epithelia. Polar- £

ized cells presented with nontyphoidal Salmonella on the apical cell surface
exhibit similar features to enterocytes in the guinea pig model: microvilli be-
come disassembled, and the resulting membrane extrusions internalize the
bacteria (Finlay and Falkow, 1990; Finlay et al., 1988). Thus, S. typhimurium
contacting the apical plasma membrane were observed to induce ruffling
of the membrane at sites of bacterial-epithelial cell contact, providing the
driving force for bacterial internalization. The ability of S. typhimurium to
induce contact-dependent membrane ruffling as a means of gaining entry
into the host cell suggests that the bacteria recapitulate a process resembling
phagocytosis in these normally nonphagocytic cells. This process has been
termed macropinocytosis to reflect its resemblance to pinocytosis, or fluid up-
take into cells, but with the engulfment of much larger particles (Francis
et al., 1993). Interestingly, although Salmonella initially interact with their
animal hosts at the apical surface of the intestine, studies with the T84 cell
polarizing cell line have revealed that they can invade from the basolateral cell
surface at the same frequency as from the apical surface (Criss et al., 2003).

o
u
u

However, the significance of this observation in the in vivo setting remains
to be determined.

S. typhimurium invasion is not restricted to epithelial cells. In vivo the bac-
teria also invade macrophages, and in vitro they infect a variety of eukaryotic
cells, except yeast and erythrocytes (Finlay et al., 1991). Subsequent invasion
assays with HeLa cells and the Chinese hamster ovary (CHO) fibroblast cell
line demonstrated that less polarized cells are more effectively infected by S.
typhimurium, suggesting that the rigid cytoarchitecture of polarized epithelial
cells is a hindrance to bacterial internalization in vivo. These studies implied
that S. typhimurium utilize the same strategy to enter both polarized and
nonpolarized cells, and the latter has gained widespread use for studies of
the molecular regulation of S. typhimurium invasion.

INVOLVEMENT OF THE HOST CELL CYTOSKELETON IN
S BACTERIAL INTERNALIZATION

^ The distinct morphologic changes occurring to the apical enterocyte

x membrane upon binding of S. typhimurium suggested that host cell microfil-

£ aments (composed of actin) or microtubules might be involved in the forma-

tion of membrane ruffles. Finlay and Falkow were the first to report that treat-
ment with cytochalasins, drugs that prevent F -actin polymerization, inhibits
Salmonella invasion of multiple cultured cell lines. In contrast, microtubule-
depolymerizing agents do not block bacterial internalization, suggesting that
the actin cytoskeleton, but not the microtubual network, plays an active role
in bacterial entry into host cells (Finlay and Falkow, 1988). Moreover, pre-
treatment with cytochalasin D does not prevent bacterial attachment to the
host cell surface, indicating that actin-dependent cytoskeletal rearrangements
and membrane ruffling follow initial bacterial binding (Francis et al, 1993).
Immunofluorescence microscopy later demonstrated that bacteria recruit fil-
amentous actin to sites of active bacterial invasion. Confocal laser scanning
microscopy revealed that several actin-binding proteins, including a-actinin,
tropomysin, and talin, are recruited to the S. typhimurium-induced ruffles in
cultured cells (Finlay et al., 1988). Remarkably, Salmonella do not disrupt the
actin cytoarchitecture in other regions of the cell, including cortical actin bun-
dles or stress fibers (Finlay et al., 1991). S. typhi also induce actin-dependent
ruffling during invasion, suggesting that this aspect of bacterial invasion is
conserved regardless of eventual disease outcome (Mills and Finlay, 1994).
Because ruffle formation is essential to the invasion process, understanding
the development of these structures is critical to understanding Salmonella
pathogenesis as a whole.

GTP

GDP

RadlGD^

Figure 1.3. Nucleotide cycling of monomelic GTPases: In the resting state, the
monomeric GTPase (shown here as Racl) is in the GDP-bound, inactive conformation.
Upon stimulation, a GEF catalyzes the release of GDP from the GTPase, followed by
binding of GTP. This places the GTPase in the active conformation, where it can interact
with effector proteins. To turn off the signal, a GAP enhances the GTPase's intrinsic
hydrolysis rate, leading to GTPase inaction. S. typhimurium encodes two related GEFs for
Rho GTPases, that is, SopE and SopE2, as well as one GAP for these GTPases, that is,
SptP.

INVOLVEMENT OF RHO GTPase IN S. TYPHIMURIUM INVASION
OF NONPHAGOCYTIC CELLS

Over the past 10 years, it has been demonstrated that the formation
of actin-based cytoskeletal structures, which occurs in response to growth
factors and other extracellular stimuli, is regulated by monomeric guano-
sine triphosphatases (GTPases) of the Rho family (Hall, 1998; van Aelst and
D'Souza-Schorey, 1997). Rho proteins are members of the Ras superfamily
of monomeric GTPases, and, like all Ras superfamily members, they cycle
between active (GTP-bound) and inactive (GDP-bound) conformations (Fig.
1.3). Members of this family include RhoA-B-C-D-E-G, Racl-2, Cdc42, and
TC10; however, RhoA, Racl, and Cdc42 have been the most extensively stud-
ied. In vitro, both GTP binding and hydrolysis activities of the GTPases are
extremely low; therefore, accessory factors are required to facilitate these pro-
cesses. Guanine nucleotide exchange factors (GEFs) catalyze the release of
GDP and binding of GTP, which activates the GTPase, while GTPase activat-
ing proteins (GAPs) stimulate the GTP hydrolysis rate, thereby promoting
their inactivation (Fig. 1.3). In fibroblasts, activation of RhoA promotes for-
mation of stress fibers and focal contacts; Racl activation yields lamellipodia

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>

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g

w
n

X
>

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►fl

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H

and dorsal ruffles; and Cdc42 activation leads to the extension of filopodia
(KozmaetaL, 1995; Nobes and Hall, 1995; Ridley and Hall, 1992). During cell
spreading, Rho family members function sequentially, with initial activation
of Cdc42 followed by Racl and RhoA (Nobes and Hall, 1995; Ridley and Hall,
1992). In other actin-dependent processes, distinct subsets of Rho GTPases
become activated, often in a cell-type specific manner.

The involvement of Rho GTPases in S. typhimurium invasion was initially
examined in nonpolarized cell lines of both epithelioid (HeLa and COS-1) and
fibroblastic lineages. In these cells, Chen et al. (1996) demonstrated that inva-
sion of Salmonella was primarily dependent on Cdc42. In this model, expres-
sion of a point mutant of Cdc42 unable to bind GTP (which acts in a dominant
inhibitory manner) prevented bacterial entry, whereas expression of domi-
nant negative Racl partially inhibited internalization but not as effectively as
g the Cdc42 mutant. The result of this study correlated with previous analysis

s of Rho GTPases during Fc receptor-mediated phagocytosis in macrophages.

8 Particularly, expression of dominant negative mutants of either Rac or Cdc42 ,

^ but not Rho, blocks phagocytosis of IgG-opsonized particles by having unique

x but complementary effects on localized actin polymerization at the plasma

£ membrane (Caron and Hall, 1998; Cox et al., 1997; Massol et al., 1998).

Moreover, in their activated form, Cdc42 and Rac have been shown to in-
duce actin polymerization through the activation of N-WASP and the Arp2/3
complex. At present it is not known whether S. typhimurium direct their mor-
phological changes in the actin cytoskeleton by using a similar activation
strategy.

Nonetheless, as a result of the unique structure of the enterocyte brush
border, the cytoskeletal regulatory factors co-opted by Salmonella during in-
vasion in polarized epithelia are different from those identified in studies
with nonpolarized cells (Criss et al., 2001). Dominant negative Racl, but not
Cdc42, significantly inhibited bacterial entry at the apical aspect of polarized
cells. In this in vitro model of Salmonella - enterocyte interaction, the bacteria
elicit actin reorganization and membrane ruffling at the apical surface in a
manner that is morphologically indistinguishable from ruffling in nonpo-
larized cell lines. However, during entry at the apical pole of epithelial cells,
Salmonella encounter a complex, highly organized actin cytoskeleton unlike
any other cell surface they invade. At the apical domain, polymerized actin
is organized into rigid microvilli and the underlying terminal web, a cross-
linked meshwork of actin filaments that attaches to intercellular junctional
complexes (Fath et al., 1993). Accordingly, the ability of Salmonella to reorga-
nize the apical plasma membrane and its underlying actin architecture may
require the mobilization of a unique set of cellular regulatory factors.

S. TYPHIMURIUM GENES THAT REGULATE EPITHELIAL
CELL INVASION

Several S. typhimurium gene products secreted via the SPI-1 encoded
TTSS have been found to participate in the process of bacterial uptake by
epithelial cells. These gene products fall into two categories: those that affect
Rho GTPase activity, and those that directly affect host actin dynamics.

SopE/SopE2

SopE was first identified as a protein secreted by the SPI-1 TTSS of
S. dublin, and it was subsequently found in S. typhimurium. Initial studies
determined that deletion of sop E reduces invasiveness to 40%-60% of wild-
type levels, presumably as a result of a reduced capacity of the pathogen to

elicit plasma membrane ruffling, which can be rescued by complementation 3

of the sopE locus (Wood et al., 1996; Hardt et al., 1998). Subsequently, S. s

typhimurium SopE was found to have GDP-GTP nucleotide exchange activity *

on Rho family GTPases in vitro (i.e., it acts like a GEF; see Hardt et al., 1998; »

Rudolph et al., 1999). Ectopic expression of SopE protein in mammalian cells >

elicits membrane ruffling over the surface of the cell in a Racl- and Cdc42- £

dependent manner (Hardt et al., 1998). SopE is not encoded within SPI-1 £

but is instead found on a lysogenic bacteriophage, which is only possessed g

by a subset of Salmonella spp. However, possession of the SopE phage does §

not correlate with invasiveness or pathogenicity (Mirold et al., 1999). Since §

this initial report, it was found that S. typhimurium possesses a homolog of £

SopE called SopE2, which has approximately 69% identity to SopE and is also
secreted by the SPI-1 TTSS. A mutant strain deleted in SopE2 has reduced
invasiveness relative to wild-type bacteria, but, unlike SopE, SopE2 is found
in all pathogenic strains of Salmonella examined (Bakshi et al., 2001; Stender
et al., 2000). These findings implicate SopE/E2 in the formation of the actin
rearrangements necessary for membrane ruffling on the host cell surface
and subsequent bacterial internalization.

It is interesting to note that SopE can activate Cdc42 despite its lack of
sequence similarity to Dbl-like proteins, the Rho-specific eukaryotic GEFs.
Recent investigations focusing on the mechanism by which SopE mediates
guanine nucleotide exchange have determined that SopE binds to and locks
the switch I and switch II regions of Cdc42 in a conformation that promotes
guanine nucleotide exchange (Buchwald et al., 2002). Although this confor-
mation resembles that of Racl in a complex with the eukaryotic Dbl-like
exchange factor Tiam 1, the catalytic domain of SopE has an entirely differ-
ent architecture from that of Tiam 1; furthermore, it interacts with the switch

regions by means of different amino acids. In this regard, SopE is the first
example of a non-Dbl-like protein capable of inducing guanine nucleotide
exchange in Rho family proteins.

SopB

SopB exhibits potent phosphoinositide phosphatase activity in vitro and
is capable of mediating pronounced inositol phosphate fluxes in vivo (Galyov
et al., 1997). In addition, SopB has been found to stimulate Cdc42-dependent
rearrangements of the actin cytoskeleton that are a prerequisite for cellular
invasion. The ability of SopB to activate Cdc42 is dependent on its phos-
phatase activity, because a phosphatase-defective SopB in which a critical
active-site cysteine residue was changed to serine lost its ability to activate
g Cdc42. Because inositol -based molecules can directly affect Cdc42 activity, it

s is thought that SopB activates Cdc42 and Racl indirectly by fluxing cellular

8 phosphoinositides (Zhou et al., 2001).

^ The activation of Cdc42 and Racl triggers a series of signal transduction

X events that lead to actin cytoskeleton rearrangements. Despite their different

£ biochemical activities, SopE/E2 and SopB exert at least partially redundant

functions during Salmonella invasion. Thus, introduction of a loss-of- function
mutation in the genes that encode either one of these proteins results in a
minor defect in Salmonella entry. However, the simultaneous inactivation of
SopE/E2 and SopB results in a very severe entry defect.

SptP

Cells infected with S. typhimurium quickly recover from the dramatic
actin cytoskeletal rearrangements and regain their normal cellular architec-
ture. SptP was identified as a S. typhimurium protein with homology in its
carboxy-terminal to both prokaryotic and eukaryotic phosphatases, and it was
demonstrated to possess tyrosine phosphatase activity (Kaniga et al., 1996).
Although SptP mutants do not have an invasion deficiency, cells infected with
sptP-deficient S. typhimurium do not exhibit normal recovery of their actin
cytoskeleton following bacterial entry. Sequence scanning of SptP revealed
a region in its amino terminus with homology to GAPs for Rho proteins,
which is also possessed by other bacterial pathogens (ExoS of Pseudomonas
spp. and YopE of Yersinia spp.), as well as by eukaryotes. SptP behaves as a
GAP for Cdc42 and Racl, but not Rho A or Ras. A mutation of arginine to ala-
nine within the proposed catalytic arginine finger abrogated GAP activity (Fu
and Galan, 1999). These results suggest that SopE/E2 and SptP coordinately

control the GDP-GTP cycle of Rac and Cdc42 in host cells, thereby modu-
lating the actin cytoskeleton. Thus, SptP's GAP activity opposes the Cdc42
and Racl activating function of SopE, SopE2, and SopB to help the host cell
rebuild its actin cytoskeletal network. How these proteins are regulated in
vivo so that their activities do not nullify each other is not yet clear, but it may
be due to differential secretion or activation of SptP by its chaperone, SicP
(Fu and Galan, 1998). In addition to its GAP activity located within the amino
terminus, the carboxy-terminal domain of SptP possesses potent tyrosine
phosphatase activity. Such tyrosine phosphatase activity of SptP is not only
involved in reversing the MAP kinase activation that results from Salmonella
invasion but also targets the intermediate filament vimentin, which is re-
cruited to the membrane ruffles stimulated by Salmonella (Murli et al., 2001) .

SipC

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>

SipC has been reported to nucleate and bundle actin in vitro. The z

bundling and nucleation activities are located at different domains of SipC. w

The precise role of these activities in vivo is unknown because the necessary >

experiments to address this important issue are hampered by the fact that £

SipC is required for the translocation of effector proteins into host cells. SipC £

has been identified along with SipB as a general chaperone for the transloca- c*

tion of other SPI-1 type III secreted effector proteins into the host cell (Carlson Eg

o
and Jones, 1998). In addition, SipC becomes translocated into the host cell, g

where it has a bipartite ability to modulate actin polymerization directly. In £

vitro, the C -terminus of SipC aids in the nucleation of new actin filaments
(the rate-limiting step in actin polymerization), whereas the N-terminal half
facilitates filament bundling. Accordingly, microinjection of purified SipC
protein into HeLa cells induces actin polymerization, but rather than induc-
ing ruffles like SopE, it promotes the condensation of filamentous actin into
large aggregates (Hayward and Koronakis, 1999). The physical function of
these aggregates is unclear.

SipA

SipA is also encoded within and secreted by the SPI-1 TTSS. It is thought
that SipA affects actin dynamics in cells by initiating actin polymerization at
the site of Salmonella entry by lowering the critical concentration of actin
required for polymerization (Zhou et al., 1999a). A sip A mutant strain of S.
typhimurium has a minor invasion deficiency that is only detectable at very
early time points (up to 20 min) of bacterial entry. Furthermore, although the

sip A mutant elicits actin-dependent membrane ruffling, these ruffles are less
localized to sites of internalization than those induced by wild-type bacteria
(Zhou et al., 1999a). In additon, SipA was found to bind filamentous actin
in an in vitro binding assay and induce formation of actin bundles at sites of
bacterial internalization (Hayward and Koronakiz, 1999). In vivo, SipA may
additionally affect actin dynamics by binding to, and enhancing the activity
of, the bundling protein T-plastin (Zhou et al., 1999a, 1999b). Furthermore,
McGhie et al. (2001) recently determined that SipA potentiates the effects of
SipC on filamentous actin nucleation and bundling in vitro. Thus, it appears
that SipA modulates the internalization process by decreasing the critical
concentration for actin polymerization, inhibiting depolymerization of actin
filaments, and increasing the bundling activity of T-plastin.

SipA is also unique in that interaction of this effector protein at the apical
g surface of intestinal epithelial cells is sufficient to initiate the proinflamma-

s tory signal transduction pathway that leads to polymorphonuclear leukocyte

8 (PMN) transepithelial migration. The recruitment of PMN to the intestinal

^ epithelium is a key virulence determinant underlying the development of

x Salmonella-elicited enteritis. Purified SipA applied to the apical surface of

£ intestinal epithelial cells initiates an ADP ribosylating factor 6 (ARF6) lipid-

signaling cascade, which, in turn directs the activation of protein kinase C
(PKC) and subsequent PMN transepithelial migration (Lee et al., 2000; Criss
et al., 2001). This demonstrates that some SPI-1 effector proteins involved in
the invasion of epithelial cells may have additional roles that do not require
their introduction directly into the cytosol of the host. Moreover, the signifi-
cance of these results has been confirmed by the finding that SipA plays an
important role in eliciting proinflammatory responses, such as PMN influx,
during Salmonella infection of calves - a relevant in vivo model system used
to study human enterocolitis (Zhang et al., 2002).

ROLE OF SPI-1 IN PATHOGENESIS

To understand the role of invasion in Salmonella pathogenesis, re-
searchers have investigated the in vivo phenotypes of invasion gene mutants.
Most in vivo studies have used the murine model of typhoid fever, in which
orally introduced S. typhimurium causes a systemic illness in Balb/c mice.
To induce systemic illness in these animals, S. typhimurium first colonize
the distal ileum, and, after successful colonization, a subpopulation of S. ty-
phimurium can be found in the gut-associated lymphatic tissues. Still later,
host death can occur in response to high numbers of bacteria found within
deep lymphoid-rich organs such as the spleen and liver (Carter and Collins,

1974). Invasion, per se, has long been thought to be important for this pro-
cess because mutants that are noninvasive in vitro are less able to reach the
spleen and liver and so are attenuated in orally infected mice. However, if
introduced systemically, noninvasive mutants are as virulent as the wild-type
(Galan and Curtiss, 1989; Ahmer et al., 1999). Many groups have interpreted
such data to mean that invasion of nonphagocytic cells allows S. typhimurium
to access the lymphatics, especially through the Peyer's patches that underlie
M cells in the distal ileum (Penheiter et al., 1997).

In spite of this, several recent observations suggest that invasion, per se,
is not always required for S. typhimurium to access privileged sites within
the host. For example, S. typhimurium can reach the spleen in an invasion-
independent manner by residing inside CD 18+ macrophages (Vazquez-
Torres et al., 1999). In yet another example, it has been postulated that CD18-
exp res sing phagocytes are involved in an alternate route for bacterial invasion 5

(Rescigno et al., 2001). Among CD18+ cells, dendritic cells are migratory and &

phagocytic cells that are ideally located for antigen sampling in tissues that in- z

terface with the external environment, where they perform a sentinel function w

for incoming pathogens. With the use of polarized monolayers of the intesti- >

nal epithelial cell Caco-2, it has been shown that dendritic cells are able to K

open up the tight junctions between epithelial cells, send dendrites outside £

the epithelium, and directly sample bacteria. Because dendritic cells express c*

tight junction proteins (i.e., claudin-1, occludin, and ZO-1) the integrity of §

o
the mucosa is preserved. This unique cell-cell interaction allows dendritic g

cells to sample the environmental microorganisms without compromising £

the barrier function and to deliver the organisms to the lymphoid tissues
where an efficient immune response can be mounted. Thus, this identifies
a new mechanism for bacterial uptake in the mucosa tissue.

In a different study it was demonstrated that S. typhimurium lacking
the entirety of SPI-1 cannot invade tissue cells but nevertheless still dis-
seminates to systemic organs in Balb/c mice following intragastric infection
(Murray and Lee, 2001) . This observation highlights a new important concept
in Salmonella pathogenesis establishing that TTSS-1 may also have activities
aside from inducing invasion-associated rearrangements inside nonphago-
cytic epithelial cells. It is now apparent that S. typhimurium attracts, kills,
and parasitizes different immune cells, and some of these activities require
TTSS-1.

Therefore, the in vivo significance of invasion is likely to vary depending
on the particular host-bacterial interaction. For instance, invasion may be re-
quired for some aspects of virulence but not for access to deeper tissues. It is
also likely that there are situations in which invasion-independent TTS S S P I - 1

phenotypes contribute significantly to salmonellosis and others in which
invasion-dependent systemic dissemination is more critical. Although inva-
sion can be uncoupled from some pathogenesis-associated phenotypes in
vitro, it is not currently feasible to uncouple invasion from other TTSS SPI-1
phenotypes in vivo. Thus, the relative contribution of different SPI-1 pheno-
types remains to be elucidated.

HISTORICAL PERSPECTIVE OF SPI-1

The genes that comprise the SPI-1 are not present in the genome of
Escherichia coli K-12, but groups of similarly organized genes with related
sequences occur on the virulence plasmids of the invasive enteric pathogens
Shigella and Yersinia and in the genome of certain plant and animal pathogens
2 of the genera Erwinia, Pseudomonas, and Xanthomonas (Li et al., 1995). And,

rt as already mentioned, there are similarities between certain SPI-1 genes and

u loci involved in biogenesis of flagella in a variety of bacteria.

^ Perhaps the best characterized example of the functional and structural

h conservation in TTSS-1 is between Salmonella and Shigella. The inv/spa genes

of SPI-1 are homologous to the Shigella mxi/spa genes; as a consequence, it
is not surprising that the Salmonella and Shigella TTSS not only exhibit signi-
ficant similarities in the primary sequence of their determinants (Hermant
et al., 1995) but also complement each other functionally for secretion in
vitro (Rosqvist et al., 1995). Perhaps they even share essentially the same
macro molecular structure. Moreover, there is also significant structural and
functional conservation between the Sip proteins encoded on the SPI-1 of
S. typhimurium and the Ipa proteins encoded on the Shigella mxi/spa locus,
suggesting that the entry processes engaged by these two enteric pathogens
are promoted by similar effectors (Hermant et al., 1995).

In consideration of the base compositions, genomic locations (i.e., chro-
mosome vs. plasmid), and phylogenic distribution of these genes, it is un-
likely that the SPI-1 TTSS complex was ancestral in the Enterobacteriaceae.
In addition, given their relatively low G + C content in S. enterica (46%),
Li et al. (1995) proposed that the SPI-1 genes were horizontally transferred
from Yersinia. However, because of their occurrence in all the subspecies of
S. enterica and the overall similarity of their evolutionary diversification to
that of housekeeping genes (Li et al., 1995), it is more likely that they were
already present in the last common ancestor of the contemporary lineages
of the salmonellae. Thus, it is generally agreed that Salmonella, Yersinia, and
Shigella independently acquired these genes from another source.

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CHAPTER 2

Shigella invasion

Chihiro Sasakawa

INVASION

Shigella invasion and the host inflammatory responses

Shigella cause bacillary dysentery (shigellosis), a disease provoking a se-
vere inflammatory diarrhea in humans and primates. In tropical areas of
developing countries, shigellosis is endemic and a major killer of children
under 5 years of age. Shigellosis occurs following ingestion of a very small
number (100-1000) of bacteria, thus permitting easy spread of the disease by
person-to-person contact as well as by the drinking of contaminated water.

Shigella, a Gram-negative bacillus, comprises four species - S. dysenteriae,
S.flexneri, S. boydii, and S. sonnei (Pupo et al., 2000; Lan and Reeves, 2002).
Shigella is now recognized as a member of Escherichia coli; however, the group
of bacteria causing shigellosis is idiomatically called Shigella in this chapter.
Shigellosis is also caused by enteroinvasive E. coli (EIEC), a pathogenic E. coli.
Shigella and EIEC possess a large 210- to 230-kb plasmid on which the major
virulence functions are encoded. Because Shigella has neither adhesins for
upper GI tract cells nor flagella, after infection by means of the fecal-oral
route the bacteria reach the colon and rectum directly, where they translocate
through the epithelial barrier by means of the M cells overlaying the solitary
lymphoid nodules (Fig. 2.1; also see Wassef et al., 1989; Sansonetti et al.,

1991, 1996). Once they have reached the underlying M cells, Shigella infect
the resident macrophages and multiply. Within the macrophages Shigella
secrete IpaB, which specifically binds to, cleaves, and activates caspase-1,
thus leading to macrophage cell death through apoptosis (Zychlinsky et al.,

1992, 1994, 1996).

The stimulation of caspase-1 in infected macrophages causes the pro-
duction of large amounts of lL-l/3 and IL-18, thus eventually leading to an

actin tail

bacteria

Mcell

enterocyte

i— i

X
i— i

X
u

apical side

AIWWMWWVW1

nucleus

basolateral side

macrophage

Figure 2.1. A simplified model for the infection of colonic epithelial cells by Shigella (refer
to the text for details).

increase in the permeability of the epithelial barrier to Shigella and migra-
tion of polymorphonuclear leukocytes (PMNs; see Zychlinsky et al., 1992;
Zychlinsky and Sansonetti 1997). Meanwhile, the bacteria released from the
dead macrophages immediately enter surrounding enterocytes from the ba-
solateral surface by directing large-scale membrane ruffling, which finally
leads to phagocytic events. Though the invading bacterium is entrapped by
a phagocytic membrane, Shigella immediately disrupts the membrane and
escapes into the cytoplasm (Fig. 2.1; also see Sansonetti et al., 1986). Within
the cytoplasm, Shigella multiply and induce actin polymerization at one pole
of the bacterium, by which the intracellular bacterium can gain a propulsive
force to move intracellularly and intercellularly (Fig. 2.1; also see Bernardini
etal., 1989).

Internalized Shigella release a large amount of lipopolysaccharide (LPS)
into the host cytoplasm, where the LPS binds Nodi, a member of the CED4/
Apaf-1 superfamily, eventually leading to activation of NF-/cB by means of
the stimulation of the bipartite CARD-kinase protein, RICK (Inohara et al.,
2000; Girardin et al., 2001). In response to the activation of NF-/cB, colonic
epithelial cells express a large array of proinflammatory cytokines, especially
IL-8, thus further promoting local inflammation and attracting more PMNs
(Zychlinsky and Sansonetti, 1997). Therefore, the predominant pathogenic
feature of Shigella is the ability to invade macrophages as well as epithelial
cells, including subsequent dissemination into adjacent epithelial cells. In

<
>

this chapter, I mostly focus on the bacterial system involved in the invasion
of epithelial cells and subsequent intracellular and intercellular spreading
processes.

Basolateral entry into polarized cells

Shigella infection of polarized epithelial cells such as Caco-2 or Madin
Darby Canine Kidney (MDCK) cells reveals that the bacteria invade from the
basolateral surface into the cells (Mounier et al., 1992). This characteristic
entry can be seen when Shigella infect rabbit ligated ileal loops, where bac-
teria move to the basolateral surface by means of the M cells (Wassef et al.,
1989; Sansonetti et al., 1991, 1996), indicating that Shigella have an affin-
ity to the basolateral surface of the polarized enterocytes to effectuate entry.
Shigella are capable of inducing a highly dynamic rearrangement of actin 8

and tubulin cytoskeletons during entry, and this leads to a large-scale phago- £

cytic event. Shortly after coming into contact with epithelial cells, Shigella
induce the formation of focal adhesion-like actin-dense patches beneath the
bacterial contact point (Tran Van Nhieu and Sansonetti, 1999) and trigger
local destruction of the microtubule structure (Yoshida et al., 2002), which
is followed by the protrusion of large-scale membrane ruffles. The invading
Shigella are finally enclosed by a large membrane vacuole, but the pathogens
immediately escape into the host cell cytoplasm, where they elicit intracellular
and intercellular movement (Fig. 2.1).

Invasion-associated genes on the large plasmid

The invasion of epithelial cells by Shigella requires many genes mostly
confined to the 31-kb pathogenicity island (PAI) on the large virulence plas-
mid, which is highly conserved among Shigella spp. (Sasakawa et al., 1988;
also see Fig. 2.2). The PAI of S.Jlexneri contains 28 genes bracketed by several
IS elements and vestigial DNA sequences, where the 28 genes are arranged in
several transcribed regions, encoding the components of the type III secretion
system (TTSS), secreted effector proteins, chaperone proteins, and regulatory
proteins (Fig. 2.2; also see Buchrieser et al., 2000; Venkatesan et al., 2001).
ipaBCDA encode secreted effectors such as IpaA, IpaB and IpaC required for
invasion of epithelial cells, whereas mxi and spa mostly encode components
of the TTSS including the TTSS-associated secreted proteins. ipgA, ipgC, and
spa 15 encode IpgA, with IpgC and Spa 15 that act as chaperones for IcsB,
IpaB/IpaC, and IpgBl/IpaA, respectively (Menard et al., 1994a, 1994b; Page
et al., 2002; Ogawa, unpublished results). The PAI possesses two regulatory

'h^ 1

•9

2 s -

.2
+■»

CD
Q.

ns
o

Figure 2.2. {facing page). The genetic structure of the ipa/mxi/spa PAI encoding the
Shigella TTSS. (A) A simplified structure of the ipa/mxi/spa PAI. Arrows represent IS
elements and the vestigial DNA sequences. (B) The genetic constitution of the ipa/mxi/spa
PAI. (For details refer to the text and to Buchrieser et al., 2000 and Venkatesan et al., 2001.)

genes, virB and mxiE, required for the expression of the virulence-associated
genes in the 31-kb PAI (Adler et al., 1989; Dorman and Porter, 1998; Mavris
etal.,2002).

In addition, another plasmid-encoded gene, virF, codes for the essential
regulatory protein VirF, an AraC-like transcriptional regulator, which directly
binds the virB promoter to activate the virB gene. The VirB protein in turn
activates the promoters for several transcribed regions in the 31-kb PAI. On
the large plasmid, there is another effector gene called virA (Uchiya et al.,
1995), located near the virG (icsA) gene; together these form a PAI (Venkate-
san et al., 2001). VirA has recently been shown to be essential for evoking
membrane ruffling in epithelial cells and promoting Shigella entry into host
cells (Yoshida et al., 2002). Recently the whole genomic sequence of the large
plasmid (pWRlOO) ofS.flexneri serotype 5 was determined (Buchrieser et al.,
2000; Venkatesan et al, 2001). g

Examination of the repertoire of proteins secreted by means of the £

TTSS under conditions that activate the TTSS revealed that 15 proteins
(IcsB, IpaH9.8, IpaH7.8, IpaH4.5, MxiC, MxiL, Spa32, OspCl, OspB, IpgBl,
OspDl, OspEl, OspF, OspG, and VirA) plus IpaA, IpaB, IpaC, IpaD, and
IpgD can be secreted from Shigella by means of the TTSS into the medium
(Buchrieser et al., 2000; Ogawa, unpublished results). Although the proteins
potentially secreted by means of the TTSS such as Spa32, MxiC, or MxiL
(Tamano et al., 2002; Tamano unpublished results) do not necessarily serve
as effectors, studies have clearly indicated that Shigella secrete a diverse array
of effectors into the external medium and target host cells.

Type III secretion system

The TTSS is a highly sophisticated bacterial effector protein delivery
system. Upon contact with the host cells, a set of effector proteins is delivered
from the infecting bacteria to the host cells by means of the TTSS. These
translocated proteins have a variety of effects on host cells and are necessary
for bacterial attachment, invasion, trafficking, and avoidance from the host
defense systems. Although the mechanisms of protein export by means of
the TTSS, including the biosynthesis of the secretion machinery, are still to

<
>

2 A Scale bar, 100 nm

Figure 2.3. The supramolecular structure of the Shigella type III secretion machinery.
(A) An electron micrograph of the purified type III secretion machinery from S.jiexneri.

be elucidated, some common characteristics of this secretion system have
emerged (Hueck, 1998).

Genetic and functional studies have indicated that the TTSS is encoded
by more than 20 genes. The subset of genes together with other genes coding
for the secreted effectors, chaperones, and regulators exist as a PAI, which
potentially transposes horizontally in different species of bacteria, thus dis-
tributing among many Gram-negative pathogenic bacteria, where some of
the PAI are located on the chromosome and some on a plasmid. In fact,
there is considerable homology between the proteins of the TTSS in differ-
ent pathogens (Hueck, 1998). Of note, some of the proteins of TTSSs also
share significant similarity to the components of the bacterial flagella ex-
port machinery. For example, some of the putative components of the type
III secretion complexes such as S.jiexneri MxiJ, Spa47, Spa33, Spa24, Spa9,
Spa29, Spa40, and MxiJ share significant similarity to FlhA, Flil, FliN, FliP,
FliQ, FliR, FlhB, and FliF of the Salmonella flagellar export system, respec-
tively (Hueck, 1998; Cornells and Van Gijsegem, 2000; Piano et al., 2001).

Furthermore, the TTSS is functionally and structurally similar to the
flagellar export system. Secretion of a set of proteins by means of the TTSS

effector

cytoplasmic membrane

host cell cytoplasm

iff!

IpaC

needle

IpaB

upper rings

outer membrane

periplasm

inner membrane

bacterial cytoplasm

Spa47 ATPase

Figure 2.3. (cont.) (B) Model of the structure of the type III secretion machinery, including
the translocation of effectors from bacterial cytoplasm into the host cell cytoplasm.

ir,
H

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>

is dependent on the energy supply mediated by the Fl-type ATPase asso-
ciated with the secretion apparatus. The same is true for secretion of the
extracellular flagellar components, which form the hook, cap, and flagella
filament by means of the flagella export system. The supramolecular struc-
tures of the TTSS of Salmonella typhimurium and S.flexneri have recently
been elucidated, and they share similarity with that of the flagellar basal body
(Fig. 2.3B; also see Kubori et al., 1998; Blocker et al., 1999; Tamano et al.,
2000). As already mentioned for the Shigella TTSS, the expression of genes
encoding the TTSS as well as the flagella export system is under stringent
control that is mediated by complicated regulatory networks in each of the
bacteria.

Despite the structural and functional similarities of the TTSS in each
pathogen, the proteins delivered by means of the TTSS are quite diverse.
For example, Shigella potentially deliver ~20 proteins by means of the TTSS;
some share similarity to secreted proteins from different pathogens, whereas
others are unique to Shigella. Studies of the proteins secreted from Yersinia,

Salmonella, and Shigella have indicated that some have a role in linking the
secretion complex to the target host plasma membrane, whereas others serve
as effectors to modulate host cell functions.

The purified type III secretion complexes from S. typhimurium and S.
flexneri as reported by Kubori et al. (1998) and Tamano et al. (2000), respec-
tively, contained four major components. The Salmonella type III secretion
complex contained InvG, PrgH, PrgI, and PrgK proteins, whereas the Shigella
type III secretion complex contained MxiD, MxiG, MxiH, and MxiJ. Recently,
these type III secretion complexes have been shown to contain an additional
component, which is PrgJ in Salmonella and Mxil in Shigella. The supramolec-
ular structures of the type III secretion complexes of each bacterium observed
by electron microscopy are similar, being composed of two distinctive parts,
the needle and basal parts (Fig. 2.3B). The needle of the S. typhimurium and
g S. flexneri type III secretion complexes consists mainly of PrgI and MxiH,

% respectively (Kubori et al., 2000; Tamano et al., 2000). The basal part of the S.

< typhimurium type III complex is composed of InvG, PrgH, and PrgK, whereas

§ that of the S. flexneri complex is composed of MxiD, MxiG, and MxiJ. Further-

x more, the supramolecular structures of the basal portion of both type III com-

plexes share significant similarity to that of the Salmonella flagella basal body.
Indeed, the basal part of the type III secretion complex possesses two
pairs of rings, referred to as the upper and lower rings (Fig. 2.3B). Because
the basal portion was observed by electron microscopy to be embedded in the
osmotic-shocked bacterial envelope, similar to the flagellar basal complex,
the two pairs of rings are thus assumed to be anchored to the inner and
outer membranes of bacteria. The flagellar hook forms a curved protruding
structure from which a long flagella filament is extended; the type 1 1 1 secretion
complex forms a straight needle protruding from the basal part. The length
of the type III needle of wild type S. flexneri is estimated to be 45 nm and
distributed in a narrow range with a standard deviation of 3.3 nm (Tamano
et al., 2000). The length of the basal body of the Shigella type III secretion
complex is estimated to be approximately 31 nm, which is consistent with
the thickness of the Gram-negative bacterial envelope (approximately 25 nm)
(Tamano et al., 2000). This suggests that the type III secretion complex spans
both the outer and inner membranes. Although the number of needles per
bacterium has not been accurately determined, on the basis of the distribution
of the type III secretion structures in a field of osmotically shocked bacterial
envelope as observed by electron microscopy, it is estimated to be around
50-60 per bacterium.

Genetic and functional studies of TTSSs have strongly suggested that the
basic morphological features displayed by Salmonella and Shigella would be

conserved among other pathogens. Indeed, recently studies have shown that,
in enteropathogenic E. coli (EPEC), a long (50-700 nM) filamentous structure
protrudes from the tip of the TTSS needles; it is composed of EspA, which is
encoded by the espA gene located on the locus of enterocyte effacement (LEE)
PAI, downstream of the region encoding genes of the TTSS (Sekiya et al.,
2001; Daniell et al., 2001). Previous studies indicated that EspA forms a fila-
mentous structure that assembles as a physical bridge between the bacteria
and host cell surface, which then functions as a conduit for the transloca-
tion of bacterial effectors into host cells (Knutton et al., 1998). In fact, the
espA mutant of EPEC has been shown to be deficient in forming a long fila-
mentous structure and delivering effectors such as Tir, EspB, and EspD into
the host cells, thus becoming a nonadherent mutant. Similarly, some plant
pathogens such as Pseudomonas syringae and Ralstonia solanacearum form
a filamentous appendage called the Hrp-pilus, which consists of HrpA in %

P. syringae or HrpY in R. solanacearum (Van Gijsegem et al., 2000). S

The effectors delivered from Shigella trigger host cellular signal pathways
to direct its own internalization event (Fig. 2.4). One signal transduction
pathway is linked to the interaction of secreted IpaB and IpaC with putative
host surface receptors, and the others are evoked by intracellular effectors
such as IpaA, IpaB, IpaC, IpgD, and VirA. Although the roles of IpaB and
IpaC during invasion are important, their functions are complicated. For
example, IpaB and IpaC act as secreted effector proteins in the target host
cells to stimulate caspase-1 and Rho GTPases, respectively, whereas IpaB and
IpaD act as a molecular plug for the TTSS at the tip of the TTSS needle (Figs.
2.3B and 2.4; also see Menard et al., 1996; Tran Van Nhieu and Sansonetti,
1999). Like Yersinia YopB and YopD (Hakansson et al., 1996) or Salmonella
SipB and SipC (Collazo and Galan, 1996), upon contact between Shigella and
the host cell, IpaB and IpaC serve as a membrane pore located at the tip
of the TTSS needle in the host cell plasma membrane, thus allowing the
translocation of secreted effector proteins into the host cells (Fig. 2.3B; also
see Blocker etal., 1999).

The IpaB and IpaC proteins secreted into the culture supernatant
form high molecular matrix-like structures, which promote Shigella invasion
through interaction with CD44 (the hyaluronan receptor belonging to the
immunoglobulin superfamily) and a 5 /3i integrin (Fig. 2.4; also see Skoudy
et al., 2000; Watarai et al., 1996). Because both molecules are distributed on
the basolateral surface of the polarized epithelial cells, these interactions are

Secreted effector proteins induce host cellular signaling &

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thus assumed to contribute to the basolateral entry of Shigella into the epithe-
lial cells by mediating outside-in signaling to induce local rearrangement of
the actin cytoskeleton (Skoudy et al., 2000). as Pi integrin localizes at focal ad-
hesions, and the cytoplasmic domain of the fi\ integrin acts as a cytoskeleton
linker by means of focal adhesion proteins; the cytoplasmic moiety of CD44
also acts as a cytoskeleton linker by means of association with ERM (ezrin-
radixin-moesin) proteins. Ezrin is shown to be recruited at the periphery of
the extended membrane ruffles at the point of Shigella entry (Skoudy et al.,
1999). A recent study has shown that IpaB can bind CD44, where IpaB parti-
tions during Shigella invasion within specialized membrane microdomains
enriched in cholesterol and sphingolipids, called rafts (Lafont et al., 2002).
CD44 is known to participate in signaling responses regulating the reorgani-
zation of the cytoskeleton (Hirao et al., 1996). Early in the invasion (~15 min
after contact), an accumulation of cholesterol and raft-associated proteins %

such as GPI-anchored proteins can be observed at Shigella entry foci. £

In agreement with this, bacterial entry is impaired upon cholesterol de-
pletion with methyl-/? -cyclodextrin at the site of bacterial entry (Lafont et al.,
2002). Therefore, the binding of IpaB at the tip of the TTSS needle to clus-
tered CD44 is thought to increase binding affinity; thus rafts and clustered
CD44 seem to be crucial for efficient Shigella entry. Meanwhile, a 5 /?i integrin
accumulates together with F-actin, vinculin, talin, a-actinin, and tyrosine-
phosphorylated FAK, which are major scaffolding components of focal ad-
hesions (Watarai et al., 1996, 1997). However, these interactions alone are
insufficient to elicit the phagocytic events required for bacterial uptake by
epithelial cells (Menard et al., 1996). For induction of large-scale actin re-
arrangements and large membrane protrusions sufficient to engulf several
bacterial particles simultaneously, cellular signals evoked by Shigella effectors
such as IpaA, IpaC, IpgD, and VirA appear to be necessary (Fig. 2.4).

In addition, Shigella require the activation of FAK and Src tyrosine ki-
nase to induce cytoskeletal rearrangements during entry (Watarai et al., 1997;
Dumenil et al., 1998). Overexpression of a dominant interfering form of
pp60 c " Src leads to inhibition of Shigella-induced cytoskeletal rearrangements
and decreases the phosphorylation of cortactin (Dumenil et al., 1998), a
Src substrate recruited at the site of Shigella entry (Dehio et al., 1995). Because
focal adhesion formation is dependent on the activation of Rho and Src, an
early cellular event triggered by Shigella contact may be associated with such a
cellular function (Adam et al., 1996; Menard et al., 1996; Watarai et al., 1997).

Figure 2.4. (facing page). A model for cytoskeletal rearrangements induced during Shigella
invasion of epithelial cells, and the roles of effector proteins (refer to the text for details).

I pa A

IpaA secreted via the TTSS upon cell contact has been indicated to mod-
ify the Shigella-induced entry foci, and an ipaA mutant induced disorganized
filopodial protrusions (Fig. 2.4; also see Bourdet-Sicard et al., 1999). IpaA has
a high affinity for N-terminal residues 1-265 of vinculin. In cosedimentation
and solid-phase assays, IpaA binding to vinculin increases the association of
vinculin with F-actin, which in turn promotes depolymerization of F-actin
associated with the IpaA-vinculin complex. Vinculin is a cytoskeletal protein
present at focal adhesion and cell-cell adhesion structures, involved in cell ad-
hesion to the extracellular matrix, cell motility, and tumorigenesis (Jockusch
and Rudiger, 1996). Vinculin is composed of N-terminal head and C-terminal
tail domains. It has multiple functional domains; talin and a-actinin inter-
act with the N-terminal domain, whereas F-actin binds to the C-terminal
| tail domain. Importantly, the interactions with proteins are determined by

gj the conformation of vinculin. The unfolded form is the activated form and

w interacts with talin, a-actinin, and F-actin through the exposed binding do-

g mains, whereas, in the folded conformation, the tail domain interacts with

£ the head domain, resulting in the masking of the binding domains (Jockusch

and Rudiger, 1996). Importantly, the 1-258 domain of vinculin has been in-
dicated to be involved in the intramolecular association with the C-terminal
tail domain.

The binding of IpaA to vinculin was found to be strong, with a Kd of
5 nM; therefore, it is likely that the binding of IpaA to the head domain dis-
rupts the head-tail interaction, thus allowing vinculin to open up and link
to F-actin. The vinculin-IpaA complex promotes F-actin depolymerization
in vitro and in vivo. Indeed, microinjection of IpaA into HeLa cells induces
a rapid (within 40 s) cell retraction with the disappearance of actin stress
fibers. When IpaA is comicroinjected with the MBP-fused vinculin 1-265
moiety, cell retraction induced by IpaA can be blocked and actin stress fibers
along with vinculin-containing focal complexes are still intact, indicating
that the vinculin-IpaA binding domain has a functional role in inducing
the IpaA-induced cytoskeletal rearrangements. Although the mechanism for
stimulating F-actin depolymerization is speculative, the activity of IpaA for
induction of actin depolymerization by means of the vinculin-IpaA com-
plex seems to be important in regulating the formation of protrusions that
might promote detachment of Shigella. Alternatively, the IpaA-induced acti-
vation of vinculin and recruitment of focal adhesion components may lead
to the formation of a specific focal adhesion-like structure that might help to
maintain bacterial contact with the epithelial surface (Bourdet-Sicard et al.,
1999).

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IpaC

IpaC has an activity to modulate actin dynamism, because the forma-
tion of filopodia and lamellipodia is induced when purified IpaC protein is
added to semipermeabilized Swiss 3T3 cells or a ipaC clone is transfected into
HeLa cells (Tran van Nhieuet al., 1999). The IpaC-induced membrane protru-
sions have been implicated in the activation of Cdc42, which in turn activates
Racl, suggesting that IpaC can somehow act as the effector for promoting
Shigella invasion of epithelial cells (Fig. 2.4). In addition, a recent study has
indicated that Shigella direct their own entry into macrophages by exploit-
ing IpaC to stimulate macrophage phagocytic activity (Kuwae et al., 2001).
Indeed, Shigella invade murine macrophages such as J 774 more efficiently
than the noninvasive ipaC mutants. Wild type Shigella can induce large-scale
lamellipodial extensions including ruffle formation around the bacteria. In
contrast, when macrophages are infected with the noninvasive ipaC mutant, S

the invasiveness and induction of membrane extension are dramatically re- g

duced. Shigella infection of J 774 cells causes tyrosine phosphorylation of
several proteins, including paxillin and c-Cbl, and the profile of phosphory-
lated protein is distinctive from that stimulated by S. typhimurium or phorbol 5

ester. Upon addition of a recombinant IpaC into the external medium of
J 774, membrane extensions were rapidly induced, and this also promoted
uptake of E. coli. Importantly, the exogenously added IpaC was shown to be
integrated into the macrophage plasma membrane. An analysis of the IpaC
sequence (382 amino acids) with TMpred, a program for the prediction of pu-
tative membrane-spanning regions, predicts that the residues encompassing
121-139 and 169-191 are the putative transmembrane domains, whereas the
remaining N-terminal and C-terminal regions are predicted to be presented
on the external side of the plasma membrane.

With the use of three IpaC antibodies that recognize three distinctive
regions in IpaC, it has been suggested that the residues 140-168 of IpaC
exist as the cytoplasmic loop, whereas the preceding N-terminal portion
of TM1 (transmembrane 1) and the following C-terminal portion of TM2
(transmembrane 2) exist as the external membrane domains (Kuwae et al.,
2001) . Although the mechanisms underlying the IpaC-mediated macrophage
spreading event are still to be elucidated, some surface receptors such as
Mac-1 (oiuPi integrin) and Fey R on macrophages seem to be involved in the
phagocytic event.

Incubation of J774 cells with wild type Shigella, but not with the ipaC
deletion mutant, generates Mac-1 and FcyR foci at the site of bacterial
contact. Importantly, the Mac-1 foci were observed along the periphery of
the extended cell membrane, strongly indicating that an integrin-dependent

adhesion event, which is a prominent feature of the Mac- 1 -mediated
macrophage adherence, had occurred. The observed macrophage response is
consistent with the study by Renesto et al. (1996), in which PMNs suspended
in medium became adherent onto serum-coated wells when wild type Shigella
but not the ipaC mutant was added to the external medium.

Clustering of Mac-1 and FcyR has been indicated to stimulate protein
tyrosine phosphorylation and local rearrangement of the actin cytoskeleton.
Because some truncated versions of IpaC capable of associating with the host
plasma membrane are able to stimulate macrophage cell spreading (Kuwae
et al., 2001), IpaC might have an association with or effect on some pu-
tative host receptor(s), such as otuPi integrin, mediating the induction of
membrane extension by means of activation of a cellular signal transduction
pathway such as the activation of Cdc42. The IpaC-induced membrane pro-
g trusions from HeLa cells can be inhibited by a dominant interfering form of

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m Cdc42 , whereas a dominant interfering form of Rac results in inhibition of the

< lamellipodium formation, further supporting the notion that IpaC has activ-

§ ity to stimulate Cdc42 activity. This in turn causes Racl activation (Mounier

et al., 1999; Tran van Nhieu et al., 1999). Importantly, recent studies have
suggested that activated Racl stimulates WAVE2, a WASP (Wiskott-Aldrich
syndrome protein) family protein, via IRSp53, a substrate for the insulin re-
ceptor, by forming a Racl-IRSp53-WAVE2 complex, which recruits Arp2/3
complex including profilin, thus evoking membrane ruffling (Mild et al.,
2000; Takenawa and Miki, 2000; Krugmann et al, 2001). Because WAVE2
can be detected around the area of protruded membrane ruffles induced
by Shigella (Suzuki, unpublished data), the Racl-IRSp53-WAVE2 complex
might take part in the formation of membrane protrusion induced by Shigella
invasion (Fig. 2.4).

IpgD

IpgD, a 69-kDa protein encoded by ipgD and located upstream of
ipaBCDA, is secreted by the TTSS in amounts similar to the Ipa proteins. Like
the Ipa proteins, IpgD is stored in the Shigella cytoplasm unless the TTSS
is stimulated such as by incubation in conditioned medium (Niebuhr et al.,
2000). The storage of IpgD in the cytoplasm requires its association with
a cytoplasmic chaperone, IpgE, encoded by the gene located immediately
downstream of ipgD. Interestingly, after secretion, IpgD forms a complex
with IpaA in the extracellular medium, although the biological significance
is unclear. An ipgD mutant still enters host cells; however, in comparison
with that directed by the wild type, the morphology of the membrane ruffle is
altered. For example, scanning electron microscopic analysis reveals that the

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>

ipgD mutant provokes fewer actin rearrangements and less membrane ruf-
fling on the target cell surface, suggesting that IpgD is involved in the process
of invasion of epithelial cells and that the protein serves as a translocated ef-
fector (Niebuhr et al., 2000). A protein homologous to IpgD, SopB (also called
SigD), has been identified in S. duhlin, and it is involved in invasion, because
a sigD mutant affected Salmonella invasion of CHO and HEp-2 cells (Galyov et
al., 1997; Hong and Miller 1998). SopB has sequence homology with mam-
malian inositol polyphosphate 4-phosphatase, and recombinant SopB pro-
tein prepared from S. dublin shows inositol phosphate phosphatase activity
required for promoting membrane fission during invasion (Norris et al.,
1998; Terebiznik et al., 2002).

Similarly, the sequence of IpgD has been suggested to have the active
site of mammalian inositol polyphosphate 4-phospatase, which specifically
dephosphorylates PtdIns(4,5)P 2 into PtdIns(5)P (Niebuhr et al, 2000, 2002). g

In mammalian cells this enzyme plays key roles in many processes, includ- £

ing reorganization of the actin cytoskeleton, and cytoskeleton-plasma mem-
brane linkage. The importance of the enzymatic activity encoded by IpgD has
been shown in promoting detachment of the plasma membrane from the cy-
toskeleton to facilitate extension of membrane filopodia and ruffles evoked
by Shigella invasion of epithelial cells. Indeed, continuous ectopic expression
of IpgD in HeLa cells increases membrane detachment and causes formation
of membrane blebs (Niebuhr et al., 2002).

VirA

An examination of the cytoskeletal architecture around invading Shigella
by confocal microscopy indicated that the local microtubule network beneath
the protruding ruffles undergoes remarkable destruction (Yoshida et al.,
2002). This finding, together with the increase in Shigella invasiveness in
host cells treated with microtubule -de stabilizing agents such as nocodazole,
suggests that the bacteria have the ability to modulate tubulin dynamics.
VirA has activity to trigger microtubule dynamic instability in vitro and in
vivo, which can stimulate Racl activity, thus leading to membrane ruffling
(Fig. 2.4).

VirA is a 45-kDa protein composed of 401 amino acids; it is able to
bind a/3-tubulin dimers but not microtubules. In an in vitro tubulin poly-
merization assay system, purified VirA showed activity to inhibit the poly-
merization of tubulin and stimulate microtubule de stabilization. Interest-
ingly, a portion of VirA, encompassing residues 224 to 315, involved in
the interaction with tubulin heterodimers shares significant (>40%) amino
acid homology with a portion of EspG encoded by the espG gene in the

LEE of EPEC or enterohemorrhagic E. coli, as well with as some other un-
characterized bacterial proteins such as NMB0928 (Neisseria menigitidis) or
Cjl457c (Campylobacter jejuni) . Indeed, the expression of EspG in a Shigella
virA mutant can rescue invasiveness, suggesting that EspG and VirA share
an essential function (Elliott et al., 2001). The expression of VirA in mam-
malian cells such as HeLa, COS-7, or Swiss3T3 cells allows for the forma-
tion of membrane ruffling, though the scale of ruffles is smaller than that
evoked by Shigella. Microinjection of VirA into HeLa cells also induces a
localized membrane ruffling in a few minutes, whereas overexpression of
VirA in host cells causes the destruction of microtubules and protruding
membrane ruffles. Importantly, the VirA-induced membrane ruffling is de-
pendent on the host Racl activity, because when VirA is coexpressed with a
dominant negative Racl mutant in the cells, the appearance of ruffles can be
g shut off.

m In agreement with this, wild type S. jlexneri, but not the virA mutant,

< stimulates Racl and induces the formation of membrane ruffles in infected

§ HeLa cells (Yoshida et al., 2002). These observations suggest that the desta-

i bilization of microtubules induced by VirA secreted from Shigella into host

u cells can provoke the formation of membrane ruffles, thus stimulating bacte-

rial entry (Fig. 2.4). Although it is still unclear whether or not other invasive
bacteria are able to stimulate host microtubule dynamic instability, a simi-
lar activity to Shigella VirA may also be found to be involved in some other
bacterial infections of host cells.

The microtubule network is dynamic in migrating or growing cells in
which the microtubules undergo growth and shortening, called microtubule
dynamic instability, which is mediated by various factors including micro-
tubule stabilizing and destabilizing factors. For migrating cells, the interplay
between the microtubule and actin cytoskeletal systems is though to be cru-
cial. Indeed, recent studies have strongly indicated that microtubule growth
and shortening participate in the activation of Racl and RhoA signaling,
respectively, to control actin dynamics.

Waterman- Storer et al. (1999) revealed that when microtubule growth
in host cells is stimulated by pretreatmenting with nocodazole followed by
washing out the drug, Racl is activitated, thus leading to the formation of
lamellipodial protrusions in fibroblasts. Enomoto (1996) originally showed
that microtubule disruption by colcemid or vinblastine, but not taxol, rapidly
and reversibly induced the formation of actin stress fibers and focal adhe-
sions, which was accompanied by activated cell motility. Consistent with
that study, Krendel et al. (2002) have observed that RhoA activity in fibro-
blasts can be stimulated by nocodazole. These studies have suggested that
the depolymerization or shortening of microtubules can somehow trigger the

stimulation of Rho activity, such as by releasing factors bound to the micro-
tubules into the cytosol, and these factors are then required for activating Rho
GTPases beneath the plasma membrane (Fig. 2.4).

Of note, this notion has recently been supported by the finding of func-
tional involvement of microtubules in regulating the Rho guanine nucleotide
exchange factor GEF-H1 and Rho activity itself (Krendel et al., 2002). Al-
though the association of microtubules and Racl or GEF-H1 has been in-
dicated, the function of this interaction is only recently becoming clear. In-
terestingly, recent studies have strongly indicated that Racl and RhoA have
some functional linkage to each other, where the enhancement of one ac-
tivity downregulates activity of the other. Therefore, the cross-talk between
Racl and RhoA activities may account for the microtubule instability-induced
membrane ruffling in mammalian cells as well as for the ruffling induced
by Shigella VirA (Fig. 2.4). £

CELL-CELL SPREADING

After escaping from phagocytic vacuoles, Shigella multiply and move
within the cytoplasm. The ability of Shigella to move within the host cyto-
plasm, and the subsequent cell-cell spreading, is a prerequisite for shigel-
losis. Intracellular Shigella exploit actin polymerization at one pole of the
bacterial surface, through which the bacterium gains a propulsive force to
spread within the cytoplasm and into adjacent epithelial cells (Fig. 2.5). Under
optimum conditions, intracellular bacterial motility is around 1 5-20 /xm/min
(Mimuro et al., 2000). The actin-based motility of Shigella is dependent on
VirG (IcsA) encoded by the virG gene on the large plasmid (Makino et al.,
1986; Bernardini et al, 1989; Lett et al, 1989).

VirG

VirG (IcsA) is a surface-exposed outer membrane protein, which accu-
mulates at one pole of the bacterium (Fig. 2.6; also see Goldberg et al., 1993;
Goldberg, 2001). VirG is composed of 1102 amino acids and contains three
distinctive domains: the N-terminal signal sequence (residues 1-52), the 706-
amino-acid a -domain (residues 53-758), and the 344-amino-acid C-terminal
/3-core (residues 759-1102; see Goldberg et al., 1993; Suzuki et al., 1995).
The a-domain is exposed on the surface of bacteria, whereas the /?-core is
embedded in the outer membrane to form a membrane pore. The a -domain
is translocated through the membrane pore onto the bacterial surface by a
typical autotransporter mechanism as represented by the IgA protease of
N. gonorrhoeae (Pohlner et al., 1987).

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Figure 2.5. Electron micrograph of motile Shigella in epithelial cells. (A) Bacteria escaping
from the phagocytic vacuoles. (B) Multiplied bacteria within the host cell cytoplasm. (C) A
motile bacterium forming a long actin tail. (D) A motile bacterium entering the neighbor
host cell.

The asymmetric distribution of VirG along the bacterial body is a pre-
requisite for the polar movement of Shigella in mammalian cells, including
bacterial spreading between epithelial cells (Goldberg et al., 1993; Suzuki
et al., 1995; Goldberg, 2001). Although the mechanisms are still specula-
tive, recently studies have suggested that the unipolar localization of VirG
results from its direct targeting of the pole following diffusion laterally in
the outer membrane (Goldberg, 2001; Charles et al., 2001; Robbins et al.,
2001). Interestingly, when a VirG-GFP fusion protein is expressed in E. coli,
S. typhimurium, Yersinia pseudotuberculosis, or Vibrio cholerae, the protein al-
ways localizes at one pole, suggesting that the mechanism of polar targeting
for VirG is not unique to Shigella. Several factors including its own VirG a
portion have been implicated in the establishment or maintenance of the
asymmetric distribution (Suzuki et al., 1995; Steinhauer et al., 1999). The
N -terminal two thirds of the VirG a -domain, which contains six glycine-rich
repeats, is essential for mediating actin assembly, because the domain serves

Shigella

profilin

▲

profilin

G-actin

direction of
bacterial movement

Arp2/3

Figure 2.6. Current model for VirG-induced actin polymerization on Shigella in infected
epithelial cells. VirG accumulated at one pole of bacterium recruits vinculin and N-WASP.
The vinculin recruits profilin by means of binding to VASP; the N-WASP activated upon
binding by Cdc42 allows recruitment of the Arp2/3 complex, with profilin. The activated
Arp2/3 complex, with the aid of profilin, can catalyze rapid actin nucleation and
elongation.

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to interact with host proteins such as vinculin and neural WASP (N-WASP;
see Suzuki et al., 1996, 1998; Suzuki and Sasakawa, 2001). The C-terminal
one third of the a -domain is required for VirG to distribute asymmetrically,
because S.flexneri expressing a VirG mutant with a deletion in this region no
longer displays polar movement; instead, it is surrounded by an actin cloud
(Suzuki etal, 1996).

Interestingly, LPS plays a role in either the establishment or maintenance
of VirG at one pole of Shigella (Okada et al., 1991). A number of genes

involved in the biosynthesis of LPS have been shown to affect the localization
of VirG (Rajakumar et al., 1994). Indeed, removal of the O-side chain from
the LPS of S.flexneri results in an aberrant localization of VirG, causing a
circumferential distribution over the whole bacterial body (Goldberg, 2001).
SopA (also called IcsP), an outer membrane protease, has also been indicated
to be involved in the asymmetric distribution of VirG by cleaving laterally
diffused VirG protein along the bacterial body (Egile et al., 1997; Shere et al.,
1997). Finally, the absence of OmpT, an outer membrane protease encoded
by the ompT gene in E. coli, is crucial for VirG to be maintained on the cell
surface, because OmpT specifically cleaves at Arg758-Arg 7 59 of VirG, causing
degradation of the a -domain of VirG on bacteria (Nakata et al., 1993). In fact,
none of the Shigella and EIEC strains examined has the ompT region, thus
ensuring the VirG a-domain is expressed and maintained on the bacterial
g surface (Nakata et al., 1993).

i— i

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VirG ligands

VirG can interact with at least two host proteins, vinculin and N-WASP
(Fig. 2.6). Vinculin, a protein linking focal adhesions and actin filaments,
interacts directly with a portion of the VirG a -domain that spans residues
103-508. As already mentioned (see the subsection on IpaA), the function
of vinculin in mammalian cells is regulated by PtdIns(4,5)P2. In the inac-
tive state, the N -terminal globular head domain interacts with the C -terminal
elongated tail domain, and this interaction is disrupted by the binding of
PtdIns(4,5)P2- The exposed head and tail domains become activated to inter-
act with other molecules. In epithelial cells infected with Shigella, vinculin
is recruited to the bacterial surface as well as to the actin comet tail elon-
gated from motile bacteria in infected cells (Suzuki et al., 1996). Laine et al.
(1997) revealed that the recruited vinculin is cleaved, leaving the head portion,
which interacts with VirG along with vasodilator stimulating phosphoprotein
(VASP) and pro film. Thus, the complex formed in the vicinity of the bac-
terium seems to contribute to enhancing the growth of barbed ends of actin
filaments. Microinjection of the vinculin head portion into Shigella-infected
cells stimulates the bacterial motility (Laine et al., 1997). In fact, the speed at
which E. coli expressing VirG induce formation of the actin tail in vinculin-
depleted Xenopus egg extracts is shown to be significantly decreased to less
than 30% of the original level (Suzuki, unpublished data). Thus, vinculin
appears to contribute to Shigella-mducing actin assembly such as through
interaction with VASP, because VASP recruits profilin (Fig. 2.6). Alterna-
tively, existing actin filaments bound by vinculin at the bacterial surface may

N-WASP

WASP

WAVE2

Basic

VCA

Figure 2.7. Functional domains of N-WASP, WASP, and WAVE2 and molecules that
interact with these proteins (Takenawa and Miki, 2000).

facilitate actin nucleation mediated by the Arp2/3 complex interacting with
the VirG-N-WASP complex.

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N-WASP

N-WASP is a member of the WASP and WAVE family, which includes
human WASP, Saccharomyces cerevisiae WASP-like protein Lasl7p/Beelp,
and WAVE1, WAVE2, and WAVE3 proteins (Takenawa and Miki, 2000).
WASP and WAVE family proteins integrate upstream signaling events with
changes in the actin cytoskeleton by means of the Arp2/3 complex. Fig-
ure 2.7 shows the functional structures of N-WASP, WASP, and WAVE2,
and molecules to which they bind (Takenawa and Miki, 2000). The expres-
sion of WASP is limited to hematopoietic cells, whereas N-WASP and WAVEs
are ubiquitously expressed in host cells including epithelial cells. N-WASP
has been implicated both in the formation of filopodia and in the actin-based
motility of intracellular Shigella, whereas WAVEs are involved in formation
of lamellipodia and membrane ruffling. N-WASP and WASP possess several
distinctive domains: a homology (WH1) domain that binds PtdIns(4,5)P 2 , a
domain composed of basic amino acids, a GTPase binding domain (GBD)

that binds Cdc42 , a proline-rich region ( P RR) , a G-actin-binding verprolin ho-
mology (V) domain, a domain (C) with homology to the actin-depolymerizing
protein cofilin, and finally a C-terminal acidic (A) segment (Fig. 2.7). The
C-terminal VCA domain mediates the interaction with the Arp2/3 com-
plex, by which the Arp2/3 complex is activated, thus mediating actin
polymerization.

In Shigella-infected cells, N-WASP, but not the other members of the
WASP family, accumulates at the pole of the intracellular bacterium assem-
bling an actin comet tail (Suzuki et al., 2002). Functional assays using the
ectopic expression of dominant negative N-WASP in mammalian cells or
immunodepletion in Xenopus egg extracts revealed that N-WASP is an essen-
tial host component for mediating the actin-based motility of intracellular
Shigella (Suzuki et al., 1998). Of note, none of the WASP family proteins as-
g sociate with the surface of intracellular I. monocytogenes including the actin

i— i

X
u

m tails (Suzuki et al., 2002). The binding of Shigella VirG to WASP family pro-

< teins is limited to only N-WASP. With the use of a series of chimeras obtained

§ by swapping the N-WASP and WASP domains, the specificity of VirG to in-

teract with the N-terminal WH1 region of N-WASP was found to serve as
the critical ligand (Suzuki et al., 2002). Consistent with this, hematopoietic
cells such as J 774 cells (mouse macrophages), human monocytes, PMNs, or
platelets express WASP predominantly but not N-WASP, which cannot sup-
port the actin-based movement of intracellular S.flexneri (Egile et al., 1999;
Suzuki et al., 2002). This was also confirmed by use of N-WASP-deficient
embryos (Snapper et al., 2001).

Cdc42

In vitro studies have indicated that activation of N-WASP in cells re-
quires the binding of Cdc42 to the GBD motif of N-WASP (Mild et al., 1996;
Rohatgi et al., 1999, 2000). This binding inhibits the intramolecular interac-
tion between the C-terminal acidic amino acids and the basic amino acids
near the GBD, thus causing the unfolding of N-WASP into the activated form.
Furthermore, when N-WASP interacts with a fragment of VirG encompass-
ing residues 53-503 of VirG, the N-WASP-Arp2/3 complex-mediated actin
nucleation can also be stimulated without Cdc42 in vitro (Egile et al., 1999).
The ability of VirG to activate N-WASP without Cdc42 was also observed by
using Clostridium difficile Tcd-10463, which inhibits Rho GTPases; however,
the actin tails are significantly shorter in the presence of the exotoxin than
in infected cells without the toxin, implying that actin assembly by Shigella
is partly affected by toxin (Mounier et al., 1999). A later study, however,
strongly indicated that cellular Cdc42 is required for the actin-based motility
of Shigella in infected cells (Suzuki et al., 2000). Microinjection of activated

Cdc42 accelerates Shigella motility, whereas inhibiting Cdc42 activity, for
example by adding Rho GDI, a guanine nucleotide dissociation inhibitor,
into cell extracts greatly reduces bacterial motility. In pyrene actin polymer-
ization assays, VirG-N-WASP-Arp2/3 complex is insufficient to express the
full activity for polymerizing actin; rather, in the presence of activated Cdc42,
the actin nucleation activity is remarkably stimulated. In fact, Cdc42 can be
accumulated at one pole of Shigella in the process of initiating movement
in infected cells. Importantly, Cdc42 is not accumulated on motile Shigella
possessing an actin tail in the infected cells, implying that Cdc42 seems to be
no longer necessary after a steady speed has been reached, at which stage the
VirG-N-WASP-Arp2/3 complexes would be constitutively activated. These
studies have thus indicated that Cdc42 takes part in initiating the actin-based
motility of intracellular Shigella in epithelial cells.

Co
H

Arp2/3

VirG expressed on the bacterial surface in host cells can directly recruit <

and activate N-WASP, which in turn recruits and activates the Arp2/3 com-
plex. Consequently, the VirG-N-WASP-Arp2/3 complex formed at one pole
of the bacterium can mediate actin nucleation and elongation (Fig. 2.6). To
initiate actin nucleation, the Arp2/3 complex is activated upon physical inter-
action with the VCA region of N-WASP. With the aid of other host factors, the
VirG-N-WASP-Arp2/3 complex mediates rapid actin filament growth at the
barbed end, including cross-linking between the elongated actin filaments.
In this way Shigella can gain a propulsive force in the host cytoplasm, where
some motile bacteria impinge on the host plasma membrane, leading to the
extension of membranous protrusions. Through these protrusions that pen-
etrate neighboring cells, Shigella further move into adjacent epithelial cells by
disrupting the double membranes with the secreted proteins (Suzuki et al.,
1994; Schuch et al., 1999). In a reconstitution experiment supporting the
actin-based motility of Shigella with pure proteins, host factors required
for Shigella movement were confirmed to include actin, Arp2/3 complex,
and N-WASP (Loisel et al., 1999). In addition, actin depolymerization factor
(ADF)/cofilin, capping protein, and profilin are also indicated to be involved
in the regulation of actin turnover and stabilization of the actin tail (Loisel
et al., 1999; Mimuro et al., 2000). Several other actin-associated proteins, such
as plastin (fimbrin), filamin, VASP, zyxin, ezrin, CapZ, Nek, and WASP in-
teracting protein (WIP), have also been identified as being localized to the
actin tail or at the posterior end of intracellular bacteria. However, whether
or not these host factors are functionally required for Shigella movement in
infected cells awaits further study.

Profilin

Profilin binding to actin facilitates the formation of ATP-actin
monomers, the form of actin to be assembled into the free barbed ends of
actin filaments. Profilin can interact with various proteins, notably proteins
with a proline-rich sequence, such as N-WASP, VASP, MENA, pl40mDia,
WAVE/Scar, and Arp2/3 complex (Suzuki and Sasakawa, 2001). Inareconsti-
tution assay in vitro, profilin and VASP (for Listeria) were shown to enhance
bacterial motility but were not essential, suggesting that recruited profilin
helps to increase the local concentration of ATP-actin (Loisel et al., 1999) . Pro-
filin exists in two isoforms in mammalian cells, profilin I and II, with profilin
I having a greater affinity for N-WASP (Kd = 60 nM) than profilin II (Kd = 400
nM). Hence, the role of profilin I in the actin-based motility of intracellular
Shigella has recently been investigated (Mimuro et al., 2000). Upon overex-
| pression of a profilin H133S mutant defective in interaction with the PRR of

gj N-WASP including poly-L-proline, Shigella motility is significantly decreased.

w Similarly, the depletion of profilin from Xenopus egg extracts results in a de-

g crease in bacterial motility that is rescued by adding back profilin I but not

£ H133S mutant. Consistent with this, on overexpression of an N-WASP mu-

tant lacking the PRR unable to interact with profilin, the actin tail formation of
intracellular Shigella is abolished. In N-WASP-depleted extracts, the addition
of wild type N-WASP but not the N-WASP mutant restores bacterial motility,
indicating that profilin associated with N-WASP is an essential host factor for
supporting rapid movement of intracellular Shigella (Mimuro et al., 2000).

CONCLUSION

Invasion of epithelial cells by Shigella is a highly dynamic cellular event
that occurs through the complicated interaction between bacterial effectors
and target host factors. Shigella-directed internalization requires large-scale
membrane ruffling, which eventually leads to a phagocytic event. In pro-
moting the host cellular events including the subsequent infectious steps,
the roles of effectors such as IpaA, IpaB, IpaC, IpgD, and VirA delivered
by means of the TTSS are crucial. However, many other putative effectors
secreted by the TTSS (Buchrieser et al., 2000) must also be involved in al-
most the entire stage of infection, including modulation of host immune
responses. Furthermore, it is thought that targeting of the host factors by
bacterial effectors during infection would appropriately be operated by the
pathogen, for which the timing and amounts of effectors to be secreted by
means of the TTSS may be stringently controlled at a posttranslational or

cotranslational level, depending on the stage of Shigella infection (Blocker et
al., 2003) . Clearly, we must await further study to elucidate the role of all of the
effectors in each stage of Shigella infection, including the secretion control
system. Such information is needed for better understanding of the sophis-
ticated bacterial infectious strategy and host inflammatory responses, which
are prerequisite for the development of both a novel safer Shigella vaccine
and a suitable animal model to study the disease.

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I am grateful to Drs. Toshihiko Suzuki and Reiko Akakura for their
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Immun. 64, 5357-5365.

CHAPTER 3

How Yersinia escapes the host: To Yop
or not to Yop

Geertrui Denecker and Guy R. Cornells

The genus Yersinia contains three species of Gram-negative bacteria that are
pathogenic for humans: Y. pestis, the agent of bubonic plague; Y. pseudotu-
berculosis, causing mesenteric adenitis and septicemia; and Y. enter ocolitica,
causing gastrointestinal syndromes (enteritis and mesenteric lymphadeni-
tis) . Bacteria from these three species have a tropism for lymphoid tissues and
share the common capacity to resist the innate immune response. Whereas
Y. pestis is generally inoculated by a fleabite or aerosol, Y. enterocolitica and
Y. pseudotuberculosis are foodborne pathogens, which gain access to the under-
lying lymphoid tissue (e.g., Peyer's patches) of the intestinal mucosa through
M cells (Fig. 3.1; see Autenrieth and Firsching, 1996; Perry and Fetherston,
1997). Once Yersinia has entered the lymphoid system, it overcomes the pri-
mary immune response of the host by using the type III secretion system
(TTSS) (Cornells et al., 1998; Cornells, 2002). TTSS is a sophisticated viru-
lence mechanism by which Gram-negative pathogens inject effector proteins
directly into host cells. Currently, more than 20 different TTSSs have been
described in animal, plant, and even insect pathogens (Hueck, 1998; Galan
and Collmer, 1999; Cornells, 2000; Buttner and Bonas, 2002).

Depending on the effectors injected, the employment of the TTSS will
have a different outcome. Some, like the Mxi-Spa system of Shigella Jlexneri
or the Salmonella pathogenicity island 1 (SPI-1) system of Salmonella enterica,
make use of the innate immune system of the host to enhance the proinflam-
matory response and to trigger phagocytosis by normally nonphagocytic cells,
whereas others, such as the pathogenic Yersinia Ysc-Yop system, essentially
paralyze the innate immune response of the host (Galan, 2001; Sansonetti,
2001; Cornells, 2002; Juris et al., 2002). The Yersinia TTSS becomes acti-
vated upon contact with eukaryotic cells and directs effector proteins - called
Yops - over the bacterial membranes. Some of the Yops form a kind of

Y. pestis

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Antiphagocytosis:
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Macrophages

Local and

systemic dissemination

Macrophage apoptosis,
Inhibition of proinflammatory
response
3 YopP, -H

Figure 3.1. Model showing the interaction of Yersinia with an eukaryotic cell.
At 37° C and upon contact of Yersinia with its eukaryotic target cell, the adhesins YadA or
Inv interact with the ^i-integrins and other extracellular matrix proteins at the cell surface,
and Yersinia attaches tightly to the cell membrane. The Ysc injectisome is installed and
the Yop translocators and effectors, some of which are intrabacterially capped with a
chaperone, are transported through the bacterial inner and outer membranes by the Ysc
injectisome. The translocators Yops, YopB, YopD, and LcrV form a pore in the target cell
membrane, through which the effector Yops are translocated into the cell cytosol. Four
effector Yops with different enzymatic functions (YopE, a Rho GTPase-activating protein,
YopT, a cysteine protease, YopO/YpkA, a serine/threonine kinase, and YopH, a pro-
tein tyrosine phosphatase) will cooperatively lead to the destruction of the actin cytoskeleton,

(cont.)

translocation pore in the eukaryotic target cell membrane, whereas the other
Yops are effector proteins that are delivered through this pore into the cy-
tosol of the target cell. At least six different Yop effectors are injected by
the secretion translocation apparatus, five of which have been shown to play
an important role in the defense against the innate immune response (Fig.
3.2). In this chapter we discuss how Yersinia escapes the host immune re-
sponse after initial invasion of the intestinal mucosa by (i) its strong resis-
tance to phagocytosis, which is caused by the concerted action of Yop E, YopH,
YopO (called YpkA in Y. pseudotuberculosis and Y. pestis), and YopT, (ii) its ca-
pacity to block the proinflammatory response induced by different immune
cells, caused by the action of Yop P (called Yop J in Y. pseudotuberculosis and
Y. pestis) and YopH, and (iii) its ability to induce cell death of the macrophage,
promoted by YopP/J (DeVinney et al., 2000; Aepfelbacher and Heesemann,
2001; Cornells, 2002; Juris et al, 2002; Orth, 2002). g

Figure 3.1. (cont. ) and by doing so contribute to the antiphagocytic action of Yersinia. Two
Yops (YopH and YopP/J) are involved in the downregulation of the proinflammatory
response of the immune cells, and YopP/J will also lead to the induction of apoptosis in
macrophages. YopM, a protein containing several leucine-rich repeats, is translocated to
the nucleus; however, its function remains unclear. PP = Peyer's patches.

ir,

THE YERSINIA VIRULENCE SYSTEM

Ysc-Yop III secretion system 5j

The Ysc-Yop type III secretion machinery present in all pathogenic

Yersinia is encoded by a 70-kDa virulence plasmid, which harbors the genes jjj

for the Ysc (for Yop secretion) secretion apparatus or Ysc injectisome, for g

an array of proteins secreted by this apparatus - called Yops (for Yersinia ri

outer proteins) - and for a set of proteins controlling the system (Cornells ^

et al., 1998). The Ysc injectisome is composed of a large dual-ring structure *

o
spanning the bacterial inner and outer membrane, which resembles the flag- *

ellum basal body. This structure is associated with a needle-like complex that §

extends outside the bacterium. It mediates the secretion of the Yop effector o

proteins (YopE, YopH, YopO/ YpkA, YopT, YopP/J, and YopM), a structural
component of the needle (YscF), and the components of a translocation appa-
ratus, which are YopB, YopD, and LcrV (Cornells et al., 1998; Cornells, 2002;
also see Fig. 3.2). The latter proteins are inserted into the host membranes
and form a kind of pore, which allows the delivery of the Yop effector proteins
into the cytosol of the cell. Whether the Ysc injectisome and the translocation

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Yersinia

Figure 3.2. Schematic representation of entry routes during Yersinia infection in humans.
Y. pestis is generally inoculated by fleabites or aerosol and enters the bloodstream directly.
Y. enterocolitica and Y. pseudotuberculosis are both foodborne pathogens and enter the
underlying lymphoid tissue (e.g., Peyer's patches) of the intestinal mucosa through M
cells. Once Yersinia has reached the lymphoid system, the plasmid-encoded effector Yops
allow the bacteria to avoid phagocytosis and actively downregulate the proinflammatory
response in order to promote their extracellular survival. OM = outer membrane;
P = periplasm; IM = inner membrane; LRR = leucine-rich repeat.

pore form a continuous channel connected by the needle is currently being
investigated.

Type Ill-dependent protein secretion in Yersinia is a tightly regulated
process, and several regulatory circuits control both the expression of the
injection system and the injection of the Yop effector proteins itself. The first
level of regulation involves temperature. Although growth of Yersinia is un-
affected by low temperatures, such as those found in contaminated food
(Y enterocolitica and Y. pseudotuberculosis) or the stomach of the fleas
(Y pestis), the expression of Yop effector proteins is repressed at these low
temperatures. It is only at 37° C that a stock of intracellular Yops is synthe-
sized and the Ysc injectisome is installed. However, the injectisome remains
closed and a mechanism of feedback inhibition prevents a deleterious accu-
mulation of Yops (Cornells et al., 1987). This first level of regulation involves
at least two proteins: a plasmid-encoded transcriptional activator, VirF, and
a chromosome-encoded histone-like protein, YmoA (Cornells et al., 1991;

Lambert de Rouvroit et al., 1992; Rohde et al., 1999). A second level of regu-
lation is close contact with the host cell membrane, which is established by
the bacterial adhesins Inv, YadA, and Ail (Pettersson et al., 1996). It is only at
37° C and upon contact with the eukaryotic cell that the injectisome is opened,
the negative feedback regulation is relieved, and Yersinia starts to inject its
effectors into the cytosol (Francis et al., 2002; Miller, 2002). Yop proteins
destined to be secreted have no classical signal sequence that is cleaved off
during secretion, but nevertheless their N-terminal part (~15 amino acids
or codons) contains the information that is necessary for secretion (Michiels
et al., 1990; Sory et al., 1995; Anderson and Schneewind, 1997).

Furthermore, some secreted proteins require the binding of a special-
ized cytosolic chaperone, called Syc (specific Yersinia chaperone), to be se-
creted (Wattiau et al., 1996). The loss of a chaperone results in the inefficient
secretion of its cognate partner, while the secretion of other proteins re- §

mains unaffected. Chaperones do not share a general homology, but they ^

do share common properties of being small (less than 20 kDa), being acidic 8

(pi ~4-5), and possessing a C-terminal amphipatic helix. The Syc chaper- 2

ones have been proposed to act as (i) bodyguards, preventing degradation or "

premature association of their target; (ii) secretion pilots, being part of the %

signal for recognition of their substrates by the export machinery; (iii) hier- h

archy factors, establishing a hierarchy for Yop delivery into its host cell; or
(iv) antifolding factors, maintaining their substrate in a secretion-competent
state (Frithz-Lindsten et al., 1995; Cheng and Schneewind, 1999; Boyd et al., h

Other virulence mechanisms and TTSS present
in Yersinia species

In addition to the 70-kb virulence plasmid encoding the Ysc-Yop TTSS,
Y. pestis harbors two unique plasmids encoding essential virulence determi-
nants. The 9.5-kb plasmid (pPst/pPCPl) contains the Pla protease, which
enables the spread of Y. pestis from subcutaneous infection sites into the
circulation (Perry and Fetherston, 1997). Pla has been shown to exhibit
coagulase activity and can also activate plasminogen into plasmin. It has
also been reported that Pla can serve as an adhesion-promoting factor for
Y. pestis (Cowan et al., 2000). The 100- to 110-kb plasmid (pFra/pMTl) en-
codes the murine toxin Ymt, a phospholipase D family member, and the
fraction 1 (Fl) capsule-like protein (Perry and Fetherston, 1997). Ymt has

on
H

2000; Stebbins and Galan, 2001; Lee and Schneewind, 2002; Wulff-Strobel g

et al., 2002; Feldman et al., 2002). However, at this stage it is premature to o

decide which of these functions accounts for the need of chaperones. 2

H
O

recently been shown to be an essential factor for the survival of Y. pestis
in the midgut of the flea (Hinnebusch et al., 2002), and Fl has been sug-
gested to be involved in the antiphagocytic activity of Y. pestis and to re-
duce the number of bacteria that interact with the macrophages (Du et al.,
2002).

In mammals, the level of free iron is too low to sustain bacterial growth;
therefore, pathogens possess siderophores that can solubilize the iron bound
to host proteins and transport it to the bacteria. Y. pestis, Y. pseudotuberculosis,
and high-virulence 7. enterocolitica strains carry a chromosomally encoded
high pathogenicity island (H PI), which comprises genes involved in the syn-
thesis of a siderophore called yersiniabactin (Heesemann et al., 1993; Carniel,
2001). This capacity to acquire iron is an essential virulence determinant for
the invading Yersinia bacteria, and it endows them with the ability to multiply
in the host and cause systemic infections.
S Recently a second TTSS of Y. enterocolitica, called Ysa (for Yersinia

secretion apparatus) and its substrates for secretion - Ysp proteins - has

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& been described (Haller et al., 2000; Foultier et al., 2002). Interestingly, the

g chromosome-encoded Ysa-Ysp TTS S of Y. enterocolitica is similar to the Mxi-

^ Spa TTSS of Shigella and to the SPI-1 encoded TTSS of S. enterica, but it

g is different from another chromosome-encoded TTSS of Y. pestis (Parkhill

g et al., 2001). In addition, the ysa locus is only present in the high -virulence

biotype IB strains of Y. enterocolitica and, at least in laboratory conditions,

h is only operational at low temperature (Haller et al., 2000; Foultier et al.,

S 2002) . Whether this Ysa TTSS plays a role in the high -virulence phenotype of

Y. enterocolitica or in a yet to be identified cold-blooded host is unclear at the
moment and awaits further in vivo experiments.

FIRST CONTACT

Interaction of the enteropathogenic Y. enterocolitica and
Y. pseudotuberculosis with M cells

Y. enterocolitica and Y. pseudotuberculosis possess two different adhesins:
the chromosomally encoded Inv (Invasin) and the pYV plasmid-encoded
YadA (Yersinia adherence protein A; see Boland and Cornells, 2000) . They me-
diate initial adhesion, uptake, and translocation of the bacteria through the
M cells, covering the Peyer's patches, to the underlying lymphoid tissues,
where the bacteria remain extracellular, multiply, and eventually migrate to
deeper tissues such as liver and spleen (Fig. 3.1; also see Sansonetti, 2002).
The Inv protein has been shown to be important for the initial step of inva-
sion by its strong interaction with host /^-integrin expressed on the apical

membranes of the M cells (Pepe and Miller, 1993; Berton and Lowell, 1999;
Schulte et al. , 2000) . The cytoplasmic domain of integrins will transmit signals
to the cell cytoskeleton that mediate internalization of Yersinia by a "zipper-
ing" process (Isberg et al., 2000). As epithelial cells only express integrins at
their basal membrane, the enterocytes are not expected to be heavily invaded
during oral infection. Indeed, an analysis of intestines of infected mice shows
that Y enterocolitica is only found in sections that contain Peyer's patches.
This indicates that M cells, rather than enterocytes, form the major port of
entry for Yersinia.

After this initial step of invasion, the YadA protein seems to be the pre-
dominant adhesin, mediating adherence through interaction with extracel-
lular matrix proteins such as fibronectin and collagen (El Tahir and Skurnik,
2001). YadA also protects Y. enterocolitica against the bactericidal and opso-
nizing action of complement by binding complement factor H (China et al., §
1993). Once the dome is reached, Yersiniae survive attack by professional ^
macrophages by injecting antiphagocytic Yops (see what follows) that disrupt £
the cytoskeleton. Yersinia will thus essentially remain extracellular, which al- 2
lows its survival and possible Inv-mediated entry into nonphagocytic cells, "
but this is not well documented. %

on
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Y. pestis enters the bloodstream immediately g

Y. pestis is a pathogen primarily affecting rodents, which is usually trans- h

mitted to humans by a fleabite (Fig. 3.1). When a flea ingests a blood meal har- g

boring Y. pestis, the ingested Yersinia secretes a coagulase that clots the blood o

and thus prevents the flea from swallowing the bacteria. Ymt, a plasmid- 2

encoded and intrabacterially expressed phospholipase D, protects the bac- h

terium from a cytotoxic digestion product of blood plasma in the flea gut g

(Hinnebusch et al., 1998; Hinnebusch et al., 2002). After multiplying in the
clotted blood, Y. pestis is transmitted efficiently into a human host when
the hungry flea repeatedly attempts to feed and the blood clot is regurgi-
tated into the host (Perry and Fetherston, 1997; Cole and Buchrieser, 2001).
The bacterium then spreads from the site of infection to the regional lymph
nodes, where it grows to high numbers and causes swelling of the lymph
node (bubo), resulting in bubonic plague. If the lymphatic system becomes
overwhelmed, the infection rapidly spreads into the lymphstream and blood-
stream, causing fatal blood poisoning, followed by colonization of all the main
organs (including the lungs). It is notable that Y pestis lacks functional YadA
and Inv, which are present in its enteropathogenic relatives. However, some
studies indicate that Y. pestis may invade and cause systemic infection from
digestive and aerogenic routes of infection.

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Figure 3.3. (facing page). Structural diagrams of the Yop effectors and their enzymatic
function. Except for YopM, which is an LRR protein of unknown function, the enzymatic
function of all the effector Yops has been identified, as indicated. The amino acids
important for their catalytic function are depicted. All six Yop effectors have a short
N-terminal secretion signal (~15 amino acids or codons), which is necessary for their
secretion. Three of them (YopE, YopT, and YopH) also contain a chaperone-binding site.

HOW YERSINIA ESCAPES THE HOST: TO YOP OR NOT TO YOP
Inhibition of phagocytosis

When a nonpathogenic bacterium enters the host organism, it is usually
engulfed by professional phagocytes, such as macrophages, neutrophils, or
dendritic cells (May and Machesky, 2001; Underhill and Ozinsky, 2002a).
Phagocytosis of a bacterium is usually preceded by the activation of many
signaling pathways, causing rearrangement of the actin cyto skeleton, exten-
sion of the plasma membrane, and finally engulfment. Members of the Rho
family GTPases (Cdc42, Rac, and Rho) play a central role in this process, as
they are key regulators of the actin cytoskeleton dynamics associated with ad-
hesion, membrane ruffling, and stress fiber formation (Hall, 1998; Bar-Sagi
and Hall, 2000; Chimini and Chavrier, 2000). Furthermore, the formation of
focal adhesion complexes, mediated by the action of paxillin, pl30Cas, and g

focal adhesion kinase (FAK) , at points of contact with bacteria may also play a ^

role in phagocytosis (Greenberg et al., 1993; Allen and Aderem, 1996; Berton g

and Lowell, 1999). Finally, the phosphoinositide 3-kinase (PI3K), phospholi- 2

pase C (PLC), and protein kinase C (PKC) signaling pathways are integration S

points for regulating phagocytosis. Pathogenic Yersiniae subvert several of £

these pathways by injecting YopE, YopT, YopO/YpkA, and YopH. This en- h

sures an extracellular lifestyle and propagation in the host. It was recently M

x
demonstrated that deletion of either YopE, YopT, YopO/YpkA, or YopH ren- g

ders Yersinia more susceptible to phagocytosis by macrophages and PMNs, h

and thus that the concerted action of all four antiphagocytic Yops is necessary g

for full protection against phagocytosis (Grosdent et al., 2002). o

z
o

H
H

Translocation of YopE into mammalian cells leads to a cytotoxic re- *

sponse, characterized by rounding up of the cells and detachment from the
extracellular matrix (Rosqvist et al., 1990). YopE is one of the earliest iden-
tified Yop effectors that has an inhibitory effect on the actin cytoskeleton by
inactivation of the Rho family GTPases (Figs. 3.3 and 3.4A; also see Black and

I— I

1-1
w

O
U

w
W

u
w

w
Q

i— i

&
Pi

w
w

GTP

Rho YopT
Cdc42
Rac

Actin

YopE
YopT
YpkA

Inhibition of
phagocytosis

Rho

Filopodia

and

microspikes

GTP . GTP

Rac Cdc42

Lamellipodia

and

membrane ruffles

Stress fiber
formation

Actin-based
Cytoskeletal rearrangements

Figure 3.4. Molecular mechanism of the Yop effectors in the host cell. (A) Antiphagocytic
action of YopE, YopT, and YopO/YpkA. Upon contact of Yersinia with a phagocytic
receptor, the Rho family members (Rho, Rac, and Cdc42) are targeted to the cell
membrane and converted to their GTP-activated state, which promotes actin
polymerization and facilitates phagocytosis. YopE, acting as a GAP, will transiently

(cont.)

Bliska, 2000; Von Pawel-Rammingen et al., 2000). Rho GTPases are reg-
ulated at different levels (Hall, 1998; Ridley, 2001). Cytosolic GDP-bound
Rho proteins are normally posttranslationally modified by prenylation at the
C-terminus, which is important for their translocation to the cell membrane.
The guanine nucleotide exchange factors (GEFs) induce the release of bound

Figure 3.4. (cont. ) downregulate Rho, Rac, and Cdc42 by converting them into the

inactive GDP-bound state. The YopT cysteine protease cleaves the C-terminus of Rho,

Rac, and Cdc42, removing the prenyl group of the Rho GTPases and liberating them from

the plasma membrane. The YopO/YpkA serine/threonine kinase becomes

autophosphorylated upon contact with actin and interacts with Rho and Rac; however, its

real cellular target is unknown. The concerted action of these three Yops will lead to a

destruction of the actin cytoskeleton network and in this way inhibit phagocytosis. FR, ^

phagocytic receptor; ECM, extracellular matrix. (B) Antiphagocytic and anti-inflammatory ^

action of YopH. The interaction between Yersinia and the eukaryotic cell surface causes a 3

rapid tyrosine phosphorylation of adhesion complexes to mediate uptake of Yersinia. To j3

exert its antiphagocytic role, the phosphotyrosine phosphatase YopH is targeted to the £

focal adhesion complexes, where it dephosphorylates proteins such as the FAK, pl30Cas,

n
>
and paxillin, and to other not yet well-characterized adhesion-regulated complexes in g

macrophages to dephosphorylate Fyb and SKAP-HOM. By inhibition of the PI3K/Akt h

signaling pathway, YopH also contributes to the downregulation of the inflammatory M

response. Upon infection, Akt is activated and will phosphorylate several proteins that are o

involved in apoptosis, cellular proliferation, and cytokine/chemokine production, such as ^

glycogen synthase kinase 3 (GSK3) or the transcription factors of the forkhead family (e.g., ^

o
FKHR), and locks them in their phosphorylated inactive state. Inhibition of this pathway ^

by YopH is presumably responsible for the inhibition of T-cell proliferation, IL-2 w

production, and MCP-1 production. PTEN, phosphatase and tensin homologue deleted on §

chromosome 10; PDK1, phosphoinositide-dependent kinase-1; RTK, receptor tyrosine o

kinase; TCR, T-cell receptor. (C) Model showing the anti- inflammatory and proapoptotic o

role of YopP/J. YopP/J downregulates of the inflammatory response by binding to and

preventing the activation of members of the MAP kinase kinase (MKK) family and of

IKK/3. By blocking both these pathways, YopP/J efficiently shuts down multiple kinase

cascades and the cytokine induction required by the host cell to respond to a bacterial

infection. YopP/J is also responsible for the induction of apoptosis of macrophages, which

probably involves both the downregulation of survival genes and the activation of the

apoptotic cascade upstream of Bid, presumably by interfering with a signaling pathway

triggered from the TLRs. The cysteine protease activity of YopP/J is necessary for both the

downregulation of the inflammatory response and the induction of apoptosis of

macrophages. However, exactly how the YopP-de-sumoylating (de-ubiquitinylating?)

activity is interrelated with the inhibition of the MKKs and IKK/) and the induction of

apoptotic pathways awaits further research. See color section.

GDP and thereby allow binding of GTP. This results in the activation of the
Rho proteins at the cell membrane and binding to their downstream target.
Inactivation of Rho GTPases is regulated by guanine nucleotide dissociation
inhibitors (GDIs) and GTPase-activating proteins (GAPs).

The former produce an inactive complex with the Rho proteins in the
cytosol by masking the prenyl group, and the latter induce the hydrol-
ysis of the bound GTP to GDP, thereby returning the Rho proteins to
their inactive form. During phagocytosis the reorganization of the actin cy-
toskeleton is orchestrated by these Rho family GTPases: Rho controls stress
fiber formation and actin-myosin-based contractility; Cdc42 drives the for-
mation of actin-rich filopodia; and Rac promotes the formation of lamel-
lipodia and membrane ruffles (Hall, 1998). Therefore, they represent ideal
targets for bacterial virulence factors, as their inactivation would block phago-
cytosis and allow the extracellular survival of bacterial. The C-terminal effec-
S tor domain of YopE mimics the activity of eukaryotic GAP, which results

in a transient downregulation of the Rho GTPases, and in this way leads to

I— I

h-l

O
U

P the disruption of the actin cytoskeleton and consequently the inhibition of

g phagocytosis (Black and Bliska, 2000; Von Pawel-Rammingen et al., 2000).

^ However, although it was shown in vitro that YopE has GAP activity

g toward Rho, Rac, and Cdc42, whether these three Rho proteins are all inacti-

g vated in every cell type, or whether there might be other Rho family members

that could be in vivo substrates, awaits further analysis. One clue for the speci-

h ficity of YopE came from a study on human umbilical vein endothelial cells,

S where it was shown that Yop E acted selectively on the Rac-mediated pathways

but had no effect on Cdc42- or Rho-dependent signaling (Andor et al., 2001).
It should be noted that YopE shares a high degree of structural similarity
with the GAP domains of Exoenzyme S (ExoS) of Pseudomonas aeruginosa
and SptP from S. typhimurium, but it has no obvious structural similarity
with known mammalian functional GAP homologs (Evdokimov et al., 2002),
suggesting that they could have evolved separately.

As mentioned before, the Yersinia type III weapon includes a pore, nec-
essary to translocate the effectors into the host (Cornells et al., 1998; Cornells,
2002). In a current model the translocation pore is filled by the Yop effectors
themselves (Hakansson et al., 1996b). However, recently it has been pro-
posed that apart from its antiphagocytic role, injected YopE would also play
a role in minimizing plasma membrane damage caused by pore formation
(Viboud and Bliska, 2001). The GAP function of YopE was demonstrated to
be necessary in preventing pore formation, suggesting that pore formation
itself needs the activation of Rho GTPases.

YopT

The most recently identified effector modulating the Rho family of
GTPases is YopT (Figs. 3.3 and 3.4A). Infection of mammalian cells with
a Y. enterocolitica strain only expressing the YopT effector leads to rounding
up of the cell and disruption of the cytoskeleton, which contributes to the
antiphagocytic activity of YopT (Iriarte and Cornells, 1998; Grosdent et al.,
2002). Translocation of YopT into host cells leads to a modification of RhoA,
resulting in an acidic shift in its pi and redistribution of membrane-bound
RhoA toward the cytosol (Zumbihl et al., 1999). In addition, incubation of
purified cell membranes or artificial lipid vesicles containing RhoA with
purified YopT leads to the release of RhoA to the supernatant (Sorg et al.,
2001). The mechanism of action of YopT was recently unraveled by Shao and
coworkers, who demonstrated that Yersinia YopT, as well as its homologue
AvrPphB from P. aeruginosa, belong to a family of cysteine proteases (Shao o

et al., 2002). YopT recognizes the posttranslational modified Rho GTPases -<

(Rho, Rac, and Cdc42) and proteolytically cleaves them near the C-terminus, <*

which leads to their release from the cell membrane. This cleavage removes £

the prenyl group of the Rho GTPases and results in an irreversible inactiva- g

tion of the targeted Rho GTPases, whereas the action of YopE (GAP) can be £

reverted by the GEFs within the cell. g

YopO/YpkA h

The YopO/YpkA effector is an autophosphorylating serine/threonine o

protein kinase that modulates the cytoskeleton dynamics (Figs. 3.3 and 3.4A) o

and also contributes to resistance to phagocytosis (Galyovet al., 1993; Hakans- g

son et al., 1996a; Grosdent et al., 2002). YopO/YpkA is produced as an in- h

active kinase, which becomes activated after translocation into the host cell g

upon binding to actin (Dukuzumuremyi et al., 2000; Juris et al., 2000). The
N-terminal part of YopO/YpkA contains the kinase domain, whereas the C-
terminal part of the kinase binds to actin. The C-terminal part also contains
sequences that bear similarity to several eukaryotic RhoA-binding kinases,
and it binds to RhoA and Rac but not to Cdc42 (Barz et al., 2000; Dukuzu-
muremyi et al., 2000). The kinase domain is required to localize YopO/YpkA
to the plasma membrane, whereas the C-terminal part is responsible for its
effect on the actin cytoskeleton in Yersmia-infected cells (Dukuzumuremyi
et al., 2000; Juris et al., 2000). However, although YopO/YpkA has a clear
effect on the actin cytoskeleton, its real cellular target is unknown and awaits
further investigation.

YopH

The fourth antiphagocytic Yop is the multifunctional YopH. The 51-kDa
YopH effector protein is composed of two functional domains: the C-terminal
part (residues 206-468) has a structure similar to the one of mammalian
phosphotyrosine phosphatases (PTPases; see Guan and Dixon, 1990), and the
N-terminal part (residues 1-130) is the binding site of YopH to its substrate
(Fig. 3.3; also see Black etal., 1998). Part of this latter domain (residues 20-69)
is also the binding domain for its chaperone SycH, necessary for translocation
into the eukaryotic cell (Wattiau et al., 1996). Upon interaction of the Yersinia
surface protein Inv with fi\ -integrin on the cell surface of epithelial cells, there
is a rapid tyrosine phosphorylation of proteins of focal adhesion complexes
(Perssonetal, 1997).

Translocation of YopH into epithelial cells leads to the dephosphory-
lation of proteins from these focal adhesion complexes, such as the dock-
| ing proteins pl30Cas and paxillin and the FAK (Black and Bliska, 1997;

u Persson et al., 1997). Similarly, in macrophages, contact also induces tyrosine

g phosphorylation of proteins of adhesion complexes (Andersson et al., 1996).

§ Targeting of YopH into macrophages also leads to a rapid dephosphorylation

rt of pl30Cas and paxillin (Hamid et al., 1999). In addition, in macrophages

u YopH will also lead to dephosphorylation of the Fyn -binding protein (FBP)

I— I

h-l
w

and the scaffolding protein SKAP-HOM, which have been shown to be part of
a novel adhesion-regulated signaling complex (Hamid et al., 1999; Black et al.,

h 2000) . Dephosphorylation of these proteins contributes to the antiphagocytic

g activity of YopH (Fig. 3. 4B).

Besides its role as an antiphagocytic factor, YopH has also been shown
to interfere in other signaling pathways of the immune defense system, such
as downregulating the Fc-mediated oxidative burst in macrophages and neu-
trophils (Bliska and Black, 1995; Ruckdeschel et al., 1996) and blocking cal-
cium signaling in neutrophils (Andersson et al., 1999). Finally, during infec-
tion of macrophages with Y. enter ocolitica, the PI3K/Akt pathway is rapidly
activated and then inactivated in a YopH -dependent way, which possibly con-
tributes to an anti-inflammatory role of YopH (Fig. 3.4B; also see following
subsection; also Sauvonnet et al., 2002a).

Inhibition of the inflammatory response and
induction of apoptosis

When a pathogen interacts with a mammalian cell, multiple receptors
will simultaneously recognize these pathogens both through direct binding
and by binding to opsonins on the microbe surface (Underhill and Ozinsky,

2002a) . One of the key mediators of microbe detection is the Toll-like re-
ceptor (TLR) family, which plays an important role in signaling toward in-
flammation and apoptosis (Akira et al., 2001; Imler and Hoffmann, 2001).
Ten mammalian TLRs now have been identified. Five of them (TLR2,
TLR4, TLR5, TLR6, and TLR9) have been shown to respond to an array of
different microbial components, such as lipopolysaccharide (LPS), lipopep-
tides, peptidoglycans (PGNs), lipoteichoic acid (LTA), flagellin, and CpG
motifs in DNA. By using a combination of different invariant TLRs, the im-
mune system can recognize a broad spectrum of pathogens. Stimulation of
TLR2 and TLR4 leads to the recruitment of the adaptor molecule MyD88 and
the serine kinase I L-l -receptor-associated kinase (IRAK; see Underhill and
Ozinsky, 2002b). Together with TRAF-6, this multiprotein assembly me-
diates the activation of (i) the I/cB kinase (IKK) complex, which leads to
activation of the nuclear factor /cB (NF-/cB), and (ii) the mitogen-activated §

protein kinase (MAPK) kinase family (MKKs), which also leads to the ^

activation of different transcription factors, such as activator protein- 1 £

(AP-1). |

YopP/J

M
oo

W
O

As a Gram-negative bacterium, Yersinia is endowed with several compo- h
nents capable of activating the TLR system and stimulating a proinflam-
matory response. Indeed, during Yersinia infection of macrophages, the

proinflammatory response is initially upregulated; however this is quickly h

counteracted by the YopP/J effector protein (Ruckdeschel et al., 1997, 1998). g

YopP/J has been shown to cause a variety of anti-inflammatory effects in vitro, o

such as suppression of tumor necrosis factor-a (TNF-a?) and interleukin-6 (IL- 3

6) and IL-8 production; downregulation of intercellular adhesion molecule- 1 h

(ICAM-1) expression; and blocking of the activation of MAPK pathways, in- g

eluding extracellular signal-regulated kinases (ERK) , p38, jun amino-terminal
kinase (JNK), and NF-/cB (Boland and Cornells, 1998; Palmer et al., 1998;
Schesser et al., 1998; Palmer et al., 1999; Denecker et al., 2002). The first
clue as to how YopP/J could simultaneously block these multiple signaling
pathways was elucidated by Orth et al. (1999; also see Fig. 3.4C).

In a two-hybrid system, YopJ from Y. pseudotuberculosis bound multi-
ple members of the MAPK kinase superfamily, including MKKs and IKK/3,
thereby preventing their phosphorylation and subsequent activation. The in-
teraction of its counterpart YopP from Y. enterocolitica was confirmed by
coimmunoprecipitation experiments in HEK293T cells and macrophages
(Denecker et al., 2001; Ruckdeschel et al., 2001a). By blocking both the con-
served family of MKKs and IKK/?, YopP/ J efficiently shuts down multiple

kinase cascades, resulting in a downregulation of the inflammatory response
of its host cells.

The second clue about the mechanism of action of YopP/J was based on
structural similarities. It has been suggested that YopP/J belongs to a family
of cysteine proteases related to the ubiquitin-like protein proteases (Figs. 3.3
and 3.4B; also see Orth et al., 2000; Orth, 2002). Amino acid alignment of the
adenoviral protease AVP, YopP/J, its effector homologues, and Ulpl, a yeast
ubiquitin-like protease, revealed the catalytic triad necessary for the cysteine
protease activity, which is conserved between all these proteins. Mutation
of the YopP/J hypothetical catalytic cysteine- 172, which presumably results
in the loss of its cysteine protease activity, hampers its capacity to inhibit
the NF-/cB and MAPK signaling cascades (Orth et al., 2000; Denecker et al.,
2001). Ubiquitin-like protein proteases cleave the C-terminus of an 11-kDa
small ubiquitin-related modifier, SUMO-1 (Yeh et al., 2000). YopJ has been
S shown to reduce the cellular concentration of SUMO-1 -conjugated proteins

in an overexpression experiment; however, no direct substrate of YopJ has

I— I

h-l

O
U

& been identified (Orth et al., 2000). Furthermore, it was recently suggested by

g Orth (2002) that overexpression of YopJ also leads to a decrease in ubiquiti-

^ nated proteins. Thus the exact mechanism of blockage of phosphorylation of

g MKKs and IKK/? through the cysteine protease function of YopP/J remains

fc unresolved.

In addition, it is possible that an additional cytosolic factor is needed for

h functional activity: (i) with the use of in vitro kinase reaction experiments,

S it was not possible to demonstrate that YopJ could prevent phosphorylation

of MKK1 (Orth et al., 1999); (ii) recombinant protein preparations of YopJ
were catalytically inactive when assayed with a variety of radiolabeled or flu-
orometric peptides (Orth et al., 2000); and (iii) the viral AVP protease also
requires an additional cofactor (Mangel et al., 1993). Besides cysteine-172,
it was recently demonstrated that arginine-143, present in all high -virulence
Y. enterocolitica strains and in YopJ ( Y. pestis and Y. pseudotuberculosis) , plays a
major role in determining the inhibitory impact of YopP on the suppression
of NF-/cB activation and survival of macrophages (Ruckdeschel et al., 2001b;
Denecker et al., 2002).

YopP/J is not only responsible for the inhibition of the inflammatory
response of the host but also induces apoptosis in macrophages, although
not in other cell types (Mills et al., 1997; Monack et al., 1997). Two different
mechanisms of YopP/J-dependent apoptosis have been proposed. In the first
hypothesis, YopP/ J would act as a direct activator of the cell death machinery,
which involves an early - presumably caspase-8-dependent - cleavage of Bid

to its proapoptotic truncated form, followed by the release of cytochrome c
from the mitochondria, leading to the activation of procaspase-9, -3, and -7
(Denecker et al., 2001). The point at which YopP/J interferes with the apop-
totic signaling cascade is a subject for future studies. In the second hypothesis,
YopP/ J -induced apoptosis of macrophages would merely result from its in-
hibition of NF-/cB activation, thus blocking the host cell survival pathways, in
combination with TLR stimulation (Ruckdeschel et al., 1998, 2001a, 2002).
The two hypotheses could be combined in a model in which the YopP/ J
targets both antiapoptotic and proapoptotic pathways in macrophages (Fig.
3.4C). In this model, YopP/J might downregulate the expression of some
antiapoptotic genes and at the same time alter a TLR-dependent signaling
cascade in a manner allowing procaspase-8 activation and subsequent Bid
processing.

YopH |

YopH could also contribute to the downregulation of the inflammatory |

response (Fig. 3.4B). Indeed, YopH has recently been shown to suppress 5

the Yersinia-induced activation of the PI3K/Akt signaling pathway, which *t

could be correlated with the downregulation of monocyte chemoattractant h
protein-1 (MCP-1) mRNA levels (upregulation of MCP-1 is dependent on the
PI3K/Akt signaling cascade; see Alberta et al., 1999; Scheid and Woodgett,

2001; Sauvonnet et al., 2002a). By inhibition of MCP-1 production, YopH h

would inhibit the recruitment of other macrophages to the site of infection, g

which would allow Yersinia to colonize the lymphoid system. Besides its role o

in the innate immune system, YopH also contributes to the downregulation 3

of the adaptive immune response. It was demonstrated that T-cell cytokine h

production and proliferation, and expression of the B-cell costimulatory re- g

ceptor B7.2, in response to antigen stimulation were inhibited after transient
exposure to Yersinia (Yao et al., 1999; Sauvonnet et al., 2002a). This inhibition
of antigen-specific T- and B-cell activation occurred in a YopH -dependent way
by interfering with the phosphorylation of tyrosine-phosphorylated compo-
nents associated with the T- and B-cell antigen receptor signaling complex
(e.g., Fyb and SKAP-HOM), and most probably also by interfering with the
PI3K/Akt pathway (Fig. 3.4B; also see Hamid et al., 1999; Yao et al, 1999;
Black et al., 2000; Sauvonnet et al., 2002a). Thus, Yersinia possesses different
elements that have the capacity to downregulate the inflammatory response.
The relevance of the anti -inflammatory role played by these different ele-
ments during infection is a matter for future in vivo studies.

on
H

Effectors other than Yops that help to defeat the
immune response

Analysis of the transcriptome alterations in infected mouse macrophages
revealed that several genes involved in the inflammatory response of a
macrophage to a bacterial infection are downregulated by the action of pYV-
encoded factors other than YopP (Sauvonnet et al., 2002b). As already men-
tioned, YopH is a good candidate (Yao et al., 1999; Sauvonnet et al., 2002a). In
addition, LcrV may represent another factor, as it was recently demonstrated
that LcrV-induced IL-10 release could inhibit TNF-a? production in zymosan
A-stimulated macrophages (Sing et al., 2002). According to the currently ac-
cepted type III-secretion-translocation model (Cornelis et al., 1998), LcrV is
part of the translocation machinery that delivers Yop effectors into the eu-
karyotic cell, but this does not exclude a possible role of its own, independent
£ of the rest of the injectisome.

g In addition, for the induction of cell death, YopP might not be the sole

* factor. For Y. pseudotuberculosis it was recently demonstrated that the Inv

^ protein could cause a rapid apoptotic-necrotic caspase-independent cell death

^ in T lymphocytes (Arencibia et al. , 2002) . This process was mediated by means

w of ^1-integrins and was independent of the Yop-Ysc TTSS of Yersinia.

2 YopM

h YopM belongs to a growing family of type III effectors that has several

£ representatives in Shigella (ipaH multigene family) and Salmonella (SspH; see

Kobe and Kajava, 2001). It is a strongly acidic protein composed almost en-
tirely of 20/22 residue leucine-rich repeats (LRR; see Fig. 3.3). The repeating
LRR unit of YopM is the shortest among all LRRs known to date, and, depend-
ing on the Yersinia species, the amount of copies can vary between 13 and 20
repeats. The crystal structure has revealed that the LRRs, consisting of paral-
lel p -sheets, form a crescent shape, which is flanked by an a -helical hairpin at
the N-terminus (Evdokimov et al., 2001). The latter domain has been shown
to be part of the signal necessary to target YopM for translocation into eukary-
otic cells (Boland et al., 1996). Intriguingly, individual YopM molecules form
a tetramer in the crystal, creating a hollow cylinder with an inner diameter

of 35 A (Evdokimov et al., 2001). YopM has been shown to traffic to the nu-
cleus via a vesicle-associated pathway, but its action in the nucleus remains
unknown (Skrzypek et al., 1998). New insights about the role of YopM came
from a recent study in which a transcriptome analysis of Yersima-infected
macrophages revealed that YopM may control the expression of genes in-
volved in the cell cycle and in cell growth (Sauvonnet et al., 2002b).

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After initial invasion of its host cells, pathogenic Yersinia remain extra-
cellular because they have the capacity to resist phagocytosis. This resistance
depends on the type III Ysc-Yop system, which upon close contact with a
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i_i

down multiple kinase cascades, and in this way it may be responsible for the ffi

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of apoptosis of macrophages. °

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into single key issues, but one Yop may have different effects. Surprisingly, §

LcrV, one of the proteins that are involved in translocation of the effectors o

across the host cell membrane, has an anti-inflammatory role on its own, %
without being injected into the cell cytosol.

o
3

ir,

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CHAPTER 4

Stealth warfare: The interactions of EPEC
and EH EC with host cells

Emma Allen- Vercoe and Rebekah DeVinney

Although the Gram-negative bacterium Escherichia coli is normally consid-
ered to be a harmless commensal of the gastrointestinal flora, there are some
exceptions to this rule. In the past few decades it has become increasingly
evident that there are serotypes off. coli that may cause disease in susceptible
hosts. Disease states range from the invasive infections of the urinary tract
caused by uropathogenic E. coli (UPEC) to the more typical diarrheal disease
caused by several groups of E. coli serotypes, including enteropathogenic
E. coli (EPEC) and enterohemorrhagic E. coli (EHEC). There is an increas-
ing realization that bacteria-host interactions with pathogenic E. coli are far
more complex and intricate than originally imagined. Studying the ways that
bacteria such as EHEC and EPEC are able to subvert host cell functions to
their own ends can be thought of as a "window" through which we are able
to view the inner workings of the eukaryotic cell. In this chapter we examine
some of the mechanisms by which EHEC and EPEC are able to coerce their
host; we also examine the sequelae of these interactions.

EPEC usually refers to E. coli serotypes 055:[H6], 086:H34, 0111:[H2],
OH4:H2, OH9:[H6], 0127:H6, OU2:H6, and 0142:H34, where square
brackets indicate the occurrence of nonmotile strains (Nataro and Kaper,
1998). Infection with EPEC typically causes a chronic watery diarrhea, ac-
companied by a low-grade fever and nausea. These symptoms can lead to
rapid dehydration of the patient, and, because of this, EPEC is a leading
cause of infant mortality, particularly in developing countries where rehydra-
tion therapy may be difficult. In the Western world, isolated incidences of
EPEC infection are often associated with daycares and nurseries (Nataro and
Kaper, 1998), where young children closely associate with one another and
facilitate the spread of infection.

EHEC usually refers to serotype 0157:H7 and less commonly to serotype
OllliH - . An EHEC infection is often heralded by the onset of watery diar-
rhea, which progresses rapidly to severe bloody diarrhea (hemorrhagic coli-
tis) in many patients, regardless of age (Nataro and Kaper, 1998). In the very
young and very old, as well as in immunocompromised patients, the dis-
ease can be complicated by the onset of hemolytic-uremic syndrome (HUS),
which is characterized by hemolytic anemia, thrombocytopenia, and renal
failure. HUS is caused by the secretion by EHEC of shiga-like toxin (SLT),
a potent cytotoxin with a predilection for human kidney cells. A description
of the mechanisms of action of SLT and the many effects on the host cell is
beyond the scope of this chapter but is reviewed by O'Loughlin and Robins-
Browne, 2001. The onset of HUS, even with rapid treatment, can prove fatal
to a patient. Outbreaks of EHEC infection are becoming increasingly high
g profile in North America and Europe. The low infectious dose required to

g cause infection may allow the organism to spread rapidly. A recent outbreak

g of EHEC 0157:H7 in Walkerton, Ontario infected close to 2000 people and

3 led to seven deaths (Kondro, 2000).

g Neither EPEC nor EHEC are generally regarded as invasive pathogens;

rt however, some limited evidence has suggested that EPEC in particular may,

5j in fact, be able to invade host cells under the right conditions (Czerucka et al.,

o 2000). Additionally, both EPEC and EHEC use a highly specialized mecha-

n nism that allows the bacteria to adhere tightly to the epithelial cell surface and

i subvert normal host cell functions by direct interaction. In this chapter, the

jj mechanisms and consequences of both EPEC and EHEC infections of the

2 host are discussed, and the subtle differences between EPEC and EHEC inter-

w action with host cells, which are beginning to emerge as research progresses,

are highlighted.

ADHERENCE MECHANISMS REQUIRED FOR INITIAL
ATTACHMENT OF EPEC AND EHEC TO HOST CELLS

EPEC appear to have a predilection for the human ileum (Nataro and
Kaper, 1998), and the first stage of adherence of EPEC to host cells is thought
to involve bundle -forming pili (BFP; see Giron et al., 1991). BFP are type-4
pili encoded by a cluster of 14 genes found on a 92 -kb plasmid known as the
EPEC adherence factor plasmid (EAF; see Tobe et al., 1999) . EPEC-expressing
BFP form dense microcolonies on the surface of tissue culture monolayers
in a pattern known as localized adherence (LA). Whether BFP are involved
more in interbacterial adherence or adherence to the host cell is currently
under debate. However, there is no doubt that BFP expression represents a

significant virulence factor for EPEC. Mutant EPEC unable to produce BFP
are significantly reduced in their ability to adhere to tissue culture monolayers
(Frankel et al., 1998). Furthermore, human volunteers given an inoculum of
mutant EPEC defective for the expression of BFP were far less likely to develop
diarrhea than were volunteers given the wild-type strain (Bieber et al., 1998).
Together, these factors indicate that the initial adherence of EPEC to host
cells is a critical step in the subsequent development of disease.

The flagella of Gram-negative bacteria are being increasingly recognized
as effectors of bacterial cell adhesion to host cells, such as in the binding
of Salmonella to chick gut explants (Allen-Vercoe and Woodward, 1999). Re-
cently, the flagella of EPEC have also been shown to be important in the
adherence of the bacteria to epithelial cells in vitro, and experimental evi-
dence suggested that a eukaryotic signal can induce flagellar expression and
thus enhance adhesion (Giron et al., 2002). Additionally, it was found that h

EPEC serotypes previously classified as nonmotile could, in fact, elaborate g

flagella under the correct conditions (Giron et al., 2002). ^

In addition to its role in pedestal formation (discussed in what follows), g

the EPEC and EH EC outer membrane protein intimin may play a role in g

adherence host cells. The family of intimins from attaching/effacing (A/E) g

lesion-forming bacteria has been divided into at least five distinct types that
differ from each other at the amino acid level, particularly across the putative 8

binding domain located at the C-terminus (Frankel et al., 1995). EPEC and n

EH EC encode different intimin types, depending on their serotype. Intimin- °

a and intimin-/? are usually associated with EPEC strains, whereas EHEC

0157:H7 specifically expresses intimin-y . Interestingly, intimin may be re- «

sponsible for the tissue tropism exhibited by different pathogenic E. coli. n

Infection with an EHEC strain expressing EPEC intimin-a shows a pattern §

of adherence to human gut explants that resembles that of EPEC (Fitzhenry £

etal.,2002). <j

The eukaryotic receptor(s) for intimin has yet to be identified. However, h

two targets have been suggested: /3i-integrins (Frankel et al., 1996) and nu- ffi

cleolin (Sinclair and O'Brien, 2002). The expression of intimin by EPEC and h

EHEC is an important virulence determinant. Inhuman volunteers, an EPEC ?

intimin - mutant was shown to be significantly attenuated compared with its
wild-type parent strain (Donnenberg et al., 1993). In addition, antibodies to
both EPEC and EHEC intimins are often found at a high titer in individuals
who have been infected with these pathogens (Loureiro et al., 1998; Jenkins
etal.,2000).

The preferred site for colonization of EHEC is the colon, and the initial
adherence of EHEC to host cells is thought to be quite different from that

of EPEC. EHEC does not possess the EAF plasmid and thus does not elabo-
rate BFP. Hence, the pattern of EHEC adherence to epithelial cells in tissue
culture is more diffuse than that of EPEC, and no microcolonies are formed.
In the absence of BFP, the Tir (translocated intimin receptor) -independent
adhesion mediated by intimin and described herein is likely to become more
important to EHEC for colonization of host cells than it is for EPEC. Indeed,
an intimin - mutant of EHEC 0157:H7 failed to colonize human intestinal
explants (Fitzhenry et al., 2002).

More recently, further EHEC adhesins have been proposed. Iha (IrgA
homologue adhesin) is an outer membrane protein (OMP) with externally
directed domains. Its corresponding gene, iha, confers an adhesive phenotype
when transferred to a normally nonadherent E. coll K12 laboratory strain,
although its role in EHEC adhesion remains to be elucidated (Tarr et al.,
£ 2000). The chromosomal gene efal (EHEC factor for adherence) has also been

g recently implicated in the initial adherence of non-0157 EHEC to host cells,

g and by virtue of its low G + C content may represent part of a pathogenicity

3 islet acquired by horizontal transmission. The product of the efal gene, Efal,

g has been shown to play a role in influencing the colonization of the bovine

rt gut in vivo by non-0157 EHEC strains (Stevens et al., 2002). Although first

5j characterized in EHEC, an Efa 1 homologue, LifA, has also been found in

o EPEC (Nicholls et al., 2000), where it confers upon the bacteria the ability to

n modulate host mucosal immunity in the gut (Klapproth et al., 1996; Malstrom

i and James, 1998; also discussed later in this chapter).

i-i

s INTIMATE ATTACHMENT OF EHEC AND EPEC TO HOST CELLS,

AND THE TYPE III SECRETORY APPARATUS

A hallmark of disease caused by EHEC and EPEC is the formation of
A/E lesions (Fig. 4.1). These lesions are defined by the intimate attachment
of the bacteria to the host cell surface and the localized destruction (efface-
ment) of the brush border microvilli (Moon et al., 1983). The bacteria induce
an accumulation of actin beneath their site of attachment, and this results
in the formation of a pseudopod, or pedestal, upon which the bacteria are
located (Fig. 4.2). A/E lesion-forming bacteria such as EHEC and EPEC uti-
lize a Gram-negative specific mechanism, called a type III secretion system
(TTSS) , in order to inject effector molecules directly into the host cell (Hueck,
1998).

For EPEC, a region of the chromosome known as the locus for entero-
cyte effacement (LEE) is sufficient for A/E lesion formation. The LEE is a
36-kb region containing 41 open reading frames (ORFs) organized into five

EPEC

J V

12 3 4

Figure 4.1. Simplified schematic of pedestal formation by EPEC. (1) EPEC uses BFP to
adhere to the host cell microvilli, leading to effacement of the brush border (2),
whereupon the bacterium uses its TTSS to inject Tir (black bulb shape), into the host cell,
where it is phosphorylated (asterik) (3) Tir presents its intimin-binding domain to intimin
expressed on the bacterial surface. The binding of Tir to intimin leads to a cascade of
signaling events (4), which act to stimulate actin polymerization. A pedestal is formed on
the host cell membrane, on which the bacterium resides.

polycistronic operons, termed LEE1-LEE5 (Elliott et al., 1998; Mellies et al.,
1999). LEE1-LEE3 encode genes encoding the TTSS apparatus. LEE4 con-
tains the genes required for pedestal formation. These include tat, which
codes for intimin; tir, which codes for Tir; and a gene that codes for the
molecular chaperone for Tir, cesT (Abe et al., 1999). LEE 5 contains genes for
the type Ill-secreted proteins EspA, EspB, and EspD, which are necessary for
pedestal formation. EspB and EspD are thought to form pore-like structures
in the host cell membrane (Warawa et al., 1999; Kresse et al., 1999; Wachter
et al., 1999) that interact with the filamentous EspA structures on the bac-
terial cell to allow the close association of the type III secretory apparatus
with the host cell membrane, and the subsequent insertion of Tir (Knutton
et al., 1998). Both EHEC and EPEC EspB have recently been found to interact
directly with the host protein a-catenin, a cytoskeleton-associated molecule,
in a Tir-independent way, and the formation of A/E lesions has been found
to be dependent on this interaction (Kodama et al., 2002).

Recently, the supermolecular structure of the assembled EPEC TTSS
apparatus has been elucidated by electron microscopy (Sekiya et al., 2001). It

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Figure 4.2. A cluster of pedestal structures induced by EPEC on the surface of a HeLa cell.
Indirect immunofluorescence microscopy was used to demonstrate the formation of
pedestal structures by EPEC. Long stalks of actin (green) are capped at the tip by Tir (red),
upon which a single bacterium (blue) sits. The boxed region has been enlarged to clearly
show a single pedestal. See color section.

was reported that the EspA protein, which has no homology to any known
protein in other bacterial TTSSs other than that for EH EC, may be a major
component of a sheath -like structure that is assembled by the bacteria to act
as a physical bridge between bacteria and host cells. This, in turn, indicates
that the assembly of EHEC and EPEC type III secretons may be carried out
somewhat differently compared with other pathogens.

The Tir protein, once introduced into the host cell membrane by means
of the TTSS, serves as the receptor for intimin and allows the intimate as-
sociation of bacteria with host. Whereas EPEC adherence by means of BFP
permits the bacteria to exist within 100-300 nm of the host cell surface, the
distance between bacterial cell membrane to host cell membrane during Tir-
intimin attachment can be as little as 10nm(NisanetaL, 1998). EPEC Tir was
originally thought to be a host cell protein, and its identification as a bacterial
protein, injected by the bacteria in order to effect intimate adhesion by in-
timin, represents the first discovered example of host cell subversion of this
type (Kenny et al., 1997). Work done in animal infection studies with the nat-
ural rabbit pathogen, REPEC O103:H2, has demonstrated that intimin, Tir,
EspA, and EspB are essential for diarrheal disease (Abe et al., 1998; Marches
etal.,2000).

Whereas some of the components of the TTSS are highly conserved be- h

tween EPEC and EHEC, eae, espA, espB, espD, and tir are much less highly g

conserved, although the mechanism of Tir translocation is thought to be ^

identical for both pathogens (Frankel et al., 1998). However, although it is g

possible to induce nonpathogenic laboratory strains of E. coli to form A/E le- g

sions by introducing into them plasmids carrying the LEE region from EPEC g

(McDaniel and Kaper, 1997), the reciprocal experiment with the EHEC LEE
region does not support either A/E lesion formation or Esp protein secretion, 8

suggesting that, for EHEC, further factors are involved that are outside of the n

LEE (Elliott et al., 1999; DeVinney et al., 2001). These unidentified factors °

required by EHEC are a current focus for research into EHEC pathogenicity.

The first gene of LEE1 encodes the Ler regulator, an H-NS-like protein «

that activates all other genes in the LEE for both EPEC and EHEC, and is n

required for pedestal formation (Friedberg et al., 1999; Elliott et al., 2000). §

The PerABC proteins encoded by the pEAF plasmid of EPEC positively reg- £

ulate BFP expression (Donnenberg et al., 1992) and may also regulate the 2

transcription of LEE genes through ler (Mellies et al., 1999). h

The regulation of the LEE appears to be quite different for EPEC and ffi

EHEC. Both EHEC and EPEC utilize two quorum-sensing systems, termed h

1 and 2, central to which are proteins termed autoinducer (AI)-l and AI-2, ^

respectively. During transition from the late exponential to the stationary
growth phase, both EPEC and EHEC LEEs are activated by AI-2 (Sperandio
et al., 1999). However, during the stationary phase, EHEC AI-1 appears to be
downregulated by the expression of LEE-encoded genes through the action
of a quorum-sensing regulator called SdiA (Kanamaru et al., 2000). In con-
trast, the AI-1 of EPEC does not appear to be affected in this manner (Abe
et al., 2002). The differences in EPEC and EHEC LEE gene expression can

be illustrated by the finding that, in vitro, EH EC Tir synthesis and secretion
requires specific growth conditions that do not apply to EPEC Tir expression
(DeVinneyetaL, 1999).

HOST CELL RESPONSES TO EHEC AND EPEC INFECTION
Pedestal formation

The formation of actin pedestals beneath adherent A/E bacteria is a host
response so unusual that the presence of actin pedestals in infected tissue cul-
ture monolayers can be used as a diagnostic test for EPEC and EHEC (Knutton
et al., 1989). Pedestals may grow to 10 /im above the host cell surface, at a
rate of around 1 /xm/min, and appear to be able to shorten and lengthen
£ while remaining attached to the host cell (Rosenshine et al., 1996; Sanger

g et al., 1996). It has been found that pedestals themselves are able to pro-

g pel their attached bacteria along the host cell surface at an estimated rate

3 of 70 nm/s (Sanger et al., 1996). The exact purpose of an actin pedestal is

g puzzling. It may serve as a mechanism for the bacteria to avoid the host di-

rt arrheal response, or as an avoidance tactic for internalization into the host

§ cell. However, in tissue culture, cells infected with A/E bacteria eventually

o round up and detach, suggesting that host cells may not be able to tolerate

n prolonged contact with these pathogens (Baldwin et al., 1993).

i A chief effector of pedestal formation, Tir, is a 72- to 78-kDA protein that

jj is inserted into the host cell membrane by means of the TTSS apparatus.

2 EPEC and EHEC strains that are unable to secrete Tir do not form pedestals

w (DeVinney et al., 1999). Tir contains two predicted transmembrane domains

that are required for the stable insertion of the protein into the host cell
plasma membrane (Gauthier et al., 2000). The amino- and carboxy-termini
of Tir are intracellular to the host cell, and intimin binds to a hydrophobic
extracellular domain called the intimin-binding domain (IBD; see DeVinney
et al., 1999; Kenny, 1999; Luo et al, 2000).

The process of pedestal formation is best understood for EPEC (Fig. 4.3).
EPEC Tir is tyrosine phosphorylated at position 474 (Y474) after insertion
into the host cell membrane. Whereas Tir phosphorylation is required for
pedestal formation (Kenny, 1999; Goosney et al., 2000), Tir translocation is
thought to be completely independent of host cell modifications. In studies of
EPEC attachment to red blood cells (RBCs), tyrosine phosphorylation of Tir
and pedestal formation was not seen, although Tir was inserted correctly into
the RBC membrane and was able to bind intimin (Shaw et al., 2002). The Tir
chaperone, CesT, functions to direct Tir to the translocation apparatus and
may help to maintain Tir in a secretion-competent state (Abe et al., 1999).

Pedestal
elongation

Figure 4.3. Simplified schematic of EP EC-induced signaling events leading to pedestal
formation. The binding of intimin to translocated Tir leads to tyrosine phosphorylation of
the protein at position 474. This phosphorylation is required for the direct binding of the
cellular adaptor Nek, which signals N-WASP recruitment to Tir, and the subsequent
recruitment of the Arp2/3 complex to allow pedestal formation. The cytoskeletal proteins
a-actinin, villin, cortactin, and vinculin all bind directly to Tir and are thought to play a
role in pedestal formation through their F-actin binding sites.

Several host cell structural proteins have been implicated in pedestal
formation on the basis of observations made by use of immunofluorescence
microscopy. Pedestals are predominantly formed from filamentous actin,
with micro filament-associated proteins such as a-actinin, talin, ezrin, and
villin also being found along the length of the pedestal (Goosney et al., 2001).
The actin-stabilizing proteins tropomyosin and nonmuscle myosin II can be
seen at the base of a pedestal (Sanger et al., 1996). The Tir protein serves
directly as a conduit between host cell and bacteria, and much research has
been focused on the mechanisms that enable this interaction, because this
enigmatic protein offers us a unique window into the dynamics of host cell
cytoskeletal rearrangement.

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Acquired

immunity

Tyrosine phosphorylation of Tir plays an essential role in pedestal for-
mation by facilitating the direct binding of the cellular adaptor protein Nek
(Gruenheid et al., 2001; Campellone et al., 2002). Nek binding leads to the re-
cruitment of N-WASP, and the Arp2/3 complex, which in turn allows pedestal
elongation (Kalman et al., 1999) . Cultured cells defective for the expression of
N-WASP do not support the formation of E PEC actin pedestals, highlighting
the central role played by this protein (Lommel et al., 2001). The critical role
played by N-WASP suggests an involvement of Rho family GTPases. Sur-
prisingly, these do not appear to play a role in pedestal formation (Ben-Ami
etal, 1998; Ebel et al., 1998).

Tir has also been demonstrated to bind directly to the focal adhesion (FA)
proteins a-actinin, talin, and vinculin. FAs are anchoring structures that al-
low the close association of epithelial cells and fibroblasts to the extracellular
matrix (ECM) . Tir binds directly to a-actinin by means of its amino terminus h

(Goosney et al., 2000; Huang et al., 2002), in a manner that is tyrosine phos- ^

phorylation independent (Goosney et al., 2000). Tir can also bind to talin, ^

and this association has been shown to be essential for correct F-actin focus- g

ing beneath adherent cells, possibly through the nucleation of nascent actin g

filaments (Cantarelli et al., 2001). Vinculin has also been found to bind to g

Figure 4.4. (facing page). EPEC-induced signaling events in the host cell. The interaction
of EPEC effector molecules (gray boxes) with host proteins (white boxes) leads to the
exploitation of various signaling pathways, and an array of outcomes for the host (rounded
boxes). For details of individual pathways, see text. (For clarity, some less understood
pathways have been omitted.)

Tir directly (Freeman et al., 2000). The association of FA proteins with the

pedestal structure supports the notion that pedestals are composites of FAs 8

and microvilli (Freeman et al., 2000; Goosney et al., 2001). Recent research n

has shown that EPEC can disrupt FAs, by specifically dephosphorylating the °

FA kinase, FAK. FAK dephosphorylation requires a functional TTSS and is

enhanced, but not absolutely dependent on, pedestal formation (Shifrin et al., «

2002). Cortactin, an F-actin-binding protein, has been shown to accumulate n

underneath adherent EPEC in its tyrosine-dephosphorylated form (Cantarelli §

et al., 2002) . In this form, cortactin is able to efficiently bind other cytoskeletal £

proteins and to cross-link F-actin (Huang et al., 1997), and it may thus play 2

an important role in pedestal formation. h

Several signaling molecules are thought to be involved in pedestal forma- ffi

tion (Fig. 4.4). Phospholipase C (PLC), phosphtidyinositol 3-kinase (PI3K), h

and 5 -lipoxygenase have all been shown to be important for the accumulation g
of a-actinin beneath adherent EPEC, because inhibitors of these molecules

also inhibited a-actinin accumulation in a concentration-dependent man-
ner (Johnson-Henry et al., 2001). Recent evidence also suggests that the F-
actin-binding protein, annexin 2, is recruited underneath the sites of EPEC
attachment in epithelial monolayers (Zobiack et al., 2002) and may act as a
bridge between actin and the host cell membrane (Gerke and Moss, 1997).
Recruitment of annexin 2 by EPEC was found to be Tir independent, rais-
ing the possibility that cytoskeletal rearrangements by EPEC may occur by at
least two distinct pathways - one that is Tir mediated and another that is not
(Zobiack etal, 2002).

Although EH EC and EPEC both induce pedestal formation, EH EC uses
methods distinct from EPEC to recruit the actin-nucleating machinery. EHEC
Tir is less than 60% identical to EPEC Tir, with the C-terminal domain
showing the least homology (Paton et al., 1998). Indeed, the absence of
£ the tyrosine residue that is targeted for phosphorylation in EPEC Tir cor-

g relates with the finding that EHEC is able to induce pedestal formation in

g the absence of Tir tyrosine phosphorylation (Ismaili et al., 1995; DeVinney

3 et al., 2001). Although EHEC Tir is not tyrosine phosphorylated and does not

g bind Nek, both N-WASP and the Arp2/3 complex are still recruited to the

rt EHEC pedestal (Goosney et al., 2001), which suggests that EHEC and EPEC

5j modulate different pathways in order to form pedestals (DeVinney et al.,

o 2001).

n A further major difference is that EHEC, but not EPEC, can form

i pedestals with mutant EPEC Tir in which the Y474 has been replaced by

jj phenylalanine (DeVinney et al., 2001; Kenny, 2001), suggesting that addi-

2 tional EH EC-specific bacterial factor(s) may be involved in pedestal forma-

w tion by EHEC that is independent of Tir tyrosine phosphorylation (DeVinney

et al., 2001). Although only a small proportion of EHEC strains, including
EHEC 0157, form pedestals that do not contain tyrosine-phosphorylated Tir,
several non-0157 serotypes that are designated as EHEC have Tir sequences
similar to that of EPEC and are tyrosine phosphorylated in a similar way to
EPEC Tir (DeVinney et al., 2001). Because the 0157:H7 serotype correlates
closely with human EHEC infections , it is tempting to speculate that serotypes
that form pedestals in a tyrosine-independent manner represent the more ef-
ficient pathogens, as they are less reliant on the signaling capacities of the
host cell in order to subvert host cell processes to their own ends.

Inhibition of phagocytosis

Several A/E pathogens, including EPEC, EHEC, and RE PEC, are able to
inhibit uptake by specialist phagocytic cells such as M cells and macrophages,

and they can even colonize the niches rich in these cells (Inman and Cantey,
1983; Kresse et al., 2001). An ability to inhibit phagocytosis would clearly be
advantageous to the bacteria by delaying the host immune response.

The EPEC antiphagocytic phenotype has been demonstrated to be reliant
on a functional TTSS and is independent of pedestal formation (Goosney
et al., 1999). This study demonstrated that host protein tyrosine dephospho-
rylation could be observed in type III secretion-competent EPEC, and that
mutants that had nonfunctional type III secretion systems were unable to
elicit host protein modifications, suggesting that antiphagocytosis is reliant
on a bacterial protein effector that is secreted through the type III mecha-
nism. More recent research was able to show that EPEC blocks its uptake by
inhibition of a PI3K-mediated pathway (Celli et al., 2001). Whether EHEC
and other A/E pathogens utilize the same mechanism for antiphagocytosis
is unknown (although considered likely). EPEC, however, has the distinction h

of being the first pathogen for which an antiphagocytic pathway of this type
has been described.

Inhibition of mitosis

Certain strains of RE PEC and EPEC (although not the reference strain

M
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E2 348/69) are able to induce in HeLa cells an irreversible cytopathic effect, 3

characterized by the progressive recruitment of stress fibers and FAs and n

associated with the prevention of cell proliferation (De Rycke et al., 1997; 2

Nougayrede et al., 1999). Such effects are not related to any toxin secretion,

are dependent on a functional TTSS, and require the secreted proteins EspA, «

EspB, and EspD, but not Tir or intimin (De Rycke et al., 1997; Nougayrede n

et al., 1999; Marches et al., 2000). A closer examination of the observed cyto- §

static effect revealed that the cell cycle is arrested in the G2 /M phase, and that £

this mitotic inhibition is not likely to be a consequence of the cytoskeletal 2

arrangements themselves (Nougayrede et al., 2001). h

The host cell protein, Cdkl, together with the protein cyclin Bl, forms a ffi

complex that governs the transition from G2 to mitosis. In normal cells, Cdkl h

is present at a constant level throughout the cell cycle, and its dephosphory- ?

lation is required for entry into mitosis (Norbury and Nurse, 1992). Strains
of RE PEC and EPEC that exert a cytostatic effect appear to affect Cdkl de-
phosphorylation by an as yet unknown mechanism, arresting the cell cycle
in the G2 phase (Nougayrede et al., 2001). Whether mitotic inhibition plays a
role in EPEC pathogenesis, and by which exact method, can only be guessed
at pending further research into this effect. However, it has been suggested
that a cell-cycle arrest of the stem cells that supply cells to the intestinal villi

may delay the shedding of the epithelia, thus allowing the bacteria to remain
attached to its host for a prolonged period (Nougayrede et al., 2001).

Disruption of tight junctions

Although as yet incompletely understood, the pathogenesis of EPEC-
induced diarrhea may involve not only the formation of A/E lesions but also
disruption of epithelial barrier function. Tight junctions are specialized inter-
cellular structures that form "gaskets" around epithelial cells that prevent the
leakage of fluid through the intercellular gaps, and the resultant diffusion of
membrane proteins. Several studies have demonstrated that EPEC infection
of host cells in tissue culture consistently leads to a significant decrease in
transepithelial resistance (TER), a measure of tight junction integrity, in a
£ time-dependent manner (Spitz et al., 1995). This host response has also been

jz;

g shown to be dependent on a functional TTSS (Canil et al., 1993; Philpott
g et al. , 1 996) , although attachment of bacteria mediated by Tir-intimin interac-
ts tions was not in itself sufficient to trigger host cell membrane depolarization
g (Stein etal, 1996).

rt EPEC-induced phosphorylation of myosin light chain (MLC) seems to

§ play a central role in the pathway that leads to eventual loss of cell membrane

o integrity (Manjarrez-Hernandez et al., 1996). Both EPEC and EHEC infec-

n tions in vitro have been shown to activate protein kinase C (PKC), which is

i likely to lead to MLC phosphorylation (Crane and Oh, 1997; Philpott et al.,

jj 1998). Activation of MLC-kinase (MLCK) leads to further phosphorylation of

2 MLC (Manjarrez-Hernandez et al., 1996) and contraction of the cytoskeletal

w rings underlying tight junctions, and thus it leads to a loss of tight junction

integrity.

The host cell protein occludin is thought to play an important role in
maintaining the integrity of tight junctions (McCarthy et al., 1996). EPEC
infection has been shown to result in dephosphorylation of occludin and its
resulting dissociation from tight junctions into the intracellular space, which
would very likely contribute to tight junction permeability (Simonovic et al.,
2000). Whether this occludin dephosphorylation is linked to the pathway in-
volving the phosphorylation of MLC, or whether the two pathways are sepa-
rate, is unknown. Occludin is one of many proteins identified in the makeup
of tight junctions, and whether EPEC targets any other tight junction pro-
teins has not yet been determined. Interestingly, EHEC has been shown to
alter the distribution of another important tight junction-associated protein,
ZO-1, again through the action of PKC (Philpott et al., 1998), which directly
targets ZO-1 for phosphorylation (Stuart and Nigam, 1995).

Recently it was found that EPEC mutants deficient in their ability to ex-
press EspF, a protein that is translocated into the host cell cytoplasm by the
TTSS, were deficient in their abilities to disrupt electrical resistance across
polarized epithelial cells (McNamara et al., 2001). EspF appears to rely on a
chaperone protein, CesF, for full translocation efficiency (Elliott et al., 2002).
Once translocated into the host cell, EspF is not found distributed homoge-
nously; instead it appears to be sequestered, perhaps by the host cell protein(s)
with which it interacts (McNamara et al., 2001). Whether EspF does indeed
modulate host functions through interaction with a host cell protein remains
to be elucidated.

Upregulation of chloride secretion and adenosine
triphosphate (ATP) release

on
H
w

One of the hallmark symptoms of EPEC infection is the onset of severe, g

watery diarrhea. PKC activation in the intestine triggers a rapid secretion ^

of ions (particularly chloride ions, Cl~) and fluid into the lumen (Beubler g

and Schirgi-Degen, 1993). Thus it was not surprising to find that EPEC is g

able to activate PKC (Crane and Oh, 1997), which is likely to result in the g

upregulation of Cl~ secretion and the subsequent development of watery ~

diarrhea. 3

A further mechanism whereby watery diarrhea might be induced in- n

volves the galanin-1 receptors found on the surface of epithelial cells lining g

the human GI tract. These receptors, on activation, cause Cl~ secretion. Ex-

pression of the galanin-1 receptor is transcriptionally regulated by nuclear «

factor (NF)-/cB, which is in turn activated during infection with both EH EC n

and EPEC (Savkovic et al., 1997; Dahan et al., 2002). Indeed, it was demon- §

strated that infection with both EH EC and EPEC increased the number of £

galanin-1 receptors by means of the activation of NF-/cB, and thus elevated 2

Cl~ secretion (Hecht et al., 1999), a factor that contributes to the etiology of h

infectious watery diarrhea. ffi

In addition to NF-kB-PKC activation, a further hypothesis for the patho- h

genesis of diarrhea promoted by EPEC infection has emerged. It was recently ?
demonstrated that polarized cells could be induced to release a large concen-
tration of ATP into the apical culture medium upon infection with EPEC
(Crane et al., 2002). This release of ATP is TTSS dependent, and, in partic-
ular, the EspF protein appears to play an important role (Crane et al., 2002).
ATP release in response to EPEC infection may be a direct result of leakage
from the ATP-rich cytosol through pores formed by the bacterial TTSS. The

pore created by EPEC in the host cell membrane is between 30 and 50 A (Ide

et al., 2001), which would allow for easy passage of the ATP molecule. ATP
release may also be effected by the modulation by EPEC of the cystic fibrosis
transmembrane regulator (CFTR; see Crane et al., 2002), which acts as a cel-
lular "valve" to allow for the normal release of ATP from the host cell cytosol
during cell stretching or swelling or as a response to cAMP stimulation (Jiang
etal., 1998).

Once extracellular, ATP is rapidly broken down into less phosphory-
lated nucleotides and adenosine. Adenine nucleotides released from EPEC-
infected cells could spread to neighboring cells and may trigger a fluid se-
cretory response. This could explain the apparent paradox that EPEC prefer-
entially adheres to the villi of the gut epithelia, yet it is the crypt cells of the
gut that have the capacity to generate watery diarrhea through ion secretion
(Crane et al., 2002). The exact mechanism of the stimulation of ATP release
g by EPEC, and whether the same mechanism is utilized by EH EC, remains to

fc be elucidated.

g The secretion of Cl~ and the subsequent development of watery diarrhea

3 may be considered an innate host defense mechanism that has evolved to

g rid the host of enterobacterial pathogens such as EHEC and EPEC. For the

rt pathogen itself, this defense mechanism has the advantage that it may allow

5j for increased spread of the bacteria from host to host.

O
U

* The Ca 2 + controversy

jj Early research into the effects on host cells of A/E lesion-forming EPEC

2 demonstrated that near to the sites of bacterial attachment there seemed

w to be a localized elevation of Ca 2+ within the host cell (Baldwin et al., 1991),

coupled with the phosphorylation of (among other proteins) MLC (Manjarrez-
Hernandez et al., 1991). Subsequent to this, increased levels of inositol
triphosphate (IP) were detected in EPEC-infected cells (Dytoc et al., 1994;
Foubister et al., 1994), and thus it seemed plausible that the PLC pathway,
which generates IP and in turn a release of Ca 2+ from intracellular stores,
was being stimulated by EPEC infection. Additionally, it was found that the
buffering of intracellular free Ca 2+ by use of the chelating agent BAPTA
could prevent A/E lesion formation by EPEC (Baldwin et al., 1991; Dytoc
etal., 1994).

Recent work using more sensitive methods has been able to measure
both temporal and spatial measurements of Ca 2+ in live cells infected with
both EPEC and EHEC (Bain et al., 1998). In contrast to the earlier studies
just described, no increase in Ca 2+ levels could be measured, and the addi-
tion of BAPTA to host cells was not seen to prevent the formation of A/E
lesions (Bain et al., 1998). It was suggested that the alterations in cell Ca 2+

levels seen in previous studies was related to the cytotoxic effects of E PEC
(Bain et al., 1998). Clearly, more research has to be done in this area to deter-
mine whether or not EPEC and EH EC can modulate host intracellular Ca 2+
levels.

Activation of mitogen-activated protein (MAP) kinase cascades

The MAP kinases group includes extracellular signal-related protein ki-
nases 1 and 2 (ERK1 and ERK2), p38 MAP kinase, and c-Jun N-terminal
kinase (JNK). As well as the classical mitogenic response, MAP kinases
have also been implicated in the regulation of stress responses, cytokine
and chemokine responses, and cytoskeletal reorganization (Garrington and
Johnson, 1999). Indeed, EH EC is able to induce the expression of the
chemokine interleukin-8 ( I L-8) in host cells via MAP kinase pathways ( Dahan h

et al., 2002; discussed later). g

MAP kinases play a role in the host cell response to infection with inva- ^

sive bacteria such as Salmonella (Hobbie et al., 1997). Although invasion is g

>
not thought to be a central process required for EPEC pathogenicity, EPEC g

was found to be able to invade T84 epithelial cells in culture, and this inva- g

sion relied on the activation of ERK1/2 MAP kinase (Czerucka et al., 2000).

Additionally, it was found that the intimate attachment of EPEC to host cells 8

was able to activate ERK1/2, p38, and JNK signaling pathways in a manner n

that was dependent on both a functional TTSS and the expression of intimin °

(Czerucka et al., 2001) . The MAP kinase-activated pathways proceed through

a cascade of phosphorylation events leading to the transcriptional activation «

of certain genes. Recently EPEC was found to upregulate the transcription n

of the gene coding for the early growth response factor-1 (Egr-1) through §

the ERK1/2 pathway, the p38 pathway, or both (de Grado et al., 2001). Egr-1 £

is a protein that is activated in most cell types in response to stress, and it 2

was found that the mouse egr-1 gene was upregulated during in vivo infection h

with the EPEC-related pathogen, Citrobacter rodentium (de Grado et al., 2001), ffi

demonstrating that this effect is not isolated to cultured cells. The functional h

consequences of upregulation of Egr-1 production by infected cells remain ?
to be determined and may prove to be a key part of the pathogenic strategy
of these bacteria.

Modulation of apoptosis

The induction of apoptosis plays an important role in the regulation of the
immune response to bacterial infections, although the process of apoptosis
may be hijacked by the pathogen to its own advantage (Muller and Rudel,

2001). Several enteric pathogens are known to trigger apoptosis in host cells
both in vitro and in vivo (Muller and Rudel, 2001).

For EPEC, the induction of apoptosis in host cells would seem to be
counterintuitive because the bacteria rely on their adhesion to the host ep-
ithelia in order to effect A/E lesion formation. Several studies have found
that EPEC may actually act to slow down the apoptotic process during in-
fection. For example, although EPEC-infected cells eventually succumbed to
cell death that had hallmark features of apoptosis, such as early expression of
phosphatidylserine (PS) on host cell surfaces and internucleosomal cleavage
of DNA, the time taken for this effect to become apparent was much greater
than for apoptosis induced by invasive bacteria (Crane et al., 1999). More
recently, a comprehensive in vivo study in rabbits infected with RE P EC 103
demonstrated that although apoptosis physiologically occurs in the rabbit
g ileum, particularly at the tips of the absorptive villi, infection with REPEC

g did not promote the rate of apoptosis but may actually have diminished it

g (Heczko et al., 2001). Thus the modulation of apoptosis during infection by

3 EPEC and EH EC could be an important bacterial strategy used both to in-

g crease the likelihood of bacterial attachment and to control the rate of (and

* downstream effects of) host cell death.

5j The stimulation of tyrosine kinases, PKC, and the transcription factor NF-

o k B all suppress cell death, and E PEC is able to effectively activate these factors

n (Crane and Oh, 1997; Savkovic et al., 1997). Interestingly, an EPEC secreted

i protein, EspF, has been shown to effect cell death in a manner that has fea-

jj tures compatible with pure apoptosis (Crane et al., 2001). Conversely, EPEC

2 BFP may play a role in the induction of apoptosis, because nonpathogenic

w E. coli strains expressing BFP genes induced significant levels of apoptosis

in host epithelial cells (Abul-Milh et al, 2001).

The role that EHEC plays in the modulation of apoptosis has not been
so extensively studied, although from the limited data available it appears
that EHEC is also able to effect apoptosis in a manner that is dependent on
bacterial attachment and occurs in a similar timeframe to apoptotic cell death
induced by EPEC (Barnett Foster et al., 2000). The best-characterized toxin of
EHEC, SLT, is also known to induce apoptosis (Jones et al., 2000), although
the rate of this toxin-mediated apoptosis is much slower than that triggered
by EPEC and EHEC attachment (Barnett Foster et al., 2000). Interestingly,
this study also pointed to a rationale for EPEC and EHEC adhesion, because
one of the physiological effects of apoptotic cell death is the upregulation of
levels of phosphatidylethanolamine (PE) in the cell membrane, a molecule
used by both EPEC and EHEC for bacterial attachment (Barnett Foster et al.,
1999).

Disruption of mitochondrial membrane potentia

In agreement with the observation of host cell apoptotis in response to
EPEC infection, it has recently emerged that the gene orfl9, upstream of tir,
codes for a protein that seems to interfere with the mitochondrial ability to
maintain its membrane potential, a proapoptotic trigger (Kenny and Jepson,
2000). The precise mechanism of action of the Orfl9 protein (since renamed
Map, for mitochondrial-associated protein) and the consequences of its in-
teractions with mitochondria have yet to be elucidated, although the delivery
of Map into the host cell cytoplasm by means of the TTSS and its subsequent
targeting directly to the mitochondria represents the first discovered exam-
ple of bacteria-host cell interactions of this type (Kenny and Jepson, 2000).
Whether EH EC is able to target mitochondria in a similar manner remains to
be elucidated, but this seems likely if one considers the possession by EH EC
of a Map homologue (Perna et al., 2001). >

Although it may seem counterintuitive for EPEC to possess mechanisms x

that act both to prevent and induce apoptosis, in fact it demonstrates the >

extraordinary ability of this pathogen to control a central host cell function. >

The downstream effects of modulation of apoptosis are diverse and may be ^

advantageous to the bacterium at different stages of infection. It is possible M

that bacterial genes that affect apoptosis are regulated in response to diverse g

environmental stimuli encountered during the disease process. g

H
i— i

Modulation of Cdc42 activity °

on
H

Early during infection, EPEC is able to trigger transient filopodia-like n

cytoskeletal rearrangements distinct from those stimulated by Tir-intimin §

pedestal formation (Kenny et al., 2002). Interestingly, it was found that if £

Map expression was increased by expression of orfl 9 from a plasmid, then 2

the formation of filopodia in infected cells was stimulated, although this stim- h

ulation was independent of any observable effect on the mitochondria ( Kenny x

et al., 2002). This second role for Map was demonstrated to be dependent on h

the activation of the host GTPase, Cdc42 (Kenny et al., 2002), a molecule that ?

is not required for pedestal formation triggered by Tir and intimin (Kalman
et al., 1999), although it plays a role in cytoskeletal modification in uninfected
cells.

Surprisingly, the transient nature of filopodia formation was found to be
a result of further interactions of Cdc42 with Tir, which serve to deactivate
the host GTPase. In this respect, Tir may also have a dual function, behaving
as a GTPase-activating protein (GAP) and a signal-transducing molecule,

i— i
>
m

as well as a receptor for intimin. To modulate Cdc42 activity, Tir seems to
require its interaction with intimin, although this function of the Tir-intimin
complex is separate from its ability to induce pedestal formation (Kenny et al.,
2002).

This fascinating interplay between two bacterial proteins that seem to act
on host cell functions in an opposing manner appears to be counterintuitive
at first. It is thought that the Cdc42 -dependent signaling mediated by Map
is inhibitory to pedestal formation (Kenny et al., 2002), and because pedestal
formation is important to virulence, a bacterial mechanism to downregulate
the Cdc42 -mediated pathway is necessary. Why Map induces Cdc42 in the
first place is a mystery that remains to be solved, although Map is undoubtedly
important to the infection process (because, if it were not, its function would
likely have been abolished by natural selection) .

Modification of host immune responses

x Innate immunity

g Infection of host cells with microbial pathogens, including EPEC and

rt EHEC, leads to a proinflammatory response by means of the triggering of

§ various cellular signals that activate the NF-/cB pathway and stimulate ex-

o pression of IL-8 (Kresse et al., 2001; Savkovic et al., 2001). EPEC has been

n found to exploit the ERK1/2 pathway and to induce inflammation via these

g routes (Czerucka et al., 2000, 2001; Savkovic et al., 2001). EHEC infection in

jj vitro results in phosphorylation of ERK1/2 as well as two further groups of the

2 MAPK family, p38 and JNK, which activate NF-/cB as well as the transcription

w factor, AP-1 (Dahan et al., 2002). These transcription factors in turn regulate

the expression of IL-8. It is thought that the stimulation of these pathways is

likely to be effected by the secretion of as yet unknown bacterial factors by

means of the EPEC-EHEC TTSS (Dahan et al, 2002).

When histological specimens from the intestines of animals infected
with EPEC are examined, there is evidence for a dramatic infiltration by
inflammatory cells, in particular, polymorphonuclear leukocytes (PMNs)
(Moon et al., 1983). The exact advantage to the pathogen of PMN attraction
to the site of infection remains to be elucidated, although it is known that
recruited PMNs release prostaglandins, which in turn increase the activity of
adenylate cyclase in intestinal cells. As a result, cAMP levels increase, leading
to a release of Cl~ by the cells. As already discussed, release of Cl~ contributes
to the development of watery diarrhea, a hallmark of both EPEC and EHEC
infection and a likely mechanism by which the bacteria facilitate their spread
from host to host.

One further advantage of PMN infiltration, to SLT-producing EHEC in
particular, has recently been described. The genes encoding SLT in EHEC are
carried as part of lysogenic bacteriophages, and it is thought that the induc-
tion of these phages during infection contributes to EHEC pathogenesis by
increasing the transcription and copy number of the phage genes (Plunkett
et al., 1999). It was recently found that phage induction during EHEC infec-
tion may be due, at least in part, to the hydrogen peroxide released by neu-
trophils as a component of their antibacterial arsenal (Wagner et al., 2001).
Indeed, SLT has been shown to inhibit neutrophil apoptosis (Liu et al., 1999),
which may prolong H2O2 release and promote production of SLT.

Adaptive immunity

The severity of E PEC and EHEC infections tends to correlate with the age
of the patient, with infants and young children being most at risk for the de- h

velopment of disease . It has been found that infants are more likely to develop g

diarrhea during their first colonization with EPEC than they are during sub- ^

sequent exposures to the pathogen (Cravioto et al., 1990), although whether g

this is also true of EHEC infections of infants is unknown. In adult volun- g

teers infected with EPEC, there was no specific effect of prior EPEC infection g

on the incidence of diarrhea, although disease severity was reduced in indi- ~

viduals who were reinfected with a homologous EPEC strain (Donnenberg 3

et al., 1998). Effects such as these may be the result of acquired (adaptive) n

immunity in the host. °

Several studies have been undertaken in an effort to identify the antibody

responses generated to specific EPEC and EHEC antigens during the acute «

and later stages of disease. Given the key roles that EHEC and EPEC factors, n

such as Esps, Tir, intimin, and (for EPEC) BFP, play in the virulence of §

these pathogens, studies have usually focused on the immune responses £

generated to these bacterial factors in both natural and volunteer infection
studies.

Human volunteers experimentally infected with EPEC developed a x

strong IgG response against intimin (Nataro and Kaper, 1998), although in h

subsequent studies of natural infections of both EHEC and EPEC, an anti- ?

body response to intimin has varied in strength from strong to undetectable
(Li et al., 2000). Immune responses to EspA and EspB were similarly variable
in patients naturally infected with EPEC (Martinez et al., 1999). In sera from
patients infected with EHEC0157:H7, there was little reactivity to EspA and
EspB during the acute phase of illness, although this titer was increased in
the later stages of disease. Tir was shown to induce a significant antibody
response in EHEC infections (Li et al., 2000), and this response was found

to persist after infection (Li et al., 2000). BFP was found to elicit an IgG res-
ponse, but not an IgA response, in children infected with BFP + EPEC strains
(Martinez et al, 1999).

Interestingly, a study of secretory IgA purified from breast milk from
mothers who were living in areas where EPEC was endemic showed that
these antibodies prevented the adherence of EPEC to cultured epithelial cells
(Cravioto et al., 1991). The antibodies reacted to many EPEC proteins, several
of them unknown (Manjarrez-Hernandez et al., 2000) . This illustrates, in part,
the complexity of the human immune response to EPEC (which is likely to be
reflected by that to EH EC) and indicates that there is still much to be learned
before a suitable molecular target is identified for vaccine production.

Lysates of both EPEC and EHEC are known to inhibit lymphokine pro-
duction by lymphoid cells from multiple sites (Malstrom and James, 1998).
£ The nature of this inhibition was recently partially characterized when it

g was found that a large gene present in EPEC encoded a toxin that specifi-

g cally acted to inhibit lymphocyte proliferation, and IL-2, IL-4, and gamma

3 interferon (IFN-y) production (Klapproth et al., 2000). The toxin was named

g lymphostatin and is one of the largest bacterial toxins known. Homologues of

rt lifA (encoding lymphostatin) are found in most strains of EPEC, some strains

5j of EHEC (but not 0157 strains) where it is designated Efal, and C. rodentium

o (Klapproth et al., 2000), correlating with an A/E ability of the pathogen.

n The mechanism of action of lymphostatin is yet to be determined because

i it is difficult to purify (Klapproth et al., 2000). However, its effects are limited

jj to lymphokine expression and appear to cause no changes in the epithelial cell

2 cytoskeleton (Klapproth et al., 2000) . A potential consequence of lymphostatin

w expression may be the suppression of an adaptive immune response to the

bacteria, thus prolonging the infection and increasing the likelihood that the

pathogen will be passed on to new hosts.

CONCLUSIONS

By now, it should be clear that the pathogenesis of EPEC and EHEC
infection is not a simple process. EPEC and EHEC have evolved myriad
strategies to subvert host cell functions that, when combined, give rise to the
etiology of the diseases that they cause. The host is undoubtedly not passive
during infection with these bacteria, but the outcome of infection rests on
the delicate balance of host cell processes, some of which are competing in a
molecular tug of war controlled by the host on one side and the bacteria on
the other. With the advent of increasing bacterial resistance to antibiotics, we
can no longer rely on antimicrobial compounds for the effective treatment of

bacterial infections. To design better therapeutics, we will need to target the
mechanisms the bacteria use to control their hosts.

Although we have been forced to find alternative methods to treat in-
fections with bacteria such as EPEC and EH EC, this coercion has not been
without benefit. The study of bacteria-host cell interactions has given new
dimension to the field of cell biology, and as we learn more about the ways
in which bacteria interact with us, we will undoubtedly discover levels of
complexity within the host cell far beyond those we have studied to date.
Pathogens themselves have become the cell biologist's working tools and are
providing fascinating insights into the workings of the eukaryotic cell.

ACKNOWLEDGMENTS

As a result of space limitations, we could not reference original sources h

for all data cited in this review; original sources can be found in the reviews g

cited. Research in the DeVinney Lab is supported by Canadian Institutes ^

for Health Research, and the Alberta Heritage Foundation for Medical Re- g

search (AHFMR). E. Allen-Vercoe is an AHFMR postdoctoral fellow, and g

R. DeVinney is an AHFMR scholar. g

I— I

Z
H

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fc diminishes barrier function. Am. J. Physiol. 268, G374-G379.

3 Stein, M.A., Mathers, D.A., Yan, H., Baimbridge, K.G., and Finlay, B.B. (1996).

2 Enteropathogenic Escherichia coli markedly decreases the resting membrane

w potential of Caco-2 and HeLa human epithelial cells. Infect. Immun. 64, 4820-

4825.
Stevens, M.P., van Diemen, P.M., Frankel, G., Phillips, A.D., and Wallis, T.S.
(2002). Efal influences colonization of the bovine intestine by Shiga toxin-
producing Escherichia coli serotypes 05 and Olll. Infect. Immun. 70, 5158-
5166.
Stuart, R.O. and Nigam, S.K. (1995). Regulated assembly of tight junctions by

protein kinase C. Proc. Natl. Acad. Sci. USA 92, 6072-6076.
Tarr, P. I., Bilge, S.S., Vary, J.C. Jr., Jelacic, S., Habeeb, R.L., Ward, T.R.,
Baylor, M.R., and Besser, T.E. (2000). Iha: a novel Escherichia coli 0157:H7
adherence -conferring molecule encoded on a recently acquired chromoso-
mal island of conserved structure. Infect. Immun. 68, 1400-1407.
Tobe, T., Hayashi, T., Han, C.G., Schoolnik, G.K., Ohtsubo, E., and Sasakawa,
C. (1999). Complete DNA sequence and structural analysis of the entero-
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5455-5462.

Wachter, C, Beinke, C, Mattes, M., and Schmidt, M.A. (1999). Insertion of
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Wagner, P.L., Acheson, D.W., and Waldor, M.K. (2001). Human neutrophils
and their products induce Shiga toxin production by enterohemorrhagic Es-
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Warawa, J., Finlay, B.B., and Kenny, B. (1999). Type III secretion-dependent
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CHAPTER 5

Molecular ecology and cell biology of
Legionella pneumophila

Maelle Molmeret, Dina M. Bitar, and Yousef Abu Kwaik

Legionella pneumophila, a Gram-negative bacillus that is ubiquitous in aquatic
environments, is responsible for Legionnaires' disease. It is a facultative in-
tracellular pathogen that can replicate within eukaryotic host cells such as pro-
tozoan and macrophages. In water, I. pneumophila grows within protozoan
hosts. There are at least 13 species of amoebae and 2 species of ciliated pro-
tozoa that support intracellular replication of I. pneumophila (Fields, 1996).
Among the most predominant amoebae in water sources are hartmannellae
and acanthamoebae, which have also been isolated from water sources associ-
ated with Legionnaires' disease outbreaks (Fields, 1996). Interaction between
L. pneumophila and protozoa is considered to be central to the pathogenesis
and ecology of L. pneumophila (Rowbotham, 1986; Harb et al., 2000). In hu-
mans, L. pneumophila reaches the lungs after inhalation of contaminated
aerosol droplets (Fields, 1996; Fliermans, 1996; also see Fig. 5.1). The main
sources of contaminated water droplets are hot water and air-conditioning
systems, but the bacteria have been isolated from fountains, spas, pools, den-
tal and hospital units, and other man-made water systems (Fliermans, 1996;
also see Fig. 5.1). No person-to-person transmission has been described. Once
in the lungs, L. pneumophila are ingested in alveolar macrophages, the major
site of bacterial replication. This results in an acute and severe pneumonia. In
addition to Legionnaires' disease, L. pneumophila also causes Pontiac fever,
which is a self-limiting flu-like illness that is not well understood but is not
lethal. Approximately one half of the 48 species of Legionella have been asso-
ciated with human disease. L. pneumophila is responsible for 90% of cases
of Legionnaires' disease. However, all the Legionella species under appro-
priate conditions may be capable of intracellular growth and infliction of
human disease. Infections that are due to less common species of legionellae

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are not frequently diagnosed and reported, and they are less studied than
L. pneumophila (Fields et al., 2002).

The unique intracellular fate of L. pneumophila is one of the interesting
aspects of this organism. Unlike phagosomes containing inert particles or
avirulent bacteria, the I. pneumophila-containing vacuoles avoid the "normal"
endocytic pathway, recruiting rough endoplasmic reticulum (RER) and mito-
chondria, to reside in a specialized vacuole allowing intracellular replication
(Horwitz and Silverstein, 1980; Horwitz, 1983a, 1983b, 1984; Horwitz and
Maxfield, 1984; also see Fig. 5.2). The formation of this specialized vacuole is
directed by the type IV secretion system encoded by the dot/icm genes. The dot
(defect in organelle trafficking) /icm (intracellular multiplication) loci consist
of 23 genes located in two chromosomal loci of I. pneumophila. These genes
have been identified independently by two different laboratories (Segal et al.,
1998; Segal and Shuman, 1998; Vogeletal., 1998). An analysis of the predicted §

amino acid sequences of the dot/icm genes has revealed several characteris- g

tics that indicate a role in conjugal transfer of DNA, which has been con- £

firmed by conjugation studies (Segal and Shuman, 1998; Vogel et al., 1998). w

This apparatus has also been shown to be involved in proper maturation °

of the I. pneumophila-containing phagosome in mammalian and protozoan 8

cells, directing the biogenesis of the specialized vacuole in which Legionella *

replicate (Swanson and Isberg, 1995b; Segal and Shuman, 1999; Molmeret g

et al., 2002a). The dot/icm genes are also required for macropinocytosis in £

A/ J mice macrophages (Watarai et al., 2001b), upregulation of phagocytosis g

in human-derived macrophages (Hilbi et al., 2001), induction of apoptosis g

in macrophages (Gao and Abu Kwaik, 1999a, 1999b; Zink et al., 2002), and §

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; ; *

Figure 5.1. (facing page). The environmental life of I. pneumophila within protozoa. S

(1) L. pneumophila from biofilms with other bacteria, or in suspension, infecting protozoa. ^

(2) Following entry, L. pneumophila resides in a membrane-bound vacuole that recruits m

host cell organelles, such as the mitochondria and the rough endoplasmic reticulum, and ^

o or q

does not fuse with lysosomes. Mutants such as dot/icm mutants fuse to lysosomes (3b); S

(-I
L. pneumophila replicates within specialized vacuoles and reaches large numbers (3a). £

(4) The infectious particle is not known but may include excreted Legionella-fiWed vesicles,
intact Legionella-fiWed amoebae, or free legionellae that have lysed their host cell.

(5) Transmission to humans occurs by mechanical means, such as faucets and
showerheads. Infection in humans occurs by inhalation of the infectious particle and
establishment of infection in the lungs. (6) Legionellae that have escaped their host cell
may survive in suspension for long periods of time, reinfect other protozoa, or recolonize
biofilms. (This figure was taken from ASM News 66 (10): 609-616, 2000.)

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Figure 5.2. Transmission electron micrographs of the infection of Acanthamoeb a
polyphaga (top panel) and U937 macrophages (bottom panel) by L. pneumophila. Coiling
phagocytosis (A and B); formation of the RE R- surrounded phagosome (C and D); and late
stages of the infection (E and F). Note that in E and F there is no intact phagosomal
membrane. (This figure was adapted from ASM News 66 (10): 609-616, 2000.)

pore -formation-mediated cytotoxicity in both protozoan and mammalian cells
(Alii et al, 2000; Gao and Abu Kwaik, 2000; Molmeret et al, 2002a, 2002b).

ECOLOGY OF LEGIONELLAE

After isolation of I. pneumophila from the air-conditioning system during
the first outbreak in Philadelphia, Pennsylvania (1976) , the bacteria have been
isolated from numerous sources in the environment. Legionellae species have
been repeatedly shown to be ubiquitous, particularly in aquatic environments
(Fields, 1996).

In the environment, legionellae species cannot multiply extracellularly,
and they have been shown to be parasites of protozoa (Harb et al., 2000). In
1980, Rowbotham was the first to describe the ability of I. pneumophila to
multiply intracellularly within protozoa (Rowbotham, 1980). The 13 species
of amoebae and 2 species of ciliated protozoa that allow intracellular bacterial
replication have been shown to be potential environmental hosts for legionel-
lae species (Abu Kwaik et al., 1998). This rather sophisticated host-parasite
interaction indicates a tremendous adaptation of legionellae to parasitize pro-
tozoa. This host-parasite interaction is also central to the pathogenesis and
ecology of these bacteria.

There are now at least 46 species of legionellae [Benin, et al., 2002].
In addition, 12 phylogenetic groups of bacteria belonging to 5 species have
been designated as Legionella-like amoebal pathogens (LLAP; Adeleke et al.,
1996). The LLAPs are genetically related to legionellae and many of them §

have been associated with Legionnaires' disease (Birtles et al., 1996; Marrie g

et al., 2001). In contrast to legionellae species, the LLAPs cannot be cultured £

in vitro on artificial media. The LLAPs are isolated by coculture with protozoa w

(Fields, 1996). LLAPs have been isolated from sputum samples derived from °

patients with Legionnaires' disease on the basis of the ability of these bacteria 8

to multiply in protozoa, because they cannot be grown on artificial media %

o
(Adeleke et al. , 1996) . The recent developments in using the polymerase chain g

reaction for bacterial identification in environmental samples will facilitate £

better identification of legionellae species and LLAPs. g

o
Many strategies have been used to eradicate legionellae from sources of g

infection in water and plumbing systems that have been associated with dis- 2

ease outbreaks. These strategies include chemical biocides such as chlorine, g

overheating of the water, and UV irradiation (Biurrun et al., 1999; Kool et §

al., 1999; Muraca et al., 1987). Such interventions have been successful for c

short periods of time after which the bacteria are again found in these sources g

(Yamamoto et al., 1991; Biurrun et al., 1999). Thus, eradication of L. pneu- c

mophila from the environmental sources of infection requires continuous o

treatment of the water with agents such as monochloramine or copper-silver 2

ions in addition to maintenance of the water temperature above ^55°C [Kool
et al., 1999; Kusnetsovet al., 2001]. It is clear that the sophisticated association
of legionellae with protozoa is a major factor in continuous presence of the
bacteria in the environment. Compared with L. pneumophila grown in vitro,
amoebae-grown bacteria have been shown to be highly resistant to chemical
disinfectants and to treatment with biocides (Barker et al., 1992). Amoebae-
grown L. pneumophila have been shown to manifest a dramatic increase in
their resistance to harsh environmental conditions, such as fluctuation in

temperature, osmolarity, pH, and exposure to oxidizing agents (Abu Kwaik
et al., 1997). Protozoa have been shown to release vesicles containing L. pneu-
mophila that are highly resistant to biocides (Berk et al., 1998).

The ability of I. pneumophila to survive within amoebic cysts further con-
tributes to the resistance of I. pneumophila to physical and biochemical agents
used in bacterial eradication (Barker et al., 1992, 1995). It is most likely that
eradication of the bacteria from the environment should start by preventing
protozoan infection, an integral part of the infectious cycle of I. pneumophila.
Further characterization of the mechanisms of bacterial invasion into pro-
tozoa may allow the design of strategies to block the protozoan receptor
from attachment to legionellae and thus prevent bacterial entry. Extracellular
L. pneumophila will be more susceptible to environmental conditions and will
not be protected from biocides and disinfectants. Furthermore, blockage of
w bacterial entry into amoebae will render bacteria less infective and virulent

g to mammalian cells. Alternatively, treatment of water sources contaminated

§ with I. pneumophila with "safe" agents that block certain essential bacterial

ph metabolic pathways, such as the peptidoglycan biosynthesis pathway, may

z prove to be useful (Harb et al., 1998).

Q It has been proposed that the infectious particle for Legionnaires' disease

< is amoebae infected with the bacteria (Rowbotham, 1980; also see Fig. 5.1).

< Although this has not yet been proven, there are many lines of evidence to
* suggest that protozoa play major roles in the transmission of I. pneumophila.

< First, many protozoan hosts have been identified that allow intracellular bac-
S terial replication, which is the only means of bacterial amplification in the
^ environment (Fields, 1996; Abu Kwaik et al, 1998; Harb et al., 2000). Sec-
§ ond, in outbreaks of Legionnaires' disease, amoebae and bacteria have been
| isolated from the same source of infection and the isolated amoebae support
3 intracellular replication of the bacteria (Fields et al., 1990) . Third, as discussed,

< following intracellular replication within protozoa, L. pneumophila exhibit a
dramatic increase in resistance to harsh conditions, such as high temperature,
acidity, and high osmolarity, which may facilitate bacterial survival in the envi-
ronment (Abu Kwaik et al., 1997). Fourth, intracellular L. pneumophila within
protozoa are more resistant to chemical disinfection and biocides compared
with bacteria grown in vitro bacteria (Barker et al., 1992, 1993, 1995). Fifth,
protozoa have been shown to release vesicles of respirable size that contain
numerous L. pneumophila. The vesicles are resistant to freeze-thawing and
sonication, and the bacteria within the vesicles are highly resistant to biocides
(Berk et al., 1998). Sixth, following their release from the protozoan host, the
bacteria exhibit a dramatic increase in their infectivity for mammalian cells
in vitro (Cirillo et al., 1994).

In addition, it has been demonstrated that intracellular bacteria within
Hartmanella vermiformis are dramatically more infectious and are highly
lethal in mice (Brieland et al., 1997). Seventh, the number of bacteria iso-
lated from the source of infection of Legionnaires' disease is usually very low
or undetectable, and thus enhanced infectivity of intracellular bacteria within
protozoa may compensate for the low infectious dose (O'Brein and Bhopal,
1993). Eighth, viable but nonculturable L. pneumophila can be resuscitated
by coculture with protozoa (Steinert et al., 1997). This observation may
suggest that failure to isolate the bacteria from environmental sources of
infection may be due to this "dormant" phase of the bacteria that cannot be
recovered on artificial media. Ninth, there has been no documented case of
bacterial transmission between individuals. The only source of transmission
is environmental droplets generated from many human-made devices,
such as shower heads, water fountains, whirlpools, and cooling towers of §

ADHERENCE AND ENTRY MECHANISMS

protozoan hosts

air-conditioning systems (Fields, 1996). £

n
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Initial interactions between L pneumophila and its primitive >

o
n

Bacterial attachment to H. vermiformis is mediated by adherence to

a protozoan receptor that has been characterized as a galactose/N -acetyl- 2

o
galactosamine (Gal/GalNAc) lectin with similarity to the fii integrin-like g

Gal/GalNAc lectin of the pathogenic protozoan Entamoebae histolytica (Mann 2

et al., 1991; Venkataraman et al., 1997; Harb et al., 1998). Integrins are het- g

erodimeric protein tyrosine kinase receptors that undergo tyrosine phospho- §

rylation upon engagement to ligands, which subsequently results in recruit- c

ment and rearrangements of the cytoskeleton. Interestingly, attachment of 5

L. pneumophila to the Gal/GalNAc of H. vermiformis triggers signal transduc- c

tion events in H. vermiformis that are manifested in dramatic tyrosine dephos- o

phorylation of the lectin receptor and other proteins (Venkataraman et al., 2

1997). Moreover, in addition to these manipulations of the signal transduc-
tion of H. vermiformis by L. pneumophila, bacterial invasion is also associated
with specific induction of gene expression in the protozoa, and inhibition
of this gene expression blocks entry of the bacteria (Abu Kwaik et al., 1994).
Following this initial host-parasite interaction, uptake of I. pneumophila by
protozoan cells occurs by both conventional and coiling phagocytosis (in
which the bacterium is surrounded by a multilayer coil -like structure; Abu
Kwaik, 1996; Bozue and Johnson, 1996; also see Fig. 5.2).

Invasion of H. vermiformis by I. pneumophila requires host protein syn-
thesis, because eukaryotic protein synthesis inhibitors (cycloheximide) block
the entry process (Abu Kwaik et al., 1994). The uptake of L. pneumophila into
H. vermiformis mainly occurs through cup-shaped invaginations (or zipper
phagocytosis) on the surface of the amoeba, although some coiling phagocy-
tosis also occurs (Venkataraman et al., 1998). Such invaginations are known
to be microfilament dependent (Venkataraman et al., 1998). However, the
entry of the bacteria into H. vermiformis is not inhibited by microfilament
inhibitors such as cytochalasin D (King et al., 1991; Harb et al., 1998). Methy-
lamine, which is an inhibitor of receptor-mediated endocytosis, inhibits the
entry of L. pneumophila into H. vermiformis (King et al., 1991). Apparently,
infection of Acanthamoeha polyphaga by L. pneumophila occurs through a dif-
ferent mechanism. It is not inhibited by galactose or N-acetylgalactosamine
« (Harb et al., 1998). The 170-kDa Gal/GalNAc-inhibitable lectin is only mildly

g dephosphorylated in A. polyphaga upon attachment of I. pneumophila (Harb

§ et al., 1998). Furthermore, host protein synthesis by A. polyphaga is not re-

ph quired for invasion by I. pneumophila (Harb et al., 1998). The uptake of the

s bacteria is not inhibited by cytoskeleton-disrupting agents. The role of this

Q form of phagocytosis in the intracellular fate of I. pneumophila is not fully

< understood, because human macrophages are able to phagocytose heat- or

< formalin-killed I. pneumophila by coiling phagocytosis (Horwitz, 1984).

i— i

2 ATTACHMENT AND ENTRY TO MAMMALIAN CELLS

w Invasion and intracellular replication of I. pneumophila within pul-

§ monary cells in the alveoli is the hallmark of Legionnaires' disease (Abu

° Kwaik, 1998b). These alveolar cells include macrophages, and type I and

3 II epithelial cells. Attachment of I. pneumophila into macrophages has been

< shown to be mediated, at least in part, through the attachment of complement-
is

coated bacteria to the complement receptor (Payne and Horwitz, 1987), al-
though non-complement-mediated uptake also occurs (Elliott and Winn,
1986; Rodgers and Gibson, 1993). The host cell receptor involved in non-
complement-mediated uptake in macrophages and epithelial cells is not
known.

Uptake of I. pneumophila by monocytes and macrophages has been
shown to occur through conventional and coiling phagocytosis (Horwitz,
1984; Weinbaum et al., 1984; Elliott and Winn, 1986; Rechnitzer and Blom,
1989; Dowlingetal., 1992; also see Fig. 5.2). Because heat-killed and formalin-
killed I. pneumophila are also taken up by coiling phagocytosis (Horwitz, 1984)
but are targeted to the lysosomes (Horwitz and Maxfield, 1984), this mode of

uptake may not play a role in subsequent pathogenicity of the bacteria. Many
clinical isolates of L. pneumophila have been shown to be taken up exclusively
by conventional phagocytosis (Elliott and Winn, 1986; Rechnitzer and Blom,
1989). In addition, other species of legionellae, such as L. micdadei, which
is the second most common species of legionellae that causes Legionnaires'
disease, is taken up exclusively by conventional phagocytosis (Rechnitzer and
Blom, 1989). The bacterial ligand that mediates the coiling mode of phagocy-
tosis is not known. Moreover, the phagocytic receptor that binds the bacteria
seems to play some role in determining the fate of the intracellular bacteria
because opsonization with antibodies reduces intracellular growth (Horwitz
and Silverstein, 1981a; Nash et al., 1984; Payne and Horwitz, 1987).

Studies have focused on the genetic aspects of the uptake of I. pneu-
mophila in its host cells. I. pneumophila mutants impaired in different loci,
such as rtxA and enhC, display significantly reduced entry into host cells, §

compared with wild-type bacteria (Cirillo et al., 2000). Recently, it has been g

shown that the enhanced phagocytosis of I. pneumophila by mammalian cells £

is dot/icm dependent (Hilbi et al., 2001). Interestingly, the dot/icm genes de- w

lay uptake and induce macropinocytosis in A/J mice macrophages (Watarai °

et al., 2001b). Macropinosomes containing L. pneumophila are induced tran- 8

siently and shrink rapidly (5-15 min; Watarai et al., 2001b), and this mode of %

o
uptake is linked to the Ignl locus on chromosome 13 of mice (Watarai et al., g

2001b) . With the exception of A/J mice, most of the inbred mouse strains are £

not permissive to L. pneumophila infection; neither are macrophages isolated g

o
from these mice (Yamamoto et al., 1992; Beckers et al., 1995). The differ- g

ence between these two mice strains is located on chromosome 13 and is 2

linked to a single locus, Ignl (Dietrich et al., 1995; Beckers et al., 1997). In g

macrophages of nonpermissive strains of mice, the macropinocytic uptake §

of I. pneumophila is reduced (Watarai et al., 2001b). The Ignl allele makes the c

bacteria behave as if they are lacking the dot/icm system (Watarai et al., 2001b) . g

Thus, the Ignl allele is required for dot/icm-dependent macropinocytosis and c

is;

delayed uptake by mice macrophages (Watarai et al., 2001b). o

INTRACELLULAR TRAFFICKING

Intracellular survival and replication within host cells

During the first few minutes after entry into amoebae, the bacterium is
enclosed in a phagosome surrounded by mitochondria and host cell vesicles
(Abu Kwaik, 1996; also see Fig. 5.2). The bacterial phagosome is blocked
from fusion to the lysosomes (Bozue and Johnson, 1996). In addition, the

phagosome is surrounded by a multilayer membrane derived from the RER
of amoebae (Abu Kwaik, 1996; also see Fig. 5.2). Following formation of this
phagosome within protozoan cells, bacterial replication is initiated. The 4-h
period prior to initiation of intracellular replication may be the time required
to recruit these host cell organelles that may be required for replication. Al-
ternatively, the 4-h period may be a lag phase of metabolic and environmental
adjustment of the bacteria to a new niche.

Similar to the protozoan infection, within 5 min following entry of the
bacteria into macrophages and monocytes, the I. pneumophila phagosome
is surrounded by host cell organelles such as mitochondria, vesicles, and
the RER (Horwitz, 1983b; Swanson and Isberg, 1995a; Tilney et al., 2001;
also see Fig. 5.2). Also similar to the trafficking of I. pneumophila within
protozoa, the phagosome within mammalian macrophages does not fuse to

* lysosomes (Horwitz, 1983a, 1984; Horwitz and Maxfield, 1984; also see Fig.
g 5.2). The role of the RER in the intracellular infection is not known, but the

§ RE R is not required as a source of protein for the bacteria (Abu Kwaik, 1 998a) .

ph Interestingly, examination of the intracellular infection of macrophages, alve-

z olar epithelial cells, and protozoa by another Legionella species, I. micdadei,

Q showed that, within all of these host cells, the bacteria were localized to RER-

< free phagosomes (Gao et al., 1999) . Whether other Legionella species replicate

< within RER-free phagosomes is still to be determined.

* Macrophages, peripheral blood monocytes, and alveolar epithelial cells

< support intracellular replication of I. pneumophila (Nash et al., 1984; Gao
S et al., 1998b). Although alveolar epithelial cells, which constitute more than
w 95% of the alveolar surface (Gao et al., 1998b), have been shown to allow
§ intracellular replication of L. pneumophila, their role in the pathogenesis has
° been largely overlooked.

w
i-i
i-i

s Role of the dot/icm genes in evasion of the endocytic pathway

The Dot/icm type IV secretion system is the main virulence system of
L. pneumophila. Because the Dot/icm secretion system is ancestrally related
to type IV secretion systems that mediate conjugal DNA transfer between
bacteria (Christie and Vogel, 2000), I. pneumophila may utilize this trans-
porter to transfer macromolecules into the host cell to evade endocytic fusion
(Roy and Tilney, 2002). The dot/icm loci may be involved in the insertion
of a pore in the host cellular membrane to transfer the effector proteins
(Kirby and Isberg, 1998; Kirby et al., 1998). The effector molecules involved
in intracellular trafficking and evasion of the lysosomal fusion within mam-
malian cells are limited to the phagosome harboring the bacterium, which

does not alter the biology of endocytic fusion in the rest of the cell (Coers
et al., 1999). With few exceptions, the function of individual Dot/Icm pro-
teins is unknown.

The Dot A protein was the first to be described (Berger et al., 1994). It is a
polytopic inner membrane protein with eight hydrophobic transmembrane
domains (Roy and Isberg, 1997). dotA mutants are defective in all virulence
activities that require the Dot/Icm complex (Berger and Isberg, 1993; Berger
et al, 1994; Swanson and Isberg, 1995a, 1995b; Kirby et al., 1998; Coers
et al., 2000). These data are supported by the fact that the DotA sequence
possesses significant similarities with that of TraY (Segal et al., 1999;
Komano et al., 2000), a component of the type IV transporter required for
the conjugal transfer of plasmids ColIbP9 and R64. However, Nagai and
Roy have shown that the DotA protein is also secreted through the Dot/Icm
transporter into the culture supernatant during growth of I. pneumophila in §

liquid broth by means of a functional type IV secretion system (Nagai and g

Roy, 2001) . Electron micrographs also show that DotA is part of the oligomer £

that could be the membrane channel (Roy and Isberg, 1997; Nagai and Roy, w

2001) . Mutants defective in DotA protein expression are unable to form pores °

in host cell membranes (Coers et al., 2000). 8

As DotH/IcmK and DotO/IcmB in growing I. pneumophila cultures are %

o
mainly associated with the membranous fraction, and as dot/icm products g

may be required during direct contact with host cells, the location on the £

surface of L. pneumophila of DotH and DotO proteins has been examined g

o
(Watarai et al., 2001a) . These proteins are surface exposed and associated with g

a fibrous structure on L. pneumophila after exposure to bone-marrow-derived 2

macrophages. In contrast, during broth culture, this fibrous structure seems g

to be absent (Watarai et al., 2001a). However, with the use of dotA, dotB, dotH, §

and dotO mutants, it has been shown that the exposure of DotO/DotH on the c

bacterial surface is not dependent on other Dot/Icm proteins, including the g

DotH protein, for the DotO exposure and vice versa (Watarai et al., 2001a). c

This result shows that the surface exposure of DotH and DotO after contact o

with macrophages is not dependent on an intact Dot/Icm secretion system 2

(Watarai et al., 2001a). The surface exposure of these two proteins does not
involve bacterial contact with the target cell because bacteria incubated in
medium that have been conditioned by bone-marrow-derived macrophages
for 24 h yield almost identical results (Watarai et al., 2001a).

In addition, DotH and DotO surface exposure on L. pneumophila re-
quires intracellular growth of the bacteria in macrophages and is observed
late in the infection process, mostly when there are more than 30 bacteria per
phagosome (Watarai et al., 2001a). In fact, surface-exposed DotH and DotO

disappear after uptake but reappear following intracellular growth. The expo-
sure of these two proteins increases L. pneumophila uptake into cells (Watarai
et al., 2001a) and may be necessary to promote bacterial escape from the
phagosome and to facilitate the initiation of a new infection in macrophages
(Watarai et al., 2001a). These data may also suggest that macrophage com-
ponents are able to induce changes in the L. pneumophila envelope, and
DotH/DotO export may occur as a response to the target macrophages just
before uptake to allow efficient initiation of intracellular growth.

Dot/Icm proteins such as IcmR, IcmQ, IcmX, or IcmW do not present
sequence homology to other protein components of the type IV secretion sys-
tem (Segal and Shuman, 1998; Vogel et al., 1998; Christie and Vogel, 2000).
IcmX, a periplasmic protein, is required for pore-forming activity (Matthews
and Roy, 2000). The icmXgene is also required for biogenesis of the replica-

* tive phagosomes containing I. pneumophila (Matthews and Roy, 2000). A
g truncated IcmX product is secreted into culture supernatants by wild-type

§ L. pneumophila growing in liquid media, but the transport of the protein into

ph eukaryotic host cells has not been detected (Matthews and Roy, 2000).

s icmS and icmW mutants are not defective in pore-forming activities, but

Q their phagosomes fuse to lysosomes (Coers et al., 2000). IcmS and IcmW

< are small proteins required for the trafficking of the Legionella-containmg

< phagosome, and they may function as chaperones to help the secretion of

* proteins through the type IV secretion system (Coers et al., 2000; Nagai and

< Roy, 2001). It has also been shown that IcmS and IcmW interact, suggesting
S that they may be components of a protein complex that is required for modu-
le lating phagosome biogenesis (Coers et al., 2000). Interestingly, phagosomes
pi

g harboring icmS and icmW mutants still recruit host vesicles, including the

,_i

2 RER. It is still to be confirmed whether recruitment of host vesicles does not

3 prevent the phagosome from lysosomal fusion (Roy and Tilney, 2002).

< The IcmR protein may also be a chaperone (Coers et al., 2000). An icmR
mutant has undetectable pore-forming activity and can partially evade the
endocytic pathway, but it eventually fuses with the lysosomes (Coers et al.,
2000). These data suggest that IcmR may be a cofactor for another protein
effector involved in evasion of lysosomal fusion (Roy and Tilney, 2002). The
icmR mutant that evades endocytic fusion does not recruit host vesicles, and
the phagosome is not surrounded by the RER (Roy and Tilney, 2002). Thus,
effector molecules that recruit host cell vesicles may be different from the
ones involved in evasion of the endocytic pathway (Roy and Tilney, 2002).

Similar to dot A and icmX mutants, an icmQ mutant is defective in all
virulence activities (Coers et al., 2000). Furthermore, the icmQ gene, like
the icmR and icmS genes, encodes soluble protein. The IcmR and IcmQ

proteins interact as protein chaperone-substrate. The presence of IcmR,
which has chaperone characteristics (it is acidic, small, and predicted to have a
hydrophobic alpha-helix in its C-terminal domain) affects the physical state of
IcmQ directly (Dumenil and Isberg, 2001). IcmR prevents IcmQ from partic-
ipating in the formation of high-molecular-weight complexes by dissociating
IcmQ homopolymers (Dumenil and Isberg, 2001).

It has been shown that most of the dot/icm genes required for intracel-
lular growth within human cells are also required for intracellular growth
in the protozoan host A. castellanii (Segal and Shuman, 1999). In addition,
enhanced phagocytosis by A. castellanii of wild-type L. pneumophila has also
been demonstrated to be dependent on dot/icm genes (Hilbi et al., 2001).
Although some loci have been shown to be essential only for the intracellu-
lar growth of I. pneumophila in macrophages (Gao et al., 1998a), numerous
loci have been identified as essential for survival and intracellular replica- §

tion of I. pneumophila in A. polyphaga or H. vermiformis and in macrophages £

(Cianciotto and Fields, 1992; Gao et al, 1997; Stone et al, 1999). £

Another host, Dictyostelium discoideum, a unicellular organism that lives w

in soil, has been shown to support the intracellular multiplication of L. pneu- °

mophila (Solomon and Isberg, 2000; Solomon et al., 2000). In the amoebal 8

form, the cells are highly motile and are very active in phagocytosis. This %

o
model is interesting because genetic tools are available for analysis of host- g

pathogen interactions, such as the existence of plasmids replicating in this £

haploid organism as well as extensive sequence DNA information (Solomon g

o
and Isberg, 2000; Solomon et al., 2000). The intracellular fate of I. pneu- g

mophila is very similar in infected D. discoideum to that in macrophages, 2

including the recruitment of RER and evasion of lysosomal fusion (Solomon g

et al., 2000) . The growth of I. pneumophila in D. discoideum depends on dot/icm §

gene functions (Solomon et al. , 2000) . The analysis of growth of wild-type and c

three isogenic dot/icm mutant strains of I. pneumophila in D. discoideum indi- g

cates that intracellular growth requires the products of multiple loci, because c

is;

dotH, dot I, and dotO mutants all failed to grow and lost viability over the o

course of 4 days. (Solomon et al., 2000). The similarity between the infection 2

by L. pneumophila of different protozoa supports the idea that the ability of L.
pneumophila to parasitize macrophages and hence to cause human disease is
a consequence of its prior adaptation to intracellular growth within protozoa.

RECRUITMENT OF RER

In 1982, Katz and Hashemi showed that the L. pneumophila-containmg
phagosome resembles the ER membrane. By means of electron microscopy,

replicating L. pneumophila within macrophages were visualized located
within organelles morphologically identical with the RER (Katz and Hashemi,
1982; also see Fig. 5.2). Later, several studies demonstrated that upon
internalization of L. pneumophila by the host cell, the Legionella-containmg
vacuole recruits organelles such as vesicles, mitochondria, and ER (Fig. 5.2;
also see Katz and Hashemi, 1982; Horwitz, 1983b; Bozue and Johnson, 1996).
With the use of fluorescent markers specific for the ER, it has been
shown that the L. pneumophila-containing vacuoles may resemble nascent
autophagosomes (Swanson and Isberg, 1995b). Autophagy is a physiologi-
cally important cellular process for the degradation of unwanted organelles
and cellular components, during which the autophagosome is formed from
RER and fuses to lysosomes (Dorn et al., 2002). The hypothesis that L. pneu-
mophila exploits the autophagy machinery in host cells and establishes an

* intracellular niche favorable for replication (Swanson and Isberg, 1995b) has
g been challenged recently (see the paragraphs that follow; also Tilney et al.,

g 2001; Roy and Tilney, 2002).

ph Recent studies suggest that fusion (Roy and Tilney, 2002), or exchange

=; of lipid bilayer with E R vesicles on the L. pneumophila-containing phagosome

Q (Tilney et al., 2001) occurs, allowing the phagosomal membrane to become

< as thin as the ER membrane, with similar characteristics (Tilney et al., 2001;

< Roy and Tilney, 2002). It has been shown that, within 5 min of uptake, host

* vesicles come into contact with wild-type Legione^a-containing phagosomes

< and flatten along the surface of the phagosome, and this process is com-
S pleted within 15 min (Tilney et al., 2001). This does not occur in dot/icm

w mutant-containing phagosomes (Tilney et al., 2001). This is consistent with

g earlier studies that have shown that, after 4 h of infection, there are only

,_i

° few vesicles associated with the phagosomal membrane, but there are ribo-

3 somes studding the phagosomal membrane (Horwitz, 1983b). Interestingly,

< the thickness of the phagosome membrane becomes similar to the ER mem-
brane within 15 min (Tilney et al., 2001). Thus, within 15 min of infection,
the phagosomal membrane resembles that of the ER. The ribosomes at 6 h
stud the phagosomal membrane, and I. pneumophila is thought to be located
within the RER (Tilney et al., 2001) . However, these studies rely completely on
the thickness of the membranes of the ER and the phagosome membranes to
provide evidence that L. pneumophila is located within the RER (Tilney et al.,
2001). Immunocytochemical studies should be performed to confirm these
observations. It is likely that the recruitment of the ER may be involved in
the biogenesis of the phagosome that is dependent on the type IV secretion
system, because the dot/icm mutants are unable to recruit RER and their
phagosomes fuse to the lysosomes (Swanson and Isberg, 1995b).

Interestingly, a recent study showed that recruitment of RER to matur-
ing phagosomes may be part of regular phagoytosis (Gagnon et al., 2002).
Electron micrographs of latex beads and pathogens such as Salmonella within
macrophages have shown that ER membranes fuse with plasmalemma, un-
derneath the phagocytic cup, and successive waves of ER are recruited to
the phagosomes of latex beads and bacteria. In neutrophils, the bacteria are
quickly killed and the ER is not involved in the turnover of membrane for
phagocytosis (Gagnon et al., 2002). However, because the dot/icm system is
essential for RER recruitment (Swanson and Isberg, 1995b), it is likely that
L. pneumophila utilizes a specific pathogen-regulated process to recruit vesi-
cles and that this process is distinct from regular phagocytosis.

THE ARF PROTEIN g

The protein ADP ribosylation factor-1 (ARF1), a highly conserved small £

GTP-binding protein that acts as a key regulator of vesicle traffic from ER to £

Golgi, is found on phagosomes that contain wild-type I. pneumophila but not w

dot/icm mutants (Nagai et al., 2002) . These data suggest that a protein injected °

through the type IV secretion system may be required for ARF1 recruitment. 8

Searching the L. pneumophila genome for proteins that have homology to z

ARF-specific guanine nucleotide exchange factors (GEFs), Nagai et al. have Q

identified a protein, RalF, that has a sec7-homology domain, known to be £

sufficient to stimulate the exchange of GDP for GTP (Nagai et al., 2002). It g

has been shown that RalF is injected through the phagosomal membrane g

by a process that requires the dot/icm system (Nagai et al., 2002). However, 2

phagosomes containing ralF mutants that do not recruit ARF1 evade fusion g

to lysosomes, and the bacteria replicate intracellularly within macrophages §

and amoebae (Nagai et al., 2002). Thus, RalF is not essential for transport of c

L. pneumophila to the ER (Nagai et al., 2002; Roy and Tilney, 2002). g

Pore-forming activity g

Kirby et al. were the first to describe the pore-forming ability of I. pneu-
mophila in host cell membranes (Kirby et al., 1998). This ability was doc-
umented by lysis of macrophages and red blood cells, which is dependent
on the Dot/icm secretion system (Kirby et al., 1998). Because dot/icm mu-
tants are defective in evasion of lysosomal fusion, it has been proposed that
the pore-forming activity is required for export of effector molecules nec-
essary for evasion of the lysosomal fusion (Kirby et al., 1998). Upon initial
contact with the host cell, I. pneumophila may insert a pore into the plasma

membrane to deliver bacterial effector molecules into the host cell (Kirby
and Isberg, 1998; Kirby et al., 1998). This concept is supported by the fact
that many dot/icm mutants are defective in both intracellular replication and
pore -formation-mediated cytotoxicity (Kirby and Isberg, 1998; Kirby et al.,
1998). Moreover, some dot/icm mutants are defective in trafficking and intra-
cellular replication but not in pore-forming activity (Zuckman et al., 1999).
Thus, the pore-forming activity is not sufficient for phagosomal trafficking
(Zuckman etal., 1999).

We have identified five spontaneous mutants that are unable to egress
from the macrophages but are able to grow as well as the wild-type strains
within these cells (Alii et al., 2000). These mutants, designated rib (release
of intracellular bacteria) , are defective in the pore-forming toxin activity as
shown by the contact-dependent hemolysis assay and by permeability to

* propidium idodide upon infection of macrophages and epithelial cells (Alii
g et al., 2000). The rib mutants are also defective in acute cytotoxic lethality to

§ A/ J mice and fail to cause alveolar inflammation (Alii et al., 2000; Molmeret

ph et al., 2002a), thus indicating a key role for the pore-forming toxin in the

z pulmonary pathology of the bacterium. We have further documented that

p the Rib toxin is not required for intracellular trafficking and replication (Alii

< et al., 2000; Gao and Abu Kwaik, 2000; Molmeret et al, 2002b).

< In addition to defects in evasion of lysosomal fusion, dot/icm mutants are

* also defective in induction of apoptosis (Zink et al., 2002), enhancement of

< phagocytosis by human-derived macrophages (Hilbi et al., 2001), and indue -
S tion of macropinocytic delayed uptake by A/J mice macrophages (Watarai

m et al., 2001b). In contrast, rib mutants are defective in pore-forming toxin

g but are not defective in evasion of lysosomal fusion, intracellular replica-

,_i

° tion, or induction of apoptosis (Alii et al., 2000; Molmeret et al., 2002b; Zink

3 et al., 2002) . These observations may suggest that there are at least two pores

< inserted into host membranes through the type IV secretion system at differ-
ent stages of the infection (Molmeret and Abu Kwaik, 2002). The first pore,
is the "invasion and trafficking pore," which is inserted upon initial contact
with the host cell to deliver effectors into the host cell cytoplasm and allow
the establishment of the intracellular infection. The dot/icm genes necessary
for the assembly of the secretion apparatus are constitutively expressed and
are required for this step (Hales and Shuman, 1999). The second pore is the
"egress pore," which is turned off during exponential intracellular replica-
tion but is triggered upon cessation of replication and is essential for lysis of
the host cell (Figs. 5.3 and 5.4; also see Alii et al., 2000). This is consistent
with the fact that, upon transition into the postexponential growth in vitro,
L. pneumophila becomes cytotoxic (Byrne and Swanson, 1998). Thus, the rib

24 h

48 h

8 '^tfSFfA^'

5 mB**

■•■y

Figure 5.3. The rife mutants' defect in cytolysis of the host cell is due to a defect in
necrosis-mediated killing. Representative transmission electron micrographs of infected
U937 macrophages at 24 h and 48 h postinfection by the wild-type strain AA100 and the
GN229 mutant. The original magnifications were 7,000x and 5,000x for the 24-hp and
48-h infections, respectively. (This figure was adapted from Alii et al., Infect Immun.
68 (11): 6431-6441, 2000.)

mutants retain the "invasion and trafficking pore" but are defective in the
"cytolysin/egress pore," because they replicate within but fail to egress from
the host cells (Figs. 5.3 and 5.4).

The phenotype of the rib mutants is attributable to a point deletion in the
icmT gene that is predicted to result in a truncated protein of 54 amino acids
instead of the 86 amino acids of the native protein (Molmeret et al., 2002a,
2002b). In contrast, an icmT null mutant is defective in both intracellular
replication and pore formation (Molmeret et al., 2002a, 2002b). It has been
shown that icmT expression is induced at the stationary phase compared
with the exponential phase (Gal-Mor et al., 2002). We have shown that IcmT
is bifunctional and that the carboxy terminus is essential for the pore-forming
"cytolysin/egress pore" activity and the amino terminus is essential to export
effectors involved in various pathogenic traits (Molmeret et al., 2002a, 2002b).

We hypothesized that, upon transition to the postexponential phase of
growth, the Rib toxin activity is triggered, resulting in insertions of pore first
in the phagosomal membrane, leading to its disruption and bacterial egress

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into the cytoplasm ( Figs .5.3 and 5 .4) . To test this hypothesis , we examined late
stages of the intracellular infection of macrophages and amoebae by electron
microscopy. Our data showed that disruption of the phagosomal membrane
was detectable 12 h postinfection in both A. polyphaga and macrophages (un-
published data). After 12 and 18 h postinfection in both host cells, vesicles
and organelles from the host cytoplasm entered into the LegioneHa-containing
phagosome. Between 18 h and 24 h, most of the bacteria present in this dis-
rupted phagosomal membrane are surrounded by cytoplasmic organelles,
and not by distinct phagosomal membrane (unpublished data) . These data
show that the phagosomal membrane is disrupted first; there is no simulta-
neous lysis of both the phagosomal and the plasma membranes. Whether this
disruption is the result of a mechanic process or is linked to the pore-forming
activity of the type IV secretion system (Kirby et al., 1998; Molmeret and Abu
Kwaik, 2002; Molmeret et al., 2002b) is not known. Our data are consistent ^

and outer membrane protein profiles (Barker et al., 1993). After growth in
host cells, L. pneumophila becomes more resistant to biocides and antibiotics
(Barker et al., 1992, 1995), more invasive to host cells, and more virulent in
the guinea pig model (Cirillo et al., 1994, 1999; Brieland et al., 1997).

In contrast to exponentially growing bacteria, I. pneumophila obtained
from postexponential cultures expresses traits that are correlated with viru-
lence, including sodium sensitivity, cytotoxicity, osmotic resistance, motil-
ity, and the capacity to evade phagosome-lysosome fusion (Rowbotham,
1980, 1986; Byrne and Swanson, 1998; Hammer and Swanson, 1999). Dur-
ing the replication phase within host cells, I. pneumophila organisms are

M
M

with a previous study in which Katz and Hashemi (1982) had observed on £

electron micrographs that, when present in macrophages at numbers greater £

than 25 per cell, I. pneumophila was usually dispersed within the cytoplasm w

of the host cell. °

>
GENE REGULATION IN L PNEUMOPHILA £

Growth phase and gene regulation

The phase of growth has a dramatic effect on the phenotype of L. pneu- g

mophila, whether the bacteria are cultivated in host cells or in microbiological 2

medium (Rowbotham, 1986; Byrne and Swanson, 1998). Robowtham was the g

first to observe in amoeba two distinct phenotypic phases of L. pneumophila, §

the replicative phase and the "active infective phase" (Rowbotham, 1986). £

When I. pneumophila is released from eukaryotic cells, it is short, thick, and g

highly motile, with a smooth and thick cell wall (Rowbotham, 1986) . The cells c

have a different composition of membrane fatty acids, and they differ in LPS o

sodium resistant and aflagellated (Rowbotham, 1980, 1986; Byrne and
Swanson, 1998; Hammer and Swanson, 1999). When I. pneumophila bacteria
egress from host cells, they are flagellated and sodium sensitive (Rowbotham,
1980, 1986; Byrne and Swanson, 1998; Hammer and Swanson, 1999). It has
been hypothesized that amino acid limitation induces the virulent phenotype
(Rowbotham, 1980, 1986; Byrne and Swanson, 1998; Hammer and Swanson,
1999). When L. pneumophila enters into the postexponential growth phase
or is subjected to amino acid limitation, the bacteria accumulate the strin-
gent response signal, guanosine S'^'-bispyrophosphate (ppGpp), through
the ppGpp synthetase, RelA (Bachman and Swanson, 2001). When relA from
Escherichia coli is expressed in L. pneumophila, ppGpp accumulates and the
bacteria express virulent traits independent of nutrient supply or cell den-
sity (Bachman and Swanson, 2001). The accumulation of ppGpp increases

* the amount of the stationary-phase sigma factor RpoS, which triggers the
g expression of the stationary-phase genes (Bachman and Swanson, 2001).

§ A rpoS mutant replicates within monocyte HL 60 and THP-1 cells but

ph is attenuated in A. castellanii (Hales and Shuman, 1999). Therefore, some

z RpoS -regulated traits could be critical for efficient transmission or infection

Q in this amoebae model (Hales and Shuman, 1999). Sodium sensitivity and

< maximal expression of flagellin also requires RpoS (Bachman and Swanson,

< 2001). I. pneumophila in the postexponenetial phase becomes cytotoxic by

* an RpoS-independent pathway (Bachman and Swanson, 2001). To replicate

< efficiently in macrophages, both an RpoS-independent and RpoS-dependent
S mechanism are utilized by L. pneumophila (Bachman and Swanson, 2001).
m Thus, when nutrient levels and other conditions are favorable, L. pneumophila
§ replicates within host cells, and when amino acids become rare, intracellular
o bacteria express several traits that permit escape from the host cell, survival

3 in the environment, and transmission to a new host.

i-i

The transmission phenotype

Because flagellin is expressed at the postexponential phase, Hammer
et al. (2002) screened for L. pneumophila mutants deficient in flagellin ex-
pression in order to identify genes involved in the phenotypic transition at
the postexponential phase. Five activators of virulence have been identified:
LetA and LetS, a two-component regulator homologous to GacA/S of Pseu-
domonas, or SirA/BarA of Salmonella, that represses the flagellar regulon; the
stationary-phase sigma factor RpoS; the flagellar sigma factor FliA, required
for both motility and contact-dependent cytotoxicity (Heuner et al., 2002) ; and
a novel locus, letE (Hammer et al., 2002).

In the postexponential phase, mutants in letA, letS, JliA, and letE are
nonmotile or show poor motility, are not cytotoxic nor sodium sensitive (ex-
cept JliA), and are not efficient at infecting macrophages (except letE; Ham-
mer et al., 2002). In contrast, intracellular growth is independent of these
genes (Hammer et al., 2002). Amino acid depletion or ppGpp accumulation
triggers a LetA/ LetS expression and RpoS -dependent cellular differentiation
(Hammer et al., 2002). The letE locus does not appear to produce any protein
and may encode for a regulatory RNA that may act as a regulator of letA /let S
expression (Hammer et al., 2002).

The relA mutant, producing undetectable levels of ppGpp in the cells
during the stationary phase, is unable to produce pigment as it becomes
flagellated. Although a previous study has shown that RpoS, which ac-
cumulates when RelA is activated, is required for intracellular growth in
A. castellanii (Hales and Shuman, 1999). Zusman et al. (2002) have shown §

that relA gene product is dispensable for intracellular growth in H L-60-derived g

human macrophages and in A. castellanii. Moreover, it has also been shown £

that RelA and RpoS have minor effects on expression of some of the dot/icm w

genes (icmT, icrnR, icmQ icmF, icmM, icm], icmF, icmV, and icmW; see °

Zusman et al., 2002). Thus, the role of RpoS in intracellular infection seems 8

to be specific to the host cell. %

w
I- 1

DNA regulatory elements of the dot/icm genes 2

Several studies showed that dot/icm expression is regulated during the g

intracellular infection of L. pneumophila within the host cells. The dot/icm 2

system upregulates phagocytosis of I. pneumophila (Hilbi et al., 2001), and g

surface exposure of DotO/DotH on L. pneumophila is induced at the earlier §

and later stages of the infection (Watarai et al., 2001a). c

Gal-Mor et al. (2002) examined DNA regulatory elements that may con- g

trol the expression of the dot/icm genes, using a promoter fusion to lacZ in c

I. pneumophila. They showed that the expression levels of different dot/icm o

genes are distinct from one another. The icmR, icmF, icmV, and icm W genes 2

have high levels of expression in both the exponential and postexponential
phase of growth, whereas icmR and icmF have higher levels in the stationary
phase than in the exponential phase. The icmT, icmP, icmQ icmM, and icm]
genes show low levels of expression in both exponential and postexponential
phases of growth. However, icmT and icmF have a higher level of expression
at the stationary phase.

A total of 12 regulatory elements have been identified (Gal-Mor et al.,
2002); 10 promoter elements of icmT, icmF, icmQ, and icmM genes have low

expression levels. These promoters contain 6-bp putative consensus sequence
TATACT, located from 32 to 74 bp from the ATG codons, which is essential
for their expression. — 10 promoter elements of icm V, icmW, and icmR genes
that have high expression levels have also been identified. The icmR locus con-
tains at least three regulatory elements, and regulatory elements have been
also identified for icmW, icmV, and icmF that have high expression levels. A
9-bp putative consensus sequence CTATAGTAT has been observed. In addi-
tion, icm V and icm W have an overlapping regulatory region (Gal-Mor et al.,
2002).

The reduced effect of an insertion in rpoS or relA on expression of icmP
only among the 9 icmr.lacZ fusions tested has shown that these two genes are
not involved in the regulation of the dot/icm genes (Hales and Shuman, 1999;
Zusman et al., 2002) and that there may be other factors necessary for this

* regulation. The —10 promoter elements found in some dot/icm genes have
g extensive homology to one another and are probably recognized by I. pneu-

§ mophila RpoD (Gal-Mor et al., 2002). An examination of the I. pneumophila

ph genome sequence has shown that I. pneumophila contains homologs of at

z least six sigma factors (RpoD, RpoH, RpoF, RpoE, RpoS, and RpoN). The

Q promoter sequences of RpoH and RpoF are different from the — 10 promoter

< sequences of the dot/icm genes (Gal-Mor et al., 2002). In addition, the pro-

< moters recognized by the factors RpoE and RpoN are also different from the

* promoters of the dot/icm genes (Gal-Mor et al., 2002). As RpoS is not involved

< in the expression of the dot/icm genes except for the moderate expression of
S icmP (Zusman et al., 2002), it has been proposed that these —10 regulatory
w elements of the dot/icm genes are recognized by the vegetative sigma factor
3 RpoD (Gal-Mor et al, 2002).

w
1-1

: 3 APOPTOTIC OR NECROTIC CELL DEATH

Induction of apoptosis by L pneumophila in mammalian
but not protozoan host cells

Apoptosis requires a cascade of activation of a family of cysteine proteases
(caspases) that specifically cleave proteins after aspartate residues (Anderson,
1997) . Among them, caspase-3 plays a central role, allowing caspase-activated
DNase to enter the nucleus and degrade chromosomal DNA (Enari et al.,
1998). Muller et al. (1996) have shown that L. pneumophila induce apoptosis
in HL-60 human-derived macrophages after 24-48 h, at a multiplicity of
infection (MOI) of 10-100. The induction of apoptosis in mammalian cells is
mediated by activation of caspase-3 that is dose dependent and is maximal at

3 h postinfection at MOI 50 (Gao and Abu Kwaik, 1999a, 1999b; also see Fig.
5.4). In alveolar epithelial cells and macrophages, the induction of apoptosis
is dose dependent but not largely growth phase regulated and can be induced
by extracellular bacteria (Gao and Abu Kwaik, 1999a, 1999b). The dot/icm
mutants of L. pneumophila are defective in inducing caspase-3 activation and,
thus, apoptosis (Gao and Abu Kwaik, 1999a; Zink et al., 2002). Therefore,
the Dot/icm type IV secretion system of I. pneumophila is essential for the
induction of apoptosis in mammalian cells (Gao and Abu Kwaik, 1999a;
Zink et al., 2002). The pore-forming toxin is not required for the induction of
apoptosis, but upon entry into the postexponential growth phase it enhances
the ability of the bacteria to induce apoptosis (Zink et al., 2002).

Interestingly L. pneumophila induces apoptosis in human phagocytes
but not in protozoan host cells such as A. castellanii (Hagele et al., 1998;
Gao and Abu Kwaik, 2000). Moreover, although A. polyphaga is capable of §

undergoing apoptosis, upon stimulation by actinomycin D, I. pneumophila g

does not induce apoptosis in A. polyphaga (Gao and Abu Kwaik, 1999b, £

2000). u

Induction of necrosis by L pneumophila in mammalian >

and protozoan cells °

M
M

The dot/ icm-regulated Rib pore-forming toxin is essential for L. pneu-
mophila induction of necrosis, killing, and exiting the host cells (Alii et al., g
2000; Gao and Abu Kwaik, 2000; Molmeret et al., 2002a). At high MOI, mu- g
tants defective in the Rib pore-forming toxin replicate like the wild-type strains 2
within the protozoan and mammalian cells but are trapped within these cells g
and cannot be released ( Figs .5.3 and 5 .4; also see Alii et al. , 2000; Gao and Abu §
Kwaik, 2000). The expression of the pore-forming activity by L. pneumophila c
grown in vitro and within macrophages is completely repressed during expo- 5

nential growth but is temporally activated upon entry into postexponential c

is;

phase (Fig. 5.4; also see Alii et al., 2000). All five rib mutants mentioned o

earlier that possess identical point deletions following a poly(T) stretch in 2

the icmT gene are also defective in acute lethality in A/ J mice (Alii et al.,
2000; Molmeret et al., 2002b). Therefore, the Rib pore-forming activity is
not required for phagosomal trafficking and intracellular multiplication of
L. pneumophila within the cells, but it is essential for induction of cytolysis of
the infected macrophages (Alii et al., 2000; Gao and Abu Kwaik, 2000). Thus
the C-terminus of IcmT is essential for pore-formation-mediated cytolysis.

L. pneumophila kills A. polyphaga through temporal induction of necro-
sis, which is mediated by the Rib pore-forming activity of L. pneumophila.

Wild-type intracellular I. pneumophila causes necrosis-mediated cytolysis of
A. polyphaga within 48 h after infection (Gao and Abu Kwaik, 2000) . However,
in A. polyphaga, at low MO I the rib mutants are defective in intracellular mul-
tiplication (Molmeret et al., 2002b). These results show that the C-terminus
of IcmT is essential for pore formation and intracellular trafficking and mul-
tiplication within A. polyphaga, as confirmed by fusion of phagosomes har-
boring the rib mutants to lysosomes (Molmeret et al., 2002a). Experiments
performed with an IcmT null mutant that is defective in intracellular traf-
ficking, intracellular multiplication, and egress from both protozoan and
mammalian cells suggest that IcmT is bifunctional (Molmeret et al., 2002a,
2002b). The C-terminus of IcmT is essential for pore-formation-mediated cy-
tolysis in mammalian cells, and the N-terminus is required for intracellular
trafficking (Molmeret et al., 2002a). It is unlikely that IcmT is an effector

* or a common regulator, but it is possible that this protein is a cofactor in-
g volved in the export of different substrates with roles in pore-forming toxicity

§ and intracellular trafficking (Molmeret et al., 2002a). Effectors of this type IV

ph secretion system remain to be identified.

z The ability to lyse host cells and to egress from them is a fundamental step

Q in the pathogenic cycle of intracellular bacteria and determines the overall

< consequences of the infection of an organism. Apoptosis and necrosis are

< the two commonly observed types of cell death. Necrosis is characterized

i— i

* by physical damage that causes cell death. Apoptosis is a regulated suicide

< program of the cell that manifests morphological and biochemical features
S distinct from those of necrosis (Cohen, 1993). Killing of mammalian cells

w by L. pneumophila has been proposed to occur in two phases (Gao and Abu

g Kwaik, 1999a, 1999b; also see Fig 5.4). In the first phase, L. pneumophila

° induces apoptosis in macrophages, monocytes, and alveolar epithelial cells

3 during the early stages of the infection (Hagele et al., 1998; Gao and Abu

< Kwaik, 1999b), which is mediated through the activation of caspase-3 (Gao
and Abu Kwaik, 1999a). Induction of apoptosis is largely independent of the
bacterial growth phase (Gao and Abu Kwaik, 1999a). The second phase is
mediated through rapid induction of necrosis by L. pneumophila upon entry
into the postexponential phase of growth, when the bacteria become cytotoxic
(Fig. 5.4). Our working model of bacterial egress can be presented in three
steps (Fig. 5.4). First, upon exiting the exponential phase of intracellular
growth, an "egress pore" is inserted into the phagosomal membrane, leading
to its disruption. Second, the bacteria egress into cytoplasm. Third, disruption
of organelles and the plasma membrane occurs, culminating in lysis of the
host cells and bacterial egress.

IMMUNE RESPONSE TO L PNEUMOPHILA INFECTION

When L. pneumophila is inhaled into the lungs, acute alveolitis and bron-
chiolitis can be observed (Winn and Myerowitz, 1981). The alveolar exudate
typically consists of polymorphonuclear cells and macrophages, red blood
cells, and cellular debris (Glavin et al., 1979). I. pneumophila are mainly in-
tracellular, and the fate of the bacteria depends on the host cells. Within
macrophages, most of the bacteria are intact in large vacuoles containing
a large number of bacilli (Glavin et al., 1979). Within neutrophils, L. pneu-
mophila most often appears degraded (Katz and Hashemi, 1982).

The role of alveolar epithelial cells in Legionnaires' disease has not been
well studied. L. pneumophila has been shown to replicate in a phagosome
within many epithelial cells in vitro, including type I and II alveolar epithelial
cells (Gao et al. , 1998b) . There is no reason why L. pneumophila is not expected
to replicate at the foci of infection within alveolar epithelial cells, particularly
because these constitute most of the alveolar surface, where the foci of infec- 8

tion are recognized. In addition, these cells are potential sites of intracellular j*

replication during activation of macrophages by IFN-y, which inhibits in- g

tracellular replication of I. pneumophila within monocytes and macrophages o

(Byrd and Horwitz, 1989) . >

Although the bacteria bind complement component C3, they are resis- °

tant to complement-mediated killing (Horwitz and Silverstein, 1981a, 1981b; m

Payne and Horwitz, 1987) . I. pneumophila is also resistant when treated with 2

complement and specific antibodies in presence of polymorphonuclear cells 5

(Horwitz and Silverstein, 1981a, 1981b). Different results have been obtained ^

L_J

with monocytes in experiments performed at a high multiplicity of infection h

(Horwitz and Silverstein, 1981a). In the presence of both antibody and com- £

plement, phagocytosis of L. pneumophila is more efficient and monocytes 2
kill approximately half of the inoculum of the bacteria (Horwitz and Silver-

•fl

stein, 1981a). In addition, antibodies promote fusion of the infected vacuole §

to lysosomes as compared with cells treated with complement alone (Horwitz is;

and Maxfield, 1984). However, adherence studies of I. pneumophila to U937 §

macrophages, guinea pig alveolar macrophages, and MRC-5 cells in absence
of serum have shown that neither complement nor antibody is required for
binding (Gibson et al., 1993). Overall, L. pneumophila is relatively resistant
to innate and humoral immune responses and the infection is most likely
controlled by cell-mediated immunity.

Important roles of Thl-type cytokines, such as tumor necrosis factor
alpha (TNF-a), IFN-y, and interleukin-12 (IL-12), have been demonstrated

in the murine A/J model of L. pneumophila infection (Brieland et al., 1994,
1995; Gebran et al., 1994b). Susa et al. (1998) have examined cytokines and
the role of CD4 and CD8 cells in Legionnaires' disease. After injection of
10 6 CFU of I. pneumophila in A/J mice, a challenge that allows the survival
of the infected animals, inflammatory cells are recruited into the lung on
the second day, and, by the third day of infection, macrophages, B cells, NK
cells, and large mononuclear cells are mainly present (Susa et al., 1998).
T lymphocytes infiltrate subsequently (Susa et al., 1998). The L. pneumophila
infection results in a rapid upregulation of systemic concentrations of IFN-
y, TNF-a, IL-1/3, IL-4, and IL-6 (but not IL-2); then these levels decrease on
the third day (Susa et al., 1998). Recruitment of T cells is necessary for the
clearance of the bacteria. The depletion of CD4+ and CD8+ T cells leads to
increased lethality to mice. Moreover, another study has shown that nitric ox-

* ide (NO) produced by numerous cells, such as macrophages and neutrophils,
g may regulate IL-6 production in L. pneumophila-infected macrophages

§ (Yamamoto et al., 1996). Indeed, when macrophages were primed with IFN-

ph y , bacterial replication was inhibited and NO was produced in large amounts

=; (Yamamoto et al., 1996) . An examination of cytokine levels in L. pneumophila-

Q infected macrophages primed with IFN-y revealed a moderate increase of

< IL-6 production (Yamamoto et al., 1996).

< After intratracheal injection of I. pneumophila, the increase of bacteria in

* the lungs by 48 h is accompanied by a massive accumulation of neutrophils

< (Tateda et al., 2001b). Neutrophils are recruited early during the infection of
S L. pneumophila in animal models and patients (Brieland et al., 1995). How-

m ever, Legionella are partially resistant to neutrophil killing, particularly when

g the bacteria are opsonized (Horwitz and Silverstein, 1980, 1981a; Katz and

° Hashemi, 1983). Neutrophils have the ability to produce immunoregulatory

3 cytokines-chemokines, including IL-12, which may affect Thl/Th2 host re-

< sponses (Cassatella, 1995). Previous studies have shown a protective role
for Thl cytokines such as IFN-y and IL-12 during L. pneumophila infection
(Gebran et al., 1994b; Brieland et al., 1995). In contrast, the Th2 cytokine,
IL-10, facilitates growth of L. pneumophila in macrophages through the IL-
10-mediated suppression of Thl cytokines (Tateda et al., 2001a). A recent
study shows that the CXC chemokine receptor 2, a receptor for chemokine-
mediated neutrophil accumulation, may play a role in host defense against
L. pneumophila, because blockade of this receptor enhances mortality in the
A/J mouse model (Susa et al., 1998). The neutrophils may have a protec-
tive effect through immunomodulatory actions in L. pneumophila infection
(Tateda et al., 2001b). Early recruitment of neutrophils may contribute to Tl
polarization in a murine model (Tateda et al., 2001a).

IFN-y is recognized as an important cytokine in both innate and cell-
mediated immune responses. Previous studies have shown that endogenous
IFN-y is induced in response to Legionella infection (Brieland et al., 1994;
Susa et al., 1998). Moreover, treatment with IFN-y is able to inhibit the
intracellular replication of L. pneumophila (Nash et al., 1988). When the cul-
tures are supplemented with iron-saturated transferrin, the IFN-y effect is
neutralized (Byrd and Horwitz, 1989). Administration of IFN-y in a murine
model of L. pneumophila induces the expression of IFN-y and IL-12 from
natural killer cells (Deng et al., 2001). Moreover, intrapulmonary adenovirus-
mediated IFN-y gene therapy, in a nonlethal murine model of I. pneumophila
pneumonia, results in a 10-fold decrease in lung bacterial CFU at 48 h postin-
fection, compared with controls that did not receive the gene. Thus, even
in immunocompetent hosts, expression of IFN-y promotes L. pneumophila
clearance, independent of cell recruitment and proinflammatory cytokine §

induction. Alveolar macrophages from uninfected animals treated in vivo g

with the adenovirus -mediated IFN-y inhibit the intracellular growth of £

I. pneumophila (Deng et al., 2001). Therefore, IFN-y -secreting cells such w

as T cells and NK cells may directly contribute to bacteriostasis or killing, ei- °

ther by downregulating transferrin receptors (Byrd and Horwitz, 1989, 2000; 8

Gebran et al., 1994a) or by endogenous NO that may regulate IL-6 production %

CONCLUSION

L. pneumophila is an intracellular pathogen that utilizes protozoa in
aquatic environments to replicate within and to be protected from adverse
conditions. Legionnaires' disease has become a threat to humans after our
industrialization and production of man-made devices that generate aerosols,
which allows transmission of the bacteria to humans. Thus, the normal life
cycle in protozoa stops when the bacteria encounter human host cells such
as macrophages. Although I. pneumophila utilizes different ligands to enter
the host cells and is unable to induce apoptosis in protozoan cells such as
A. polyphaga or A. castellanii, the remarkable similarities in the intracellular

o
(Yamamoto et al., 1996). TNF-a? also helps to control the infection by means g

of endogenous NO. As a consequence of the I. pneumophila infection, a rapid £

nonspecific immune response is followed by a slower-developing specific im- g

o
mune response necessary for final eradication of the infection. CD4+ and g

CD8+ T cells play a role in both phases. During the first phase, T cells might 2

produce I FN and IL-6, whereas in the second phase they support humoral g

immunity and specific T-cell-mediated immunity (Kaufmann, 1993; Susa §

etal, 1998).

o
HI

infection of the two evolutionarily distant host cells (macrophages and proto-
zoa) suggest that I. pneumophila may utilize similar molecular mechanisms
to manipulate processes of these host cells (Gao et al., 1997). It has been
hypothesized that L. pneumophila has evolved as a protozoan parasite in the
environment and its adaptation to this primitive phagocytic unicellular host
was sufficient to allow the bacteria to survive and replicate within the biologi-
cally similar phagocytic cells of the more evolved mammalian host (Cianciotto
and Fields, 1992; Abu Kwaik, 1996).

The type IV secretion system is the key virulence factor of this organism,
allowing it to invade the host cells, replicate, evade the endocytic pathway,
induce apoptosis, and egress from the host cells (Gao and Abu Kwaik, 1999a,
1999b, 2000; Alii et al, 2000; Hilbi et al, 2001; Watarai et al., 2001b; Molmeret
et al., 2002a, 2002b; Zink et al, 2002). Not all the functions of the Dot/Icm
* proteins have been understood, and no effectors other than RalF have yet

g been identified. However, understanding these host-parasite interactions at

§ the molecular level will be of considerable help in controlling the replication of

ph this bacterium, both environmentally in the protozoa and in human infection.

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>
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i— i

%
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Pi
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l-l
1-1

CHAPTER 6

Listeria monocytogenes invasion and
intracellular growth

Kendy K.Y. Wong and Nancy E. Freitag

Studies focused on the Gram-positive, facultative intracellular bacterial path-
ogen Listeria monocytogenes have provided valuable insights into many facets
of biology, including cell-mediated immunity, cell physiology, and bacterial
pathogenesis. The bacterium invades and replicates within a wide variety
of cell types, and is capable of infecting an astonishing diversity of hosts,
including mammals, fish, and insects (Gray and Killinger, 1966). The well-
established use of murine and tissue culture models of infection, the ease of
growing L. monocytogenes within the laboratory, and the existence of numer-
ous genetic tools for the generation and analysis of bacterial mutants have
helped to make L. monocytogenes a powerful model system for the exploration
of the molecular basis of host-pathogen interactions. Because of its ubiquity
within the environment and robust survival skills, this important foodborne
pathogen remains a constant concern for public health departments and the
food industry.

Listeriae are noncapsulated, nonspore-forming, facultative anaerobic
bacilli. They are 0.4 /xm by 1 to 1.5 /xm in size and are motile at 10° C to 25° C
through the expression of polar flagellae (Farber and Peterkin, 1991; Lorber,
1997; Vazquez- Boland et al., 2001b). There are six species in the Listeria
genus: L. monocytogenes, I. ivanovii, L. innocua, L. seeligeri, L. welshimeri, and
L. grayi (Collins et al., 1991; Sheehan et al., 1994). Only L. monocytogenes and
I. ivanovii are considered pathogenic and are responsible for the disease
known as listeriosis. L. ivanovii is mainly an animal pathogen, whereas
L. monocytogenes has been reported to cause infections in humans, other ani-
mals, birds, and fish (Gray and Killinger, 1966; McLauchlin, 1987; Alexander
et al., 1992; Chand and Sadana, 1999; Ryser, 1999; Wesley, 1999). L. mono-
cytogenes is the major listerial species pathogenic for humans; although rare,
L. ivanovii has caused human infections, and at least one report of human

listeriosis has been associated with L. seeligeri (Rocourt et al., 1986; Cummins
et al., 1994; Lessing et al., 1994; Ramage et al., 1999). The Listeria spp. are
widespread in nature and can be isolated from a variety of sources such as wa-
ter, soil, food, and feces of animals (Schuchat et al., 1991; Fenlon, 1999). The
natural habitat for Listeria organisms is believed to be decaying vegetation, but
the two pathogenic species can invade and survive within a variety of hosts and
host cell types. L. monocytogenes was first characterized in 1926 (Murray et al.,
1926), but it was not until the early 1980s that a clear link was established
between listeriosis and the ingestion of food contaminated with the organ-
ism (Schlech et al., 1983). Serious infections occur in immunocompromised
individuals, neonates, the elderly, and pregnant women, usually through
the ingestion of contaminated food such as dairy products and ready-to-eat
deli meats (Gray and Killinger, 1966; Farber and Peterkin, 1991; Rocourt,
^ 1996; Ryser, 1999). The infection of healthy individuals has been reported to

w cause gastroenteritis, a condition thought to be underdocumented because

* routine clinical testings do not search for listeriae (Hof, 2001). Although

g I. monocytogenes does not usually pose a high risk for healthy individuals,

< the mean mortality rate of human listeriosis is 20-30% (Farber and Peterkin,

g 1991; Schuchat et al., 1991; Rocourt, 1996). The ability of I. monocytogenes to

tolerate high salt concentrations and low pH' and to multiply at refrigerated

o temperatures makes it a difficult and constant challenge for the food industry

p\ (Lammerding and Doyle, 1990; Lou and Yousef, 1999). This chapter briefly

>* reviews the current knowledge regarding L. monocytogenes invasion of host

§ cells and the resulting course of infection following host cell entry.

CLINICAL PRESENTATIONS OF LISTERIOSIS: AN OVERVIEW OF
THE DIVERSITY OF CELL TYPES AND TISSUES THAT SUPPORT

BACTERIAL REPLICATION

Among the first cells encountered by I. monocytogenes following inges-
tion of the bacterium by the host are the epithelial cells lining the gut. In
the mouse, L. monocytogenes has been observed to translocate the intestine
without causing gross lesions, and rapid listerial translocation occurs in rat
ileal loop models of intestinal infection (Marco et al., 1992; Pron et al., 1998).
On the basis of these findings, it has been suggested that bacterial replica-
tion in the intestinal mucosa is probably not required for the establishment
of systemic infection, although L. monocytogenes is able to proliferate at this
location. I. monocytogenes can cross the intestinal barrier by means of M
cells of Peyer's patches, and the Peyer's patches appear to be preferential

multiplication sites for the bacteria, which can then invade neighboring cells
by direct cell-to-cell spread (Havell et al., 1999; Daniels et al., 2000; Hof,
2001). Dendritic cells are also early listerial targets in Peyer's patches (Pron
et al., 2001). Gross intestinal lesions and gastroenteritis appear to occur only
following the ingestion of large numbers of bacteria. The risk for gastroen-
teritis also appears to increase in the presence of small amounts of mucosal
damage within the intestine; such damage may happen periodically in hu-
mans (MacDonald and Carter, 1980; Pron et al, 1998; Daniels et al, 2000).
Once across the intestinal barrier, L. monocytogenes is translocated to the liver
and spleen within minutes, where most of the organisms are then cleared
(Conlan and North, 1991; Pron et al., 1998; Cousens and Wing, 2000; Daniels
et al., 2000). In susceptible hosts, surviving L. monocytogenes multiplies,
mainly in hepatocytes, and spreads through the liver parenchyma. Spleno-
cytes are also target cells for L. monocytogenes, although they appear less
permissive for bacterial replication than hepatocytes. In both organs, mi- g

croabscess formation is observed and neutrophils are rapidly recruited to the g

infection foci; however, L. monocytogenes-infected spleen cells appear to be §

o
less susceptible to lysis by neutrophils during early infection (Conlan and Q

North, 1994; Rogers et al., 1996). L. monocytogenes has been demonstrated to §

induce apoptosis in hepatocytes and Caco-2 cells (Rogers et al., 1996; Valenti ; -

et al., 1999), and in dendritic cells apoptosis was recently shown to be trig- g

gered by listeriolysin O (LLO; see Guzman et al., 1995). If I. monocytogenes >

infection is not resolved within the liver and spleen, the bacteria enter into g

circulation and disseminate to other tissues. £j

L. monocytogenes appears to have a tropism for the placenta of pregnant

women and for the central nervous system (CNS). The organism has the 3

ability to cross both the feto-placental barrier and the blood-brain barrier S

(Armstrong and Fung, 1993; Lorber, 1997; Greiffenberg et al., 1998; Parkassh g

et al., 1998) . At the placenta, L. monocytogenes causes the formation of numer- »

ous microabscesses and the necrosis of placental villi (Parkassh et al., 1998; g

Vazquez- Boland et al., 2001b). L. monocytogenes infects the developing fe- h

tus and causes chorioamnionitis, which often leads to abortion or stillbirth.
Alternatively, fetal listeriosis may lead to the birth of a baby with granu-
lomatosis infantiseptica, characterized by microabscesses all over the body
and internal organs (Klatt et al., 1986; Schuchat et al., 1991; Lorber, 1997).
The mother may be asymptomatic or may only have mild flu-like symp-
toms, and CNS infection rarely occurs in pregnant women. For the establish-
ment of CNS infections, active actin-based intra-axonal movement may facil-
itate L. monocytogenes spread (Otter and Blakemore, 1989; Antal et al., 2001).

Listeria-infected phagocytes and dendritic cells have also been shown to play a
role in bacterial dissemination and in the initiation of CNS infection (Drevets
et al., 1995, 2001; Guzman et al, 1995; Drevets, 1999; Pron et al, 2001). The
growth of L. monocytogenes in brain tissues appears to be unrestricted, and the
bacterium has been found to infect the epithelial cells of the choroids plexus,
ependymal cells, microglial cells and neurons, and brain parenchymal tissue
(Kirk, 1993; Schluter et al., 1999).

In nonpregnant individuals, meningitis and encephalitis are common
manifestations of I. monocytogenes disease. Clinical features often seen with
listeriosis include fever, headache, nausea, vomiting, movement disorders,
altered consciousness, and seizures. Bacteremia is also frequently observed in
adult listeriosis (Lorber, 1997; Vazquez- Boland et al., 2001b). Some unusual
forms of listerial infections include endocarditis, hepatitis, myocarditis, ar-
^ teritis, pneumonia, sinusitis, conjunctivitis, ophthalmitis, joint infection, and

w skin infection (Vazquez- Boland et al., 2001b and references therein). Febrile

* gastroenteritis has also been described in recent years (Salamina et al., 1996;

g Dalton et al., 1997; Miettinen et al, 1999; Aureli et al, 2000). This wide spec-

< trum of I. monocytogenes disease manifestations reflects the diversity of cell

g types and tissues that can harbor the bacterium.

As L. monocytogenes can invade many types of cells and tissues within

o a host, a number of tissue culture cell model systems have been developed

^ to study bacteria-host cell interactions, including epithelial cells, such as

!* Caco-2, Henle 407, Vero, HeLa, Chinese hamster ovary (CHO) cells, and

§ potoroo rat kidney (PtK2) cells; endothelial cells, such as human umbilical

vein endothelial cells (HUVEC) and human brain microvascular endothelial

cells (HBMEC); fibroblast cells; macrophages (primary and macrophage cell

lines such as J774); hepatocytes; and dendritic cells (Gaillard et al., 1987;

Sun et al., 1990; Dramsi et al., 1995; Drevets et al., 1995; Guzman et al.,

1995; Mengaud et al, 1996; Greiffenberg et al, 1998; Parida et al, 1998;

Kolb-Maurer et al., 2000; Suarez et al., 2001). The mechanisms governing

the invasion of these various cell types by L. monocytogenes are the focus of

the following section.

INVASION OF HOST CELLS BY L MONOCYTOGENES

The intracellular life cycle of I. monocytogenes has been extensively stud-
ied. The overall process resulting in intracellular bacterial replication and cell-
to-cell spread includes the following: (1) bacterial adherence or attachment
to the host cells; (2) bacterial internalization by phagocytic cells, or bacterial
induced-uptake into nonprofessional phagocytic cells; (3) bacterial escape

Entry

(inlA, inlB: Internalins InlA, InlB)

Vacuole lysis f *

(lily: Listeriolysin O/LLO, J\\
pic A: PlcA/PI-PLC)y^>.

Lysis of two-membrane
vacuole

(plcB: Lecithinase
PlcB/PC-PLC)

Intracellular v

movement

(act A: ActA)

r; , x Cell to cell
spread

(act A : ActA)

Figure 6.1. Overview of I. monocytogenes infection of host cells. The stippled regions
represent host actin filaments. The major gene products that contribute to intracellular
growth and cell-to-cell spread are indicated. Adapted from Tilney and Portnoy (1989) with
permission from the Journal of Cell Biology.

from the phagosome; (4) bacterial multiplication and actin polymerization-
based movement within the host cell cytosol; and (5) spread of the bacte-
ria to adjacent cells and escape from a double-membrane-bound vacuole
(Fig. 6.1).

Adherence and internalization mechanisms for nonprofessional
phagocytic cells

The first steps in I. monocytogenes infection following bacterial inges-
tion are bacterial adherence and invasion of the gastrointestinal epithelium.
Following adherence, the internalization of I. monocytogenes proceeds by
means of a zipper-like mechanism in which the plasma membrane of the
host cell closely enwraps the bacterium until it is completely engulfed into
a vacoule. This process is strikingly different from the trigger mechanism
induced by Salmonella and Shigella spp., which leads to the formation of dra-
matic membrane ruffles in host cells during uptake (Swanson and Baer,
1995; Dramsi and Cossart, 1998; Kuhn and Goebel, 2000). I. monocyto-
genes has been found to enter polarized cells through the basolateral surface,
and its internalization is increased following the disruption of intercellular
junctions in the host cells (Gaillard and Finlay, 1996). Of the various liste-
ria! ligands described to participate in bacterial attachment and invasion of

%
o
n

hi
%

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M
M
M

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to
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ffi

nonprofessional phagocytic host cells, the internalins (encoded by the inl
genes) appear to be the major players and have been the best described.

The inlAB gene products

The inlAB locus was identified following the isolation of bacterial
transposon-insertion mutants that were defective for invasion of Caco-2 cells
(Gaillard et al., 1991). L. monocytogenes inlAB mutants were found to be
severely impaired in host cell invasion, and the expression of InlA and InlB
on the surface of latex beads or on the surface of normally noninvasive bac-
teria (such as L. innocua and Enterococcus faecalis) was sufficient to induce
internalization (Lecuit et al., 1997; Braun et al., 1998).

I. monocytogenes InlA promotes entry into human intestinal epithelial
SJ cell lines such as Caco-2 cells and into other cells expressing the E-cadherin

n receptor ligand (Mengaud et al., 1996). E-cadherin is a 110-kDa transmem-

* brane glycoprotein specific to epithelial tissues, such as those in the diges-

g tive tract and in the cells lining the choroid plexus and placental chorionic

< villi. The protein is predominantly expressed at the adherens junctions and

g on the basolateral face of cells, where it mediates calcium-dependent cell-

cell adhesion and helps maintain tissue cohesion. It has been suggested that

o I. monocytogenes breaches the intestinal barrier through M cells at the Peyer's

p\ patch surface to gain access to the basolaterally located E-cadherins. Alter-

>* natively, the E-cadherin receptor might be transiently accessible when crypt

§ cells migrate to the tips of intestinal villi for exfoliation (Mengaud et al., 1996;

Vazquez- Boland et al., 2001b; Cossart, 2002). The cytoplasmic domain of E-
cadherin interacts with proteins known as catenins, and these associations
ultimately stimulate the cytoskeletal rearrangements within the host cell that
result in L. monocytogenes uptake (Cossart and Lecuit, 1998; Kuhn and Goebel,
1998; Vazquez-Boland et al., 2001b).

InlA is an approximately 80-kDa protein that is targeted to the bacterial
surface by an N-terminal signal sequence. The InlA C-terminus contains a
hydrophobic stretch of 20 amino acids preceded by a cell wall anchor motif,
LPXTG (Fig. 6.2). This motif is a signature of many Gram-positive surface pro-
teins and is necessary for their covalent linkage to the peptidoglycan (Fischetti
et al., 1990). A putative transamidase known as sortase, encoded by srtA,
was recently identified and shown to be required for the cell wall anchor-
ing of InlA (Bierne et al., 2002). Moreover, L. monocytogenes mutants lack-
ing functional sortase were shown to be less invasive in vitro and atten-
uated in a mouse model of infection (Garandeau et al., 2002). Similar
to other members of the internalin family, the N-terminus of InlA has a
series of leucine-rich repeats (LRRs) consisting of a string of 22 amino

signal

peptide

InlA
(800 aa)

LPTTG

15 LRRs

E-Cadherin
binding

Cell wall
anchor

signal
peptide

InlB
(630 aa)

8 LRRs

GW GW GW

gClq-R,

MET binding

Cell surface
anchor

Figure 6.2. Schematic illustration of InlA and InlB. LRR represents leucine-rich repeats,
and GW represents the GW repeats present in InlB that anchor the protein to the bacterial
cell surface. The LPTTG motif is involved in cell wall anchoring of InlA. The receptors for
InlA and InlB are indicated below the LRR binding regions.

acids containing leucine or isoleucine residues at seven fixed positions (at
residues 3, 6, 9, 11, 16, 19, and 22; see Dramsi et al., 1997; Engelbrecht et al.,
1998a, 1998b; Raffelsbauer et al, 1998). There are 15 successive LRR units
in InlA; each LRR is composed of a beta strand that alternates with a more
flexible antiparallel helix, and the repeats are connected by coils. An intact
LRR region is necessary and sufficient to induce bacterial entry, whereas the
inter-repeat region has been suggested to play a role in the proper folding
and stabilization of the LRRs (Lecuit et al., 1997). The InlA protein is thought
to interact by means of the LRR regions with the extracellular domain of
E-cadherin, whose cytoplasmic domain then leads to rearrangement of the
host cell actin cytoskeleton for bacterial internalization. The proline residue
at position 16 of E-cadherin has been shown to be critical for InlA interac-
tion. Interestingly, the mouse and rat E-cadherins have glycine in place of
proline at this position and do not facilitate InlA-dependent internalization
of I. monocytogenes. The position 16 proline of E-cadherin thus contributes
some measure of host specificity for I. monocytogenes (Lecuit et al., 1999).
InlA has also been shown to contribute to the attachment and internalization
of L. monocytogenes by macrophages (Sawyer et al., 1996).

InlB is another L. monocytogenes surface protein that mediates bacterial
invasion of a broader range of host cells, including CHO cells, Vero cells, hep-
atocytes, and several endothelial, epithelial, and fibroblast cell lines (Dramsi
et al., 1995; Lingnau et al, 1995; Ireton et al, 1996; Greiffenberg et al., 1998;

%
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n

hi
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M
M

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ffi

Parida et al., 1998). Two host receptors have been identified for InlB. One,
gClq-R, is expressed in most tissues and is the receptor for the globular
part of the complement component Clq (Braun et al., 2000; Galan, 2000).
The gClq-R protein has no transmembrane domain and no cytoplasmic tail;
thus a co-receptor may be required to mediate the effects of InlB-gClq-R
interactions. The other receptor for InlB is Met, a 145-kDa tyrosine kinase
that is also a high-affinity receptor for hepatocyte growth factor (Shen et al.,
2000). InlB-mediated listerial entry into host cells leads to the stimulation
of phosphoinositide 3-kinase (PI-3 kinase; see Ireton et al., 1996). In ad-
diton, the MEK-1, ERK-1, and ERK-2 protein kinases are activated during
I. monocytogenes invasion of HeLa epithelial cells (Tang et al., 1998).

InlB is a 71-kDa member of the internalin family and has a signal se-
quence followed by 8 LRRs at the N-terminus. However, InlB does not possess
^ the Gram-positive surface anchor LPXTG motif at its C-terminus. Instead,

w a "GW" motif, containing repeats led by the sequence GW, functions as the

* cell surface anchor domain (Fig. 6.2; also see Braun et al., 1997). InlB is

g only loosely associated with lipoteichoic acid in the cell wall by means of the

< GW motif, and some InlB is released into the supernatant (Jonquieres et al.,

g 1999). The GW motif also binds cellular glycosaminoglycans, and this inter-

action appears to be required for efficient InlB-mediated bacterial invasion by

o enhancing the internalization triggered by the N-terminal LRRs (Jonquieres

>; etal.,2001).

>* Besides inlAB, six additional genes encoding putative surface-anchored

§ internalins have been identified in L. monocytogenes. Similar to InlA, these six

members (InlC2, InlD, InlE, InlF, InlG, and InlH) all have signal sequences
and various numbers of LRRs at the N-terminal region, plus the LPXTG
motifs at the C-terminus (Dramsi et al., 1997; Engelbrecht et al., 1998a;
Raffelsbauer et al., 1998). These internalins were recently shown to enhance
InlA-mediated invasion of I. monocytogenes into Caco-2 cells and into HB-
MEC. However, InlB-triggered bacterial internalization into these cell lines
did not require any of the other internalin proteins (Bergmann et al., 2002).
InlC is an unusual member of the internalin protein family. It is small
(~30 kDa) and is secreted by L. monocytogenes instead of being anchored
to the cell wall. The potential role of InlC in L. monocytogenes pathogenesis
is still unclear. It has been suggested to be involved in the dissemination
of L. monocytogenes infection; however, cell-to-cell spread is not affected in
in/C mutant strains (Engelbrecht et al., 1996; Lingnau et al., 1996; Domann
et al., 1997). Southern blot and genome analysis has indicated the presence
of internalin homologues in the nonpathogenic L. innocua, and it is possible
that these homologues might have functions that are distinct from those
required for invasion (Gaillard et al., 1991; Glaser et al., 2001).

Other possible L monocytogenes adherence and
internalization factors

In addition to the internalin protein family, a number of other L. monocy-
togenes surface proteins have been implicated in bacterial adherence and inva-
sion of host cells (Kuhn and Goebel, 1989; Alvarez- Dominguez et al., 1997b).
The L. monocytogenes surface protein ActA, which mediates the polymeriza-
tion of host actin filaments required for bacterial motility within the host
cytosol, has been shown to recognize host cell heparan sulfate proteoglycans
(HSPG) and may contribute to epithelial cell invasion (Alvarez- Dominguez
et al., 1997b; Suarez et al., 2001). A major extracellular protein, p60 (en-
coded by lap) , has been reported to be required for bacterial attachment and
invasion of mouse fibroblasts. However, it is possible that the decrease in
invasion of I. monocytogenes p60 mutants was due to septation defects asso- H

ciated with the disruption of the p60 murein hydrolase activity (Bubert et al., h

1992; Wuenscher et al., 1993). Bacterial adherence to Caco-2 cells seems to be £

independent of p60 and rather mediated by a 104-kDa surface protein known §

as the Listeria adhesion protein (Lap; see Pandiripally et al., 1999). There is o

also a fibronectin-binding protein that has been identified in L. monocytogenes g

(Gilotetal, 1999, 2000). £

The completion of the I. monocytogenes genome sequence has revealed £

that, among the bacterial genomes currently sequenced, L. monocytogenes con- §

tains the largest number of proteins with the cell wall anchor motif LPXTG
(Glaser et al., 2001). It is possible that some of these putative surface proteins

L monocytogenes entry into professional phagocytes

Macrophages actively engulf I. monocytogenes, and several host cell com-
ponents have been shown to participate in macrophage binding and uptake

M
M

>
are involved in adherence and internalization. In addition to protein ligands, o

other surface components, such as lipoteichoic acid, appear to be impor- *

tant for L. monocytogenes adhesion to various cell lines. The D-alanylation of £

the lipoteichoic acids, catalyzed by products expressed from the dlt operon,

was recently shown to be required for adhesion to murine macrophages and

hepatocytes as well as human Caco-2 epithelial cells (Abachin et al., 2002). A o

virulent I. monocytogenes strain possessing surface alpha-D-galactose residues g

was shown to bind through lectin-like interactions to a human hepatocarci- ffi

noma cell line, which expresses a well-characterized carbohydrate receptor

for alpha-D-galactose at the surface (Cowart et al., 1990). Agglutination by

lectins suggests the presence of other carbohydrate-binding ligands on the

surface of I. monocytogenes (Cottin et al., 1990; Facinelli et al., 1998).

of the bacterium. The complement component C3b is involved in mediating
listerial interactions with macrophages by means of CR3, the complement
receptor type 3 (Drevets and Campbell, 1991; Croize et al., 1993; Drevets
et al., 1993). I. monocytogenes also binds Clq in a specific, saturable, and
dose-dependent manner. Enhanced uptake of Clq-bound L. monocytogenes
was mediated by Clq complement receptors expressed on the macrophage
surface (Alvarez- Dominguez et al., 1993). Nonopsonic interactions may also
be involved, as L. monocytogenes uptake is efficient in the absence of serum
(Pierce et al., 1996). The type I and type II macrophage scavenger receptors
can recognize and bind a wide range of polyanions, and these receptors have
been shown to bind listerial lipoteichoic acids (Dunne et al., 1994; Greenberg
et al., 1996; Ishiguro et al., 2001). N-acetylneuraminic acid (NAcNeu) has
also been suggested to play a role in the attachment of I. monocytogenes to
o murine macrophages (Maganti et al., 1998). Host nuclear factor /cB (NF-/cB)

w is activated when I. monocytogenes adheres to the surface of macrophage -like

* cell lines; long-lasting NF-/cB activation appears to require LLO and products

g of the lecithinase operon (mpl-actA-plcB; see Hauf et al., 1994).

| LIFE AFTER INVASION: THE PHAGOSOME IS NO PLACE FOR

% L MONOCYTOGENES TO LIVE

pi Inside activated macrophages, most of the internalized I. monocytogenes

p* are killed within minutes after uptake (Davies, 1983; Raybourne and Bunning,

§ 1994). Nevertheless, L. monocytogenes appears well adapted to establish and

maintain a niche inside various types of nonbactericidal host cells. L. mono-
cytogenes mediates escape from the host cell phagosome, multiplies within
the cytosol, and moves to infect adjacent cells by means of the expression of
a number of protein products that function together to enable the bacterium
to exploit the host cell environment.

L monocytogenes escape from the phagosome

Host cell invasion ultimately provides access to a rich growth medium
for L. monocytogenes: the host cytosol. Following host cell uptake of the bac-
terium, L. monocytogenes is enclosed in a primary phagosomal vacuole that
becomes quickly acidified. L. monocytogenes prevents further maturation of
this phagosome and enhances its own survival by escaping into the cytosol
after disruption of the phagosomal membrane (Alvarez- Dominguez et al.,
1997a). In mouse bone marrow-derived macrophages, bacterial-mediated
perforation of the phagosome membrane is detectable within minutes

(Beauregard et al., 1997). Complete disruption of the phagosomal membrane
within infected Caco-2 cells is observed in approximately 30 minutes (Gaillard
et al., 1987). The major factor contributing to phagosomal escape is LLO, first
described following the isolation of bacterial transposon-insertion mutants
that had a nonhemolytic phenotype on sheep blood agar plates (Gaillard et al.,
1986; Kathariou et al., 1987). LLO, encoded by hly, was the initial virulence
factor to be identified as essential for I. monocytogenes virulence (Gaillard
et al, 1986; Geoffroy et al, 1987; Kathariou et al, 1987; Mengaud et al, 1988;
Cossart et al., 1989); bacterial mutants lacking LLO do not mediate phagoso-
mal escape in most cell lines examined and are highly attenuated in mouse
models of infection (Portnoy et al., 1992). The production of LLO by the non-
pathogenic soil bacterium Bacillus subtilis is sufficient to mediate bacterial
escape from macrophage phagosomes following uptake of B. subtilis into
tissue culture cells (Bielecki et al., 1990).

LLO is a 58-kDa cytolysin belonging to a large family of cholesterol- g

dependent, pore-forming toxins that includes streptolysin O (SLO) and per- g

fringolysin O (PFO), secreted by Streptococcus pyogenes and Clostridium per- §

o
fringens, respectively (Palmer, 2001). Similar to other family members, LLO Q

has a conserved tryptophan-rich undecapeptide (ECTGLAWEWWR) at its C- §

terminus. This region appears to be important for the cholesterol-dependent g

membrane binding of these cytolysins (Iwamoto et al., 1990; Boulnois et al., g

1991; Sekino-Suzuki et al., 1996; Palmer, 2001). LLO and its family mem- >

bers mediate membrane disruption through the formation of oligomeric g

ring-shaped structures that form membrane pores that are approximately £j

20-30 nm in diameter (Palmer, 2001). The crystal structure of PFO has been

solved, revealing four protein domains; this structure has provided valuable 3

information regarding structure-function relationships within this family of S

proteins (Rossjohn et al., 1997). A recent study demonstrated that two vari- g

ants of LLO, comprising domains 1-3 or domain 4 alone, could be secreted »

efficiently and reassembled into a functionally active protein when the do- g

mains were expressed simultaneously (Dubail et al., 2001). Another study h

demonstrated that domains 1-3 of LLO were essential for cytokine induc-
tion, whereas domain 4 was important for binding to membrane cholesterol
(Kohdaetal., 2002).

LLO is unique among the family members because of its acidic pH
optimum (Geoffroy et al., 1987). LLO is active at pH 4.5 to 6.5, an ideal range
for the acidic phagocytic vacuole, the pH of which is approximately 5. The
cytolysin PFO, which does not have an acidic pH optimum, is toxic to host
cells when expressed by I. monocytogenes Ahly mutants, possibly as a result
of PFO-mediated host cell plasma membrane damage (Jones and Portnoy,

1994). Mutations that result in single amino acid substitutions within PFO
can apparently convert the protein into a vacuole-specific hemolysin similar
to LLO, but L. monocytogenes strains expressing these PFO variant molecules
are still highly attenuated in mouse models of infection (Jones et al., 1996). A
single leucine located at position 461 within LLO has been recently identified
to confer the acidic pH activity (Glomski et al., 2002). The substitution of
leucine 461 with a threonine found at a similar position within PFO increased
the activity of LLO at a neutral pH. I. monocytogenes mutants expressing
the hly L461T variant were cytotoxic to infected host cells (Glomski et al.,
2002). In addition, a PEST-like sequence in the N-terminus of LLO has been
suggested to target LLO for rapid degradation within the host cytosol (Decatur
and Portnoy, 2000). L. monocytogenes mutants producing LLO molecules that
lack the PEST sequence accumulate the protein within the host cytosol, and
u the mutants are extremely cytotoxic for host cells and highly attenuated for

w virulence in mice. Moreover, the addition of the PEST sequence to PFO

* can convert it into a protein variant that is nontoxic to host cells (Decatur

g and Portnoy, 2000) . An independent study has shown that L. monocytogenes

< strains that express an LLO variant with substitutions for all of the P, E, S, and

g T residues is also highly attenuated for virulence in mice (Lety et al., 2001). It

thus appears that multiple mechanisms exist to restrict LLO activity within

o the host cytosol, thereby compartmentalizing the activity of the protein to the

p\ phagosome and preventing LLO-mediated damage to the host cell plasma

p* membrane. The existence of multiple mechanisms that serve to regulate LLO

§ activity highlights how extraordinarily suited L. monocytogenes has become for

life within the host cytosol.

As previously discussed, LLO plays an important role in mediating the
escape of I. monocytogenes from the phagosome of many cell types; however,
its activity is not required for phagosome disruption within all cells. Non-
hemolytic I. monocytogenes mutants lacking functional LLO are still capable
of replicating within the cytosol of Henle 407 human epithelial cells, which
suggests that other factors can contribute to phagosomal membrane disrup-
tion (Portnoy et al., 1988). I. monocytogenes encodes two phospholipases: a
33-kDa phosphatidylinositol-specific phospholipase C (PI-PLC) that is en-
coded by plcA, and a broad-specificity phospholipase that is encoded by plcB
(Camilli et al., 1991; Geoffroy et al., 1991; Leimeister-Wachter et al., 1991;
Mengaud et al., 1991; Vazquez- Boland et al., 1992).

PI-PLC can hydrolyze moieties that anchor many eukaryotic membrane
proteins to the plasma membrane, and the secreted enzyme has an acidic
pH optimum (Goldfine and Knob, 1992). PI-PLC is thought to be active in
the phagocytic vacuole and to mediate lysis of the vacuolar membrane in
conjunction with LLO (Goldfine and Knob, 1992). It has been suggested that

the initial pores formed by LLO provide access to PI-PLC membrane sub-
strates. Cooperation between the LLO hemolysin and the phospholipases was
also suggested to increase the membrane affinity of LLO, thereby facilitating
membrane lysis (Goldfine et al., 1995; Sibelius et al., 1996; Stachowiak and
Bielecki, 2001). Recently, the combined activities of LLO and PI-PLC were
shown to affect bacterial entry and vacuolar escape in J 774 cells by influenc-
ing the translocation of protein kinase C and calcium signaling (Wadsworth
and Goldfine, 2002).

The 29-kDa lecithinase or phosphatidylcholine-phospholipase C (PC-
PLC) encoded by plcB is another factor that contributes to the escape of
L. monocytogenes from cell phagosomes. This enzyme has a wide pH optima
and a broad substrate spectrum (Geoffroy et al., 1991; Goldfine et al., 1993).
PlcB is synthesized as an inactive proenzyme pro- PlcB, a regulatory step that
has probably evolved to prevent PlcB -mediated bacterial membrane damage.
The N-terminal propeptide of PlcB is cleaved by a zinc-dependent metallo- g

protease encoded by mpl (Poyart et al., 1993). Interestingly, L. monocytogenes g

appears to sequester pools of inactive pro-PlcB during bacterial replication §

o
within the host cytosol, and active enzyme is then rapidly released when Q

pH decreases below 7.0 (Marquis and Hager, 2000). PlcB is required for the §

efficient escape of I. monocytogenes from the secondary double-membrane g

vacuoles formed during the process of cell-to-cell spread (discussed in the g

following section); however, the protein can functionally replace LLO to me- >

diate escape from the primary phagosome in human epithelial cells (Marquis g

etal., 1995).

In addition to the factors mentioned herein, a previously unknown

surface protein designated SvpA (surface virulence-associated protein) was 3

recently suggested to promote phagosomal escape and thus intracellular S

survival of L. monocytogenes (Borezee et al., 2001). A svpA mutant was shown g

to be confined within the phagosomal vacuole and was attenuated in a »

>
O

ffi

mouse model of infection with decreased bacterial growth in the liver and g

spleen (Borezee et al., 2001). L. monocytogenes therefore appears to possess a
variety of factors that may work in concert to facilitate efficient escape from
the phagosome so that the bacterium can reach the promised land: the host
cytosol.

THE GOOD LIFE: L MONOCYTOGENES AT HOME IN
THE CYTOSOL

The doubling time for L. monocytogenes within the host cytosol is
very similar to the doubling time observed for bacterial cultures grown in
rich broth media; therefore, the cytosol appears to be a very permissive

environment for I. monocytogenes proliferation (Portnoy et al., 1988). Intra-
cellular growth of L. monocytogenes does not require the induction of stress
proteins, and several auxotrophic mutants of I. monocytogenes were able to
replicate in the cytosol of the macrophage-like cell line J 774 and the human
epithelial cell line Henle 407 with growth rates similar to those of wild-type
strains (Marquis et al., 1993; Hanawa et al., 1995). The expression of three
genes involved in purine and pyrimidine biosynthesis (purH, purD, and pyrE ) ,
and a gene encoding an ATP-dependent arginine transporter (arpj), is in-
duced in bacteria located within host cells; however, L. monocytogenes strains
carrying mutations in these genes were not affected for rates of intracellu-
lar growth and were fully virulent in a mouse model of infection (Klarsfeld
et al., 1994). Other in vivo-induced genes that encode metabolic enzymes and
nutrient transport systems have been identified through the use of a variety of
o genetic screens (Dubail et al., 2000; Gahan and Hill, 2000; Autret et al, 2001;

w Wilson et al., 2001). It has been suggested that some nutrients, such as nU-

* cleotides and certain amino acids, are not at limiting concentrations within

g the cytosol but are nonetheless at low concentration, and I. monocytogenes

< therefore induces the expression of genes required for the uptake and biosyn-

g thesis of various metabolites to facilitate efficient intracellular proliferation

o (Klarsfeld et al, 1994; Sheehan et al., 1994; Vazquez- Boland et al, 2001b).

£ —

o Recent evidence has implicated Hpt, a L. monocytogenes homologue of

pi the mammalian glucose-6-phosphate (G-6-P) translocase, in facilitating rapid

>* intracellular proliferation of bacteria through utilization of hexose pilos-

is phates obtained from the host cytosol (Chico-Calero et al., 2002). The mam-

malian G-6-P translocase transports G-6-P from the cytosol into the endoplas-
mic reticulum. L. monocytogenes Hpt may function in an analogous manner to
transport G-6-P from the cytosol into the bacterium; bacterial mutants lack-
ing the G-6-P translocase were defective for intracellular proliferation and
were attenuated for virulence in mice (Chico-Calero et al., 2002).

The acquisition of iron is important for L. monocytogenes, as it is for all
other living cells. Iron is an essential cofactor for a wide variety of enzymatic
processes. Iron stimulates I. monocytogenes growth in vitro, and it increases
bacterial proliferation in vivo (Sword, 1966; Stelma et al., 1987; Payne, 1993).
Increased concentrations of iron have been shown to upregulate the expres-
sion of inlAB and enhance L. monocytogenes invasion of Caco-2 cells (Conte
et al., 1996). In macrophages, iron is required for antimicrobial responses in-
volving the generation of reactive oxygen and nitrogen intermediates. How-
ever, humans with iron overload are more susceptible to listeriosis; thus a
balance of iron must be maintained to appropriately respond to L. mono-
cytogenes infection (Schuchat et al., 1991; Fleming and Campbell, 1997). In

mammalian hosts, iron is normally sequestered by serum transferrin and is
also complexed by intracellular heme compounds; it is therefore not freely
available to L. monocytogenes. Unlike many pathogens, L. monocytogenes does
not appear to secrete the high-affinity iron-binding siderophores; however,
the organism can use exogenous siderophores by means of an extracellu-
lar ferric iron reductase. L. monocytogenes also possesses a citrate-inducible
iron transport system and a cell-surface-associated trans ferrin-binding pro-
tein (Adams et al., 1990; Hartford et al., 1993; Deneer et al., 1995; Barchini
and Cowart, 1996; Coulanges et al., 1997).

THE GRASS IS ALWAYS GREENER: SPREAD OF
L MONOCYTOGENES TO ADJACENT HOST CELLS

The cytosol may be a tasty smorgasbord for replicating L. monocytogenes,
but it pays to move onto greener cell pastures. Each cell can only sustain a g

finite amount of bacterial replication, and evidence suggests that I. mono- g

cytogenes needs to spread to new host cells as a means of outrunning host §

o
cellular immune responses (Auerbuch et al., 2001). The necessity of cell-to- Q

cell spread as a means of obtaining fresh supplies of nutrients and of avoiding §

humoral and cellular immune responses was discussed in a recent review g

(O'Riordan and Portnoy, 2002), in which the authors also noted that although g

there are not many cytosolic bacterial pathogens, most of them are capable >

of cell-to-cell spread.

L monocytogenes-d'irected actin-based motility §

L. monocytogenes cytosol motility and bacterial spread to adjacent cells S

is accomplished by the exploitation of host actin polymerization machinery. g

Observations of infected host cells by electron microscopy indicate that cy- »

tosolic L. monocytogenes is soon surrounded by a dense cloud of host-derived g

F-actin, which eventually forms a comet-tail-like structure at one bacterial h

pole (Tilney and Portnoy, 1989). Approximately 2h after internalization,
I. monocytogenes begins to move in the host cytosol with a velocity of 0.1-0.4
/xm/s, a rate approximately proportional to the rate of actin polymerization
(Theriot et al., 1992). The L. monocytogenes surface protein ActA is absolutely
necessary for actin-based motility within the host cytosol.

ActA was identified following the isolation of I. monocytogenes mu-
tants that lacked the ability to polymerize actin and spread to adjacent cells
(Domann et al., 1992; Kocks et al., 1992). I. monocytogenes actA mutants in-
vade host cells, escape from the phagosome, and replicate within the cytosol,

'■5
<

1*1

► \ <i

< \

D-

Q-

\<]

\ t-

\ "

\ T

-5V O

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but the bacteria are unable to use actin polymerization as a motile force and
thus accumulate as microcolonies within infected host cells. L. monocytogenes
actA deletion mutants are 1000-fold less virulent in mice, and infection is re-
stricted predominantly to the gut (Domann et al., 1992; Kocks et al., 1992;
Manohar et al., 2001). ActA appears to be the only L. monocytogenes protein
required to nucleate actin polymerization; actin-based motility and the for-
mation of actin comet tails is observed in cell extracts for both streptococci
and beads that have been asymmetrically coated with purified ActA (Smith
etal., 1995; Cameron etal., 1999). L. innocua, a nonpathogenic Listeria species,
does not polymerize actin when added to cell extracts, but the expression of
the actA gene product on the L. innocua surface enables the bacteria to form
actin tails and move (Kocks et al., 1995).

The length of L. monocytogenes actin tails reflects the rates of bacterial
movement (Theriot et al., 1992; Sechi et al., 1997), and the rate of move-
ment is likely to affect the efficiency of cell-to-cell spread. Interestingly, there g
seems to be an optimal window of ActA production required for normal g

movement and cell-to-cell spread. A single additional copy of the actA gene §

o
integrated within the L. monocytogenes chromosome can reduce the efficiency Q

of cell-to-cell spread, apparently because of increased ActA production (Lauer §

et al., 2002). Conversely, recent research in our laboratory indicates that g

a reduction in actA expression appears to proportionally reduce cell-to-cell g

spread. A series of actA promoter mutants, demonstrated to express de- >

creasing amounts of actA as measured through the use of actA-gus reporter g

gene fusions, produced correspondingly smaller plaques in monolayers of £j

mouse L2 fibroblast cells (Fig. 6.3; K. Wong and N. Freitag, unpublished

data). A linear relationship was found to exist between actA expression and 3

plaque size. Decreasing actA expression was also associated with decreasing S

actin tail length as visualized by staining for filamentous actin in monolay- g

ers of infected PtK2 cells in tissue culture (Fig. 6.3). These data support the »

Figure 6.3. (facing page). Correlation of actA expression levels with L. monocytogenes *

cell-to-cell spread. (A) Diagram of the actA promoter region. The dark arrow represents

the transcript initiation site, SD indicates the ribosome binding site, and the PrfA binding

site is indicated by the presence of the PrfA box. Targeted actA deletions introduced into

the L. monocytogenes chromosome are shown, with the numbers representing the size of

the deletion and the distance from the ATG start codon. (B) Actin staining of PtK2

epithelial cells infected with L. monocytogenes wild type (NF-L476) or the actA promoter

mutant strains. Filamentous actin is shown in green, and bacteria are shown in red. (C)

Correlation between levels of actA expression and cell-to-cell spread ability of I.

monocytogenes. See color section.

premise that an optimal concentration of ActA at the bacterial surface is
critical for function (Moors et al., 1999).

The mature ActA protein contains 610 amino acids after the cleavage of
a 29-residue signal sequence from its N-terminus. ActA can be divided into
three domains: an N-terminal domain (1-234) rich in cationic residues, a
central domain (235-394) containing proline-rich repeats, and a C-terminal
domain (395-610) containing a stretch of hydrophobic residues (585-606)
that anchors the protein to the surface of L. monocytogenes. The N-terminal
domain contains all the information necessary to mediate actin polymeriza-
tion and support actin-based motility (Lasa et al., 1997). The cationic residues
(129-153) within this domain appear to be critical for actin assembly; residues
21-97 are responsible for the continuity of the assembly process; and residues
117-121 contribute to tail formation (Lasa et al., 1997).
u A key function of the ActA N-terminal domain is the recruitment of

w the host Arp2/3 complex, which is critical for the nucleation step initiat-

* ing actin filament formation (Welch et al., 1997). The N-terminus of ActA

g shares homology with the Wiskott-Aldrich syndrome protein (WASP) fam-

< ily in mammalian cells (Skoble et al., 2000; Boujemaa-Paterski et al., 2001),

g which have been shown to stimulate actin polymerization through activation

of Arp2/3 and to connect the cytoskeleton with signaling pathways that me-

o diate actin rearrangement (Welch et al., 1997; Machesky and Insall, 1998;

p\ Goldberg, 2001). ActA binds two actin monomers and three of the seven

p* subunits of the Arp2/3 complex (Zalevsky et al., 2001). A mutational analy-

§ sis of the ActA N-terminus indicates that the actin-binding region spans 40

residues (31-72); a separate region of ActA (136-231) contributes to the actin
cloud-to-tail transition that affects intracellular motility rates (Lauer et al.,
2001).

The central domain of ActA contains four short proline-rich repeats
(PRRs) with consensus sequence DFPPPPTDEEL. These PRRs are similar to
PRRs present in mammalian cytoskeletal proteins, and they serve as binding
sites for the host tetrameric vasodilator phospho-protein (VASP; see Reinhard
etal., 1995; Niebuhretal., 1997; Dramsi and Cossart, 1998). VASP binds host
profilin, an actin-sequestering protein that recruits actin monomers for the
elongation of the actin filaments. Both VASP and profilin have been shown to
co-localize at the interface between I. monocytogenes and the actin tails (The-
riotetal., 1994; Chakrabortyetal., 1995). Interestingly, I. monocytogenes is still
capable of actin recruitment and intracellular movement in the absence of
the PRRs, VASP, or profilin, although the organism moves at a significantly
lower rate (Lasa et al., 1995; Smith et al., 1996; Niebuhr et al., 1997; Loisel

: s ActA dimer £7 actin monomer ^ profilin
i tiitm actin filament^ capping protein ▼ cofilin
W VASP ^ Arp2/3 complex J ct-actinin

Figure 6.4. Model of actin nucleation and polymerization as directed by I. monocytogenes
(based on previous models described by Cossart and Bierne, 2001 and Skoble et al., 2000).
VASP binds to the four proline-rich repeats present in the central domain of ActA. The
N-terminal domain of ActA contains three regions that contribute to actin binding and the
activation of the Arp2/3 complex.

et al., 1999). This suggests a stimulatory role for the ActA PRRs and VASP
in actin-based motility.

Several other host proteins are involved in L. monocytogenes-directed actin
polymerization. Alpha-actinin is distributed over the actin tail and is respon-
sible for stabilizing the tail by cross-linking actin filaments (Dabiri et al., 1990;
Loisel et al., 1999) . Although alpha-actinin is not required for I. monocytogenes
actin-based motility, the tails are less rigid and the bacteria tend to drift in its
absence. Capping protein blocks actin assembly by binding to barbed ends of
actin filaments. This protein was shown to be required in the reconstitution
of L. monocytogenes motility in vitro. The absence of this protein or its pres-
ence at low concentrations results in a fishbone-like web of actin filaments,
which is inefficient at providing motile force (Marchand et al., 1995; Loisel
et al., 1999; Pantaloni et al., 2000). Cofilin depolymerizes actin and may play
a role in generating pools of actin monomers (Carlier et al., 1997); it is also
required for reconstituting bacterial movement in vitro (Loisel et al., 1999).
Taken together, a model depicting L. monocytogenes actin-based movement is
shown in Fig. 6.4.

%
o
n

hi
%

>
o
z

M
(- 1
M

o
to
o

ffi

L monocytogenes invasion of adjacent cells

As I. monocytogenes moves through the cell and contacts the plasma
membrane, it appears to push out the membrane and form a pseudopod-like
(or Listeria-pod-\ike) structure that is somehow recognized or taken up by
the neighboring cell. The bacterium is then enclosed in a double-membrane-
bound secondary vacuole, and lysis of this compartment is facilitated by the
phospholipase PC-PLC (Vazquez- Boland et al., 1992). The plcA gene product
(PI-PLC) appears to function synergistically with PC-PLC, as I. monocyto-
genes mutants lacking both enzymes have a more serious defect in cell-to-cell
spread than either of the single mutants (Marquis et al., 1995). LLO is also
required for escape from the double-membrane vacuoles (Gedde et al., 2000) .
Through the coordinated activities of the hly, plcA, and plcB gene products,
I. monocytogenes disrupts the double -membrane vacuole and makes a fresh
eh home within the cytosol of the new cell victim. In this way, the bacterium

g gains new pastures without exposure to the humoral immune responses of

« the host.

% COORDINATING THE BACTERIAL DANCE STEPS OF INVASION,

t REPLICATION, AND CELL-TO-CELL SPREAD

p\ I. monocytogenes is similar to other bacterial pathogens in that many of

^ the gene products that contribute to infection are expressed preferentially

§ within host cells. The expression of virulence determinants by I. monocyto-

genes appears to be tightly regulated and can be triggered by specific host cell
compartment environments, such as the phagosome or cytosol. The process
of L. monocytogenes virulence gene regulation in response to host cell envi-
ronments is a complex and fascinating story, with many of the players still
waiting to be discovered.

Most of the virulence genes identified thus far in L. monocytogenes are
located within a 10-kb chromosomal region known as the Listeria pathogenic-
ity island 1 (LIPI-1; see Vazquez- Boland et al., 2001a). In addition to the
aforementioned virulence genes plcA, hly, mpl, actA, and plcB, a positive
transcriptional regulator known as prfA is located within this cluster, and
LIPI-1 is commonly referred to as the PrfA regulon (Fig. 6.5). The LIPI-1
is only present in pathogenic Listeria species, although an apparently inacti-
vated form is also present in I. seeligeri. This region is located between prs
and Idh, two housekeeping genes coding for phosphoribosyl-pyrophosphate
synthetase and lactate dehydrogenase, respectively. DNA sequences in the
prs-ldh intergenic region of the nonpathogenic listeriae were shown to have

+ +

[prfA — ( plcA

hly > mpl > act A / plcB

lkb

Figure 6.5. The L. monocytogenes LIPI— 1, also referred to as the PrfA regulon;
+ designates promoters whose expression is increased by PrfA binding, and
— designates promoters for which PrfA appears to decrease expression.

homology with integration consensus sequences for a transposon, support-
ing the hypothesis that horizontal gene transfer might be involved in the
evolution of this gene cluster (Gouin et al., 1994; Cai and Wiedmann, 2001).
PrfA is essential for L. monocytogenes virulence, and it is required for
the expression of all the other genes within the LIPI-1. The prfA gene was 3

first identified during the characterization of a spontaneous nonhemolytic ^

L. monocytogenes mutant that was found to contain a small deletion in an §

open reading frame downstream of pic A. Complementation of the mutation Q

with the complete open reading frame led to increased transcription from §

genes located within LIPI-1 and demonstrated the pleiotropic effects of the g

prfA gene product (Leimeister-Wachter et al., 1989, 1990). The expressions g

of several other virulence genes, such as inlA, MB, hpt, and bsh (encoding a >

bile salt hydrolase) , have also subsequently been shown to be completely or g

partially dependent on PrfA (Kreft and Vazquez-Boland, 2001; Chico-Calero £j
et al., 2002; Dussurget et al., 2002). Other I. monocytogenes genes, such as

clpC, whose gene product contributes to stress responses, said flaA, encoding 3

the flagellin subunit, appear to be negatively regulated by PrfA (Ripio et al., S

1997, 1998). In contrast, there are virulence genes that are PrfA-independent, g

such as svpA and the inlGHE operon (Lingnau et al., 1995; Ripio et al., 1997; *

Raffelsbauer et al., 1998; Borezee et al., 2001). §

Although the expression of many L. monocytogenes virulence genes is h

dependent on PrfA, the patterns of gene expression vary for individual genes
during bacterial growth and during the course of host cell infection. inlA and
MB expression is readily detectable during bacterial growth outside of host
cells, whereas hly, pic A, and plcB have been reported to be induced within the
phagosome (Bubert et al., 1999). The expression of mpl and actA is highly
upregulated within the host cytosol, whereas actA expression alone appears
downregulated within the secondary vacuoles that are formed following the
spread of I. monocytogenes to adjacent cells (Bohne et al., 1994; Bubert et al.,
1999; Freitag and Jacobs, 1999). The patterns of virulence gene expression

observed appear to correspond perfectly with the functional roles ascribed
to the different products. However, it is unclear why plcB transcripts were
detected in the phagosome in the absence of detectable actA mRNA, as the
two genes are cotranscribed with no apparent intergenic promoter (Vazquez-
Boland et al., 1992). It has been suggested that the actA mRNA region and
the plcB mRNA region may differ in message stability.

The 27-kDa PrfA protein is a member of the Crp/Fnr family of tran-
scriptional activators, most members of which have been identified in Gram-
negative bacteria (Lampidis et al., 1994; Ripio et al., 1997). In Escherichia coli,
Crp (also known as Cap) and Fnr are involved in carbon utilization and anaer-
obic growth responses, respectively (Spiro and Guest, 1990; Kolb et al., 1993).
The PrfA sequence is only approximately 20% identical and 30% similar to
that of Crp; however, the two proteins appear to share significant structural
u similarities (based on the reported Crp structure and on sequence structure

w predictions for PrfA). Both proteins contain an N-terminal /3-roll structure

* and a C -terminal DNA-binding helix-turn -helix (HTH) motif. The activation

g regions that are involved in the interaction of Crp with RNA polymerase may

< also be conserved in PrfA. Perhaps most intriguing in regard to the similar-

g ities between Crp and PrfA is the existence of mutationally activated forms

of the proteins, known as Crp* and PrfA*. Crp requires the presence of a

o cofactor, cAMP, for full activity, and two mutations within Crp have been

^ described, G145S and A144T, that lead to cofactor-independent Crp activa-

>* tion (Crp* mutants; see Garges and Adhya, 1988). Ripio et al. (1997) found

§ that L. monocytogenes isolates that expressed high amounts of LLO or PC-PLC

contained a single amino acid substitution within PrfA located in a region
(G145S) similar to the mutations found to confer the Crp* phenotype to Crp.
The PrfA* G145S substitution causes constitutively high expression of all
PrfA-regulated genes, suggesting that, like Crp, a cofactor (which does not
appear to be cAMP) may be required for PrfA activation (Lampidis et al.,
1994; Ripio et al., 1997; Goebel et al., 2000).

Our laboratory has recently identified two additional mutations within
PrfA that appear to confer a PrfA* phenotype (Shetron-Rama et al., 2003).
L. monocytogenes PrfA E77K contains a glutamate -to -lysine substitution
within the /3-roll structure of PrfA, whereas PrfA G155S contains a glycine-
to-serine substitution near the location of the original PrfA* mutation
(G145S). Both mutations confer high-level expression of PrfA-regulated
genes, and strains containing either mutation appear hyper-invasive for ep-
ithelial cell lines. Interestingly, the PrfA G155S mutant strain is approx-
imately fivefold more virulent in a mouse model of infection than the

Environmental signals that influence virulence gene expression

wild-type strain, suggesting that constitutive activation of PrfA is not detri-
mental to virulence.

PrfA is a site-specific DNA-binding protein that recognizes a 14-bp palin-
drome known as the "PrfA-box" within the -40 region of PrfA-dependent
promoters. The binding affinity of PrfA for its target promoters is influenced
by the nucleotide sequence of the PrfA box. PrfA binds with high affinity to
the perfectly symmetrical PrfA box (TTAACA-NN-TGTTAA) shared by the
divergently transcribed hly and plcA promoters. The PrfA boxes of the mpl
and act A promoters contain single base pair asymmetries, and the inlA PrfA
box has two base pair asymmetries. These PrfA box asymmetries have been
postulated to cause PrfA to bind with lower affinity to these target promoters,
and thus it has been speculated that the relative amount of PrfA present at
any time within the cell determines the level of expression of the various
virulence genes (Freitag et al., 1993; Goebel et al., 2000; Kreft and Vazquez-
Boland, 2001). Recent studies have shown that although the expression of g

some genes appears to follow the PrfA binding affinity rule (hly, mpl), other g

PrfA-dependent promoters, such as the act A promoter, do not (Williams et §

o
al., 2000), and that even the increased expression of the mutationally acti- Q

vated prfA* allele is not sufficient to raise levels of actA expression to those §

observed for bacteria located within the host cell cytosol (Shetron-Rama et al., *

2002; Greene and Freitag, 2003) . Our laboratory has isolated I. monocytogenes g

mutants that express high levels of actA during growth in broth culture, and >

these mutations map at least 40 kb outside of the PrfA regulon, indicating g

the participation of additional factors in the induction of actA expression £j

(Shetron-Rama et al., 2003).

z
n

M
M
M

Multiple conditions have been reported to influence I. monocytogenes vir- »

ulence gene expression in culture, including temperature, available carbon g

sources, nutrient deprivation, and iron (Kreft and Vazquez- Boland, 2001) . Vir- h

ulence gene expression has been reported to be optimally activated at 37°C,
the body temperature of mammalian hosts (Leimeister-Wachter et al., 1992),
and the expression of the key regulatory protein PrfA appears to be under
the control of a thermosensitive mechanism of translation in which optimal
translation occurs at 37° C (Johansson et al., 2002). However, L. monocyto-
genes virulence gene expression has been observed within infected insect cell
lines maintained at room temperature (Mansfield et al., 2003 (in press)),
indicating that the appropriate cell environment can stimulate bacterial

gene expression even at low temperatures. Iron and available carbon sources
influence the expression of I. monocytogenes virulence genes, and bacterial
gene products involved in iron uptake or in the utilization of hexose sug-
ars are upregulated by intracellular L. monocytogenes (Sword, 1966; Payne,
1993; Klarsfeld et al, 1994; Borezee et al., 2001; Chico-Calero et al, 2002).
Interestingly, the proximal prfAP2 promoter was recently demonstrated to be
regulated by the stress-responsive alternative sigma factor a B ; however, the
loss of a B function only modestly reduces the virulence of I. monocytogenes
(Nadon et al., 2002) . As L. monocytogenes is a bacterium that survives in a mul-
titude of environments, regulation of gene expression has probably evolved
in order to respond to a variety of signals that tell the bacterium whether its lo-
cation lies inside or outside of a multicellular organism. When the bacterium
determines that it is indeed inside of a prospective host organism, additional
^ signals may then serve to direct the bacterium to its replicative niche within

m the cytosol.

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CHAPTER 7

A/, gonorrhoeae: The varying mechanism of
pathogenesis in males and females

Jennifer L. Edwards, Hillery A. Harvey, and Michael A. Apicella

Neisseria gonorrhoeae, the gonococcus, is the causative agent of gonorrhea,
one of the oldest human diseases on record. Biblical references to gonorrhea
in Leviticus (15:1-15:19) showed that the infectious nature of the disease was
recognized even at that time. Probably, the best description of gonorrhea in
a man in the preantibiotic era can be found in the writings of Bos well, who
described in detail each of his 19 episodes of infection (Ober, 1970). These
descriptions also allude to the asymptomatic nature of the disease in women,
as many of the contacts from whom he acquired the infection were without
symptoms of disease. Today, it is estimated that greater than 1 million cases of
N. gonorrhoeae infection occur in the United States, and 60 million cases
are reported annually worldwide. Hence, N. gonorrhoeae infection remains
prevalent in the general population, despite the fact that antibiotic therapy is
readily available. The high incidence of this disease remains a major concern
in lower socioeconomic groups in the United States; however, the highest
incidence of infection and of complications resulting from infection occur
in developing countries. In underdeveloped nations it has been shown that
patients with gonorrhea are at much higher risk for contracting human im-
munodeficiency virus (HIV). It is proposed that this increased susceptibility
to HIV infection results from the inflammatory response generated by infect-
ing gonococci with the subsequent disruption and shedding of the mucosal
epithelium.

The gonococcus was classically considered an extracellular pathogen that
colonized the epithelial surfaces of the human genital tract. This was in spite
of the fact that over the past three decades the pathogenesis of gonococci in
eukaryotic cells has been studied extensively in patient exudates and in tissue
and organ culture. Electron microscopic studies by Evans (1977) of biopsies
of infected females show that N. gonorrhoeae colonize and invade epithelial

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Figure 7.1. A cross section of a biopsy derived from the squamocolumnar junction of the
uterine cervix of an infected female demonstrates the invasive nature of N. gonorrhoeae.
Extensive cytoskeletal rearrangements (CR; e.g., filopodia, lamellipodia, and ruffles) of the
mucosal epithelium are elicited by gonococcal infection and invasion and are visible on
the apical cell surface. Gonococci (GC) are internalized in spacious vacuoles, which are
denoted by arrows.

cells of the cervical squamocolumnar junction (Fig. 7.1). Studies by Apicella
and coworkers (1996) show that urethral epithelial cells in exudates from
infected men contain numerous intracellular organisms (Fig. 7.2). It is now
understood that the ability of this organism to gain entry into host cells is
crucial to its capability to establish and sustain infection.

The gonococcus can infect a number of different epithelial surfaces,
thereby causing an array of clinical syndromes. Anorectal and pharyngeal
infection usually results in an asymptomatic or a subclinical disease state.
Conjunctival infection generally results in a severe conjunctivitis with corneal
ulceration, although mild disease is occasionally reported. The gonococcus
can also cause disseminated infection. However, disseminated gonococcal
infection (DGI) occurs less frequently (1-3%) in both men and women. DGI
results from gonococcal bacteremia and is commonly associated with un-
treated asymptomatic gonococcal infection or, less frequently (less than 3%),

Figure 7.2. An exfoliated urethral epithelial cell obtained from the exudate of a male with
documented gonococcal urethritis. Gonococci (GC) are labeled with a gold-bead
antibody-conjugate that is specific for lipooligosaccharide. Arrows denote the intimate
association of N. gonorrhoeae with the apical surface of the urethral epithelial cell and
pedestal formation.

with complement (C) deficiency. The majority of DGIs are found in women,
in which the onset of clinical symptoms is often associated with menses.
Arthritis-dermatitis syndrome is the most common manifestation of DGI,
but severe cases can lead to endocarditis and meningitis.

In men, uncomplicated gonococcal infection is usually presented as an
acute urethritis but can result in an acute epididymitis. Symptoms of disease
primarily result as the consequence of the inflammatory response directed
at the invasive gonococcus. However, experimental gonococcal infections in
male human volunteers show that there is a delay between infection and

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Figure 7.3. A 30-min experimental infection of a primary urethral epithelial cell with N.
gonorrhoeae demonstrates filopodia extension induced by the gonococcus. The diplococcus
arrangement, characteristic of N. gonorrhoeae, is visible at the upper left. Arrows denote
filopodia engulfing bacteria.

the onset of clinical symptoms (Schneider et al., 1995). During this time,
gonococci cannot be cultured from the urethra. Microscopy studies of ure-
thral exudates from men with gonorrhea demonstrate that N. gonorrhoeae
is an intracellular pathogen (Apicella et al., 1996). These studies also re-
veal that intracellular invasion by the gonococcus occurs through the inti-
mate association between the gonococcus and the urethral cell membrane
(Fig. 7.3), followed by internalization of gonococci within vacuoles. These ob-
servations, in conjunction with data obtained from experimental infections
of men, suggest that viable gonococci are later released from the epithelial
cells back into the lumen of the urethra. The organism's ability to gain ac-
cess to and to survive the intracellular environment and the mechanisms
that allow its subsequent release to the extracellular environment are poorly
understood.

If left untreated, gonococcal infection of most men will resolve over a pe-
riod of several weeks, but resolution may be mistaken for an asymptomatic
carrier state. The occurrence of asymptomatic infection in men, however, is
very low. Less than 3% of (infected) men exhibit asymptomatic gonococcal
infection. In striking contrast to gonococcal infection of men, 50-80% of
women exhibit asymptomatic genital tract infection, and 70-90% of women

with DGI lack symptoms of genital tract involvement (Densen et al., 1982).
Additionally, many women exhibit subclinical manifestations of N. gonor-
rhoeae infection; consequently, medical treatment is often not sought, or
gonococcal infection is undiagnosed.

The high incidence of asymptomatic gonorrhea in women contributes
substantially to the prevalence of N. gonorrhoeae in the general population
and leads to acute and chronic complications in these women. Women with
gonorrhea often exhibit multiple sites of N. gonorrhoeae colonization, which
is usually attributed to contamination by gonococci-containing cervical secre-
tions. Infection in women is further compounded by coinfection with other
sexually transmittable organisms and a number of host physiological factors
that, cooperatively, complicate disease (Bolan et al., 1999). Of the women
who exhibit symptomatic gonorrhea, the severity of clinical presentation of
disease is widely variable. Ascending gonococcal infection occurs in up to
45% of infected women and can result in pelvic inflammatory disease (PID), g

which can cause permanent fallopian tube scarring and blockage with sub- §

sequent infertility and ectopic pregnancies. One in 10 women suffer from §

PID (Aral et al., 1991), of which N. gonorrhoeae is the etiological agent in 40% jj

of all reported cases (Sweet et al., 1986). Greater than 25% of women with g

PID will exhibit long-term complications of disease, 20% will suffer chronic <

pelvic pain, and 20% will become infertile. In contrast, men with a history of %

gonorrhea very rarely become infertile because of N. gonorrhoeae infection. o

An additional 10% of women with PID will have an ectopic pregnancy, which m

is the leading cause of death of women in their first trimester of pregnancy. jj

Pregnancy is also considered to put a woman at an increased risk for acquir- £

ing DGI. The association of DGI with asymptomatic infection is reflected by o

the disproportionate percentage (80%) of DGIs that occur in women. jg

As with other bacterial pathogens, the widespread emergence of K

antibiotic-resistant strains of N. gonorrhoeae has increased the urgency of 2
vaccine development. The hope of a vaccine, however, requires a greater un-

derstanding of both the human and the bacterial factors that contribute to a
diseased state. Previous attempts at vaccine development have met with fail- s

ure or with limited success. The propensity of gonococcal surface proteins to S

undergo both phase and antigenic variation has contributed to the ill success §

of vaccine development. Recent analysis of the N. gonorrhoeae genome has *

identified 100 gonococcal genes that exhibit phase variation (Snyder et al., >

2001). Gonococcal infection was more recently shown to elicit only a mild 5

humoral immune response, and no immunological memory has been ob-
served. This suggests gonococcal subversion of the immune response by an
as yet undefined mechanism. These difficulties may potentially be overcome
by the use of genetically engineered hybrid proteins.

N. GONORRHOEAE CONSTITUENTS IMPLICATED
IN PATHOGENESIS

Pili

Pili (or fimbriae) are filamentous appendages that extend from the sur-
face of a bacterial cell. N. gonorrhoeae express the type IV-A class of bacterial
pili, which are characterized by the presence of a N-methyl-phenylalanine
amino acid residue at the first position in the mature pilin protein. An individ-
ual pilus fiber exists as a polymer composed of individual subunits (i.e., pilin)
of approximately 18-22 kDa. Individual pilin subunits contain conserved and
semivariable (SV) and hypervariable (HV) regions. Pilus assembly requires
a repertoire of proteins that are highly conserved between bacterial species.
Several levels of control regulate the expression of type IV pilus in the gono-
coccus. Multiple copies of the pilin gene can be found, scattered, throughout
the gonococcal genome. Usually only one copy (but sometimes two copies),
< termed pilE (E for expressed), contains a promoter region and, thus, is ex-

2 pressed. The remaining copies, termed pilS (S for silent), provide genetic

E material for nonreciprocal recombination events, which leads to antigenic

g variation between strains of gonococci.

g Pili are also subject to phase variation that can result from alterna-

tive cleavage of the N-terminal hydrophobic region of PilE to produce sol-

i-i

w
u

% uble S-pilin or from the production of aberrant or excessively long (L-)pilin

K ( Forest and Tainer, 1997). Phase and antigenic variation provide mechanisms

^ of immune avoidance, which allows the gonococcus to proliferate within a

S particular microenvironment of its human host. The importance of pili to

W N. gonorrhoeae colonization of epithelial cells has been demonstrated by sev-

g eral laboratories, and it is generally accepted that pili facilitate adherence

^ by allowing the gonococcus to overcome electrostatic repulsion that occurs

m . with the host cell. PilC, a pilus-associated protein, and which is also associated

g with the bacterial outer membrane (Rahman et al., 1977) , may facilitate pilus-

£ mediated adherence as a pilus tip-adhesin. Phase variation of PilC expression

w occurs by frameshift mutations within a polyguanine region upstream of the

two alleles (pilCl and pi\C2) encoding PilC (Dehio et al., 2000).

The absence of pilus does not appear to influence adhesion of the gono-
coccus to polymorphonuclear cells (PMNs), and it is suggested that the pre-
sence of pili may confer resistance to phagocytosis by these cells (Dilworth
et al., 1975). Pili also mediate genetic transformation and agglutination,
and they are postulated to provide a mechanism by which bacteria are
able to move across and colonize mucosal epithelia through twitching
motility. The gonococcal pilus is covalently modified with an O-linked,

N-acetylglucosamine(o;l-3) galactose (Galofl-3GlcNac) disaccharide, which
(to date) makes it unique among all prokaryotic proteins (Parge et al.,
1995). A trisaccharide, galactose)/? 1-4) galactose^ l-3)2,4-diacetimido-2,4,6-
trideoxyhexose (Gal/3 l-4Galal-3-DATDH; see Stimsonet al., 1995) is present
in this same position of meningococcal pilin (Marceau et al., 1998). Removal
of the meningococcal glycosyl residue confers a hyperadherent phenotype to
mutant organisms (Marceau et al., 1998), suggesting that pilin glycosylation
modulates pilin function and its interaction with epithelial cells. The role of
phosphate (Forest et al., 1999) and phase -variable phosphorylcholine (Weiser
et al., 1998) posttranslational modifications to pilus function are, currently,
not known.

The role of pili in gonococcal pathogenesis is implicated in vivo by ex-
amination of gonococcal isolates from patients with gonorrhea. In addition,
multiple in vitro studies have demonstrated the significance of pili in gono-
coccal adherence to epithelial cells. Complement receptor type 3 (CR3; see g
Edwards et al., 2001) and the C regulatory protein, CD46 (or membrane cofac- §

Porin

tor receptor; see Kallstrom et al., 1997), can serve as receptors for gonococcal §

pilus. CD46 is a human-specific transmembrane protein that is expressed by jj

all nucleated cells. The association of pili with CD46 results in a rapid cal- g

cium influx within the host cell (Kallstrom et al., 1998) . This suggests that pili <

may modulate host cell signaling mechanisms to aid gonococcal epithelial %

invasion. However, Tobiason and Seifert (2001) demonstrated that the pres- o

ence of CD46 on several epithelial cell lines is inversely related to gonococcal m

association with these cells. Additionally, CD46 does not appear to play a role jj

in the CR3-mediated initial colonization of the uterine cervix (Edwards et al., K

2002). Consequently, discernment of the role of CD46 in N. gonorrhoeae col- o

onization will require further examination. jg

H
O

%
M

Porin (P.I; 32-39 kDa) accounts for greater than 60% of the total weight ~

of proteins present in the outer membrane of the gonococcus, making it the s

most abundant outer membrane protein. Gonococcal nomenclature regard- S

ing porin is based on homology to meningococcal PorB. A gonococcal homo- %

logue of N. meningitidis PorA was not thought to exist; however, a gene show- *

ing homology to the PorA gene sequence has recently been identified (Dehio >

et al., 2000). Unlike pili, opacity-associated outer membrane proteins (Opa), 5
and lipooligosaccharide (LOS), all of which exhibit phase or antigenic varia-
tion or both, porin expression is stable within a given strain of N. gonorrhoeae.
Antigenic variation between strains, however, has provided one system of

N. gonorrhoeae serotyping. Two isotypes of porin are found in gonococci, P. I A
and P. IB. Isotypes P.IA and P. IB can be further differentiated into serovars
(classes 1-9) by their ability to react with a panel of monoclonal antibodies.
For any N. gonorrhoeae, only one porin isotype is produced; however, anoma-
lous P. I A/ P. IB hybrid porins have been clinically isolated on rare occasion
(Cooke et al., 1998). Gonococci exhibiting a P.IA isotype are associated with
an increased risk for DGI and are thought to exhibit increased resistance to
killing by normal human serum (i.e., serum-resistance).

Gonococcal porin resembles porins expressed by other Gram-negative
bacteria in that it forms a water-filled channel in the bacterial outer mem-
brane through which small solutes may cross. By analogy to Escherichia coli
porins, gonococcal porin is suggested to exist in the bacterial outer mem-
brane as a homotrimer, with each monomer forming a channel. Each porin
S monomer possesses 16 membrane-spanning regions, resulting in the expo-

S sure of eight antigenically variable loops in the extracellular environment. The

< longest of these extracellular loops express the greatest antigenic variability;

hj they are immunodominant and serve as docking sites for bactericidal anti-

X bodies. Although porin is highly immunogenic, its intimate association with

g reduction-modifiable protein (Rmp or P. 1 1 1) and with LOS within the outer

§ membrane makes it inaccessible to immunological attack in the presence of

^ LOS- or Rmp -directed blocking antibody.

% In contrast to other Gram-negative porins, which exhibit cation selec-

E tivity, gonococcal porins resemble mitochondrial porins in their anion se-

g lectivity. Another distinguishing property of gonococcal and mitochondrial

Jj porins is their ability to interact with the adenosine and guanosine triphos-

e phates (ATP and GTP, respectively) as a means to regulate pore size, voltage-

g dependent gating, and ion selectivity. Also unique among Gram-negative

^ porins is the ability of gonococcal porin to translocate into eukaryotic host

" cell membranes. Within the eukaryotic cell membrane, porin it is thought

£ to form a voltage-gated channel that is modulated by the host cell ATP and

I GTP.

El The role of porin in potentiating disease by N. gonorrhoeae is thought to

be multifactorial. Porin appears to modulate host cell function. Additional di-
verse functions of porin suggest that it is an important virulence factor for the
gonococcus. Studies using PMNs have indicated that porin changes the mem-
brane potential of these cells upon PMN membrane insertion (Haines et al.,
1988). A change in membrane potential results in degranulation inhibition
without altering the respiratory burst associated with oxidative killing. In con-
trast, gonococcal porin increases formyl-methionine-leucine-phenylalanine
(fM LP) -mediated hydrogen peroxide production in PMN (Haines etal., 1991).

Further modulation of host cell function includes the ability of porin to in-
hibit phagosome maturation and downregulation of immunologically impor-
tant cell surface receptors, such as immunoglobulin G (IgG), Fc receptors II
(FcyRII) and III (FcyRIII), and complement receptor 1 (CR1) and CR3, in
professional phagocytic cells.

Although porin appears to inhibit PMN actin polymerization in response
to chemoattractants (Bjerknes et al., 1995), in epithelial cells porin acts as an
actin-nucleating protein (Wen et al., 2000). In this respect porin may facil-
itate the cytoskeletal rearrangements required for actin-mediated entry of
the gonococcus into its target host cell. Porin also modulates apoptosis of
epithelial cells by inducing a calcium influx and, consequently, calpain and
caspase activity within these cells (Dehio et al., 2000). Recent data have sug-
gested that the ability of porin to induce apoptosis in epithelial cells may
play a role in the cytotoxicity observed in fallopian tube organ culture (FTOC)
and in shedding of epithelial cells (Dehio et al., 2000), which occurs in vivo g

during mucosal infection. Porin may also aid Opa-heparin sulfate proteo- §

glycan (H SPG) -mediated invasion and Opa-independent invasion of Chang §

cells in the absence of phosphate (van Putten et al., 1998). In cooperation with jj

gonococcus-bound iC3b and gonococcal pilus, porin also acts as an adhesin, g

mediating adherence to the cervical epithelium (Edwards et al., 2002).

<
>

Opacity-associated outer membrane proteins g

Opa (P. II or class 5) proteins comprise a family of closely related in- jj

tegral outer membrane proteins. Opa proteins were originally identified by £

their ability to confer opacity and color changes to colonies of N. gonorrhoeae o

(grown on translucent agar) when viewed under diffused light with a stere- jg

omicroscope. Subsequent studies revealed that opacity is the result of Opa- K

LOS interactions that occur between adjacent bacteria. Opa proteins are heat 2

modifiable, exhibiting altered electrophoretic mobility subsequent to heating 5

at 1 00° C. Molecular mass (M r ) for individual Opa proteins ranges from 24 kDa ~

to 30 kDa at 37°C and from 30 kDa to 32 kDa at 100°C. Gel filtration suggests s

that purified Opa proteins are trimers or tetramers; however, Opa isolation S

has proven difficult because of its high (one third) content of hydrophobic %

amino acids and, thus, its insolubility in the absence of detergents. *

Structural models predict eight membrane-spanning regions with four >

membrane loops exposed on the extracellular face of the bacterium. The 5
first three loops correspond to the SV, HV1, and HV2 regions; the fourth
(C-terminal) loop is highly conserved. There are 11 or 12 opa loci that oc-
cur throughout the chromosome of a given N. gonorrhoeae strain. Expression

of an individual opa gene occurs independently of other opa genes; conse-
quently, a single gonococcus may exhibit none, one, or more than one Opa
protein simultaneously. Each opa locus is subject to antigenic and phase
variation; therefore, a given strain of N. gonorrhoeae may be highly hetero-
geneous with respect to Opa expression. Opa phase variation results from
slipped-strand mispairing during replication in which alteration in the num-
ber of pentameric (CTCTT) repeat elements causes translational frameshifts
(Lemon and Sparling, 1999).

A correlation has been made between the presence (and absence) of
Opa proteins on N. gonorrhoeae clinical isolates with the site of isolation.
In one human volunteer study, gonococci recovered from males infected with
Opa - organisms had shifted to Opa + (Schneider etal., 1995). Clinical isolates
obtained from men tend to express Opa proteins as do isolates recovered from
3 human volunteer studies . Similarly, cervical isolates obtained from women at

S the time of ovulation (i.e., midcycle) also exhibit Opa expression. Translucent,

< Opa - organisms predominate in cervical isolates obtained during menses; in

hj the fallopian tube; in genital, blood, and joint fluid obtained from patients with

X DGI; and in asymptomatic men. Opa proteins facilitate adherence to PMNs

g and, consequently, may play a role in potentiating gonococcal urethritis. A

§ recent study has proposed a novel role for Opa proteins. Williams et al. (1998)

>; suggested that Opa proteins may contribute to the intracellular survival of

% gonococci by binding host cell pyruvate kinase in order to acquire intracellular

ffi pyruvate, which is required for growth.

1-1
1-1

I— I

Lipooligosaccharide

g LO S is an amphipathic glycolipid that constitutes a significant proportion

^ of the N. gonorrhoeae outer membranes. In addition to differences in biosyn-

" thesis genes, the major difference between LOS and lipopolysaccharide (LPS),

£ which is prevalent among Gram-negative bacteria, is its lack of a repeating

£ O-antigen. LOS is composed of a lipid A moiety, a biantennary or trianten-

*L. nary core region composed of two L-glycero-D-manno-heptopyranose (hep-

tose) and two 2-keto-3-deoxyoctulosonic acid (KDO) residues, and variable

oligosaccharide side chains (Mandrell and Apicella, 1993; also see Fig. 7.4).

The lipid A moiety anchors the LOS molecule within the outer membrane

and consists of a dihexosamine backbone linked to four hydroxymeristic

acids. These are substituted on the 3' position by lauric acid. KDO molecules

are juxtaposed to the lipid A and one KDO serves as the site of the dihep-

tose addition. The heptose molecules serve as docking sites for short (6-10

sugar moieties) oligosaccharide addition(s). Additionally, the second heptose

NANA a 2 -> 3GlcNAc pl^ 3Gal pi-> 4Glcl -> 4Hepl->5KD0 2 -> Lipid A

T
l

GlcNAcl -^2Hep
3

PEA

Figure 7.4. A model of the sialylated LOS glycoform from N. gonorrhoeae strain 1291 is
shown. This glycoform is represented as containing the sialyl-lactosamine found on the
organism in the presence of CMP-NANA. Over 97% of gonococcal isolates express this
lactosamine-containing glycoform that is important as a ligand to the human
asialoglycoprotein receptor.

(which is not directly liked to KDO) bears an N-acetylglucosamine residue.

Oligosaccharide substitutions exhibit interstrain and intrastrain variability. g

Interconversion of LOS oligosaccharides occurs spontaneously and is §

dependent on the presence or absence of available substrates for, and the en- §

zymes involved in, LOS biosynthesis. Several genes involved in LOS biosyn- £

thesis have been identified. The Igt locus, which encodes the genes for syn- g

thesis of lacto-N-neotetraose (LNnT) and digalactoside moieties, has recently <

been described. Several of these genes contain a polyguanine region that, 3

in a manner similar to PilC phase variation, can result in frameshift mu- o

tations with concurrent loss (or gain) of certain oligosaccharide moieties m

available for LOS assembly. Phosphate, phosphoethanolamine or pyrophos- jj

phoethanolamine, and O -acetyl substitutions of the core region along with the £

addition of sialic acid to terminal galactose residues confer further variation o

to the LOS structure. jg

LOS oligosaccharide side chains terminate in epitopes that mimic oligo- K

saccharide moieties of mammalian glycosphingolipids (including P , i, and £

paragloboside), regardless of oligosaccharide chain length. This form of mo-
lecular mimicry not only provides the bacterium with a method of immune

avoidance but also allows the bacterium to use host-derived molecules that g

normally associate with the mimicked structure. The presence of a terminal S

LNnT epitope (Gal^l-4GlcNac^l-3Galygl-4Glc) on LOS, which mimics hu- %

man paragloboside, allows the gonococcus to invade the urethral epithelium *

of men by adherence to the asialoglycoprotein receptor (ASGP-R; see Harvey >

et al., 2001), and this may facilitate disease transmission by adherence to the 5

ASGP-R on human sperm (Harvey et al., 2000).

An analysis of N. gonorrhoeae strains demonstrates the predominance
of the LNnT epitope among gonococci (Campagnari et al., 1990). These

observations were confirmed in situ in that the LNnT epitope is selected
for in men in human volunteer studies and in men with naturally acquired
gonococcal urethritis. Schneider et al. (1995) further demonstrated that se-
lection of the paragloboside epitope significantly enhances the ability of the
gonococcus to cause disease in human volunteers. The predominance of a
paragloboside moiety may enhance gonococcal survival in two ways: first, by
avoidance of immune recognition by molecular mimicry of the LNnT, and
second, by an increased serum-resistance by sialylation of the terminal galac-
tose residue of the LNnT epitope. Thus, a lower infectious dose is required to
establish disease, because a greater proportion of the inoculum would survive
and proliferate.

The prevalence of the paragloboside moiety among N. gonorrhoeae is con-
sistent with the finding that most clinically isolated gonococci initially exhibit
S serum-resistance; however, this property is lost with subsequent subculture.

S This unstable form of serum-resistance is attributed to sialylation of the ter-

< minal lactosamine of the LNnT moiety present on LOS. LOS and LPS stimu-

hj late cytokine production, which is associated with cytotoxicity. Stimulation of

X cytokine production by gonococcal LOS may allow this bacterium to access

g subepithelial tissues while increased serum-resistance and the predominance

§ of human-like epitopes on the bacterium surface enhances survival. Sialyla-

^ tion of the gonococcus surface inhibits its ability to be phagocytosed by PMNs

% and may increase the survival of those gonococci that are phagocytosed. Sia-

E lylation of the gonococcus, however, significantly inhibits its ability to invade

g urethral epithelial cells (Harvey et al., 2001) and to initiate disease in human

3 volunteer studies (Schneider et al., 1996).

e Studies that explored the importance of LOS sialylation during infection

g and that used human volunteers and in vitro tissue culture models led to the

^ hypothesis that although LOS sialylation may protect extracellular organisms

" from complement-mediated killing, these organisms become desialylated

£ prior to internalization by host cells. Immunoelectron microscopy studies

£ of urethral exudates from men with gonococcal urethritis show that in vivo

El sialylation of gonococcal LOS Gal/? 1-4-GlcNAc residue occurs during human

infection and that although the majority of the LOS of intracellular gonococci

is sialylated, approximately 10% of LNnT-terminal LOS remain unsialylated

(Apicella et al., 1990). Studies by other groups later showed evidence that

gonococci with sialylated LOS are less invasive for immortalized tissue culture

epithelial cells than unsialylated gonococci (van Putten and Robertson, 1995) .

Conversion (by phase variation) of LOS to epitopes unable to be sialylated

allows the gonococcus to adapt to environmental changes associated with

progressive disease. LOS significantly contributes to the pathogenicity of the
gonococcus in that it not only functions as an adhesin but also facilitates
disease transmission, modulates bacterial invasion, and modulates disease
progression.

Additional virulence factors

Through coevolution with its sole human host, the gonococcus has devel-
oped an impressive repertoire of gene products that allow it to exert redundant
mechanisms by which it is able to infect, colonize, and proliferate within the
hostile and benign microenvironments of the human body. Completion of
the N. gonorrhoeae (strain FA1090) genome project and continued research
will undoubtedly result in the identification of (as yet unknown) additional
factors that contribute to gonococcal virulence. Several researchers have de-
scribed factors that may contribute to virulence; however, their actual role in g
human disease is less well defined. §

Many mucosal pathogens escape immunological killing by their abil- §

ity to produce, and secrete, proteases that inactivate IgAl and its secretory £

counterpart (slgAl), the principal antibody isotype of the mucosa and mu- g

cosal secretions. Cleavage of these antibodies within their hinge region yields <

Fab a monomeric fragments that are separate from their effector Fc a counter- 3

parts; consequently, they are rendered inactive. Serine, cysteine, and metallo o

IgA proteases have been identified among those mucosal pathogens that pro- m

duce these endopeptidases. N. gonorrhoeae, N. meningitidis, and Haemophilus jj

influenzae produce serine proteases that cleave the IgA hinge region between £

proline and serine residues (i.e., type 1 activity) or between proline and thre- o

onine residues (i.e., type 2 activity). All N. gonorrhoeae and N. meningitidis jg

strains examined to date have been found to constitutively express either a K

type 1 or a type 2 IgAl protease. In gonococci the type of IgAl protease pro- 2

duced correlates with auxotype and the serovar of porin produced. Gonococci

that possess a nutritional requirement for arginine, hypoxanthine, and uracil «

(i.e., AHU auxotype) and a P. I A class 1 or 2 porin serovar produce a type 1 s

IgAl protease. Type 2 IgAl protease activity is prominent in gonococci that S

are excluded from the AHU/P.IA1/2 serotype. %

The role of neisserial IgAl protease activity in disease processes is unde- *

termined. It is generally presumed that inactivation of potentially bactericidal >

IgAl by a mucosal pathogen potentiates colonization and, therefore, potenti- 5
ates disease caused by IgA protease-producing microbes. However, infection
with N. gonorrhoeae IgAl null mutants in human volunteer studies resulted in

infection and disease indistinguishable from that observed with the parental
wild-type strain. Analysis of cervical secretions yielded similar findings in
that the level of IgAl protease activity in samples obtained from women with
gonococcal cervicitis were comparable with those obtained from women who
were not infected. These observations suggest that IgAl protease does not
significantly influence colonization of the (uro) genital epithelia.

In epithelial cells, cleavage of lysosome-associated membrane proteins 1
(LAM PI) with concurrent phagosome modulation has been proposed as an
alternative function for IgAl protease activity. However, LAMP1 of profes-
sional phagocytes are resistant to (gonococcal) IgAl protease activity. Within
professional phagocytes LAM PI are heavily glycoslyated; consequently, it is
thought the hinge region is protected from protease cleavage. These studies
suggest that IgAl protease activity is limited to epithelial cells. This apparent
S conundrum pertaining to gonococcal IgAl protease activity has to be exam-

S ined further before a role for IgAl protease activity in gonococcal pathogen-

< esis can be resolved.

hj The ability of pathogenic organisms to sequester iron from their host

X animal is well established to contribute significantly to a diseased state. The

g gonococcus is no exception, and gonococci possess multiple mechanisms by

§ which they are able to scavenge iron (Schryvers and Stojiljkovic, 1999) . Neisse-

^ via do not produce siderophores; however, they do produce siderophore recep-

% tors that are homologous to siderophore receptors of other bacterial species.

E Expression of siderophore receptors allows N. gonorrhoeae to use siderophores

g produced by other bacterial species with which they may share an ecological

Jj niche. In addition to siderophore receptors, most pathogenic Neisseria also

E produce receptors for transferrin (Tf), lactoferrin (Lf), hemoglobin (Hb),

g heme, and haptoglobin-hemoglobin (Hp-Hb). Lf is found in higher concen-

^ tration on mucosal surfaces than is Tf and, therefore, was presumed to be the

" preferred iron source for pathogenic Neisseria spp. However, some gonococci

£ do not produce receptors for Lf, and mutants defective in Lf acquisition retain

£ their virulence in human volunteer studies (Cornelissen et al., 1998).

|z;

El In contrast, however, gonococci deficient in Tf acquisition are avirulent

in human volunteer studies (Cornelissen et al., 1998), suggesting that Tf is
required for gonococcal colonization of (at least) the male urethral epithelium.
The ability of HmbR (the heme and Hb receptor), HpuAB (the Hb and Hp-
Hb receptor), and TonB mutants to grow in the presence of heme as a sole
iron source has presented the idea that heme may passively diffuse across
the gonococcal outer membranes as a result of its hydrophobic character.
Alternatively, porin may facilitate heme uptake (Schryvers and Stojiljkovic,
1999). However, recent data indicate that PilQ may serve a dual function,

allowing pilin transport (with subsequent pilus assembly) out of, and heme
passage into, the bacterial cell body (Chen et al., 2002).

The ability of gonococci to use heme, Hb, and Hp-Hb has been postu-
lated to be responsible for the increased risk observed in women to develop
PID and DGI during menses. However, fluctuations in hormone levels and
associated changes to the female reproductive tract may also be responsible
for the increased risk of complicated gonococcal infection with menses. En-
vironmental fluctuations may allow gonococci of a particular isotype to gain
access to subepithelial tissues or ascend the female genital tract and, thereby,
initiate DGI or PID, respectively.

Interaction of the gonococcus with the lutropin receptor (LHr) is sug-
gested to confer a contact-inducible phenotype to this bacterium that in-
creases its invasive character, thereby allowing it to invade epithelia by ad-
herence to the LHr (Spence et al., 1997). Identification of a LHr-gonococcus £
interaction involved the use of several immortal cell lines. Because LHr is 2
upregulated in immortal cells and because ligand binding downregulates §
LHr surface expression, the significance of a LHr-gonococcus interaction in §
vivo within the lower female genital tract (e.g., the ectocervix and endocervix >
and the distal endometrium) remains to be determined. Gorby et al. (1991) g

demonstrated that gonococci bind to the LHr on nonciliated cells in FTOC, <

suggesting that the LHr may be paramount to colonization of the fallop- %

ian epithelia. The presence of the LHr on human uterus, placenta, decidua, o

fetal membrane, and fallopian tube tissues, in conjunction with increased m

expression of this receptor during menses, suggests that a gonococcus-LHr jj

interaction could facilitate ascending infection in women. £

s
Although speculative, a gonococcus-LHr interaction occurring on de- o

cidua and placental membranes could potentially result in severe compli- jg

cations and may, in part, contribute to the increased risk of spontaneous K

abortion associated with N. gonorrhoeae infection. The inhibitory effect of 2

human chorionic gonadotropin (hCG), a ligand for LHr, on the observed

LHr-gonococcus interaction suggests that gonococci possess an unidenti-
fied surface molecule that mimics hCG; however, the gonococcal LHr ligand s

has yet to be identified. The interaction of the gonococcus with the LHr ap- S

pears to be a significant factor in colonization of fallopian tube tissue and %

may pose a serious risk to a developing fetus in pregnant women. *

Several gonococcal gene products appear to be induced under conditions >

of limited oxygen. Because the oxygen tension is presumed to be low in 5

some sites of gonococcal infection (and under certain circumstances), the
contribution of these gene products, such as AniA (or Panl; see Householder
et al., 1999), Pan2, and Pan3 (Clark et al., 1987) to progressive infection and

h-l

disease cannot be overlooked. Currently, however, there is little or no evidence
to confirm or negate the contribution of these anaerobically induced proteins
to the pathogenesis of N. gonorrhoeae in males or females.

COMPLEMENT AND PATHOGENIC NEISSERIA

Coevolution of the human C system with potentially pathogenic micro-
organisms has allowed several human pathogens to adapt mechanisms by
which they are able to avoid C'-mediated eradication (Vogel and Frosch, 1999) .
Methods evolved by microorganisms that allow them to escape the potentially
lethal effects of C activation include the following: (1) development of an an-
tiphagocytic capsule; (2) the development of surface structures that inhibit
C3-convertase activity; (3) evasion of the lytic activity of the membrane at-
tack complex (MAC), mediated by cell surface structures; (4) mimicry of C'
3 components; and (5) exploitation of C' components to facilitate infection.

< The pathogenic Neisseria, that is, N. meningitidis and N. gonorrhoeae, are strict

hj human pathogens that remain prevalent in the general population, in part

X because of their exquisite capability to avoid host defense mechanisms. In

§ addition to the generalized defense mechanisms of phase and antigenic varia-

§ tion, pathogenic Neisseria have evolved highly specialized immune avoidance

£ mechanisms that allow them to persist within their (sole) human host.

% N. meningitidis (the meningococcus) produces a capsule composed pri-

E marily, or entirely, of sialic acid. The presence of an extracellular capsule

g prohibits the interaction between cell-wall-deposited opsonins and host cells

Jj by providing a physical barrier impermeable to the phagocyte; that is, it is

e antiphagocytic. Meningococci of serogroup B possess a sialic acid capsule

g that is structurally similar to neural cell adhesion molecules (NCAM). This

^ NCAM-like capsule confers further protection to serogroup B meningococci

" by rendering these bacteria nonimmunogenic. Sialic acid is widely distributed

£ upon host cells and thus is recognized as an indigenous (or nonactivating)

£ surface. In the absence of an effective antibody response (as already noted),

*L. direct recognition of a potential pathogen becomes critical to bacterial clear-

ance. Within the alternative pathway (AP) of the C system, the presence of
sialic acid on a target surface favors binding of factor H (f H) (with C inactiva-
tion) over binding of factor B (f B), which would otherwise result in activation
of the C system.

Although N. gonorrhoeae does not possess a capsule, it does share with
the meningococcus the ability to sialylate its LOS. Gonococci that bear sialic
acid moieties on their LOS have been found in cell-free systems to be resis-
tant to C'-mediated killing; that is, they are serum-resistant. Serum-resistance

conferred by sialylated LOS is attributed to the ability of LOS to bind a specific
site within short consensus repeats (SCRs) 16-20 of fH (Ram et al., 1998).
This results in C inactivation through the cofactor activity of f H in factor I (fl)-
mediated inactivation of C3b. Because gonococci are not capable of producing
cytosine S'-monophosphate N-acetylneuraminic acid (CMP-NANA, the pre-
cursor for sialic acid synthesis), sialylation of a gonococcal LOS requires the
use of an exogenous (i.e., host derived) substrate. In contrast, N. meningitidis
is capable of synthesizing its own sialic acid and, therefore, incorporates en-
dogenous sialic acid into its capsular and LOS structures. Significantly less
f H is deposited upon capsulated meningococci when compared with strains
that lack a capsule (Estabrook et al., 1997), suggesting that other factors may
play a role in fH deposition on encapsulated organisms. An additional role
for sialic acid has also been elucidated. The presence of sialic acid moieties
can lead to a decrease in C9 deposition (Jarvis, 1995) upon the gonococcal
surface, thereby prohibiting MAC assembly and, thus, its associated ability g

to lyse these cells. §

Serum resistance of gonococci is also attributed to the expression of one §

of two isotypes ofporin, PI. A and Pl.B. Isotype PI. A is found more frequently jj

on gonococci in people with DGI. Expression of PI. A by the gonococcus g

is attributed to increased serum resistance in comparison with gonococcal <

strains that express the Pl.B isotype. Although porin is intimately associated %

with gonococcal LOS, binding ofporin to f H occurs independently of LOS, o

at a site distinct from that defined for sialic acid (Ram et al., 1998). Within m

the classical pathway (CP) of the C system, C4bp functions in an analogous jj

manner to f H of the AP. C4bp does not bind to the surface of sialylated £

gonococci; however, C4bp can bind gonococci when it is not sialylated (Ram o

et al., 2001). Binding of C4bp occurs on gonococci that express a Pl.B isotype. jg

Several studies have indicated that gonococcal killing is mediated primarily K

through the action of the CP; consequently, the ability of certain gonococcal 2
strains to directly bind C4bp, which allows fl-mediated inactivation of C4b

deposited on the gonococcal surface, may be critical to their survival within

their human host. g

In vitro N. gonorrhoeae infection studies and an examination of clini- S

cally isolated N. gonorrhoeae reveal that inactive C3b (i.e., iC3b, in com- §

parison with active C3b) is predominant on the surface of this bacterium. *

iC3b opsonization allows the gonococcus to invade primary cervical epithe- >

lial cells in a process mediated by the action of gonococcal porin and pilus 5

(Edwards et al., 2002), and which occurs independently of the sialylation sta-
tus of the bacterium (Edwards and Apicella, 2002). Because endocytosis that
is mediated by CR3 occurs independently of a proinflammatory response,

internalization of the gonococcus within cervical epithelial cells may con-
tribute to the asymptomatic nature associated with gonococcal infection in
women.

The membrane-associated fl co-factor, CD46, may serve as a receptor
for gonococcal pilus on some cell lines (Kallstrom et al., 1997). Although the
physiological relevance of a CD46-pilus interaction on the urogenital epithe-
lium remains controversial, pilus binding to CD46 present on PMNs could
facilitate fl -mediated inactivation of C3b or C4b on an opsonized bacterium.
Another family of surface molecules expressed by the pathogenic Neisse-
ria is the Opa proteins. Opa proteins serve as ligands for members of the car-
cinoembryonic antigen-related family of cell adhesion molecules (CEACAM
or CD66). CEACAM molecules can serve as coreceptors for the leukocyte
integrins, including CR3 and CR4. Interaction of CEACAM with CD18 (the
S beta subunit shared by CR3 and CR4) is thought to inhibit the respiratory

S burst that occurs with ligand binding and phagocytosis by PMNs. This may

< promote the intracellular survival of PMN phagocytosed Neisseria.

^ An additional role for Opa proteins relies on their ability to bind heparin.

X Gonococcal binding of heparin by means of their Opa proteins increases the

g serum-resistance observed by these bacteria (Chen et al., 1995). Although

§ the mechanism by which serum-resistance is conferred has yet to be defined,

the ability of heparin to augment the regulatory role of vitronectin in the inhi-

rt bition of MAC assembly provides one explanation for the observed increased

ffi serum-resistance.

g Despite the redundant ability of the pathogenic Neisseria to evade C-

Jj mediated killing, the C system is still capable of eliciting an efficient an-

e timicrobial defense. This becomes most evident in individuals with various

g C deficiencies that are prone to recurrent bacterial infections, most notably

^ with Neisseria spp. Individuals deficient in C components of the MAC exhibit

" an increased incidence of neisserial infections. In particular, deficiencies of

£ C5, C6, C7, and C8 lead to a much greater risk for recurrent, and commonly

£ systemic, infection with Neisseria spp. Despite the increased risk for systemic

*L. neisserial infections, a decreased mortality associated with infection is also

observed and is thought to be attributable to a decrease in endotoxin released
in the absence of a functional MAC.

People who are deficient in C9 are also at a much greater risk for Neisseria
spp. infection. This has led to the suggestion that the bactericidal action of
normal human serum is critical to controlling Neisseria spp. infection. Sim-
ilarly, greater than 50% of people who lack the AP components fB, factor D
(fD), and properdin exhibit recurrent neisserial infections. Studies focusing
on neisserial serum resistance have primarily involved cell-free systems in

ular host cell target and microenvironment.

Infection of the male urethra

which serum-resistance has been measured as the ability of these organisms
to survive the lytic action of pooled human serum. However, mucosal mem-
branes serve as the primary site for neisserial infection. Increasing evidence
suggests that AP C' components are produced by epithelial cells, albeit at a
much lower level than is observed in human serum. Therefore, the ability
of the pathogenic Neisseria to survive within their human host may reside in
their ability to evade AP ([/-mediated killing at the level of the mucosal ep-
ithelium with a subsequent transient carrier-like state. Progressive infection
with dissemination into areas of the human body where specific antibody
and C serum concentrations would far exceed those observed at the mucosal
epithelium may lead to C -mediated Neisseria eradication.

THE GONOCOCCUS AS AN INTRACELLULAR PATHOGEN ^

Despite the historic prevalence of N. gonorrhoeae within the human pop- g

ulation and the considerable severity and frequency with which adverse com- §

plications accompany disease (most notably in women), it has only been in §

recent years that we have begun to understand gonococcal pathogenesis as it >

occurs within its (sole) human host. Discernment of gonococcal pathogene- g

sis has been hindered by the lack of a good animal model by which to study <

the pathology of N. gonorrhoeae infection. Although several animal models %

have been described, no animal model exists that mirrors the full spectrum o

of human disease caused by the gonococcus. Consequently, researchers have m

relied on the use of human volunteers, tissue and organ cultures, and im- jj

mortalized or malignant tissue culture cell lines by which to study gonococcal £

pathogenesis. Microscopic analyses of clinical biopsies and male urethral ex- o

udates have allowed extrapolation of successful gonococcus infection as it jg

occurs in vivo with the realization that the gonococcus exists as an intracel- K

lular pathogen. However, more recent studies indicate that the mechanisms 2
used by the gonococcus to obtain an intracellular status vary with the partiC-

>
t- 1
W

The development of a primary, human, male urethral epithelial cell cul- *

ture system (Harvey et al., 1997) has greatly enhanced analysis of N. gonor- >

rhoeae infection of the urethral epithelium. Within the urethral epithelium of 5

men, infection is thought to occur as a sequential process that is initiated by
attachment to and an intimate association with the targeted host cell that re-
sults in pedestal formation of the urethral cell membrane beneath an adherent

1-1
w

I— I

<
u

w
1-1
i-i
i— i

Q
w

i-j

Pi
w

Ph
i— i

Figure 7.5. Polystyrene beads coated with LOS isolated from N. gonorrhoeae bind to the
ASGP-R on human sperm as denoted by the arrow. This LOS glycoform harbors a
terminal lactosamine as shown in Fig. 7.4. The LOS-ASGP-R interaction between bacteria
and sperm may facilitate disease transmission.

gonococcus. Gonococci are internalized within vacuoles by actin-dependent
(Giardina et al., 1998) and clathrin-dependent (Harvey et al., 1997) processes,
where they replicate prior to their release to the extracellular milieu. Epithelial
cells are subsequently shed. The intimate association observed between the
gonococcus and the urethral cell surface is mediated by the ASGP-R (Harvey
et al., 2001). This receptor binds to the terminal galactose of the gonococcus
LOS. Consequently, LOS sialylation greatly impairs the ability of gonococci
to associate with the male urethral epithelium. However, the presence or
absence of sialic acid on gonocococal LOS or the presence of the ASGP-R on
the cervical epithelium does not appear to contribute to lower female genital
tract colonization. The ASGP-R is also present on human sperm (Harvey
et al., 2000), suggesting that an LOS-ASGP-R interaction may also facilitate
disease transmission (Fig. 7.5).

Studies in fallopian tube explants

Similar processes to those observed with infection of the male urethra
have been demonstrated to occur in FTOC. In this model, however, gono-
coccal adherence occurs selectively on the nonciliated cells of fallopian tube
tissue (McGee et al., 1981). Adherence is mediated by the LHr in a contact-
inducible manner (Gorby et al., 1991). Following endocytosis of gonococci,
bacteria-containing vacuoles are trancytosed to the basolateral surface of the
infected epithelial cell, where they are released to the extracellular space. In
this manner invasive bacteria are able to access the subepithelial tissues.
Although the gonococcus selectively invades nonciliated cells, it is the ad-
jacent ciliated cells that exhibit gonococcus -mediated cytotoxicity and ulti-
mately results in the complete loss of ciliary action. The release of LOS and
peptidoglycan by the gonococcus is thought to facilitate cytotoxicty of cili- ^

ated cells. Cytotoxicity occurs either directly or indirectly by the induction o

of increased production of the inflammatory cytokine, tumor necrosis factor §

(TNF).

Infection studies of the uterine cervix

Analysis of clinical N. gonorrhoeae isolates obtained from the fallopian §

tube as well as FTOC infection studies suggest that gonococcal pili also facili- •

tate fallopian tube colonization. Additionally, gonococci that exhibit a translu- S

cent phenotype (Tr), and, therefore, lack Opa outer membrane proteins, are >

more invasive in FTOC. The prevalence of Tr gonococci in clinical isolates g

o
from the fallopian tube and in genital, blood, and joint fluid of DGI patients g

supports this observation. Expression of the LHr increases in an ascending g

manner from the endometrium to the fallopian tubes. Conversely, surface z

level expression of CR3 decreases progressively from the ectocervix to the g

upper female genital tract. Although it is currently not known if the expres- *

'■d

sion of CR3 within the cervical epithelia exhibits cyclic variation, synthesis of in

C3 and surface expression of the LHr is upregulated at the time of menses. g

In a concerted action these molecules may contribute to an increased risk of 2
complicated gonococcal infection.

>
t- 1
w

In contrast to the pathology of N. gonorrhoeae infection observed for the *

male urethra and the upper female genital tract, lower genital tract (i.e., the >

uterine cervix) infection does not provoke a proinflammatory response. Re- "

cent data suggest that this subclinical or asymptomatic condition may be
the result of the ability of the gonococcus to subvert the AP of the C sys-
tem. Primary cervical cells produce all of the alternative pathway C proteins

1-1
w

I— I

<
<

<
u

w
1-1
i-i
i— i

Q
w

i-j

Pi
w

Ph
i— i

Figure 7.6. Gonococci (denoted by arrows) elicit membrane ruffles upon their association
with the cervical epithelium. Membrane ruffling is the result of extensive host cytoskeletal
rearrangements and results in bacterial internalization within macropinosomes.
Membrane ruffles induced on ectocervical cells (left) exhibit a long ribbon-like
morphology; ruffles formed by endocervical cells (right) are spherical in nature.

required for C activation and its inactivation. C protein C3 is activated upon
N. gonorrhoeae infection to form C3b, which is deposited upon the lipid A
portion of gonococcal LOS and is rapidly inactivated to form iC3b (Edwards
and Apicella, 2002). The presence of iC3b on the gonococcal surface allows
binding to CR3 in a cooperative manner with gonococcal pilus and porin
(Edwards et al., 2002). Several studies demonstrate that ligand binding to
the I -(or inserted) domain of CR3 does not invoke a proinflammatory re-
sponse in immune cells. Therefore, the cooperative binding of iC3b, pilus,
and porin to this region of CR3 may contribute to the asymptomatic nature
of gonococcal cervicitis. Once the gonococcus adheres to CR3, a complex
signal transduction cascade is initiated that results in extensive cervical cell
cytoskeletal rearrangement. Large protrusions of the cervical cell membrane,
termed membrane ruffles (Fig. 7.6), engulf the bacterium, resulting in gono-
cocci internalization within macropinosomes (Fig. 7.7) in an actin-dependent
manner (Edwards et al., 2000).

The interaction of the gonococcus with human neutrophils

The gonococcus, being a strict human pathogen, exquisitely senses its
particular microenvironment (within its sole human host) and alters its phys-
iological response to that environment in such a manner as to promote its
own survival. This is evident by the finding that the interaction of the gono-
coccus with PMNs, which express CR3, occurs independently of a CR3 asso-
ciation (Farrell and Rest, 1990). The gonococcus-PMN association requires

Figure 7.7. A cross section of polarized cervical cells experimentally infected with N.
gonorrhoeae reveals that gonococci (GC) reside within spacious macropinosomes (denoted
by arrows). Gonococci have been labeled with a gold-bead antibody-conjugate to
gonococcal porin. A large membrane protrusion, indicative of a membrane ruffle (MR),
can be seen on the right.

the presence of Opa outer membrane proteins; however, pilin proteins are
not required. In contrast, gonococcal adherence to primary cervical and male
urethral epithelial cells occurs independently of Opa proteins but requires
gonococcal pilin.

Opa proteins are thought to confer distinct cellular trophisms to individ-
ual gonococci by the ability of different Opa proteins to differentially recog-
nize host cell surface molecules. Two broad classes of Opa proteins exist that
are represented by Opaso (i.e., Opa proteins that recognize host cell HSPG)
and Opa52 (i.e., Opa proteins that recognize CEACAM). The interaction of
Opa proteins with HSPG is dependent on the presence of vitronectin or fi-
bronectin (van Putten et al., 1998), which functions as a bridging molecule
between the gonococcus and its target receptor(s). Association with an in-
tegrin coreceptor (a v /3i or a w Ps for vitronectin-mediated adherence or a w P\

o
to

X
w

<
>

w
n

%
i— i

X
o

M
in

>
t- 1
w

for fibronectin-mediated adherence) triggers a signaling cascade within the
target cell that is dependent on the activation of protein kinase C (PKC).

A second, distinct, interaction between Opaso and HSPG is demonstrated
in Chang conjunctiva epithelial cells. Interaction of Opaso with HSPG initi-
ates a signaling cascade that activates phosphatidylcholine-dependent phos-
pholipase C (PC-PLC). Generation of diacylglycerol (DAG) in turn activates
acidic spingomyelinase (ASM), which results in ceramide production from
spingomyelin. Ceramide is speculated to modulate the cytoskeletal rearrange-
ments required for endocytosis of the cell-associated gonococcus.

Variable CEACAM isoforms are differentially expressed by a variety of
different cell types, including PMNs and epithelial cells. Activation of the host
cell Src tyrosine kinases, Hck and Fgr, results in the subsequent activation
of Rac, which results in epithelial cytoskeletal rearrangement and internal-
S ization of the gonococcus. Although a single, primary receptor has not been

S identified that modulates the gonococcus-PMN interaction, the presence of

< the C E AC AM , integrin, and H S PG receptors on PM Ns suggests that any or all

hj of these molecules may play a role in gonococcus phagocytosis. Additionally,

X binding of specific antibody to the gonococcus may facilitate phagocytosis by

means of Fey receptors.

s A COMPREHENSIVE MODEL OF N. GONORRHOEAE

< PATHOGENESIS IN MEN AND WOMEN

g The male urethra and the female uterine cervix are the primary sites

Jj of the majority of gonococcal infections. These sites are illustrative of the

E varying environments the organism must face in its pathogenesis, as these

g are entirely different epithelial surfaces that are derived from distinct em-

^ bryological origins. The urethra in the male and female is derived from the

" initial division of the internal cloaca to form the urogenital sinus. The in-

£ termediate section of this sinus becomes the pars pelvina, which ultimately

£ becomes the female urethra, a portion of the vestibule and the distal vagina

*L. in the female, and the prostatic urethra in the male. The caudalmost portion

of the urogenital sinus becomes the membranous and penile portions of the
urethra in the male and a portion of the vestibule in the female. In contrast,
the uterine cervix is derived from the paramesonephric ducts. It is, therefore,
not surprising that the surface receptors interacting with the organism on ep-
ithelial surfaces of these structures are different. The gonococcus has adapted
very well to these different conditions; consequently, it is not surprising that
distinct pathogenic mechanisms occur with N. gonorrhoeae infection in men
and women.

N. gonorrhoeae possesses an impressive array of mechanisms that allow
it to gain entry into host cells and to evade the host immune response. The
importance of virulence factors such as pilus and Opa adhesins in gonococ-
cal association with host cells is well established both in vitro and in vivo.
In addition, although gonococcal LOS is implicated in the inflammatory re-
sponse associated with gonococcal infection, clinical studies support in vitro
evidence that gonococcal LOS plays a role in gonococcal adherence to and
invasion of host cells. LOS and LPS play a role in invasion of host cells by
other pathogenic bacteria. All of these virulence factors, which are located
on the surface of the gonococcus, are capable of phase or antigenic variation
or both. Because an association between the gonococcus and the host cell
surface is a prerequisite for entry, it is believed that pili, Opa, LOS, and porin
play an important role in the initiation of disease. Multiple studies show
that the process of adherence to, followed by the invasion of, host cells in-
volves the interaction of a repertoire of bacterial and host constituents. Given g
that gonorrhea is a sexually transmitted disease and reflecting upon what is §
known about gonococcal pathogenesis, assembling a model in the context of §
the male and female urogenital tracts may promote an understanding of this >
process at the level of human disease (Fig. 7.8). g

Gonococci enter and survive within cervical epithelial cells. Because the <

intracellular environment is a source of CMP-NANA, the model shown in %

i— i

Fig. 7.8 presumes that gonococci with sialylated LOS are released from o

cervical epithelial cells to the extracellular environment. LOS sialylation is m

mediated by gonococcus-encoded sialyltransferase, but with host-derived jj

CM P-NANA. Cooperativity between the gonococcus and other microbes that £

produce sialidases in the vaginal microflora results in desialylation of gono- o

coccal LOS. Loss of the sialic acid moiety on gonococcal LOS, while in res- jg

idence within the lower female genital tract, may prime the gonococcus for K

infection of the male urethra (where the presence of a sialic acid moiety 2
would inhibit urethral interaction with ASGP-R). Upon transfer to the male

partner, these desialylated gonococci can interact with the ASGP-R through

the now available lactosamine residue on the nonreducine end of the LOS g

>
oligosaccharide. This initiates clathrin-mediated endocytosis of the organism, S

facilitating entry into the urethral epithelial cell. §

Experiments examining the mechanism of gonococcal internalization *

involving LOS and the ASGP-R show that, although the number of ASGP- >

Rs at the epithelial cell surface increases after 4 h following infection with 5

terminal lactosamine-expressing N. gonorrhoeae, no difference in the levels
of total cellular ASGP-R protein or ASGP-R mRNA could be detected. This
suggests cellular ASGP-R traffics to the cell surface during infection rather

than being newly synthesized. Considering these findings together with what
is known about the ASGP-R, one can propose a model in which extracellular
gonococci bind ASGP-R and are internalized in clathrin-coated vesicles.

Clathrin-coated vesicles, containing receptor-ligand complexes, fuse
with other clathrin-coated vesicles. In this early endosome, the clathrin coat
is shed in an ATP-dependent process, and a pH drop results in uncoupling
of the receptor-ligand complex. The ASGP-R is then recycled back to the cell
surface where it is again available for binding ligand. In addition to the recy-
cling back of ASGP-R to the cell surface in the absence of ligand, evidence is
presented that a large fraction of ASGP-R is recycled to the cell surface prior
to ligand dissociation, a process termed diacytosis, which may play a role in
potentiating gonococcal urethritis.

Signaling mechanisms that are required for recruitment of cellular
ASGP-R to the cell surface, whether or not fusion of gonococci-containing
vesicles occurs in vivo, and the intracellular fate of the gonococcus remain to g

be determined. Human volunteer studies, in vitro invasion assays, and mi- §

croscopy studies suggest that gonococci survive intracellularly. Lysosomes are §

considered the endpoint of ASGP-R-mediated endocytosis, but it is not known jj

if gonococci internalized by means of ASGP-R escape lysosomal degradation. g

Pathogenic Neisseria species secrete an IgA protease, which cleaves host cell <

LAM PI. LAM PI is involved in maturation of the endosome; consequently, %

the ability of the gonococcus to cleave this molecule is believed to contribute o

to gonococcal survival in the intracellular environment. m

° n

However, recent studies with a gonococcal IgAl protease mutant showed jj

that, compared to the wild type, this mutant was not compromised in its ability £

Figure 7.8. (facing page). The different pathogenic interactions of the gonococcus in men d

and women. In women, infection of the cervical epithelial cells CEC occurs by membrane o

ruffling, which is an actin- dependent process. Once within the epithelial cell, the §

organism acquires the substrate for sialylation and sialylates its LOS (•). The £

inflammatory response is minimal, and as cells are shed they are released into the vaginal 2

environment, which is rich in sialidases (\) produced by endogenous microflora that >

results in a desialylation of the LOS (•). Organisms passed to men are predominately ^

desialylated and capable of entering urethral epithelial cells (UEC) by the interaction of 2

the terminal lactosamine on the LOS and the asialoglycoprotein receptor on the urethral JJ

cell by a clathrin-dependent receptor-mediated endocytosis. Once within the cell, the >

organisms become resialylated. As cells are shed, organisms are released and "

phagocytosed by PMNs. Infected epithelial cells and PMNs are transferred to the female

in the ejaculate. Uptake by the cervical cell through CR3 is unaffected by the sialylation

state of the organism. Nonsialylated gonococci can also bind to human sperm and may aid

in infection spread. See color section.

to cause urethritis in human volunteers. Whether the organism can prolifer-
ate within the host cell is still a matter for study, although exfoliated urethral
epithelial cells in exudates from infected men show numerous organisms.
This suggests that gonococcus replication very likely occurs within host (ep-
ithelial) cells. Within urethral epithelial cells, host-derived CMP-NANA is
again available as a substrate for sialylation of gonococcal LOS. By an as yet
undefined mechanism, gonococci traffic to the surface of the epithelial cell
and are released from the surface. Once on the surface, these sialylated gono-
cocci cannot reenter urethral epithelial cells or interact with the ASGP-R on
sperm until the bacteria have divided in this CMP-NANA-free environment
and the terminal lactosamine of their LOS becomes free of sialic acid.

In males, gonococcal infection likely results in inflammatory cytokine
release by urethral epithelial cells. Few studies have addressed the question
S of epithelial cell signal transduction resulting from gonococcal adherence

S and invasion of these cells. However, in vitro studies using primary and im-

< mortal epithelial cells demonstrate that infection with N. gonorrhoeae elicits

hj the production of the cytokines, TNF-a, interleukin (IL)-6, and IL-1/3 and the

X chemokine, IL-8, which triggers PMN influx and significantly contributes to

g local inflammation within the male urethra. It is also shown that, in exper-

§ imental gonococcal infection in men, the cytokines, IL-6, IL-8, and TNF-a,

^ are released at the local site of infection, the urethral mucosa. This is in con-

% trast with findings observed in women with gonococcal cervicitis where local

E levels of IL-1, IL-6, and IL-8 are not elevated (Hedges et al., 1998). Therefore,

g the release of inflammatory cytokines by the cervical epithelium in response

3 to N. gonorrhoeae infection remains under question.

e In both males and females, gonococcal infection usually results in shed-

g ding of urethral and cervical epithelial cells, respectively. PMNs containing

^ gonococci are also found in exudates from males with gonorrhea. Similar

" to the urethral epithelial cells, PMNs could also serve as a source of inflam-

£ matory cytokines and CMP-NANA and may release viable gonococci to the

£ extracellular environment for disease transmission or reentry into urethral

^ epithelial cells following desialylation by host sialidases. An Opa-CEACAM

interaction on PMNs may allow survival of some intracellular gonococci, be-
cause engagement of CE AC AM initiates a priming signal within these cells
that results in activation of adhesion receptors without the release of inflam-
matory mediators or the induction of a respiratory burst.

The majority of gonococci that are transmitted from males to their female
partners have sialylated LOS. However, the presence or absence of sialic acid
on gonococcal LOS does not influence the association of the gonococcus
with the cervical epithelium (Edwards and Apicella, 2002). In this model of

cervical invasion, AP C components are produced by the cervical epithelium
and subsequently released to the extracellular milieu. Upon transmission of
gonococci to the lower female genital tract, C3 deposition occurs on the lipid A
determinant of gonococcal LOS. Expression of an LOS (as opposed to an LPS)
structure may increase fH activity and, consequently, the conversion of C3b
to iC3b on the gonococcus surface. Similarly, the resemblance of gonococcal
LOS to human paraglobosides and glycosphingolipids (some of which serve
as synthetic precursors of cervical mucins) may favor C inactivation. The
interaction of gonococcal pilin with the I -domain may initially serve as an
anchor to CR3 that allows this bacterium to overcome repulsive forces that
occur with the host cell membrane and, thus, position the gonococcus at the
cervical cell membrane where the concentration of C components may be
expected to be sufficient to allow an efficient interaction with CR3.

Porin is very closely associated with LOS on the gonococcal surface, and
its role in CR3-mediated endocytosis may be multifactorial; however, the di- g

rect association of porin with the I-domain of CR3 is crucial to cervical cell §

invasion. The juxtaposition of porin to LOS within the outer gonococcal mem- §

brane may spatially favor porin and iC3b adherence to the CR3-I-domain. >

Additionally, pilin- and porin-mediated binding of the gonococcus to CR3 g

may elicit a biphasic calcium flux in cervical epithelial cells that increases <

the affinity and avidity of iC3b-, porin-, and pilin-mediated CR3 adherence, %

as well as the release of additional effector proteins that might augment the o

eonococcus-CR3 interaction. m

° n

Membrane ruffling in professional phagocytic cells is attributed to the jj

activity of the Rho family of GTPases. Although ruffling has not been pre- £

viously observed in response to CR3 activation, similar responses probably o

occur within the cervical epithelium in that the inhibition of Rho activation jg

also inhibits invasion of cervical epithelial cells. Jones et al. (1998) recently K

reported that fMLP- and Fey coreceptor-induced stimulation of CR3 results 2
in the activation ofp21 -activating kinase (PAK1). Activation of PAK1 can elicit

lamellipodia and ruffle formation, as well as vinculin accumulation, through

a signaling cascade involving the PAK-interacting guanine nucleotide ex- s

change factor (PIX) and Rac. The ability of porin to inhibit fMLP-induced S

signaling events in neutrophils suggests that these molecules may share §

effector proteins; however, it is unknown if binding of gonococcal porin to *

CR3 elicits membrane ruffling in primary cervical cells by augmenting signal >

transduction of PIX and PAK1.

Binding of CR3 triggers a cascade of events that may involve the activation
of PIX and PAK1 and that results in ezrin and vinculin focal complex forma-
tion and the cytoskeletal changes required for ruffle formation. Engagement

t- 1

of CR3 by the gonococcus is sufficient to elicit membrane ruffling; however,
other fluid phase molecules may facilitate gonococcal internalization. Once
membrane ruffles are formed, endocytosis ensues, allowing gonococcal inter-
nalization within macropinosom.es. These ideas are consistent with the wide
variability associated with the incubation period, the clinical manifestations,
and the high prevalence of asymptomatic gonococcal cervicitis in women.
Clearly, coevolution with its human host has allowed the gonococcus to care-
fully orchestrate a mechanism of CR3 adherence in which constituents of its
outer membrane, that is, porin and pilus, function cooperatively with host
cell factors (i.e., AP proteins) to ensure its increased survival.

The deposition of iC3b on the gonococcal lipid A core may confer a
survival advantage to the gonococcus by displacing the LOS oligosaccharide
chain such that it is unable to bind to the lectin-binding domain of CR3.
S iC3b-mediated adherence to CR3 occurs independently of a proinflamma-

S tory response; however, binding of the lectin domain stimulates a respiratory

< burst through the generation of a second signal within the eukaryotic (host)

hj cell. Consequently, a CR3 association mediated through the lectin domain

X of this receptor may result in decreased survival of the invasive organism.

g Additionally, the presence of iC3b on the lipid A core may obstruct the asso-

§ ciation of the lipid A core region with CD 18. It, therefore, appears that C3b

^ deposition on lipid A facilitates gonococcal infection and may promote bac-

% terial survival in that any or all of these phenomena would favor adherence to

E the I -domain of CR3 and subsequent bacterial internalization in the absence

g of a respiratory burst. iC3b-mediated epithelial adherence, in the absence of

Jj a respiratory burst, may allow the gonococcus to attain a carrier-like state

e within the cervical epithelium. Ascent to the upper female reproductive tract

g may subsequently occur as the result of hormonal changes that alter host

^ cell epithelia, molecules available for gonococcal use, or the expression of

" gonococcal virulence factors. These ideas are supported by the increased risk

£ to women for PID and DGI that is associated with menses and with the cor-

£ relation made between the presence or absence of Opa proteins and the site

*L. of gonococcus isolation. (Transparent gonococci are predominantly isolated

from the cervix at the time of menses and from the fallopian tubes; from

asymptomatic men; and from blood, joint, and genital fluids of DGI patients.

Opa-expressing gonococci are predominantly isolated from men and from

the cervix at the time of ovulation.)

Surface level expression of CR3 decreases progressively from the ecto-
cervix to the upper female genital tract. Conversely, expression of the LHr
increases in an ascending manner from the endometrium to the fallopian
tubes (where it serves as a receptor for gonococcal adherence; see Gorby et al.,

1991). Although it is currently not known if the expression of CR3 within the
cervical epithelia exhibits cyclic variation, synthesis of C3 and surface ex-
pression of the LHr is upregulated at the time of menses. In a concerted
action, these molecules may contribute to an increased risk of complicated
gonococcal infection.

The level of sialic acid found within the cervical milieu exhibits cyclic
variation and is inversely related to C3 production. Additionally, although
sialidase activity is present within cervical mucus throughout the menses
cycle, cyclic variation also exists with the ability of this enzyme to use endoge-
nous or exogenous substrates. With regard to the increased susceptibility to
ascending gonococcal infection correlated with menses, the decreased preva-
lence of sialic acid within the cervical milieu at the time of menses might
suggest that sialylated bacteria are impaired in their ability to ascend the fe-
male genital tract, although no discernable difference exists in the ability of
sialylated or unsialylated gonococci to interact with cervical epithelia. How- g

ever, removal of sialic acid from the mucosal surfaces of the female genital §

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i-i

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<
X

i-i
i-i
i— i

Q
w

i-i

Pi
w

Ph
i— i

!zi

CHAPTER 8

Group A streptococcal invasion of host cells

Harry S. Courtney and Andreas Podbielski

Group A streptococci (GAS), Streptococcus pyogenes, are beta-hemolytic, Gram-
positive, pyogenic cocci that usually grow in chains. S. pyogenes is classified as
group Abased on serological reactions with its C carbohydrate, which consists
of polymers ofrhamnose substituted with N-acetylglucos amine (Lancefield,
1933). For epidemiological purposes, GAS are further classified into more
than 100 different types based on serological reactions with the variable do-
mains of M proteins, or, more recently, based on 5' emm gene sequences
(Beall et al., 1996). Other typing schemes based on serological reactions with
serum opacity factor, T proteins, and R proteins are also used (Johnson and
Kaplan, 1993).

S. pyogenes is almost exclusively associated with humans and commonly
causes a variety of diseases, including pharyngotonsillitis, impetigo, scarlet
fever, and more severe infections, such as puerperal sepsis, myositis, necro-
tizing fasciitis, and toxic shock syndrome. Among several of the nonsuppu-
rative complications of group A streptococcal infections are acute rheumatic
fever and acute glomerulonephritis, which are usually preceded by infections
of the throat and skin, respectively. These sequelae are thought to be due to
autoimmune T- and B-cell responses induced by streptococcal products. Ac-
cumulating evidence also suggests that group A streptococcal infections may
lead to other autoimmune diseases, such as obsessive compulsive disorders,
or they may exacerbate others such as guttate psoriasis (reviewed by Cun-
ningham, 2000) .

ADHESION: PRELUDE TO INVASION?

To establish these infections, the streptococcus must first attach to the ep-
ithelium of the host. This attachment is accomplished by specific interactions

1-1
w
I— I
PQ
Q
O
Ph

<
W

<
Q

<
w

Pi
<

Figure 8.1. An electron micrograph of streptococcal adhesion to a human buccal
epithelial cell.

between surface structures of the streptococcus (adhesins) and receptors on
host cells (reviewed by Courtney et al., 2002). Attachment of streptococci to
a human buccal epithelial cell is shown in Fig. 8.1. Note that the epithelial
cell has a ruffled surface that is due to convoluted ridges that cover its sur-
face and that appear as projections in electron micrographs of thin sections.
Similar ridges are found on the surfaces of human pharyngeal and tonsil-
lar epithelial cells (Stenfors et al., 2000). Streptococcal attachment to these
cells occurs primarily by interactions with receptors on these ridges, and ad-
hesion is often mediated by multiple interactions resulting in high-avidity
binding.

Early adhesion experiments demonstrated that streptococci did not at-
tach to all buccal cells but instead attached primarily to those cells coated
with fibronectin (Abraham et al., 1983; Simpson and Beachey, 1983). These
findings coupled with additional research confirmed that fibronectin was a
receptor for GAS (Courtney et al., 1986). Lipoteichoic acid (LTA) was the first
streptococcal adhesin to be identified that reacted with fibronectin (Beachey
and Ofek, 1976; Courtney et al., 1986). Subsequently, at least 24 other ad-
hesins have been described, many of which react with fibronectin or other

Table 8.1. Putative streptococcal adhesins-invasins and receptors

Adhesin-Invasin Receptor

References

C5a peptidase

C -carbohydrate

•

Collagen-binding

collagen

protein

Fba(FbaA)

FbaB

FBP54

Fn, Fgn

Galactose-binding

}

protein

G3PDH(orSDH)

Fn, Fgn, 30-kDa

protein

Hyaluronic acid

CD44

Lbp

Iaminin

Lsp

Iaminin

Lipoteichoic

acid

scavenger receptor,
CD14

M protein

CD46, galactose,
Fn, Iaminin
fucose/fucosylated
glycoprotein
sialic acid-containing
receptors

PFBP

Protein Fl/Sfbl

integrins

Cheng et al., 2002
Botta, 1981
Podbielskietal., 1999;

Visai et al., 1995
Teraoetal.,2001
Teraoetal.,2002
Courtney et al., 1996
Gerlachetal., 1994

Pancholi & Fischetti,

1992, 1997
Ashbaughetal., 2000;

Cywes et al., 2000;

Schrager et al., 1998;

Wessels and Bronze, 1994
Teraoetal.,2002
Eisner et al., 2002
Beachey and Ofek, 1976;

Courtney et al., 1986
Dunne etal., 1994

Courtney et al., 1994;

Okadaetal., 1994, 1995;

Perez-Casal et al., 1995;

Berkower et al., 1999;

Waostromauo Tylewska,

1982; Wang and Stinson,

1994

Ryan etal., 2001
Rocha & Fischetti, 1999
Hanski & Caparon, 1992;

Selaetal., 1993;

Molinari et al., 1997;

Talayetal., 1993
Ozeri et al., 1998;

Okadaetal., 1998

(cont.)

a
o

in
H
JO
M

H
O

o
n
n

<
>

I— I
O

X
o

on
H

M
M
M

Table 8.1. (cont.)

Adhesin-Invasin

Receptor

References

caveolin-1

Rohde et al., 2003

Fn-collagen interactions

Dinklaetal., 2003

Protein F2

Jaffe et al., 1996

Pullulanase

thyro globulin, mucin, fetuin

Hytonenetal., 2003

R28

•

Stalhammar-Carlemalm
et al., 1999

Sell, (SclA)

•

Rasmussen et al., 2000;

Scl2 (SclB)

•

Rasmussen & Bjorck,
2001; Whatmore, 2001;

l— i

Lukomski et al., 2001

1-1

SpeB

integrin

Stockbauer et al., 1999;

I— i
M

laminin, mucin,

fetuin,

Hytonenetal., 2001

Q
O

thyrogobulin

Sfbx

Jengetal., 2003

SOF

Kreikemeyer et al., 1995;

4h
<

Rakonjacetal., 1995

Vn-binding protein

Valentin -Wiegand et al.,

1988

28-kDa protein

Courtney et al., 1992

o
u

•

cytokeratin

Tamura & Nittayajarn, 2000

•

heparin sulfate

Duensing et al., 1999

Notes: Fn = fibronectin, Fgn = fibrinogen, Vn = vitronectin, PFBP = S. pyogenes
Fn-binding protein, FBP54 = Fn-binding protein 54, G3PDH = glyceraldehyde-
3-phosphate-dehydrogenase, SOF = serum opacity factor, Scl = streptococcal
collagen-like protein, Sfbx = streptococcal Fn-binding protein x, Lbp = laminin-
binding protein, Fba = Fn-binding protein a, FbaB = Fn-bindin protein B, and
Lsp = lipoprotein of S. pyogenes, ? = Unknown

components of the extracellular matrix (Table 8.1). It has been proposed that
adhesion of streptococci is a multistep process initiated by LTA, which facili-
tates the binding of a second adhesin, such as M protein or protein F (Hasty
et al., 1992). The secondary adhesin provides host-cell specificity and leads
to high-affinity attachment. The list of adhesins in Table 8.1 emphasizes the
fact that GAS utilize multiple adhesins to mediate attachment to host cells.
There are conflicting reports on some of these adhesins.

Figure 8.2. Scanning electron micrograph of GAS interacting with HEp-2 cells. M type 49
S. pyogenes (A and B) or its nra mutant (C-E) were added to monolayers of HEp-2 cells.
The attachment and internalization of streptococci are shown after 1 h (A) and 3 h (B) of
infection. The microvilli were in close contact with bacteria (A) and no significant
morphological changes were apparent in HEp-2 cells incubated with wild-type
streptococci (B). The nra mutant exhibited a similar interaction pattern after 1 h of
infection (C), whereas drastic changes were observed after 3 h of infection (D and E). The
nra mutant induced large invaginations and was found to enter one such invagination and
exit through another. No such alterations were noted with the wild-type parent.
(Reproduced with permission from Molinari et al., 2001.)

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Some investigators found a role for M protein-CD46 interactions or
hyaluronate-CD44 interactions in streptococcal adhesion to keratinocytes
(Table 8.1), whereas others reported that inactivation of a variety of viru-
lence factors, including M protein and capsule, did not decrease adhesion
or colonization (Darmstadt et al., 2000; Jadoun et al., 2002; Ji et al., 1998).

Although there is, as yet, no entirely satisfactory explanation for all of these

discrepancies, it is important to note that the findings for one serotype of

GAS cannot necessarily be extrapolated to all other serotypes. Moreover, not

all serotypes of GAS have the same array of adhesins and not all adhesins will

be expressed simultaneously (Mclver et al., 1995) . Which adhesin or adhesins

that will be used by a particular serotype of GAS will depend on its repertoire

of adhesin genes, on environmental signals, and on the receptors expressed

by a particular type of host cell.

Some, but not all, of the adhesins listed in Table 8.1 will initiate invasion

of the host cells by streptococci. Whether or not a host cell is invaded will

depend on the physiological status of the host cell and on the nature of the

adhesin-receptor interactions. For example, the adhesion depicted in Fig. 8.1

probably will not lead to invasion because most of the cells in the superficial

5 layer of the buccal epithelium are dead and ready to be desquamated. HOW-

w ever, GAS have been found to invade human tonsillar cells in vivo (Stenfors

g et al., 2000) and to invade many types of tissue culture cells (Table 8.1). An

example of adhesion that leads to internalization of streptococci is shown in

3 Fig. 8.2.

< PATHWAYS FOR INFECTION

h There are several possible pathways for infection that streptococci may

o follow once they have attached to the epithelium (Fig. 8.3). One outcome is

^ the persistence of the streptococci on the host epithelia. Under such circum-

g stances, the bacteria will eventually face the activation of nonspecific, innate,

ffi and specific host defense mechanisms. Such effects, as well as a potential

induction of bacterial "apoptosis," can result in the complete clearance of
the bacteria from the infected host; thus the self-limiting nature of the in-
fection. Alternatively, the individual may become an asymptomatic carrier
of the streptococci. Although the phenotype of streptococci within carriers is
not known, certain virulence factors are probably downregulated during car-
riage, resulting in less damage to the host and less activation of host defense
mechanisms (Podbielski et al., 2003).

A second pathway is the invasion of tissues and the metastatic spread
of the streptococci. This may be accomplished by the release of degradative
factors and toxins that can destroy both the intercellular substances and eu-
karyotic cells, resulting in the spreading of the bacteria to new anatomical
sites in the host. At the new sites, the bacteria are exposed to more favor-
able conditions and can multiply rapidly and continue to spread to adjacent
tissues.

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A third pathway that can occur during the infectious process is the inter-
nalization of the adherent bacteria by the host cells. In the intracellular space,
the bacteria will survive provided they can evade or inhibit the activation of
phagolysosomes. Thus, the intracellular space can be regarded as a sanctu-
ary where the streptococci can avoid the defenses of the host or antibiotics.
Indeed, the internalization of streptococci by host cells has been proposed as
an explanation for recurrent infections after antibiotic treatment (Osterlund
et al., 1997). Once inside the host cell, the streptococci probably downreg-
ulate degradative and lytic factors while upregulating "persistence" factors.
In response to a signal, as yet unknown, GAS will escape from their intra-
cellular sanctuary by inducing an apoptotic response in host cells. Another
form of invasion has been described for S. agalactiae, which can transverse
a polarized epithelium without altering its structural integrity (Kallman and

S Kihlstrom, 1997). However, no such transcytosis has been noted with GAS.

w There are varying amounts of supporting data for each of these pathways .

o That GAS initiate adhesion and persist at a local site for some time before

S being eliminated has been observed since the days of ancient medicine and is

£ regarded as a textbook standard (Bisno and Stevens, 2000). Similar observa-

* tions have long been made on the invasion of host tissues and the metastatic

§ spread of streptococci throughout the body of the host.

* However, the concept of an intracellular location and the intracellular
h persistence of GAS is relatively new and does not have extensive, supporting,
o clinical data. In fact, there are only three studies in which the intracellular

w presence of S. pyogenes was directly demonstrated in tissue specimens from

g humans (Osterlund and Engstrand, 1997; Osterlund et al., 1997; Podbielski

ffi et al., 2003). There are a number of studies in which clinical isolates of GAS

were tested for their in vitro capability to enter host cells, and even more groups
have demonstrated the ability of GAS to be internalized by cultured cell lines
(see Cleary and Cue, 2000, for review). The epidemiologic and experimental
investigations have focused, so far, on the effect of an intracellular location
on the persistence of GAS in upper respiratory tract infections. There is no
evidence that persistent and recurrent GAS infections of the skin, especially
of the lower legs, are associated with an intracellular location of the bacteria.

EXTRACELLULAR MECHANISMS OF TISSUE INVASION

As just described, streptococci can invade tissues and can be internalized
by cells within these tissues (LaPenta et al., 1994). Tissue invasion and inter-
nalization are not mutually exclusive and may occur simultaneously. We first
discuss the extracellular mechanisms utilized by GAS to invade tissues, and

then we discuss the mechanisms required for internalization. One way that
streptococci could invade tissues is to blast their way through the epithelial
barrier by secreting cytolytic toxins that induce apoptosis of the host cells,
thereby clearing a pathway to deeper tissues. Adhesion of streptococci to the
host cell would help to effectively deliver the toxin(s) to the desired target
(Ofek et al., 1990). Just such a mechanism has been described by Madden
et al. (2001). These investigators found that the M -protein-mediated at-
tachment of streptococci to keratinocytes was required for the insertion of
streptolysin O (SLO) into cholesterol-rich domains in the cell membranes.
The pore formed by SLO facilitated the delivery of streptococcal NAD-
glycohydrolase (SPN) into the host cell, resulting in the lysis of host cells.
There are several pathways whereby SPN may contribute to virulence. SPN
can cleave /3-NAD, resulting in a decrease in the intracellular pools of NAD

and an increase in nicotinamide, a vasoactive compound. SPN also forms £

cyclic ADP-ribose and has ADP-ribosylating activities (Stevens et al., 2000). g

Expression of both SLO and SPN were required for lysis of host cells. Interest- £

ingly, spn is located adjacent to slo, and these genes appear to be cotranscribed £

from a common promoter (Madden et al., 2001). o

Other factors, in addition to SLO and SPN, are involved in the lysis of °

host cells. Sierig et al. (2003) reported that both SLO and SLS (streptolysin fc

I— i

S) contributed to the lysis of keratinocytes. These investigators also found %

that expression of SLO, but not SLS, impaired the killing of nonencapsulated S

S. pyogenes by neutrophils. The reason why SLO did not impair the killing of *

encapsulated streptococci is not known. ^

Another form of extracellular invasion, termed paracellular translocation, ™

was recently described by Cywes and Wessels (2001). In this case, adhesion 2

to human keratinocytes was mediated by interactions between the hyaluronic w

capsule of S. pyogenes and CD44, an integrin expressed on several cell types
(for an integrin review, see Hynes, 2002) . The adhesion of encapsulated strep-
tococci induced membrane ruffling and disruption of intercellular junctions.
In some instances, the cell membranes curled back and lifted away from adja-
cent cells, exposing a passage for streptococci around the cells and into deeper
spaces. The soluble form of hyaluronate also induced similar responses in
host cells. It was suggested that the binding of hyaluronate to CD44 acti-
vated Racl, a member of the Rho family of GTPases, to phosphorylate a yet
unidentified protein that recruited ezrin, an actin linker protein. These inter-
actions triggered the cytoskeletal movements resulting in alterations of cell
morphology and intercellular junctions.

In addition to the membrane effects described herein, this activation
of the cytoskeleton also caused the formation of lamellipodia. Encapsulated

streptococci were found to primarily associate with these lamellipodia, but
such interactions did not lead to internalization of the streptococci. Interest-
ingly, an acapsular mutant did not induce such changes, and these strep-
tococci bound primarily to membrane sites devoid of the lamellipodia. The
fates of these two different phenotypes of streptococci were also different.
Instead of remaining extracellularly, the nonencapsulated streptococci were
internalized by the keratinocytes. It was concluded that the hyaluronic acid
capsule contributes to translocation by two mechanisms (Cywes and Wessels,
2001). In one, the hyaluronic acid capsule disrupts intercellular junctions and
thereby facilitates the passage of streptococci into deeper tissues. In the sec-
ond, the capsule prevents streptococci from being internalized and trapped
within epithelial cells. As a corollary, the acapsular mutant failed to be effi-
ciently translocated, because it became internalized and trapped within ep-
S ithelial cells. It is interesting to note that the acapsular mutant was more

w cytolytic than the encapsulated wild type, presumably because the acapsular

o mutant was internalized more efficiently.

S Another intriguing hypothesis concerning the role of ezrin and the strep-

£ tococcal inhibitor of complement (SIC) in the fate of streptococci that invade

* human tissues was recently proposed by Hoe et al. (2002). Inactivation of

§ the sic gene enhanced adhesion to host cells, suggesting that the SIC protein

x interfered with adhesion in some manner. SIC was found to rapidly enter

h epithelial cells and to react with ezrin and moesin. The mode of entry is not

o known, but perhaps the cytolysin-mediated transfer mechanism described

w by Madden et al. (2001) may have a role. Because ezrin, radixin, and moesin

g (ERM) are involved in the formation of lamellipodia, filopodia, and microvilli

<3 _

ffi through linkages with actin filaments (Bretscher et al., 2002), it was sug-

gested that the binding of SIC to ERM alters actin-mediated formation of
microvilli at the cell surface. The implication is that such disruptions may
also prevent receptor clustering at lamellipodia, which may be required for
efficient adhesion.

SIC was also found to impair phagocytosis of the Ml serotype by neu-
trophils. It was proposed that the ability of SIC to impede adhesion provided
a survival advantage within the host (Hoe et al., 2002). It is also possible that
SIC could have a role in the dissemination of streptococci. Adhesion may
occur while SIC is downregulated, and upregulation of SIC may release the
streptococci and enhance their ability to spread to other tissues within the
host or to other hosts. In this regard, it would be interesting to determine if
the application of SIC to animals that are colonized with S. pyogenes would
attenuate or enhance the virulence of the streptococci. It should be noted that
sic has been found only in Ml, M12, M55, and M57 serotypes (Akessonetal.,
1996; Hartas and Sriprakash, 1999).

Although the effects of hyaluronic acid and SIC on adhesion were dif-
ferent, their effect on internalization of the streptococci was the same, that
is, inhibition of uptake. In each case, the ERM proteins were found to be
intimately involved. It is not yet clear how hyaluronate recruits actin to help
in the formation of lamellipodia yet prevents actin from participating in the
uptake of streptococci. The effects of hyaluronic acid and SIC on epithelial
cells are similar to those seen in cells in which the gene for moesin was in-
activated (Speck et al., 2003), which also resulted in the loss of polarity and
intercellular junctions. In addition, these moesin-deficient cells were found
to detach and migrate. This finding suggests an intriguing possibility - that
the invasion of host cells by GAS could result in the detachment of a portion
of these cells and their migration, resulting in the spread of the internalized
streptococci to other tissues.

MECHANISMS FOR INTERNALIZATION OF STREPTOCOCCI BY
HOST CELLS

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Several different mechanisms have been described for the internalization o

o
n

of S. pyogenes by host cells . One of the most common mechanisms is that me- °

diated by adhesins that interact with extracellular matrix proteins (Table 8.1). fc

Our discussion is limited primarily to adhesins that are known to be involved %

>
in the internalization of the streptococci. Future research will undoubtedly S

reveal additional adhesins that contribute to the uptake of streptococci by *

host cells. ^

in
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Protein F/Sfbl p

Protein Fl/Sfbl is a large adhesin that is anchored to the bacterial cell
surface by an LPXXG motif. Although the first description of this protein used
the designation Sfbl (Talay et al., 1991), most publications have used protein
Fl, which is the designation that is used in the following sections. Protein Fl
promotes adhesion and invasion of host cells by interactions with fibronectin.
The binding of fibronectin to protein Fl involves two binding domains - a
repeating peptide domain that is immediately preceded by an upper binding
domain (UBD) . The effect of these domains on binding is sequentially exerted
(Talay et al., 2000). The second binding step appears to trigger a conforma-
tional change in fibronectin that exposes its cell attachment domain (RGD),
which is cryptic in soluble fibronectin (Tomasini-Johansson et al., 2001). The
exposed RGD domain of fibronectin can then interact with integrins on the
surface of the host cell and trigger internalization of the bacterium. Protein-
Fl -mediated invasion of host cells may also require interactions between the

UBD of protein Fl and the collagen-binding domain of fibronectin for the
optimal uptake of streptococci (Dinkla et al. 2003). Recently, an alternative
internalization mechanism involving protein Fl and caveolin-1 on the mem-
branes of host cells and the formation of omega-shaped cavities was identified
(RohdeetaL, 2003).

Although the in vitro data on the involvement of protein Fl in the in-
ternalization process are very convincing, the epidemiological data are not
as strong. Protein Fl is not an absolute requirement, because up to 48%
of the GAS strains do not carry a prtFl gene (Natanson et al., 1995). Some
investigators found an association between the expression of protein Fl and
internalization (Neeman et al., 1998), whereas others found no such associ-
ation (Brandt et al, 2001).

2 M proteins

I— I
pq

g M proteins are alpha-helical, coiled-coil proteins that are one of the major

w virulence factors of the organism and contribute to the ability of streptococci

£ to evade phagocytosis and to attach to host cells (reviewed by Fischetti, 1989;

5 Cunningham, 2000). A number of M proteins are adhesins (Table 8.1) and

§ at least two of the M proteins, Ml and M3, bind fibronectin (Schmidt et al.,

g 1993; Cue et al., 1998) . An M type 1 S. pyogenes bound fibronectin and invaded

h human lung cell cultures, whereas an M -negative mutant had a reduced ca-

o pacity to bind fibronectin and to invade these cells (Cue et al., 1998) . Laminin

was also found to promote invasion by the same M type 1 S. pyogenes, indicat-
% ing that a single strain can utilize multiple pathways that lead to invasion of

ffi host cells. It was suggested that invasion is mediated by interactions between

M protein and either fibronectin or laminin. The bound fibronectin then re-
acts with integrins on the cell surface that induce actin polymerization and
uptake of streptococci by a zipper-like mechanism (Dombek et al., 1999). The
M3 protein also mediates adhesion to HEp-2 cells and HaCat keratinocytes
(Berkower et al., 1999). The expression of the M3 protein led to an increase in
the invasion of HaCat cells, but the M3-protein-mediated invasion of HEp-2
was dependent on the presence of serum. Although it was not determined
what component of serum was responsible for this invasion, it is likely that
fibronectin will play a role similar to that found for Ml protein.

It is clear that certain M proteins that bind fibronectin can initiate adhe-
sion and participate in the invasion process. However, most M proteins do
not bind fibronectin. Do these M proteins contribute to invasion of host cells?
Only a few such types of M proteins have been investigated for their role in
the invasion process. The most widely investigated is the type M6 protein,

but the results and conclusions of different investigators varied consider-
ably. Fluckiger et al. (1998) found that inactivation of emm 6 had no effect on
streptococcal adhesion but dramatically reduced invasion of Detroit 565 pha-
ryngeal cells. However, a more common finding is that protein F is the major
component responsible for invasion and that the M6 protein contributes to
a lesser degree (Jadoun et al., 1997).

One of the ways that the M6 protein can contribute to invasion is to in-
crease the numbers of adherent streptococci, which can lead to an increase in
the numbers of streptococci that invade the host cells by a protein- F-mediated
mechanism. This possibility is supported by the findings of Okahashi et al.
(2003), who reported that inactivation of emm 6 reduced adhesion to mouse
osteoblasts by ~90% but only reduced invasion of these cells by ~ 35%. In
contrast, inactivation of prtf had no effect on adhesion but reduced invasion

by ~90%. In addition, the introduction and expression of emm6 in S. gor- £

donii led to an increase in adhesion but not invasion of HEp-2 tissue culture g

cells (von Hunolstein et al., 2000). However, the introduction and expression £

of emm 6 in Enterococcus faecalis led to an increase in invasion of host cells £

(Okadaetal, 1998). §

From the foregoing paragraphs, it appears that the background in which °

M proteins are presented can have a dramatic impact on its role in the inva- fc

I— i

sion of host cells. To evaluate the roles of several different M proteins in the %

same genetic background, Berkower et al. (1999) introduced emm3, emm6, S

and emm 18 into an isogenic M -negative and protein- Fl -negative mutant and *

tested the recombinant strains in adhesion and invasion assays. Their results ^

indicated that M3, M18, and M6 proteins do mediate adhesion to HEp-2 cells 3

and to HaCat keratinocytes. These findings are similar to those of Court- 2

ney et al. (1997a), who found that introduction of emm 18 and emmS into an w

M -negative strain restored adhesion to HEp-2 cells. However, the role of M
proteins in the invasion of host cells appears to depend on the type of host
cells and on the serotype of the M protein. Berkower et al. (1999) reported
that the expression of the M6 protein, but not M3 and M18 proteins, restored
the ability to invade HEp-2 cells. In contrast, expression of both M6 and M3
proteins, but not the M18 protein, led to invasion of HaCat keratinocytes.
The effect of serum on invasion was dependent on the M type. M6- and M 18-
protein-mediated invasion of HEp-2 cells was unaffected by serum, whereas
invasion by streptococci expressing the M3 protein was enhanced by serum.
These data indicate that the ability of M proteins to act as adhesins and in-
vasins can be dependent on M type; that is, it will depend on the variable
sequences in the N-terminus of the molecule (Fig. 8.4). Furthermore, strep-
tococci expressing different M proteins can bind to different receptors on the

1-1
w
I— I
PQ
Q
O
Ph

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W

<
Q

<
w

Pi
<

M-type-dependent adhesion

Hypervariable Domain

Variable Domain

M-type-independent adhesion

Conserved Domain

Cell wall spanning domain
LPXTG anchor motif

Figure 8.4. Schematic of M proteins and their role in adhesion and invasion of host cells.
M protein is the primary virulence factor of S. pyogenes and confers the abilities to resist
phagocytosis in blood and to attach to host cells. M protein is an a -helical, coiled-coil
protein that emanates from the surface of the bacteria. The number and sequence of the A
and B repeats will vary depending on the M type. The C repeats are conserved among
different M types. Adhesion to host cells can be mediated by either the variable domain or
the conserved domain, depending on the receptors that are expressed by host cells. Host
cells expressing CD46 can react with the C-repeat domain and mediate adhesion that is
independent of the M type. Adhesion and invasion mediated by the variable domain will
depend on the type of M protein expressed by the streptococcus and on the kinds of
receptors expressed by the targeted tissues. The binding of fibronectin to M proteins (and
therefore the internalization of streptococci by host cells) is mediated by the variable
domain of M proteins. (Reproduced with permission from Courtney et al., 2002.)

same host cell. Berkower et al. (1999) found that recombinant strains express-
ing the M6 protein did not inhibit adhesion-invasion of HaCat keratinocytes
by a recombinant strain expressing the M18 protein, implying that the M6
and M18 proteins bind to different receptors on these keratinocytes.

Frick et al. (2000) described another property of M proteins that promotes
adhesion and invasion. They found a 19 amino acid consensus sequence in
certain M and M-like proteins that promotes interbacterial aggregation and
the attachment of microcolonies to host cells. Such attachment also enhanced
invasion of host cells. A synthetic peptide copying this aggregative sequence
not only inhibited adhesion and invasion of host cells but also dramatically
increased the survival of mice challenged with M type 1 S. pyogenes.

FbaA

FbaA, originally termed OrfX (Podbielski et al., 1996), is a 38-kDa protein
with an LPXTG cell-anchoring motif (Terao et al., 2001). FbaA is expressed
by M serotypes 1, 2, 4, 22, 28, and 49, but not by other M serotypes or by

C5a peptidase

streptococcal groups B, C, and D. It has three or four proline-rich repeat do-
mains that are highly homologous to the fibronectin-binding repeats found
in FbpA. Similar to many other fibronectin-binding proteins, expression of
FbaA promoted the streptococcal invasion of HEp-2 tissue culture cells. Al-
though it was not determined how the binding of fibronectin to FbaA led
to invasion, it is likely that fibronectin served as a cross-linking agent as
described herein. A comparison of invasion of a multigene activator (Mga)-
negative mutant and a FbaA-negative mutant with the parental Ml strain
suggested that other surface proteins under the control of Mga may also be
involved. This finding is not surprising, because others have reported that
the Ml protein is under control of Mga and that it mediates adhesion and
invasion of host cells (see earlier paragraphs) . The FbaA-negative mutant was
also less virulent in a mouse model than its wild-type parent, suggesting that

FbaA is a virulence factor. £

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The scpA gene encodes the C5a peptidase, which degrades the chemoat- o

tractive complement factor C5a and thereby inhibits migration of neutrophils °

into an infected area. The C5a peptidase also binds fibronectin and is involved fc

in adhesion and invasion of host cells by group B streptococci (Cheng et al., %

2002) . Whether it functions as an invasin in GAS remains to be demonstrated. S

The scpa gene is cotranscribed with the JbaA gene. Transcription attenuation *

in GAS was first demonstrated in the scpa promoter (Pritchard and Cleary, ^

1996) and later in the downstream JbaA promoter (Podbielski et al., 1996). 3

There are no known stimuli that derepress this transcription attenuation. 2

FbaB and other RGD-containing proteins

FbaB is a newly described 79-kDa, fibronectin-binding protein found only
in M 3 and M 18 serotypes of S. pyogenes (Terao et al., 2002) . FbaB has a repeated
peptide that binds fibronectin and has homology to the fibronectin-binding
repeats of other streptococcal proteins. In addition, the N-terminal domain of
FbaB has ^45% homology with an N-terminal region of protein Fl, protein
F2, and Cpa. Inactivation of the gene for FbaB resulted in decreased adhesion
to and invasion of HEp-2 epithelial cells. The FbaB-negative mutant was
less virulent in mice-challenged IP, indicating that FbaB is another virulence
factor. Relative to its virulence, the FbaB protein was expressed mainly by type
M3 and M 18 S. pyogenes isolated from toxic-shock-like syndrome patients. No
expression of FbaB was found in pharyngeal isolates of M types 3 and 18.

h-l

These findings suggest that FbaB may contribute to the invasive potential of
GAS.

One interesting structural feature of this protein is that, in addition to
its fibronectin-binding repeat peptide, FbaB also contains the RGD peptide.
As already described, many of the adhesins interact with fibronectin, which
then interacts, by means of its RGD domain, with integrins on host cell mem-
branes. Thus, a streptococcal surface protein that contains the RGD motif may
be able to bind directly to integrins without the need for a cross-linking agent.
For example, a variant of SpeB that contained the RGD sequence is expressed
by a highly virulent clinical isolate of M type 1 S. pyogenes (Stockbauer et al.,
1999). This speB variant, called mSpeB2, was found to interact with human
integrins a Y p$ and ot\\hP?> and to mediate adhesion to host cells. However, it
is not known if the RGD sequence in FbaB or mSpeB2 has a direct role in
the invasion of host cells.
w Another RGD-containing protein of GAS is the Mac protein (Lei et al.,

o 2002). This protein was so named because of its homology to the human

S Mac-1 leukocyte integrin, but it has also been termed IdeS for its IgG de-

£ grading activity (von Pawel-Rammingen et al., 2002). The streptococcal Mac

* protein is secreted and is involved in the resistance of streptococci to phago-

§ cytosis. Variants of Mac that contained the RGD sequence were found to bind

x to different integrins and to block uptake of streptococci by neutrophils, but

h it is not known if the secreted Mac interferes with adhesion and invasion of

o host cells other than professional phagocytes.

< Alternative mechanisms for internalization

Invasion of host cells can also be triggered by interactions that do not
involve integrins or fibronectin. Such alternative pathways could be serotype
specific and involve the formation of large invaginations (Molinari et al.,
2000). For the majority of strains, the responsible mechanisms remain to be
elucidated. One potential bacterial receptor candidate is the Lsp (for lipopro-
tein of S. pyogenes) surface protein.

Lsp is a recently described surface molecule involved in adhesion and
invasion of host cells by GAS ( Eisner etal., 2002). Lsp is a member of the Lral-
lipoprotein family that is expressed by a variety of streptococci. Lsp was found
to bind directly to laminin, suggesting that laminin-Lsp interactions may
have a role in adhesion and invasion. Lsp-negative mutants neither attached
as well nor invaded host cells as well as the parental M49 strain. However,
the Lsp-negative mutant also demonstrated reduced binding of fibronectin.

Table 8.2. Responses of host cells to group A streptococci

Response of

M type

Host cells

host cells

References

keratinocytes

IL-la, IL-1/3,
IL-6, IL-8

Wang et al., 1997

prostagland in E 2

Ruiz et al., 1998

M24

HEp-2 cells

IL-6

Courtney et al., 1997b

HEp-2 cells

NF-/cB

Medina et al., 2002

M49, M52

keratinocytes

p defensin 2

Dinulos et al., 2003

M22, M54,

mouse

cathelicidin

Dorschner et al., 2001

M59

keratinocytes

A-549, HEp-2 cells

apoptosis

Tsaietal., 1999

human blood cells

CDlllb, ROS, I
CD62L

Saetre et al., 2000

human mononuclear

IL-1/3, IL-6, TNFcx,

Miettinenetal., 1998

cells

IL-12,
IL-18, IFN-y

Detroit 562, FaDu

phosphorylation

Pancholi & Fischetti,

cells

1997

HEp-2 cells

caspase 9, apoptosis

Nakagawa et al., 2001

M49

HEp-2 cells,

annexin V, caspases

Kreikemeyer et al.,

keratinocytes

2,3,7

2003

Because Lsp does not react directly with fibronectin, this suggested that, in
addition to Lsp, the expression of other surface proteins may have been altered
in the Lsp-negative mutant. Thus, the role of Lsp in streptococcal adhesion
and invasion remains to be clarified.

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RESPONSES OF THE HOST TO STREPTOCOCCAL ADHESION
AND INVASION

The attachment of GAS to host cells is known to stimulate responses from
host cells (Table 8.2) . For example, the adhesion of an M type 24 S. pyogenes to
HEp-2 cells resulted in the release of IL-6, whereas an isogenic, M24-protein-
negative mutant did not attach to these cells and failed to stimulate the release
of IL-6 (Courtney et al., 1997). Similarly, the adhesion of a type M6 S. pyogenes

to cultures of HaCat keratinocytes caused a dramatic increase in the release
of IL-la, IL-1/3, IL-6, IL-8, and prostaglandin E 2 (Wang et al, 1997; Ruiz
et al., 1998). In contrast, an M6 protein-negative mutant either caused no
such increase or did so at later time points.

Wang et al. (1997) reported that adherent streptococci caused injury to
keratinocytes that coincided with the release of cytokines. Subsequent re-
search by Ruiz et al. (1998) indicated that both SLO expression and adhesion
were required for the stimulation of IL-6, IL-8, and prostaglandin E 2 expres-
sion in HaCat keratinocytes. The role of SPN in the SLO-mediated stimulation
of the cytokines was not determined. Interestingly, expression of SLO had
no impact on the stimulation of IL-1 by adherent streptococci (Ruiz et al.,
1998), suggesting that other streptococcal products may stimulate the IL-1
response. Indeed, there are a number of streptococcal products including
S LTA, DNA, and a variety of streptococcal pyrogenic exotoxins that can evoke

w cytokine responses in the host. These responses are thought to be a central

o cause of morbidity and mortality associated with toxic shock syndrome and

S necrotizing fasciitis.

£ Streptococci can also stimulate phosphorylation of proteins in host cells.

* SDH (streptococcal dehydrogenase) was identified as one streptococcal pro-

§ tein that can initiate phosphorylation events in host cells (Pancholi and

x Fischetti, 1992, 1997). SDH is a 35.8-kDa glyceraldhyde-3-phosphate dehy-

h drogenase that is expressed on the surface of streptococci. It also has ADP-

o ribosylating activity and binds to a variety of mammalian proteins. SDH was

w found to react with 32-kDa membrane proteins in Detroit 562 and FaDu

g tissue culture cells derived from human pharyngeal carcinomas. The inter-

ffi action of SDH and M type 6 S. pyogenes with these pharyngeal cells stimulated

the phosphorylation of histone H3. SDH did not induce phosphorylation in
other tissue culture cells such as Chang cells, Chinese hamster ovarian cells,
and human epitheloid carcinoma cells. It was postulated that the induction of
tyrosine kinases and protein kinase C may regulate actin polymerization and
invasion of host cells by streptococci. The findings that genistein, a tyrosine-
kinase inhibitor, and staurosporine, a serine/threonine-kinase inhibitor, had
no inhibitory effect on adhesion, yet almost completely blocked invasion,
clearly indicate that phosphorylation of host proteins is required for invasion
(Pancholi and Fischetti, 1997).

The adhesion of streptococci induces other host cell responses, including
the production of IL-la andTNF-a (Table 8.2). Darmstadt etal. (1999) demon-
strated that, in addition to their proinflammatory role, these cytokines inhib-
ited the adhesion of S. pyogenes to keratinocytes. The mechanism whereby
IL-la and TNF-a blocked adhesion is not known.

The interaction of streptococci with host cells also enhanced the expres-
sion of the transcription factor known as nuclear factor kappa B (NF-/cB;
Medina et al., 2002; Okahashi et al., 2003). Adhesion induced an initial spike
of transcription, whereas internalization of the streptococci resulted in a sus-
tained NF-a:B response that was blocked by cytochalisin D. LTA is one of the
streptococcal components that can contribute to this response. LTA induced
the expression of NF-/cB and COX 2 expression in a human pulmonary cell
line, and this expression was blocked by genistein (Lin et al., 2002), again
indicating a role for tyrosine kinases in host cell responses to streptococci
and their products.

Bacterial interactions with host cells can also induce the expression of
antibacterial peptides, which is part of the innate immune defenses. There
are presently four main groups of antibacterial peptides: a-defensins, f5-

defensins, 0-defensins, and cathelicidins. GAS stimulated the production £

of cathelicidin in vivo in mouse keratinocytes (Dorschner et al., 2001). Cathe- g

licidin in the \-iuM range inhibited the growth of S. pyogenes, indicating that £

it has the potential to prevent or mitigate streptococcal infections. To eval- £

uate this potential, Nizet et al. (2001) compared the invasiveness of GAS in o

Cnlp -null mice and wild-type mice by using a subcutaneous model of in- °

fection. Cnlp encodes the antimicrobial peptide CRAMP, the mouse form fc

of cathelicidin. The lesions induced by S. pyogenes in wild-type mice were %

almost completely resolved by day 8 but were still readily apparent in Cnlp- S

null mice. These investigators also showed that a CRAMP-resistant strain of z

S. pyogenes caused greater necrosis than wild-type S. pyogenes in their sub- ^

cutaneous model of infection. These data indicate that cathelicidins are an 3

important part of the innate immune response to streptococcal infections of 2

the skin. w

GAS also induced, albeit poorly, the production of /3-defensin 2 in hu-
man keratinocytes (Dinulos et al., 2003). That S. pyogenes is a poor stimulator
of /?-defensins is somewhat unexpected, because S. pyogenes induces the ex-
pression of NF-/cB (Medina et al., 2002), which can upregulate the expression
of defensins. However, it was suggested that this lack of stimulation of f5-
defensins could contribute to the virulence of S. pyogenes in skin infections,
because ^-defensin 2 is a potent killer of S. pyogenes.

RESPONSES OF GAS TO INTERACTIONS WITH HOST CELLS

The interaction of streptococci with host cells not only induces a response
in the host cell but also induces a response in the streptococci. Broudy et al.
(2001, 2002) reported that coculturing a type M6 S. pyogenes with Detroit 562

pharyngeal cells induced the expression of a phage-encoded streptococcal
pyrogenic exotoxin C (SpeC) and DNase (called Spdl). SpeC and Spdl are
coexpressed from the same transcript. The coculturing of S. pyogenes strain
CS112 with pharyngeal cells also resulted in the induction of lysogenic bacte-
riophage particles. These investigators went on to show that a factor, released
by the pharyngeal cells, was responsible for these responses. The soluble fac-
tor was <10 kDa and resistant to heat and proteolytic degradation.

The expression of a variety of virulence factors is upregulated when GAS
invade the host. The bacterial factors regulated in response to invasion versus
those regulated in response to internalization with host cells have to be dis-
tinguished from each other. Investigators have long known that expression
of M protein increases when the bacteria enter the bloodstream. Gryllos et al.
(2001) found that introduction of GAS into the pharynx of baboons induced
S expression of the hyaluronic acid capsule. A similar increase in capsule pro-

w duction was found when the streptococci were introduced into the blood and

o the peritoneal cavity of mice. However, it is not entirely clear if this induction

S of virulence factors streptococci is in response to signals from the host or is

£ due to the growth phase of the streptococci. The expression of the capsule

* and a variety of other virulence factors are upregulated during the exponential

§ growth phase and downregulated during the stationary phase (M elver and

i Scott, 1997; Gryllos et al., 2001).

h Only recently, some data on bacterial virulence gene expression were

o collected from GAS during their internalization or intracellular persistence.

^ During such behavior, it seems favorable for the bacteria to suppress the

g expression of secreted lytic factors. This suppression is at least partially con-

ffi trolled by the negative global regulator Nra (Podbielski et al., 1999) , a member

of the RALP (RofA-like proteins) regulator family (Fogg et al., 1997; Granok
et al., 2000) . Inactivation of Nra in an M49 strain greatly increased the cytolysis
of HEp-2 cells. Utilizing whole genome array hybridization, Nra was shown
to regulate between 120 and 290 genes depending on the growth phase and
filtering levels used (Podbielski et al., unpublished results). Approximately
two thirds of the genes are negatively regulated by Nra and include the cap-
sule synthesis operon has, the genes for surface factors such as protein F2,
Cpa, ScpA, SOF/Sfbll, SfbX, SclA, and SclB; and genes for secreted proteins
such as the CAMP factor, SLS, SpeB, and SpeA. Consequently, inactivation
of nra resulted in an increase in the expression of these genes. However,
the expression of SLO was unaffected. It is interesting to note that the Nra-
negative mutant induced large invaginations in the membranes of HEp-2
cells, and chains of streptococci were found to enter one such invagination
and resurface through another (Fig. 8.2). No such phenomenon was seen
with the parent.

Another predominantly negative regulator is encoded by the Fas-regulon.
It comprises two histidine kinases, FasB/FasC, the response regulator, FasA,
and an effector RNA, FasX. Judged by its sequence, its temporal expression
pattern with a maximum at the end of the exponential growth phase, and the
type of dependent genes (positively regulated secreted factors such as SLS,
streptokinase, and SpeB; negatively regulated surface proteins-adhesins such
as the M-related protein, SCPA, and FBP54), the Fas-regulon resembles the
Staphylococcus aureus Agr-regulon. However, unlike the Agr-regulon, the Fas-
regulon has not been demonstrated to be involved in a cell-density-dependent
type of regulation (Kreikemeyer et al., 2001). A recent whole genome array
hybridization analysis revealed that 18 to 90 genes are influenced by a func-
tional Fas-regulon. This fact, as well as the potential convergence of several
signaling pathways in the two histidine kinase sensors of the Fas-regulon,

indicate that there is probably more than growth-phase-dependent regulation §

o
that is exerted by this regulon. g

By using various Fas-regulon mutants and complemented strains, re- ^

searchers recently showed that the Fas-regulon exerts a negative control of £

bacterial fibronectin binding and adhesion to keratinocytes and H Ep-2 epithe- o

Hal cells - albeit at different time points of the growth curve (keratinocytes, °

exponential phase; HEp-2 cells, stationary phase). Inactivation of the Fas- £

I— i

regulon not only enhanced adhesion but also increased internalization of %

the streptococci. Once internalized, the mutants exhibited a strongly reduced ~

induction of apoptotic pathways as measured by annexin V, and caspase-2, *

-3, and -7 expression (Kreikemeyer et al., unpublished data). The GAS iso- %

late used for this analysis was a serotype M49 strain. Utilizing a serotype 3

M6 strain, Nagakawa et al. (2001) found a somewhat different pattern of 2

apoptosis induction that relied on caspase-9 activation and subsequent se- w

lective degradation of mitochondrial functions. Because the M6 strain also
carried a typical Fas-regulon, there could more than one way that GAS induce
apoptosis.

GAS exhibit the highest tendency for internalization once they enter
the stationary phase. The Fas-regulon and the nra gene are maximally tran-
scribed during the transition from exponential to stationary growth phase.
This temporal expression profile fits well into the concept of FasBCAX and
Nra (and other RALPs, Beckert et al., 2001) as central regulators that keep
the delicate balance between extracellular aggressiveness and intracellular
"tameness" that would be necessary for the bacteria to best survive in these
different environments.

Voyich et al. (2003) used a gene array encompassing the whole genome
of an Ml serotype of GAS to probe gene expression during phagocytosis by
human neutrophils. A two-component regulatory system, Ihb-Irr regulon,

was identified that was expressed during phagocytosis. The Ihb-Irr system
played a fundamental role in the survival of GAS within phagocytes. In addi-
tion, the in gene was highly expressed by multiple serotypes of GAS during
infections of the human pharynx, indicating that it regulates gene expression
in response to interactions with the host in vivo.

INTRASPECIES AND INTERSPECIES SIGNALING BETWEEN
STREPTOCOCCI

Cell-to-cell signaling among bacteria is a relatively new discovery and
has been referred to as quorum sensing to indicate that thresholds of signals
are achieved only when a sufficient density of bacteria is reached. These sig-
naling peptides have also been called pheromones because of their role in
2 regulating competence and conjugation. For many Gram-positive bacteria,

w this signal will consist of small, linear, or cyclic peptides (reviewed by Dunny

g and Leonard, 1997). Upton et al. (2001) described a lantibiotic peptide, sail-
er varicin A (SalA), that is secreted by streptococci and whose expression is
£ autoregulated. The secreted form of this peptide, called SalAl in GAS, not
5 only upregulated expression of salAl in S. pyogenes but also upregulated the

§ expression of salA in S. salivarius. Similarly, SalA upregulates expression of

g salAl in S. pyogenes. In addition to autoregulation, SalA/ SalAl also induces

h the expression of an immunity factor that is required for resistance to the

o antibiotic effect of SalA. Thus, strains of streptococci that do not produce

SalA/SalAl are sensitive to its antibiotic effect. Hence, strains of streptococci
% that express SalA/SalAl could modulate the bacterial population in the host.

X Although the effect of adhesion on secretion of SalA has not been determined,

adhesion may enhance secretion as a result of an increase in the density of
the bacterial population. In addition, adherent bacteria can multiply and form
microcolonies that are very dense.

Another factor that seems to be involved in interbacterial signaling with
effects on the GAS virulence in animals, as well as on a putative natural com-
petence of the bacteria, is the Sil/Blp-like system (Hidalgo-Grass et al., 2002;
Smoot et al., 2002). The silA-E genes encode a regulatory secreted peptide,
a potential processing machinery, a two-component regulatory system, and
another protein with an unknown function. The sil locus resembles the S. au-
reus agr and S. pneumoniae com quorum-sensing regulons. Inactivation of silC
reduced the invasiveness of an emml4 strain of S. pyogenes in a mouse model.
Coinjection of the silC mutant with an avirulent emm 14- negative mutant re-
stored virulence and invasiveness. It was suggested that the emm 14-negative
mutant complemented the defect of the silC mutant by secreting a signaling

peptide that activated silA/B, which then activated other genes required for
invasion. So far, sil has been identified in only two serotype strains, M14 and
M18. It apparently resides on a 13.8-kb genomic island that is not contained
in two other genomes of S. pyogenes. Exactly how the various components of
the sil locus interact with each other, and with other genes involved in the
infectious process, remains to be elucidated. Sil may be a target for novel
therapies because of its involvement in the regulation of factors required for
invasion.

ASSAY FOR INTERNALIZATION

The most widely used assay for internalization of bacteria by host cells is
the antibiotic protection assay. This assay is based on the assumptions that

internalized bacteria will not be killed by the addition of antibiotics to the %

medium, and that only those bacteria on the surface will be killed. A measure g

of both adhesion and internalization can be obtained by determining the £

total number of CFU before and after treatment with antibiotics. However, £

investigators utilizing this assay should be aware of a potential problem that o

has been described by Molinari et al. (2001). These investigators found a low °

rate of internalization of streptococci using the antibiotic protection assay, £

I— i

but double-staining, immunofluorescent, microscopic analysis indicated that %

the internalization rate was actually high. It was suggested that the host S

cell membrane is damaged during invasion, allowing antibiotics to enter *

host cells and kill the bacteria. Thus, investigators need to ensure that the ^

integrity of the host cell membrane is maintained during the assay to obtain 3

valid results. The antibiotic protection assay also cannot distinguish between 2

the rate of uptake of bacteria and the rate of intracellular multiplication, or w
between adherent bacteria and those that have been internalized and released
by apoptosis.

FUTURE CONSIDERATIONS

It is clear that many types of eukaryotic cells can internalize streptococci
and that GAS can invade host cells in vivo. Some of the molecular mechanisms
involved in these processes have been identified. Additional research has to
be done to sort out the adhesins and mechanisms of invasion that are truly
involved in streptococcal infections from those that have a role only under in
vitro conditions. Much progress has been made in these directions, but there
are many remaining questions. How long can GAS persist intracellularly in
humans? Do intracellular GAS multiply or are they dormant? Do L-forms

of GAS have a role in intracellular persistence? Do intracellular GAS have
the means to initiate their efflux from the cell without apoptosis? If so, are
there specific stimuli for the bacterial switch between persistence and efflux?
What are the genes involved in persistence versus those involved in tissue
invasion?

A hint to the answers of some of these questions has come from re-
search on bacterial regulation, which has identified a number of genes that
are regulated in response to internalization. Whole genome gene arrays in
concert with real-time reverse transcription polymerase chain reaction (RT-
PCR) analyses hold great promise in identifying genes required for inva-
sion of tissues, metastatic spreading, internalization, persistence, and resis-
tance to innate defense mechanisms. These approaches are only possible
as a result of the genome sequencing work of Ferretti et al. (2001), Smoot
5 et al. (2002), and Beres et al. (2002). Currently being conducted are gene

w array analyses of the genomes of Ml, M3, M18, and M49, and real-time

Q RT-PCR analyses of fas and nra at three different phases of growth (Pod-

S bielski et al, unpublished work). Undoubtedly, these types of investigative

£ approaches will generate a wealth of information, a better understanding

3 of streptococcal virulence, and novel directions to pursue for preventive

§ therapies.

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CHAPTER 9

Invasion of oral epithelial cells by
Actinobacillus actinomycetemcomitans

Diane Hutchins Meyer, Joan E. Lippmann, and Paula Fives-Taylor

ACTINOBACILLUS ACTINOMYCETEMCOMITANS: ADHERENCE
MECHANISMS REQUIRED FOR INVASION

Colony phase variation

A. actinomycetemcomitans produces three distinct colonial morphologies
on solid medium. A rough colony phenotype is typically generated by organ-
isms upon isolation from the gingiva. These are small (~0.5-l mm in diam-
eter), translucent circular colonies with rough surfaces and irregular edges
(Fig. 9.1). An internal star-shaped or crossed cigar morphology that embeds
the agar is a distinguishing characteristic that gives A. actinomycetemcomitans
its name (Zambon, 1985). In liquid culture, the rough colony phenotype cells
form aggregates on the vessel walls, resulting in a clear medium (Fig. 9.1).
Repeated subculture on agar of rough phenotypic isolates yields two distinct
colonial variants; one is smooth surfaced and transparent, and the other is
smooth surfaced and opaque (Slots, 1982; Scannapieco et al., 1987; Rosan
et al., 1988; Inouye et al., 1990). The transparent smooth- surfaced variants
appear to be an intermediate between the transparent rough-surfaced and
opaque smooth-surfaced types (Inouye et al., 1990). In broth, the smooth-
surfaced opaque type grows as a turbid homogeneous suspension, whereas
the smooth-surfaced transparent type aggregates and adheres to the vessel
walls (Inouye et al., 1990). In general, isolates undergo a rough-to-smooth
variant transition soon after culture in vitro. In contrast, a smooth-to-rough
variant transition that appears to be associated with nutritional requirements
occurs only rarely during in vitro culture (Inouye et al., 1990; Meyer et al.,
1991; Meyer, unpublished observation).

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Figure 9.1. Micrographs of A. actinomycetemcomitans phenotypes. Rough (A and C) and
smooth (B and D) colonial variants cultured in agar (top panel) and broth (lower panel). A
unique characteristic of the rough colony morphology is the lovely star form that is
embedded in the agar (A). In broth culture, the rough phenotype forms tight aggregates
(C).

Surface-associated molecules and organelles

Bacterial colonial variation is indicative of the differential expression
of cell surface components (Braun, 1965). A. actinomycetemcomitans rough
colony variants express 43- and 20-kDa outer membrane proteins, rough
colony protein A and B, respectively, that are not expressed in smooth colony
variants (Haase et al., 1999). These proteins are encoded by genes that have
homology to genes known to encode fimbriae-associated proteins. In that
regard, A. actinomycetemcomitans rough colony variants are heavily fimbri-
ated, whereas smooth colony variants have few or no fimbriae (Scannapieco
et al., 1987; Rosan et al., 1988; Inouye et al., 1990; Meyer and Fives-Taylor,
1994) . In accordance, rough colony variants adhere better than smooth colony
variants in vitro to epithelial cells (Meyer and Fives-Taylor, 1994). In contrast,
smooth colony variants invade epithelial cells in vitro significantly better than
do rough variant colonies (Meyer et al., 1991). A. actinomycetemcomitans strain
SUNY 465, the invasion prototype, exhibits a smooth -to -rough variant shift
after anaerobic growth on agar that is accompanied by cell fimbriation and in-
creased adherence to KB oral epithelial cells (Meyer and Fives-Taylor, 1994).
Although the role of the phenotypic variation is not known, it has been pro-
posed that it may play some role in the episodic nature of periodontitis (Meyer
etal., 1991).

A. actinomycetemcomitans fimbriae are peritrichous, ~2 /xm in length
and 5 nm in diameter, and frequently occur in bundles (Holt et al., 1980;

Figure 9.2. Scanning electron micrographs of vesicles (blebs) associated with the A.
actinomycetemcomitans surface. Vesicles may vary in number and occur in different forms
or shapes and sizes. The vesicles associated with the organism in panel A (35,000x) are
relatively short and peritrichous (arrows), whereas those in panel B (70,000x) are
extremely long and essentially wrap around the organism (arrows).

Scannapieco et al., 1983, 1987; Preus et al., 1988; Rosan et al, 1988). They
are composed of a 54-kDa fimbrial subunit (Inouye et al., 1990). Adhesion
of A. actinomycetemcomitans-fimbrisLted strains to both buccal epithelial cells
and the Gin-1 fibroblast cell line is inhibited by antiserum to the 54-kDa pro-
tein, indicating a role for it in adhesion. Fimbrial-associated protein (fap), an
A. actinomycetemcomitans attachment factor, is expressed in fimbriated, but
not in nonfimbriated, strains (Ishihara et al., 1997). Flp, a 6.5-kDa protein
which is a component of A. actinomycetemcomitans fimbriae, exhibits some
amino acid sequence similarity to type IV pilin (Inoue et al., 1998). Clearly,
there is a correlation between A. actinomycetemcomitans fimbriation and ad-
hesion. However, A. actinomycetemcomitans cells devoid of fimbriae exhibit
adhesiveness as well, indicating that nonfimbrial components also function
in adhesion (Inouye et al., 1990; Meyer and Fives-Taylor, 1994).

Membraneous vesicles (blebs) are a prominent feature of the surface of
A. actinomycetemcomitans. These structures, which are thought to be predom-
inantly lipopoly saccharide in nature, are extensions of the outer membrane
that either remain attached to or bud off from the cell surface (Fig. 9.2).
Large numbers of vesicles are released into the external environment dur-
ing culture (Holt et al., 1980). The formation and morphology of vesicles are
altered by growth conditions (Meyer and Fives-Taylor, 1993). Cells grown
on agar have vesicles characterized by thick fibrils with knob-like ends. A.

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actinomycetemcomitans vesicles contain endotoxin, bone resorption activity,
actinobacillin (a bacteriocin) , and leukotoxin (Hammond et al., 1981, 1987;
Nowotny etal., 1982; Stevens etal., 1987; Lucia etal., 2002). Highly leukotoxic
A. actinomycetemcomitans strains have an abundance of vesicles, whereas low-
leukotoxic or nonleukotoxic strains have few or no vesicles (Lai et al., 1981).
The leukotoxicity of vesicles associated with highly leukotoxic strains is 5
to 10 times greater than that of minimally leukotoxic strains (Kato et al.,
2001).

A. actinomycetemcomitans vesicles also demonstrate adhesiveness. The
addition of vesicles to weakly adherent or nonadherent strains significantly
increases the ability of those strains to attach to epithelial cells (Meyer and
Fives-Taylor, 1993). The adhesive nature of the vesicles prompted the hy-
pothesis that these organelles function in A. actinomycetemcomitans as deliv-
g ery vehicles for the toxic materials that they harbor (Meyer and Fives-Taylor,

5 1993). Scanning electron microscopy of the process of invasion of A. actino-

£ mycetemcomitans into epithelial cells revealed that bacteria in contact with,

E and in the process of being internalized by, the cells have surface-associated

g vesicles. In contrast, bacteria not in contact with epithelial cells do not pos-

* sess vesicles, suggesting that the internalization process may be a trigger

5 for vesicle formation (Meyer et al., 1996). The role of vesicles in the A. acti-

| nomycetemcomitans internalization process is not known, but clearly further

2 investigation is warranted.

S The surface of A. actinomycetemcomitans may also be associated with an

w extracellular amorphous material (ExAmMat) that can embed groups of cells

< in a matrix (Holt et al., 1980) . Whereas an early study reported that cells grown

rt in liquid medium lacked amorphous material, other reports indicate that Ex-

n AmMat can occur on cells grown in liquid culture (Wilson et al., 1985; Meyer

co and Fives-Taylor, 1994). Expression of ExAmMat has some association with

£ growth in tryptone-based medium (Wilson et al., 1985). Therefore, similar

g to fimbriae and vesicles, culture conditions can modify expression of ExAm-

w Mat. It has been determined that ExAmMat is proteinacious, most likely a

$ glycoprotein, and has adhesive properties (Meyer and Fives-Taylor, 1993).

ExAmMat is not affixed firmly to the cell surface, as the washing of cells

with phosphate-buffered saline removes ExAmMat and results in reduced

adhesion to epithelial cells (Meyer and Fives-Taylor, 1993). Moreover, weakly

adherent A. actinomycetemcomitans strains adhere strongly to epithelial cells

after suspension in ExAmMat, a process termed conveyed adhesion. The

surfaces of A. actinomycetemcomitans suspended in ExAmMat are associated

with large amounts of material, indicating that the conveyed adhesion is the

result of a direct transfer of ExAmMat onto the bacterial surface (Fives-Taylor

etal., 1995).

Specific adhesion to epithelial cells

Most A. actinomycetemcomitans strains that have been tested to date ad-
here to epithelial cells strongly; however, the adhesiveness of strains does
vary (Meyer and Fives-Taylor, 1994). The rapid process reaches saturation
levels within lh of infection (Mintz and Fives-Taylor, 1994). Adhesion is
affected by growth conditions (Meyer and Fives-Taylor, 1994), which likely
determine the expression of specific adhesins. Cell surface entities that me-
diate adherence include fimbriae (Rosan et al., 1988; Meyer and Fives-Taylor,
1994), ExAmMat (Meyer and Fives-Taylor, 1994), and vesicles (Meyer and
Fives-Taylor, 1993). Trypsin and protease treatment of smooth, nonfimbri-
ated strains reduces adhesion of A. actinomycetemcomitans to epithelial cells,
indicating that these nonfimbriae types of adhesins are proteinacious (Mintz
and Fives-Taylor, 1994).

Recently, a surface-associated protein determined to be an autotrans- $

porter was shown to be involved in A. actinomycetemcomitans adhesion to o
epithelial cells (Rose et al., 2003). The adhesin, called Aae for adhesion to o
epithelial cells, is encoded by a gene (aae) that is homologous to autotrans- 2
porter genes of Haemophilus influenzae and Neisseria species (St. Geme et £
al., 1994; St. Geme and Cutter, 2000). A unique feature of aae is a 135-base q
repeat sequence that varies in number from strain to strain (Rose et al., 2003). «
Four alleles have been identified to date. Lactoferrin in human milk whey £
was shown to decrease epithelial cell binding of A. actinomycetemcomitans
strain 29523, but not of SUNY 465, a strain having an allele with fewer re-
peats (Rose et al., 2003). On the basis of these results it was suggested that >
the repeats may play a role in the binding of lactoferricin, the peptide on the g
N -terminus of lactoferrin that interacts with and causes damaee to the cells' t»

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outer membrane (Yamauchi et al., 1993). Fewer copies of the repeats would

effectively reduce the opportunity for binding. S

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In summary, the adhesion of A. actinomycetemcomitans to epithelial cells q

is multifactorial, with several adhesins and mechanisms playing a role. §

HOST CELL SIGNALING PATHWAYS MODULATED IN RESPONSE g

TO THE BACTERIAL CHALLENGE §

Several lines of evidence suggest that A. actinomycetemcomitans infec- §

tion and subsequent internalization is associated with activation of host cell
signaling pathways. An active metabolic state and novel protein synthesis by
both A. actinomycetemcomitans and the epithelial cell are required for invasion
(Sreenivasan et al., 1993). In addition, entry is associated with an elevation of
intracellular Ca 2+ levels (Fives-Taylor et al., 1996), a process associated with

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Figure 9.3. Real-time video microscopy frames of the interaction of A.
actinomycetemcomitans strain SUNY 465 with KB epithelial cells. KB microvilli (arrow)
become highly active and seek out bacteria (arrowhead) soon after infection (A). A
microvillus makes contact with a bacterium (B). The microvillus seizes the A.
actinomycetemcomitans and draws it toward and eventually into the KB cell (C).

signaling events (Berridge, 1995) and characteristic of invasion by some bac-
teria (Baldwin etal., 1991; Pace etal., 1993; Izutsuetal., 1996). It has also been
shown that staurosporin, a wide-spectrum protein kinase inhibitor, decreases
A. actinomycetemcomitans internalization, whereas genistein, a specific in-
hibitor of tyrosine protein kinase, increases internalization (Fives-Taylor and
Meyer, 1998). These results led to the hypothesis that staurosporin may be
modulating a signaling pathway involved in the A. actinomycetemcomitans
internalization process, whereas genistein is modulating one involved in its
egression from the host cell.

In related studies it was shown that an infection of KB cells produces
changes in both its SDS-PAGE tyrosine-phosphorylated protein profile and
immunofluorescence microscopy phosphotyrosine-labeling pattern (Fives-
Taylor and Meyer, 1998). The fact that cross talk or signaling is an absolute
requirement for A. actinomycetemcomitans invasion was ascertained by use of 5

real-time video immunofluorescence microscopy (Fig. 9.3). Uninfected KB &

cells and those subjected to nonviable A. actinomycetemcomitans are abso- z

lutely quiescent. In contrast, infection with viable A. actinomycetemcomitans *

arouses the KB cells; their microvilli become highly active — seeking out, se- >

questering, drawing in, and eventually engulfing the bacterium (Lippmann 3

and Fives-Taylor, 1999). x

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PHYSICAL MECHANISM OF INTERNALIZATION AND g

INTRACELLULAR LOCATION, FATE OF BACTERIA,

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AND CELL-TO-CELL SPREAD >

A. actinomycetemcomitans penetration of the gingival epithelium was §

demonstrated in early clinical studies (Saglie et al., 1986; Christersson et al., %

1987). Those in vivo studies revealed that A. actinomycetemcomitans occurs £

in very specific intracellular locations and exhibits a very distinctive penetra- >
tion pattern (Saglie et al., 1982, 1986, 1988). It has also been established that

intracellular A. actinomycetemcomitans is present in vivo in human buccal §

epithelial cells, where it occurs in clusters (Rudney et al., 2001). A. actino- g

mycetemcomitans invasion of epithelial cells, both in an oral cell line (Meyer g

et al., 1991) and in human primary gingival cells (Fives -Taylor et al., 1995), o

has been demonstrated by use of in vitro studies . The overall A. actinomycetem- h

comitans invasion process is dynamic and complex: it involves attachment to §

the host cell, entry in a vacuole, escape from the vacuole into the cytoplasm,
intracellular spread, and cell-to-cell spread (Meyer at al., 1996). Entry of the
epithelial cell is initiated when interaction of A. actinomycetemcomitans with
the epithelial cell triggers effacement of microvilli and formation of craters

on the epithelial cell surface (Sreenivasan et al., 1993; Meyer et al., 1996). The
majority of A. actinomycetemcomitans strains use an actin-dependent mecha-
nism for invasion, but the mode of entry of a few strains is actin independent
(Brissette and Fives-Taylor, 1998). The invasion process in strains that uti-
lize the actin-independent mechanism is not well studied. Thus, a role for
the two different mechanisms is not known; nor is it known whether actin
dependence or independence is the only characteristic that separates these
two invasion processes.

The process of invasion described here is that of the A. actinomycetem-
comitans invasion prototype, strain SUNY 465, a strain that utilizes the
actin-dependent mechanism. Attachment of A. actinomycetemcomitans to
the epithelial cell promotes rearrangement of actin from the periphery of
the epithelial cell to a focal point beneath the organism at the point of entry
g (Fives-Taylor et al., 1995). A. actinomycetemcomitans enters the epithelial cell

5 through ruffled, lip-rimmed apertures (Fives-Taylor et al., 1996; Meyer et al.,

g 1996). To date, two pathways believed to be associated with the entry of A.

E actinomycetemcomitans into epithelial cells have been identified; the transfer-

al rin receptor is implicated in one pathway, and integrins are implicated in the

* other (Meyer et al., 1997a).

5 Subsequent to the initial interaction and attachment, A. actinomycetem-

| comitans enters the host cell in a membrane-bound vacuole by receptor-

2 mediated endocytosis. In the classical endocytic pathway, macromolecules

S are taken into early endosomes and delivered to lysosomes. Internalized or-

e's ganisms are known to use a variety of means to avoid lysosomal degradative

< enzymes; these include blockage of delivery to lysosomes, inhibition of en-

rt dosome acidification, and acid activation of virulence factors that modify the

n lysosome. A. actinomycetemcomitans trafficking within the vacuole, a process

co that does not require endosomal acidification, is as follows. Within 30 min of

£ infection, 40% of internalized A. actinomycetemcomitans are in the early en-

g dosome. By 60 min, the number in the early endosome is greatly reduced and

w A. actinomycetemcomitans are also present in the late endosome. By 2h, es-

$ sentially all A. actinomycetemcomitans are associated with the late endosome,

and by 3-4 h the vacuoles are devoid of bacteria and A. actinomycetemcomi-
tans are present in both the cytoplasm and cell culture medium. Thus, the A.
actinomycetemcomitans organisms avoid degradation by lysosomal enzymes
by escaping from these organelles 3-4 h postinfection (Lippmann and Fives-
Taylor, 2000) .

The mechanism(s) used by A. actinomycetemcomitans for lysis of the host
vacuole is not known. Preliminary studies suggest a role for leukotoxin in
lysis of the vacuole (Meyer, unpublished results). A. actinomycetemcomitans
possesses phospholipase C (PLC; see Meyer et al., 1997b), a molecule used

by certain enteric pathogens for vacuole lysis (Camilli et al., 1991; Smith
et al., 1995); thus, PLC is another possibility.

Clearly, intracellular A. actinomycetemcomitans are not quiescent. A short
time after escape from the vacuole into the cytoplasm, A. actinomycetemcomi-
tans interact in a highly specific manner with host cell microtubules and
utilize a microtubule -mediated mechanism for intracellular spread, as well
as for spread to neighboring epithelial cells (Meyer et al., 1999). The spread
to neighboring cells is by means of A. actinomycetemcomitans-induced inter-
cellular protrusions, which are extensions of the host cell membrane that
extend from one epithelial cell to another (Meyer et al., 1996). Bacteria can be
seen within these protrusions by scanning, transmission, and fluorescent mi-
croscopy (Meyer et al., 1996). The cell-to-cell spread involves movement and
transfer through protrusions (Meyer et al., 1996), not engulfment of protru-
sions as is the case with both Shigella and Listeria (Tilney and Portnoy, 1989; 5
Kadurugamuwa et al., 1991). In addition to microtubules, the protrusions &

contain microfilaments that may be involved in the structural framework z

of protrusions rather than mechanistically in the movement process (Meyer *

etal, 1999). Sj

Whereas the precise means by which A. actinomycetemcomitans usurps 3

host cell microtubules for movement is not known, in vitro studies show that x

A. actinomycetemcomitans localizes exclusively with the plus ends of micro- «

tubules of taxol-induced microtubule asters (Fig. 9.4), indicating a specific A. n

actinomycetemcomitans-microtubule interaction (Rose et al., 1998, 1999) . Fur- P

thermore, both immunofluorescence microscopy and bactELISA indicate the 3

presence of a kinesin-like entity on the surface of A. actinomycetemcomitans, Q

thus implicating motor proteins in the movement along the microtubules §

(Meyer et al., 2000) . It is hypothesized that the kinesin entity on the A. actino- %

mycetemcomitans surface mediates the interaction with microtubules and that £

the bacterium is transported in a manner similar to that of organelles and >
vesicles, the usual cargo of microtubules. Whereas certain bacteria, such as

Shigella and Listeria, usurp host cell actin to move within cells and to spread §

to adjacent cells (Tilney and Portnoy, 1989; Kadurugamuwa et al., 1991), g

this provides the first evidence that host cell dispersion of an intracellular S

pathogen involves usurpation of the host cell microtubule transport system. o

Two genes have been implicated in A. actinomycetemcomitans invasion. h

One is homologous to apaH, a gene that encodes RGD, a sequence known §

to bind integrins (Saarela et al., 1999). A. actinomycetemcomitans DNA that
contains the apaH gene confers on noninvasive Escherichia coli the ability
to invade epithelial cells (Meyer et al., 1995; Saarela et al., 1999). It was also
determined that insertional inactivation of apaH in A. actinomycetemcomitans
substantially reduced its invasion (Lippmann and Fives-Taylor, unpublished

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Figure 9.4. Microtubule asters and the A. actinomycetemcomitans kinesin-like entity.
Immunofluorescent microscopy of the interaction of A. actinomycetemcomitans strain
SUNY 465 (arrows) with taxol-induced asters (panel A) and the kinesin-like motor protein
(arrowheads) associated with the A. actinomycetemcomitans surface (panel B). The A.
actinomycetemcomitans (green) bind specifically to the plus ends (periphery) of the asters
(orange) (panel A). Binding of A. actinomycetemcomitans to the microtubules is believed to
be mediated by the kinesin-like entities (yellow) on the A. actinomycetemcomitans surface
(orange) (panel B). See color section.

observation). Furthermore, RGD peptides inhibit A. actinomycetemcomitans
invasion, whereas RAD peptides have no effect on invasion (Saarela et al.,
1999). The apaH gene is a homolog of invA, ialA, and ygdP, genes that
are associated with invasion by Rickettsia prowazekii (Gaywee et al., 2002),
Bartonella bacilliformis (Mitchell and Minnick, 1995), and E. coli Kl (Bessman
et al., 2001), respectively. These genes produce proteins that are members

Figure 9.5. Schematic representation of A. actinomycetemcomitans invasion of epithelial
cells. Aae and ApaH have been identified as molecules on the surface of A.
actinomycetemcomitans that can mediate its contact with epithelial cells. ApaH interacts
with host cell integrins; the receptor to which Aae binds is not known. The transferrin
receptor is also implicated as a receptor for A. actinomycetemcomitans. Entry associated
with actin rearrangement and a Ca ++ flux occurs in a membrane-bound vacuole. The
mechanism of escape from the vacuole is unclear, but leukotoxin (Ltx) and phospholipase
C (PLC) are implicated. Once in the cytoplasm, A. actinomycetemcomitans can move within
the host cell and spread to adjacent cells by usurping host cell microtubules. Kinesin-like
motor proteins on the A. actinomycetemcomitans surface are implicated in mediating its
interaction with microtubules. See color section.

of the Nudix family of hydrolases, which catalyze the dinucleoside polyphos-
phates, a class of signaling nucleotides (Conyers and Bessman, 1999; Saarela
et al., 1999; Bessman et al., 2001). It has also been reported that A. actino-
mycetemcomitans invasion involves genes that share some homology to spa
genes, genes that are involved in the export of proteins from cells (Laing-
Gibbard et al., 1998).

In summary, it is clear that the process by which A. actinomycetemcomi-
tans enters and escapes from host cells is very complex (Fig. 9.5). Future
studies should reveal even more dynamic interactions and lead to targeting
of mechanisms suitable for therapeutic intervention.

285

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CONSEQUENCES OF INVASION WITH REGARD TO INNATE
HOST RESPONSE

The first line of defense of the host against intrusive bacteria is phagocyte
recruitment (chemotaxis) to the region. A number of steps are involved in
this process: binding of chemotactic signaling factors, upregulation of adhe-
sion receptors, binding to the endothelium, and movement of phagocytes to
the underlying tissues. Whereas invasion per se is a means by which bac-
teria can ultimately escape this response, it initially represents a significant
challenge to intrusive organisms. Thus, the ability to disrupt chemotaxis pro-
motes survival of pathogenic organisms. A. actinomycetemcomitans secretes a
low-molecular-weight protein that can inhibit polymorphonuclear leukocyte
chemotaxis (Van Dyke et al., 1982; Ashkenazi et al., 1992). It is also known
that A. actinomycetemcomitans capsular-like serotype-specific polysaccharide

2 antigen (SPA) plays an important role in its ability to resist phagocytosis and

h killing by polymorphonuclear lymphocytes (Yamaguchi et al., 1995). Expres-

> sion of chemoattractant protein 1 (MCP-1) and neutrophil chemotactic factor

<j I L-8 mRNA is increased in monocytes after stimulation with S PA (Yamaguchi

% etal, 1996).

Polymorphonuclear leukocytes can also kill bacteria by fusing with lyso-

1 somes from which they acquire potent antibacterial agents. Bacteria able to
5j inhibit the fusion or ward off the antibactericidal action are protected. A. acti-
ph nomycetemcomitans can inhibit the production by polymorphonuclear leuko-
^ cytes of some of these compounds, and it is resistant to others. A heat-stable
& protein in A. actinomycetemcomitans inhibits the production of hydrogen
SL peroxide by polymorphonuclear leukocytes (Ashkenazi etal., 1992), and many
w strains are intrinsically resistant to high concentrations of hydrogen peroxide

2 (Miyasaki et al., 1984). In addition, A. actinomycetemcomitans is resistant to a

g number of defensins, which are cationic peptides that occur in neutrophils

5 (Miyasaki etal, 1990).

x A. actinomycetemcomitans induces apoptotic cell death in murine macro-

5 phages (J 774.1 cells) in vitro. Entry of A. actinomycetemcomitans into the

macrophages is an absolute requirement for the cytotoxicity, that is, nuclear
morphology changes and an increase in the proportion of fragmented DNA
(Kato et al., 1995). Studies suggest that protein kinase C signaling regu-
lates the apoptosis (Nonaka et al., 1997). With the use of LR-9 cells, CD14-
defective mutants of J774.1 cells, it was determined that CD14 molecules
likely participate in the phagocytosis of A. actinomycetemcomitans, as well as
in the regulation of the apoptotic events (Muro et al., 1997). Both caspase-1
and caspase-3 appear to play a role in the A. actinomycetemcomitans-induced

apoptosis in macrophages (Nonaka et al., 2001). In general, infected macro-
phages kill bacteria within phagosomes by means of nitric oxide (NO). In
this regard, it has been reported that NO affords A. actinomycetemcomitans-
infected murine macrophages partial protection from apoptosis by decreasing
caspase activity (Nakashima et al., 2002). An interesting caveat that requires
further investigation is the finding that A. actinomycetemcomitans LPS stimu-
lates the production of NO, a process that involves activation of protein kinase
C and protein tyrosine kinase, as well as the regulatory control of cytokines
(Sosroseno et al., 2002).

CORRELATION AMONG INVASION, PATHOGENICITY,
AND CLINICAL PRESENTATION

A lack of an adequate animal model in which to study invasion precludes,

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by Actinobacillus actinomycetemcomitans. I. Chemotaxis, surface receptor ex-
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<
>

at this juncture, the ability to establish a correlation among A. actinomycetem- 8

comitans invasion, pathogenicity, and clinical presentation. On the basis of o

►n

the collective in vivo and in vitro observations made to date, the hypothesis g

is put forth that invasion of epithelial cells per se and the dynamic process £

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mycetemcomitans migrates to gingival and connective tissue and initiates the ™

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it,

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CHAPTER 10

Invasion by Porphyromonas gingivalis

Ozlem Yilmaz and Richard J. Lamont

Porphyromonas gingivalis cells are Gram-negative, anaerobic, nonmotile short
rods that produce black pigmented colonies on blood agar. The taxonomy of
the species dates back to 1921 when Oliver and Wherry isolated an organism
from a variety of oral and nonoral sites that they were to designate Bacterium
melaninogenicum. This heterogeneous grouping was later subdivided into
nonfermenters, weak fermenters, and strong fermenters. After a number of
status changes within the genus Bacteroides, asaccharolytic oral isolates were
assigned to the taxon P. gingivalis. The primary ecological niche of P. gingivalis
is in the subgingival crevice, the gap between the surfaces of the tooth and the
gingiva (gum); however, the organism can be found elsewhere in the mouth,
including supragingival (above the gum) tooth surfaces, the tongue, tonsils,
and buccal (cheek) mucosa. Although the species has been associated with
odontogenic abscesses and nonoral infections (discussed later), the primary
pathogenic potential of P. gingivalis is in periodontal disease. The periodon-
tal tissues include the gingiva, periodontal ligament, and alveolar bone, and
they constitute the supporting tissues of the teeth. Chronic destruction of the
periodontium, such as occurs in periodontal diseases, can eventually lead to
exfoliation of teeth and is the most common cause of tooth loss in adults.
Periodontal diseases vary in severity and age of onset, and P. gingivalis is
associated, either alone or in combination with other bacteria, with the most
severe manifestations. However, the frequent occurrence of the organism in
healthy adults and in young children indicates that a complex interplay be-
tween host and pathogen exists, and that disruption of this ecological balance
is required for disease to ensue.

In the gingival crevice the area of contact between the gingiva and the
tooth is known as the junctional epithelium, and it is characterized by a
lack of keratinization, limited differentiation, and a relatively permeable

structure. In destructive periodontal disease there is migration of the junc-
tional epithelium, resulting in enlargement of the crevice into a deeper pe-
riodontal pocket that contains inflammatory cells such as neutrophils and
T cells. The gingiva itself also contains immune cells, including B cells,
T cells, and dendritic cells. The microbiota of the gingival area in both health
and disease is complex, with at least 500 species of bacteria present in the
gingival crevice. Colonization and persistence by periodontal pathogens re-
quire, therefore, successful encounters with antecedent bacterial species and
with host eukaryotic cells. Consistent with these constraints, P. gingivalis can
bind to, invade, and survive inside a variety of host cells, including epithelial
and endothelial cells.

P. GINGIVALIS ADHESION

H
%

§ An early event in the process of bacterial internalization is adhesion to

^ the host cell surface. Attachment of bacteria is often required to trigger the

§ signaling pathways within the host cells that ultimately induce the mem-

= brane and cytoskeletal rearrangements that bring the bacteria into the cell.

* P. gingivalis is endowed with a multiplicity of adhesins that mediate adhesion

% to epithelial cells, endothelial cells, fibroblasts, and erythrocytes, and to com-

< ponents of the extracellular matrix, namely laminin, elastin, fibronectin, type

~ I collagen, thrombospondin, and vitronectin (Sojar et al., 1995, 1999; Kontani

§ et al., 1997; Nakamura et al., 1999; Dorn et al., 2000; Lamont and Jenkinson,

.g 2000). Adhesive activity has been demonstrated for fimbriae, outer mem-

brane proteins, and proteases. These molecules may function collectively,
and their activities are integrated and controlled at the transcriptional and
posttranslational levels (reviewed in Lamont and Jenkinson, 1998, 2000).

The major fimbriae of P. gingivalis are distinct from other Gram-negative
fimbriae and do not appear to belong to any of the existing classifications.
The fimbriae are composed of an ^43-kDa fimbrillin (FimA) monomer that
possesses a number of binding domains for individual substrate recognition.
The functional domain of FimA for epithelial cells has been localized to a re-
gion spanning amino acid residues 49-90, although the boundaries of the
interactive site are not known precisely (Sojar et al., 1999) . Fimbriae-mediated
binding is important for subsequent invasion, as FimA-deficient mutants are
attenuated in their ability to internalize, and both purified fimbrillin and an-
tibodies to fimbriae can block invasion (Njoroge et al., 1997; Weinberg et al.,
1997). Furthermore, fimbrillin-coated microspheres are efficiently taken
up by epithelial cells (Nakagawa et al., 2002). A number of epithelial cell
molecules have been found to function as cognate receptors for fimbrillin,
including a 48-kDa surface protein, cytokeratins, and integrins (Weinberg

et al., 1997; Sojar et al., 2002; Yilmaz et al., 2002). With regard to stimulation
of invasion, fimbriae-integrin binding may be the most significant interac-
tion, as integrin antibodies can inhibit P. gingivalis invasion of epithelial cells
(Yilmaz et al., 2002). Moreover, as integrins are initiators of signal trans-
duction pathways, the engagement of an integrin receptor by P. gingivalis
fimbriae may be a means by which the organism begins to seize control of
host cell signaling machinery. P. gingivalis fimbriae are also involved in the
adhesion-dependent invasion of endothelial cells (Deshpande et al., 1998;
Khlgatian et al., 2002), although other adhesins may work in concert (Dorn
etal.,2000).

The Jim A gene is monocistronic, although immediately downstream are
four genes whose products may be associated with the mature fimbriae
(Watanabe et al., 1996). The fimA upstream region contains a functionally
active sigma-70-like promoter consensus sequence along with a potential UP 5

element (Xie and Lamont, 1999). AT-rich sequences upstream of the RNA &

polymerase binding sites are involved in positive regulation of transcriptional z

activity. Environmental cues to which the fimA promoter responds include ^

temperature, hemin concentration, and salivary molecules (Xie et al., 1997), a

which are parameters with relevance to conditions in the oral cavity. The^mA 53

gene can also be positively autoregulated by the FimA protein (Xie et al., 2

2000). In addition, expression of FimA decreases following association of %

P. gingivalis with epithelial cells (Wang et al., 2002). Thus, after completing £

i-i

the task of inducing invasion, fimbriae may no longer be required by the §

intracellular P. gingivalis cells. As fimbrillin has a number of immunostim- g

ulatory properties, such as induction of cytokines and chemokines (Ogawa &

et al., 1994), reduced FimA expression may aid in immune avoidance by the
organism.

Although the primary function of proteinases secreted by the asaccha-
rolytic P. gingivalis is the provision of nutrients, proteinases are also involved
both directly and indirectly in adhesion. Several distinct proteinases are pro-
duced by P. gingivalis (reviewed in Potempa et al., 1995; Kuramitsu, 1998;
Curtis et al., 1999), and direct enzyme-substrate interactions may be able
to effectuate adhesion, although such binding is unlikely to persist for ex-
tended periods. Of greater importance, the C-terminal coding regions of
the Arg-X and Lys-X specific proteases RgpA and Kgp contain extensive re-
gions with hemagglutinin activity (Barkocy-Gallagher et al., 1996; Lamont and
Jenkinson, 1998; Curtis et al., 1999). These regions, therefore, can be pre-
dicted to bind directly to human cell surface receptors. Proteinases can also
contribute to adherence through the partial degradation of substrates result-
ing in the subsequent exposure of epitopes for adhesin recognition. For exam-
ple, hydrolysis of fibronectin or other matrix proteins by the Arg-X specific

proteases RgpA and RgpB displays C-terminal Arg residues that mediate
fimbriae -dependent binding (Kontani et al., 1996). Other means by which
RgpA and RgpB can contribute to adhesion are through processing the leader
peptide from the flmbrillin precursor (Nakayama et al., 1996), and by upreg-
ulating transcription of the fimA gene (Tokuda et al., 1996; Xie et al., 2000).
In addition to the hemagglutinin-associated activities of the RgpA and
Kgp proteinases, several additional hemagglutinin (hag) genes are present
in the P. gingivalis genome. The hagA gene encodes a large protein of over
230 kDa containing four contiguous direct 440-456 aa residue repeat blocks
(Han et al., 1996). Each repeat block may represent a functional hemagglu-
tinin domain, and similar hagA-hke sequences are found at multiple sites in
the chromosome. The hagB and hagC genes are at distinct chromosomal loci,
although the HagB and C proteins (~40 kDa) are very similar (Progulske-
g Fox et al., 1995). A minimal peptide motif PVQNLT has been shown to be

§ associated with hemagglutinating activity and is found within the proteinase-

hemagglutinin sequences (Shibita et al., 1999) and at multiple chromosomal

§ sites.

g Thus, P. gingivalis possesses a variety of adhesins with differing recep-

* tor specificities and affinities that can potentially impinge to varying degrees

^ upon diverse receptor-dependent host cell biochemical pathways. This may

< allow the organism to utilize more than one pathway for internalization. For

= example, although fimbriae-deficient mutants show reduced uptake into ep-

§ ithelial cells, a low level of invasion remains (Weinberg et al., 1997). Nonfim-

g brial dependent uptake, although less efficient than FimA-mediated uptake,

may result from attachment by other surface adhesins.

UPTAKE OF P. GINGIVALIS BY HOST CELLS

P. gingivalis can invade epithelial cells, endothelial cells, and dendritic
cells (reviewed in Lamont and Jenkinson, 1998; Lamont and Yilmaz, 2002).
Interestingly, although the overall mechanistic basis is similar in these cell
systems, the signal transduction pathways activated by the organism and the
intracellular locations and trafficking of the bacteria differ according to cell
type. However, in all cases invasion is an active, bacterially driven process that
has a significant impact on the phenotypic properties and fate of the host cell.

Primary gingival epithelial cells

The study of primary cultures of gingival epithelial cells (GEC) provided
the first evidence for intracellular invasion by P. gingivalis in 1992 (Lamont

et al., 1992). GEC are cultured from basal epithelial cells extracted from gin-
gival explants, and they can be maintained in culture for several generations.
Immunohistochemical staining has shown that the cells are nondifferenti-
ated and noncornified, which are characteristics that are in common with
the junctional epithelium. Thus, although not derived from the junctional
epithelium, GEC demonstrate similar properties and thus provide a relevant
ex vivo model for the events that occur at the base of the gingival crevice.

P. gingivalis lacks the components and effectors of the type III secretion
machinery that are important in the invasive processes of other organisms
and that are discussed elsewhere in this volume. However, when in contact
with GEC, P. gingivalis is induced to secrete a novel set of extracellular proteins
(Park and Lamont, 1998). Some functional equivalence to type III effectors
is implied by the finding that one of the secreted proteins of P. gingivalis
is a homologue of a phosphoserine phosphatase (Lamont and Yilmaz, 2002), 5

although the functionality of this molecule, either intracellularly or extra- &

cellularly, remains to be established. Nevertheless, despite the absence of a z

classical type III secretion apparatus, P. gingivalis invasion of GEC is swift ^

and profuse (Belton et al., 1999). a

Fluorescent microscopic imaging has shown that invasion is complete 53

within 15 min, and that all GEC exposed to P. gingivalis take up large num- 2

bers of organisms: this is invasion on a scale unsurpassed by any of the %

enteropathogens. Once inside the cells, the bacteria are not confined to a £

i-i
membrane-bound vacuole and congregate in the perinuclear region (Belton §

et al., 1999). P. gingivalis cells remain viable and capable of intracellular repli- g

cation (Lamont et al., 1995). Interestingly, despite the burden of large num- &

bers of intracellular bacteria, GEC do not undergo necrotic or apoptotic cell
death (Nakhjiri et al., 2001); however, the cells contract and there is conden-
sation of the actin cytoskeleton after prolonged cohabitation with P. gingivalis
(Belton et al., 1999).

The signaling events required for uptake into GEC are thought to ensue
primarily from fimbriae-integrin interactions, although other signal trans-
duction pathways may be operational (Yilmaz et al., 2002). Integrins have
both a structural role, in linking extracellular matrix proteins with the cellular
actin cytoskeleton in order to regulate cell shape and tissue architecture, and
a signaling role, through intracellular signals generated by integrin-receptor
coupling that regulate cell migration, gene expression, growth, survival, and
inflammatory responses. Integrins are heterodimeric transmembrane recep-
tors with no catalytic activity; therefore, the signals initiated by integrin-
ligand interactions are transduced into cells through the activation of a num-
ber of specialized cytoplasmic proteins. Enhanced tyrosine phosphorylation

1." -J

i -

•

1
< »

* 'V.

■ V

™ -

•m

/ i

•

t r \

■

Figure 10.1. (facing page). Immunofluorescence microscopy (400x) of P. gingivalis
invasion of GEC. P. gingivalis (green) induces recruitment of paxillin (red), A, and FAK
(red), B, to the cell peripheries. GEC in C and D are stained with antibodies to paxillin or
FAK, respectively, in the absence of P. gingivalis. See color section.

of these proteins plays an essential role in the perpetuation of the intracel-
lular signals created by integrins into specific targets in cells. Among those
proteins, FAK (focal adhesion kinase) and paxillin have emerged as key signal-
transducing components (Turner, 2000). Phosphorylation of paxillin is asso-
ciated with coordinate formation of focal adhesions and stress fibers. Paxillin
thereby provides a platform for efficient propagation of signals from one
component to the next. Invasion by fimbriated P. gingivalis promotes a sig-
nificant amount of tyrosine phosphorylation of paxillin in GEC during early
infection (between 5 and 20 min; Yilmaz et al., 2002).

Recent immunofluorescent studies (Yilmaz et al., 2003) have visualized
the subcellular distribution of paxillin during P. gingivalis invasion (Fig. 10.1).
Immediately after infection by P. gingivalis, paxillin aggregates at the plasma
membrane and forms microspikes or lamellipodial-like extensions at the
edges of cells. These results suggest that P. gingivalis recruits paxillin to the 5

plasma membrane and promotes formation of focal adhesion complexes &

that may potentiate efficient bacterial uptake in GEC. In addition to the early z

redistribution of paxillin, after 24 h there is colocalization of paxillin with ^

P. gingivalis in the perinuclear space. Immunofluorescence also indicates that a

FAK is activated and recruited to the membrane of GEC during early periods 53

of P. gingivalis exposure (Fig. 10.1), and later it relocates to the perinuclear 2

area with P. gingivalis. Paxillin and FAK phosphorylations, therefore, appear ^

to be important in both early and late events of P. gingivalis invasion and
intracellular trafficking.

Integrin signaling also modulates actin cytoskeleton and microtubule §

dynamics. Sensitivity of the invasion process to the inhibitors cytochalasin &

D and nocodazole provides indirect evidence that both actin microfilament
and microtubule rearrangements are required for P. gingivalis entry into the
host cell (Lamont et al., 1995). Indeed, immunofluorescent microscopy has
shown that actin is assembled into filament-rich microspikes at the periphery
of the GEC during P. gingivalis invasion. Later, both the actin microfilament
and microtubules are dramatically depolymerized and nucleated.

Signaling pathways downstream of integrin focal adhesions often funnel
through the MAP kinase family of signal transduction mediators. Consistent

with this, invasion by P. gingivalis results in activation of JNK, a stress -
activated protein kinase of the MAP kinase family (Watanabe et al., 2001).
Also consonant with integrin activation, P. gingivalis invasion is associated
with a transient increase in the intracellular Ca 2+ concentration. The cal-
cium ion increase results, at least in part, from release of calcium from
a thapsigargin-sensitive intracellular store (Izutsu et al., 1996). However,
P. gingivalis is capable of multiple independent interventions on GEC sig-
nal transduction pathways as, in contrast to JNK, internalized P. gingivalis
cause dephosphorylation of ERK1/2 (Watanabe et al., 2001). In addition, P.
gingivalis does not induce activation and nuclear translocation of the eukary-
otic transcriptional activator NF-/cB. Such an ability to selectively activate and
suppress different components of related signaling pathways signifies a de-
gree of versatility and sophistication of P. gingivalis in its dealings with GEC,
g pointing toward a long evolutionary association between the two cell types.

§ The interactions between P. gingivalis and GEC are represented schematically

* in Fig. 10.2.

Q
Pi
<
X

3 KB (HeLa) cells

Jj Another cell type used to study of P. gingivalis invasion is the KB cell. Long

s thought to be derived from the oral epithelium, KB cells are now recognized

£ to be in fact HeLa cells that contaminated the original cell culture. Invasion

3 of these transformed epithelial cells by P. gingivalis is somewhat less efficient

° than GEC, with values less than 0.1% of the initial inoculum generally re-

ported (Duncan et al., 1993; Sandros et al., 1994; Njoroge et al., 1997). This
may be a consequence of the alterations in signal transduction pathways and
surface protein expression that accompany transformation. Initial adherence
to KB cells can be mediated by both cysteine proteases (Chen et al., 2001)
and FimA (Njoroge et al., 1997). In contrast to GEC, engulfment of bacteria
then occurs by classic receptor-mediated endocytosis (Sandros et al., 1996),
and bacteria can be found both free in the cytoplasm and contained within
membrane-bound vacuoles (Njoroge et al., 1997). Features in common with
GEC include the accumulation of P. gingivalis cells in the perinuclear region
(Houalet-Jeanne et al., 2001) and subsequent bacterial replication (Madianos
etal., 1996).

Endothelial cells

Invasion of bovine and human heart and aortic endothelial cells by P.
gingivalis has been established (Deshpande et al., 1998; Dorn et al., 1999).

P. gingivalis

Ca gated chamiel(s)

CM-

inlcgnn

secreted pntfems

■ *-6-j phosphatase

<....>

stores

MAP-kinase family r-"

Ca++

proteases

phosphatase

Disruption of nuclear transcription factor activity and
modulation of gene expression (e.g., IL-8, Bcl-2)

Figure 10.2. Model of currently understood P. gingivalis interactions with primary gingival
epithelial cells. P. gingivalis cells bind through adhesins such as fimbriae to integrins on
gingival cells. FAK and paxillin are recruited, and microtubules and microfilaments are
rearranged to facilitate invagination of the membrane that results in the engulfment of
bacterial cells. P. gingivalis rapidly locate in the perinuclear area where they replicate.
Calcium ions are released from intracellular stores, which may regulate calcium-gated
pores in the cytoplasmic membrane. Other signaling molecules such as the MAP-kinase
family can be phosphorylated/dephosphorylated or degraded. Gene expression in the
epithelial cells is ultimately affected. Abbreviations: Ca = calcium; CM = cytoplasmic
membrane; IL-8 = interleukin-8; MF = actin microfilaments; MT = tubulin

microtubules; NM = nuclear membrane; P = phosphate; ► is for a pathway with

potential intermediate steps; >• is for translocation; -► is for release; «■- — > is for

reversible association.

<
>

on
O

w
*<

£
o

t-H

Invasion requires remodeling of the microfilament and microtubule cyto-
skeleton through protein phosphorylation-dependent signaling (Deshpande
et al., 1998). Once inside the cells the bacteria are present in multimem-
branous vacuoles that resemble autophagosom.es (Dorn et al., 2001). These
vacuoles are positive for the early endosomal marker Rab5, and they rapidly
acquire HsGsa7p, required for the formation of the autophagosome. The
bacteria then traffic to a late autophagosome that contains both the rough
endoplasmic reticulum protein BiP and the lysosomal protein LGP120, but
that lacks cathepsin L and late endosomal markers (Dorn et al., 2001). In en-
dothelial cells, therefore, P. gingivalis evades the endocytic pathway leading to

Q
Pi
<
X
u
i— i

Pi
Q

N
<

1-1

I— I

w
i-i
N
:Q

Figure 10.3. Transmission electron microcopy (12,500x) of P. gingivalis captured by a
dendritic cell and located within multivesiculated compartments (arrowheads). (Image
provided by C. W. Cutler.)

lysosomes, and instead it traffics to the autophagosome, where development
of this organelle is impeded. Invasion of endothelial cells by P. gingivalis could
allow access to cells of vascular walls, where the induction of autophagocytic
pathways may alter the properties of the cells. The persistence of P. gingivalis
could then exacerbate the immune response along the vasculature. Invasion
of vascular endothelial cells may also provide a portal for bacterial entry into
the bloodstream with subsequent systemic spread.

Dendritic cells

Dendritic cells are antigen-presenting cells that can activate lymphocytes,
including, distinctively naive T cells (Banchereau and Steinman, 1998). Den-
dritic cells increase in number at sites of diseased oral epithelium that contain
intragingival bacteria. P. gingivalis can internalize within cultured dendritic
cells (Fig. 10.3), and invasion is associated with sensitization and activation
of the dendritic cells (Cutler et al., 1999). This process has parallels with

contact hypersensitivity responses, and hence such reactions may play a role
in periodontal diseases. The mechanistic basis for invasion in these cells has
yet to be investigated.

CONSEQUENCES OF P. GINGIVALIS INVASION
Epithelial cells

The presence of large numbers of intracellular P. gingivalis would, at first
glance, appear to represent an insult that could be terminal for the epithe-
lial cells. Indeed, after several hours of infection, GEC begin to shrink and
round up, features indicative of apoptotic cell death. However, the cells do
not detach from the substratum and remain capable of trypan blue exclusion,
calcein hydrolysis, and maintenance of physiologic intracellular calcium ion 5

concentrations (Bel ton et al., 1999). An examination of molecular markers &

o
of apoptosis showed that at early time points of P. gingivalis invasion there is 2

an increase in proapoptotic molecules such as Bax. However, after extended ^

incubation the Bax levels decline, and there is an increase in the expres- a

sion of the antiapoptotic molecule Bcl-2 (Nakhjiri et al., 2001). Furthermore, 2j

P. gingivalis is even capable of blocking apoptosis induced by the human §

topoisomerase I inhibitor, camptothecin. These results can be interpreted in %

terms in which the initial response of GEC to the P. gingivalis onslaught is one £

1-1

of activation of programmed cell death. P. gingivalis, however, having located §

in a nutritionally rich, immune-privileged site, blocks the cell death pathways g

in order to maintain its intracellular lifestyle. Furthermore, the proteases of &

P. gingivalis may protect the organism from /3-defensins, which are intra-
cellular antimicrobial peptides produced by epithelial cells (Devine et al.,
1999). Unconstrained replication of P. gingivalis may be prevented, how-
ever, by calprotectin, an S100 calcium-binding protein with broad-spectrum
antimicrobial activity produced inside epithelial cells (Nisapakultorn et al.,
2001).

The interaction between P. gingivalis and GEC is more than a life or death
struggle for supremacy and can result in more subtle phenotypic changes in
the host. Transcription and secretion of interleukin (IL)-8 (a potent neutrophil
chemokine) by GEC is inhibited following P. gingivalis invasion. Moreover,
P. gingivalis can antagonize IL-8 secretion following stimulation of epithelial
cells by common plaque commensals (Darveau et al., 1998). Reduced ex-
pression of epithelial cell intercellular adhesion molecule (ICAM)-l may also
contribute to downregulation of the innate host response (Madianos et al.,
1997). Regulation of matrix metalloproteinase (MMP) production by gingival

epithelial cells is disrupted following contact with P. gingivalis (Fravalo et al.,
1996), thus interfering with extracellular matrix repair and reorganization.

Endothelial cells

Although the ultimate metabolic fate of endothelial cells and their in-
ternalized organisms remains to be determined, it is becoming apparent
that many important properties of endothelial cells are modulated by P. gin-
givalis infection. Invasion by P. gingivalis leads to upregulation of ICAM-1
and vascular cell adhesion molecule (VCAM)-l, along with P- and E-selectins
(Khlgatian et al., 2002). The effect appears to be mediated through the major
fimbriae, as it can be blocked with fimbrial antibodies and does not occur with
a fimbriae-deficient mutant. Increased expression of these molecules can be
£ predicted to elevate the levels of leukocytes recruited to sites of P. gingivalis

^ infection. Levels of IL-8 and MCP-1 are also modulated in response to P.

ha gingivalis. In these cases, however, whole cells of P. gingivalis abolish normal

pj IL-8 and MCP-1 responses, whereas isolated outer membrane components

u and fimbrillin can stimulate production (Nassar et al., 2002). The inhibitory

Q effect of whole cells is not invasion dependent, as it occurs in the absence

< of detectable internalized P. gingivalis. Indeed, under certain conditions, en-

s dothelial cells can be induced to secrete MCP-1 by internal P. gingivalis (Kang

i-i

£ and Kuramitsu, 2002). Thus endothelial cell responses to P. gingivalis are

w complex and multithreaded, possibly a means for facilitating the long-term

: o association of the organism with the host cell.

RELEVANCE TO HEALTH AND DISEASE
Periodontal diseases

The pathogenesis of periodontal diseases involves multiple bacteria with
a range of virulence factors interfacing with a variety of host cells and im-
mune effector molecules. Within this framework, P. gingivalis invasion could
play a number of important roles in the disease process. An intracellular
location will shelter the bacteria from the ravages of the immune system,
and it may allow the organisms to increase in number to exceed a threshold
required to initiate disease. The phenotypic changes in epithelial cells that are
related to P. gingivalis invasion also have the potential to impinge on several
aspects of pathogenesis (Lamont and Yilmaz, 2002).

The gene encoding the neutrophil chemokine IL-8 is transcriptionally
downregulated by P. gingivalis even in the presence of otherwise stimulatory

organisms such as Fusobacterium nucleatum (Darveau et al., 1998; Huang
et al., 1998, 2001). In clinically healthy tissue, IL-8 forms a concentration
gradient that increases from the gingival tissue toward the surface (Tonetti et
al., 1994) and will thus direct neutrophils to sites of bacterial accumulation.
Hence, low level expression of IL-8 is considered to be important in ensuring
gingival health by controlling bacteria and preventing neutrophil-mediated
damage in the tissues. Inhibition of IL-8 accumulation by P. gingivalis at
sites of bacterial invasion could impede innate host defense at the bacteria-
epithelia interface, as the host would no longer be able to detect the presence
of bacteria and direct neutrophils for their removal. The ensuing overgrowth
of bacteria would then contribute to a burst of disease activity.

Nonetheless, host polymorphonuclear neutrophils and other defense
mechanisms do eventually become mobilized, as evidenced by the inflamma-
tory nature of P. gingivalis-as sociated periodontal diseases. The overgrowth of 5
subgingival plaque bacteria, or of P. gingivalis itself, that ensues after initial &
immune suppression may therefore reactivate the immune response. Indeed, z
epithelial cells can be induced to secrete IL-8 by P. gingivalis, depending on ^
the conditions of stimulation (Huang et al., 2001). Moreover, the encounter a
with different host cells as the infection progresses may also stimulate the im- 53
mune response. For example, P. gingivalis invasion of dendritic cells results 2
in maturation, increased costimulatory molecule expression, and stimula- %

tory activity for T cells (Cutler et al., 1999) . The migration and proliferation of £

i-i

P. gingivalis-speciFic effector T cells could be one means by which the im- j*

mune system gears up in periodontal disease (Saglie et al., 1987; Cutler et al., $

1999).

In addition to effects on immune modulators, invasion by P. gingivalis can
impinge on the activity of MMP enzymes. MMPs are members of a family of
zinc-dependent endopeptidases with a broad spectrum of proteolytic activity
that collectively can degrade all of the components of the extracellular matrix
(DeCarlo et al., 1998). MMPs are required for epithelial cell migration and to
detach the cells from the underlying matrix, and they are also involved in tis-
sue remodeling by facilitating the removal of damaged tissue. These activities
are important in maintenance of the integrity of the epithelial layer. How-
ever, destruction of the extracellular matrix, which is a feature of periodontal
lesions, may be caused by elevated MMP activity (Tonetti et al., 1994; DeCarlo
et al., 1997, 1998), and a higher percentage of tissues from periodontitis sites
have mRNA for MMPs as compared with tissue from healthy sites (Tonetti
et al., 1994). Control of MMP activity is, therefore, important in sustaining
gingival health. Invasion of P. gingivalis disrupts the expression of MMPs by
gingival epithelial cells (Fravalo et al., 1996), which could contribute both to

tissue destruction and to failure to repair a periodontal lesion. These activities
are distinct from, but probably complementary to, the direct action of pro-
teolytic enzymes that will be delivered in close proximity to their substrates
during the adhesion and entry process. Indeed, P. gingivalis proteinases can
activate and upregulate the transcription of MMP enzymes (DeCarlo et al.,
1997, 1998). Furthermore, P. gingivalis proteinases can degrade IL-8 and other
cytokines along with occludin, cadherins, catenins, and integrins (Fletcher
et al., 1997; Darveau et al., 1998; Yun et al., 1999; Zhang et al, 1999; Katz
et al., 2000), which are proteins that are important in maintaining the barrier
function of the epithelium.

Systemic diseases

g Although tissue destruction in periodontal diseases is limited to the sup-

§ porting structures of the teeth, epidemiological evidence is emerging for an

^ association between periodontal infections and serious systemic diseases,

§ including coronary artery disease (Scannapieco and Genco, 1999). Several

= observations provide a credible, though still preliminary, basis for a causal

* link between infections with periodontal organisms such as P. gingivalis

% and heart disease. P. gingivalis has been detected in carotid and coronary

< atheromas (Chiu, 1999; Haraszthy et al., 2000), and the organism can induce

~ platelet aggregation, which is associated with thrombus formation ( Herzberg

§ et al., 1994). Furthermore, P. gingivalis infection accelerates the progression

.g of atherosclerosis in a heterozygous apolipoprotein E -deficient murine model

(Li et al., 2002). Although common dental procedures, even vigorous tooth
brushing, can lead to the presence of oral bacteria in the bloodstream, it is
also possible that tissue and cell invasion by P. gingivalis in the highly vascu-
larized gingiva may be a means by which these bacteria can gain access to the
circulating blood and establish infections at remote sites. Once located at sites
such as the heart vessel walls, the invasion of endothelial cells (Deshpande
et al., 1998; Dorn et al., 1999) could constitute a chronic insult to arte-
rial walls that may increase susceptibility to tissue damage. In addition,
modulation of factors that regulate recruitment of leukocytes (ICAM-1,
VCAM-1, MCP-1, and selectins) could enhance arthrosclerosis progression
(Kuramitsu et al., 2001; Khlgatian et al., 2002; Nassar et al., 2002). For exam-
ple, monocytes migrating through the endothelial layer into the subendothe-
lial spaces may produce foam cells (Kang and Kuramitsu, 2002), a feature of
both early and late artherosclerotic lesions. P. gingivalis can also directly in-
duce foam cell production in a murine macrophage cell line (Kuramitsu et al.,
2001).

It is likely that we are only beginning to uncover the full range of con-
sequences of the interactions between invasive oral bacterial and host cells.
It is also important to consider that P. gingivalis is present in the mouths
of healthy individuals and can be found in high numbers inside epithelial
cells (Rudney et al., 2001). An intracellular location may initially serve as an
integral part of the interactions that maintain a balance between host and
pathogen. Disruption of this balance, either by the action of the internal bac-
teria or by other pathogen or host factors, may be required to activate the
disease process.

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Index

Acanthamoeba castellanii, 135, 142-143, 145
Acanthamoeba polyphaga, 126, 135, 145-146
Actinobacillus actinomycetemcomitans, 275-285

actinobacillin, 278

adhesion, 275-278

adhesion, Aae adhesin and, 279, 285

adhesion, conveyed, 278

adhesion, specific to epithelial cells, 279

autotransporter genes, 279

bone resorption activity, 278

calcium, 279, 285

CD14, 286

cell-cell spread, 283

colony phase variation, 275

egression, from host cell, 281

endocytic pathway, classical, 282

endotoxin, 278

epithelial cells (micrograph), 280

extracellular amorphous material
(ExAmMat), 278

fimbriae, 276-277, 279

IL-8 and, 286

integrins, 282

internalization process, 278, 281-283

intracellular-location mechanisms, 283

invasion, 283-285

invasion gene homologs, E. coli, 284

invasion, host response to, 286

invasion/pathogenicity correlation, 287

lactoferrin, 279

leukotoxin, 278, 282, 285

Listeria, 283

motor proteins/kinesin-like entity,

283-285
nitric oxide (NO), 287
periodontitis, episodic nature of, 276, 287
phenotype /variant shifts, 287
phenotypes (micrographs of), 276
phospholipase C (PLC), 282, 285
RGD, 283-284

rough colony phenotypes, 275-276
serotype-specific polysaccharide antigen

(SPA), 286
Shigella, 283

signaling pathways of host cell, 279-281
smooth colony phenotypes, 275-276
staurosporin, 281
surface-associated molecules /organelles,

276
transferrin receptor, 282, 285
type-IV pilin, 277
vesicles, 277-278
ampicillin, 2
apoptosis

bundle-forming pili (BFP), 104

cancer, xii

dendritic cells and, 163

dot/icm genes, 125-126

Escherichia coli, 103-105

gingival epithelial cells (GEC), 299

Group A streptococci, 244, 246-247, 259,

261

X
w

apoptosis (cont.)

Legionella pneumophila, 144-145,
149-150

Legionella pneumophila (figure), 140

Listeria monocytogenes, 163

macrophages, 74-75, 125-126

necrosis, distinguished from, 146

Neisseria gonorrhoeae, 211

porin, 211

Porphyromonas gingivalis, 305

REPEC, 104

Shigella, 25-26

Yersinia, inhibited by, 72-73

Yersinia outer proteins (Yop), 74-75
arp2/3 complex, 43, 45

Escherichia coli, 95, 97-98

Listeria monocytogenes, 178-179

profilin, 48

Shigella, 38, 45-47

See also N-WASP; VirG-N-WASP
complex
arthritis-dermatitis syndrome, 205
asialoglycoprotein receptor (ASGP-R), 213,

222, 227-230
attaching/effacing (A/E) lesions, 90-91,
93-94, 99, 102-103

bacteria

minimizing damage to host, xi-xii

number on human body, xi
bacteroides-epithelium development

interactions, xii
blood-brain barrier, 163-164
Boswell, 203

bubonic plague, 59, 65. See also Y. pestis
bundle-forming pili (BFP)

apoptosis, 104

Escherichia coli, 88-91, 93, 104

Clq, 170

C3, 170, 218-220, 223-224, 231-233

C4, 219-220

C5 through C8, 220

C9, 219-220

calcium

Actinohacillus actinomycetemcomitans, 279,
285

attaching /effacing (A/E) lesions, 102-103

Escherichia coli, 102-103

neutrophils, 72

Porphyromonas gingivalis, 302, 305

YopH, 72
calprotectin, 305
cancer, xii
CD4, 147-149
CD8, 147-149
CD14, 286
CD18, 17, 220, 232
CD44, 33-35, 241, 243, 247
CD46, 209, 220, 241, 243, 252
CD62, 255
CD66, 220
CD111, 255
Cdc42, 43

EPEC, 105-106

GTPase activating proteins, 14

IpaC, 37, 38

N-WASP, 46-47

Salmonella, 11-14

Shigella, 37-38, 46-47

sopB, 14

sopE, 13-14

sopE/E2, 14-15

SptP, 14-15

VirG, 46-47

Yersinia, 67-70
Cdkl, 99

cephalosporins, 2
chlorine, 127-128
complementation

complement receptors, 170

See also specific components, receptors
conjunctivitis, 164
CR1, 211
CR3, 170, 209, 211, 219-220, 223-224, 231,

233
CR4, 220
cystic fibrosis transmembrane regulator

(CFTR), 102
cytoskeletal structures. See specific molecules,
species

defect in organelle trafficking (dot) genes. See
dot/icm genes

dendritic cells

apoptosis of, 163

CNS infection, 163-164

Listeria monocytogenes, 163-164

Porphyromonas gingivalis, 296, 304-305,
307

Salmonella, 17

sampling of bacteria, 17
Dictyostelium discoideum, 135
dot/km genes, 125, 131-132, 135-139,
143-144, 150

DNA regulatory elements of, 143-144

macrophages and, 125-126

macropinocytosis, 125-126

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