MiniReview

Shigella spp. and enteroinvasive Escherichia coli pathogenicity factors

Claude Parsot

*

Unite´ de Pathoge´nie Microbienne Mole´culaire, INSERM U389, Institut Pasteur, 25 rue du Dr Roux, 75724 Paris Cedex 15, France

Received 23 June 2005; accepted 17 August 2005

First published online 15 September 2005

Edited by I. Henderson

Abstract

Bacteria of Shigella spp. (S. boydii, S. dysenteriae, S. flexneri and S. sonnei) and enteroinvasive Escherichia coli (EIEC) are

responsible for shigellosis in humans, a disease characterized by the destruction of the colonic mucosa that is induced upon bacterial
invasion. Shigella spp. and EIEC strains contain a virulence plasmid of

220 kb that encodes determinants for entry into epithelial

cells and dissemination from cell to cell. This review presents the current model on mechanisms of invasion of the colonic epithelium
by these bacteria and focuses on their pathogenicity factors, particularly the virulence plasmid-encoded type III secretion system.
Ó 2005 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.

Keywords: Invasion; Secretion; Chaperone; Regulation; Pathogenesis; Virulence; Plasmid; Type III secretion

1. Introduction

Bacteria of Shigella spp. and enteroinvasive Esche-

richia coli (EIEC) are responsible for shigellosis in hu-
mans. The burden of this disease was estimated to 150
million cases and 1 million deaths per year in the devel-
oping world

[1]

. Shigellosis is characterized by the

destruction of the colonic epithelium provoked by the
inflammatory response that is induced upon invasion
of the mucosa by bacteria. Shigella is divided in four
groups (or species), S. boydii, S. dysenteriae, S. flexneri
and S. sonnei. However, these bacteria are so closely re-
lated to each other and to E. coli strains that, in fact,
they all belong to the species E. coli

[2]

. Most studies

have been conducted on S. flexneri and, notwithstand-
ing strain specificities, results probably apply to other
Shigella spp. and EIEC. As compared to commensal
and other pathogenic E. coli strains, characteristic fea-
tures of Shigella spp. and EIEC strains are the presence
of a virulence plasmid (VP) of

220 kb and their ability

to induce their entry into epithelial cells and disseminate
from cell to cell. This review briefly presents the model
on mechanisms of invasion of the epithelium by bacteria
of Shigella spp. and focuses on their pathogenicity fac-
tors, particularly the VP-encoded type III secretion
(TTS) system. This system consists of a TTS apparatus
(TTSA) that spans the bacterial envelope, translocators
that transit through the TTSA and insert into the host
cell membrane to form a pore (translocon), effectors that
transit through the TTSA and the translocon and are in-
jected within the eukaryotic cell, chaperones that bind to
translocators and some effectors prior to their transit
through the TTSA, and transcription activators. Due
to space limitations, priority for references was given
to recent publications in which readers will find refer-
ences to older, but not less interesting, works.

2. Pathogenesis

The current model on mechanisms of pathogenesis

induced by bacteria belonging to Shigella spp. are

0378-1097/$22.00

Ó 2005 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.

doi:10.1016/j.femsle.2005.08.046

*

Tel.: [phone]; fax: [phone].
E-mail address:

[e-mail]

.

www.fems-microbiology.org

FEMS Microbiology Letters 252 (2005) 11–18

derived from in vitro and in vivo studies using various
cell types (epithelial cells, macrophages, monocytes,
fibroblasts and red blood cells) and animal models of
infection, such as the cornea in guinea pigs, ligated ileal
loops in rabbits and lungs in mice, in which bacteria in-
duce an inflammatory response leading to destruction of
the corresponding epithelium. In the colonic mucosa,
bacteria are proposed to cross the epithelial layer by
invading M cells overlaying lymphoid follicles, which al-
lows them to reach the basolateral pole of epithelial cells
where they induce their uptake (

Fig. 1

). Entry into epi-

thelial cells involves rearrangements of the cell cytoskel-
eton that extend beyond the zone of contact between the
bacterium and the cell membrane, leading to membrane
ruffling and engulfment of the bacterium within a vacu-
ole

[3]

. Once internalized by epithelial cells, bacteria rap-

idly lyse the membrane of the entry vacuole and gain
access to the cell cytoplasm where they multiply with a
generation time of

40 min. By inducing actin polymer-

ization at one of their poles, intracellular bacteria move
within the cytoplasm of infected cells. This movement
generates the formation of protrusions that contain
one bacterium at their tip and are engulfed by adjacent
epithelial cells, thereby allowing bacteria to disseminate
from cell to cell without being exposed to the external
milieu. Peptidoglycan fragments released by intracellu-
lar bacteria are detected by the Nod1 pathway, leading
to phosphorylation and degradation of IjB, transloca-
tion of NF-jB to the nucleus and activation of NF-jB
regulated genes

[4,5]

. Analysis of the transcriptome of

infected epithelial cells showed, in particular, increased
expression of the gene encoding IL-8, a potent chemoat-
tractant for neutrophils

[6]

. Thus, epithelial cells actively

participate in the detection and signaling of invasive
bacteria to host defences. Bacteria released from M cells
(after their initial uptake) or epithelial cells (after intra-

cellular multiplication) interact with macrophages, es-
cape

from

the

phagocytic

vacuole

and

induce

apoptosis of infected cells. Apoptotic macrophages re-
lease pro-inflammatory cytokines, including IL-1 and
IL-18, which, together with IL-8 released from infected
epithelial cells, leads to recruitment of polymorphonu-
clear cells (PMN) at the site of infection. Transmigration
of PMN destabilises the epithelial barrier and facilitates
further invasion by luminal bacteria. Interactions of
bacteria with host cells, innate and adaptive immune re-
sponses induced upon infection and vaccine develop-
ments are presented in recent reviews

[7–9]

.

3. Chromosomal pathogenicity islands and black holes

The genomic sequence of two S. flexneri 2a strains,

CP301 and 2457T, was recently determined

[10,11]

.

Genomes of these strains are very similar in size
(4 Mbp), sequence and organization. As compared to
E. coli K-12 strain MG1655, major differences are (i)
the number of insertion sequences (IS), with 400 com-
plete or partial IS in CP301 vs. 40 in MG1655; (ii) the
number of genes that are inactivated or missing, 900
genes of MG1655 being absent or inactivated by frame-
shift mutations or insertion of IS in 2457T; and (iii) the
presence of 195 genes specific to S. flexneri. Most spe-
cific genes are carried by islands that either encode an
integrase, are located at tRNA sites, or are associated
with IS or prophage remnants. A number of these genes
encode proteins predicted to be membrane-associated or
secreted or exhibiting similarities to adhesins. Genes car-
ried by islands involved in pathogenesis include sit genes
encoding an iron uptake system

[12]

, sigA encoding an

extracellular protease

[13]

, gtr genes encoding proteins

involved in glucosylation of the O antigen

[14]

, shiA

Fig. 1. Model of pathogenesis induced by Shigella spp. Bacteria cross the epithelium barrier by entering into M cells (1). They are delivered to
resident macrophages, in which they induce apoptosis (2), and reach the basolateral pole of epithelial cells (3), in which they induce their entry (4).
Movement of intracellular bacteria (5) leads to the formation of protrusions and dissemination of bacteria within the epithelium (6). Release of
cytokines and chemokines, including IL-1 by apoptotic macrophages (A) and IL-8 by infected enterocytes (B), promotes recruitment of monocytes
that migrate through the epithelial barrier (C), facilitating entry of luminal bacteria into epithelial cells (D) and increasing invasion of the epithelium
(E).

12

C. Parsot / FEMS Microbiology Letters 252 (2005) 11–18

encoding a membrane-associated protein of unknown
function

[15]

and stx genes encoding the Shiga toxin

(in S. dysenteriae strains). Many chromosomal genes
that are not specific of Shigella spp. are required for
virulence, including those encoding the transcription ter-
mination factor Rho, the DNA topoisomerase I, nucle-
oid-associated proteins H-NS and FIS

[16]

, tRNA

modifying enzymes

[17]

, the superoxide dismutase SodB,

the protease DegP, the outer membrane protein OmpC,
the periplasmic disulfide bond catalyst DsbA and pro-
teins involved in lipopolysaccharide biosynthesis or cell
division.

4. Overview of the VP

Sequence analysis of pWR100 and pCP301 from S.

flexneri 5 and 2a strains, respectively, indicated that
the VP is composed of a mosaic of

100 genes and

numerous IS, these latter representing one-third of the
VP

[10,18,19]

. The 30-kb region (designated the entry

region) essential for entry into epithelial cells carries
genes encoding components of a TTSA, translocators
and some effectors, chaperones and transcription activa-
tors (

Fig. 2

). Genes encoding other effectors are scat-

tered on the VP. The TTS system comprises 50 genes
exhibiting a similarly low G+C content (34% G+C),
indicating that it was acquired by lateral transfer. Two
gene clusters exhibit a G+C content of 42%, one encod-
ing IcsA/VirG (involved in movement of intracellular
bacteria), a truncated PapC, UshA (a periplasmic
UDP-sugar hydrolase) and PhoN1 (a periplasmic acid
phosphatase) and the other encoding Orf185 and
Orf186 (of unknown function), VirK (involved in pro-
duction or localization of IcsA) and MsbB2 (an acyl

transferase modifying the lipid A). The organization
and G+C content of these two regions suggest that they
were acquired from the same source, different from the
entry region, and that they initially encoded Pap pili.
The sepA gene encoding a secreted protease has a
G+C content of 49%, suggesting that it came from a
different source. The VP contains several gene clusters
involved in plasmid replication, partition and post seg-
regation killing and an incomplete transfer region. The
presence of two seemingly intact and one incomplete
partition systems exhibiting G+C contents that are dif-
ferent from one another and different from that of the
replication region suggests that the VP carries elements
from four different plasmids

[18]

. pWR100 and pCP301

differ by the presence or absence 16 fragments, most of
which correspond to IS and deletions involving IS or di-
rect repeats. This suggests that the common ancestor of
these two VP contained more intact genes and less IS.
Genes detected on one VP are not present on all VP:
impCAB genes of pCP301 are absent on pWR100 and
sepA and several other genes of pCP301 and pWR100
were not detected on the VP of a S. flexneri 6 strain

[20]

. Many S. flexneri strains carry two small plasmids,

one of which encodes the Cld protein controlling the
number of O antigen repeats, and often more than
one large plasmid. For example, a 165-kb plasmid
exhibiting 99.7% identity with plasmid R27 from Salmo-
nella enterica serovar Typhi was present in strain 2457T
used for sequencing

[11]

.

5. Expression of VP genes

The regulation of expression of VP genes has been

extensively studied

[21]

. Transcription of genes of the

Fig. 2. Genetic map of the entry region and electron microscopy analysis of the TTSA. Top panel: schematic genetic map of the 30-kb entry region
(in two parts); genes encoding transcription activators are in purple, effectors in red, chaperones in blue, components of the needle complex in green
and innermembrane proteins in yellow. Bottom panel: structural analysis of the TTSA; (A) negative staining of one TTS apparatus on osmotically
shocked bacteria; (B) deduced projection density map of images as shown in A; (C) negative staining of one purified needle complex; (D) average
image of the major class of needle complexes (as shown in C); (E) surface representation of the volume of the needle complex, assuming cylindrical
symmetry (adapted from

[32,34]

).

C. Parsot / FEMS Microbiology Letters 252 (2005) 11–18

13

entry region is regulated by temperature and under
the control of two VP-encoded proteins, VirF, a mem-
ber of the AraC family of transcription activators, and
VirB, a member of the ParB family of partition pro-
teins. Binding of H-NS to virF and virB promoters
prevents transcription of these genes at 30

°C

[22]

.

At 37

°C, changes in DNA conformation lead to in-

creased transcription of virF and activation of the virB
promoter by VirF and production of VirB

[23]

. Acti-

vation of VirB-dependent promoters, including those
of the entry region, might involve oligomerization of
VirB and displacement of H-NS

[24]

. In addition to

genes of the entry region, VirB controls transcription
of

15 VP genes

[25]

, most of which are part of the

TTS system. Inactivation of orf81 encoding a tran-
scription activator of the AraC family did not affect
expression of any VP gene

[25]

. Since orf81 is inacti-

vated or absent in other S. flexneri strains, this gene
might be a remnant of a regulatory circuit that is
no longer used.

The TTSA assembled by bacteria growing in broth at

37

°C is only weakly active and is activated upon contact

of bacteria with epithelial cells (and deregulated in some
mutants). TTSA activation (or deregulation) leads to in-
creased transcription of 12 VP genes encoding TTSA
substrates

[25–28]

. Increased transcription of these genes

in conditions of secretion is controlled by MxiE, a tran-
scription activator of the AraC family produced by tran-
scriptional frameshifting from two overlapping ORF

[29]

. The cis-acting element required for increased tran-

scription of MxiE-controlled promoters is the 17-bp
MxiE box (GTATCGTTTTTTTANAG) located be-
tween positions

49 and 33 with respect to the tran-

scription start site

[30]

. There are 9 MxiE boxes on the

VP and MxiE boxes are present in the 5

0

region of chro-

mosomal ipaH genes encoding TTSA substrates

[25]

. To

be active, MxiE requires IpgC, the chaperone of the
translocators IpaB and IpaC, acting as a co-activator

[27]

. In conditions of non-secretion, IpaB and IpaC

are associated independently with IpgC, which titrates
IpgC. In addition, MxiE is associated with the TTSA
substrate OspD1 and its chaperone Spa15, which pre-
vents MxiE from being activated by free IpgC

[31]

.

Upon TTSA activation, transit of IpaB and IpaC liber-
ates IpgC, but this is not sufficient to activate MxiE as
long as OspD1 is present in the cytoplasm. Following
transit of OspD1 through the TTSA, MxiE is liberated
and an interaction between MxiE and IpgC would
allow MxiE to bind to and activate transcription at
MxiE-controlled promoters. On the basis of their
expression profiles, TTSA substrates can be classified
into three categories (

Fig. 3

): (i) those that are expressed

independently of the TTSA activity; (ii) those that are
expressed in conditions of non-secretion and induced
in conditions of secretion; and (iii) those that are
expressed only in conditions of secretion

[25]

.

6. The TTS apparatus

The TTSA encoded by mxi and spa genes of the entry

region has been visualized by transmission electron
microscopy on osmotically shocked bacteria

[32,33]

and scanning electron microscopy on intact bacteria

[14]

. The core of the TTSA, designated the needle com-

plex, consists in a needle, composed of MxiH and MxiI,
and a platform, composed of MxiG, MxiJ, MxiD and
presumably MxiM

[33,34]

(

Fig. 2

). The platform can

be assembled in the absence of MxiH and MxiI, but is
not functional, i.e., unable to secrete TTSA substrates.
In the needle, MxiH subunits are arranged in a helical
fashion to form a 500-A

˚ cylindrical structure with an

outer diameter of 70 A

˚ and a central channel of 20 A˚

[35]

. Inner membrane proteins MxiA, Spa9, Spa24,

Spa29 and Spa40 are not present in the needle complex,
the purification of which involves extraction with deter-
gents. Incorporation of MxiH and MxiI into the needle
completes the TTSA and requires Spa47, the ATPase
providing energy for transit of substrates through the
TTSA. Steady-state amounts of MxiH and MxiI are re-
duced in secretion defective mutants, indicating that, in
contrast to other TTSA substrates stored when the
TTSA is not active, needle components are degraded
when they are not delivered to the TTSA. MxiK and
MxiN, both of which interact with Spa47, are required
for transit of needle components, but not Ipa proteins
(when the TTSA is artificially assembled by overexpres-
sion of MxiH or MxiI in mxiK or mxiN mutants)

[36]

.

Inactivation of spa32 led to assembly of needles that
were two times longer than those of the wild-type strain
and inability to secrete Ipa proteins upon TTSA activa-
tion

[37,38]

, suggesting that Spa32 is required to control

the needle length and switch the TTSA substrate speci-
ficity from needle components to other substrates.
Spa32, Spa33 and MxiC transit through the TTSA.
Analysis of a spa33 mutant indicated that Spa33 is re-
quired for IpaB and IpaC secretion, but the involvement
of Spa33 in completion of the needle complex is not
known

[39]

. Sequence similarities suggest that, like

YopN of pathogenic Yersinia spp., MxiC might be in-
volved in control of TTSA activity.

The TTSA is activated upon contact of bacteria with

epithelial cells or exposure to the dye Congo red and is

Fig. 3. Repertoires of TTS effector genes controlled by VirB and/or
MxiE. Transcription of genes controlled by VirB is independent upon
the TTSA activity, whereas transcription of genes controlled by MxiE
is activated (or increased) in condition of secretion (adapted from

[25]

).

14

C. Parsot / FEMS Microbiology Letters 252 (2005) 11–18

deregulated, i.e., constitutively active, by inactivation of
ipaB or ipaD. The mechanism by which IpaB and IpaD
control TTSA activity is not known, one possibility
being that these proteins are required to form a complex
that plugs the TTSA. It was recently shown that choles-
terol-containing membranes trigger TTSA activity

[40]

.

Genes whose expression is regulated by TTSA activity
are induced upon entry into epithelial cells and no
longer transcribed in bacteria growing in the intracellu-
lar compartment, suggesting that TTSA activity is
turned on during entry and off in the cell cytoplasm

[26]

. The involvement of the TTS system in lysis of the

cell membrane that surrounds bacteria in protrusions
during cell-to-cell spread suggests that the TTSA is re-
activated in protrusions

[41,42]

.

7. TTS chaperones, translocators and effectors

Due to regulation of TTSA activity by external sig-

nals, TTSA substrates are stored in the bacterial cyto-
plasm prior to their transit through the TTSA.
Dedicated chaperones stabilize translocators and some
effectors and/or maintain them in a secretion-competent
state while they are stored in the bacterial cytoplasm

[43]

. Four chaperones are encoded by the VP, (i) IpgA,

the chaperone for IcsB

[44]

; (ii) IpgE, the chaperone for

IpgD

[45]

; (iii) Spa15, the chaperone for IpaA, IpgB1,

OspB, OspD1, OspC2 and OspC3

[46,47]

; (i) and IpgC,

the chaperone for IpaB and IpaC

[48]

. IpgC and Spa15

are also involved in the regulatory mechanism by which
TTSA activity controls transcription of a set of genes
encoding effectors

[27,31]

.

Translocators are proposed to be IpaB and IpaC as

the sequence of these proteins exhibits two hydrophobic
regions and purified IpaB and IpaC interact with lipid
membranes

[49,50]

and both proteins are essential for

contact hemolytic activity (which reflects the ability of
bacteria to insert a pore of a defined size in the mem-
brane of erythrocytes) and associate with the membrane
of erythrocytes

[32]

. IpaB interacts with numerous part-

ners, probably in a sequential order: it binds to IpgC in
the bacterial cytoplasm and probably to IpaD within the
TTSA, forms trimers

[50]

and associates with IpaC in

the external milieu. It also interacts with CDC44 on
the cell surface

[51]

, cholesterol in the cell membrane

[52]

and caspase-1 in the cytoplasm of infected macro-

phages

[53]

. Although not part of the translocon, IpaD

is probably required for insertion of IpaB and IpaC in
cell membranes

[54]

. IpaB and IpaC are required for ly-

sis of the membrane of the vacuole surrounding bacteria
internalized by macrophages and epithelial cells and the
membrane of protrusions during cell-to-cell spread

[41,42]

.

Effectors are defined as proteins that, via the TTSA

and the translocon, are injected into the cell, where they

affect cellular functions. Binding of IpaB to caspase-1
and a direct role of IpaC in promoting cellular signaling
cascades that lead to internalization of bacteria in epi-
thelial cells

[55]

suggest that translocators are also en-

dowed with effector functions. The repertoire of
effectors, identified by N-terminal sequencing of pro-
teins secreted by a TTSA-deregulated strain and se-
quence analysis of the virulence plasmid and, more
recently, the chromosome consists in

25 proteins en-

coded by the VP and 3–5 proteins encoded by the chro-
mosome (depending on strains). These latter belong to
the IpaH family and, so far, there is no evidence that
the chromosome encodes other TTSA substrates. Mem-
bers of the IpaH family are characterized by a variable
N-terminal domain containing 7–10 repeats of a LRR
motif and a constant C-terminal domain. In addition
to IpaH proteins, several VP-encoded effectors belong
to multigene families, with three ospC genes, three ospD
genes, two ospE genes and two ipgB genes

[18]

.

The roles of effectors are starting to be elucidated, but

their exact function, i.e., enzymatic or binding activities,
remains elusive in most cases. IpgD specifically dephos-
phorylates

phosphatidyl-inositol

4,5-biphosphate

(PtdIns(4,5)P2) into phosphatidyl-inositol 5-phosphate
upon entry of bacteria within epithelial cells, which
uncouples the plasma membrane from the cellular cyto-
skeleton

[56]

. IpaA binds to the focal adhesion protein

vinculin, which promotes depolymerization of actin fila-
ments

[57]

. IpgB1, directly or indirectly, stimulates activ-

ities of Rho GTPases Rac1 and Cdc42, contributing to
the formation of membrane ruffles during entry

[58]

.

VirA binds ab-tubulin dimers and destabilizes microtu-
bules, which also leads to Rac1 activation and forma-
tion of membrane ruffles

[59]

. IpgD, IpaA, IpgB1 and

VirA facilitate the formation of entry structures, indicat-
ing that several effectors act synergistically to promote
internalization of bacteria. IcsB was shown to interact
with IcsA (VirG) and this interaction was proposed to
prevent association of IcsA with the autophagy protein
Atg5

[60]

.

8. Other VP genes involved in pathogenicity

In addition of the TTS system, the VP carries several

genes involved in pathogenicity. icsA (virG) encodes the
outer membrane protein directly responsible for pro-
moting the movement of intracellular bacteria. IcsA
binds the Neural Wiskott-Aldrich syndrome protein
(N-WASP), leading to the formation of a complex with
Arp2/3 that induces actin polymerization

[61,62]

. IcsA is

asymmetrically distributed on the bacterial surface and
enriched at the old pole of the bacterium

[63]

. A certain

proportion of IcsA is released into the culture medium
following cleavage by the VP-encoded outer membrane
protease IcsP (SopA)

[64,65]

. Inactivation of virK led

C. Parsot / FEMS Microbiology Letters 252 (2005) 11–18

15

to a decreased production of IcsA by an unknown mech-
anism

[66]

. The sepA gene encodes a secreted serine pro-

tease of the IgA protease family that, like IcsA, is
transported across the outer membrane by an autotrans-
porter C-terminal domain. Inactivation of sepA in S.
flexneri 5 led to an attenuated phenotype upon infection
of rabbit ileal loops

[67]

. The absence of sepA in other S.

flexneri strains

[20]

suggests that a protein functionally

equivalent to SepA might be encoded by the chromo-
some or the VP in some strains. The VP msbB2 gene
and the chromosomal msbB1 gene encode acyl transfer-
ases that catalyze the acyl–oxyacyl linkage of a myristate
on the hydroxy-myristate attached to the 3

0

position of

glucosamide disaccharide in lipid A. This modification
contributes to lipid A toxicity. Both MsbB1 and MsbB2
are required for the modification of all lipid A molecules
and mutants in either msb1 or msbB2 are attenuated

[68]

.

9. Concluding remarks

Invasion of the colonic epithelium by bacteria in-

volves various interactions between bacteria, both extra-
and intracellular, and different cell types. Moreover,
these interactions, their outcomes and their conse-
quences are evolving during infection, as cell popula-
tions are changing, in nature, relative numbers and
states of activation. Likewise, external signals influence
repertoires of bacterial genes that are expressed by ex-
tra- and intracellular bacteria. Thus, infection is far
more complex than what is studied using homogenous
populations interacting in vitro. Nevertheless, in vitro
models of infection, imperfect as they are, have been
essential in deciphering some events that take place dur-
ing infection. In many cases, in vitro results have been
validated using animal models, mostly infection of li-
gated loops in rabbits and lungs in mice. However, we
still have much to learn on the functions of bacterial vir-
ulence factors and the responses of infected cells.

Comparison of S. flexneri 2a and E. coli K-12 gen-

omes indicates that many genes are inactivated or miss-
ing in S. flexneri strains. Inactivation of these genes
suggests that their products were detrimental to invasive
bacteria. Indeed, introduction of some E. coli K-12
genes, such as ompT and cadA, into S. flexneri interfered
with some virulence traits

[69,70]

. Acquisition, or assem-

bly, of the VP allowed an ancestral E. coli strain to
explore a new environment, but exploiting this ‘‘oppor-
tunity’’ required numerous adjustments of the chromo-
some. Understanding why these genes have been
inactivated might shed light on metabolic constraints
imposed by an intracellular lifestyle. On the other hand,
we start to have ideas on functions of genes carried by
pathogenicity islands and the VP. However, as exempli-
fied by stx genes present only in S. dysenteriae 1 strains

or by orf81 present on pWR100 and inactivated on
pCP301, genes specific to particular strains or isolates
might not be of general relevance to the invasive behav-
iour. Ongoing sequencing projects for S. dysenteriae and
S. sonnei strains should lead to a better evaluation of
genes that are conserved, or inactivated, between mem-
bers of Shigella spp. and, likewise, comparison of several
VP should help to understand the history of the VP. The
TTS system is central to the pathogenicity of Shigella
spp. and EIEC strains, being essential for entry of bac-
teria in epithelial cells and killing of infected macro-
phages and dendritic cells. The large repertoire of
effectors suggests that the TTS system is used for addi-
tional functions, possibly to modulate cell responses in-
duced by invading bacteria

[71]

. The differential

regulation of genes encoding TTS effectors suggests that
different effectors might be required at different times
following contact of bacteria with host cells. Elucidating
the function of these effectors and their role(s) at various
stages of infection is a challenging prospect.

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