Antonie van Leeuwenhoek 74: 191–197, 1998.
© 1998 Kluwer Academic Publishers. Printed in the Netherlands.

191

Molecular bases of epithelial cell invasion by Shigella flexneri

Philippe J. Sansonetti

∗

& Coumaran Egile

Unit´e de Pathog´enie Microbienne Mol´eculaire, INSERM U 389, Institut Pasteur, 28 rue du Dr Roux, F-75724 Paris
C´edex 15, France (

∗

Author for correspondence: E-mail: [e-mail])

Received 6 February 1998; accepted 20 May 1998

Key words: epithelial cell invasion, Shigella flexneri, pathogenesis

Abstract

The pathogenesis of shigellosis is characterized by the capacity of the causative microorganism, Shigella, to invade
the epithelial cells that compose the mucosal surface of the colon in humans. The invasive process encompasses
several steps which can be summarized as follows: entry of bacteria into epithelial cells involves signalling path-
ways that elicit a macropinocitic event. Upon contact with the cell surface, S. flexneri activates a Mxi/Spa secretory
apparatus encoded by two operons comprising about 25 genes located on a large virulence plasmid of 220 kb.
Through this specialized secretory apparatus, Ipa invasins are secreted, two of which (IpaB, 62 kDa and IpaC,
42 kDa) form a complex which is itself able to activate entry via its interaction with the host cell membrane. Inter-
action of this molecular complex with the cell surface elicits major rearrangements of the host cell cytoskeleton,
essentially the polymerization of actin filaments that form bundles supporting the membrane projections which
achieve bacterial entry. Active recruitment of the protooncogene pp 60

c

−src

has been demonstrated at the entry site

with consequent phosphorylation of cortactin. Also, the small GTPase Rho is controlling the cascade of signals
that allows elongation of actin filaments from initial nucleation foci underneath the cell membrane. The regulatory
signals involved as well as the proteins recruited indicate that Shigella induces the formation of an adherence plaque
at the cell surface in order to achieve entry. Once intracellular, the bacterium lyses its phagocytic vacuole, escapes
into the cytoplasm and starts moving the inducing polar, directed polymerization of actin on its surface, due to the
expression of IcsA, a 120 kDa outer membrane protein, which is localized at one pole of the microorganism,
following cleavage by SopA, a plasmid-encoded surface protease. In the context of polarized epithelial cells,
bacteria then reach the intermediate junction and engage their components, particularly the cadherins, to form
a protrusion which is actively internalized by the adjacent cell. Bacteria then lyse the two membranes, reach the
cytoplasmic compartment again, and resume actin-driven movement.

Introduction

Each year, at least a billion cases of diarrhoeal diseases
account for about three million deaths. Shigellosis is a
bloody diarrhoea caused by the invasion of the colonic
and rectal mucosa by members of the genus Shigella,
a gram-negative bacteria belonging to the family En-
terobacteriaceae. In the developing world, children
are the major victims with 600,000 deaths every year.
The symptoms are characterized by initial watery di-
arrhoea rapidly followed by fever, violent intestinal
cramps, and emission of muco-purulent and bloody
stools. Immediate complications may occur, such as

severe hypoglycemia, seizure, toxic megacolon which
may cause deadly sepsis, and the hemolytic uremic
syndrome (HUS). A chronic enteropathy may also be
observed in children with delayed treatment.

There are four species of Shigella,

(namely

Shigella boydii, Shigella dysenteriae, Shigella flexneri
and Shigella sonnei). The endemic form of the disease,
which occurs worldwide, is caused by S. sonnei and S.
flexneri (essentially serotypes 1 and 2). The epidemic
form of the disease which accounts for deadly out-
breaks in the developing world is caused by S. dysente-
riae 1, the Shiga bacillus which expresses Shiga toxin,
a potent cytotoxin. Shigellae are highly contagious

anto1021.tex; 7/12/1998; 10:43; p.1

MENNEN/SCHRIKS:Typeset: Pips Nr.:181091; Ordernr.:234000-mc (antokap:bio2fam) v.1.1

192

Figure 1. Scanning electron microscopy. Shigella entry focus into a
HeLa cell. (Photo by Roger Webf and Ariel Blocker, EMBL).

microorganisms which are transmitted directly from
person to person by hand contact, or indirectly by con-
taminated food or water. High infectious capacity and
rapid occurrence of multiple resistance to antibiotics
make prevention and treatment of shigellosis a difficult
task. No vaccine is yet available.

Shigella flexneri invades the human colonic and

rectal mucosa where it causes severe inflammation
leading to mucosal destruction. A key factor of the
pathogenesis of these bacteria is their capacity to in-
vade epithelial cells (LaBrec, 1965). Based on in
vitro assays, «invasion» encompasses: (i) the capac-
ity of bacteria to their entry into epithelial cells by
macropinocytosis (Clerc Sansonetti, 1987), (ii) their
ability to lyse the membrane of the endocytic vacuole
and to escape into the cytoplasm where unrestricted
growth occurs (Sansonetti et al., 1986), and (iii) their
power to move intracellularly and to spread from cell
to cell by an actin-dependent process (Bernardini et
al., 1989). This capacity of S. flexneri to interact with
components of the eucaryotic cell, particularly its cy-
toskeleton, is a remarkable example of molecular cross
talk between a pathogen and its target cell.

The two paradigms of bacterial entry into
epithelial cells

Two major paradigms of bacterial entry into non-
phagocytic cells have been described (Isberg, 1991).
In the «zipper» paradigm, bacteria express a surface
protein which binds, with high affinity, to an eu-
caryotic surface receptor that has a function in cell
adherence or motility. The two examples are the

Figure 2. Map of the Shigella flexneri entry loci.

Yersinia pseudotuberculosis invasin which binds β1
integrins (Tran Van Nhieu Isberg, 1993), and the Lis-
teria monocytogenes internalin, In1A, which binds
E-cadherin (Mengaud et al., 1996). High affinity of
binding and minimum reorganization of the cytoskele-
ton allow internalization of the pathogen via a process
which probably exacerbates the physiological signals
induced by the interaction of the receptors with their
physiological ligands.

In the «trigger» paradigm, the bacteria, upon con-

tact with the host cell surface, secrete a set of proteins
(i.e. invasins) which form a complex interacting with
the eucaryotic membrane, thereby eliciting signals
which massively rearrange the cell cytoskeleton by
inducing actin polymerization. This causes localized
membrane ruffling in which the bacterium is trapped
and internalized (Figure 1). The «trigger» paradigm
is characteristic of the mode of entry of Shigella and
Salmonella (Ménard et al., 1996; Francis et al., 1993).

Entry of

Shigella flexneri

into epithelial cells

This phenotype which is essential to the pathogenic-
ity of S. flexneri is encoded by a 30 kb sequence
(Maurelli et al., 1985) located on a 200 kb virulence
plasmid (Sansonetti et al., 1982, 1983) (Figure 2).
Two adjacent loci transcribed in the opposite direc-
tion are necessary and sufficient to encode and secrete
the effectors – Ipa proteins or invasins – which elicit
the formation of the entry focus, via localized actin
polymerization (Clerc Sansonetti 1987). One locus is
composed of the ipa operon (invasion plasmid antigen)
which encodes four secreted proteins: IpaB (62 kDa),
IpaC (42 kDa), IpaD (37 kDa) and IpaA (70 kDa),
which are the effectors of bacterial internalization
(Ménard et al., 1993). In the bacterial cytoplasm,

anto1021.tex; 7/12/1998; 10:43; p.2

193

IpaB and IpaC are associated with a 18 kDa chaperon,
IpgC, which prevents their aggregation and proteolytic
degradation (Ménard et al., 1994). Ipa proteins have
no signal peptide; upon contact of the bacteria with
the cell surface, they are secreted, already folded,
from the cytoplasm to the extracellular milieu with-
out a periplasmic phase. In the extracellular medium,
IpaB and IpaC form a complex (Ménard et al., 1994).
This property of secretion upon contact is based on the
expression and assembly of a type III secretory appa-
ratus composed of about 20 different proteins called
Mxi-Spa (membrane expression of antigens and se-
cretion of protein antigens) which bridges the inner
and outer membrane. This secretory system is encoded
by the mxi and spa operons which are transcribed in
the opposite direction to the ipa operon. A complex
formed by the association of IpaB and IpaD appears
to regulate the flux of Ipa proteins through this ap-
paratus (Ménard et al., 1994). Homologues of this
system are shared by several other enteric pathogens
such as enteropathogenic Escherichia coli (EPEC),
Salmonella, and Yersinia. We have recently identified
artificial compounds such as Congo red which fully
activate the Mxi-spa secretory system (Barahni et al.,
1997). This has allowed us to study the kinetics of
Ipa secretion which is completed within 15 minutes of
activation, occurs only at 37

◦

C, and is at a maximum

in exponentially-growing bacteria.

Identification of the effectors of entry is done by

combining mutagenesis of the candidate genes in the
ipa operon, and a cell biology approach designed
to study interaction of the relevant proteins with the
cell surface. Deletion of the ipaB, ipaC, and ipaD
genes causes complete inactivation of the entry phe-
notype (Ménard et al., 1993). Mutation in the ipaA
gene causes about 90% decrease in entry efficiency
(Tran Van Nhieu et al., 1997). Latex beads coated
with the IpaB-C complex are internalized into HeLa
cells (Ménard et al., 1996). The entry foci elicited by
IpaB-C-coated beads nevertheless cause limited cy-
toskeletal and membrane rearrangements, indicating
that the actual entry of the bacteria is a more complex
process, and that other proteins are likely to be directly
involved.

It can be postulated from these experiments that

IpaB and IpaC constitute the primary effectors of
Shigella entry (Ménard et al., 1996). It is not yet
definitely known whether these effectors bind surface
receptors of the epithelial cell, which may cause sig-
nals inducing the cytoskeletal rearrangements, or form
a complex inserting into the epithelial cell membrane,

thereby inducing the cytoskeletal changes by a di-
rect effect, but also by forming a protein translocator
allowing the invading bacterium to inject other se-
creted proteins into the cytoplasmic compartment of
the epithelial cell. Evidence indicates that the α5β1
integrin may be mediating interaction between a com-
plex formed by the Ipa proteins and the target epithe-
lial cell (Watarai et al., 1996). This would suggest
a link between the Ipa proteins and the triggering of
cytoskeletal rearrangements via a transmembrane re-
ceptor. However recent data indicate that β1 knock
out cells are similarly invaded by Shigella (Skoudy &
Sansonetti, unpubl.).

We have shown that IpaB can bind and recruit the

hyaluronan-binding receptor CD44 on the epithelial
cell surface. This is correlated with the local recruit-
ment and activation of ezrin, a protein involved in
linking cytoskeletal components, including actin fil-
aments, to the cell membrane, which also binds to the
cytoplasmic domain of CD44 (Skoudy et al., unpub.).
The issue of membrane receptor activation therefore is
not yet solved. The latter hypothesis, which postulates
insertion of the IpaB-C complex into the epithelial
membrane itself, is suggested by experiments show-
ing that IpaC (De Geyter et al., 1997), as well as
IpaB (Cabiaux et al. unpubl.) are able to cause lysis
of calcein-loaded liposomes. Our recent data indicate
that IpaA may be injected into the epithelial cell by
such a process in order to regulate the cytoskeletal
rearrangements (Tran Van Nhieu et al., 1997). The
constitution of a protein translocator has still to be def-
initely demonstrated in Shigella, as well as its possible
function in causing direct polymerization of actin (by-
passing receptor-mediated events). It is still possible
that a combination of these two mechanisms operates.

The signals allowing actin nucleation-polymeri-

zation and cytoskeletal rearrangements are currently
being deciphered. Two major signalling pathways are
activated during Shigella entry. The protooncogene
pp60

c

−src

is recruited at the entry site and its activa-

tion allows the tyrosine-phosphorylation of one of its
major substrates, cortactin (Dehio et al., 1995). The
actual role of cortactin in the cytokeletal rearrange-
ments is not yet defined.

Small GTPases of the Rho family (i.e. RhoA, B

and C) are also recruited at the entry site of Shigella.
Their involvement has been demonstrated by treat-
ing cells prior to infection by the C3 exoenzyme
of Clostridium botulinum which inactivates Rho by
ADP-ribosylation on Asparagin 41. This treatment
decreased the rate of infection by 90%. Instead of

anto1021.tex; 7/12/1998; 10:43; p.3

194

forming long actin filaments at the site of bacteria-cell
interaction, C3-treated cells only accumulated large
quantities of short actin filaments or nuclei (Adam et
al., 1996). These results have recently been confirmed
(Watarai et al., 1997). The role of Rho proteins there-
fore seems to be filament elongation in the filopodial
extension of the entry focus, possibly in line with their
physiological function in regulating stress cable for-
mation in cells. Once elongated, filaments are tightly
bundled by plastin (Adam et al., 1995)

Several actin-associated proteins which constitute

and regulate cell adhesion foci are present in associ-
ation with the cytoskeletal rearrangements supporting
entry (Ménard et al., 1996). This observation, in addi-
tion to the need for c-src and Rho, suggests that entry
is similar to plaque formation. This led to a study of
the function of plaque-associated proteins in Shigella
entry. Vinculin is essential to allow efficient entry
(Tran Van Nhieu et al., 1997). Shigellae barely in-
vade vinculin-deficient cells, and their entry is restored
by transfection with vinculin-expressing cDNA. The
IpaA protein is also required for entry; mutagenesis
of the ipaA gene decreases entry by about 90%. This
protein, which appears to be injected while the bac-
terium is internalized, binds vinculin. This interaction
allows activation of vinculin and its binding to cy-
toskeletal proteins which organize adhesion plaques,
such as α-actinin, constructing a stable structure that
allows optimal entry into cells. Entry of Shigella into
epithelial cells therefore appears to be the result of
a massive rearrangement of the cytoskeleton induced
by a complex of Ipa proteins which is concurrently
remodeled by at least another Ipa protein. These ex-
periments confirm that entry is a stepwise process
constantly regulated by bacterial products.

Escape of

Shigella flexneri

into the cell cytoplasm

Once intracellular,

S. flexneri rapidly lyse the

membrane-bound vacuole and escape into the cyto-
plasm (Sansonetti et al., 1986). Ipa proteins account
for this lysis (High et al., 1992). IpaB is the main
effector but recent evidence indicates that IpaC may
also contribute to vacuole lysis. This purified pro-
tein, as already mentioned, is able to lyse lipid vesi-
cles in a pH-dependent manner (De Geyter et al.,
1997). Once cytoplasmic, shigellae can grow freely in
this rich medium and reach numbers of several hun-
dreds per infected cell. This behaviour is different
from Salmonella which need to adapt to the hostile

metabolic conditions that prevail inside the phagocytic
vacuole, which they are unable to lyse, before they
are able to grow at a rate similar to Shigella (Garcia
del Portillo et al., 1993). This difference may explain
differences in pathogenic behaviour observed between
members of these two genera.

Intracellular motility of

Shigella flexneri

and cell

to cell spread

After escape from the vacuole, bacteria can move
along the stress fibers that radiate from adhesion
plaques to the nucleus. This movement has been
termed organelle-like movement in fibroblasts (Olm)
(Vasselon et al., 1992); its molecular basis is still
unknown. In epithelial cells, the Olm movement is
hard to detect, instead bacteria move in the cytoplasm
by an actin-based movement which is caused by the
formation of an F-actin comet tail (Figure 3) at one
pole of the bacteria (Bernardini et al., 1989). Bacterial
movement inside the cytoplasm is random and rapid
(6–60 µm/min). It occurs optimally at the stage of
bacterial division (Goldberg et al., 1993). Observation
of the comet tail in transmission electron microscopy
after S1 myosin decoration of actin filaments shows
short filaments with their ’barbed’ ends oriented at the
surface of the bacteria, indicating that addition of new
actin monomers occurs at the bacterial surface. These
newly formed filaments are released and crosslinked
into a tail structure providing the motile force that
achieves bacterial movement.

The icsA gene, located on the large virulence plas-

mid, accounts for F-actin comet tail formation and
movement (Bernardini et al., 1989). IcsA was discov-
ered as a protein promoting tissue dissemination of
Shigella, and was also named VirG (Makino et al.,
1986). Virulence of a

M

icsA mutant is strongly at-

tenuated in vivo thereby making this mutation a basis
for the development of live attenuated Shigella vaccine
candidates (Sansonetti et al., 1991).

IcsA is a 120 kDa outer membrane protein. It

is composed of a C-terminal transporter domain,
IcsAβ, which allows translocation and anchorage of
the N-terminal domain, IcsAα, at the outer mem-
brane (Suzuki et al., 1996). IcsAα accounts for actin
polymerization. It doet not share any homology with
any known protein, neither with cytoskeletal proteins,
nor with ActA, its counterpart in L. monocytogenes
(Domann et al., 1992; Kocks et al., 1992). On its N-

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195

Figure 3. Double fluorescence labelling of intracellular Shigella showing actin driven motility of the bacteria. Bacteria are labelled by anti-LPS
serum/rhodamin and F-actin that constitutes comet tails by fluorescent NBD-phallacidin (Photo Coumaran Egile).

terminus, IcsAα has a series of 6 repeats of 32 to 34
residues, which are very rich in glycine.

IcsA is both necessary and sufficient to induce

actin polymerization and bacterial motility which can
be reproduced with the same efficiency in cell-free ex-
tracts, such as cytoplasmic extracts of Xenopus laevis
eggs or human cells. Surface expression of IcsA into
E. coli K12 in these extracts causes actin polymer-
ization and formation of actin comets correlated with
motility of the bacterial bodies (Kocks et al., 1995;
Goldberg & Thériot, 1995).

The surface distribution of IcsA is unusual. In wild

type bacteria, it is asymetrically distributed, since it is
seen only at the bacterial pole opposite to the septa-
tion furrow in dividing bacteria. Inside infected cells,
IcsA determines the site of actin assembly and the di-
rection of movement. Two factors contribute to polar
localization: (i) a yet uncharacterized intrinsic prop-
erty of IcsA may promote adhesion of the protein to
the bacterial pole, although a significant proportion is
still expressed over the entire cell surface: (ii) cleavage
of 50% of the total amount of expressed IcsA occurs
at the junction of its α and β domains (Fukuda et al.,
1995) on a sequence – S756SRRASS762 – which is
also a phosphorylation site for PKA (d’Hauteville &
Sansonetti, 1992).

A bacterial surface protease, SopA, cleaves IcsA

(Egile et al., 1997). SopA activity is sufficient to elim-

inate the proteins located around the bacterial body,
except at the pole where IcsA concentration is natu-
rally higher. IcsA cleavage by SopA allows a transition
between circumferential expression with polar rein-
forcement to exclusively polar location. A

M

sopA

mutant is impaired in its intracellular motility, both in
cell assay systems and in animal models of Shigella
infection, and remains trapped in the actin subcortical
network of infected epithelial cells.

The mechanism of actin polymerization induced

by IcsA is for the most part still unknown. The re-
cruitment of a cytosolic protein complex in the bac-
terial vicinity may create appropriate conditions for
actin polymerization thereby causing formation of the
comet. One may anticipate the following steps: (i)
actin nucleation; (ii) elongation of actin filaments; (iii)
capping of the filaments’ pointed ends thereby ex-
plaining the shortness of filaments, and (iv) bundling
of the filaments achieving formation of the comet.

Several proteins have been detected in the Shigella

actin tail such as α-actinin, plastin, VASP and vin-
culin. Recently, vinculin was identified as the first
ligand of IcsA (Suzuki et al., 1996). Vinculin has been
shown to interact in vitro with IcsA by its globular
head domain. Recruitment of vinculin at the pole of
Shigella occurs before any accumulation of F-actin.
VASP, another focal adhesion protein is a ligand for
ActA (Niebuhr et al., 1993) and for vinculin (Rein-

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196

hard et al., 1996). These results suggest that Listeria
and Shigella have evolved a similar motility mecha-
nism involving VASP and an ActA analog, vinculin.
Nevertheless, Shigella motility appears to be unaf-
fected in a vinculin-deficient murine cell line (Gold-
berg, 1997). Whether another functional homolog of
vinculin is recruited by IcsA in this cell line is not
known. Thus the relevance of IcsA-vinculin interac-
tion in the actin-based motility of Shigella still awaits
further biochemical analysis. The role of cellular pro-
teins involved in Listeria actin-based motility (VASP,
Arp2/Arp3 complex and cofilin) and Ics-A mediated
motility is currently not known. It seems that IcsA
is composed of different domains which may either
participate in the early nucleating event that initiates
comet formation, or contribute at different stages of
the process by offering modules that are sequentially
used to mature the actin-driven motor.

When contact occurs between the moving organ-

ism and the inner face of the cytoplasmic membrane, a
protrusion is formed which is eventually phagocytosed
by the adjacent cell (Prévost et al., 1992; Sansonetti
et al., 1994). Some of these bacteria appear to es-
tablish interactions with components of the cellular
junction which allow their passage via an actin-driven
protrusion, into the adjacent cell. Expression of cad-
herins is a prerequisite to allow phagocytosis of these
protrusions by the adjacent cells (Sansonetti et al.,
1994). The molecular nature of the interaction be-
tween Shigella and cell junctional components is un-
der study. Once absorbed by the adjacent cell, the
bacteria find themselves trapped inside a pocket sur-
rounded by a double membrane which is eventually
lysed. A key factor in this lytic process is IcsB, a
57 kDa protein which is encoded by a gene located
upstream of the ipa genes in the locus encoding the
effectors of entry (Allaoui et al., 1996).

Conclusions

In summary, in the context of epithelial cells, the
invasive phenotype of Shigella can be considered as
an efficient mean of intracellular colonization by the
pathogen. It encompasses entry into non-phagocytic
cells, intra-cellular growth, intracellular motility and
cell to cell spread. This multifactorial process is a
spectacular example of integration of several defined
steps leading to progression of infection in a sanctu-
ary protected against some effectors of the immune

response. The in vivo counterpart of these in vitro-
established phenotypes is being actively studied.

Acknowledgements

We wish to thank all the members of Unité de
Pathogénie Microbienne Moléculaire whose excellent
work made this review possible. C.E. is a graduate
student supported by the Foundation Louis Jeantet de
Médecine (Geneva, Switzerland). Recent work from
our laboratory reviewed in this article has been sup-
ported by a Direction des Recherches et Techniques
(DRET) contract n

◦

94 092. We are also very grateful

to Colette Jacquemin for editing this manuscript.

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