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The virulence mechanisms that allow pathogens to colonize the intestine remain unclear. Here, we show that germ-free animals are unable to eradicate Citrobacter rodentium, a model for human infections with attaching and effacing bacteria. Early in infection, virulence genes were expressed and required for pathogen growth in conventionally raised mice but not germ-free mice. Virulence gene expression was down-regulated during the late phase of infection, which led to relocation of the pathogen to the intestinal lumen where it was outcompeted by commensals. The ability of commensals to outcompete C. rodentium was determined, at least in part, by the capacity of the pathogen and commensals to grow on structurally similar carbohydrates. Thus, pathogen colonization is controlled by bacterial virulence and through competition with metabolically related commensals.

Key Points The mouse pathogen Citrobacter rodentium is a useful model to investigate important human intestinal diseases, including enteropathogenic Escherichia coli (EPEC) and enterohaemorrhagic E. coli (EHEC) infections, Crohn's disease, ulcerative colitis and colon tumorigenesis. Whole-genome sequencing of multiple pathogenic attaching and effacing (A/E) bacteria has led to the identification of many genes that are involved in pathogenesis, including gene families that encode effector proteins of the type III secretion system (T3SS). The functions of many putative virulence genes have been evaluated in the C. rodentium model, which has improved our understanding of pathogenesis and the corresponding host responses. C. rodentium elicits robust inflammasome-dependent responses in a caspase 1- and caspase 11-dependent manner. Type I interferon signalling is a key factor that regulates inflammasome activation in C. rodentium infection. The intestinal microbiota is crucial for coordinating mucosal immune responses to C. rodentium infection, including the development of IgA + plasma cells, group 3 innate lymphoid cells (ILC3s; also known as inducible T helper (iT H ) cells), T H 17 cells and T H 22 cells. Defined dietary components, such as vitamin D, vitamin E, selenium, ligands from cruciferous vegetables and polyunsaturated fatty acids (PUFAs), as well as the intestinal microbiota, directly modify mucosal immune responses and epithelial barrier function in response to C. rodentium infection. Future research using the C. rodentium model will focus on quantitative proteomics, metabolomics and four-dimensional (4D) imaging studies to unravel pathogen–host–microbiota interactions in unprecedented detail. The mouse pathogen Citrobacter rodentium has long been used as a model for investigating the pathogenesis of the important enteric human pathogens, enterohaemorrhagic Escherichia coli (EHEC) and enteropathogenic E. coli (EPEC). In this Review, Frankel and colleagues discuss the infection cycle of this pathogen, the mucosal immune response that is elicited and the role of the gut microbiota in preventing colonization. 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.

Pathogens sense and respond to several small molecules within the GI tract to modulate expression of their virulence repertoire. Indole is a signaling molecule produced by the gut microbiota. Here we show that indole concentrations are higher in the lumen, where the microbiota is present, than in the intestinal tissue. The enteric pathogens EHEC and C. rodentium sense indole to downregulate expression of their virulence genes, as a read-out of the luminal compartment. We also identified the bacterial membrane-bound HK CpxA as an indole sensor. This regulation ensures that EHEC and C. rodentium express their virulence genes only at the epithelial lining, which is the niche they colonize. Microbial establishment within the gastrointestinal (GI) tract requires surveillance of the gut biogeography. The gut microbiota coordinates behaviors by sensing host- or microbiota-derived signals. Here we show for the first time that microbiota-derived indole is highly prevalent in the lumen compared to the intestinal tissue. This difference in indole concentration plays a key role in modulating virulence gene expression of the enteric pathogens enterohemorrhagic Escherichia coli (EHEC) and Citrobacter rodentium . Indole decreases expression of genes within the locus of enterocyte effacement (LEE) pathogenicity island, which is essential for these pathogens to form attaching and effacing (AE) lesions on enterocytes. We synthetically altered the concentration of indole in the GI tracts of mice by employing mice treated with antibiotics to deplete the microbiota and reconstituted with indole-producing commensal Bacteroides thetaiotaomicron ( B. theta ) or a B. theta Δ tnaA mutant (does not produce indole) or by engineering an indole-producing C. rodentium strain. This allowed us to assess the role of self-produced versus microbiota-produced indole, and the results show that decreased indole concentrations promote bacterial pathogenesis, while increased levels of indole decrease bacterial virulence gene expression. Moreover, we identified the bacterial membrane-bound histidine sensor kinase (HK) CpxA as an indole sensor. Enteric pathogens sense a gradient of indole concentrations in the gut to probe different niches and successfully establish an infection. IMPORTANCE Pathogens sense and respond to several small molecules within the GI tract to modulate expression of their virulence repertoire. Indole is a signaling molecule produced by the gut microbiota. Here we show that indole concentrations are higher in the lumen, where the microbiota is present, than in the intestinal tissue. The enteric pathogens EHEC and C. rodentium sense indole to downregulate expression of their virulence genes, as a read-out of the luminal compartment. We also identified the bacterial membrane-bound HK CpxA as an indole sensor. This regulation ensures that EHEC and C. rodentium express their virulence genes only at the epithelial lining, which is the niche they colonize.

Parkinson’s disease is a neurodegenerative disorder with motor symptoms linked to the loss of dopaminergic neurons in the substantia nigra compacta. Although the mechanisms that trigger the loss of dopaminergic neurons are unclear, mitochondrial dysfunction and inflammation are thought to have key roles 1 , 2 . An early-onset form of Parkinson’s disease is associated with mutations in the PINK1 kinase and PRKN ubiquitin ligase genes 3 . PINK1 and Parkin (encoded by PRKN ) are involved in the clearance of damaged mitochondria in cultured cells 4 , but recent evidence obtained using knockout and knockin mouse models have led to contradictory results regarding the contributions of PINK1 and Parkin to mitophagy in vivo 5 – 8 . It has previously been shown that PINK1 and Parkin have a key role in adaptive immunity by repressing presentation of mitochondrial antigens 9 , which suggests that autoimmune mechanisms participate in the aetiology of Parkinson’s disease. Here we show that intestinal infection with Gram-negative bacteria in Pink1 −/− mice engages mitochondrial antigen presentation and autoimmune mechanisms that elicit the establishment of cytotoxic mitochondria-specific CD8 + T cells in the periphery and in the brain. Notably, these mice show a sharp decrease in the density of dopaminergic axonal varicosities in the striatum and are affected by motor impairment that is reversed after treatment with l -DOPA. These data support the idea that PINK1 is a repressor of the immune system, and provide a pathophysiological model in which intestinal infection acts as a triggering event in Parkinson’s disease, which highlights the relevance of the gut–brain axis in the disease 10 . In mice lacking PINK1, bacterial infection in the intestine results in mitochondrial antigen presentation and generation of CD8 + T cells, and infected mice develop motor impairments, suggesting that PINK1 suppresses autoimmunity.

The gut microbiome has major roles in modulating host physiology. One such function is colonization resistance, or the ability of the microbial collective to protect the host against enteric pathogens 1 – 3 , including enterohaemorrhagic Escherichia coli (EHEC) serotype O157:H7, an attaching and effacing (AE) food-borne pathogen that causes severe gastroenteritis, enterocolitis, bloody diarrhea and acute renal failure 4 , 5 (haemolytic uremic syndrome). Although gut microorganisms can provide colonization resistance by outcompeting some pathogens or modulating host defence provided by the gut barrier and intestinal immune cells 6 , 7 , this phenomenon remains poorly understood. Here, we show that activation of the neurotransmitter receptor dopamine receptor D2 (DRD2) in the intestinal epithelium by gut microbial metabolites produced upon dietary supplementation with the essential amino acid l -tryptophan protects the host against Citrobacter rodentium , a mouse AE pathogen that is widely used as a model for EHEC infection 8 , 9 . We further find that DRD2 activation by these tryptophan-derived metabolites decreases expression of a host actin regulatory protein involved in C. rodentium and EHEC attachment to the gut epithelium via formation of actin pedestals. Our results reveal a noncanonical colonization resistance pathway against AE pathogens that features an unconventional role for DRD2 outside the nervous system in controlling actin cytoskeletal organization in the gut epithelium. Our findings may inspire prophylactic and therapeutic approaches targeting DRD2 with dietary or pharmacological interventions to improve gut health and treat gastrointestinal infections, which afflict millions globally. In a mouse model of enteric pathogen infection, tryptophan metabolites protect against infection via activation of dopamine receptor D2 and regulation of actin cytoskeletal organization in intestinal epithelial cells.

Citrobacter rodentium uses a type III secretion system (T3SS) to induce colonie crypt hyperplasia in mice, thereby gaining an edge during its competition with the gut microbiota through an unknown mechanism. Here, we show that by triggering colonie crypt hyperplasia, the C. rodentium T3SS induced an excessive expansion of undifferentiated Ki67-positive epithelial cells, which increased oxygénation of the mucosal surface and drove an aerobic C. rodentium expansion in the colon. Treatment of mice with the -secretase inhibitor dibenzazepine to diminish Notch-driven colonie crypt hyperplasia curtailed the fitness advantage conferred by aerobic respiration during C. rodentium infection. We conclude that C. rodentium uses its T3SS to induce histopathological lesions that generate an intestinal microenvironment in which growth of the pathogen is fueled by aerobic respiration.

Enteropathogenic and enterohemorrhagic are diarrheagenic bacterial human pathogens that cause severe gastroenteritis. These enteric pathotypes, together with the mouse pathogen , belong to the family of attaching and effacing pathogens that form a distinctive histological lesion in the intestinal epithelium. The virulence of these bacteria depends on a type III secretion system (T3SS), which mediates the translocation of effector proteins from the bacterial cytosol into the infected cells. The core architecture of the T3SS consists of a multi-ring basal body embedded in the bacterial membranes, a periplasmic inner rod, a transmembrane export apparatus in the inner membrane, and cytosolic components including an ATPase complex and the C-ring. In addition, two distinct hollow appendages are assembled on the extracellular face of the basal body creating a channel for protein secretion: an approximately 23 nm needle, and a filament that extends up to 600 nm. This filamentous structure allows these pathogens to get through the host cells mucus barrier. Upon contact with the target cell, a translocation pore is assembled in the host membrane through which the effector proteins are injected. Assembly of the T3SS is strictly regulated to ensure proper timing of substrate secretion. The different type III substrates coexist in the bacterial cytoplasm, and their hierarchical secretion is determined by specialized chaperones in coordination with two molecular switches and the so-called sorting platform. In this review, we present recent advances in the understanding of the T3SS in attaching and effacing pathogens.

In this study, we identify and characterize a novel phage-inducible chromosomal island (PICI) found in commensal Escherichia coli MP1. This novel element, EcCIMP1, is induced and mobilized by the temperate helper phage vB_EcoP_Kapi1. EcCIMP1 contributes to superinfection immunity against its helper phage, impacting bacterial competition outcomes. Genetic analysis of EcCIMP1 led us to uncover a putative transcriptional repressor, which silences virulence gene expression in the murine pathogen Citrobacter rodentium. We also found a putative excisionase encoded by EcCIMP1 which paradoxically does not promote excision of EcCIMP1 but rather supports excision of the helper phage. Another putative excisionase encoded by a presumed integrative conjugative element can also support the excision of vB_EcoP_Kapi1, demonstrating crosstalk between excisionases from multiple classes of mobile genetic elements within the same cell. Although phylogenetically distant from other characterized PICIs, EcCIMP1 and EcCIMP1-like elements are prevalent in both pathogenic and commensal isolates of E. coli from around the world, underscoring the importance of characterizing these abundant genetic elements.

The colonic epithelium provides an essential barrier against the environment that is critical for protecting the body and controlling inflammation. In response to injury or gut microbes, colonic epithelial cells produce extracellular hydrogen peroxide (H₂O₂), which acts as a potent signaling molecule affecting barrier function and host defense. In humans, impaired regulation of H₂O₂ in the intestine has been associated with early-onset inflammatory bowel disease and colon cancer. Here, we show that signal transduction by H₂O₂ depends on entry into the cell by transit through aquaporin-3 (AQP3), a plasma membrane H₂O₂-conducting channel. In response to injury, AQP3-depleted colonic epithelial cells showed defective lamellipodia, focal adhesions, and repair after wounding, along with impaired H₂O₂ responses after exposure to the intestinal pathogen Citrobacter rodentium. Correspondingly, AQP3−/− mice showed impaired healing of superficial wounds in the colon and impaired mucosal innate immune responses against C. rodentium infection, manifested by reduced crypt hyperplasia, reduced epithelial expression of IL-6 and TNF-α, and impaired bacterial clearance. These results elucidate the signaling mechanism of extracellular H₂O₂ in the colonic epithelium and implicate AQP3 in innate immunity at mucosal surfaces.

Niche-adaptation of a bacterial pathogen hinges on the ability to recognize the complexity of signals from the environment and integrate that information with the regulation of genes critical for infection. Here we report the transcriptome of the attaching and effacing pathogen Citrobacter rodentium during infection of its natural murine host. Pathogen gene expression in vivo was heavily biased towards the virulence factor repertoire and was found to be co-ordinated uniquely in response to the host. Concordantly, we identified the host-specific induction of a metabolic pathway that overlapped with the regulation of virulence. The essential type 3 secretion system and an associated suite of distinct effectors were found to be modulated co-ordinately through a unique mechanism involving metabolism of microbiota-derived 1,2-propanediol, which dictated the ability to colonize the host effectively. This study provides novel insights into how host-specific metabolic adaptation acts as a cue to fine-tune virulence. Infection of mice with Citrobacter rodentium is a common model of infection with attaching-and-effacing pathogens. Here, Connolly et al. analyse the transcriptome of C . rodentium during mouse infection, showing host-induced coordinated upregulation of virulence factors and 1,2-propanediol metabolism.