Citrobacter rodentium: infection, inflammation and the microbiota.

REVIEWS

Citrobacter rodentium: infection, inflammation and the microbiota James W. Collins1, Kristie M. Keeney2, Valerie F. Crepin1, Vijay A. K. Rathinam3, Katherine A. Fitzgerald3, B. Brett Finlay2 and Gad Frankel1

Abstract | Citrobacter rodentium is a mucosal pathogen of mice that shares several pathogenic mechanisms with enteropathogenic Escherichia coli (EPEC) and enterohaemorrhagic E. coli (EHEC), which are two clinically important human gastrointestinal pathogens. Thus, C. rodentium has long been used as a model to understand the molecular basis of EPEC and EHEC infection in vivo. In this Review, we discuss recent studies in which C. rodentium has been used to study mucosal immunology, including the deregulation of intestinal inflammatory responses during bacteria-induced colitis and the role of the intestinal microbiota in mediating resistance to colonization by enteric pathogens. These insights should help to elucidate the roles of mucosal inflammatory responses and the microbiota in the virulence of enteric pathogens.

Crohn’s disease A chronic inflammatory disease of the gastrointestinal tract; it primarily affects the ileum and colon and causes recurrent abdominal pain, fatigue, weight loss, blood and mucus in the faeces, and diarrhoea.

Medical Research Council (MRC) Centre for Molecular Bacteriology and Infection, Department of Life Sciences, Flowers Building, Imperial College, London SW7 2AZ, UK. 2 Michael Smith Laboratories, University of British Columbia, Vancouver, British Columbia V6T 1Z4, Canada. 3 Division of Infectious Diseases and Immunology, University of Massachusetts Medical School, Worcester, Massachusetts 01655, USA. Correspondence to G.F. e‑mail: [email protected] doi:10.1038/nrmicro3315 Published online 4 August 2014 1

The mouse-restricted pathogen Citrobacter rodentium and the human enteric pathogens enteropathogenic Escherichia coli (EPEC) and enterohaemorrhagic E. coli (EHEC) colonize the intestinal mucosa via the formation of attaching and effacing (A/E) lesions1–3 (FIG. 1), which are characterized by intimate bacterial attachment to the intestinal epithelium, effacement of the brush border microvilli and the formation of pedestallike structures underneath the adherent bacterium (reviewed in REFS 4,5). The formation of A/E lesions distinguishes EPEC and EHEC from other pathogenic and commensal E. coli strains. EPEC is a major cause of infantile diarrhoea, which leads to high rates of morbidity and mortality in developing countries6. EHEC, particularly serotype O157:H7, which expresses the highly potent Shiga toxin (Stx) that causes kidney failure, is prevalent in developed countries6. C. rodentium shares 67% of its genes with both EPEC and EHEC7, including the locus of enterocyte effacement (LEE) pathogenicity island, which encodes several effector proteins and a type III secretion system (T3SS) that functions as a molecular syringe to inject effector proteins into host cells. Importantly, it has recently become clear that, in addition to a conserved set of core T3SS effectors (which includes the translocated intimin receptor (Tir), mitochondrial-associated protein (Map), EspF, EspG, EspH, EspI, EspJ, EspL, EspZ, NleB, NleC, NleD, NleE, NleF, NleG and NleH), different clinical isolates of EPEC and

EHEC encode strain-specific combinations of auxiliary effectors (for example, EspM, EspT, EspV, EspW and EspFU (also known as TccP)) that might expand host range, contribute to colonization efficiency and worsen the outcome of infection7–10. C. rodentium expresses many of the same effectors and a type IV pilus (known as colonization factor Citrobacter (CFC))11, which is also found in typical EPEC strains6, and two T6SSs7 that are present in EHEC. Therefore, C. rodentium infection of mice has become the ‘gold standard’ small-animal model for investigating the virulence mechanisms of A/E pathogens. Moreover, a C. rodentium strain that expresses Stx (known as C. rodentium λstx2dact) was recently constructed, which provides a convenient smallanimal model for studying how the toxin contributes to pathogenesis in vivo12. In addition, C. rodentium infection is used to model several important human intestinal disorders, including Crohn’s disease, ulcerative colitis and, more recently, colon tumorigenesis13,14. Following C. rodentium infection, mice develop colitis15, and this causes a pronounced dysbiosis that is characterized by an overgrowth of C. rodentium and a consequent reduction in the abundance and overall diversity of the resident microbiota16. C. rodentium levels reach 1–3% of the total intestinal microbiota16–18 and 109 colony forming units (CFUs) per g in the colon19. Importantly, C. rodentium-induced colitis is influenced by the genetic background of the host: in some mouse strains, infection is fatal, whereas

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REVIEWS Ulcerative colitis A chronic inflammatory disease of the colon and rectum that is characterized by recurrent abdominal pain, chills, fever, colitis and diarrhoea.

in others it causes self-limiting inflammation of the caecum and colon20–22. The composition of the intestinal microbiota has recently been recognized as an important factor that influences susceptibility to C. rodentium infection and the subsequent immune response23–25. The origin, characterization and molecular pathogenesis of C. rodentium has already been reviewed in

C. rodentium Intimin

Tir-M IRTKS or IRSp53

Tir

NPY motif

P Y471 NCK

? PRD Inactive N-WASP WH1 domain

WIP

PRD Active N-WASP

WH1 ARP2

ARP3

Polymerized actin Enterocyte

Figure 1 | The role of Tir signalling pathways in the formation of attaching and Nature Reviews | Microbiology effacing lesions. During Citrobacter rodentium infection of the caecal or colonic epithelium, loosely adherent bacteria translocate Tir (translocated intimin receptor) into the infected enterocyte via the type III secretion system (T3SS), which is then integrated into the plasma membrane in a hairpin-loop topology116. This results in the exposure of an extracellular domain, known as Tir-M, which functions as a receptor for the outer membrane adhesin intimin. The binding of intimin induces clustering of Tir, assembly of signalling complexes and actin polymerization via three distinct pathways (reviewed in REF. 35). Phosphorylation of the carboxy‑terminal Y471 of Tir is the major actin polymerization pathway in vitro36,117. This leads to the formation of a binding site for the mammalian adaptor protein non-catalytic region of tyrosine kinase adaptor protein (NCK)118. NCK is then thought to activate the neural Wiskott–Aldrich syndrome protein (N‑WASP) by interacting with its proline-rich domain (PRD), which in turn recruits the actin-related protein 2/3 (ARP2/3) complex, causing actin polymerization (reviewed in REF. 35). In addition, NCK can also activate N‑WASP by interacting with the WASP-interacting protein (WIP), which binds to the WH1 domain of N-WASP8,119. Tir is also known to promote weak actin polymerization in vitro in an NCK-independent manner and involves the C‑terminal Y451 of Tir120, which is found within a conserved NPY motif121. The NPY motif recruits the adaptor protein insulin receptor tyrosine kinase substrate (IRTKS) or the insulin receptor substrate protein of 53 kDa (IRSp53), which in turn activates N-WASP122,123. IRTKS recruitment in vivo is dependent on Y451 of Tir, although its presence is not essential for N‑WASP recruitment or attaching and effacing (A/E) lesion formation36. Strikingly, mutation of both Y471 and Y451 inhibit Tir-induced actin polymerization in vitro, but N‑WASP activation and the subsequent formation of A/E lesions still occur ex vivo and in vivo, which suggests that there is another unknown Tir-induced actin polymerization pathway at mucosal surfaces36.

detail2,15,21. In this Review, we discuss our current understanding of the mechanisms that are used by C. rodentium to colonize the mouse large intestine, recent insights into the molecular basis of C. rodentium-induced colitis, intestinal mucosal immune responses to infection and the effects of diet and the composition of the intestinal microbiota on resistance to C. rodentium colonization.

The infection cycle of C. rodentium C. rodentium is transmitted via the faecal–oral route and causes colitis (also known as transmissible murine crypt hyperplasia (TMCH) (BOX 1)). Disease severity ranges from self-limiting colitis and sterilizing immunity to severe inflammation and potentially lethal dehydration14,15,26,27. Most infection studies involve the inoculation of mice by oral gavage with laboratory-cultured bacteria, which results in a highly reproducible infection cycle15,21,28. C. rodentium initially colonizes the major lymphoid structure in the caecum, which is known as the ‘caecal patch’; however, most inoculated bacteria pass straight through the gastrointestinal tract19,27. Adherent bacteria adapt to the gastrointestinal environment and undergo a virulence switch that facilitates colonization of the distal colon and rectum. Following a peak in the bacterial load19,27,29, the infection begins to clear as colonized epithelial cells are shed into the intestinal lumen — first from the caecum and then from the colon, until complete clearance of the pathogen in the stool occurs 2–3 weeks post infection19,27. The bacteria that are shed at the peak of infection are ‘hyperinfectious’ and are efficiently spread via coprophagy to uninfected littermates30,31. Hostadapted bacteria are highly infectious, as they can initiate an infection with 1000‑fold fewer CFUs and bypass the initial tropism for the caecum, as bacteria directly colonize the colon30. The exact mechanisms that underlie this phenomenon are currently unknown; however, host-adapted C. rodentium show higher expression of T3SS genes compared with laboratory-cultured bacteria and are hyperadherent in vitro32. Collectively, these data suggest that laboratory-cultured C. rodentium might require signals from the intestinal microbiota or from the host to regulate pathogenesis and facilitate colonization of the colon. C. rodentium virulence determinants The filamentous T3SS effectors. Colonization of intestinal epithelial cells by C. rodentium is thought to occur in three distinct stages: transient loose attachment (mediated by fimbriae, the T3SS EspA filaments or other adhesion factors), followed by translocation of bacterial effectors into the cell via the T3SS and the formation of an A/E lesion (following disassembly of the T3SS). As mentioned above, the ability to form A/E lesions is conferred by the expression of the evolutionary divergent LEE pathogenicity island33, which consists of 41 genes that are clustered into five operons: LEE1, LEE2, LEE3, LEE5 and LEE4 (REFS 33,34). The LEE encodes transcriptional regulators (such as ler, glrA and glrR), structural components of the filamentous T3SS, effectors and their respective chaperones and the outer membrane adhesion

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REVIEWS Box 1 | Transmissible murine crypt hyperplasia

a Uninfected tissue

b Infection of susceptible mice

Shedding of luminal cells

c Infection of resistant mice

C. rodentium

MYD88

Intestinal lumen ↓ Slc26a3 ↓ Car4

Goblet cell

A/E lesion

TLR2

TLR4

H2O

Lamina propria MEK, ERK and NF-κβ pathways

IFNγ

Crypt elongation

Proliferation of TA cells

β2 integrin γδ T cell

TA cell WNT Pericryptal myofibroblast

Microbiota/ probiotics

Proliferation and inflammation of TA cells

Lgr5+ stem cell

FGF7 R-spondin 2

IL-11 Other signals

Transmissible murine crypt hyperplasia (TMCH) is a hallmark pathological feature of Citrobacter rodentium infection; it accompanies intestinal inflammation during colitis and is defined by a thickening of the colonic mucosa, which is caused by excessive induction of epithelial regeneration and repair mechanisms1,2,15,28. In uninfected mice (see the figure, part a), colonic epithelial cells are continually renewed by Lgr5+ progenitor stem cells, which are found at the base of the crypt. The production of Wingless (WNT) by pericryptal myofibroblasts triggers asymmetric cell division of the colonic stem cells, which forms a replacement stem cell and an undifferentiated transit amplifying (TA) cell108 . Following stimulation, TA cells undergo a limited number of cell divisions and migrate from the bottom of the crypt to the luminal surface, where they terminally differentiate and are later lost by shedding and replaced by new cells108. The progression of TMCH varies between mouse strains that are resistant to C. rodentium and those that are susceptible to C. rodentium. Susceptible mouse strains encode the C. rodentium infection 1 (CRI1) locus on chromosome 15, which is ~4 mb and encompasses five genes (rspo2 (which encodes R‑spondin 2), eif3e, gm10373, ttc35 and tmem74)20,109. During C. rodentium infection of susceptible mice (see the figure, part b), pericryptal myofibroblasts secrete the mitogen R‑spondin 2, which targets Lgr5+ stem cells and undifferentiated TA cells near the base of the crypts and triggers an excessive intestinal epithelial repair response, which results in an accumulation of undifferentiated colonocytes at the luminal surface and a reduction in the number of goblet cells20. The large increase in TA cell number, combined with bacterial inhibition of colonocyte detachment, causes dramatic crypt elongation44 and characteristic thickening of the colonic mucosa. Importantly, the undifferentiated colonocytes at the luminal surface have decreased expression of the solute carrier family 26, member 3 (SLc26a3) HCO3–Cl− exchanger and carbonic anhydrase IV (Car4), which causes impaired electrolyte absorption and profuse

diarrhoea that results in death20,22. In resistant mice (see the figure, part c), TMCH typically lasts 2–3 weeks and follows a biphasic response, with Nature | Microbiology progressive and regressive phases28,110. During the Reviews progressive phase, mitogens (such as fibroblast growth factor 7 (FGF7)111) and cytokines (for example, Toll-like receptor 2 (TLR2)‑dependent production of IL‑11 (REF. 100)) produced by pericryptal myofibroblasts, and other uncharacterized signals trigger cell proliferation via activation of WNT–β-catenin20, STAT3 (signal transducer and activator of transcription 3)100 and phosphatidylinositol 3‑kinase–AKT–β‑catenin signalling pathways112. TMCH is triggered by the loss of epithelial barrier integrity and the transit of bacteria into the sterile lamina propria100,108. In mice that have a disrupted colonic epithelium as a result of intrarectal administration of ethanol, heat killed bacteria that express β‑intimin trigger TMCH in an interferon-γ (IFNγ)‑dependent process that is mediated by interactions between the bacterium and β1 integrins on T cells in the lamina propria111. In response to infection-induced epithelial damage, TLR2 (REFS 57,100), TLR4 (REFS 58,100) and myeloid differentiation primary-response protein 88 (MYD88)55 induce inflammation via the MEK, ERK and nuclear factor κB (NF-κB)110 pathways in both epithelial cells and pericryptal myofibroblasts. These innate immune responses, including epithelial repair mechanisms, are modulated by environmental factors, including diet110, probiotic treatment and undefined microbial species that are present in the colonic microbiota of resistant mice25. The regressive phase involves pathogen clearance, the resolution of colitis and inflammation and a return to normal intestinal homeostasis28,110. Importantly, the epithelial regenerative and repair pathways that are associated with TMCH replicate the pathology and cellular signalling events that are observed in human diseases such as inflammatory bowel disease (IBD), ulcerative colitis and colon tumorigenesis13,14. Thus, TMCH is a suitable model for the study of such diseases. Figure modified from REF. 20, Nature Publishing Group.

intimin33,34. One of the best characterized T3SS effectors is Tir, which inserts into the plasma membrane in a hairpin-loop topology, where it functions as a receptor for intimin. Following the clustering of intimin, Tir

triggers localized actin polymerization by three different pathways, all of which rely on the activation of the nucleation promoting factor neural Wiskott–Aldrich syndrome (N‑WASP) and actin-regulated protein 2/3



REVIEWS

Sterilizing immunity An immune response that completely prevents an infection.

Coprophagy The consumption of faeces.

H‑NS family (Histone-like nucleoidstructuring family). A family of DNA-binding proteins that bind to AT‑rich double-stranded DNA and are involved in transcriptional silencing and bacterial chromosome organization.

Integration host factor (IHF). A histone-like DNA-binding protein that binds to consensus sites and bends the DNA to form a nucleoprotein complex that promotes transcription.

AHL-type quorum sensing system (N-Acyl-homoserine lactone quorum sensing system). A dedicated communication system that is present in Gram-negative bacteria and is used to regulate specific genes in response to population density via the production of autoinducer 1.

LuxS quorum sensing system A communication system that is found in both Gram-positive and Gram-negative bacteria; it controls the expression of virulence genes in a cell density-dependent manner via the production of the signalling molecule autoinducer 2 by luxS.

C3H/HeJ mice A substrain of the widely used laboratory mouse strain C3H; they carry a mutation in Tlr4 and are resistant to endotoxin exposure.

BarA–SirA two-component regulatory system A two-component system that is present in Salmonella enterica subsp. enterica serovar Typhimurium. BarA encodes a histidine kinase and SirA encodes the response regulator. Following activation, the system triggers a signalling cascade that results in increased expression of virulence genes and decreased expression of motility genes.

(ARP2/3) complex (FIG. 1). The main pathway in vitro involves phosphorylation of the Tir residue Y471 and subsequent recruitment of the mammalian non-catalytic region of tyrosine kinase adaptor protein (NCK), which in turn activates N‑WASP35. Although the Tir mutation Y471A leads to loss of NCK recruitment, N‑WASP still accumulates underneath attached bacteria in vivo. Thus, N‑WASP is recruited independently of Tir Y471, but phosphorylation of this residue is essential for the recruitment of NCK36. Furthermore, Tir can also induce weak actin polymerization via the insulin receptor tyrosine kinase substrate (IRTKS)–insulin receptor substrate protein of 53 kDa (IRSp53) pathway and via an unknown pathway (FIG. 1). In addition to LEE, other pathogenicity islands and mobile genetic elements, including prophages and insertion sequences, encode non-LEE effectors34,37. The non-LEE effectors do not seem to be required for the formation of A/E lesions, but they do promote bacterial colonization. Following translocation, LEE and non-LEE effectors cooperate in subverting multiple signalling pathways in the host cell, including actin polymerization36 (FIG. 1), tight junction integrity38,39, apoptosis40, endosomal trafficking41, phagocytosis42 and innate immune responses43, as well as epithelial cell shedding and detachment44. Importantly, without Tir3, intimin1 or a functional T3SS7, C. rodentium is completely avirulent and has colonization dynamics that are similar to those of commensal E. coli7,27,45, whereas deletion mutants of nleA (also known as espI)46 and nleB37 are highly attenuated. Other virulence factors. Relatively few non‑T3SS C. rodentium virulence factors have been characterized in vivo so far. The pili operons CFC11 and Kfc (K99 Fimbral Cluster), as well as the adhesin AdcA, which is involved in diffuse C. rodentium adhesion47, have been suggested to have a role in colonization. However, the mechanisms by which these adhesins contribute to the overall infection cycle of C. rodentium, such as conferring tissue tropism or involvement in initial attachment prior to the formation of A/E lesions, are unclear. In addition to adhesins, two putative T6SSs (known as CTS1 and CTS2) have been identified7. Following the artificial induction of cts1 from a heterologous promoter, this operon was recently shown to form a functional T6SS48. As CTS1 was found to target competitor bacterial cells, this suggests that the CTS loci might contribute to the colonization of mucosal surfaces by outcompeting commensal bacteria. Regulation of virulence. The expression of virulence factors, particularly the T3SS apparatus genes, is stringently regulated to reduce the metabolic burden on the bacterial cell. Ler is a member of the H-NS family (histone-like nucleoid-structuring family) of DNA-binding proteins and promotes the transcription of the five LEE operons (LEE1–5) and the grlRA operon by disrupting transcriptional silencing by other H‑NS proteins34. The products of the grlRA operon are transcriptional regulators that have opposing effects: GrlA interacts directly with the

LEE1 promoter and stimulates the initiation of transcription, whereas GrlR represses the LEE1 promoter. Activation of grlA transcription by Ler and activation of LEE1 transcription by glrA initiates a strong positivefeedback loop (reviewed in REF. 49), which is the target of other H‑NS or integration host factor (IHF) proteins that regulate LEE expression in response to environmental signals in the host49. RegA, which is an AraClike transcriptional regulator, also binds to the grlAR promoter and activates the Ler–GrlA regulatory loop47. Importantly, the deletion of ler results in a complete loss of virulence45. Using a ler–luxCDABE fusion construct, ler was shown to be expressed at high levels in the caecum and colon 5 days post-infection and was undetectable 14 days post-infection45. Interestingly, bicarbonate ions, which are normally present at high levels in the duodenum, were found to activate RegA by an unknown mechanism. This triggers C. rodentium virulence, including the expression of the genes that encode Kfc and AdcA, which suggests that bicarbonate ions have a role in promoting early adhesion and the subsequent formation of A/E lesions47,49. Quorum sensing systems have been implicated in regulating the T3SS and other virulence factors in several enteric pathogens50. C. rodentium has an AHL-type quorum sensing system (N‑acyl homoserine lactonetype quorum sensing system), known as CroIR, that produces N‑butanoyl-l‑homoserine lactone (BAL) and small amounts of N‑hexanoyl-l‑homoserine lactone, in addition to a LuxS quorum sensing system that produces and responds to autoinducer 2 (REFS 7,51). However, the T3SS of C. rodentium is not regulated either by BAL or by N‑hexanoyl-l‑homoserine lactone, and no role in virulence has been identified for the luxS system 51. By contrast, the CroIR system regulates adhesion in vitro, and deletion of croIR causes a marked increase in the mortality of infected C3H/HeJ mice51. Although further studies are required to define the basis of these observations, the CroIR quorum sensing system might be involved in regulating adaptation to the host. It is currently unknown whether C. rodentium regulates virulence in response to the levels of shortchain fatty acids (SCFAs) in the gastrointestinal tract; Salmonella enterica subsp. enterica serovar Typhimurium regulates T3SS expression in response to SCFA levels via the BarA–SirA two-component regulatory system52. We anticipate that the homologues BarA and UvrY in C. rodentium are important regulators of LEE expression, and they were probably not identified by previous signature-tagged mutagenesis screens owing to the complex and redundant regulatory systems that control LEE expression.

Immune responses during C. rodentium infection Innate mucosal immune responses. The innate immune compartment of the intestinal mucosa, which is a vital interface between the microbiota and the host, has a pivotal role in mucosal homeostasis and in antimicrobial immunity (FIG. 2). Myeloid differentiation primaryresponse protein 88 (MYD88) is a key adaptor molecule in innate immune signalling downstream of the Toll-like



REVIEWS a

b C. rodentium

Intestinal lumen

Expression of REGIIIβ and REGIIIγ

NOD

MYD88 TLR2 TLR4 Immune cell recruitment

Dendritic cell

γδ T cell

IL-17F

ILC3 cell

IFNγ TH17 cell

NLRP3

Caspase 11 Macrophage

Caspase 1 IL-1β IL-18

IL-6 IL-12 IL-23 TNF

TH22 cell IL-22 TH1 cell

TNF IL-17A

Neutrophil recruitment Lamina propria

Neutrophil

Figure 2 | Mucosal immune responses to Citrobacter rodentium. In the intestines, mucosal-associated and Reviews | Microbiology luminal C. rodentium that have breached the epithelial barrier trigger inflammation owing Nature to the recognition of lipopolysaccharide (LPS), the type III secretion system (T3SS) and peptidoglycan as pathogen-associated molecular patterns (PAMPS). a | C. rodentium and/or its associated PAMPS are recognized by myeloid differentiation primary-response protein 88 (MYD88)‑dependent Toll-like receptor (TLR) signalling54, which is mediated by TLR2 (REF. 57) and TLR4 (REF. 58) on the surface of epithelial and myeloid cells, and by nucleotide-binding oligomerization domain-containing (NOD) proteins within epithelial cells, which leads to the activation of nuclear factor-κB (NF-κB) and the production of the pro-inflammatory cytokines interleukin-6 (Il‑6), IL‑12, IL‑23 and tumour necrosis factor (TNF) by innate immune cells, including dendritic cells, macrophages and neutrophils. C. rodentium also induces the production of IL‑1β and IL‑18 by macrophages and dendritic cells via the caspase 1‑dependent NLRP3 inflammasome, which requires the activation of caspase 11 by intracellular LPS (not shown62,66). b | C. rodentium infection induces a diverse T cell effector response, including interferon-γ (IFNγ)-producing γδ T cells and T helper 1 (TH1) cell responses59,70,73. TH17 cells59 and TH22 cells99 have a central role in driving host resistance to C. rodentium infection via the production of the pro-inflammatory cytokine IL‑17A, which recruits neutrophils74,90, and the anti-inflammatory cytokine IL‑22, which upregulates the expression of antimicrobial peptides (such as REGIIIβ and REGIIIγ) in colonocytes101. In addition, TH17 cells can be induced by epithelial cell apoptosis in an IL‑6‑dependant manner. In the caecum, group 3 innate lymphoid cells (ILC3s; also known as iTH17 cells) are induced early during infection in a NOD-dependent manner, are an important source of IL‑17 and IL‑22 and contribute to the clearance of C. rodentium infection.

iNOS (Inducible nitric oxide synthase). A cytosolic enzyme that is found in multiple cell types and that produces nitrous oxide (NO) from l‑arginine, following induction by lipopolysaccharide and pro-inflammatory cytokines. It is presumed that the production of NO, in conjunction with superoxide radicals, leads to the formation of antimicrobial reactive nitrogen intermediates, such as peroxynitrite and nitrosothiols, which restrict the growth of invading pathogens.

receptor (TLR) and the interleukin‑1 receptor (IL1R) superfamilies, both of which regulate the transcriptional activation of several immune-related genes53. MYD88 signalling controls C. rodentium infection via several mechanisms, including the recruitment of neutrophils, macrophages and dendritic cells to the mucosa, the expression of iNOS (inducible nitric oxide synthase) and triggering the proliferation of epithelial cells (known as colonic hyperplasia)54,55. Consequently, MYD88 deficiency compromises the ability of the host to restrict bacterial replication. Although MYD88 is a signalling adaptor for the TLR and IL1R superfamily pathways, TLR2 and TLR4 signalling seem to be the primary contributors to MYD88‑mediated protective innate responses against C. rodentium, and they control the pathogen by inducing the the upregulation of iNOS and the production of the pro-inflammatory cytokines keratinocyte

chemoattractant (KC), tumour necrosis factor (TNF) and interleukin-6 (IL‑6)54–56 (FIG. 2a). C. rodentium infection of TLR2-deficient mice leads to severe colonic pathology, rapid weight loss and accelerated mortality57. Although TLR4 signalling is required for chemokine responses and infiltration of the intestinal mucosa by neutrophils and macrophages, it is dispensable for clearance of the infection58. Surprisingly, TLR4‑mediated responses promote colonization of the colon by C. rodentium during the early stages of infection, which suggests that lowlevel inflammation is advantageous for the pathogen. Moreover, the intracellular pattern recognition receptors nucleotide-binding oligomerization domain-containing protein 1 (NOD1) and NOD2, which have been implicated in promoting susceptibility to inflammatory bowel diseases (IBDs) in humans, contribute to triggering host innate responses against C. rodentium59 in mice.



REVIEWS

Inflammasomes Macromolecular complexes that are found in the cytosol of haematopoietic cells and are assembled in response to a range of microbial and endogenous danger signals, which leads to the proteolytic activation of the effector protein caspase 1. Inflammasomes typically consist of a receptor, an adaptor molecule (apoptosisassociated speck-like protein containing caspase activation and recruitment domain), and the effector caspase 1.

Specific pathogen-free mice Mice that are provided by laboratory animal vendors or are generated in a home laboratory, that have a guaranteed health status and are free of particular pathogens.

Faecal microbiota transplantation (FMT). A transplantation process in which the faecal material (including the faecal microbiota) from a healthy donor is transferred into a recipient. Patients are often treated by enema infusion or the consumption of capsules containing donor faeces.

Inflammasomes regulate mucosal immune homeostasis60 and have recently emerged as a central defence mechanism against bacterial pathogens61. Inflammasomes are macromolecular scaffolds in the cytosol of immune cells and are responsible for proteolytic maturation of caspase 1 and the IL‑1 family of cytokines, including IL‑1β and IL‑18 (REF. 62). C. rodentium triggers IL‑1 responses in a NLRP3‑dependent manner, and in vitro studies have shown that C. rodentium-mediated NLRP3‑dependent caspase 1 maturation and IL‑1 responses also require the related inflammatory protease caspase 11 (REF. 63) (FIG. 2a). The type I interferon signalling pathway, which is driven by TRIF (an adaptor molecule that functions downstream of TLR4), is also an important regulator of caspase 11‑dependent immune responses during C. rodentium infection64–66. Interestingly, recent studies have revealed a novel TLR4‑independent mechanism for innate immune recognition of intracellular lipopolysaccharide (LPS), which seems to be important for the control of caspase 1 activation in cells that are infected with C. rodentium and other Gram-negative bacterial pathogens; however, the LPS receptor and the composition of the non-canonical inflammasome remain to be elucidated67. Caspase 1-mediated mucosal responses are crucial for host resistance to C. rodentium, and caspase 1-deficient mice have increased bacterial loads, aberrant inflammatory and chemokine responses, severe immunopathology and rapid weight loss68. Although NLRP3‑deficient mice display the same defects, the phenotypes are milder in NLRP3-null mice than in caspase 1-null mice, which suggests that another inflammasome pathway has a role in C. rodentium infection in vivo. The NLRC4 inflammasome recognizes components of the T3SS and bacterial flagellin (which is not expressed by C. rodentium69) and seems to contribute to protection against infection, although it is not required for C. rodentium-driven IL‑1β production in macrophages68.

Adaptive mucosal immune responses. C. rodentium has been widely used as a tool to investigate adaptive mucosal immune responses to bacterial infection21. B cells and CD4+ T cells are crucial for the development of sterilizing immunity and resistance to C. rodentium infection70,71, and mice that lack CD4+ T cells or B cells (but not CD8+ T cells) show hypersusceptibility to mucosal inflammation, colonic pathology and systemic dissemination of the bacterium70,71. CD4+ T celldependent humoral immunity is essential for clearance of the pathogen71 and C. rodentium colonization elicits a robust T helper 1 (TH1) cell response in the gut, which is characterized by the production of interferon-γ (IFNγ) and tumour necrosis factor (TNF)14 (FIG. 2b). The hypersusceptibility of CD4+ T cell-deficient mice, and to a lesser degree, IFNγ-null mice, is shown by increased bacterial loads and a compromised ability to limit the systemic dissemination of C. rodentium72,73. C. rodentium has been key to unravelling the function of TH17 cells, which are a type of CD4+ T helper cell that produce IL‑17 and comprise a distinct branch of mucosal immune defences74. In addition to traditional TH17 cells, a new subset of innate lymphoid cells that

produce IL‑17, known as group 3 innate lymphoid cells (ILC3s; also known as iTH17 cells)75, have been identified59. C. rodentium triggers a potent TH17 cell response, which is stronger than the TH1 cell response76 1 week after oral challenge; these responses collectively contribute to pathogen control. The development of the TH17 cell response is linked to C. rodentium-induced epithelial cell apoptosis, as blocking apoptosis during infection impairs the TH17 cell response in the lamina propria77. Transforming growth factor β (TGFβ) and IL‑23, which have a role in the differentiation and proliferation of TH17 cells, are essential for protection against C. rodentium76. Moreover, IL‑17A and IL‑17F, which are important effector molecules of TH17 cells and some innate immune cells, are also important for the control of C. rodentium infection, as deficiencies of these cytokines result in increased mucosal inflammation and ulceration, increased bacterial burden and systemic spread of the bacterium59,76,77. Interestingly, ILC3s are induced in the caecum early during C. rodentium infection and require priming by the intestinal microbiota for their development59. In addition to IL‑17, populations of CD4+ T cells and ILC3s produce the anti-inflammatory cytokine IL‑22, which is essential for protection against C. rodentium infection (BOX 2).

The role of the microbiota and nutrition The generation of ILC3s in response to infection requires the intestinal microbiota (FIG. 3), as germ-free mice have substantially fewer IL‑17A+CD4 +TCRβ + lymphocytes in their caecal lamina propria during S. Typhimurium infection than specific pathogen-free mice59. Faecal microbiota transplantation (FMT) is an emerging treatment for ulcerative colitis and Clostridium difficileassociated diarrhoea in humans, and it has been used to test the role of the microbiota in mediating resistance to C. rodentium infection23–25,78. C57Bl/6 mice that were purchased from two different vendors of laboratory mice had vastly different TH17 cell numbers in the small intestinal lamina propria24. FMT and co-housing studies revealed that the mice that had higher TH17 cell numbers had higher levels of segmented filamentous bacteria (SFB) in their microbiota, which in turn stimulated CD4+ T helper cells to produce and release IL‑17 and IL‑22 into the lamina propria, and increased IL‑17 and SFB levels correlated with increased resistance to C. rodentium24. Two recent investigations have shown that the transfer of the microbiota from resistant mice to mice that are susceptible to infection results in the transfer of host resistance to C. rodentium infection23,25. In one of these studies, resistance to infection was associated with an increase in IL‑22‑mediated innate defences, including production of the antimicrobial peptides RegIIIγ and RegIIIβ (BOX 2). Furthermore, immunoneutralization of IL‑22 reduced the protective effect of FMT23. Resistance to infection was correlated with an increase in Bacteriodetes23,25 and a decrease in Firmicutes23 at the phylum level, and at the family level, a decrease in Porphyromonadaceae23 and an increase in Lachnospiraceae, Bacteroidaceae and an unclassified family of Clostridiales23. Unlike another study, in



REVIEWS Box 2 | IL‑22-mediated defence against Citrobacter rodentium infection In addition to interleukin-17 (IL‑17), group 3 innate lymphoid cells (ILC3s) also produce IL‑22, which is a recently discovered cytokine that belongs to the IL‑10 cytokine family. Other populations of CD4+ T cells and innate lymphoid cells are equally important sources of IL‑22 in the intestinal mucosa. Infection with C. rodentium triggers the expression of IL‑22 in the gut and IL‑22 provides protection against C. rodentium113 by inducing the production of RegIII antimicrobial peptides and by promoting epithelial barrier integrity. During C. rodentium infection, IL‑22 is produced in a bimodal manner in which innate lymphoid cells function as an early source, whereas CD4+ T cells (including T helper 17 (TH17) cells and TH22 cells) function as late sources99. During the later stages of infection, IL‑22‑producing TH22 cells seem to be more protective than IL‑22‑secreting TH17 cells. Interestingly, the production of IL‑22 during both the early and late stages of infection is absolutely required for the complete resolution of infection99. The IL‑22 response to C. rodentium infection seems to be dependent on IL‑23 and a lymphotoxin pathway101,102. Lymphotoxin is a membrane-bound molecule that belongs to the tumour necrosis factor (TNF) family and is expressed by intestinal lymphocytes. The biologically active heterotrimeric form of lymphotoxin (LTα1β2) is crucial for the control of C. rodentium infection, as mice readily succumb to the infection in the absence of LTα1β2 signalling. LTα1β2 positively regulates the production of IL‑22 by innate lymphoid cells in response to C. rodentium. Overall, C. rodentium elicits a well-coordinated host immune response that involves multiple components of the mucosal defence system, and as such, this bacterium continues to function as a key tool for investigating the complexities of the defence programme against attaching and effacing (A/E) pathogens.

Intestinal lymphoid follicles A type of lymphoid tissue that consists of aggregates of B cells, CD4+ T cells and IgA-producing plasma cells, which are found directly underneath the associated epithelium. These follicles are induced following environmental cues from the intestinal microbiota and dietary components.

RegIII antimicrobial peptides Secreted antimicrobial peptides that are produced in the gastrointestinal tract and pancreas and consist of a signal peptide and a single C‑type lectin domain; they bind to peptidoglycan and are bactericidal for Gram-positive bacteria.

which SFB were implicated in the induction of a protective IL‑22 response24, in the study by Willing et al.23, SFB levels were not increased in the mice that received the protective microbiota from resistant mice, and SFB levels were not depleted in the mice that received the microbiota that was associated with decreased IL‑22 expression. The fact that SFB were present in mice that had the IL-22-deficient microbiota suggests that other bacterial species are involved23. Moreover, IgA+ plasma cells, which produce TNF and iNOS, are induced by the microbiota, and when they are absent, the composition of the microbiota is altered, such that the levels of SFB are reduced and the levels of Bacillus spp., Clostridium coccoides (cluster IVa) and Clostridium leptum (cluster IV) are increased, which promotes C. rodentium colonization79. Collectively, these data show that the composition of the microbiota influences host susceptibility to C. rodentium infection by modulating the development of intestinal TH17 cells and the subsequent production of IL‑22 and IL‑17 in response to bacterial infection. Probiotics and nutrition. Probiotics, which are live microorganisms that, when administered in adequate quantities, confer health benefits to the host, are increasingly used to prevent and treat acute diarrhoea that is caused by antimicrobial-resistant intestinal pathogens. A probiotic mixture that contains Lactobacillus rhamnosus and Lactobacillus helveticus has been shown to prevent C. rodentium-induced mortality in neonatal mice, to diminish the expression of proinflammatory cytokines that are involved in the TH1 and TH17 cell responses (FIG. 3) and to induce an anti-inflammatory state80. Similarly, administration of Bifidobacterium breve UCC2003 to mice also reduced C. rodentium colonization81. This protective effect was dependent on the expression of a cell surface-associated exopolysaccharide

(EPS) by B. breve, which was linked to downregulation of the immune response and evasion of adaptive B cell responses81. The mechanism that is involved has yet to be characterized, but EPS-negative B. breve UCC2003 showed impaired colonization, induced proinflammatory responses in the mucosa and promoted C. rodentium colonization81. A recent study has shown that daily treatment of mice with fermented dairy products (FDPs) that contain the probiotics L. rhamnosus and two strains of Lactobacillus paracasei, reduces TMCH but not C. rodentium colonization 82. Interestingly, although the underlying molecular mechanisms are not fully understood, protection against TMCH required the presence of live bacteria in the FDPs and was associated with blocking a reduction in the abundance of Turicibacter spp. and Ruminococcus spp. in the microbiota at the peak of C. rodentium infection. Loss of both Turicibacter spp. and Ruminococcus spp. is associated with susceptibility to dextran sodium sulphate (DSS)induced colitis83. These data suggest that live probiotics that are found in FDPs may modify the intestinal microbiota to confer health benefits, such as preventing TMCH, although further study is required to support this hypothesis82. Several nutrients have recently been implicated in modulating C. rodentium colonization. Dietary ligands, such as those that are found in cruciferous vegetables from the family Brassicaceae induce the expression of the aryl hydrocarbon receptor (AhR), which regulates intestinal homeostasis via the formation of isolated inducible intestinal lymphoid follicles. This process is blocked in AhR-deficient mice, which are more easily infected by C. rodentium, suggesting that dietary ligands regulate intestinal homeostasis, and as a result, affect the ability of C. rodentium to colonize the host84. The antioxidant vitamin D increases susceptibility to C. rodentium colonization by suppressing the host TH17 cell response, which leads to increased pathogen burden and exaggerated tissue pathology85. By contrast, mice that are fed a diet that is deficient in the antioxidants selenium and vitamin E are also highly susceptible to C. rodentium infection and show increased pathogen burden, exaggerated inflammation and tissue pathology86. The mechanisms by which dietary antioxidants affect C. rodentium infection are currently unknown; however, it is likely that these metabolites regulate the differentiation of immune cells in the lamina propria, in addition to scavenging damaging free radicals that are produced during inflammation. Importantly, a lack of selenium and vitamin E in the diet could be an important risk factor for infection with gastrointestinal pathogens. Polyunsaturated fatty acids (PUFAs) that are derived from dietary oils are also known to increase susceptibility to C. rodentium infection and exacerbate colitis87 (FIG. 3). It was recently shown that dietary supplementation with omega-6 PUFA increased the levels of Enterobactereriaceae, SFB and Clostridium spp. in mice, all of which have been implicated in detrimental pro-inflammatory responses and IBDs88–90. Compared with omega-3 PUFA-treated mice, infection of omega-6 PUFA-treated mice with C. rodentium leads to increased



REVIEWS a Resistance to colonization

b Susceptibility to colonization C. rodentium

Antibiotics Healthy microbiota

Altered microbiota

Probiotics

TH17 cell IL-22

IgA plasma cell +

C2 GPR41 GPR43 C3

Colitis

A/E lesion

ILC3 cell

Brassicaceae ligand Macrophage Thin mucus layer

IL-17A

Neutrophil

Full goblet cell

Natural killer cell

Thick mucus layer

Omega-3 PUFA, Omega-6 PUFA, Vitamin D

Modulation of mucosal immunity

Empty goblet cell

Healthy enterocytes

Inflamed enterocytes

Figure 3 | The role of the microbiota and nutrition in modulating resistance to Citrobacter rodentium colonization. a | In mice that have increased resistance to colonization by C. rodentium, the intestinal microbiota directly influences the development and/or activity of intestinal immune cells, including iNOS (inducible nitric oxide synthase)- and tumour necrosis factor (TNF)‑producing IgA+ plasma cells79, nucleotide-binding oligomerization domain-containng protein 1 (NOD1)- or NOD2-dependent mucosal group 3 innate lymphoid cells (ILC3s; also known as inducible T helper 17 (iTH17) cells)59,75 and IL‑17- and IL‑22‑producing TH17 cells in the lamina propria24. The intestinal microbiota can also indirectly effect the activation of intestinal immune cells by the production of the short-chain fatty acids acetate (C2) and propionate (C3), which interact with G protein-coupled receptor 41 (GPR41) and GPR43 on the surface of enterocytes and prime the mucosa to respond to bacterial infection by stimulating an increase in the proliferation of TH1 cells and TH17 cells and by promoting neutrophil recruitment103. The consumption of probiotics, including Lactobacillus rhamnosus, Lactobacillus helveticus, and Bifidobacterium breve UCC2003 have also been shown to prevent C. rodentium colonization81 and/or to prevent the mortality of infected mice80. Dietary compounds containing brassicaceae ligands modify the mucosal immune response by promoting intestinal lymphoid follicle development via activation of the aryl hydrocarbon receptors (AhR) on ILC3s84. b | Perturbation of the intestinal microbiota by antibiotic treatment or by modification of the diet can confer increased susceptibility to C. rodentium infection and exacerbate colitis. Alteration of the microbiota with some antibiotics increases susceptibility to C. rodentium infection by deregulating mucosal homeostasis and promoting the development of a pro-inflammatory environment, which is associated with increased proliferation of natural killer cells and macrophages in the lamina propria93. In addition, metronidazole treatment decreases goblet cell function and thins the mucus layer, which directly promotes pathogen attachment and increases access of the altered microbiota to the intestinal mucosa, triggering additional inflammation93. Nutritional supplementation with omega-6 polyunsaturated fatty acids (PUFAs), omega-3 PUFAs87 or vitamin D85 directly modulates mucosal immune responses. Supplementation with omega-6 PUFA and omega-3 PUFA reduces C. rodentium-induced inflammation but inhibits epithelial intestinal alkaline phosphatase (which detoxifies C. rodentium lipopolysaccharide (LPS) to limit inflammation), causing increased mortality87. Supplementation of the diet with monosaccharides provides a carbon source for pathogen growth, which enables C. rodentium to outcompete the resident microbiota44.

Nature Reviews | Microbiology

intestinal damage, immune cell infiltrate, prostaglandin E2 expression and dissemination of C. rodentium to extra-intestinal sites87. However, combining omega-3 PUFA with the omega-6 PUFA supplement reduces the pro-inflammatory response and increases the proportion of Lactobacillus spp. and Bifidobacteria spp. in the microbiota during C. rodentium infection. This combination also leads to a reduction in the abundance of intestinal alkaline phosphatase-expressing cells, which are involved in the detoxification of LPS, and increased mortality from C. rodentium infection87.

In addition, monosaccharides were recently suggested to aid C. rodentium colonization of germ-free mice45. In this model, C. rodentium is readily outcompeted by E. coli but not Bacteroides thetaiotaomicron or Bacteriodes vulgatus, possibly owing to competition for monosaccharides, which are the preferred carbon source for the Enterobacteriaceae. These data suggest that access to nutrients and the ability to outcompete the microbiota for nutrients in the gastrointestinal tract is an important first step in pathogen colonization.



REVIEWS

Trefoil factor A secretory protein that contains a trefoil motif and is produced by goblet cells in the gastrointestinal mucosa; it is thought to protect against mucosal damage by stabilizing the mucus layer.

Resistin-like molecule B A protein that is produced by intestinal goblet cells and is induced by microorganisms; it is thought to regulate innate mucosal immune responses, such as macrophage activation and antimicrobial lectin expression.

Altered Schaedler flora (ASF). A cocktail of eight culturable bacterial strains that is used to colonize the gastrointestinal tract of germ-free mice, thereby generating a defined low-complexity microbiota. Notably, different variations of ASF are commercially available.

Effects of antibiotic treatment. Antibiotic treatment frequently makes a host more susceptible to pathogen colonization91; for example, treatment of mice with streptomycin (20 mg per mouse) prior to C. rodentium infection reduces the total commensal microbiota and results in a consequent 10–50-fold increase in the abundance of C. rodentium92. Similar effects are observed following the treatment of mice with metronidazole (at doses that are used to treat humans), which results in a reduction in the levels of Porphyromonadaceae and Bacteroidetes and an increase in the level of Lactobacillus spp.93. Subsequent infection by C. rodentium in this altered intestinal environment facilitates increased pathogen attachment to colonic epithelial cells and infiltration of macrophages and natural killer cells into the lamina propria (FIG. 3). In addition, metronidazole treatment causes impaired goblet cell function, including a reduction in the expression of intestinal trefoil factor and resistin-like molecule B, as well as a compromised inner mucus layer93. Mice lacking the main intestinal mucin, Muc2, are highly susceptible to C. rodentium infection and have a 10–100-fold increase in pathogen load compared with wild-type mice. In Muc2‑deficient mice, the mucus layer is completely disrupted, which enables the microbiota and invading pathogens to gain direct access to the normally sterile colonic epithelium, resulting in severe inflammation92 (FIG. 3). Importantly, there is evidence to suggest that members of the microbiota, such as the Bacteroides, indirectly promote the production of Muc2 (REF. 94) to facilitate colonization of the outer mucin layer, which improves epithelial barrier function and prevents pathogen attachment. The pattern recognition receptor NLRP6 has recently been identified as a crucial coordinator of mucin granule exocytosis in intestinal goblet cells95. Goblet cells of NLRP6‑deficient mice exhibit impaired autophagy, which leads to reduced mucin secretion into the intestinal lumen, and as a consequence, mice are unable to clear C. rodentium from the mucosal surface95. Collectively, these studies suggest that the composition of the microbiota modulates the thickness of the inner mucus layer and that the production of mucin is used as an intestinal defence mechanism to flush pathogens away from the mucosal surface92,93,95.

Conclusions and future prospects C. rodentium has become a model microorganism to study how enteric A/E pathogens subvert normal host cell functions, such as innate immunity, intestinal cell shedding and apoptosis, to colonize the host8,15,96,97. Alternative models of EPEC infection include intestinal epithelial cell culture, in vitro organ culture and in vivo models, including nematodes, insects, rabbits, cattle and human volunteers, which are summarized in a recent review98. C. rodentium infection of mice has proven to be an excellent model to study intestinal inflammation, particularly the mucosal immune responses to infection14,54,56,57,70,71,99–102, and has even led to the discovery of new immune cell subsets in mice (such as ILC3s and IgA+ plasma cells)59,79. This Review also highlights the importance of the intestinal microbiota in modulating the ability of enteric pathogens to colonize the intestinal mucosa and in coordinating mucosal immune responses to infection24,25,59,79–81. In addition, dietary compounds, such as vitamin D85, vitamin E86, selenium86, cruciferous vegetable ligands84, PUFAs87 and monosaccharides, as well as the metabolic activity103 and composition of the intestinal microbiota, directly affect mucosal immune responses to infection25. Given the paucity of new antibiotics reaching the market and the substantial increase in the number of infections that are caused by drugresistant pathogens, a new class of antimicrobial agents that promote resistance to colonization by modulating the intestinal microbiota (particularly its metabolic activity and composition), combined with modified nutrition, might be a viable alternative. Importantly, reports of changes in the microbiota of mice that are infected with enteric pathogens are largely descriptive (increase or decrease in microbial populations based on sequencing) and are often contradictory. However, this is probably due to differences in the microbiota of the mice that were used, differences in mouse strains and differences in 16S rRNA sequencing pipelines (such as DNA extraction, PCR conditions, sequencing, bioinformatics and data interpretation; reviewed in REF. 104). The field of host–microbiota interactions is rapidly evolving, and it is becoming increasingly important to show causal links between alterations

Box 3 | New tools for Citrobacter rodentium research Whole-genome sequencing of multiple pathogenic and commensal bacteria has led to the identification of many genes that are related to pathogenesis, including families of effector proteins that are conserved among different attaching and effacing (A/E) pathogens, which suggests that they have common cellular targets and shared infection strategies7. However, genomics alone provides little information on the identity of novel virulence factors, and more importantly, their functional roles in pathogenesis. There has recently been a paradigm shift and many research groups are now focusing on the use of various high-throughput proteomics114,115 and four-dimensional (4D) imaging30 methodologies to investigate the molecular mechanisms of virulence factor function and the responses of the host to bacterial infection. These include the use of metabolomics to understand the chemical language between pathogens and the host, as well as host cell responses and pathogen–microbiota interactions. Furthermore, the use of systematic screens for host cell effects (such as phosphorylation or other modifications) will yield a more comprehensive understanding of host cell responses to this pathogen. The field has historically studied the effect of single mutations or the transfection of single effectors to elucidate binding targets in the host and other responses; however, tools to examine the infection as a dynamic process now exist, and the use of strains that contain multiple mutations will enable us to obtain a much more comprehensive understanding of the disease process. It is likely that the type III effectors of C. rodentium function in a coordinated manner and the role of one effector may be dependent on the prior activity of another, which may help us to understand additional effector mechanisms.



REVIEWS Monocolonized mice Former germ-free mice that have been colonized with a single bacterial strain.

Humanized microbiota A term used to describe human microbiota colonizing the mouse gastrointestinal tract.

Metabolome The complete profile of small-molecule metabolites, such as amino acids, nucleotides, antioxidants, organic acids, vitamins, hormones, drugs and food components that are found within a cell, tissue, organ or entire organism.

in the microbiota and specific phenotypes. This could be considered to be a microbiota Koch’s postulate, in which bacterial species are isolated, identified and re‑introduced into mice that have a defined microbiota (that is, mice that have altered Schaedler flora (ASF), monocolonized mice, mice that have humanized microbiota or germ-free mice) to re‑capitulate any observed pheno types. One caveat to this approach is that many bacterial species in the gut are currently deemed ‘unculturable’; however, we anticipate that a ‘renaissance’ in basic microbiology, coupled with metagenomics, will facilitate the culture of many new bacterial species from this site78,105, reveal their metabolic requirements and revolutionize the way we study host–pathogen–microbiota interactions. Other aspects of C. rodentium–micobiota interactions have received surprisingly little, if any, attention. So far, the effect of C. rodentium infection on the mouse intestinal metabolome and the metabolome of the intestinal microbiota remains unexplored. SCFAs are by‑products

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Acknowledgements

The authors are supported by the Canadian Institutes for Health (CIHR) operating grants (to K.M.K. and B.B.F.), US National Institutes of Health (NIH) grant AI083713 (to K.A.F.), NIH grant AI085761 (to K.M.K.), the Wellcome Trust and the UK Medical Research Council (MRC) (to G.F.).

Competing interests statement

The authors declare no competing interests.



Dynamic changes in mucus thickness and ion secretion during Citrobacter rodentium infection and clearance.

Dietary Chitosan Supplementation Increases Microbial Diversity and Attenuates the Severity of Citrobacter rodentium Infection in Mice.

Influence of NleH effector expression, host genetics, and inflammation on Citrobacter rodentium colonization of mice.

Attenuation of intestinal inflammation in interleukin-10-deficient mice infected with Citrobacter rodentium.

DOCK2 confers immunity and intestinal colonization resistance to Citrobacter rodentium infection.

IL-6-dependent manner.

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Fermented dairy products modulate Citrobacter rodentium-induced colonic hyperplasia.

Type 3 Muscarinic Receptors Contribute to Clearance of Citrobacter rodentium.

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Selective enrichment of commensal gut bacteria protects against Citrobacter rodentium-induced colitis.