Shigella's ways of manipulating the host intestinal innate and adaptive immune system

REVIEW

Shigella’s ways of manipulating the host intestinal
innate and adaptive immune system: a tool box
for survival?

Armelle Phalipon and Philippe J Sansonetti

Shigella, a Gram-negative invasive enteropathogenic bacterium, causes the rupture, invasion and inflammatory destruction of the
human colonic epithelium. This complex and aggressive process accounts for the symptoms of bacillary dysentery. The so-called
invasive phenotype of Shigella is linked to expression of a type III secretory system (TTSS) injecting effector proteins into the
epithelial cell membrane and cytoplasm, thereby inducing local but massive changes in the cell cytoskeleton that lead to
bacterial internalization into non-phagocytic intestinal epithelial cells. The invasive phenotype also accounts for the potent pro-
inflammatory capacity of the microorganism. Recent evidence indicates that a large part of the mucosal inflammation is initiated
by intracellular sensing of bacterial peptidoglycan by cytosolic leucine-rich receptors of the NOD family, particularly NOD1, in
epithelial cells. This causes activation of the nuclear factor kappa B and c-JunNH

-terminal-kinase pathways, with interleukin-8

appearing as a major chemokine mediating the inflammatory burst that is dominated by massive infiltration of the mucosa by
polymorphonuclear leukocytes. Not unexpectedly, this inflammatory response, which is likely to be very harmful for the invading
microbe, is regulated by the bacterium itself. A group of proteins encoded by Shigella, which are injected into target cells by the
TTSS, has been recently recognized as a family of potent regulators of the innate immune response. These enzymes target key
cellular functions that are essential in triggering the inflammatory response, and more generally defense responses of the
intestinal mucosa. This review focuses on the mechanisms employed by Shigella to manipulate the host innate response in order
to escape early bacterial killing, thus ensuring establishment of its infectious process. The escape strategies, the possible direct
effect of Shigella on B and T lymphocytes, their impact on the development of adaptive immunity, and how they may help
explain the limited protection induced by natural infection are discussed.
Immunology and Cell Biology advance online publication, 9 January 2007; doi:10.1038/sj.icb.7100025

Keywords: Shigella; manipulation; host responses; instestinal inflammation; mucosal immunity; type III secretion system

Shigellosis, or bacillary dysentery, is a dysenteric syndrome caused by
Shigella, a Gram negative, enteroinvasive bacterium belonging to the
family Enterobacteriacae. Shigella is divided into four subgroups,
namely, S. flexneri, S. sonnei, S. dysenteriae and S. boydii. Adding to
the diversity of subgroups, several serotypes are responsible for
infection. Serotypes are defined on the basis of the carbohydrate
composition of the O-antigen, the polysaccharide part of the lipo-
polysaccharide (LPS), the major bacterial surface antigen.

S. flexneri

and S. sonnei are responsible for the endemic form of the disease,
whereas S. dysenteriae 1, which produces a highly cytotoxic toxin
called Shiga toxin, accounts for devastating epidemics.

This exclusively human disease is transmitted directly via the feco-

oral route from an infected patient or indirectly through contami-
nated food and water. It is a highly contagious infection, capable of
transmission with as few as 100 microorganisms.

Infection produces

a spectrum of symptoms, from watery diarrhea to severe dysentery.
Severe dysentery is characterized by fever, abdominal cramps and

acute permanent bloody and mucoid stools. These symptoms largely
reflect bacterial invasion of the colonic and rectal mucosa, which
elicits acute inflammation leading to massive mucosal destruction.

Shigellosis represents a significant public health burden in devel-

oping countries, with about 160 million cases occurring annually,
predominantly in children under the age of 5 years, leading to possibly
one million deaths per year worldwide.

The disease also exists in

industrialized countries. Shigella represents an index of the level of
hygiene in a given population. Whereas S. flexneri and S. dysenteriae
1 are prevalent in developing countries, S. sonnei is most often
associated with shigellosis outbreaks in developed countries. Compli-
cations may occur and account for the most severe cases that often
lead to death, particularly in toddlers and children.

These complica-

tions encompass hypoglycemia, bacteremia or septicemia with possible
septic shock, dehydration due to fever and acute diarrhea leading to
hypovolemia and acute renal failure, uremic and hemolytic syndrome
(a complex acute renal failure leading most often to death in the

Received 25 September 2006; revised 6 November 2006; accepted 7 November 2006

Unite´ de Pathoge´nie Microbienne Mole´culaire, INSERM U786, Institut Pasteur 25, Rue du Dr Roux, Paris, France
Correspondence: Dr A Phalipon, Unite´ de Pathoge´nie Microbienne Mole´culaire, INSERM U786, Institut Pasteur, 75015 Paris, France.
E-mails: phalipon@pasteur.fr or psanson@pasteur.fr

Immunology and Cell Biology (2007), 1–11
&

www.nature.com/icb

absence of resuscitation) and toxic megacolon (a paralytic syndrome
leading to lower intestinal occlusion, possibly accompanied by per-
foration and peritonitis). Shigella also causes a level of morbidity due
to secondary malnutrition, possibly leading to severe growth retarda-
tion in young children. In developing countries, bacillary dysentery is
recognized as one of the major causes of malnutrition.

In contrast to

other enteric infections such as cholera, bacillary dysentery does not
benefit from oral rehydration alone. Antibiotic treatments are efficient,
although multidrug resistance is now prevalent in Asia, Africa or
South America, a major concern in particular for S. flexneri and
S. dysenteriae 1.

5–15

These strains are generally multiresistant to the most

common, ‘first-line’ antibiotics (ampicillin, tetracycline, sulfonamides,
chloramphenicol, nalidixic acid, sulfamethoxazole-trimethoprin), lead-
ing to the use of more expensive, new-generation antibiotics such as
fluoroquinolones for which resistance is already spreading.

16–18

As with

all diarrheal diseases, disease prevention relies on sanitary improvement,
which is unfortunately unrealistic for many countries with increasing
population. Therefore, vaccination appears to be the only rational
prophylactic approach to control shigellosis. Unfortunately, no vaccine
is available so far, although several vaccine candidates have undergone or
are currently undergoing Phase I and II and even, for a few of them,
Phase III clinical trials.

19–21

Shigella initially crosses the epithelial layer via M cells overlaying

lymphoid follicles associated with the colo-rectal mucosa. M cells are
specialized epithelial cells (EC) within the follicle-associated epithe-
lium (FAE) capable of transcytosing lumenal antigens into the sub-
epithelial space for processing by phagocytic cells and presentation
to the gut-associated lymphoid tissue, leading to the generation of a
specific immune response.

From this port of entry, Shigella interacts

with (1) resident macrophages and dendritic cells (DC) present in the
dome of the lymphoid follicle, and (2) intestinal EC (IEC) via their
basolateral pole. The consequence is induction of proinflammatory
cytokine and chemokine production that initiate the inflammatory
process, leading to aphtoid-like inflammatory ulcers overlaying the
rectal lymphoid follicles.

Inflammation, accompanied by further

bacterial invasion, then spreads progressively to involve a large part
of the mucosal surface, but remains confined to the large bowel.

The

spread of inflammation throughout the bowel initially causes edema,
erythema and production of an adherent mucopurulent exudate,
followed by abscess formation and mucosal hemorrhages.

Shigella infection represents an interesting paradigm of imbalance

of the host immune mechanisms that regulate inflammation, and of
bacterial strategies developed to escape killing by host immune cells
that are equipped with a large repertoire of anti-microbial weapons to
control microbial infection. This review will focus on the most recent
advances in our understanding of the mechanisms induced by Shigella
to manipulate host innate and adaptive immune responses in order to
escape early killing, and therefore ensure its survival in the infected
host. The tools that Shigella uses to subvert the cytoskeleton to gain
entry and spread intra- and intercellularly has been the subject of
recent reviews and will not be discussed here.

19,24,25

EXPERIMENTAL MODELS OF INFECTION
Analysis of molecular, cellular and tissular mechanisms leading to the
development of the infectious process has benefited from studies using
both in vitro models of interaction of Shigella with epithelial cells (EC)
and immune cells such as macrophages, dendritic cells or neutrophils,
and several in vivo models of infection. The commonly used models
are summarized here. The most relevant model is the macaque
monkey model. Indeed, macaque monkeys infected intragastrically
(i.g.) with Shigella develop shigellosis, similarly to humans, except that

the needed inoculum is much higher than that required for human
infection (10

and 100 CFU, respectively).

26,27

This model has been

mainly used to assess the immunogenicity, and possibly protection
induced by orally administered, live attenuated vaccine candidates.
However, high cost and ethical issues render its routine use almost
impossible. Rabbits are poorly susceptible to oral infection with
Shigella,

but infection of ligated ileal loops leads to bacterial invasion

accompanied by induction of acute inflammation and subsequent
tissue destruction.

This model allows the study of Shigella interac-

tion with the intestinal barrier and subsequent induction of an innate
response. It has been extensively used for phenotype characterization
of Shigella mutants. However, due to a limited availability of specific
immunological tools, this model has been underexploited. In guinea
pigs, the keratoconjunctivitis assay, known as the Sereny test, is
useful to assess invasiveness, intercellular spread and virulence of
Shigella strains. Indeed, Shigella invades the corneal epithelium and
spreads to contiguous cells, with the more virulent strains causing
ulcerative keratoconjunctivitis.

30,31

Despite its usefulness for immuno-

genicity studies and protective efficacy of Shigella vaccine candidates,
a detailed analysis of host immune responses remains difficult
in this model.

In fact, a murine model of shigellosis would be an ideal model

to decipher the mechanisms underlying both innate and adaptive
immunity to Shigella infection. Unfortunately, adult mice orally or
i.g. infected with Shigella do not develop a disease similar to
shigellosis. Why mice are refractory to Shigella infection is still
unknown. Since induction of inflammation at early times post-
infection is crucial for the establishment of the infectious process,

32,33

the leading hypothesis to explain the absence of infection in mice
is that mice are defective in some of the steps leading to acute
inflammation, such as sensing of Shigella and/or expression of
effectors required for PMN recruitment. For pathogen sensing,
differences between mouse and human sensor molecules have been
recently reported. In the case of the intracellular sensor of peptido-
glycan (PG), Nod1, murine Nod1 efficiently detects a four amino-acid
muropeptide, whereas the human ortholog recognizes a muramyl-
tripeptide.

Interestingly, Gram-negative human pathogens such as

Shigella, Salmonella or pathogenic Escherichia coli all have in common
a high tetrapeptide/tripeptide ratio (up to 10/1) in their PG,

suggesting that they may be efficiently recognized by murine Nod1.
It is therefore unlikely that the absence of Shigella infection in mice
relies on a sensing deficiency. An interesting difference between mice
and human is that mice, in contrast to humans, are deficient in
interleukin-8 (IL-8) production due to the deletion of the gene
encoding this major neutrophil chemoattractant.

However, the

mouse chemokine receptor CXCR2 for KC and MIP-2 neutrophil
chemoattractants also recognizes human IL-8 and, therefore, mice are
responsive to human IL-8. IL-8 plays a key role in the Shigella-induced
pro-inflammatory process. It is mainly produced by Shigella-infected
intestinal epithelial cells (IEC) following bacterial intracellular sensing
by Nod1 and subsequent nuclear factor kappa B (NF-kB) activation.

Interestingly, intra-rectal infection with Shigella in the presence of
recombinant human IL-8 induces tissue damage in contrast to IL-8-
treated, non-infected control mice, suggesting that the refractory state
of mice could be attributed to the absence of IL-8 production.

further investigate the key role of IL-8, and maybe eventually obtain
access to a murine model of shigellosis, the development of transgenic
mice expressing human IL-8 is currently ongoing (T Pedron and
PJ Sansonetti, personal communication).

Despite the lack of a relevant murine model strictly mimicking

human infection, several murine models are available that allow one to

Shigella’s ways of manipulating the host immune system

A Phalipon and PJ Sansonetti

Immunology and Cell Biology

address specific issues of the infection process. In the model of
pulmonary infection,

38,39

mice intranasally infected with Shigella

develop an acute broncho-pneumonia characterized by a massive
neutrophil infiltrate, thus mimicking the acute inflammation induced
during shigellosis. Recently, this murine model was shown to repro-
duce the downregulation of INF-g production induced by Shigella
in infected patients at the acute phase of the disease

(J Gamelas-

Magalhaes et al., in preparation). In contrast to adult mice that are
resistant to oral infection with Shigella, newborn mice i.g. adminis-
tered with Shigella are susceptible to infection, in an age-dependent
manner. In 4-day-old newborns, Shigella invades the intestinal tissue,
inducing massive inflammation that leads to death of the infected
pups within 4–6 h postinfection. In contrast, 5-day-old newborns
become refractory to infection.

This model is therefore useful

to study the early interactions of Shigella with the intestinal mucosa,
and to decipher the molecular and cellular mechanisms leading to
resistance/susceptibility to infection. Upon partial elimination of local
flora following a streptomycin treatment, Shigella colonizes the intes-
tine of adult mice when administered i.g. The absence of PMN
recruitment, and, therefore, of tissue destruction, reveals some of
the very early steps of interactions of Shigella with the mucosal
immune system, such as the mucosa-associated lymphoid structures
that appear to expand with a high apoptotic index upon Shigella
infection.

Finally, a SCID mouse–human intestinal xenograft model

is available to study interactions between Shigella and human intes-
tine. Inoculation of Shigella into human intestinal xenografts causes
severe inflammation and mucosal damage.

Although laborious,

this model holds enormous potential to characterize the pheno-
type of Shigella mutants at the molecular level (for instance tran-
scriptome analysis) in the context of a ‘reconstituted’ human intestinal
barrier.

INVASIVE PHENOTYPE AND BACTERIAL VIRULENCE
EFFECTORS
The ability of Shigella to invade the colonic epithelium, and conse-
quently to trigger inflammation, is a key determinant in the disease
process. Expression of the invasive phenotype requires the presence of
a 213-kb virulence plasmid encoding bacterial virulence effectors.

Sequence analysis of the virulence plasmid indicated that it is
composed of a mosaic of approximately 100 genes and numerous
insertion sequences, the latter representing one-third of the virulence
plasmid.

Genes required for entry of bacteria into EC and induction

of apoptosis in infected macrophages are clustered on a 31 kb-region
(designated the entry region) of the virulence plasmid. This region
encodes components of a type III apparatus (TTSA) (or Mxi-Spa
TTSA), substrates of this secretion apparatus (the translocators IpaB
and IpaC and the effectors IpaD, IpgB1, IpgD and IcsB) and their
dedicated chaperones (IpgA, IpgC, IpgE and Spa 15) to which they
bind when stored in the bacterial cytoplasm before being secreted, and
two transcriptional activators (VirB and MxiE).

46–48

The TTSA is a needle-like structure dedicated to the translocation

of Shigella effector proteins from the bacterial cytoplasm to the
membrane and cytoplasm of the host cell. It assembles in a structure
spanning both the inner and outer bacterial membranes and extends
a 60-nm needle into the external milieu.

49–51

Contact of bacteria with

host cells constitutes a secretion signal, upon which a rapid burst of
protein secretion occurs. The translocators IpaB and IpaC insert into
the host cell membrane to form a pore, through which effectors will
transit to reach the host cell cytoplasm.

The ultrastructure of the

needle complex encompasses (i) an external needle, (ii) an upper ring
doublet, the transmembrane neck domain; and (iii) a basal bulb.

Detailed organization of this peculiar structure comprising 20 proteins
has been recently reviewed.

53–55

The regulation of expression of virulence plasmid genes has been

extensively studied.

46,56

Briefly, transcription of genes of the entry

region is regulated by temperature, these genes being expressed at
371C but not at 301C and under the control of VirF, a member of the
AraC family of transcription activators, and VirB, a member of the
ParB family of partitioning proteins. The TTSA assembled by bacteria
growing in broth at 371C is only weakly active and is activated upon
contact of bacteria with EC. TTSA activation leads to increased
transcription of 12 virulence plasmid genes encoding TTSA substrates.
Increased transcription of these genes in conditions of secretion is
controlled by MxiE, a transcription activator of the AraC family (for
a review see Le Gall et al.

and Parsot

Other substrates of the TTSA are encoded by genes scattered

throughout the virulence plasmid, such as virA, ospB-G and ipaH
genes.

Several of these putative effectors are encoded by multigens

families, with five ipaH, four ospC, three ospD and two ospE genes
carried by the virulence plasmid. On the basis of their expression
profiles, TTSA substrates can be classified into three categories:
(i) those that are expressed independently of the TTSA activity
(under the control of VirB); (ii) those that are expressed only in
conditions of secretion (under the control of MxiE) and (iii) those
that are expressed in conditions of non-secretion and induced
in conditions of secretion (thus under the control of both VirB
and MxiE).

In addition, the virulence plasmid encodes at least five other

proteins that are involved in virulence, including IcsA, IcsP, VirK,
MsbB2 and SepA. IcsA (VirG) is an outer-membrane protein directly
involved in promoting actin polymerization at one pole of intra-
cellular bacteria.

57–59

IcsP (SopA) is an outer-membrane protease

involved in the release of a certain proportion of surface-exposed
IcsA.

60,61

VirK is required for the production of IcsA by an unknown

mechanism.

MsbB2 is an acyl transferase that, in conjunction with

the product of the chromosomal gene msbB1 gene, acts to produce full
acyl-oxy-acylation of the myristate at the 3¢ position of the lipid A
glucosamine dissacharide.

SepA is a secreted serine protease of the

IgA1 protease family whose substrate is not known.

Although a partial scheme emerges from examining those effectors

that are co-regulated, the function of many bacterial virulence
effectors remains to be elucidated.

ESCAPE FROM THE FIRST LINE OF NONSPECIFIC IMMUNE
DEFENSES WITHIN THE GASTROINTESTINAL TRACT
In addition to its function in digestion, nutrient transport, water and
electrolyte exchange, and endocrine and paracrine hormone produc-
tion, the gastrointestinal tract, and in particular the intestinal epithe-
lium, has a role in defining the barrier between the host and the
external environment. This barrier is devoted to permanently protect-
ing the body against invasion and systemic dissemination of both
pathogenic and commensal microorganisms.

Few data are available on the mechanisms allowing Shigella survival

in the gastrointestinal environment. As Shigella species require a
uniquely small inoculum for causing dysentery, one explanation is
that the bacteria are better able to survive the acidic conditions
encountered in the stomach than are other enteric pathogens. Indeed,
most isolates of Shigella survive acidic treatment at pH 2.5 for at least
2 h whereas none of the Salmonella isolates are able to do so.

Antimicrobial peptides are essential effector molecules of the innate

immune system and are of great importance to bacterial host defense,
in particular within the intestinal environment.

There are two major

Shigella’s ways of manipulating the host immune system
A Phalipon and PJ Sansonetti

Immunology and Cell Biology

classes of antimicrobial peptides in humans, the defensins and the
cathelicidins.

67,68

Recently, it has been reported that Shigella has

evolved a strategy to inhibit the production of some of these
antimicrobial peptides. Indeed, expression of the human antimicrobial
peptide LL31 and human b-defensin-1 (HDB1) was shown to be
downregulated at the early time of Shigella infection.

Interestingly,

butyrate treatment results in upregulation of LL-37 in colonic EC
in vitro and decreases the severity of inflammation in experimental
shigellosis. Recent data obtained in rabbits have shown that expression
of CAP-18, the homolog of LL-37 in rabbit, is upregulated upon
butyrate-oral treatment. In addition, in Shigella-infected, butyrate-
treated rabbits, elimination of bacterial infection is more efficient than
in Shigella-infected, untreated control animals.

These findings

suggest that oral butyrate treatment in shigellosis may be of clinical
value to promote elimination of Shigella.

Lactoferrin, a glycoprotein present in most human mucosal secre-

tions, including human milk, is bacteriostatic in low iron media and,
in some settings, bactericidal. Lactoferrin at a concentration normally
found in human colostrum has been shown to block the development
of Shigella-induced inflammation in a rabbit model of enteritis.

Lactoferrin impairs the ability of S. flexneri to invade HeLa cells, by
inducing degradation of IpaB and, to a lesser extent, IpaC, the two key
proteins responsible for bacteria-directed phagocytosis by mammalian
cells. Lactoferrin does not directly degrade previously released proteins
but works by making IpaB/C susceptible to breakdown by surface-
expressed proteases.

It remains to be demonstrated whether such a

mechanism takes place in vivo or whether Shigella has developed a
strategy to escape it.

INDUCTION AND CONTROL OF THE INFLAMMATORY
RESPONSE BY SHIGELLA OR THE SHIGELLA YIN-AND-YANG
Innate immunity to Shigella infection is characterized by the induction
of an acute inflammation with massive recruitment of PMN, and
subsequently massive tissue destruction. In humans, analysis of
cytokine expression in rectal biopsies of infected patients at the
acute phase of the disease has revealed upregulation of proinflamma-
tory genes such as those encoding IL-1b, IL-6, IL-8, TNF-a and b,
although anti-inflammatory genes encoding IL-10 and TGF-b are also
upregulated.

Here are summarized numerous studies aimed at

characterizing the molecular and cellular mechanisms induced by
Shigella to trigger, but also control, the inflammatory process.

Escape from phagocytic vacuole and induction of macrophage
apoptosis
After crossing of the epithelial lining, bacteria encounter phagocytic
cells, particularly macrophages that are present in the follicle dome.
Thus, depending on the capacity to survive in the presence of
macrophages, the outcome of infection is dramatically affected.
During the early stages of infection, Shigella is phagocytosed by
resident macrophages and DC, which are highly abundant in the
subepithelial dome. In vitro studies have shown that similar to what
occurs in EC, Shigella escapes from the macrophage phagocytic
vacuole into the cytosol via Ipa-mediated lysis of the vacuole mem-
brane.

Secreted IpaB then activates host capsase-1 to induce rapid

apoptotic cell death associated with early loss of membrane integrity.

A similar outcome occurs, at least in vitro, with Shigella-infected
human monocyte-derived DC.

Large numbers of apoptotic phago-

cytic cells are observed both in the subepithelial dome in the rabbit
intestinal model of shigellosis

and in colo-rectal biopsies of infected

patients.

In addition to caspase-1, another endogenous protease, the

cytoplasmic serine protease tripeptidyl peptidase II, a component of

the apoptotic pathway located upstream of caspase-1, is involved in
the apoptotic cascade elicited by Shigella.

IpaB-dependent activation of caspase-1 in Shigella-infected macro-

phages and DC has important consequences for the inflammatory
response. Activated caspase-1 cleaves pro-IL-1b and pro-IL-18 that are
released from the dying cells in a mature active form.

IL-1b levels are

elevated in the rabbit ileal loop model of shigellosis and, moreover,
this is associated with an early decrease in endogenous IL-1 receptor
antagonist (RA) levels, further leading to unopposed IL-1 action.

There is good evidence that IL-1b is detrimental to the host in
shigellosis. Blocking IL-1 activity using recombinant IL-1 RA in the
rabbit-ligated ileal loop model efficiently prevents neutrophil influx,
particularly into the crypt lamina propria. Notably, bacterial invasion
is also decreased, implying that IL-1-dependent inflammation facil-
itates bacterial invasion.

In support to this conclusion, the early

inflammatory response in the murine lung model of infection was
decreased in IL-1b knockout mice, compared with wild-type mice, yet
bacterial clearance was similar.

Thus, in both models of infection,

IL-1b is pro-inflammatory, but this inflammation is not beneficial for
the host in terms of bacterial clearance or prevention of invasion.
Surprisingly, in other animal models of Gram-negative infection,
IL-1b generally increases protection against the disease.

82,83

This

implies that the pathological consequences of elevated IL-1 may
have some specificity to induction by IpaB in the context of shigellosis.

Caspase-1 activation promotes processing and secretion of pro-

inflammatory cytokines and is regulated by a protein complex called
the NALP3-inflammasome.

Recent work indicates that the NALP3-

inflammasome can be activated by endogenous ‘danger signals’ as well
as compounds associated with pathogens.

84,85

However, the NALP3-

dependent activation of caspase-1 by bacterial PAMPs remains
controversial.

84,86

Shigella escape from autophagy
The degradation of undesirable cellular components or organelles,
including invading microbes, by autophagy is crucial for cell survival.
As a component of the innate immune response, autophagy exerts
selective pressure on intracellular microbes. L. pneumophila slows
autophagosome maturation and C. burnetii exploits the specialized
autophagic compartment as its replicative niche.

87–89

Therefore, it is

likely that other bacteria such as Shigella that thrive in cytoplasm will
also have acquired mechanisms to inhibit autophagy or modify
bacteria-containing autophagic compartment.

In line with this

view, Shigella was found to be able to escape autophagy by secreting
IcsB through the type III secretion system. Mutant bacteria lacking
IcsB were trapped by autophagy during multiplication within the host
cells.

Therefore, these findings exemplify the diversity of the strate-

gies developed by Shigella, through its type III-secreted proteins, to
gain a foothold for survival within the host and avoid recognition by
the innate immune system.

Manipulation of EC by Shigella or the reprogramming of gene
expression in infected EC
After crossing of the intestinal barrier via M cells and escape from
macrophage killing in the dome region of the mucosa-associated
lymphoid follicles, Shigella may gain access to the basolateral pole of
EC from which it can invade or at least interfere with some
intracellular signaling pathways through injection into the cell cyto-
plasm of type III secretory system (TTSS)-secreted bacterial effectors.

To better characterize the global response of IEC to Shigella, the

transcriptome of the human colonic epithelial cell line Caco-2,
infected by invasive or non-invasive S. flexneri, was analyzed using

Shigella’s ways of manipulating the host immune system

A Phalipon and PJ Sansonetti

Immunology and Cell Biology

Affymetrix microarrays (12 000 genes). Results indicate that, besides
genes similarly expressed by both strains, the major differences in the
invasive Shigella-induced transcriptome reside in the induction of
early genes (i.e. mostly transcription factors expressed within the first
45 min of invasion) and late genes (i.e. after 1 h of invasion) whose
pattern was strongly biased towards stimulation of granulopoiesis,
chemoattraction, activation and adherence of PMN. Thus, invasive
Shigella programs IEC to recruit a PMN infiltrate to the mucosa. The
outstanding increase in IL-8 gene transcription, compared to other
genes, points to this chemokine as the major molecule orchestrating
mucosal inflammation in shigellosis.

Extra- and intracellular recognition of Shigella by EC
The intestinal barrier is endowed with detection and defense mechan-
isms to achieve tolerance to commensal microorganisms and protec-
tion against invading microorganisms.

Invasion by extracellular and

intracellular pathogens is sensed by various signaling pathways that
converge to activate NF-kB, involved in the activation of a large
number of genes including IL-8, in response to pathogens, stress
signals and proinflammatory cytokines.

Under non-stimulating

conditions, NF-kB is retained in the cytoplasm through its association
with inhibitory proteins (IkBs). A variety of signaling pathways
activate IkB kinases to phosphorylate IkBs, leading to ubiquitination
of phospho-IkBs and degradation by the proteasome.

93,94

NF-kB is

then allowed to translocate to the nucleus and activates NF-kB-
regulated genes, thus establishing an inflammatory response.

Survival of Shigella within the intestinal mucosa, and particularly

within IEC, leads to activation of the innate immune system by
bacterial products, of which LPS is thought to be the most important.
LPS is an amphiphilic molecule composed of hydrophobic lipid A, an
essential structural component of the bacterial outer membrane, and
a hydrophilic polysaccharide, the O-antigen (O-Ag) that extends from
the bacterial surface. Both components play an important role in
shigellosis. The O-Ag provides resistance to complement-mediated
lysis.

Bacteriophage-encoded glucosylation of Shigella O-Ag, the

basis of different serotypes, has been recently shown to shorten the
LPS molecule by around half. This enhances TTSS function without
compromising the protective properties of the LPS. Thus, LPS
glucosylation promotes bacterial invasion and evasion of innate
immunity.

The lipid A component is involved in stimulating the

inflammatory response leading to epithelial damage.

Lipid A is an

important inducer of the inflammatory response to Gram-negative
bacteria, and thus is not peculiar to disease pathogenesis of Shigella.
A unique feature of shigellosis is the fact that Shigella survives in
the tissues, presumably providing a continuous source of LPS for
prolonged activation of the inflammatory response. Considerable
advances have improved our understanding of the molecular mechan-
isms of LPS-induced signaling. In monocytes and macrophages, LPS is
shuttled from the extracellular environment to membrane-bound
CD14 by a serum protein, LPS-binding protein, which together with
an accessory molecule MD-2 presents LPS to a cell surface Toll-like
receptor (TLR), TLR4. TLR4 activation ultimately leads to transloca-
tion of cytosolic NF-kB to the nucleus and increased transcription of
multiple genes, including pro-inflammatory cytokine genes. In con-
trast, IEC lack membrane expression of CD14, express low levels of
TLR4 and MD-2, and are therefore refractory to activation by
extracellular LPS

or non-invasive Gram-negative bacteria.

98,99

This

presumably prevents the unwanted activation of IEC by commensals
bacterial components within the bowel lumen during a non-inflam-
matory situation. Nevertheless, inflammatory cytokines can increase
expression of CD14 and MD-2 within IEC, greatly enhancing the IL-8

response to LPS.

The ability of cells to recognize bacterial LPS via a

CD14-dependent mechanism is important for host protection in a
number of bacterial infection models, including shigellosis. In the
rabbit-ligated ileal loop model, blocking CD14 with an anti-CD14
antibody increases Shigella invasion and tissue destruction without
affecting neutrophil recruitment or bactericidal activity.

100

It is not

clear whether the CD14-dependent protective effect is dependent on
phagocytic cells or IEC, although in these experiments, CD14 expres-
sion could only be demonstrated on infiltrating monocytes, neutro-
phils and endothelial cells.

The discovery of the intracellular receptor Nod1 has allowed a

better understanding of how IEC, by sensing the presence of intra-
cellular microbial compounds such as PG, may activate the NF-kB
pathway and trigger the inflammatory response.

Nod1 belongs to

the recently described Nod-like receptors (NLR) family of intracellular
molecules that contribute to the sensing of microbial products.

101

Nod1, which has homology with a family of plant cytosolic pathogen
resistance proteins called R-proteins, has an N-terminal caspase-
recruitment domain (CARD), a nucleotide-binding domain and a
C-terminal regulatory domain containing multiple leucine-rich
repeats (LRR) homologous to those found in TLRs. Highlighting
their key role as intracellular PRMs, Nod1 and Nod2 have been shown
to be activated directly by invasive bacterial pathogens, such as
S. flexneri, enteroinvasive Escherichia coli and Streptococcus pneumo-
niae.

101

S. flexneri infection induces Nod1 oligomerization via a

homophilic CARD–CARD interaction allowing transient recruitment
of RICK and IKK, which phosphorylates IkB, leading to prolonged
activation of NF-kB.

Increasingly, studies are now implicating Nod1

and/or Nod2 in the antimicrobial response to a variety of different
pathogens.

101

Importantly, the intracellular location of NLRs seems

not to strictly exclude sensing of extracellular pathogens. Thus, it was
shown that the non-invasive pathogen Helicobacter pylori can deliver
PG via its type IV secretion system into the host cells where it activates
Nod1.

102

Whether the delivery of bacterial PAMPs by type IV secretion

systems, and perhaps also type III systems, is a common theme awaits
further investigation. Nevertheless, PG fragments can reach the cytosol
even in the absence of bacterial invasion or delivery through type III/
IV systems. In human IEC, the transmembrane protein hPepT1, which
mediates the uptake of di- and tripeptides, was shown to be a
transporter of bacterial muramyl dipeptide (MDP) leading to Nod2-
dependent IL-8 release.

101

Recently, the microbial motifs sensed by Nod1 and Nod2 have been

characterized. Both Nod1 and Nod2 recognize PG; however, each
requires distinct molecular motifs to achieve sensing. Nod1 recognizes
a naturally occurring muropeptide of PG that presents a unique
amino acid at its terminus called diaminopimelic acid.

103

This

amino acid is found mainly in the PG of Gram-negative bacteria
designating Nodl as a sensor of Gram-negative bacteria. In contrast,
Nod2 can detect the minimal bioactive fragment of PG, called
MDP.

104

Thus Nod2 is a general sensor of bacterial PG. Interestingly,

mutations in the gene encoding Nod2 were recently shown to be
associated with Crohn’s disease,

105

suggesting possible insight to the

mechanisms of this chronic inflammatory bowel disease.

Role of IL-8 in neutrophil recruitment
Previous studies have revealed the central role played by IL-8 in
experimental shigellosis. In the rabbit-ligated ileal loop model, sys-
temic injection of a neutralizing anti-IL-8 antibody before infection
inhibits neutrophil recruitment and epithelial damage. This is, how-
ever, at the expense of a threefold increase in bacterial invasion that
extends beyond the epithelial surface, diffusing into the lamina

Shigella’s ways of manipulating the host immune system
A Phalipon and PJ Sansonetti

Immunology and Cell Biology

propria. In addition, the number of bacteria in the efferent venous
blood from the infected loop was much higher.

106

Immunohisto-

chemical staining of infected mucosal tissues shows strong epithelial
staining for IL-8 in loops exposed to wild-type Shigella, and weak
staining in loops infected with a non-invasive Shigella mutant.
Importantly, IL-8 was expressed by IEC that were clearly not infected
with Shigella, implying that a significant proportion of IL-8 is
produced in response to extracellular activation either by LPS or
indeed by inflammatory cytokines.

IL-8 staining was also only

weakly present in lamina propria leukocytes, suggesting that they
are a less important source of this cytokine during infection.

106

The

inflammatory response to IEC-derived IL-8 has also been modeled
in vitro using a polarized IEC monolayer overlying neutrophils.
Activation of the monolayer with LPS in the presence of serum caused
IL-8 release predominantly from the basolateral side to the apical
side.

107

It will be important to determine the extent to which IL-8

produced in response to intracellular and extracellular activation
contributes towards the inflammatory response. Whether IL-8 pro-
duced from either stimulus is equally beneficial in preventing bacterial
invasion or equally detrimental in terms of tissue damage remains to
be assessed. However, observations made with the icsA mutant shed
some light on the relative importance of these pathways. The ability of
the icsA mutant to invade EC and lymphoid follicles and to induce
macrophage cytotoxicity and IL-1b production is essentially the same
as the wild-type strain. This mutant is compromised only in its ability
to spread from cell to cell and thus decreases IL-8 production
mediated by the Nod1-dependent intracellular pathway. The icsA
mutant is highly attenuated in its ability to cause intestinal inflamma-
tion and remains localized to lymphoid follicles,

108

supporting the

notion that Nod1 signaling is a pathological proinflammatory event.
Perhaps, in turn, IEC-derived IL-8 stimulated via the extracellular
pathway will be the component of the IL-8 response important for
prevention of invasion.

Shigella-mediated control of induced inflammation
Although inflammation is clearly important in the pathogenesis of
shigellosis, there is presumably an optimal level of inflammation for
Shigella. In order to attain this optimum, Shigella may need to encode
anti-inflammatory effectors since an overly exuberant inflammatory
response may ultimately be detrimental to the bacteria. A rapid,
massive influx of PMN to the site of infection may serve to completely
destroy the bacteria before they have the opportunity to establish an
infection, thereby decreasing their overall survival fitness. Alterna-
tively, an excessive inflammation may kill the host before the bacteria
have the opportunity to disseminate. The integrated functioning of
virulence effectors may allow Shigella to achieve an optimal level of
inflammation, where they can stave off the immune response long
enough to multiply, but still cause significant diarrhea to spread into
the environment.

Control of inflammation by Shigella virulence effectors has been

recently reported. ShiA, a protein encoded by the SHI-2 pathogenicity
island localized on a region of the chromosome linked to the
induction of inflammation, has been identified as an effector involved
in the downregulation of inflammation.

109

Accordingly, SHI-2

deletion mutants induce a stronger inflammatory response than
wild-type Shigella as measured by increased villus blunting, increased
PMN infiltration and induction of apoptosis in a rabbit ileal loop
model of shigellosis. Mutational analysis mapped the hyper-inflam-
matory phenotype to a single gene, shiA. Similar to SHI-2 deletion
mutants, infection with a shiA mutant strain induces dramatically
elevated levels of inflammation when compared to the wild-type

strain.

109

The ShiA-mediated mechanism of action remains to be

elucidated.

An effector encoded by the virulence plasmid and secreted by the

type III secretion apparatus has also been reported as a modulator of
Shigella-induced inflammation. OspG, a 196-residue protein whose
production is regulated by secretion activity,

110

is a protein kinase

that binds ubiquitinylated ubiquitin-conjugating enzymes (E2s),
including UbcH5 which belongs to the stem cell factor SCF

b-TrCP

complex promoting ubiquitination of the phosphorylated inhibitor
of NF-kB type a (phospho-IkBa). Transfection experiments indicated
that OspG can prevent phospho-IkBa degradation and NF-kB
activation induced by TNF-a stimulation. Infection of EC by
S. flexneri wild-type strain, but not an ospG mutant, led to accumu-
lation of phospho-IkBa consistent with OspG inhibiting SCF

b-TrCP

activity. Upon infection of ligated ileal loops in rabbits, the ospG
mutant induced a stronger inflammatory response than the wild-type
strain. This finding indicates that OspG negatively regulates the
host innate response induced by S. flexneri upon invasion of the
epithelium. Recent findings emphasize the idea that ubiquitination
events are ciritcal for regulating immune signals and also raise several
new issues about ubiquitin-dependent signaling cascades involving
mitogen-activated protein (MAP) kinases and the transcription
factor NF-kB. Ubiquitination of signaling proteins regulates both
their stability and activity, thereby controlling the intensity and
duration of inflammatory signaling cascades in immune cells.

Further studies will reveal whether other virulence effectors are
dedicated to the elegant fine-tuning of the inflammatory reaction
that allows Shigella to optimize its successful rate of infection. For
instance, several strategies have been reported for Yersinia virulence
effectors.

111

CONTROL OF PRIMARY INFECTION: ANOTHER WAY FOR
SHIGELLA TO MANIPULATE INNATE IMMUNITY?
Escape from a bactericidal Th-1 type IFN-c-mediated response
Gamma interferon (IFN-g) is an important cytokine in the host
defense against infection by viral and microbial pathogens, inducing
a variety of physiologically significant responses that contribute to
immunity.

112

Control of Shigella invasion in IEC is dependent on

IFN-g.

113

In the murine model of pulmonary infection, natural killer

(NK) cell-mediated IFN-g production was shown to be essential for
controlling bacterial proliferation upon primary Shigella infection,
promoting bacterial killing by macrophages.

114

Lymphoid cells, parti-

cularly T cells representing another source of IFN-g, are recruited to
the rectal mucosa of infected patients during the acute phase of
shigellosis.

115–117

However, their contribution to the control of

primary infection was unknown. Using an alymphoid mouse strain
(Rag

gc) devoid of B, T and NK cells and the mouse pulmonary model

of Shigella infection, Rag

gc mice were shown to be highly susceptible

to S. flexneri infection in comparison with wild-type (wt) mice.

118

Whereas PMN recruitment upon infection was similar, macrophage
recruitment and production of proinflammatory cytokines were
significantly decreased in Rag

gc mice compared with wt mice.

Upon selective engraftment of Rag

gc mice with polyclonal abT

cells, but not with abT cells from IFN-g

mice, S. flexneri infection

could be subsequently controlled. The role of NK cells was confirmed
by using Rag

mice devoid of B and T cells, but harboring NK cells,

that were shown to control infection. Local production of IFN-g by T
and NK cells recruited to the lung was demonstrated in S. flexneri-
infected wt mice. These data demonstrate that both abT cells and NK
cells contribute to the early control of S. flexneri infection through
amplification of an inflammatory response. This cellular lymphocyte

Shigella’s ways of manipulating the host immune system

A Phalipon and PJ Sansonetti

Immunology and Cell Biology

redundancy guarantees IFN-g production, which is central to innate
immunity against Shigella infection.

118

These data are consistent with

the fact that local production of IFN-g is associated with the
convalescent stage of shigellosis when bacteria are no longer detectable
in the feces of infected patients.

119,120

During the convalescent stage,

enhanced expression of the IFN-g receptor is comparable to the
constitutive level of expression in healthly subjects. Thus, recovery
from shigellosis correlates with an upregulation of IFN-g and IFN-g
receptor expression.

In fact, in the course of shigellosis, modulation of IFN-g production

is observed in infected patients characterized by systemic downregula-
tion of IFN-g production at the acute stage of the disease, compared
to the convalescent stage and to the status of healthly controls.

This suggests that Shigella has developed a strategy to escape from a
bactericidal Th-1 type IFN-g-mediated response, to establish a suc-
cessful infection in the host. Interestingly, the modulation of IFN-g
production observed in humans is mimicked in the murine model of
pulmonary infection (J Gamelas-Magalhaes and A Phalipon, unpub-
lished). Similar to what is observed at the acute phase of infection
in humans, the IFN-g-mediated Th1 type response is abrogated
in Shigella-infected mice at the early stage of infection, that is from
a few hours to 3 days postinfection (J Gamelas-Magalhaes and
A Phalipon, unpublished). Studies using different Shigella mutants
revealed that the IFN-g downregulation is mediated by a type III-
secreted bacterial effector, which interferes with the production of
IL-12, one of the most potent inducers of IFN-g production
(J Gamelas-Magalhaes et al., in preparation).

Neutrophils: the ultimate barrier to control Shigella infection
Soon after Shigella invasion, mucosal tissues become infiltrated pre-
dominantly by neutrophils, but also by monocytes. An early in vitro
study noted that neutrophils and monocytes could kill Shigella by
antibody-dependent cell-mediated antibacterial activity.

121

In response

to inflammatory stimuli, neutrophils migrate from the circulating
blood to infected tissues, where they efficiently bind, engulf and
inactivate bacteria. How neutrophils control pathogenic bacteria
expressing virulence factors that manipulate host cells was, until
recently, unclear. In 2002, Zychlinsky and his group demonstrated
that, in contrast to other cells, such as macrophages, neutrophils
prevent the escape of Shigella from phagocytic vacuoles in which the
bacteria are killed. Human neutrophil elastase (NE) was identified as
a key host defense protein: NE degrades Shigella virulence factors at
a 1000-fold lower concentration than that needed to degrade other
bacterial proteins. In neutrophils in which NE is inactivated pharma-
cologically or genetically, Shigella escapes from phagosomes, increasing
bacterial survival. NE also preferentially cleaves virulence factors of
Salmonella and Yersinia. Therefore, NE is the first described neutrophil
factor targetting bacterial virulence proteins.

122

Neutrophil-phagocytosed bacteria are killed rapidly by proteolytic

enzymes, antimicrobial proteins, and reactive oxygen species.
Neutrophils also degranulate, releasing antimicrobial factors into the
extracellular medium. Besides the classical intracellular pathway
(engulfment of bacteria by neutrophils and killing of the microorgan-
isms when the antimicrobial granules fuse with the phagosome), the
existence of a new extracellular pathway has been recently character-
ized. Neutrophils generate extracellular fibers, or neutrophil extra-
cellular traps (NETs), which are structures composed of granule
and nuclear constituents that disarm and kill bacteria extracellularly,
even before the microorganisms are engulfed by neutrophils.

123

This

extracellular structure amplifies the effectiveness of secreted anti-
microbial substances by ensuring their high local concentration.

In addition to their antimicrobial properties, NETs may serve as a
physical barrier that prevents further spread of bacteria. Moreover,
sequestering the granule proteins into NETs may keep potentially
noxious proteins like proteases from diffusing away and inducing
unwanted damage in tissue adjacent to the site of inflammation. NETs
are abundant in vivo in experimental dysentery and spontaneous
human appendicitis, two examples of acute inflammation. Thus,
NETs appear to be a form of innate response that binds microorgan-
isms, prevents them from spreading and ensures a high local
concentration of antimicrobial agents to degrade virulence factors
and kill bacteria.

It is noteworthy that some pathogens have evolved strategies to

escape neutrophil-mediated host defenses (for a review, see Urban and
Zychlinsky

124

). For instance, Francisella tularensis, a Gram-negative

bacterium and the causative agent of tularemia, escapes neutro-
phil-intracellular killing by disrupting the oxidative burst and escaping
the phagosome.

125

S. pneumoniae, the most common cause of com-

munity-acquired pneumonia, are trapped but, unlike many other
pathogens, not killed by NETs. Escaping NETs was shown to promote
spreading of pneumococci from the upper airways to the lungs and
from the lungs into the bloodstream during pneumonia.

126

serotype strains of the pathogen group A Streptococcus (GAS) are
associated with invasive infections including necrotizing fasciitis (NF)
and express a potent DNase, Sda1, which is both necessary and
sufficient to promote GAS neutrophil resistance and virulence in
a murine model of NF. Using this experimental model, Buchanan
et al. demonstrated a direct link between NET degradation and
bacterial pathogenicity. Indeed, inhibition of GAS DNase activity
enhanced neutrophil clearance of the pathogen in vitro and reduced
virulence in vivo.

127

Our

current

knowledge

Shigella-neutrophil

cross-talks

illustrates the dual role that innate immune responses may play
throughout the course of an infection. At early time postinfection,
neutrophils favors Shigella invasion by destabilizing the integrity of
the epithelial barrier whereas, at later stages of the infectious process,
they ultimately play a crucial role in bacterial clearance and sub-
sequent resolution of infection. How Shigella survives the early first
wave of neutrophil infiltration whereas succombing later on remains
to be elucidated. A hypothesis would be that some virulence effectors
are dedicated to Shigella escape from neutrophil-mediated bacterial
killing, with a finely tuned regulation of their expression, that is
upregulation and downregulation at the early and later stages of
the infectious process, respectively. In line with this idea, human
neutrophils undergo necrosis 2 h after Shigella infection with the
requirement for a functional TTSS.

128

Therefore, the disruption of

the epithelial barrier integrity at the early stage, which promotes
Shigella invasion, is probably due to both infiltrating neutrophils
and release of tissue injuring granular proteins upon their killing
by necrosis.

IMPACT OF SHIGELLA-INDUCED MODULATION OF HOST
INNATE IMMUNITY ON THE DEVELOPMENT OF ADAPTIVE
IMMUNITY
Shigella-specific immunity elicited upon natural infection is charac-
terized by the induction of a humoral response. Local secretory IgA
and serum IgG are produced in infected hosts. Both are directed
against LPS and some protein effectors (for a review see Phalipon and
Sansonetti

). Although T-cell recruitment and activation seem to

occur locally,

117,129,130

the contribution of these cells to protection

remains elusive. In the murine model of pulmonary infection, T cells,
in contrast to B cells, do not seem to account for protection against

Shigella’s ways of manipulating the host immune system
A Phalipon and PJ Sansonetti

Immunology and Cell Biology

re-infection.

131

In fact, protective immunity is serotype specific,

pointing out the O-Ag, the polysaccharide part of LPS, whose
carbohydrate composition defines the serotype, as the target for
protective antibodies. Recently, the serotype-specific oligosaccharide
determinants carried by the O-Ag of S. flexneri serotype 2a have been
identified.

132

A pentadecasaccharide has been shown to mimic

accurately the native O-Ag: upon its coupling to a carrier protein
and immunization of mice, a protective serotype-specific anti-LPS
antibody response is elicited

132

(A Phalipon, unpublished).

Natural protective immunity arises only after several episodes of

infection, is of short duration, and seems to be poorly efficient in
limiting re-infection, in particular in young children.

133,134

Consider-

ing the instructive role of innate immunity in the acquired immune
response,

135–137

the modulation of host innate immune responses

by Shigella certainly accounts for the type of protective adaptive
immunity developed in Shigella-infected patients, as discussed below.

Cytokines and chemokines are key players in linking innate and

adaptive immunity.

138,139

The proinflammatory cytokine/chemokine

network induced at the onset of Shigella infection

72,91,140

(J Gamelas

Magalhaes and A Phalipon, unpublished) is accompanied by the
production of anti-inflammatory mediators such as IL-10 and
TGF-b in order to limit tissue destruction and, therefore, avoid host
death. However, IL-10 and TGF-b are potent immunosuppressive
cytokines that impair the development of an efficient Th1-type
immunity.

141

Moreover, as previously cited, Shigella via the expression

of a type III-secreted bacterial effector directly inhibits the develop-
ment of an IL-12/IFN-g-mediated Th1-type response (J Gamelas
Magalhaes and A Phalipon, unpublished).

In addition, acute inflammation is accompanied by massive cell

death. Indeed, apoptotic cell death, including macrophage, DC, T and
B cells, in the lamina propria of infected patients is markedly
upregulated at the acute stage of Shigella where an increased number
of necrotic cells is also seen.

76,77,115

Phenotypic analysis of apoptotic

cells revealed that about 40% of T cells underwent apoptosis. Induc-
tion of apoptosis at the local site in the early phase of infection is
associated with a significant upregulation of Fas/Fas-L and perforin
and granzyme A expression and a downregulation of Bcl-2 and IL-2,
which promote cell survival.

Besides the direct effect of such massive

cell death on potent immune cells, recognition and engulfment of
apoptotic cells by professional APCs, such as DC, and their interaction
with effector immune cells have been recently described to result in
apoptotic cell-derived antigen specific tolerance.

142

Beyond its role in induction of acute-phase antimicrobial defense

genes, NF-kB is also a major regulator of the adaptive immune
response. Mice lacking individual NF-kB proteins show defects in
B- and T-cell proliferation and activation, cytokine production, and
isotype switching.

143

NF-kB also controls the production of factors,

including IL-18, IFN-g, IL-12 and co-stimulatory molecules such as
CD80/CD86, which are required for the development of an efficient T
helper-1 response against invasive pathogens.

144,145

By interfering with

NF-kB activation, a bacterial effector such as OspG

110

might also play

a role in preventing this switch, leading to a less efficient and shorter-
lasting anti-Shigella Th-2 type response.

In summary, the emerging scheme is that manipulation of innate

immunity by Shigella to promote its survival in the infected host
drives the acquired immunity towards immunosuppressive responses.
Further investigation aimed at studying specific immunity, with a
particular interest in the analysis of cross-talks between Shigella and
DCs or B and T cells, is needed to unravel the mechanisms mediated
by the virulence effectors and/or the host factors that account for the
poorly efficient anti-Shigella natural immunity.

IS THERE A NEED FOR SHIGELLA TO DIRECTLY TARGET THE
CELLULAR EFFECTORS FOR ADAPTIVE IMMUNITY?
Although every pathogen must contend with the onslaught of innate
immune responses, it is not necessarily the case that every pathogen
must counteract the acquired immune response to fulfill its replication
program. For most pathogens that cause acute infections, their life
cycle within the host is most often over by the time the naive host can
mount a meaningful acquired immune response capable of controlling
the infection. On the other hand, pathogens that cause persistent
infections might be under strong evolutionary pressure to evolve
specific mechanisms to avoid acquired immune responses.

Recently, several bacterial toxins such as anthrax toxins and the

Yersinia YopH bacterial effector have been shown in vitro to directly
disrupt immune cell functions by interfering with B- and T-cell
signaling.

111,146

However, it remains to be established in vivo whether

these activities are relevant during infection and whether these toxins
have specifically evolved to disrupt the adaptive immune system.

147

We may speculate that functionally impairing antigen-presenting cells
is the most convenient and efficient way for achieving control of
adaptive immunity. In vivo data recently revealed that in the case
of Y. pestis, DCs, macrophages and neutrophils are injected most
frequently with Yop proteins, whereas B and T lymphocytes are rarely
selected.

148

Whether inhibition of T- and/or B-cell signaling by

bacterial toxins or bacterial type III-secreted virulence effectors,
including those secreted by Shigella, should be added to the list of
the pathogenic mechanisms specifically evolved to encounter the
acquired immune response awaits further in vivo experimentation.

CONCLUSIONS
Higher organisms have evolved in the continuous presence of various
microbes and, therefore, developed a variety of host defense mechan-
isms to control resident microflora and prevent invasive microbial
diseases. Conversely, microbial organisms have coevolved with their
hosts to overcome protective host barriers, developing a variety of
outstanding strategies to manipulate host responses. Bacterial patho-
gens are able to avoid host recognition, dampen immune activation
or benefit from stimulation of the inflammatory response, which
disrupts the epithelial barrier and facilitates bacterial invasion.

149

The

innate immune system is clearly critical to achieve early control of
bacterial replication and successful eradication of the emerging infec-
tion. It is also linked to adaptive immunity, which completes clearance
of the infection and prevents further relapse by establishing specific
memory response. Shigella possess highly sophisticated mechanisms as
an adaptation to the human intestinal cell environment in order to
successfully multiply and disseminate. During infection, Shigella use
effectors secreted via the TTSS to direct various cellular signaling
pathways and modify the innate immune activation of the host.
Although the numbers of recently discovered Shigella effectors and
host cell targets have been rapidly increasing, many uncharacterized
effectors encoded by the virulence plasmid, and perhaps on the
chromosome still remain to be characterized, implying that Shigella
still possess unidentified strategies for infection and survival. There-
fore, effort to identify the activity of the novel bacterial effectors, their
host cell targets and the outcome of the interplay is needed to uncover
the complex interactions that occur between Shigella and the host.
Such knowledge will certainly open new routes for the development of
vaccine strategies to prevent shigellosis.

ACKNOWLEDGEMENTS

We wish to thank all the members, past and present, of the ‘Unite´ de
Pathoge´nie Microbienne Mole´culaire’ whose excellent contributions have made

Shigella’s ways of manipulating the host immune system

A Phalipon and PJ Sansonetti

Immunology and Cell Biology

this work possible. We are pleased to acknowledge Thierry Pedron and John
Rohde for a careful reading of the manuscript. PJ Sansonetti is a Howard
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