Shigellosis from symptoms to molecular pathogenesis

, October , 2007; 153 (10): 3499-3507.

Microbiology

Miguel A. Valvano and Inés Contreras

Javier A. Carter, Carlos J. Blondel, Mercedes Zaldívar, Sergio A. Álvarez, Cristina L. Marolda,

gene

wzy

RfaH-mediated transcriptional control of the

2a is growth-regulated through

higella flexneri

O-antigen modal chain length in S

2008; 76 (1): 369-379.

Infect. Immun.

Beth A. McCormick and Anthony T. Maurelli

Daniel V. Zurawski, Karen L. Mumy, Luminita Badea, Julia A. Prentice, Elizabeth L. Hartland,

Infections

Shigella flexneri

and

Escherichia coli

Transepithelial Migration, a Characteristic Shared by Enteropathogenic

The NleE/OspZ Family of Effector Proteins Is Required for Polymorphonuclear

2008; 76 (8): 3614-3627.

Infect. Immun.

A. McCormick

Karen L. Mumy, Jeffrey D. Bien, Michael A. Pazos, Karsten Gronert, Bryan P. Hurley and Beth

Neutrophils

To Induce the Transepithelial Migration of

Shigella flexneri

Typhimurium and

Serotype

Salmonella enterica

Mediate the Ability of

Distinct Isoforms of Phospholipase A

2009; 191 (4): 1132-1142.

J. Bacteriol.

Kent and Luiz E. Bermudez

Melanie J. Harriff, Lia Danelishvili, Martin Wu, Cara Wilder, Michael McNamara, Michael L.

Putative Surface Protein Interacting with Host Cell Signaling Pathways

That Play a Role in Invasion of Epithelial Cells, in Part by Their Regulation of CipA, a

Genes MAV_5138 and MAV_3679 Are Transcriptional Regulators

Mycobacterium avium

http://ajpgi.physiology.org/content/280/3/G319.full.html

, October , 2009; 155 (10): 3260-3269.

Microbiology

Miguel A. Valvano and Inés Contreras

Javier A. Carter, Juan C. Jiménez, Mercedes Zaldívar, Sergio A. Álvarez, Cristina L. Marolda,

Shigella flexneri

pHS-2

WzzB and Wzz

The cellular level of O-antigen polymerase Wzy determines chain length regulation by

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Microbes and Microbial Toxins:
Paradigms for Microbial-Mucosal Interactions
III. Shigellosis: from symptoms to molecular pathogenesis

PHILIPPE J. SANSONETTI
Unite´ de Pathoge´nie Microbienne Mole´culaire et Unite´ Institut National de la Sante´ et de la
Recherche Me´dicale 389, Institut Pasteur, 75724 Paris ce´dex 15, France

Sansonetti, Philippe J. Microbes and Microbial Toxins:

Paradigms for Microbial-Mucosal Interactions. III. Shigellosis:
from symptoms to molecular pathogenesis. Am J Physiol Gas-
trointest Liver Physiol 280: G319–G323, 2001.—Interaction of
Shigella flexneri with epithelial cells includes contact of bacte-
ria with the cell surface and release of Ipa proteins through a
specialized type III secreton. A complex signaling process in-
volving activation of small GTPases of the Rho family and c-src
causes major rearrangements of the subcortical cytoskeleton,
thereby allowing bacterial entry by macropinocytosis. After
entry, shigellae escape to the cell cytoplasm and initiate intra-
cytoplasmic movement through polar nucleation and assembly
of actin filaments caused by bacterial surface protein IcsA,
which binds and activates neuronal Wiskoff-Aldrich syndrome
protein (N-WASP), thus inducing actin nucleation in an Arp
2/3-dependent mechanism. Actin-driven motility promotes effi-
cient colonization of the host cell cytoplasm and rapid cell-to-cell
spread via protrusions that are engulfed by adjacent cells in a
cadherin-dependent process. Bacterial invasion turns infected
cells to strongly proinflammatory cells through sustained acti-
vation of nuclear factor-

␬B. A major consequence is interleukin

(IL)-8 production, which attracts polymorphonuclear leukocytes
(PMNs). On transmigration, PMNs disrupt the permeability of
this epithelium and promote its invasion by shigellae. At the
early stage of infection, M cells of the follicle-associated epithe-
lium allow bacterial translocation. Subsequent apoptotic killing
of macrophages in a caspase 1-dependent process causes the
release of IL-1

␤ and IL-18, which accounts for the initial steps

of inflammation.

dysentery; epithelium; colon; inflammation

SHIGELLAE ARE GRAM

NEGATIVE

, nonsporulating, facultative

anaerobic bacilli that belong to the family Enterobacteri-
aceae. They cause shigellosis, or bacillary dysentery, an
invasive infection of the human colon that affects a spec-
trum of clinical presentations, from short-lasting watery
diarrhea to acute inflammatory bowel disease, the classic

expression of bacillary dysentery characterized by the
triad of fever, intestinal cramps, and bloody diarrhea
with mucopurulent feces (10). The etiological agents be-
long to the genus Shigella, which comprises four different
species. S. flexneri (6 serotypes) and S. sonnei (1 serotype)
account for the endemic disease, the former being prev-
alent in the developing world, the latter in the industri-
alized world. S. dysenteriae (16 serotypes) includes sero-
type 1, the “Shiga bacillus,” which accounts for deadly
epidemics in the poorest countries, largely due to its
capacity to produce shigatoxin, a potent cytotoxin. S.
boydii (8 serotypes) remains restricted to the Indian sub-
continent. In terms of public health (13), shigellosis
shows three major characteristics: 1) it is mostly a pedi-
atric disease,

⬎60% of the cases occurring in children

between the ages of 1 and 5 yr; 2) it is a third-world
disease, with

⬃150 million cases occurring every

year, compared with 1.5 million cases in industrial-
ized countries; and 3) it is also a deadly disease, with

⬃1 million deaths every year, again mostly infants
and young children. Lack of hygiene is the major, if
not exclusive, contributing factor, the disease being
transmitted by person-to-person contact or contami-
nated food.

In addition to poverty being the primary factor fa-

voring occurrence of shigellosis, other disease-specific
parameters aggravate the public health burden of shig-
ellosis. They are essentially four, all of which deserve
increased research attention: 1) extension of antibiotic
(multi-) resistance in both endemic and epidemic areas;
2) very low infectivity, 10–100 microorganisms admin-
istered orally being able to cause the disease in adult
volunteers; 3) severity of acute complications, particu-
larly in infants and malnourished children, with com-
plex and yet often unexplained pathogeneses, some of
them being lethal, as is the case for acute hypoglyce-

Address for reprint requests and other correspondence: P. J. San-

sonetti, Unite´ de Pathoge´nie Microbienne Mole´culaire et Unite´ IN-
SERM 389, Institut Pasteur, 28 Rue du Docteur Roux, 75724 Paris
ce´dex 15, France (E-mail: psanson@pasteur.fr).

The costs of publication of this article were defrayed in part by the

payment of page charges. The article must therefore be hereby
marked ‘‘advertisement’’ in accordance with 18 U.S.C. Section 1734
solely to indicate this fact.

Am J Physiol Gastrointest Liver Physiol

280: G319–G323, 2001.

0193-1857/01 $5.00 Copyright

2001 the American Physiological Society

http://www.ajpgi.org

G319

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mia, seizures, toxic megacolon, pseudoleukemoid reac-
tion and hemolytic-uremic syndrome, intestinal perfo-
rations, peritonitis, and Gram-negative septicemia; and
4) the recently recognized importance of delayed com-
plications, characterized by a prolonged state of malnu-
trition whose pathogenesis is still unclear and indeed
poses another challenge.

This situation makes vaccination a cost-effective ap-

proach, and the World Health Organization has put the
development of a Shigella vaccine at the top of its
priority list of awaited vaccines against enteric infec-
tions (22). Understanding the pathogenesis of shigello-
sis, the basis of the innate immune response that
causes excessive inflammation leading to intestinal tis-
sue destruction, as well as the basis of the protective
adaptive immune response has become a priority topic
in several laboratories worldwide, with the aim of de-
veloping a vaccine against the disease.

MOLECULAR AND CELLULAR PATHOGENESIS
OF SHIGELLOSIS: AN UPDATE

The ability of Shigella to invade and colonize the

intestinal epithelium is a key determinant of the dis-
ease. However, the pathogenesis of shigellosis is a sub-
tle combination, particularly at the early stage of the
disease, of 1) the capacity for the bacteria to cross the
epithelium in selected areas corresponding to M cells of
the follicle-associated epithelium (FAE) that covers the
mucosa-associated lymphoid follicles, the inductive
sites of local immune responses (28); 2) the intrinsic
invasive properties of the bacteria for epithelial cells

(21); and 3) the inflammatory response achieved by the
cellular components of the intestinal barrier that dis-
rupt the coherence of this barrier and facilitate bacte-
rial invasion (37). Expression of the Shigella invasive
phenotype in the presence of the various cell popula-
tions that constitute the intestinal barrier (Fig. 1),
particularly M cells, epithelial cells, resident macro-
phages, and polymorphonuclear leukocytes (PMNs),
engages variable interactions whose result constitutes
the overall process leading to rupture, invasion, and
inflammatory destruction of the intestinal barrier.

Genetic and Molecular Basis of the Shigella
Invasive Phenotype

In S. flexneri and other Shigella species, a 214-kb

virulence plasmid contains most of the genes required to
express the key steps of the invasive phenotype (5, 26).
The coding sequences are scattered over the entire viru-
lence plasmid, essentially separated by multiple, often
incomplete, insertion sequences. One 30-kb block, how-
ever, shows a dense pattern of genes, the ipa/mxi-spa
locus that can be considered the main Shigella pathoge-
nicity island (PAI). This PAI is necessary and sufficient to
cause entry into epithelial cells via macropinocytosis,
macrophage apoptotic death, and activation of PMNs. It
primarily encodes a type III secreton, a flagella-like
structure able to deliver Shigella effector proteins, par-
ticularly Ipa proteins, straight from the bacterial cyto-
plasm into the cytoplasmic membrane of the eukaryotic
cell target or its cytoplasm.

Fig. 1. Differential expression of the Shigella invasive
phenotype depending on the cellular target.

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Interaction of Shigella with M cells and epithelial

cells. In vivo evidence indicates that expression of the
invasive phenotype is required for Shigella to translocate
at high frequency through M cells (23). It is therefore
likely that the Shigella PAI mediates efficient invasion of
both M cells and epithelial cells. However, whereas Shi-
gella can enter via the apical pole of M cells, they are very
inefficient, if not unable, in entering via the apical pole of
epithelial cells whose basolateral pole, on the other hand,
is very permissive for entry (16). M cell translocation,
therefore, is likely to allow access of shigellae to the
basolateral pole of these epithelial cells, where efficient
internalization proceeds. Bacterial signals and cell re-
sponses mediating entry of Shigella into epithelial cells
have mostly been studied in the epithelioid HeLa cell
line. These data have been summarized in a recent re-
view (33). The Shigella type III secreton allows the inser-
tion of a pore into the cytoplasmic membrane of the
eukaryotic cell target. This pore, which contains a com-
plex of the IpaB and IpaC proteins, expresses a dual
function: it induces early events of actin polymerization
via the COOH terminal domain of IpaC (32), and it is also
likely to introduce into the eukaryotic cell cytoplasm a
series of plasmid-encoded proteins (

⬃15) that are known,

in vitro, to be secreted through the type III secreton (5).
Among these, IpaA and IpgD are involved in the matu-
ration of the entry focus (4, 17), whereas the possible
function of the others is unknown.

While entering cells, Shigella causes the formation of

filopods that are quickly remodeled in lamellipods, thus
resulting in a structure that entraps the microorganism
(1). Further remodeling causes the formation of a mac-
ropinocytic vacuole that eventually completes internal-
ization. These events result from a cross-talk between the
bacterium and the host cell signaling pathways that
regulate the cytoskeleton, essentially causing actin nu-
cleation and polymerization at the eukaryotic cell mem-
brane (33). The major target of IpaC is the cascade of
small GTPases of the Rho family (11). In parallel, recruit-
ment of the protooncogene c-src (7) enhances actin poly-
merization (8), thus causing the formation of gigantic cell
extensions that need to be further controlled by IpaA to
form a structure that is productive for entry. IpaA acts
through binding to the NH

-terminal domain of vinculin

(31), thereby causing actin bundling, formation of a
pseudoadherence plaque, and subsequent depolymeriza-
tion of the filaments (4).

Once internalized, the phagocytic vacuole is quickly

lysed by the invading bacterium, thereby allowing its
escape into the host cell cytoplasm, where it nucleates
and assembles an F-actin comet at one of its poles (3).
This results in the bacterium moving inside epithelial
cells and passing from cell to cell, thereby causing a very
efficient process of intracellular colonization. Shigella
actin-based motility is mediated by a single outer mem-
brane protein, IcsA/VirG (14, 15). IcsA/VirG is unable to
directly induce actin nucleation, indicating that recruit-
ment of a cytosolic component is required. Glycine-rich
repeats in the NH

-terminal end of IcsA/VirG bind neu-

ronal Wiskoff-Aldrich syndrome protein (N-WASP) (30),
a member of the WASP family of Cdc-42-dependent me-
diators of actin nucleation via the Arp 2/3 complex. For-

mation of a complex between IcsA/VirG, N-WASP, and
Arp 2/3 at the bacterial surface is sufficient to cause actin
nucleation/polymerization in the presence of actin mono-
mers (9). Motile intracellular shigellae then engage com-
ponents of the cell intermediate junction (27) to form a
protrusion that is internalized by the adjacent cell, thus
causing cell-to-cell spread as summarized in Fig. 2.

Crossing, Disruption, and Invasion of the Epithelial
Barrier by Shigella

Shigellosis is a paradigm of a pathogen manipulat-

ing the innate immune system, thereby causing intes-
tinal inflammation, which is responsible for disruption
of the epithelial barrier’s impermeability, facilitating
further bacterial invasion of tissues (18, 19), as well as
for massive tissue destruction, causing the mucosal
abscesses and ulcers that are characteristic of shigel-
losis. Tissue destruction may be the cost to pay for
eradication of the bacteria.

After crossing M cells of the FAE, invasive shigellae

find themselves in the dome area of the lymphoid follicle,
which is essentially populated by macrophages and den-
dritic cells. Unlike Yersinia, which has selected an an-
tiphagocytic strategy, and Salmonella, which has se-
lected a strategy of survival inside macrophages, Shigella
expressing the invasive phenotype kill macrophages, es-
sentially by apoptosis (36). Apoptotic death occurs within
2 h, both in vitro and in vivo (23). Apoptotic killing of
macrophages, and probably dendritic cells as well, is
caused by secreted IpaB and occurs following activation
of the cysteine protease caspase 1 by this molecule (6).
Macrophage-induced cell death not only permits bacte-
rial survival following the crossing of the FAE but is also
central to the early triggering of inflammation. This dual
function is likely to reflect another consequence of
caspase 1 activation by IpaB, the capacity to hydrolyze
pro-interleukin (IL)-1

␤ and pro-IL-18, thereby allowing

the release of mature IL-1

␤ and IL-18 (35). Rapid killing

of macrophages by invasive shigellae may also account
for the drop in IL-1 receptor antagonist (IL-1RA), which
is observed in infected tissues in the first 4 h of infection

Fig. 2. Mechanisms of epithelial cell invasion and intracellular/
intercellular colonization by Shigella.

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(2). This causes massive decrease in the IL-1RA-IL-1
ratio, a characteristic of severe inflammatory processes.
Conversely, perfusion of IL-1RA during the course of
experimental shigellosis causes considerable attenuation
of the severity of lesions, thus emphasizing the major role
that IL-1 is playing in Shigella-mediated inflammation
(24). Recent experiments have provided evidence on the
respective roles of IL-1

␤ and IL-18 release following

caspase 1 activation by IpaB in infected macrophages
(29). Release of IL-1

␤ early after infection causes rupture

of the epithelial barrier and destabilization of tissue ho-
mogeneity, which favors bacterial diffusion, massive tis-
sue invasion, and enhancement of inflammation and tis-
sue destruction. On the other hand, the parallel release of
IL-18, which is a potent interferon (IFN)-

␥ inducer, al-

lows the innate immune system to establish proper con-
ditions for eradication of the Shigella inoculum, since
IFN-

␥ is essential for the killing of Shigella (34). In

consequence, it is likely that macrophage and, possibly,
dendritic cell apoptosis occurring early after bacteria have
crossed the FAE causes both IL-1

␤-mediated inflammation

participating in the inflammatory rupture of the epithelial
barrier and facilitation of bacterial dissemination as well as
IL-18-programmed control of Shigella growth.

Bacterial Invasion Causes Epithelial Cells to Produce
Proinflammatory Molecules: Consequences of the
Epithelium Becoming a Participant in Inflammation

In response to bacterial invasion, it is now well

established that colonic epithelial cells produce a large
array of proinflammatory cytokines and chemokines,
such as IL-8 (12). Invasion of epithelial cells by Shi-
gella activates high and sustained nuclear transloca-
tion of nuclear factor-

␬B (20), which accounts for IL-8

production by infected cells. This process that may
occur at a distance from the FAE as epithelial invasion
proceeds, once IL-1

␤ has started to disrupt integrity of

the epithelial barrier, and accounts for attraction of
PMNs in subepithelial tissues, followed by their trans-
migration through the epithelium, which extends the
zone of invasion and causes major tissue destruction.
Neutralization of PMN transmigration either in vitro
(19) or in vivo (18), using an anti-CD18 monoclonal
antibody that neutralizes binding of PMNs to epithe-
lial cells, dramatically decreases both bacterial inva-
sion and inflammatory destruction of the epithelium.
In addition, neutralization of IL-8 during experimental
infection by Shigella causes a dramatic decrease in
PMN invasion of the epithelium and epithelial destruc-
tion. On the other hand, bacteria that have been able to
cross the epithelial barrier grow freely in the lamina
propria, unchecked by PMNs, and subsequently dis-
seminate in the blood stream (25). In consequence, IL-8
production by invaded epithelial cells and their neigh-
boring cells accounts for Shigella control at the epithe-
lial level, but at the cost of massive epithelial destruc-
tion, particularly by PMNs. It is therefore likely that
the spread of invasive bacteria, at a distance from the
FAE, is constantly maintained by an influx of PMNs
that subverts epithelial integrity and facilitates fur-
ther bacterial invasion. These various elements of Shi-
gella pathogenesis are summarized in Fig. 3.

CONCLUSION

Recent research has illustrated the power of combin-

ing microbial genetics and cell biology to decipher the
cross-talks established between bacterial pathogens
such as Shigella and their eukaryotic cell targets. We
tried to summarize here some of the major features of
the Shigella invasive process that leads to rupture,
invasion, and inflammatory destruction of the intesti-
nal barrier. We have now reached the stage at which
these data must be integrated in a global scheme of
signaling studied at the level of infected tissues, with

Fig. 3. Current scheme of Shigella pathogens: rupture,
invasion, and inflammatory destruction of the intesti-
nal barrier.

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particular interest in understanding engagement of
the innate immune response and how this affects the
adaptive immune response itself. This is essential to
strengthening our future approaches aimed at devel-
oping an anti-Shigella vaccine.

I thank all of my colleagues from Unite´ de Pathoge´nie Microbi-

enne Mole´culaire who, through their research, made this review
possible and C. Jacquemin for her editing of this document. Partic-
ular thanks to A. Phalipon, C. Parsot, G. Tran Van Nhieu, and A.
Zychlinsky.

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