When a pathogenic microorganism first infects its host,
there is usually a dramatic activation of the INNATE and
ADAPTIVEimmune responses,which can result in disease
symptoms.If the pathogen and the host survive this
initial interaction,the adaptive host immune system
usually clears the invading offender.However,some
pathogenic bacteria are capable of maintaining infec-
tions in mammalian hosts even in the presence of
inflammation,specific antimicrobial mechanisms and
a robust adaptive immune response,and can therefore
be described as giving rise to persistent infection1,2(BOX 1;
see TABLE 1 for a list of bacteria that cause persistent
infections in humans).For example,Helicobacter pylori
inhabits the human gastric mucosa and persistence
of this bacterium in its host can be life-long3;
Mycobacterium tuberculosis can establish long-term
infections that can manifest as acute or chronic disease,
or can be clinically asymptomatic with the potential to
become reactivated later4,5; and Salmonella enterica
serovar Typhi (S. typhi) causes systemic infection
(typhoid fever) that involves colonization of the
RETICULOENDOTHELIAL SYSTEM (RES). Some individuals
who are infected with S. typhi become life-long car-
riers,periodically shedding large numbers of bacteria
in their stools.Persistently infected carriers serve as
the reservoir for these pathogens,and the carrierstate is
an essential feature that is required for survival of the
bacteria within a restricted host population.
Persistent colonization with these bacterial
pathogens is usually not clinically apparent.However,
even in the absence of clinical symptoms, infection
poses some risk to the host. Individuals who are
infected with M.tuberculosisare at risk of reactivation
of the pathogen to produce an active disease state that
can be life-threatening.A significant proportion of
people who are infected with H.pyloridevelop peptic or
duodenal ulcers,or even gastric cancer6.In addition,
individuals carrying S.typhihave an increased risk of
developing hepatobiliary cancer7.The long-term resi-
dence of the bacteria in a privileged host niche — such
as the MACROPHAGEvacuole or gastric mucosal layer —
poses several fundamental biological questions.For
example,what is the replicative and metabolic state of
the bacteria during persistent asymptomatic infection,
and how do these organisms manage to escape clear-
ance for so long in the presence of the host immune
response? We are only now beginning to understand
the bacterial and host factors that are involved in the
host–pathogen interaction during persistent infection,
and the answers to these questions are likely to provide
new and exciting directions for research in the fields of
microbial pathogenesis and immunology.
PERSISTENT BACTERIAL INFECTIONS:
THE INTERFACE OF THE PATHOGEN
AND THE HOST IMMUNE SYSTEM
Denise M.Monack,Anne Mueller and Stanley Falkow
Abstract | Persistent bacterial infections involving Mycobacterium tuberculosis, Salmonella
enterica serovar Typhi (S. typhi) and Helicobacter pylori pose significant public-health problems.
Multidrug-resistant strains of M. tuberculosis and S. typhi are on the increase, and
M. tuberculosis and S. typhi infections are often associated with HIV infection. This review
discusses the strategies used by these bacteria during persistent infections that allow them to
colonize specific sites in the host and evade immune surveillance. The nature of the host immune
response to this type of infection and the balance between clearance of the pathogen and
avoidance of damage to host tissues are also discussed.
INNATE IMMUNE RESPONSE
A cellular defence reaction that
counteracts invading pathogens,
such as bacteria and viruses.It
signalling and leads to the
activation of genes that are
responsible for bactericidal or
ADAPTIVE IMMUNE RESPONSE
This involves specificity and
immunological memory.It is
mediated by T and B cells
through the activation of
cytotoxic CD8+T cells for
pathogen killing or by
interaction with CD4+T cells
for antibody production.
NATURE REVIEWS | MICROBIOLOGY
VOLUME 2 | SEPTEMBER 2004 | 747
Stanford School ofMedicine,
Correspondence to D.M.M.
A diffuse system of cells that
helps the body fight infection
and eliminate cellular debris
through the action of phagocytic
cells (such as macrophages),
Kupffer cells in the liver and
reticular cells of the spleen,bone
marrow and lymph nodes.
Cells of the mononuclear-
phagocyte system that can
phagocytose foreign particulate
present in many tissues and are
important for nonspecific
cells that take up proteins and
present peptide antigens to
T cells in conjunction with
accessory molecules that
stimulate T-cell activation.
They are characterized by many
long,thin processes extending
from the cell body.
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R E V I E W S
Most individuals resolve infection with M.tuberculosis
soon after the onset of adaptive immunity12.However,
in some infected individuals,the organisms are never
completely cleared by the immune response4.
Persistently infected individuals can harbour bacteria
for many years, and even throughout their life.
Infected individuals are at risk of experiencing the
conversion of an asymptomatic infection into a highly
contagious, clinically active and potentially deadly
disease state that is known as reactivation TB.The risk
of conversion from an asymptomatic infection to one
that is clinically active is greatest soon after the initial
infection,and this occurs most often in immunolog-
ically compromised individuals, such as newborns,
the elderly and those who are infected with HIV13,14.
Mycobacterial survival at the immune interface
Persistent mycobacteria reside in granulomas.Although
the exact location of viable latent mycobacteria during
persistent infections remains controversial,bacteria are
often found inside macrophages within granulomas,
which are formed in response to persistent intracellular
pathogens15(FIG.1).Tuberculous granulomas in humans
and mice contain an organized collection of differenti-
ated macrophages,T lymphocytes,some B lymphocytes,
dendritic cells,neutrophils,fibroblasts and extracellular
matrix components16,17. Granulomas are thought to
arise initially from aggregates of mononuclear phago-
cytes that surround individual infected macrophages.
These macrophages become activated,and in many
cases several macrophages fuse to form giant cells,
which are also formed in response to other persistent
The ability to cause persistent infection is a funda-
mental aspect of the interaction between many diverse
viral, bacterial and eukaryotic pathogens and their
mammalian hosts. This review is not intended to
address all or even a significant proportion of these
pathogens. Rather, we will discuss several aspects of
three persistent bacterial pathogens:H.pylori,a pre-
dominantly extracellular pathogen,and M.tuberculosis
and S. typhi,which are both facultative intracellular
pathogens. We believe that the information that is
emerging from these studies will provide an insight into
the general features shared by all microorganisms that
have adapted to persist in the face of a highly evolved
host immune system.
Persistent mycobacterial infections
Pathogenic mycobacteria cause several long-term
infections in their respective hosts. M. tuberculosis
causes tuberculosis (TB), one of the oldest known
human infectious diseases,and this bacterium is esti-
mated to infect one-third of the global population8.
Primary infection with M.tuberculosisinvolves replica-
tion of the organism at the initial pulmonary site of
infection. This is followed by bacillaemia, in which
small numbers of bacteria are disseminated to the
extrapulmonary organs — such as the regional lymph
nodes — as well as to uninfected portions of the lung,
by a mechanism that may involve the migration of
M.tuberculosiswithin DENDRITIC CELLS9,10.Adaptive immu-
nity andrestriction of bacterial growth occurs after this
dissemination and is probably promoted by the arrival
ofbacteria in extrapulmonary lymphoid organs4,11.
Box 1 | Persistence versus commensalism
When thinking about persistent bacterial infections,it is important to keep in mind the distinction between bacteria that
are true commensals or part ofour normal flora and those that can cause disease symptoms in certain circumstances.
We believe that bacterial pathogens that are capable ofpersisting in a human host for long periods oftime fall into at
least two classes,both ofwhich have characteristics that distinguish them from commensal species.
One class is defined by a group of organisms that,after causing an initial disease state,are kept in check by an
adaptive immune response,but are not completely cleared from the host and persist in a privileged niche — perhaps
inside host cells — for long periods of time.Examples of such species are Helicobacter pylori,Salmonella enterica
serovar Typhi and Mycobacterium tuberculosis.
A second class ofpersistent bacterial pathogens are carried asymptomatically in the nasopharynx in most people
among the commensal flora,although they still have the ability to cause life-threatening disease in seemingly
immunocompetent individuals.Streptococcuspneumoniae,Neisseriameningitidisand Haemophilusinfluenzaetype B
are perhaps the best-known members ofthis group.All ofus at some time in our life are colonized,typically
asymptomatically,by these species.The host and bacterial factors that contribute to the disease state are unknown,but
the epidemiology ofmeningitis caused by S.pneumoniaeand N.meningitidisshows that it is caused by the acquisition
ofa new serotype ofthe same species,rather than by superinfection with the strain that is already present216.
How do the organisms in this second class ofpersistent bacteria colonize the human nasopharynx in an apparently
silent manner? The virulence mechanisms ofthese organisms usually involve antiphagocytic capsules,immunoglobulin
A (IgA) protease and antigenic variation ofouter-membrane proteins217.However,an intriguing hypothesis that can be
overlooked in this focus on disease is that these pathogenicity determinants have evolved to allow the bacteria to
colonize deeper tissues in the nasopharynx,and not just the mucosal surfaces as organisms ofthe normal flora do.
We propose that these organisms use virulence factors to penetrate the mucosal barrier and become resident in the
nasopharyngeal-associated lymphatic tissue (NALT)218in the same way that Salmonellapersists in the mesenteric lymph
nodes adjacent to the Peyer’s patches.In this way,the persistent bacteria in the NALT can re-seed the mucosal surface,
which is constantly exposed to host cleansing mechanisms,such as those involving neutrophils and IgA.The trigger that
causes colonization to go awry and develop into disease is presumably a combination ofboth host and pathogen
physiological and genetic factors that shifts the delicate balance.
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R E V I E W S
role in persistence in vivo,and is a feature that distin-
guishes pathogenic from non-pathogenic strains22.
As infected macrophages are the main reservoir of
infection by pathogenic mycobacteria,studies of the
biology and biogenesis of the mycobacteria-containing
phagosome have generated information that is important
for the understanding of the cell biology,immunology
and microbiology of these pathogens23.Many groups
have described the trafficking of mycobacteria within
unactivated macrophages in tissue-culture experiments.
In brief,mycobacteria interfere with phagosome matu-
ration by blocking the fusion of nascent phagosomes
with endosomal and lysosomal compartments and by
causing alterations in membrane proteins that normally
promote the formation of an acidic phagolysosome.
The steps involved in this process are beyond the scope
ofthis review,but are described in detail in REF.24
In addition to the ability to block phagosome matu-
ration in some circumstances,pathogenic mycobacteria
have also evolved mechanisms that allow them to persist
in macrophage phagolysosomes,within granulomas,in
the presence of a host immune response.A recent study
indicated that within frog granulomas ~60% of intact
bacteria of the species Mycobacterium marinum —
which is closely related to M.tuberculosis— resided in
phagolysosomes,and the level ofphagolysosomal fusion
correlated with the level of macrophage activation25.
Therefore,it is possible that mycobacteria have at least
two mechanisms of adaptation to intramacrophage
survival:restriction of phagolysosomal fusion early in
infection and adaptation to phagolysosomal fusion
within the activated macrophages of granulomas later
infections, particularly those caused by viruses15.
T lymphocytes and other immune cells are recruited
early during the process of granuloma formation18.The
lesion that is formed is sealed off from surrounding
tissue by epithelioid cells, which have tightly inter-
digitated cell membranes that form zipper-like arrays
and link adjacent cells,and which can also be fibrotic
and calcified.In the centre of granulomas there is usu-
ally an area of caseous necrosis — a region of cellular
debris that has a distinct appearance.
How does M.tuberculosissurvive within these lesions
for so many years? One hypothesis is that persistent
bacteria are either in a non-replicative state or only have
low levels of replication within the amorphous debris at
the caseous centre of the granuloma19. Evidence in
humans that the persisting bacteria are in a dormant
state comes from the results of culturing and staining
diseased tissues from patients who have undergone
chemotherapy, which might have resulted in false-
negative culturing results19-21.An alternative hypothesis
as to how a constant bacterial load is maintained is that
there is a balance between active bacterial replication
and killing by the immune system.This is an active area
ofresearch and is discussed in more detail below.
Survival within macrophages.Pathogenic mycobacteria
initiate long-term infection by entering host macro-
phages22,after which they cause extensive remodelling
of the PHAGOSOMAL environment to prevent the normal
maturation of this organelle into an acidic,hydrolytic
compartment23.The ability ofpathogenic mycobacteria
to replicate and/or survive in macrophages has an essential
cytoplasmic vacuole formed
around particles that are
ingested by phagocytosis.
Table 1 | Some persistent bacterial pathogens of humans
Likely sites of persistence
Macrophages in various sites and in
Macrophages in bone marrow, the RES
and possibly the gall bladder
Epithelial and endothelial cells
Salmonella enterica serovar
C. pneumonia causes respiratory and
cardiovascular disease; C. trachomatis
causes trachoma, genital-tract infections
and lymphogranuloma venereum
Gastritis; ulcers; gastric cancer;
Brucellosis (this can be chronic, leading to
lymphadenopathy and hepatosplenomegaly)
Cat-scratch disease; bacillary angiomatosis; Extracellular; in erythrocytes in blood
bacillary peliosis hepatitis
Genital-tract infections, which can lead to
epididymitis, pelvic inflammatory disease
Invasive infection results in meningititis
Acute otitis media; bacteraemia; meningitis
Extracellular; possibly also intracellular
in the stomach
Macrophages in the RES
Disseminated in various organs
Extracellular; intracellular at mucosal
Acute pharyngotonsillitis; pneumonia;
endocarditis; skin, soft tissue and bone
infections (necrotizing fasciitis)
Pneumonia; meningitis; bactereamia
Haemophilus influenzae type BNasopharynx; NALT?
NALT, nasopharyngeal-associated lymphatic tissue; RES, reticuloendothelial system.
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Figure 1 | Persistent mycobacterial infection and the host immune response. a | Pathogenic mycobacteria use several
receptors to enter macrophages, where they reside in a unique vacuole. The amount of mycobacterial replication is controlled
by many host immune factors. Effector T cells (including CD4+and CD8+T cells and double-negative (DN) T cells) and
macrophages participate in the control of infection. Interferon-γ (IFN-γ) and tumour-necrosis factor-α (TNF-α) produced by
T cells are important macrophage activators. Macrophage activation promotes phagosomal maturation, vacuole acidification
and the production of antimicrobial molecules, such as reactive nitrogen intermediates (RNIs) by nitric oxide synthase 2
(NOS2), reactive oxygen intermediates (ROIs), antimicrobial peptides and the NOS2-independent 47-kDa guanosine
triphosphatase protein LRG-47, which can block bacterial replication. The production of the proinflammatory cytokines
TNF-α, interleukin (IL)-18 and IL-23 by activated macrophages also contributes to controlling the intracellular replication of
mycobacteria. The ability of mycobacteria to inhibit the secretion of IL-12 by infected macrophages might contribute to
bacterial survival, as this cytokine normally functions to induce the production of IFN-γ. b | In most persistent mycobacterial
infections, the bacteria are initially contained in granulomas. Tuberculous granulomas are thought to arise from aggregates of
phagocytic cells that surround individual infected macrophages. These structures contain many T and B lymphocytes,
dendritic cells, neutrophils, fibroblasts and extracellular matrix components (for simplicity, only T cells are shown here).
Another striking feature of certain tuberculous granulomas is the presence of caseous necrosis in the centre of the
granuloma. Some of the genes that are specifically expressed by mycobacteria in granulomas encode the following proteins:
isocitrate lyase (ICL)60, an enzyme essential for the metabolism of fatty acids; outer-membrane proteins of the PE/PGRS
family that might have a role in antigenic variation; the transcriptional regulator MprA, which is involved in the regulation of
unidentified genes during adaptations that are required for persistence61; and PcaA, which encodes a cyclopropane
synthase. Mycobacteria that lack PcaA have reduced levels of persistence in the chronic mouse model63.
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important in controlling intracellular M.tuberculosis
replication34–37.However,a proportion of the bacteria
are clearly still able to survive in macrophages,perhaps
by a mechanism that involves inhibiting STAT1-
mediated IFN-γ transcriptional responses38and/or
suppressing the secretion of IL-12 — a proinflamma-
tory CYTOKINEthat acts to amplify IFN-γproduction39,40
— which may in part be mediated by the M.tuberculosis
Snm secretion pathway41.It is also likely that the ability
to resist killing by antimicrobial peptides contributes to
mycobacterial survival in macrophages42.
Animal models of mycobacterial persistence
From both a therapeutic and an epidemiological view-
point,the study of persistent mycobacterial infections
in animal models is very important given the difficul-
ties of studying latent TB in humans.Some research
has been done using guinea pigs, which are able to
arrest the initial acute phase of bacterial replication
during M.tuberculosis infection43.However,although
the resulting pathology resembles that seen in human
disease, these animals succumb to the pathological
consequences of infection43.Persistent infection can
also occur after intratracheal infection of cynomolgus
monkeys44,45.Although it is likely that this closely mim-
ics human infections,high costs limit the widespread
use of this non-human primate model. Therefore,
many research groups commonly use mouse models
of M.tuberculosis infection or frog and fish models of
M.marinuminfection.Although much information can
be gained from these models,care should be taken in
extrapolating the results obtained with these models
directly to human TB.
in infection.It is still unclear how the bacteria sense
these different intramacrophage environments; the
identification of the bacterial effector proteins that are
involved in this should provide considerable insight
into this crucial stage of mycobacterial pathogenesis.
In further support of the proposal that mycobacteria
use different strategies according to the circumstances,
recent publications indicate that mycobacteria have
temporal and immune-response-triggered differences
in gene expression both in activated macrophages in
vitro and in macrophages isolated from infected tis-
sue26–31.In addition,a recent study showed differential
mycobacterial survival in type 1 (interleukin-23 (IL-23)-
producing) and type 2 (IL-10-producing) macrophages32.
The immune status of the macrophage therefore has
an important role in bacterial persistence.Indeed,the
control of bacterial growth in murine models of latency
requires interferon-γ(IFN-γ),tumour-necrosis factor-α
(TNF-α) and nitric oxide (NO)16,all of which can alter
the environment of the bacteria-containing phagosome
and lead to killing ofthe pathogen.
IFN-γis a crucial component of immunity to TB as
it activates infected host macrophages,which directly
inhibit the replication of M.tuberculosis.The impor-
tance of this molecule in the control of mycobacterial
infections is highlighted by the discovery of IFN-γ-
related genetic mutations that predispose affected
individuals to active TB,as well as to other infections
that are caused by intracellular bacterial species,such
as Salmonellaspp.33(BOX 2; TABLE 2).IFN-γinduces the
expression of NO synthase 2 (NOS2) and of the newly
identified, NOS2-independent, 47-kDa guanosine
triphosphatase protein LRG-47,and both pathways are
that are important for
contribute to the
pathophysiology of acute and
Box 2 | Insights into host–pathogen interactions gained from genetics
Host genetic factors strongly determine the outcome ofinfectious disease.However,the molecular mechanisms of
resistance and susceptibility in humans are only just beginning to be investigated.Mouse models ofhuman infectious
disease have been used to identify and map host loci that are involved in controlling the complex aspects ofhost–pathogen
interactions (for recent reviews,see REFS 128,219).Three main approaches have been taken to identify these loci:
production ofmouse mutants by gene targeting;positional cloning ofhost-resistance genes in mutant mice;and mapping
and characterization ofquantitative trait loci (QTL) that control the complex aspects ofhost–pathogen interactions.
In some cases,the results of knockout-mouse studies have provided important information about the genetic basis
of susceptibility to bacterial infections in humans (TABLE 2).For example,mice with null mutations in the genes
encoding interferon-γ(IFN-γ) and either of the subunits of the IFN-γreceptor have been shown to be susceptible to
infection with pathogens such as Listeriamonocytogenes,Salmonella entericaserovar Typhimurium and
Mycobacterium tuberculosis,among others219,220.The investigation of a paediatric syndrome — known as idiopathic
mycobacterial infection or Mendelian susceptibility to mycobacterial infection — led to the identification of various
human mutations in the genes encoding the IFN-γreceptor,interleukin-12 (IL-12) and the IL-12 receptor.These
mutations lead to deficiencies that abrogate IFN-γ-mediated and IL-12-mediated immunity.The phenotype of
patients with this syndrome is an increase in the occurrence and severity of infections with mycobacteria that are
usually poorly pathogenic,such as the bacille Calmette–Guérin strain,and non-typhoidal salmonella,such as
Salmonellaenteritidis.IFN-γand IL-12 are therefore indispensable for bactericidal granuloma formation and
protective immunity against mycobacteria and salmonella in mice and humans.By contrast,Helicobacter pyloridoes
not seem to cause invasive or more severe disease in immunocompromised or very young individuals.For example,
IFN-γ-knockout mice are colonized with higher numbers of H.pyloriduring the first 4 weeks post-infection,but the
mice survive.Indeed,persistent H.pyloriinfection of IFN-γ-knockout mice resulted in less gastric inflammation even
in the presence of high levels of bacteria.So,IFN-γand other pro-inflammatory cytokines (TABLE 2),which are
ordinarily necessary for controlling bacterial infections,might actually contribute to the histological changes that are
associated with H.pyloriinfection,such as atrophic gastritis,intestinal metaplasia and dysplasia — conditions that
can lead to the development of H.pylori-induced gastric cancer.
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response,followed by a stable maintenance at high levels
in the lung over many months.The mice seem healthy
until the disease reactivates, which can take place as
much as 18 months later48.This chronic model resem-
bleslatency in humans in that it depends on the host
immune response to contain the infection.However,
unlike latent TB in humans,this model results in large
numbers of bacteria,which leads to pulmonary damage
that steadily accumulates in the lungs of chronically
Although these mouse models have limitations,
several groups have used them to address the meta-
bolic state of persistent mycobacteria.Rees and Hart
Mouse models of M. tuberculosis infection. Several
mouse models of TB latency have been established46–48.
The Cornell mouse model (also known as the drug-
induced model) involves partial clearance of M.tuber-
culosis infection by incomplete chemotherapy; the
infection is reduced to a point at which no bacterial
colonies are recovered46.Drug intervention is needed to
induce the latent state,which is not necessary in human
disease.The low-dose mouse model of latent TB (also
known as the chronic or plateau model) involves aerosol
infection or infection by intravenous routes.This results
in an initial acute phase of bacterial replication that is
controlled by the onset of an adaptive immune
Table 2 | Genes involved in susceptibility to M. tuberculosis, Salmonella serotypes and H. pylori
Gene product Phenotype in knockout mice*Phenotype
TNF-α p55 receptor
High susceptibility; increased granuloma necrosis
High susceptibility; increased granuloma necrosis
High susceptibility; increased granuloma necrosis
High susceptibility; defective granuloma formation
Moderate susceptibility; larger granulomas, but no
Moderate susceptibility; larger granulomas in lungs
High susceptibility; necrotic granulomatous
High susceptibility associated with mice homozygous
for the Nramp1D169/D169 allele
High susceptibility; impaired macrophage killing of
Higher susceptibility; reduced clearance of bacteria
from spleen and liver
High susceptibility; increased bacterial replication in
early phase of infection
High susceptibility associated with mice homozygous
for the Nramp1D169/D169 allele
TNF-α p55 receptorU 232
Higher numbers of bacteria early in infection;
no inflammation in persistently infected mice
More severe gastritis followed by bacterial clearance
*The phenotype listed for mice is for experimental infections with virulent bacteria.‡Human polymorphisms that are associated with higher
levels of expression have been linked to an increased risk of gastric cancer and its precursors. §Human polymorphisms that reduce
expression of the anti-inflammatory cytokine IL-10 are associated with increased risk of distal gastric cancer. I, proven susceptibility in
humans where normal individuals do not become infected with Mycobacterium tuberculosis, Mycobacterium avium, Mycobacterium bovis
bacille Calmette–Guérin, Mycobacterium smegmatis, Mycobacterium intracellulare, other non-pathogenic mycobacterial strains, Salmonella
enterica serovar Typhimurium, Salmonella enteritidis, Salmonella enterica serovar Paratyphi, and group B Salmonella. U, unknown or no
proven susceptibility in humans. IFN-γ, interferon-γ; IL, interleukin; IL-12Rβ1, IL-12 receptor β1; LPS, lipopolysaccharide; NF-IL6, nuclear
protein IL6; NOS2, nitric oxide synthase 2; TRL, tuberculosis-resistance locus; TNF-α, tumour-necrosis factor-α.
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model system to study mycobacterial pathogenesis22.
Experiments using the M.marinummodel have indi-
cated that persistent mycobacteria are in a metabolically
and replicatively active state.Bouley et al.have shown by
transmission electron microscopy in conjunction with
immunohistochemistry and acid phosphatase cyto-
chemistry that even long-term,single granulomas are
surprisingly dynamic environments,within which bac-
terial replication and phagocytic killing occur simulta-
neously25.In support ofthis,using the attenuated BACILLE
CALMETTE–GUÉRIN strain in rabbits, Dannenberg and
colleagues arrived at a similar conclusion56.These data
indicate that mycobacteria are not in an inert or ‘spore-
like’state,and that relatively constant bacterial numbers
are maintained in the presence of an active immune
response.Furthermore,studies using DIFFERENTIAL FLUO-
RESCENCE INDUCTIONto analyse M.marinumgene expres-
sion in granulomas showed that most of the promoters
that were found to be induced drive the expression of
genes that encode proteins with metabolic and synthetic
functions and that are expressed during logarithmic-
phase growth in laboratory media57.These results indi-
cate that persistent mycobacteria are metabolically
active;however,they do not prove that they are actively
dividing.Furthermore,these studies were performed
using the relatively stable reporter green fluorescent pro-
tein (GFP),so that downregulation of bacterial gene
expression within granulomas might not have been
detected.It is therefore possible that persisting bacteria
exist as a mixed population,in which some are actively
replicating and others are in an inactive state.
Mycobacterial persistence factors
In recent years,our understanding of mycobacterial
pathogenesis has advanced rapidly.Many genes that
are important for pathogenesis have been identified in
virulence expression screens and mutant screens,and
this work has been reviewed recently22.Sasseti et al.have
combined mutagenesis using the mariner transposon
with microarray technology to determine the genes that
are required for mycobacterial growth under certain in
vitroconditions (a method called TraSH,for transposon
site hybridization)58.When this method was applied to a
mouse model of infection,several genes were found to
be required at different stages after infection59.One class
ofmutants — known as persistence mutants — are able
to establish an infection to the same level as wild-type
bacteria,but are unable to maintain levels of bacteria in
the lungs to the same extent as wild-type strains.
The further characterization of these persistence
mutants awaits further studies.However,a number of
M.tuberculosisgenes have previously been indicated to
be important for persistent infection in the chronic
mouse model on the basis of gene-expression studies
and experiments using bacteria carrying knockout
mutations (reviewed in REF.5).Most notably,the results
of McKinney et al. show that persistence in mice is
facilitated by isocitrate lyase (ICL)60,an enzyme that is
essential for the metabolism offatty acids.Disruption of
the iclgene attenuated bacterial persistence and virulence
in immunocompetent mice,without affecting bacterial
compared total microscopic counts of bacteria in lungs
from infected mice with counts of viable bacteria over
a period of several months50.The number of bacteria
obtained by both techniques remained constant.One
interpretation of these findings is that the bacteria were
replicating either very slowly or not at all.However,it is
possible that any difference between the total number of
bacteria visualized and the number ofviable bacteria was
not within the limits ofdetection ofthe methods used.
More recently, other groups have used mouse
models to compare bacterial gene expression under
in vitro conditions that induce a non-replicating or
dormant bacterial state (that is,conditions of nutrient
deprivation51and oxygen depletion52) with in vivo
bacterial gene expression.These studies used either
quantitative real-time PCR (qRT-PCR) or cDNA microar-
ray techniques26,27,30.One such study of M.tuberculosis
gene expressionin infected mice showed that α-CRYSTALLIN
and genes of the DosR regulon are highly expressed in
response to host immunity mediated by type 1 HELPER T
CELLS(TH1 cells)27.DosR seems to have a crucial role in
mediating the expression of a series of hypoxia-induced
and NO-induced M.tuberculosisgenes53–55— a transcrip-
tion pattern that is characteristic ofthe non-replicating
persistence that is associated with the adaptation of
tubercle bacilli to hypoxia in vitro52.It was inferred from
this study that host immunity induces the arrest of
More recently,qRT-PCR has been used to measure
the levels of selected M. tuberculosis mRNAs during
laboratory culture,in vivo in the lungs of mice and in
lung tissue from four chronically infected humans26.In
culture,the differential expression of M.tuberculosis
mRNAs that are associated with iron limitation,alter-
native carbon metabolism and cellular hypoxia — con-
ditions that are thought to occur in granulomatous
lesions associated with TB — correlated with those that
were seen in bacteria isolated from wild-type mice.
However, in bacteria isolated from human TB lung
specimens,this set of mRNAs did not show the same
expression patterns as those seen in mice.This might
reflect host-specific differences or the fact that the
human lung tissue was not microdissected,resulting in
mixed bacterial populations from different environ-
ments.The latter hypothesis is supported by a recent
study that used DNA microarrays to examine genome-
wide expression profiles of M.tuberculosisisolated from
human lung tissue that was surgically removed from
patients who had not been treated with antibiotics,
but were infected with high levels of bacteria. The
gene-expression profiles of M.tuberculosiswere shown
to be characteristic of the site of infection (caseous
centres of granulomas,pericavities or distant lung),
indicating that M. tuberculosis actively senses and
responds to its microenvironment (H.Rachman and
Frog and fish models ofM.marinuminfection.Other
animal models have been developed more recently that
use M. marinum, which causes a TB-like disease in
ectothermic hosts such as frogs and fish and is a useful
The expression of this
chaperonin protein is
upregulated in vitroby hypoxia.
HELPER T CELLS
A subpopulation of activated
CD4+T cells that secrete
characteristic cytokines and
function primarily in cell-
mediated responses by
promoting the activation of
cytotoxic T cells and
The attenuated Mycobacterium
A selection strategy used to
identify bacterial genes that are
preferentially expressed when a
bacterium is in a particular
random pieces of bacterial DNA
in front of a promoterless green
fluorescent protein (GFP) gene,
flow cytometry can be used to
screen for genes expressed in
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R E V I E W S
of T-cell subsets in these two phases of infection4.
Whether or not this is the case, CD4+T cells have an
important but as yet undefined role in the control of
persistent infection67.Although other activities ofCD8+
T cells,in addition to IFN-γproduction,might also be
important for the control of persistent infections68-70,
further studies are needed to determine the exact roles
The production ofimmunosuppressive cytokines —
such as IL-10 and transforming growth factor-β(TGF-β)
— has been documented in humans with active TB71-73,
and IL-10 production is increased in the lungs of mice
that show chronic mycobacterial infection.This indi-
cates that the ability of IL-10 to downregulate the
immune response might contribute to the reactivation
of chronic M.tuberculosisinfection74.In animal models,
the proinflammatory cytokine TNF-αhas a key role in
host responses against TB75,76,including granuloma
formation and the containment of disease18,77.
Furthermore,treatments with antibodies that neutralize
TNF-αcause reactivation of TB in a mouse model of
latent infection78and in human latent infection, as
shown by the clinical observation of reactivation of TB
during the treatment of autoimmune disease79.TNF-α
therefore has a significant role in the control of persis-
tent M.tuberculosisinfections and,as discussed below,is
equally important in persistent Salmonellainfections.
Persistent Salmonella infections
Salmonellaserovars are responsible for human diseases
ranging from gastroenteritis to systemic infections.
Systemic Salmonellainfection is usually host-depen-
dent,and S. typhi causes only systemic infection —
typhoid fever — in humans.Salmonella entericaserovar
Typhimurium(S.typhimurium) infection of mice and
S.typhiinfection of humans is characterized by inflam-
mation at the site of bacterial entry,which is typically at
the PEYER’S PATCHES80.After Salmonellaspp.penetrate the
epithelial barrier,they preferentially infect phagocytes
within the lamina propria (FIG.2).In Salmonella gas-
troenteritis,the infection is usually self-limiting and
does not proceed beyond the lamina propria.
However, in host-adapted salmonellosis, such as
typhoid fever, Salmonella-infected phagocytes gain
access to the lymphatics and bloodstream,allowing the
bacteria to spread to the liver and the spleen81,and can
persist in the gall bladder and bone marrow82,83.
S.typhi and Salmonella enterica serovar Paratyphi
(S.paratyphi) serovars are important human pathogens
ofimmense concern to public health and with consider-
able economic impact.They are endemic in regions of
the world where drinking-water quality and sewage-
treatment facilities are poor1,84and infections remain
difficult to treat by antibiotic therapy due to the
increasing frequency of resistant bacteria85.A significant
percentage (1–6%) of typhoid patients become chronic
carriers of S.typhi,as do many people who have never
had a clinical history of typhoid fever86–88.These indi-
viduals shed bacteria in their stools and urine for peri-
ods of time that range from a year to a lifetime,without
any apparent signs of disease89.Typhoid carriers are of
growth during the acute phase of infection.These data
indicate that during late stages of infection,M.tubercu-
losis cells might convert lipids into carbohydrates
through the GLYOXYLATE-SHUNT PATHWAY60and that latent
bacteria might reside in an environment such as lung
granulomas,in which carbohydrates are limited but
lipids are available.
Several other persistence mutants have been identi-
fied.One of these affects the transcriptional regulator
MprA,which is involved in the regulation of unidenti-
fied genes during adaptive responses that are required
for persistence61.In addition,several mutants have been
identified that have alterations in their cell walls62.For
example, the M. tuberculosis pcaA mutant (pcaA
encodes a cyclopropane synthase) shows reduced levels
of persistence in the chronic mouse model63.The roles
ofthese bacterial genes in persistent infections and their
functions in immune modulation await further studies.
Immune responses to persistent mycobacteria
Substantial progress has been made in understanding
the immune-system mechanisms that are involved in
the containment of the initial phase of M.tuberculosis
infection (reviewed recently in REFS 4,5,16).However,less
is known about the immune mechanisms that are
involved during persistent mycobacterial infections.In
addition to reactivation,recent work using molecular-
fingerprinting techniques has documented the reinfec-
tion of immunocompetent individuals with new strains
of M.tuberculosis22.These data show that immunity to
TB can be incomplete,and indicate that reinfection,at
least in areas where TB is prevalent, probably has a
greater role than was previously appreciated.
Indeed, the dynamic nature of mycobacteria and
their interactions with granulomas during persistent
infection is highlighted by the recent findings of Cosma
et al. This group showed that exogenously infecting
M. marinum in zebrafish rapidly enter pre-existing
granulomas by specific mycobacteria-mediated mecha-
nisms that direct infected macrophages into granulomas64
(FIG.1).To probe the cellular dynamics of mycobacterial
reinfection in vivo,Cosma and colleagues followed the
route of superinfecting M.marinum or an unrelated
Salmonella strain — which were labelled with
macrophage-inducible GFP reporter constructs — in
the context of a previously established infection.The
superinfecting M.marinum— and not the Salmonella
strain — trafficked into pre-existing granulomas,
rapidly expressed granuloma-specific promoters and
remained in these lesions for up to 2 months64.These
findings indicate that mycobacteria rapidly adapt to the
mature granuloma environment and provide new
insights into the interactions of these bacteria with the
adaptive immune system.
The role ofthe adaptive immune response in persistent
mycobacterial infections is an area ofgreat interest.There
is some evidence that CD8+T cells secrete most of the
IFN-γthat is produced during persistent infection in the
chronic mouse model.This is in contrast to the acute
phase of disease,during which CD4+T cells produce
most ofthe IFN-γ65,66,indicating a differential activation
A biochemical pathway that is
used by plants and
microorganisms to metabolize
acetate or long-chain fatty acids
as a source of energy.
Lymphoid nodules located in the
small intestine that trap antigens
from the gastrointestinal tract
and provide sites where immune
cells — such as B and T
dendritic cells — can interact
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R E V I E W S
Mycobacteriumspp.and Leishmaniaspp.— presumably
by depriving the bacteria of divalent cations93.Nramp1
is therefore involved in the control of the exponential
growth of S.typhimurium in the reticuloendothelial
organs during the early phase (first week) of infection
in mice94.Consequently,mice carrying two copies of
the mutant Nramp1Asp169allele are significantly less
resistant to lethal S.typhimuriuminfections than mice
that harbour the wild-type Nramp1Gly169allele94.
Salmonellaclearance during the late phase of infec-
tion (3–4 weeks post-infection) seems to be influenced
by various host loci through effects on the acquired
immune response.The mouse MAJOR HISTOCOMPATIBILITY
COMPLEX(MHC;also known as H2) has an important
role in the clearance of Salmonella91,95. Similarly, in
humans,a genetic link between specific class II and class
III MHC haplotypes and relative resistance to S.typhi
has been shown96. Several studies have also shown a
requirement for both CD4+and CD8+T lymphocytes
for clearance of Salmonella infections97–100, and the
humoral response is also required for this100–102.
Historically,Salmonellapathogenesis has been inves-
tigated in Nramp1Asp169mice,which are highly sensitive
to Salmonellainfection.This reflects,in part,the fact that
death is an experimental endpoint that is readily mea-
sured,so this model is useful for assessing the relative
contributions of Salmonellavirulence factors and host
special concern from a public-health viewpoint as they
are the reservoirs for the spread of infection and disease.
From the bacterial perspective,persistent infection is
essential for microbial survival in nature.S.typhiis car-
ried for years — even in the presence of an immune
response — and chronic carriers of S.typhihave high
levels of circulating serum antibodies to the Vi antigen
and to flagellar antigens84,90.Investigating the chronic
carrier state in salmonellosis should provide an insight
into bacterial survival strategies,as well as information
that could be used to develop new approaches for the
treatment of typhoid and other persistent microbial
Mouse models of Salmonella persistence
Mouse typhoid is similar to human typhoid in a number
of ways,although different strains of mice have varying
levels ofsusceptibility to Salmonellainfection91.In mice,
a significant component ofinnate resistance or suscepti-
bility to infection with S.typhimuriumis controlled by
the gene Nramp1(also known as Slc11a1),which encodes
a proton/divalent-cation antiporter that regulates suscep-
tibility to infectious disease92. Nramp1 expression is
restricted to cells of the monocyte/macrophage lineage,
and because it localizes to the vacuolar membrane it
affects the capacity of the host to control intracellular
replication of Salmonella bacteria — as well as
A complex of genes encoding
cell-surface molecules that are
required for antigen
presentation to T cells.
lumen via bile
Figure 2 | Persistent Salmonella infection. Schematic representation of persistent infection with Salmonella enterica serovar Typhi
in humans. Bacteria enter the Peyer’s patches of the intestinal tract mucosal surface by invading M cells — specialized epithelial
cells that take up and transcytose luminal antigens for uptake by phagocytic immune cells. This is followed by inflammation and
phagocytosis of bacteria by neutrophils and macrophages and recruitment of T and B cells. In systemic salmonellosis, such as
typhoid fever, Salmonella may target specific types of host cells, such as dendritic cells and/or macrophages that favour
dissemination through the lymphatics and blood stream to the mesenteric lymph nodes (MLNs) and to deeper tissues. This then
leads to transport to the spleen, bone marrow, liver and gall bladder. Bacteria can persist in the MLNs, bone marrow and gall
bladder for life, and periodic reseeding of the mucosal surface via the bile ducts and/or the MLNs of the small intestine occur, and
shedding can take place from the mucosal surface. Interferon-γ (IFN-γ), which can be secreted by T cells, has a role in maintaining
persistence by controlling intracellular Salmonella replication. Interleukin (IL)-12 — which can increase IFN-γ production — and the
proinflammatory cytokine tumour-necrosis factor-α (TNF-α) also contribute to the control of persistent Salmonella (not shown).
756 | SEPTEMBER 2004 | VOLUME 2
R E V I E W S
trafficking of the Salmonella phagosome has been
analysed in unactivated tissue-culture macrophages and
it has been concluded that most Salmonella-containing
vacuoles do not interact extensively with late endosomes
and lysosomes113.Studies of intracellular Salmonella
gene expression in unactivated macrophages have
shown that numerous virulence and SOS-response
genes show significant changes in expression in
response to the vacuolar environment114.However,the
trafficking and gene expression patterns of persistent
intracellular Salmonella have not yet been investi-
gated.Furthermore,the fate of macrophages that are
persistently infected with Salmonella is not known,
nor is it clear how the bacteria infect new host cells
over time. It is possible that bacteria persist within
macrophages for the lifetime of the host cell and then
infect a new macrophage.However,S. typhimurium is
able to induce host-cell death in vivo115,116,providing a
potential mechanism by which Salmonella can escape
from an infected cell to infect neighbouring cells.
S.typhimuriummediates macrophage death by at least
two mechanisms. One mechanism involves rapid
macrophage death that requires the type III secretion
system (TTSS) that is encoded by the Salmonella PATHO-
GENICITY ISLANDSPI1 (REF.117).The potential role of SPI1
and SPI1-mediated macrophage cytotoxicity in persis-
tent S.typhimuriuminfections is under investigation.
S.typhimuriumcan also induce macrophage death
that occurs approximately 18 hours after infection.This
delayed macrophage death requires another TTSS that
is encoded by a second pathogenicity island,SPI2,and is
used inside host cells118,119.It is possible that dead or
dying macrophages containing S. typhimurium are
phagocytosed by other macrophages that are recruited
to the site of infection,which then serve as a safe haven
in which Salmonellacan survive while avoiding extra-
cellular host defences.It is also possible that the SPI2-
mediated mechanism of cell death is not active during
persistent infection of macrophages.Indeed,differential
expression of SPIgenes could be a strategy used by per-
sistent Salmonella120.It is clear that SPI2 is required to
avoid the effects of PHAGOCYTIC OXIDASE (PHOX) during
infection of macrophages121and to initiate systemic
infection122,123,but its role and the role of individual
SPI2-secreted effector molecules in the continuing
presence ofpersistent ofS.typhimuriumis not yet known.
Persistent Salmonella and the immune response
Mice that are persistently infected with S.typhimurium
have high anti-Salmonella antibody titres106. This
might represent a deliberate infection-associated shift
from a TH1 to a TH2 response,which might be involved
in keeping the numbers of bacteria inside each
macrophage lower in the persistent S. typhimurium
model than those reported in previous studies of acute
infections in Nramp1-susceptible mice in which the
mice died124.However,the adaptive immune response
also provides positive feedback to the innate immune
system through the synthesis of cytokines that either
increase effector-cell numbers or activate these cells to
produce an increased antibacterial response.
immune responses in an acute infection. Although
acute S.typhimuriuminfections have been well charac-
terized using this model,it is not suitable for studies of
long-term carriage,as the mice either die rapidly from
relatively low doses of Salmonella or attain sterilizing
immunity.Previous studies using specific S.typhimurium
mutant strains — such as an attenuated strain that is
unable to synthesize aromatic amino acids de novo
(aroA–strain) or a mutant in polynucleotide phospho-
rylase (PNPase) that has altered virulence gene expres-
sion — in Nramp1-deficient strains of mice have
shown that these bacterial mutants can colonize mice
for as long as 2 months103–105.Although these models
are useful for understanding the development of pro-
tective immunity to Salmonella,they have not added a
great deal to the understanding of the biology and
pathogenesis of natural persistent Salmonellainfections
with wild-type bacteria.
Persistent Salmonella infection can be effectively
studied using the 129sv mouse strain — which carries a
wild-type Nramp1 allele — and wild-type S. typh-
imurium.Oral infection of129sv mice results in systemic
infection that,in most cases,does not lead to death ofthe
host.Persistent infection in this model is characterized
by sporadic excretion ofbacteria in stools and long-term
carriage of S.typhimuriumin low numbers within clas-
sical granulomatous lesions,which arise in the spleen,
liver, gall bladder and mesenteric lymph nodes
(MLNs)106.The data obtained from studies using persis-
tently infected mice indicate that the most common site
of chronic carriage of S.typhimuriumis the MLNs106.
Indeed, this is often the only site from which viable
Salmonella can be recovered.
Chronic infections with S. typhi and Salmonella
enterica serovarDublin are classically associated with
long-term excretion of bacteria and localization in the
gall bladder107–109. Although humans that carry
Salmonellachronically often have BILIARY-TRACTdisease,
this condition is not an absolute requirement for devel-
opment of the carrier state88,110. A previous study
showed that S.typhi was carried exclusively in MLNs
50 days after oral infection of chimpanzees111.In the
case of Salmonella enterica serovar Pullorum,it was
recently shown that bacteria are carried in the spleen
and reproductive tract,specifically in the ovaries and
oviducts of hens112. In a recent study from our own
laboratory,we found the main site of chronic carriage of
S.typhimuriumin mice to be the MLNs,and not the gall
bladder106.These studies indicate that the true reservoir
ofpersistent bacterial carriage might change in response
to the host immune status and the underlying disease,a
situation that might also apply to human infections.
Persistent Salmonella in macrophages
The ability of Salmonellato survive in macrophages is
required for systemic colonization of the host.Indeed,
chronically infected humans and mice harbour
Salmonella within the reticuloendothelial system for
long periods of time,and our group has shown that the
persistent bacteria reside in low numbers within MOMA2+
MACROPHAGESresiding in the MLNs106.The intracellular
Includes the gall bladder and bile
ducts,which make and transport
bile.Bile contains salts or
detergents that disrupt bacterial
membranes;it also activates
autolysins that digest
MOMA2 is expressed in the
cytoplasm of monocytes and
macrophages can be found in
the splenic red pulp,in the
cortex of the thymus,in the
subcapsule and medullary
regions of lymph nodes and in
sites of acute and chronic
Large (10 –50-kb) insertions in
the bacterial chromosome that
encode virulence determinants.
They are thought to be acquired
by horizontal transfer.
PHAGOCYTIC OXIDASE (PHOX)
Production of reactive oxygen
intermediates,which can kill
bacteria directly or after reacting
with chlorine,is mediated by the
NADPH oxidase system located
in the membrane of the
macrophage and includes the
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genes that are specifically required for persistence.
S. typhimurium strains that are deficient for mig-14,
virK and somA are able to replicate in unactivated
macrophages and establish infections in mice; how-
ever,these mutants begin to be cleared in BALB/c mice
between 7 and 10 days post-infection134,135.The exact
functions of the proteins encoded by these genes are
not known,but they contribute both to resistance to
antimicrobial peptides,which are produced in acti-
vated macrophages136,and to replication in these cells
(I.Bradshaw,D.M.M.and S.F.,unpublished observa-
tions). The identification of other S. typhimurium
mutants that are unable to persist in mice will
increase our understanding of bacterial mechanisms
Persistent Helicobacter pylori infections
During the past 20 years,H.pylori has emerged as an
important example of a persistent bacterial pathogen.
Not only does this bacterium successfully colonize the
hostile environment of the human stomach, but the
infection regularly persists for the lifetime of the host in
the face of a constant,vigorous innate and adaptive
immune response.In most infected people,H.pylori
infection causes superficial chronic gastritis,which is
usually clinically asymptomatic,although histologi-
cally apparent. However, a significant subset of
infected individuals are at risk of the subsequent
development of duodenal and peptic ulcers,and 1% of
those that are infected will develop adenocarcinomaor
lymphoma of the stomach137.
Most basic research in the Helicobacter field has
focused on the study ofbacterial virulence determinants
(reviewed in REFS 6,138,139; TABLE 3),particularly in the
context of their association with severe gastrointestinal
sequelae of infection.Although the experimental focus
on the association of H.pylori with disease is under-
standable,it is important to point out that the ecological
niche for H.pyloriis progressively lost with the devel-
opment of ATROPHIC GASTRITIS140,and bacteria can only
rarely be cultured from seropositive patients withade-
nocarcinoma.So,from the microbial standpoint,the
progression of asymptomatic gastritis to more serious
tissue destruction can be viewed as contraryto the best
interests ofthe bacteria in terms ofevolutionary success.
Therefore,for the purpose of this review,we will shift
the focus from the minority of colonized individuals
who develop overt disease (~20%) to the majority who
remain relatively symptom-free and are likely to repre-
sent the reservoir for human infection.We focus here on
the bacterial and host characteristics that enable H.pylori
to colonize its host persistently in the face of a normal
Most of the studies we will refer to here usedanimal
models.Some H. pylori isolates establish long-term
infections in rodents or have been adapted to do so by
repeated passaging (reviewed in REF.141).Indeed,the
limiting factor for the development of animal models of
persistent H.pyloriinfection seems to be host specificity
rather than lack of persistence.The models that have
been used so far vary markedly with respect to disease
Once T cells are activated during an infection,they
produce the macrophage-activating factor IFN-γ,which
has a role in the acute Salmonella mouse model in
controlling the early phase of bacterial replication125–127.
It was recently shown that IFN-γhas an important role
in maintaining and controlling the level of bacterial
replication in persistently infected animals,perhaps by
stimulating infected macrophages to suppress bacterial
replication106.Furthermore,people who lack the IL-12
receptor — in whom the TH1 response and the pro-
duction of IFN-γare defective — are more susceptible
to infections with Salmonellaspp.128In addition to IFN-
γand IL-12,TNF-αmight also have a role in maintain-
ing andcontrolling the level of bacterial replication in
persistently infected hosts.Similar to the results seen
with M.tuberculosis,patients who were treated with
anti-TNF-α antibodies developed Salmonella septi-
caemia129.HIV-positive individuals develop chronic
bacteraemia caused by species of Salmonella that do not
normally pass beyond the MLNs in healthy individuals1
— further indicating that an intact adaptive immune
system has a role in immunity to Salmonella.
Salmonella bacteria are probably not passive
bystanders in terms of maintaining the balance between
clearance and persistence.In this regard,Salmonella
might have an active role in modulating or even directly
manipulating host responses,thereby preventing clear-
ance of intracellular bacteria.Many studies have shown
that S.typhimuriummay limit the in vivoproliferation
of CD4+and CD8+T cells,despite their activated phe-
notype130. In addition, it has been shown that active
S.typhimuriuminfection leads to immunosuppression
in mice and causes the production of large amounts of
IL-10,which has immunosuppressive activities,and
nitric oxide (NO),which has both immunosuppressive
and direct antibacterial activities126,131–133. Indeed,
S.typhimuriummutants have been identified that are
unable to persist in mice, indicating that they lack
Chronic inflammation of the
stomach with degeneration of
Table 3 | Helicobacter pylori virulence determinants
Description/potential role in
95-kDa secreted vacuolating toxin; induces
apoptosis; involved in immunomodulation
and colonization of mouse stomach
37-kb genomic fragment; contains 29
genes that encode a type IV secretion
120-kD protein; translocated into host cell by
type IV secretion apparatus encoded on Cag-
PAI; phosporylated in host cell and binds
SHP-2 tyrosine phosphatase; disrupts tight
junctions; epidemiologic link to cancer
78-kDa outer membrane protein; binds to
fucosylated Lewis B blood group antigen;
mediates adhesion to epithelial cells and
possibly stomach epithelium
Resists acidic conditions in the stomach; activates
innate immune responses during early steps
Involved in motility; essential for colonization
PAI, pathogenicity island.
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R E V I E W S
innate and adaptive immune responses;avoidance of a
strong proinflammatory response; extensive genetic
intrastrain and interstrain diversity; and a partially
intracellular lifestyle. In the following sections, we
discuss each of these strategies in turn.
H. pylori evasion of host immune responses
Evasion ofinnate responses.NO is a key component of
theinnate immune system and an effective antimicro-
bial agent143.It is produced by activated macrophages
through the action of NOS2,which uses L-arginine as a
substrate and is highly expressed both in macrophages
infected with H.pylori144and infected gastric tissues145.
In a series of elegant experiments using both cultured
and peritoneal macrophages,Gobert et al.showed that
H.pyloriprevents NO production by host cells by pro-
ducing the enzyme arginase146.Encoded by the gene
rocF,arginase — which is associated with the bacterial
cell envelope — competes with NOS2 for the L-arginine
substrate and converts it to urea and L-ornithine,rather
than NO.Mutation of the rocFgene results in efficient
killing of the bacteria in an NO-dependent manner,
whereas wild-type bacteria survive under these condi-
tions.Furthermore,the rocFmutant is mildly attenuated
in its ability to colonize mice147,indicating that arginase
expression might indeed be important for survival and
persistence in vivo.
Other bactericidal functions of macrophages also
seem to be impaired in the presence of H.pylori,and
two possible mechanisms for this have been suggested.
In one study,Allen et al.showed delayed uptake of bac-
teria into macrophages followed by the formation of
megasomes as a result of phagosome fusion. These
megasomes protect intracellular bacteria from efficient
killing148.In a second study using human blood mono-
cytes and polymorphonuclear cells, Ramarao et al.
showed that H.pylori can actively block itsown uptake,
as well as the uptake of co-cultured bacteria of other
species and latex beads149,150.Both of these phenotypes
depended on the presence of the Cag pathogenicity
island (Cag-PAI),which is a 37–40-kb stretch of DNA
that encodes a type IV secretion system (TFSS) and
which epidemiological studies have linked to more
severe disease outcomes151–153.
Evasion ofadaptive responses.H.pylori has evolved to
subvert not only the innate, but also the adaptive
immune response,which is based on MHC-class-II-
restricted — and to a lesser degree MHC-class-I-
restricted— T cells154.Antigen-dependent proliferation
of T cells is blocked specifically by H. pylori155— an
effect that is mediated by the virulence factor vacuolat-
ing cytotoxin A (VacA)156,157.VacA is a 95-kDa,secreted
protein that,among other functions,induces cellular
vacuolization in epithelial cells158,159.VacA has been
shown to act as an immunomodulator by interfering
with the IL-2signalling pathway in T cellsby blocking
Ca2+mobilization and the activity of the Ca2+/calmod-
ulin-dependent phosphatase calcineurin156,157. The
secretion of IL-2 and the surface localization of the
high-affinity IL-2 receptor (IL-2R) are necessary for
outcomes,which range from atrophic gastritis,intestinal
metaplasia and gastric adenocarcinoma to mucosa-
associated lymphoid tissue (MALT) lymphoma,
depending on the species used and the genetic back-
ground of the host141.The identification of bacterial
genes that are associated with colonization and persis-
tence of H.pylorihas been limited by the lack of animal
models that support infection by strains for which the
genomes have been completely sequenced.This obsta-
cle has recently been overcome by the development of
an IL-12-deficient mouse model that is susceptible to
infection by the sequenced strain KE26695,which should
facilitate whole-genome-based studies ofvirulence and
H.pylorihas developed a number of unique features
and strategies that enable it to persist in its host (FIG.3).
These include escape from and neutralization of the
Type IV secretory
Disruption of epithelial
barrier — nutrients
leak into mucous layer
Figure 3 | Persistent Helicobacter pylori infection. Interplay between H. pylori factors and
the host response leads to chronic gastritis and persistent colonization. H. pylori binds to gastric
epithelial cells through BabA and other adhesins249. In strains that carry the Cag pathogenicity
island (Cag-PAI), a type IV secretory apparatus allows translocation of effector molecules such
as CagA into the host cell, resulting in the production of interleukin (IL)-8 and other chemokines
by epithelial cells. The secreted chemokines lead to the recruitment of polymorphonuclear cells
(PMNs), resulting in inflammation. Injected CagA also associates with tight junctions and targets
H. pylori to them. In the long term, CagA might cause disruption of the epithelial barrier and
dysplastic alterations in epithelial-cell morphology. Disruption of junctions by CagA might also
cause leakage of nutrients into the mucous layer245and entry of bacterial VacA into the
submucosa. VacA induces apoptosis in epithelial cells by reducing the mitochondrial
transmembrane potential and inducing cytochrome c release, which might also contribute to the
disruption of the epithelial barrier. Tumour-necrosis factor-α (TNF-α)-mediated apoptosis may
also lead to disruption of the epithelial barrier. The chronic phase of H. pylori gastritis links an
adaptive lymphocyte response with the initial innate response. Cytokines produced by
macrophages, particularly IL-12, activate recruited cells — such as helper T cells (TH0, TH1 and
TH2), which respond with a biased TH1 response, and B cells. Cytokines also alter the secretion
of mucus, which contributes to H. pylori-induced disruption of the mucous layer, as they induce
changes in gastric-acid secretion and homeostasis. H. pylori inhibits the host immune response
by blocking the production of nitric oxide (NO) by macrophages and through the ability of VacA
to interfere with the IL-2 signalling pathway in T cells (and therefore T-cell activation) by blocking
transcription of the genes encoding IL-2 and its receptor, IL-2R (see main text for details).
An intracellular pool of H. pylori may repopulate the mucous layer after cycles of extracellular
clearance. Ig, immunoglobulin.
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The effect of H.pyloriLPS on gastric epithelial cells
— the other main source ofproinflammatory cytokines
in the stomach besides macrophages — is still unclear.
One study has shown that H.pylori LPS produced by
strains harbouring the Cag-PAI,but not from Cag-PAI
mutants,activates Toll-like receptor 4 (TLR4) on gas-
tric-pit cells,thereby stimulating the innate immune
responses of the gastric mucosa168.By contrast,Smith et
al.reported that TLR2 mediates the proinflammatory
effects of H. pylori on epithelial cells and show that
H.pylori LPS is a TLR2 agonist169.These authors sug-
gest that the low level of pathogenicity of H.pylori LPS
does indeed result from its failure to activate TLR4,the
receptor that mediates LPS signalling of most other
A recent study has reported similar findings for
H.pyloriflagellins.The two H.pyloriflagellins — FlaA
and FlaB — were shown to have a markedly reduced
potential to activate TLR5 compared with flagellins
of other Gram-negative bacteria, such as FliC of
S.typhimurium171.The evolution of these unique fla-
gellins,which share extensive amino-acid homology
with flagellins from other species that do stimulate
TLR5,has been proposed to preserve the essential func-
tion of the flagella during chronic colonization while
avoiding the activation ofthe innate immune system171.
In a recent study investigating a link between the
Cag-PAI and the induction of proinflammatory
responses,Philpott et al.showed that clinical isolates of
H.pylori were more likely to colonize mice if they did
nor harbour the Cag-PAI and were therefore unable to
induce such responses in cultured cells172. Mouse-
adapted variants that lacked the Cag-PAI infected mice
at higher levels and had a reduced capacity to induce
inflammatory responses in vitro compared with the
respective parental strains.Taken together,these find-
ings imply that there may be a profound in vivoselec-
tion against H.pyloristrains and variants that induce a
strong host inflammatory response172,at least in the
The relative in vivo advantages for H. pylori with
Cag+versus Cag–phenotypes in human hosts are
unclear173,174.One study of an individual infected with
multiple strains showed recombination between strains
resulting in excision of the Cag-PAI and subsequent
positive selection of a Cag–strain175. It is therefore
unclear as to how the Cag-PAI benefits the bacteria dur-
ing a persistent infection;it may be that it is important
for a specific stage of colonization,but is dispensable at
Genetic diversity in H. pylori
Extensive recombination and a PANMICTICoverall popu-
lation structure result in substantial genetic diversity
among H.pyloristrains176–179.This has been proposed
to allow the bacteria to adapt rapidly to changing con-
ditions in their current host as well as to facilitate the
colonization of new hosts180–182.Although H. pylori
populations in individuals and even families seem to be
clonal,as determined by conventional procedures —
such as RANDOMLY AMPLIFIED POLYMORPHIC DNA (RAPD) PCRand
efficient T-cell proliferation and activation.In normal
T cells,calcineurin dephosphorylates the transcription
factor NFAT (for nuclear factor of activated T cells),
which then translocates into the nucleus and activates
the transcription ofseveral genes that are involved in the
immune response.Among these are the genes encoding
IL-2 and IL-2Rα.In T cells that are infected with VacA+
H. pylori,however,nuclear translocation of NFAT is
blocked, as dephosphorylation is prevented and the
downstream genes are not expressed.
Another possible function ofVacA in subverting the
adaptive immune response is its ability to interfere with
antigen presentation mediated by MHC class II160.After
it inserts into the plasma membrane,VacA is internal-
ized by endocytosis and reaches the late-endosomal
compartment.This compartment is then converted
into large acidic vacuoles by the anion-selective chan-
nel activity of VacA161,162.In antigen-presenting cells,
the processing of proteins into peptide epitopes,
which takes place in the endocytic compartment,is
greatly reduced owing to VacA activity,indicating that
antigen presentation is abrogated in these cells160.
The importance ofVacA in establishing an infection
has been corroborated by experiments in mice,which
have shown that a null mutation of vacAcompromises
the ability of H.pylori to colonize the murine stomach
in the presence of the corresponding parental strain163.
However, the precise effect of VacA on cellular and
epithelial physiology that facilitates H.pyloricolonization
in the murine stomach is unknown.
H. pylori suppresses inflammatory responses
Several lines of evidence indicate that,to allow long-
term colonization,there has been selective pressure on
H.pylorito avoid triggering an intense inflammatory
reaction164. It has been proposed that high levels of
inflammation may lead to loss of gastric glandular
structure and function165and that H.pylori disappears
from stomachs that have developed atrophic gastritis140.
Furthermore,an increased inflammatory reaction —
as seen in IL-10-knockout mice — is associated with
clearance of the bacteria from the stomach within 8
days of the infection166.Similarly,increased inflamma-
tion due to deletion of the gene encoding PHOX
results in a marked reduction in bacterial numbers167.
All of these findings indicate that,at least in animal
models,a strong inflammatory reaction is necessary for
the elimination of H.pylori and seems to be actively
repressed by this bacterium.
But how does H.pylori modulate the host inflam-
matory response? Bacterial lipopolysaccharide (LPS)
is the main mediator of inflammation during infec-
tions with most Gram-negative bacteria because it
activates phagocytic cells,endothelial and epithelial
cells and lymphocytes. H. pylori LPS, however, has
very low biological activity when compared with
Escherichia coli LPS,at least as measured by its ability
to activate macrophages165.In fact,the minimum con-
centration of purified LPS that is required to achieve
similar responses was several thousand times greater
for H.pyloricompared with E.coliLPS165.
Mating without regard to the
genetic constitution of the mate.
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The high degree of diversity that is seen in H.pylori
is probably facilitated by its natural competence for
DNA transformation.In contrast to other bacteria,
natural competence in H.pylori is not mediated by type
IV pili or type IV pilin-like proteins,but by a TFSS that
is encoded by the comB operon193,194. The ComB8,
ComB9 and ComB10 proteins correspond to the
Agrobacterium tumefaciensVirB8,VirB9 and VirB10
proteins and constitute the basic components of a
TFSS193.H.pylori therefore possesses two functionally
independent TFSSs — one for protein secretion and
translocation into the host cell that is encoded by the
Cag-PAI,and one for DNA uptake.In addition,a third
cluster of type IV secretion genes was recently discov-
ered that seems to be present in only a subset of
strains195.The functional significance of this cluster is
unknown at present.
The identification of CRYPTIC PLASMIDSin approximately
halfofall H.pyloristrains196has given rise to speculation
that conjugative transfer of novel sequences carried on
plasmids could be another means of horizontal gene
transfer — and therefore strain diversity — in H.pylori.
In support of this hypothesis, Hofreuter et al. have
recently presented evidence that some of these plasmids
contain hot spots for site-specific recombination,
encode elements from CHROMOSOMAL PLASTICITY ZONESand
might be mobilizable197.This indicates that exchange
of genetic material between these plasmids and the
chromosome can occur,and that these plasmids might
be mobilized and spread rapidly in the population.So,
genetic diversity,which in turn leads to antigenic diver-
sity,might be an important strategy used by H.pylorito
evade immune surveillance.
Repopulation of the stomach by H. pylori
The gastric mucosa is normally well-protected against
bacterial colonization due to the acidic pH of the
lumen,the production of mucus and rapid epithelial-
cell turnover.It is therefore tempting to speculate that
the adoption of a partially intracellular lifestyle by
some members of the bacterial population might
allow H. pylori to achieve long-term persistence.
Although there is little evidence to indicate that the
bacterium is predominantly an intracellular pathogen,
numerous experimental and clinical observations of
biopsy specimens support the notion that a subpopu-
lation of H.pylori is able to invade epithelial cells both
in vitro198,199and in vivo200–202.
Using time-lapse video microscopy and gentamicin-
protection assays in a cell-culture system,Amieva et al.
have provided evidence that H.pylori residing in multi-
vesicularbodies survive in the intracellular milieu for
at least 24 hours,remain motile and retain the ability
to emerge from the cells to repopulate the extracellular
space199.Up to several dozen bacteria can be found in
one vesicle,indicating that intracellular replication does
occur,at least sporadically.These findings indicate that
the intracellular niche can potentially function as a
‘hideout’ and sustain the renewal of the population
underthe unfavourable conditions that are found in the
mammalian stomach.Nevertheless,the vast majority of
DNA sequence analysis179— more sensitive approaches
have indicated that extensive changes can and do occur
in a single host over time183.For example,fortuitously,
additional H.pylori isolates were obtained from one
patient six years after an isolate of the sequenced strain
J99 was first obtained from this individual.These new
clinical isolates were subjected to extensive molecular
comparisons with the original J99 strain.RAPD PCR
and sequencing of several unlinked loci indicated that
these isolates were undoubtedly related to the original
strain;however,microarray analysis showed differences
in genetic content that reflected both acquisitions and
losses of genomic DNA183.Approximately 3% ofJ99 loci
showed variation between isolates obtained from this
individual,compared with 22% of loci when isolates
from different individuals were compared184.Although
most of the open reading frames that were affected
represented ‘hypothetical genes’,one was predicted to
encode a protein belonging to the TraG family,other
members of which have been shown to be involved in
genetic transfer185,186.A putative similar function of
TraG implies that acquisition of this gene might con-
fer an evolutionary advantage by providing another
mechanism through which DNA exchange could occur.
In a recent similar study,three output strains from
an experimental infection of Rhesus macaques were
compared with the strain that was used for inocula-
tion187.All three of the output strains had lost expres-
sion of the babA gene,which encodes an adhesin that
binds to the LEWIS BLOOD GROUP Lebantigen188. Loss of
babA expression occurred by two different mecha-
nisms in different isolates. In some cases, babA was
replaced by the closely related babB gene,leading to
loss of babA and duplication of babB. In others, a
change in the number of CT repeats in the 5’ coding
region of babAresulted in a frameshift and subsequent
loss of Lebadhesion.H.pyloritherefore uses both anti-
genic variation and phase variation to regulate babA
expression (and possibly the expression of other outer-
membrane proteins) in vivo;however,the significance
of this observation in the context of the host immune
response remains to be shown.
Another H.pylori structure that undergoes phase
variation is its LPS,more specifically the Lewis-blood-
group determinant of the LPS O-antigen.The on/off
statuses of at least five glycosyltransferase genesdeter-
mine which LPS phase variant is expressed (reviewed in
REF.189).It was initially assumed that mimicry of the
Lewis blood group antigens of the host by H. pylori
would provide a mechanism of immune evasion and
adaptation to the host,a concept that was supported by
reports describing a link between the Lewis-blood-
group phenotype of isolates with the phenotype of the
individuals they were derived from (reviewed in REF.190).
However, this concept has been challenged by the
isolation of both LeX- and LeY-expressing strains from
the same host and the fact that even high levels of anti-
LPS antibodies do not eradicate the organism. It is
now assumed that the production of Lewis antigens
by H.pylori facilitates the colonization of the host by
mediating adhesion to gastric epithelial cells191,192.
POLYMORPHIC DNA (RAPD) PCR
A molecular technique used for
the classification and
comparison of different isolates
of the same species.This method
uses a randomly chosen
oligonucleotide to prime DNA
synthesis and results in strain-
specific patterns of DNA
LEWIS BLOOD GROUP
Antigens of red blood cells,
saliva and other body fluids that
are specified by the Le gene and
react with the antibodies
designated anti-Leaand anti-Leb.
elements that can encode
Segments of the chromosome
that are characterized by their
different G+C content,which is
a hallmark of horizontally
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M.tuberculosis and S.typhi— where they are effectively
screened from active immune surveillance.The ability
of persistent species such as M.tuberculosis,S.typhi,
Chlamydia spp. and Brucella spp. to modify the
intravacuolar environment is a common feature of
these bacteria that could clearly favour persistence and
evasion of immune responses through reduced surface-
antigen presentation or the control of apoptotic path-
ways204–208.Some persistent pathogens might seek an
intracellular location at specific times during infection.
Indeed, the ability of the mucosal-surface colonizer
H.pylorito cycle between extracellular and intracellular
locations highlights a strategy that may be crucial for
some persistent pathogens.Localized subversion ofthe
immune response is also an important feature ofpersis-
tent bacteria — for example, by interference with
cytokine signalling,as described for M.tuberculosis,or
with innate immune signalling,as described for H.pylori.
Finally,the pathological damage that results from
continued macrophage activation will at some stage
outweigh the immediate risk that is posed by the
residual bacteria, and the immune response might
turn itself off, allowing bacterial persistence.
Regulatory T cells co-expressing CD4 and CD25
markers have been shown to exert this type of control
during Leishmaniamajorinfection209.Although there
are diverse phenotypes of regulatory T cells,function-
ally they share the ability to downregulate immune
responses.One way that this is achieved is by the secre-
tion of cytokines such as IL-10 and TGF-β, which
inhibit both TH1 and TH2 responses in vivoand have a
role in controlling T-cell responses that are directed
against self-antigens210,211.Belkaid et al. have shown
that during persistent infection by L.majorin the skin,
CD4+CD25+T cells accumulate in the dermis,where
they suppress the ability of CD4+CD25– effector T cells
to eliminate the parasite from this site through both
IL-10-dependent and IL-10-independent mecha-
nisms209.Furthermore,the sterilizing immunity that is
achieved in mice with impaired IL-10 activity is fol-
lowed by loss of immunity to reinfection.Therefore,it
is possible that the equilibrium that is established
between effector and regulatory T cells in sites of
chronic infection might reflect both pathogen and
host survival strategies (FIG. 4). Whether regulatory
T cells have a role in the mechanisms that are used in
persistent Helicobacter,Mycobacteria and Salmonella
infections is unknown.However,a high proportion of
CD4+T cells that are able to release IL-10 can be
found in chronic mycobacterial infections212, and
S.typhimuriuminduces macrophage and splenic IL-10
expression213,possibly indicating the presence of reg-
ulatory T cells.It was recently shown that regulatory
T cells reduce H.pylori-induced gastritis in mice,while
allowing the bacterium to colonize the mucosa at higher
densities214,and that H.pylori-specific CD4+CD25+reg-
ulatory T cells suppress memory T-cell responses to
H. pylori in infected humans215. In line with this
observation,H. pylori infection of IL-10-knockout
mice resulted in more severe gastritis and bacterial
clearance after 8 days166.
H. pylori in the stomach are extracellular, highly
motile organisms that reside in the mucus overlying
the gastric mucosa.How the organism survives here
in the presence of high levels of antibody is one of the
most poorly understood issues in H.pylori pathogen-
esis.It is equally remarkable that individuals who are
cured ofinfection by antibiotic therapy after decades of
colonization are susceptible to re-infection,although
at a slightly lower rate203.
Many hypotheses can be proposed for the survival of a
population of microorganisms in the presence of
immune responses.The organisms could ‘hide’inside
macrophages within granulomas — as is the case for
Regulatory T cells
Innate and adaptive
Figure 4 | Balancing protective immunity and
immunopathology during persistent bacterial infections.
The balance between the immune response and infection during
persistent infections is important for both the host and the
pathogen, with the host switching off the immune response
when it becomes more harmful than the presence of the
pathogen. This is depicted by the horizontal axis between
protection and pathology on either side of the balance. The
illustration shows how complex interactions between the
pathogen and host lymphocytes and antigen-presenting cells
may drive immune responses, with regulatory T cells having an
important role in the balance between protection and
immunopathology. Host factors involved in the immune
response — such as interferon-γ(IFN-γ), tumour-necrosis factor-
α(TNF-α), nitric oxide (NO) and the interleukins IL-12 and IL-23
— contribute to both immunopathology and suppression of
bacterial persistence. Bacterial persistence is mediated by
specific bacterial persistence factors and potentially
dysregulated immune responses that could facilitate persistence
by contributing to chronic inflammation and low-level tissue
injury that facilitates bacterial survival. We postulate that bacterial
factors that are important for persistence might have an as-yet-
undetermined role in directing this balance by influencing
regulatory T cells. TGF-β, transforming growth factor-β.
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R E V I E W S
bacterial mutants and host expression profiling — as well
as laser microdissection,will allow further investigation
ofthe fundamental genetics ofbacterial persistence and
host immune responses.These findings will hopefully
lead to improvements in therapeutic approaches and,
perhaps,the elimination ofthese unwanted companions.
Although we describe some possible mechanisms of
pathogen persistence in this review,we actually know very
little about how these microorganisms survive for long
periods of time in the host in the presence of immuno-
surveillance.Future applications of genome-based tech-
niques — including array-based analysis of libraries of
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We are grateful to H. Rachman, S. H. E. Kaufmann, C. Cosma and
L. Ramakrishnan for sharing unpublished data. We thank
S. H. E. Kaufmann, C. Cosma, L. Ramakrishnan, E. Joyce,
I. Brodsky, S. Merrell, R. Haas and M. Amieva for critical reading
of the manuscript. Research by the authors is supported by
the National Institutes of Health, the Ellison Medical Foundation,
the Digestive Disease Center (to S.F.) and Deutsche
Forschungsgemeinschaft (to A.M.).
Competing interests statement
The authors declare no competing financial interests.
The following terms in this article are linked online to:
Helicobacter pylori | IFN-γ | IL-2 | IL-2R | IL-10 | IL-12 | IL-23 |
LRG-47 | Mycobacterium marinum | Mycobacterium tuberculosis |
NFAT | NOS2 | Nramp1 | S. typhi | S. typhimurium | TGF-β | TLR2 |
TLR4 | TNF-α
Infectious Disease Information:
HIV | salmonellosis | tuberculosis | typhoid fever
babA | babB | FlaA | FlaB | ICL | mig-14 | rocF | TraG | VacA | virK
Access to this interactive links box is free online.