Microbes and Infection, 2, 2000, 813−819

© 2000 Éditions scientifiques et médicales Elsevier SAS. All rights reserved

S128645790000366X/REV

Diversion of cytoskeletal processes by

Shigella during invasion of epithelial cells
Raphaëlle Bourdet-Sicard, Coumaran Egile, Philippe J. Sansonetti, Guy Tran Van Nhieu*
Unité de pathogénie microbienne moléculaire, Inserm U389, Institut Pasteur, 28, rue du Dr. Roux, 75724 Paris cedex 15, France

ABSTRACT – Shigella, the causative agent of bacillar dysentery, invades colonic epithelial cells and
moves intracellularly to spread from cell to cell. The processes of Shigella entry, determined by the Ipa
proteins, and of actin-based motility, dependent on the IcsA/VirG protein, represent different levels of
bacterial manipulation of the cell cytoskeleton. © 2000 Éditions scientifiques et médicales Elsevier
SAS
Shigella / actin / RhoGTPases / Ipa protein / IcsA

1. Introduction tact with the host cell membrane. Actin polymerization

Shigella is the causative agent of bacillar dysentery in
humans. Colonization of intestinal epithelial cells by this
pathogen induces an intense inflammatory reaction that
leads to destruction of the colonic mucosa [1]. Shigella
has the ability to enter epithelial cells and to spread from
cell to cell, properties that are key determinants of bacte-
rial virulence [2]. Upon contact with epithelial cells, Shi-
gella induces the formation of cell extensions that reach
several tens of microns in length, rise above the apical cell
surface at the site of bacterial interaction, and engulf the
bacterium in a large vacuole in a process reminiscent of
macropinocytosis [3, 4]. Once internalized, Shigella lyses
the phagosomal membrane and multiplies freely in the
cell cytosol. During this multiplication phase, Shigella
moves intracellularly by polymerizing actin at one pole of
the bacterial body [3]. Using this actin-based motility, the
bacterium induces protrusions that invade neighboring
cells. After lysis of both protrusion and recipient cell
membranes, Shigella reinitiates its intercellular cycle and
can spread within the cell monolayer without an extracel-
lular step. Both cell entry and intracellular motility are
examples of bacterial manipulation of processes control-
ling the host cytoskeletal dynamics, and will be the subject
of this review.
2. Cytoskeletal proteins of the Shigella
entry structure
During the initial phases of the entry process, Shigella
induces actin polymerization at the site of bacterial con-
* Correspondence and reprints
allows the formation of filopodial structures that fill in to
form a leaflet surrounding the bound bacterium [5]. The
organization of these extensions requires the F-actin bun-
dling protein plastin (fimbrin), and probably others such as
α-actinin or cortactin, that are massively recruited at the
level of these extensions [5]. Concomitantly, a denser
F-actin network enriched in vinculin, a focal adhesion
protein that links the cytoskeleton to the cell membrane,
forms at the intimate contact between the cell and the
bacterium [6]. Remarkably, ezrin, a member of the ERM
(ezrin-radixin-moesin) family [7], is also involved in the
formation of Shigella entry foci [8]. As opposed to other
cytoskeletal components, massive recruitment of ezrin
occurs after the peak of actin polymerization, and is most
prominent at the tip of the cell extensions where little
F-actin is detected [8]. Upon completion of bacterial entry,
polymerized actin mostly concentrates at the intimate
contact with the membrane of the nascent phagosome.
This actin coat disappears as the bacterium lyses the
vacuolar membrane and gains access to the cell cytosol.
3. Implication of Rho GTPases during
bacterial entry
Rho GTPases (Cdc42, Rac and Rho) are essential regu-
lators of cellular processes involving cytoskeletal reorga-
nization [9]. When activated, under their GTP-bound
state, Cdc42 and Rac induce actin polymerization [9, 10].
In the case of Cdc42, however, actin polymerization leads
to the formation of filopodia and microspikes, whereas
activation of Rac leads to the formation of lamellipodia or
ruffles. In general, it is believed that activation of Rho does
not induce actin polymerization, but leads to stress fiber

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2000, 813-819
Current focus: bacteria/host cell interactions Bourdet-Sicard et al.

Figure 1. Cytoskeletal rearrangements induced during Shigella entry. 1. Upon bacterium-cell contact, the Ipa proteins are secreted by the
Mxi-Spa type III secretion apparatus, and a pore containing the IpaB and IpaC proteins is inserted in host cell membranes. Membrane
projections sustained by actin filaments are induced by IpaC and require the activation of the GTPases Cdc42 and Rac. The Src tyrosine
kinase, which is activated during Shigella entry, is also required for the formation of these extensions. 2. The conversion of these membrane
projections into a structure that allows bacterial internalization involves the GTPase Rho. The recruitment of cytoskeletal proteins such as
ezrin, that are important for entry, is dependent on Rho. Rho also allows the recruitment of Src at the site of entry whose activity, in turn,
downregulates Rho activity. The Shigella protein IpaA is translocated into the cell cytosol and also participates in the conversion into a
structure that is productive for entry by associating with the focal adhesion protein vinculin and inducing depolymerization of actin
filaments.

formation through the activation of myosin. All three Rho
GTPases have been functionally involved in Shigella entry
into cells [11]. The fact that these GTPases are involved in
the formation of distinct cellular structures suggests they
play distinct roles during bacterial entry. Consistently,
analysis of Shigella-induced actin foci indicates that Cdc42
and Rac are required for actin polymerization at the site of
entry, whereas Rho appears to allow the transformation of
these extensions into a structure that is productive for
bacterial entry [12] (figure 1). In the presence of the C3
exoenzyme, a specific inhibitor of Rho, Shigella induces
polymerization of actin, but fails to recruit ezrin, or the Src
tyrosine kinase at the site of entry [12]. Similar to what has
been proposed for focal adhesion formation [13], it is
possible that the activation of Rho allows the formation of
structures required for the recruitment of cytoskeletal com-
ponents, such as ezrin, at the site of entry.
4. Role of the Src tyrosine kinase in
Shigella-induced cytoskeletal
rearrangements
A number of host cell proteins are tyrosylphosphory-
lated during Shigella entry. Among those, cortactin, a
cytoskeletal protein that bundles actin filaments and that is
814
a substrate for Src, is the most prominent tyrosylphospho-
rylated peptide, indicating that Src is activated during
bacterial internalization [14]. Furthermore, Src is func-
tionally involved in Shigella entry, because inhibition of
the Src kinase activity by overexpression of a dominant-
negative form of Src in HeLa cells results in inhibition of
Shigella-induced actin polymerization and bacterial entry
[15]. Interestingly, in cells that express a mutant form of Src
with constitutive kinase activity, Shigella-induced foci of
actin polymerization appear and downregulate more rap-
idly than in parental HeLa cells [15]. This indicates that
besides its requirement for Shigella-induced actin poly-
merization, the Src kinase activity is also implicated in the
regulation of actin polymerization induced by Shigella.
Further indications on the role of Src were obtained by the
analysis of its interdependence with Rho GTPases in actin
foci formation.
In cells expressing constitutively active Src, Shigella-
induced actin foci do not show ezrin recruitment [12]. As
a similar phenotype is observed when Rho is inhibited,
these results suggest that the Src kinase activity downregu-
lates Rho during foci formation. Consistent with a more
global effect of Src kinase activity on the downregulation
of Rho-dependent responses, inhibition of Src kinase by
herbymicin or by overexpression of a kinase inactive form
of Src led to an increase in stress fiber and focal adhesion
Microbes and Infection
2000, 813-819
Shigella signalling to actin
formation [12]. Furthermore, cells expressing constitu-
tively active Src show few stress fibers and focal adhesions
[12, 16]. This negative regulation of Rho-dependent
responses by Src can occur downstream of the GTPase,
because many focal adhesion and cytoskeletal compo-
nents are substrates of Src [17]. This regulation can also
occur upstream of Rho, through the p190RhoGAP protein
[16, 18]. Interestingly, p190RhoGAP is tyrosyl-
phosphorylated in an Src-dependent manner, and its level
of phosphorylation influences its level of association with
GTP-Rho [12, 18]. Thus, Src could downregulate Rho by
activating p190RhoGAP and favoring the transition to the
GDP-Rho inactive form. As Rho is required for recruitment
of Src at Shigella-induced actin foci, a negative regulatory
loop may occur during Shigella entry, whereby Rho acti-
vation allows recruitment of Src whose kinase activity, in
turn, downregulates Rho (figure 1).
5. The IpaC protein as the main effector
of actin polymerization during Shigella
entry
As many as thirty gene products are required for Shi-
gella entry into epithelial cells [2]. Among these, the 4
genes ipaA-D are located on an operon, and immediately
adjacent to this operon, the mxi-spa operon is devoted to
the expression of a type III secretion apparatus. This appa-
ratus is visualized as a macromolecular structure at the
surface of the bacterium by electron microscopy consist-
ing of a bulb, probably cytoplasmic, a neck, and a needle
that protrudes in the extracellular medium [19]. It has
been shown that this apparatus allows the insertion of a 25
Å-pore into host cell membranes, containing the IpaB and
IpaC proteins [19] (figure 1). This complex was shown to
have ion channeling activity [20], and it is believed that
similar to what has been proposed for other type III secre-
tion systems found in Gram-negative bacteria [21, 22], the
Mxi-Spa apparatus allows translocation of bacterial effec-
tors from the bacterium to the cell cytosol via the IpaB,
C-containing pore [19].
Thus, effectors of Shigella entry may have a direct
access to the cell machinery governing cytoskeletal
dynamics. To test for such effectors, a semi-permeabilized
cell assay was developed using proteins that are secreted
by the Mxi-Spa apparatus [23]. These proteins were iso-
lated from the culture supernatant of Shigella mutant
strains showing constitutive secretion, and added to semi-
permeabilized cells to identify Shigella proteins suscep-
tible to induce actin polymerization. Some evidence indi-
cates that IpaC is necessary and sufficient to induce actin
polymerization during entry. Purified IpaC induces actin
polymerization that leads to the formation of filopodial
structures that fill in to form leaflets at the cell periphery
[23]. These extensions are inhibited by the addition of a
monoclonal antibody that recognizes the carboxytermi-
nus of IpaC, whereas addition of an anti-IpaC aminotermi-
nal antibody results in the formation of large lamellipodial
structures [23]. These results indicate that the carboxyter-
minus of IpaC is involved in actin polymerization, whereas
its aminoterminus is involved in the conversion between
Microbes and Infection
2000, 813-819
Current focus: bacteria/host cell interactions
filopodial to lamellipodial structures. IpaC-induced exten-
sions are inhibited by a dominant-negative form of Cdc42,
whereas a dominant-negative form of Rac mostly inhibits
IpaC-induced leaflets [23]. In the presence of IpaC, inhi-
bition of Rho by C3, on the other hand, does not result in
cell rounding up and actin cable disappearance usually
observed with C3 alone, suggesting that IpaC also inter-
feres with Rho-dependent responses. Taken together, IpaC
appears to induce polymerization of actin and formation
of filopodia through the activation of Cdc42, with activa-
tion of Rac and Rho resulting from the hierarchy that links
these different GTPases.
The mechanism involved in the activation of Cdc42 by
IpaC is currently unknown. Unlike SopE, a Salmonella
protein injected in the cell cytosol during bacterial entry
[24], IpaC does not show an in vitro exchange factor (GEF)
activity on the Cdc42 or Rac GTPases (our unpublished
results). Also, as opposed to SipC, its Salmonella homolog,
IpaC does not appear to directly nucleate and polymerize
actin filaments in vitro [25].
6. IpaA induces actin depolymerization
in a vinculin-dependent manner
During Shigella entry, actin polymerization occurs dur-
ing the early stages of the entry process, but as the bacte-
rium enters the cell, actin depolymerizes. It was formerly
observed that a Shigella ipaA mutant was impaired by
tenfold for entry into epithelial cells [6]. This mutant,
however, induces actin polymerization with the same
efficiency as the wild-type strain, although the cell exten-
sion does not organize into a structure productive for
bacterial entry and actin does not depolymerize as rapidly
as for the wild-type strain [6]. Interestingly, IpaA directly
binds to the aminoterminal region of vinculin, a focal
adhesion protein that regulates anchoring of the cytoskel-
eton to the membrane [26]. Furthermore, analysis of cells
deficient for vinculin, and of vinculin-transfectants, sug-
gest that the cellular effects induced by IpaA occur through
vinculin [6]. Vinculin is a key player in the organization of
the actin cytoskeleton because of its ability to interact with
F-actin as well as with various cytoskeletal proteins, and
because this ability depends on its configuration. In its
folded or inactive state, the ligand binding sites on vincu-
lin are masked by an intramolecular interaction between
the aminoterminal (head) and carboxyterminal (tail)
domains [27, 28]. Upon activation, vinculin unfolds and
under this configuration, can simutaneously interact with
F-actin via its tail domain; via its head domain it can bind
to talin or α-actinin, two cytoskeletal proteins that associ-
ate with the cytoplasmic domain of the integrin β1 subunit
[17]. IpaA was found to induce vinculin association with
actin filaments that are stabilized with the fungus toxin
phalloidin [26]. Remarkably, IpaA represents the first
example of a protein that ’activates’ vinculin, and the
increase in vinculin association with F-actin induced by
IpaA is about an order of magnitude higher than that
observed for phosphatidylinositol (4,5)-bisiphosphate, the
only other factor described so far to have a similar effect on
vinculin [28]. When actin filaments were not stabilized,
815
Current focus: bacteria/host cell interactions
however, IpaA was found to exert a depolymerizing activ-
ity on actin filaments in vinculin-dependent manner [26].
As this activity is not observed when IpaA is incubated
with a truncated form of vinculin lacking the tail domain
that binds to F-actin, IpaA may depolymerize filamentous
actin in a two-step process whereby: i) binding of IpaA to
vinculin results in exposure of the F-actin binding site on
the vinculin tail domain; ii) the IpaA-vinculin complex
associates with actin filaments and induces actin depoly-
merization. Thus, IpaA has two types of activities that
could potentially be regulated during Shigella entry. Vin-
culin activation induced by IpaA could allow the recruit-
ment of vinculin at the intimate site of contact between the
bacterium and the host cell membrane to form a pseudo-
focal adhesion structure (figure 1). Actin depolymeriza-
tion induced by the IpaA-vinculin complex, on the other
hand, could be involved in the transformation of filopodia
into organized leaflet structures, as well as in the down-
regulation of the Shigella entry foci.
7. IcsA induces N-WASp-Arp2/3
complex-dependent actin filament
nucleation and Shigella actin-based
motility
After lysis of the phagocytic vacuole, Shigella assembles
at one end of its body an F-actin rich comet tail structure
and moves intracellularly. As for the formation of mem-
brane extensions during the entry process, actin polymer-
ization in itself provides the force that drives the bacterium
across the cell cytosol. Shigella ability to move intracellu-
larly by an actin-based mechanism is also shared with
Listeria monoytogenes, some Rickettsia sp. and the vac-
cinia virus [29].
Shigella actin-based motility is mediated by the func-
tion of a single protein, the IcsA/VirG protein [30, 31]. IcsA
is an outer membrane protein which is exported and
anchored by a C-terminal autotransporter domain IcsAβ
The N-terminal exposed domain IcsAα is responsible for
actin assembly. At the bacterial surface, IcsA exhibits an
asymmetric distribution and is present only at one pole of
the bacterial body [32]. IcsA polar distribution is essential
for the assembly of the comet tail inside infected cells and
it determines the direction of movement [33].
Early work has revealed that IcsAα does not induce
actin assembly directly and it was speculated that the
recruitment and activation by IcsA of cytoskeletal proteins
would mediate actin tail assembly and bacterial propul-
sion. Several cytoskeletal proteins have been identified in
Shigella actin comets [34, 35]. Among them, two proteins
interact directly with IcsA: vinculin and N-WASp (Neural-
Wiskott-Aldrich Syndrome protein) [34–36]. There is some
controversy about the precise role of vinculin in Shigella
intracellular motility. Although vinculin does not appear
to be essential for actin-based motility [37, 38], its possible
function in Shigella cell-to-cell spread remains an open
issue. In vitro analysis of IcsA-induced actin polymeriza-
tion, however, has revealed that the N-WASp-IcsA inter-
action by itself was necessary to induce actin nucleation
[39].
816
Bourdet-Sicard et al.
N-WASp is a member of the growing WASp/Scar pro-
tein family [40]. WASp/Scar proteins connect several sig-
naling pathways to the actin cytoskeleton [41]. Members
of the family contain different N-terminal domains, a
central proline-rich region and a C-terminal WA domain
which consists of a verprolin/WASp homology 2 (WH2)
motif and an extreme C-terminal acidic (A) motif. The WA
domain interacts with the Arp2/3 complex via the A motif
and with G-actin via the WH2 motif [42]. Among WASp/
Scar proteins, both WASp and N-WASp bind to the acti-
vated form of Cdc42 via their GBD/CRIB domain. Upon
binding of Cdc42-GTP to the CRIB domain, N-WASp
switches from its folded inactive state to an unfolded
active state where the WA domain is unmasked [43], and
can activate the Arp2/3 complex.
Interestingly, it was recently shown that binding of IcsA
to N-WASp results in the activation of the actin nucleation
activity of the Arp2/3 complex [39]. Unlike Cdc42-GTP,
however, IcsA forms a stable complex with N-WASp. This
interaction involves the glycine-rich region of IcsA, which
interacts specifically with the CRIB domain of N-WASp
(Egile et al., unpublished). Formation of an IcsA-N-WASp-
Arp2/3 ternary complex is responsible for the actin fila-
ment nucleation. How actin nucleation and polymeriza-
tion induced by IcsA converts into comet tail formation
and bacterial motility is not fully understood. The isolated
WA domain interacts with G-actin in a profilin-like fash-
ion [39, 44] and thus, probably increases shuttling of
G-actin into the barbed ends generated by the Arp2/3
complex (figure 2). Work on Listeria motility has shown
that actin filaments constantly attach to and detach from
the bacterial surface [45]. This transient association allows
de novo insertion of actin monomers at the barbed ends,
which transform actin polymerization into a propulsive
force by a gliding insertional polymerization process. The
isolated N-terminus of N-WASp binds to actin filament
and this interaction might be sufficient to mediate actin
filament association with the Shigella surface via IcsA
[39]. Recent reconstitution of Shigella motility with puri-
fied proteins demonstrated that in addition to N-WASp
and the Arp2/3 complex, proteins that regulate the fun-
nelled treamilling process of actin filament such as ADF/
Cofilin, the Capping protein and profilin are also required
for bacterial propelling [38].
8. Perspectives
Although our understanding of the mechanisms that
govern Shigella entry and actin-based intracellular motil-
ity has improved at a considerable pace, many questions
remain open. In particular, it will be interesting to establish
the various connections between the signalling pathways
involved in Shigella entry and the responses linked to the
Ipa proteins. Understanding how these responses integrate
together and act in a concerted manner to induce bacterial
entry will represent another exciting field of investigations.
Also, even though actin-based motility emerges as a
remarkably straightforward model to study the processes
of actin nucleation and polymerization in vitro, much is
Microbes and Infection
2000, 813-819
Shigella signalling to actin Current focus: bacteria/host cell interactions

Figure 2. Working model of Shigella actin-based motility. 1. N-WASp activation. Binding of N-WASp to IcsA at the bacterial surface
leads to the opening of N-WASP which unmasks the WA domain and the F-actin binding domain. 2. Actin nucleation. Formation of a
ternary complex between N-WASp, G-actin, and the Arp2/3 complex induces the actin nucleating activity of the Arp2/3 complex. 3.
Actin-based motility. Barbed end growth of the actin filaments due to the profilin-like activity of the WA domain. The growing filament
associates with the bacterial surface via its binding to the N-terminal domain of N-WASp. New filaments are cross-linked in a dense
network by α-actinin. Bacterial propelling is mediated by the capping protein, ADF and profilin that promote a funnelled treadmilling
process.

yet to be learned about the factors that regulate the dynam-
ics of actin-tail formation and bacterial motility inside the
cell.
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