Since the discovery of the bacterial aetiology of many illnesses by Robert Koch, Louis Pasteur and others, there has been much interest in understanding the pathogen-esis of infectious diseases. A major step towards this goal came from the discovery that some bacteria release tox-ins (for example, diphtheria toxin), which, when puri-fied, can reproduce the signs of disease that are caused by the respective pathogen (for example, Corynebacterium diphteriae). Studies on bacterial toxins that are responsi-ble for the degree of pathogenicity (virulence) of a micro-organism gave rise to the concept of virulence factors. Biochemical characterization of the activities of purified toxins ushered in rapid advances in our understanding of the pathogenesis of diseases caused by toxin-produc-ing (toxigenic) bacteria. The knowledge gained from these studies provided a coherent picture of the disease process that seamlessly connected research into bacte-rial pathogenesis with research into clinical infectious disease. The identification of bacterial virulence factors, such as toxins, that were directly responsible for most disease symptoms, laid the foundation for the develop-ment of novel strategies for treatment of infection with anti-toxins (neutralizing antibodies) and prevention of disease by vaccination with toxoids (heat-inactivated or chemically inactivated toxins). Research into bacterial toxins made microbiology relevant to infectious-disease physicians. The success of vaccines against diphtheria, whooping cough and tetanus is testament to the power of deriving new intervention strategies from a thorough understanding of the main bacterial factors responsible for disease.

However, the pathogenesis of infections caused by non-toxigenic bacteria proved more complicated and could not be reduced to a single factor, such as a toxin. The biology of non-toxigenic bacterial disease is complex, and involves various bacterial and host factors. Owing to the multifactorial nature of these malaises, studies on a single virulence factor frequently do little to explain how a path-ogen causes a particular symptom in a patient. As a result, findings from molecular bacterial pathogenesis research are not always of obvious relevance to the infectious-disease clinician and the fields have diverged since the heyday of toxin research. In this Review, we aim to reverse this trend and apply recent advances in our understanding of the molecular basis of host–pathogen interactions to explain the clinical presentation and pathology of a dis-ease — enteric fever — to provide an overview that bridges molecular bacterial pathogenesis and clinical infectious-disease research. We discuss the pathogenesis of the three major clinical syndromes associated with bacterial enteric infections — secretory diarrhoea, inflammatory diarrhoea and enteric fever — by focusing on pathogen biology and the clinical presentation of disease.

Major syndromes of enteric infectionHost responses are often typical for groups of pathogens, rather than being specific to individual pathogens, which suggests that some pathogens share important basic characteristics. This principle is illustrated by enteric infections, which, despite many possible causes, typically manifest as one of only three clinical syndromes1 (FIG. 1). The first two syndromes are characterized by diarrhoea or

*Department of Medical Microbiology and Immunology, School of Medicine, ‡Department of Food Science and Technology, College of Agricultural & Environmental Sciences, University of California at Davis, One Shields Avenue, Davis, 95616 California, USA. Correspondence to A.J.B. e‑mail: ajbaumler@ucdavis.edudoi:10.1038/nrmicro2012Published online 28 October 2008

From bench to bedside: stealth of enteroinvasive pathogensRenée M. Tsolis*, Glenn M. Young‡, Jay V. Solnick* and Andreas J. Bäumler*

Abstract | Bacterial enteric infections are often associated with diarrhoea or vomiting, which are clinical presentations commonly referred to as gastroenteritis. However, some enteric pathogens, including typhoidal Salmonella serotypes, Brucella species and enteropathogenic Yersinia species are associated with a clinical syndrome that is characterized by abdominal pain and/or fever and is distinct from acute gastroenteritis. Recent insights into molecular mechanisms of the host–pathogen interaction show that these enteric pathogens share important characteristics that explain why the initial host responses associated with these agents more closely resemble host responses to viral or parasitic infections. Host responses contribute to the clinical presentation of disease and improved understanding of these responses in the laboratory is beginning to bridge the gap between bench and bedside.

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No fever orlow grade fever

No white blood cellsin stool sample

Fever

White blood cellsin stool sample(neutrophils)

Fever

White blood cellsin stool sample(mononuclear cells)

Secretorydiarrhoea

Inflammatorydiarrhoea

Entericfever

Gastroenteritis

Febrile patient

Ice pack

• Typhoidal Salmonellaserotypes

• EnteropathogenicYersinia spp.

• Brucella spp.

• Campylobacter jejuni• Shigella spp.• Non-typhoidal Salmonella

serotypes• EIEC

• Vibrio cholerae• ETEC• EAggEC• EPEC• EHEC

Lamina propria Connective tissue that underlies the epithelium of mucosal surfaces.

Pattern-recognition receptor A receptor of the innate immune-surveillance system that recognizes and responds to conserved microorganism-associated molecular patterns.

Reactive microscopic pattern Microscopic pathological changes in tissue.

Exudative inflammation Reactive microscopic pattern characterized by acutely increased vascular permeability, neutrophil recruitment and the formation of tissue exudates above surfaces or within spaces.

vomiting, which are clinical presentations that are com-monly referred to as gastroenteritis. However, on closer examination it is clear that patients with gastroenteritis can be divided into two groups based on findings from a physical examination and examination of a stool sam-ple. On the one hand, the absence of faecal leukocytes, in combination with an absence of fever or a low-grade fever, is suggestive of secretory diarrhoea. On the other hand, fever and the presence of numerous leukocytes, predominantly neutrophils, in stool samples is suggestive of inflammatory diarrhoea2. Both clinical syndromes are associated with a distinct set of Gram-negative bacterial pathogens, which are listed in FIG. 1.

distinctions between secretory diarrhoea and inflam-matory diarrhoea are, at least in part, explained by the fact that the pathogens which cause each syndrome share basic characteristics that account for some of the dif-ferences in the host response. Bacterial pathogens that cause secretory diarrhoea are found in the intestinal lumen and rarely invade the intestinal mucosa. Because bacteria in the intestinal lumen do not trigger innate immune responses, intestinal inflammation is minimal and faecal leukocytes are absent from stool samples (FIG. 1). Bacteria that are associated with secretory diar-rhoea encode virulence factors that mediate intestinal colonization3–5 and bacterial toxins stimulate chloride secretion in enterocytes of the intestinal crypts, result-ing in fluid loss6. The classic example of such a toxigenic bacterium is Vibrio cholerae, a luminal pathogen that produces cholera toxin (BOX 1).

In marked contrast to pathogens that cause secretory diarrhoea, agents associated with inflammatory diar-rhoea are highly invasive7. In non-typhoidal Salmonella serotypes, Shigella species or enteroinvasive Escherichia coli, the type III secretion systems that are required for invasion of epithelial cells are major virulence fac-tors8,9. Translocation of bacteria from the intestinal lumen into the lamina propria is detected by the innate immune-surveillance system through pattern-recognition receptors, including the membrane-localized Toll-like receptors (TLRs)10 and the cytosolic nucleotide-binding and oligomerization domain-like receptors11. These pattern-recognition receptors function as ‘bar-code readers’ that enable the host to distinguish bacteria from viral and parasitic agents by recognizing characteristic combinations of conserved microorganism-associated molecular patterns (mAmPs)12. For example, TLR3, TLR7 and TLR8 recognize nucleic acids and specialize in the recognition of viral agents, whereas TLR1, TLR2, TLR4, TLR5 and TLR6 recognize products that are unique to bacteria10.

The ability to distinguish bacteria from other micro-organisms (such as viruses of parasites) enables the host to mount appropriate responses. The reactive microscopic pattern that is typically associated with bacterial infection is exudative inflammation, which is characterized by acutely increased vascular permeability and cellular infiltrates that are dominated by neutrophils, the pathological hallmark of inflammatory diarrhoea13–15. These changes result in the formation of tissue exudates that contain

Figure 1 | major syndromes of enteric infection. Host responses that distinguish the three syndromes exhibit features that are characteristic for different groups of pathogens, which are shown on the right. EAggEC, enteroaggregative Escherichia coli; EHEC, enterohaemorrhagic E. coli; EIEC, enteroinvasive E. coli; ETEC, enterotoxigenic E. coli; EPEC, enteropathogenic E. coli.

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Pyogenic bacteria Pus-forming bacteria that are associated with exudative inflammation and neutrophil recruitment.

Pyrogenic cytokine A fever-inducing cytokine, including interleukin-1 (IL-1), IL-6, tumour necrosis factor-α and interferon.

Interstitial inflammation Reactive microscopic pattern characterized by inflammatory infiltrates that are dominated by macrophages, dendritic cells and/or lymphocytes.

Atypical bacteria Bacteria that differ from pyogenic bacteria, in that they do not elicit neutrophil recruitment during infection.

Neutrophilia Increased counts of neutrophils in the blood.

bacteria, fibrinogen, necrotic cells and neutrophils (pus), leading to fluid accumulation (BOX 1) and the presence of neutrophils in stool samples2,16,17 (FIG. 1). Because of these properties, pathologists commonly refer to bacteria that are associated with exudative inflammation as pus-form-ing or pyogenic bacteria. The recruitment of neutrophils constitutes an appropriate response to bacterial invasion, because these professional phagocytes are equipped with a formidable arsenal of antibacterial host defences. In patients who are immunocompetent, neutrophil recruit-ment during inflammatory diarrhoea helps to contain bacterial dissemination, resulting in infections that remain localized to the intestinal mucosa and mesenteric lymph nodes. Fever is a nonspecific host response that is associated with inflammatory diarrhoea, which is mediated by pyrogenic cytokines such as interleukin-1β (IL-1β), IL-6, tumour necrosis factor-α (TnF-α) and interferon (IFn). These pyrogenic cytokines enter the circulation and reach the brain, where they increase the temperature set point, resulting in fever18.

Finally, enteric infection can manifest in a third clinical syndrome that is characterized by abdominal pain and fever and is distinct from acute gastro enteritis (FIG. 1). Gastrointestinal manifestations may include ulceration of the Peyer’s patches and lower-right quadrant pain that mimics appendicitis19–21. Because the entry point of the pathogen is usually the gastro-intestinal tract, the term enteric fever is used to describe this clinical syndrome1. Although enteric fever is sometimes used synonymously with typhoid fever, the term enteric fever will be used throughout this Review in a broader sense to describe the clinical syndrome caused by Salmonella enterica subsp. enterica serovar Typhi (S. Typhi), Brucella species and enteropathogenic Yersinia species. In common with pathogens that cause inflammatory diarrhoea, the Gram-negative organisms that are associated with the enteric fever syndrome are invasive, and their presence in tissue is detected by the innate immune-surveillance system, resulting in the production of pyrogenic cytokines that give rise to fever. However, exudative inflammation, which characterizes inflammatory diarrhoea, is not a feature of the intestinal pathology during enteric fever. Instead, enteric fever is

associated with interstitial (within spaces) inflamma-tion in the intestinal mucosa, which is characterized by mononuclear infiltrates2,22–25.

Interestingly, interstitial inflammation is typically asso-ciated with acute viral infections. These observations suggest that enteric fever is caused by atypical bacteria, which differ in some of their basic characteristics from the pyogenic bacteria that are associated with the exu-dative inflammation observed during inflammatory diarrhoea. Perhaps more importantly, the association of interstitial inflammation with both viruses and atypical bacteria that cause enteric fever suggests that the innate immune-surveillance system cannot efficiently distin-guish between these two groups of pathogens, despite the fact that these organisms share few similarities.

Extraintestinal manifestationsBrucella spp. and S. Typhi cause disseminated infec-tions in immunocompetent patients that involve multiple organ systems, such as the liver, spleen and bone marrow. The clinical presentation of these sys-temic infections reinforces the view that pathogens that cause enteric fevers do not elicit the typical antibacte-rial responses that are associated with infections caused by pyogenic bacteria. In this Review, we contrast the clinical presentations of typhoid fever and undulant fever with systemic infections caused by pyogenic bacteria. non-typhoidal Salmonella serotypes are well suited for such a comparison, because they are closely related genetically to S. Typhi, but are associated with inflammatory diarrhoea (FIG. 1). non-typhoidal Salmonella serotypes cause localized gastroenteritis in immunocompetent individuals, whereas patients who are immunocompromised develop a fulminant bacteraemia, the clinical presentation of which can be contrasted with typhoid fever (FIG. 2).

The presence of microorganisms in the blood usually stimulates pattern-recognition receptors that are located on the surface of, or within, macrophages and mono-cytes, which leads to the release of pyrogenic cytokines. In addition to fever induction, pyrogenic cytokines mediate a number of physiological changes that are collectively referred to as the acute phase response26. Pyogenic bacteria generally trigger a more robust induc-tion of the acute phase response than atypical bacteria or viruses, unless the viral infection is accompanied by tissue injury, which by itself is a potent inducer of this response. The presence of pyogenic bacteria in the blood is typically associated with an elevated white blood cell count (leukocytosis) that results from an IL-1β-mediated release of neutrophils from the bone-marrow stores (neutrophilia)27,28. neutrophilia is a typical antibacte-rial response that accompanies bacteraemia caused by non-typhoidal Salmonella serotypes28 (FIG. 2), but is not typically observed during infection with viruses. Interestingly, patients with enteric fever generally do not exhibit neutrophilia19,29,30, and therefore their clinical presentation most closely resembles that of a viral infection.

The robust induction of acute phase responses by pyogenic bacteria, such as non-typhoidal Salmonella

Box 1 | Fluid loss during secretory and inflammatory diarrhoea

Studies on cholera toxin that induced chloride secretion serve as a paradigm for the mechanism of fluid loss during secretory diarrhoea. Cholera toxin consists of an A subunit with enzymatic activity and five B

subunits that mediate binding to the

ganglioside GM1 receptor on the surface of erythrocytes6,95–98. After internalization of

the toxin, the A subunit is translocated into the host cell cytosol where it ADP-ribosylates and thereby activates a small GTP-binding protein that regulates host cell adenylate cyclase activity99–102. Increased cyclic AMP levels in turn result in the opening of a chloride channel in the apical membrane of enterocytes, resulting in the efflux of chloride ions into the intestinal lumen102,103. This mechanism leads to the loss of electrolytes and water, which is characteristic of secretory diarrhoea.

Inflammatory diarrhoea is characterized by exudative inflammation, accompanied by increased vascular permeability and movement of fluid from the blood to the intestinal tissue. This fluid eventually accumulates in the intestinal lumen because exudative inflammation leads to necrosis of the upper mucosa, thereby impairing the epithelial permeability barrier104. In contrast to secretory diarrhoea, inflammatory diarrhoea results in loss of serum protein in addition to loss of electrolytes and water105.

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S. Typhi

Exudativeinflammation

Interstitialinflammation

Smallintestine

Immunocompetent patients

Immunocompromised patients

Bacteraemia

Detection of LPS bymonocytes expressing TLR4

TLR4Weak stimulation of acute phase responses

Noneutrophilia

IFN-γ IL-1βTNF-α

Tissuefactor Neutrophilia

Disseminatedintravascularcoagulopathy

Septic shock

Nosepticshock

Bloodmonocyte

High serum levels of

S. Typhimurium

Bacteraemia

iNOS

Hypotension

Intravascular coagulopathy Formation of blood clots in blood vessels owing to activation of the clotting cascade.

serotypes, results from their initial interactions with tissue macrophages or blood monocytes through bacteria-specific TLRs. For example, lipopolysaccha-ride (LPS), a conserved mAmP that is present in the outer membrane of pyogenic Gram-negative bacteria, is a potent agonist of the TLR4–md-2–cd14 receptor complex12. Although signals transmitted by TLRs that recognize bacterial products generally allow the host

to better combat the intruder, the presence of pyogenic bacteria in the blood can result in excessive induction of the acute phase response, giving rise to a systemic inflammatory-response syndrome that, in severe cases, can result in septic shock28. Purified LPS is a potent inducer of endotoxic shock, a clinical syndrome that is characterized by a decrease in blood pressure (hypoten-sion) and activation of the coagulation cascade, leading to fibrin deposition in small blood vessels (microvas-cular thrombosis)31 (FIG. 2). The toxic effects of LPS are primarily due to TLR4-dependent stimulation of TnF-α production in monocytes and macrophages32. A fatal outcome of bacteraemia with non-typhoidal Salmonella serotypes is associated with patients who develop signs of septic shock33, and in a mouse model34 LPS contributes to mortality in part because it elicits the rapid TLR4-dependent production of TnF-α35,36. TnF-α, in combination with IL-1β and IFn, induces expression of the inducible nitric oxide synthase37, thereby increasing production of nitric oxide, a potent vasodilator that contributes to hypotension38,39. The coagulation system can become activated during bacteraemia owing to a TnF-α-mediated increase in tissue-factor expression on monocytes40, which results in the cleavage of serum fibrinogen to fibrin41. during sepsis, deposition of fibrin in the microvasculature results in disseminated intravascular coagulopathy, which can lead to organ failure31.

unlike the robust induction of acute phase responses observed with non-typhoidal Salmonella serotypes, S. Typhi only induces this response weakly. For exam-ple, coagulation abnormalities do not become clinically apparent in patients with typhoid fever42. Serum levels of pyrogenic cytokines, such as TnF-α and IL-1β, are elevated in patients with typhoid fever compared with healthy individuals but are low compared with cytokine levels measured in the serum of patients with Gram-negative septic shock7 (FIG. 2). Similarly, septic shock is not associated with brucellosis. Because the initial clinical presentation of enteric fever lacks the telltale signs of a bacterial infection, it is not surprising that the clinical presentations of certain viral (for example, dengue virus) or parasitic (for example, Plasmodium spp.) infections are often difficult to distinguish from that of enteric fever43,44.

These different host responses raise several impor-tant questions that are relevant for understanding the pathogenesis of enteric fever. First, why do causative agents of enteric fever elicit a nonspecific response in humans that resembles certain viral or parasitic infec-tions rather than a typical antibacterial response that characterizes diseases caused by pyogenic organisms, such as non-typhoidal Salmonella serotypes? Second, are the similarities in host responses to atypical bac-teria that cause enteric fever due to the use of similar virulence strategies by these pathogens? Third, can the atypical bacteria that are associated with enteric fever alter their ‘bar code’ to conceal their identity from the host, and if so, what are the molecular mechanisms and potential benefits? next, we discuss recent work that has begun to shed light on these questions.

Figure 2 | comparison of host responses elicited in patients with bacteraemia. Salmonella enterica subsp. enterica serovar Typhi (S. Typhi) infection is associated with interstitial inflammation in the intestine, and progresses to bacteraemic disease in immunocompetent patients, which does not typically manifest with neutrophilia or septic shock. Salmonella enterica subsp. enterica serovar Typhimurium (S. Typhimurium) infection results in exudative inflammatory changes in the intestine, and in immunocompromised patients leads to bacteraemia. In contrast to S. Typhi infection, bacteraemia with S. Typhimurium triggers a strong induction of acute phase responses, which may result in septic shock. IFN-γ, interferon-γ; IL-1β, interleukin-1β; iNOS, inducible nitric oxide synthase; LPS, lipopolysaccharide; TLR4, Toll-like receptor 4; TNF-α, tumour necrosis factor-α.

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Stealth tactics of enteric pathogensTyphoid fever: a pathogenicity island conceals identity. The host response elicited by typhoidal Salmonella serotypes, such as S. Typhi, differs markedly from that observed with non-typhoidal Salmonella serotypes, such as Salmonella enterica subsp. enterica serovar Typhimurium (S. Typhimurium) or Salmonella enterica subsp. enterica serovar enteritidis (S. enteritidis). non-typhoidal Salmonella serotypes are pyogenic and elicit a typical antibacterial host response that is characterized by exudative intestinal inflammation, neutrophilia and, during bacteraemia, septic shock (FIG. 2). By contrast, interstitial intestinal inflammation, the absence of neu-trophilia and the absence of septic shock during typhoid fever suggest that S. Typhi is an atypical bacterium that uses virulence mechanisms which prevent the generation of a classic antibacterial response.

Interestingly, an analysis of the factors that could trig-ger exudative inflammation and acute phase responses reveals no differences between non-typhoidal and typhoi-dal Salmonella serotypes. experimental evidence suggests that stimulation of TLR4 by LPS has an important role in the development of septic shock during S. Typhimurium infection34–36,45. However, LPS is also present in S. Typhi, and purified S. Typhi LPS elicits cytokine secretion in human monocytes at equal levels to those elicited by LPS purified from non-typhoidal Salmonella serotypes46. S. Typhi and non-typhoidal Salmonella serotypes express flagellin, a second mAmP that is characteristic of Gram-negative bacteria. Bacterial flagellin is recognized on the cell surface by TLR5 (ReF. 47) in a process that has been implicated in neutrophil influx in the intestinal mucosa during S. Typhimurium infection48,49. However, flagel-lins purified from S. Typhi or S. Typhimurium do not differ in their capacity to elicit cytokine production in human monocytes or intestinal epithelial cells50,51. Thus, differences in the host response to infection with S. Typhi or non-typhoidal Salmonella serotypes cannot be explained by different capacities of their LPS or flag-ellin molecules to serve as agonists for TLR4 or TLR5, respectively. As a result, the mechanisms that would explain why S. Typhi does not elicit host responses that are typical of pyogenic bacteria have long remained an enigma.

Recent analysis of a 134 kb dnA region, named Salmonella pathogenicity island 7 (SPI7)52, has helped illuminate the features that make S. Typhi an atypical bacterium7. SPI7 is present in S. Typhi, but is absent from the genomes of Salmonella serotypes that cause inflammatory diarrhoea7. Loss of SPI7 in S. Typhi is associated with increased TnF-α production in human macrophage-like cells53 and increased IL-8 secretion during infection of human colonic epithelial cells54. The SPI7 genes that are responsible for this trait are clustered in a 14 kb region called the viaB locus55 (FIG. 3). virulence factors encoded within the viaB locus enable S. Typhi to prevent recognition by TLR4 and TLR5 (ReF. 55), albeit by different mechanisms.

evasion of TLR4 recognition by S. Typhi involves the vi-capsular antigen, a linear polymer of α-1,4(2-deoxy)-2-N-acetylgalacturonic acid variably O-acetylated

at the c3 position56. The S. Typhi viaB locus contains genes that are involved in the regulation (tviA), biosyn-thesis (tviB–E) and export (vexA–E) of the vi-capsular antigen57 (FIG. 3). expression of the vi-capsular antigen markedly reduces the TLR4-dependent production of TnF-α both in vitro (in bone-marrow-derived macro-phages) and in vivo (in a mouse sepsis model)36. The mechanism by which the vi-capsular antigen interferes with TLR4 recognition is currently unclear, but it might involve masking or preventing physical access to surface structures7.

The viaB-mediated attenuation of TLR5 signalling entails a different mechanism. TviA, a regulatory pro-tein encoded by the viaB locus, represses transcription of flhC and flhD, which encode the master regulator of flagella expression. This results in reduced transcription of the flagellin gene fliC and reduced secretion of the encoded TLR5 agonist flagellin (Flic)50 (FIG. 3). Although evasion of innate immune surveillance by TLR4 and TLR5 is mediated through different mechanisms, the TviA regulatory protein is essential for both processes, because it negatively regulates flagellin production50 and positively regulates production of the vi-capsular antigen57. Thus, TviA could be viewed as a regulatory switch that enables S. Typhi to change its appearance from that of a pyogenic bacterium to that of an atypical bacterium.

As a result of TviA-mediated changes in bacterial gene expression, S. Typhi can conceal two important molecular signatures, LPS and flagellin, that identify it as a Gram-negative bacterium36,50. Without detect-ing these molecular signatures through TLR4 and TLR5, the ability of the host to identify the intruder as a Gram-negative bacterium is impaired. By display-ing an incorrect bar code, S. Typhi therefore prevents the generation of a typical antibacterial host response, which results in suppression of neutrophil recruitment in the intestinal mucosa58 and weak induction of acute phase responses during bacteraemia36. In conclusion, the S. Typhi genome carries the pathogenicity island SPI7, which confers properties that help explain why S. Typhi is an atypical bacterium.

Undulant fever: the stealthy design of Brucella species. unlike mice infected with pyogenic bacteria, such as S. Typhimurium, Brucella abortus-infected animals do not show disseminated intravascular coagulopathy, do not augment levels of fibrin breakdown products, do not exhibit neutrophilia and only induce marginally increased levels of TnF-α, IL-1β and IL-6 (ReF. 59). Because S. Typhimurium triggers these host responses through TLR stimulation34,36, the fact that they are not induced during B. abortus infection suggests that this pathogen can evade detection by TLRs59. However, the strategy used by Brucella species to evade TLR recogni-tion is fundamentally different to that used by S. Typhi. Brucella species do not conceal the presence of mAmPs by repressing flagellin expression or masking LPS with a capsule. Instead, Brucella spp. alter the chemical com-position of these surface structures such that they are no longer recognized by TLR4 and TLR5.

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VexD VexB

ABCtransporter

vexEvexDvexCvexBvexAtviEtviDtviCtviBtviA

+

flhD flhC

–

Vi biosynthesis Vi export

LPS

Vi antigen

UDP-GlcNAc

UDP-GlcUANAc

UDP-GalUANAc

viaB locus of S.Typhi

Outermembrane

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Evasion of TLR5

[α-1,4 GalUANAc]n

Cytoplasm

TviD TviE

VexAVexE

VexC

TviA

TviB

TviC

Figure 3 | Function of the viaB locus in Vi-capsule biosynthesis and evasion of detection by Tlr4 and Tlr5. Salmonella enterica subsp. enterica serovar Typhi TviA activates gene expression in the viaB locus and represses flhD and flhC, which encode the master regulator that controls the expression of flagellin, a Toll-like receptor (TLR) 5 agonist50,57. In the cytoplasm, the enzymes encoded by tviB, tviC, tviD and tviE catalyse the conversion of uridine diphosphate (UDP)-α-d-N-acetylglucosamine (UDP-GlcNAc) into a polymer that is composed of (1,4)-α-2-acetamido-2-deoxy-α-d-glucuronic acid ([α-1,4 GalUANAc]

n), which is variably O-acetylated at the C3 position56,57,106.

Transport of the polymer across the cytoplasmic membrane, the periplasmic space and the outer membrane is mediated by VexB, VexC, VexD and VexA57. The VexE protein has homology to acyl transferases and is required for attaching the Vi antigen to the cell surface57. The presence of the Vi antigen on the cell surface prevents recognition of lipopolysaccharide (LPS) by TLR4 (ReF. 36).

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OOO

NH

OH

OO

OHO

ONH

OO

O

O

P

PO

OH

–O

O–O

OH

OHOH

OHOH

OOHN

NH

OH

OO

OHO

ONH

OO

O

O

P

PO

OH

–O

O–O

OH

OHOH

OO

OO

O

OOO

NH

OH

OO

OHO

ONH

OO

O

O

P

PO

OH

–O

O–O

OH

OHOH

OO

OO

Y. enterocoliticatetra-acylated lipid A (37°C)

S. enterica and Y. enterocoliticahexa-acylated lipid A (26°C)

B. abortushexa-acylated lipid A

Brucella spp. modify the lipid A moiety of their LPS by incorporating a longer fatty acid residue (c28) com-pared with enterobacterial LPS (c12–c16), and this modification diminishes its endotoxic properties by reducing TLR4 agonist activity60 (FIG. 4). Brucella flagel-lin lacks a domain that has been shown to be essential for flagellin recognition by TLR5 (ReF. 61). As a result, Brucella flagellin is likely to be a poor inducer of TLR5-mediated inflammatory responses. elevated levels of pyrogenic cytokines, such as IFn-γ, in sera of mice62,63 are largely due to the action of a type Iv secretion sys-tem (T4SS)64. A B. abortus mutant that lacks a functional T4SS does not elicit cytokine expression in vivo, even if present in the same numbers as wild-type bacteria (early during infection), which suggests that the host can detect neither LPS nor flagellin.

The approach by which Brucella species evade TLR4 and TLR5 signalling is probably more complicated than that taken by S. Typhi, as the wide phylogenetic conservation of mAmPs is thought to be the result of an essential function of each corresponding molecule in bacterial physiology. For example, an msbB muta-tion in S. Typhimurium abrogates TLR4 signalling, but also renders the bacterium avirulent34. Therefore, multiple genetic changes are probably required to convert S. Typhimurium LPS to a fully functional, but non-signalling, molecule. Similarly, single amino-acid substitutions in the TLR5 recognition domains of S. Typhimurium flagellin also result in loss of motility, and introduction of additional compensatory mutations are required to generate flagellin derivatives that confer motility but are no longer recognized by TLR5 (ReF. 61). Thus, although evasion of TLR4 and TLR5 signalling in S. Typhi was accomplished by a single horizontal transfer event that involved the viaB locus, in Brucella species

this trait is most likely the result of a longer evolutionary adaptation that involved the incorporation of multiple genetic changes.

The ability of Brucella spp. to evade detection by TLR4 and TLR5 at the onset of infection might be essential for these pathogens to establish the persistent infection of the reticuloendothelial system that is characteristic of brucellosis. Although S. Typhi and Brucella species evade innate immune recognition by different mecha-nisms, the outcomes are similar, providing an attractive explanation for the similar clinical presentations of both typhoid fever and brucellosis.

Yersiniosis: stealth mode induced by body temperature. unlike S. Typhi and Brucella species, which are faculta-tive intracellular pathogens, enteropathogenic Yersinia species are extracellular pathogens that avoid uptake by phagocytes during growth in tissue65. Given these funda-mental differences in their pathogenic lifestyle, it seems far-fetched to assume that these organisms would share virulence strategies. However, closer examination reveals parallels in the way each pathogen evades detection by a subset of pattern-recognition receptors, which usually drive the generation of host responses that are elicited by pyogenic bacteria.

Temperature is an important signal for the con-trol of virulence gene expression in Yersinia species66. When grown at 21°c, Yersinia pseudotuberculosis and Yersinia enterocolitica express flagellin and are motile. However, growth at temperatures encountered in the mammalian host results in a major phenotypic change that is associated with loss of motility67. The biosynthe-sis of flagella is under hierarchical control of the master flagella regulator encoded by the class I genes flhD and flhC68. Flhd and Flhc control the expression of class II

Figure 4 | lipid A structures of Salmonella serovars, Brucella species and Yersinia species. Hexa-acylated lipid A produced by subspecies of Salmonella enterica and by Yersinia enterocolitica grown at 26°C is a strong Toll-like receptor 4 (TLR4) agonist60. By contrast, tetra-acylated lipid A produced by Y. enterocolitica during growth at 37°C exhibits only weak TLR4 agonist activity75,76. Incorporation of a long-chain fatty acid (C28) into the hexa-acylated lipid A of Brucella abortus might represent an alternate strategy for reducing the TLR4 agonist activity of this microorganism-associated molecular pattern60,107.

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genes, including fliA, the gene that encodes a sigma fac-tor required for expression of class III genes, including those that encode flagellins69. Temperature regulation is mediated by arresting fliA transcription during growth at 37°c70,71, which explains the lack of flagellin gene expres-sion72. It has been speculated that temperature regula-tion of flagellin secretion could enable enteropathogenic Yersinia species to avoid recognition by TLR5 when they enter their mammalian hosts73. This strategy would be similar to that used by S. Typhi, a pathogen that evades recognition by TLR5 through TviA-mediated repression of flagellin secretion50. However, although S. Typhi con-trols flagellin expression by TviA-mediated repression of flhD and flhC 50 (FIG. 3), temperature regulation of flagel-lin production in Yersinia species seems to be mediated at the level of fliA71.

After growth at 21°c, Y. pseudotuberculosis and Y. enterocolitica predominantly produce LPS with hexa-acylated lipid A, which stimulates human monocytes to secrete TnF-α74. By contrast, LPS synthesized at 37°c contains predominantly tetra-acylated lipid A75,76 (FIG. 4). unlike hexa-acylated lipid A synthesized at 21°c, the tetra-acylated lipid A synthesized at 37°c does not stimulate human monocytes to secrete TnF-α74. modifications introduced into the lipid A moiety dur-ing growth of Yersinia species at 37°c prevent TnF-α production more effectively in human macrophages than in murine macrophages77. This incomplete suppres-sion of TLR4 recognition in the mouse might explain why neutrophils are present in intestinal infiltrates elicited by Y. enterocolitica in this animal species78,79. However, intestinal infiltrates in humans are com-monly dominated by mononuclear infiltrates, whereas neutrophils are scarce80.

LPS modifications observed at 37°c suggest that Y. pseudotuberculosis and Y. enterocolitica do not produce hexa-acylated lipid A during growth in patients, and therefore it may at first seem counterintuitive that Y. ente-rocolitica can cause septic shock. A closer examination of these cases reveals that these patients develop septic shock immediately after receiving units of packed erythrocytes contaminated with Y. enterocolitica81. Retrospective anal-ysis links donors with a recent history of gastrointestinal infection to contaminated blood units, suggesting that a mild Y. enterocolitica infection in the donor gives rise to transient bacteraemia during donation. After collection of a blood unit from an infected donor, Y. enterocolitica readily grows at the storage temperature of 4°c with a generation time of 15 hours, resulting in the production of substantial amounts of LPS after prolonged storage82. Based on studies of temperature regulation, this LPS spe-cies probably represents the hexa-acylated lipid A synthe-sized by Y. enterocolitica grown at 21°c74. Introduction of large quantities of hexa-acylated LPS produced during storage at 4°c can result in the development of septic shock within minutes after transfusion83,84. Thus, septic shock caused by Y. enterocolitica results from a chain of coincidences, and these occurrences are not typical of the behaviour of the organism after an infection by the natural route, which is expected to give rise to bacteria that are not recognized by TLR4.

Similar to other atypical bacteria, such as S. Typhi and Brucella species, enteropathogenic Yersinia species therefore avoid recognition by both TLR4 and TLR5 during growth in host tissue. The picture emerging from these studies is that the ability to conceal features (flagellin and LPS) that would identify bacteria as being Gram-negative is a shared virulence strategy of agents that cause enteric fever. The use of a common virulence strategy to manipulate the induction of host responses provides an attractive explanation as to why the clini-cal presentations of yersiniosis, brucellosis and typhoid fever are similar.

ConclusionsWhat are the benefits of eliciting an inappropriate host response? Although it is clear that pathogens which cause enteric fever do not elicit classic antibacterial host responses, there are few direct experimental data that explain how avoiding these responses might affect host–pathogen interactions. neutrophilic intestinal infiltrates are an important component of a mucosal barrier that prevents bacterial dissemination to systemic sites of infection85,86. The scarcity of neutrophils in intestinal infiltrates of patients with enteric fever could facilitate bacterial dissemination, thereby partly explaining the high propensity of S. Typhi and Brucella species to cause bacteraemia. TLR-dependent mechanisms that lead to neutrophil recruitment in the intestine involve the production of IL-23 by mononuclear cells, a cytokine that stimulates T cells to release IL-17, which in turn stimulates epithelial cells to release neutrophil chemo-attractants87. Interestingly, blunted IL-17 production caused by simian immunodeficiency virus-induced depletion of intestinal T cells promotes S. Typhimurium bacteraemia in rhesus macaques86. Thus, preventing neutrophil recruitment by evading detection by TLR4 and TLR5 (S. Typhi and Brucella species in patients who are immunocompetent) or by taking advantage of T-cell depletion (S. Typhimurium in simian immuno-deficiency virus-infected rhesus macaques) allows systemic bacterial dissemination. These data suggest that neutrophils provide an important barrier to the translocation of bacteria from the gut. However, this explanation seems less satisfactory for enteropathogenic Yersinia species, which rarely cause bacteraemia in immunocompetent patients, although bacteraemia can accompany Y. enterocolitica infections in infants88,89 or patients with iron overload90,91.

A second possible benefit to the pathogen is that typical antibacterial responses that are initiated by the stimulation of TLR4 and TLR5 might be better suited to control of the intruder and to clearance of the infection than responses observed in patients with enteric fever. consistent with this idea, patients who are IRAK4 deficient, and therefore have a defect in mounting responses that are generated by bacteria-specific TLRs, suffer from recurrent infections with pyogenic bacteria92. experimental support for this hypothesis comes from studies on Yersinia pestis, the caus-ative agent of bubonic plague. Y. pestis is closely related to Y. pseudotuberculosis and both pathogens share the ability to synthesize predominantly tetra-acylated lipid A, which

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is a weak TLR4 agonist, at 37°c74,77,93. A Y. pestis strain that is genetically engineered to produce hexa-acylated lipid A (a strong TLR4 agonist) at 37°c is rapidly cleared from mice after subcutaneous infections, whereas the tetra-acylated lipid A-producing wild type causes a lethal systemic infection94. clearance of the Y. pestis mutant that produces hexa-acylated lipid A at 37°c is TLR4

dependent94, which shows that appropriate host responses to a Gram-negative infection (as indicated by TLR4 stimu-lation) are essential for effective control of the pathogen. Thus, by evading recognition through TLR4 and TLR5, pathogens that cause enteric fever could prevent the host from mounting responses that would normally result in rapid elimination of these intruders.

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AcknowledgementsThis work was supported by Public Health Service grants AI050553 (to R.M.T.), AI067676 (to G.M.Y.), AI042081 (to J.V.S.), and AI040124, AI044170 and AI079173 (to A.J.B.).

DATABASESEntrez Genome Project: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=genomeprjBrucella abortus | Corynebacterium diphteriae | Escherichia coli | S. Enteritidis | S. Typhi | S. Typhimurium | Vibrio cholerae | Yersinia enterocolitica | Yersinia pestis | Yersinia pseudotuberculosis Entrez Protein: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=proteinIFN | IL-1β | IL-6 | TLR1 | TLR2 | TLR3 | TLR4 | TLR5 | TLR6 | TLR7 | TLR8 | TNF-α

FURTHER INFORMATIONAndreas J. Bäumler’s homepage: http://www.ucdmc.ucdavis.edu/medmicro/staff/baumler.html

All links Are AcTiVe in The online pdF

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