Dopamine receptor D2 confers colonization resistance via microbial metabolites.

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 pathogens1-3, 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 failure4,5 (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 cells6,7, 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 infection8,9. 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.

Citrobacter amalonaticus Inhibits the Growth of Citrobacter rodentium in the Gut Lumen.

The gut microbiota plays a crucial role in susceptibility to enteric pathogens, including Citrobacter rodentium, a model extracellular mouse pathogen that colonizes the colonic mucosa. C. rodentium infection outcomes vary between mouse strains, with C57BL/6 and C3H/HeN mice clearing and succumbing to the infection, respectively. Kanamycin (Kan) treatment at the peak of C57BL/6 mouse infection with Kan-resistant C. rodentium resulted in relocalization of the pathogen from the colonic mucosa and cecum to solely the cecal luminal contents; cessation of the Kan treatment resulted in rapid clearance of the pathogen. We now show that in C3H/HeN mice, following Kan-induced displacement of C. rodentium to the cecum, the pathogen stably colonizes the cecal lumens of 65% of the mice in the absence of continued antibiotic treatment, a phenomenon that we term antibiotic-induced bacterial commensalization (AIBC). AIBC C. rodentium was well tolerated by the host, which showed few signs of inflammation; passaged AIBC C. rodentium robustly infected naive C3H/HeN mice, suggesting that the AIBC state is transient and did not select for genetically avirulent C. rodentium mutants. Following withdrawal of antibiotic treatment, 35% of C3H/HeN mice were able to prevent C. rodentium commensalization in the gut lumen. These mice presented a bloom of a commensal species, Citrobacter amalonaticus, which inhibited the growth of C. rodentium in vitro in a contact-dependent manner and the luminal growth of AIBC C. rodentium in vivo. Overall, our data suggest that commensal species can confer colonization resistance to closely related pathogenic species. IMPORTANCE Gut bacterial infections involve three-way interactions between virulence factors, the host immune responses, and the microbiome. While the microbiome erects colonization resistance barriers, pathogens employ virulence factors to overcome them. Treating mice infected with kanamycin-resistant Citrobacter rodentium with kanamycin caused displacement of the pathogen from the colonic mucosa to the cecal lumen. Following withdrawal of the kanamycin treatment, 65% of the mice were persistently colonized by C. rodentium, which seemed to downregulate virulence factor expression. In this model of luminal gut colonization, 35% of mice were refractory to stable C. rodentium colonization, suggesting that their microbiotas were able to confer colonization resistance. We identify a commensal bacterium of the Citrobacter genus, C. amalonaticus, which inhibits C. rodentium growth in vitro and in vivo. These results show that the line separating commensal and pathogenic lifestyles is thin and multifactorial and that commensals may play a major role in combating enteric infection.

Deprivation of dietary fiber in specific-pathogen-free mice promotes susceptibility to the intestinal mucosal pathogen Citrobacter rodentium.

The change of dietary habits in Western societies, including reduced consumption of fiber, is linked to alterations in gut microbial ecology. Nevertheless, mechanistic connections between diet-induced microbiota changes that affect colonization resistance and enteric pathogen susceptibility are still emerging. We sought to investigate how a diet devoid of soluble plant fibers impacts the structure and function of a conventional gut microbiota in specific-pathogen-free (SPF) mice and how such changes alter susceptibility to a rodent enteric pathogen. We show that absence of dietary fiber intake leads to shifts in the abundances of specific taxa, microbiome-mediated erosion of the colonic mucus barrier, a reduction of intestinal barrier-promoting short-chain fatty acids, and increases in markers of mucosal barrier integrity disruption. Importantly, our results highlight that these low-fiber diet-induced changes in the gut microbial ecology collectively contribute to a lethal colitis by the mucosal pathogen Citrobacter rodentium, which is used as a mouse model for enteropathogenic and enterohemorrhagic Escherichia coli (EPEC and EHEC, respectively). Our study indicates that modern, low-fiber Western-style diets might make individuals more prone to infection by enteric pathogens via the disruption of mucosal barrier integrity by diet-driven changes in the gut microbiota, illustrating possible implications for EPEC and EHEC infections.

Uremia Coupled with Mucosal Damage Predisposes Mice with Kidney Disease to Systemic Infection by Commensal Candida albicans.

Infections are the second major cause of mortality in patients with kidney disease and accompanying uremia. Both vascular access and non-access-related infections contribute equally to the infection-related deaths in patients with kidney disease. Dialysis is the most common cause of systemic infection by Candida albicans in these patients. C albicans also reside in the gastrointestinal tract as a commensal fungus. However, the contribution of gut-derived C albicans in non-access-related infections in kidney disease is unknown. Using a mouse model of kidney disease, we demonstrate that uremic animals showed increased gut barrier permeability, impaired mucosal defense, and dysbiosis. The disturbance in gut homeostasis is sufficient to drive the translocation of microbiota and intestinal pathogen Citrobacter rodentium to extraintestinal sites but not C albicans Interestingly, a majority of uremic animals showed fungal translocation only when the gut barrier integrity is disrupted. Our data demonstrate that uremia coupled with gut mucosal damage may aid in the translocation of C. albicans and cause systemic infection in kidney disease. Because most of the individuals with kidney disease suffer from some form of gut mucosal damage, these results have important implications in the risk stratification and control of non-access-related opportunistic fungal infections in these patients.

Anaerobic Respiration of NOX1-Derived Hydrogen Peroxide Licenses Bacterial Growth at the Colonic Surface.

The colonic microbiota exhibits cross-sectional heterogeneity, but the mechanisms that govern its spatial organization remain incompletely understood. Here we used Citrobacter rodentium, a pathogen that colonizes the colonic surface, to identify microbial traits that license growth and survival in this spatial niche. Previous work showed that during colonic crypt hyperplasia, type III secretion system (T3SS)-mediated intimate epithelial attachment provides C. rodentium with oxygen for aerobic respiration. However, we find that prior to the development of colonic crypt hyperplasia, T3SS-mediated intimate attachment is not required for aerobic respiration but for hydrogen peroxide (H2O2) respiration using cytochrome c peroxidase (Ccp). The epithelial NADPH oxidase NOX1 is the primary source of luminal H2O2 early after C. rodentium infection and is required for Ccp-dependent growth. Our results suggest that NOX1-derived H2O2 is a resource that governs bacterial growth and survival in close proximity to the mucosal surface during gut homeostasis.

STING controls intestinal homeostasis through promoting antimicrobial peptide expression in epithelial cells.

Stimulator of interferon genes (STING) has been shown to play a critical role in orchestrating immune responses to various pathogens through sensing cyclic dinucleotides. However, how STING regulates intestinal homeostasis is still not completely understood. In this study, we found that STING-/- mice were more susceptible to enteric infection with Citrobacter rodentium compared to wild-type (WT) mice evidenced by more severe intestinal inflammation and impaired bacterial clearance. STING-/- mice demonstrated lower expression of REG3γ but not ß-defensins and Cramp in IECs. Consistently, STING-/- IECs showed reduced capacity to inhibit bacterial growth. STING agonists, both 10-carboxymethyl-9-acridanone (CMA) and 5,6-dimethylxanthenone-4-acetic acid (DMXAA), promoted REG3γ expression IECs. Furthermore, STING agonists promoted WT but not REG3γ-deficient IEC bacterial killing. Mechanistically, STING agonists activated STAT3 and promoted glycolysis in IECs. Inhibition of STAT3 pathway and glycolysis suppressed STING-induced REG3γ production in IECs, and abrogated STING-mediated IEC killing of C. rodentium. Additionally, treatment with the STING ligand, 2,3-cGAMP, inhibited C. rodentium-induced colitis in vivo. Overall, STING promotes IEC REG3γ expression to inhibit enteric infection and intestinal inflammation, thus, maintaining the intestinal homeostasis.

Variations in microbiota composition of laboratory mice influence Citrobacter rodentium infection via variable short-chain fatty acid production.

The composition of the intestinal microbiota influences the outcome of enteric infections in human and mice. However, the role of specific members and their metabolites contributing to disease severity is largely unknown. Using isogenic mouse lines harboring distinct microbiota communities, we observed highly variable disease kinetics of enteric Citrobacter rodentium colonization after infection. Transfer of communities from susceptible and resistant mice into germ-free mice verified that the varying susceptibilities are determined by microbiota composition. The strongest differences in colonization were observed in the cecum and could be maintained in vitro by coculturing cecal bacteria with C. rodentium. Cohousing of animals as well as the transfer of cultivable bacteria from resistant to susceptible mice led to variable outcomes in the recipient mice. Microbiome analysis revealed that a higher abundance of butyrate-producing bacteria was associated with the resistant phenotype. Quantification of short-chain fatty acid (SCFA) levels before and after infection revealed increased concentrations of acetate, butyrate and propionate in mice with delayed colonization. Addition of physiological concentrations of butyrate, but not of acetate and/or propionate strongly impaired growth of C. rodentium in vitro. In vivo supplementation of susceptible, antibiotic-treated and germ-free mice with butyrate led to the same level of protection, notably only when cecal butyrate concentration reached a concentration higher than 50 nmol/mg indicating a critical threshold for protection. In the recent years, commensal-derived primary and secondary bacterial metabolites emerged as potent modulators of hosts susceptibility to infection. Our results provide evidence that variations in SCFA production in mice fed fibre-rich chow-based diets modulate susceptibility to colonization with Enterobacteriaceae not only in antibiotic-disturbed ecosystems but even in undisturbed microbial communities. These findings emphasise the need for microbiota normalization across laboratory mouse lines for infection experiments with the model-pathogen C. rodentium independent of investigations of diet and antibiotic usage.

Lactobacillus reuteri maintains intestinal epithelial regeneration and repairs damaged intestinal mucosa.

Little is known about the regulatory effect of microbiota on the proliferation and regeneration of ISCs. Here, we found that L. reuteri stimulated the proliferation of intestinal epithelia by increasing the expression of R-spondins and thus activating the Wnt/ß-catenin pathway. The proliferation-stimulating effect of Lactobacillus on repair is further enhanced under TNF -induced intestinal mucosal damage, and the number of Lgr5+ cells is maintained. Moreover, compared to the effects of C. rodentium on the induction of intestinal inflammation and crypt hyperplasia in mice, L. reuteri protected the intestinal mucosal barrier integrity by moderately modulating the Wnt/ß-catenin signaling pathway to avoid overactivation. L. reuteri had the ability to maintain the number of Lgr5+ cells and stimulate intestinal epithelial proliferation to repair epithelial damage and reduce proinflammatory cytokine secretion in the intestine and the LPS concentration in serum. Moreover, activation of the Wnt/ß-catenin pathway also induced differentiation toward Paneth cells and increased antimicrobial peptide expression to inhibit C. rodentium colonization. The protective effect of Lactobacillus against C. rodentium infection disappeared upon application of the Wnt antagonist Wnt-C59 in both mice and intestinal organoids. This study demonstrates that Lactobacillus is effective at maintaining intestinal epithelial regeneration and homeostasis as well as at repairing intestinal damage after pathological injury and is thus a promising alternative therapeutic method for intestinal inflammation.

Dietary L-serine confers a competitive fitness advantage to Enterobacteriaceae in the inflamed gut.

Metabolic reprogramming is associated with the adaptation of host cells to the disease environment, such as inflammation and cancer. However, little is known about microbial metabolic reprogramming or the role it plays in regulating the fitness of commensal and pathogenic bacteria in the gut. Here, we report that intestinal inflammation reprograms the metabolic pathways of Enterobacteriaceae, such as Escherichia coli LF82, in the gut to adapt to the inflammatory environment. We found that E. coli LF82 shifts its metabolism to catabolize L-serine in the inflamed gut in order to maximize its growth potential. However, L-serine catabolism has a minimal effect on its fitness in the healthy gut. In fact, the absence of genes involved in L-serine utilization reduces the competitive fitness of E. coli LF82 and Citrobacter rodentium only during inflammation. The concentration of luminal L-serine is largely dependent on dietary intake. Accordingly, withholding amino acids from the diet markedly reduces their availability in the gut lumen. Hence, inflammation-induced blooms of E. coli LF82 are significantly blunted when amino acids-particularly L-serine-are removed from the diet. Thus, the ability to catabolize L-serine increases bacterial fitness and provides Enterobacteriaceae with a growth advantage against competitors in the inflamed gut.

Citrobacter rodentium-host-microbiota interactions: immunity, bioenergetics and metabolism.

Citrobacter rodentium is an extracellular enteric mouse-specific pathogen used to model infections with human pathogenic Escherichia coli and inflammatory bowel disease. C. rodentium injects type III secretion system effectors into intestinal epithelial cells (IECs) to target inflammatory, metabolic and cell survival pathways and establish infection. While the host responds to infection by activating innate and adaptive immune signalling, required for clearance, the IECs respond by rapidly shifting bioenergetics to aerobic glycolysis, which leads to oxygenation of the epithelium, an instant expansion of mucosal-associated commensal Enterobacteriaceae and a decline of obligate anaerobes. Moreover, infected IECs reprogramme intracellular metabolic pathways, characterized by simultaneous activation of cholesterol biogenesis, import and efflux, leading to increased serum and faecal cholesterol levels. In this Review we summarize recent advances highlighting the intimate relationship between C. rodentium pathogenesis, metabolism and the gut microbiota.

Intestinal Epithelial Cells and the Microbiome Undergo Swift Reprogramming at the Inception of Colonic Citrobacter rodentium Infection.

We used the mouse attaching and effacing (A/E) pathogen Citrobacter rodentium, which models the human A/E pathogens enteropathogenic Escherichia coli and enterohemorrhagic E. coli (EPEC and EHEC), to temporally resolve intestinal epithelial cell (IEC) responses and changes to the microbiome during in vivo infection. We found the host to be unresponsive during the first 3 days postinfection (DPI), when C. rodentium resides in the caecum. In contrast, at 4 DPI, the day of colonic colonization, despite only sporadic adhesion to the apex of the crypt, we observed robust upregulation of cell cycle and DNA repair processes, which were associated with expansion of the crypt Ki67-positive replicative zone, and downregulation of multiple metabolic processes (including the tricarboxylic acid [TCA] cycle and oxidative phosphorylation). Moreover, we observed dramatic depletion of goblet and deep crypt secretory cells and an atypical regulation of cholesterol homeostasis in IECs during early infection, with simultaneous upregulation of cholesterol biogenesis (e.g., 3-hydroxy-3-methylglutaryl-coenzyme A reductase [Hmgcr]), import (e.g., low-density lipoprotein receptor [Ldlr]), and efflux (e.g., AbcA1). We also detected interleukin 22 (IL-22) responses in IECs (e.g., Reg3γ) on the day of colonic colonization, which occurred concomitantly with a bloom of commensal Enterobacteriaceae on the mucosal surface. These results unravel a new paradigm in host-pathogen-microbiome interactions, showing for the first time that sensing a small number of pathogenic bacteria triggers swift intrinsic changes to the IEC composition and function, in tandem with significant changes to the mucosa-associated microbiome, which parallel innate immune responses.IMPORTANCE The mouse pathogen C. rodentium is a widely used model for colonic infection and has been a major tool in fundamental discoveries in the fields of bacterial pathogenesis and mucosal immunology. Despite extensive studies probing acute C. rodentium infection, our understanding of the early stages preceding the infection climax remains relatively undetailed. To this end, we apply a multiomics approach to resolve temporal changes to the host and microbiome during early infection. Unexpectedly, we found immediate and dramatic responses occurring on the day of colonic infection, both in the host intestinal epithelial cells and in the microbiome. Our study suggests changes in cholesterol and carbon metabolism in epithelial cells are instantly induced upon pathogen detection in the colon, corresponding with a shift to primarily facultative anaerobes constituting the microbiome. This study contributes to our knowledge of disease pathogenesis and mechanisms of barrier regulation, which is required for development of novel therapeutics targeting the intestinal epithelium.

Dietary Arginine Regulates Severity of Experimental Colitis and Affects the Colonic Microbiome.

There is great interest in safe and effective alternative therapies that could benefit patients with inflammatory bowel diseases (IBD). L-arginine (Arg) is a semi-essential amino acid with a variety of physiological effects. In this context, our aim was to investigate the role of dietary Arg in experimental colitis. We used two models of colitis in C57BL/6 mice, the dextran sulfate sodium (DSS) model of injury and repair, and Citrobacter rodentium infection. Animals were given diets containing (1) no Arg (Arg0), 6.4 g/kg (ArgNL), or 24.6 g/kg Arg (ArgHIGH); or (2) the amino acids downstream of Arg: 28 g/kg L-ornithine (OrnHIGH) or 72 g/kg L-proline (ProHIGH). Mice with DSS colitis receiving the ArgHIGH diet had increased levels of Arg, Orn, and Pro in the colon and improved body weight loss, colon length shortening, and histological injury compared to ArgNL and Arg0 diets. Histology was improved in the ArgNL vs. Arg0 group. OrnHIGH or ProHIGH diets did not provide protection. Reduction in colitis with ArgHIGH diet also occurred in C. rodentium-infected mice. Diversity of the intestinal microbiota was significantly enhanced in mice on the ArgHIGH diet compared to the ArgNL or Arg0 diets, with increased abundance of Bacteroidetes and decreased Verrucomicrobia. In conclusion, dietary supplementation of Arg is protective in colitis models. This may occur by restoring overall microbial diversity and Bacteroidetes prevalence. Our data provide a rationale for Arg as an adjunctive therapy in IBD.

Characterization of the Citrobacter rodentium Cpx regulon and its role in host infection.

Envelope-localized proteins, such as adhesins and secretion systems, play critical roles in host infection by Gram-negative pathogens. As such, their folding is monitored by envelope stress response systems. Previous studies demonstrated that the Cpx envelope stress response is required for virulence of Citrobacter rodentium, a murine pathogen used to model infections by the human pathogens enteropathogenic and enterohemorrhagic Escherichia coli; however, the mechanisms by which the Cpx response promotes host infection were previously unknown. Here, we characterized the C. rodentium Cpx regulon in order to identify genes required for host infection. Using transcriptomic and proteomic approaches, we found that the Cpx response upregulates envelope-localized protein folding and degrading factors but downregulates pilus genes and type III secretion effectors. Mouse infections with C. rodentium strains lacking individual Cpx-regulated genes showed that the chaperone/protease DegP and the disulfide bond oxidoreductase DsbA were essential for infection, but Cpx regulation of these genes did not fully account for attenuation of C. rodentium ΔcpxRA. Both deletion of dsbA and treatment with the reducing agent dithiothreitol activated the C. rodentium Cpx response, suggesting that it may sense disruption of disulfide bonding. Our results highlight the importance of envelope protein folding in host infection by Gram-negative pathogens.

The Virulence Effect of CpxRA in Citrobacter rodentium Is Independent of the Auxiliary Proteins NlpE and CpxP.

Citrobacter rodentium is a murine pathogen used to model the intestinal infection caused by Enteropathogenic and Enterohemorrhagic Escherichia coli (EPEC and EHEC), two diarrheal pathogens responsible for morbidity and mortality in developing and developed countries, respectively. During infection, these bacteria must sense and adapt to the gut environment of the host. In order to adapt to changing environmental cues and modulate expression of specific genes, bacteria can use two-component signal transduction systems (TCS). We have shown that the deletion of the Cpx TCS in C. rodentium leads to a marked attenuation in virulence in C3H/HeJ mice. In E. coli, the Cpx TCS is reportedly activated in response to signals from the outer-membrane lipoprotein NlpE. We therefore investigated the role of NlpE in C. rodentium virulence. We also assessed the role of the reported negative regulator of CpxRA, CpxP. We found that as opposed to the ΔcpxRA strain, neither the ΔnlpE, ΔcpxP nor the ΔnlpEΔcpxP strains were significantly attenuated, and had similar in vivo localization to wild-type C. rodentium. The in vitro adherence of the Cpx auxiliary protein mutants, ΔnlpE, ΔcpxP, ΔnlpEΔcpxP, was comparable to wild-type C. rodentium, whereas the ΔcpxRA strain showed significantly decreased adherence. To further elucidate the mechanisms behind the contrasting virulence phenotypes, we performed microarrays in order to define the regulon of the Cpx TCS. We detected 393 genes differentially regulated in the ΔcpxRA strain. The gene expression profile of the ΔnlpE strain is strikingly different than the profile of ΔcpxRA with regards to the genes activated by CpxRA. Further, there is no clear inverse correlation in the expression pattern of the ΔcpxP strain in comparison to ΔcpxRA. Taken together, these data suggest that in these conditions, CpxRA activates gene expression in a largely NlpE- and CpxP-independent manner. Compared to wildtype, 161 genes were downregulated in the ΔcpxRA strain, while being upregulated or unchanged in the Cpx auxiliary protein deletion strains. This group of genes, which we hypothesize may contribute to the loss of virulence of ΔcpxRA, includes T6SS components, ompF, the regulator for colanic acid synthesis, and several genes involved in maltose metabolism.

Autophagy proteins suppress protective type I interferon signalling in response to the murine gut microbiota.

As a conserved pathway that lies at the intersection between host defence and cellular homeostasis, autophagy serves as a rheostat for immune reactions. In particular, autophagy suppresses excess type I interferon (IFN-I) production in response to viral nucleic acids. It is unknown how this function of autophagy relates to the intestinal barrier where host-microbe interactions are pervasive and perpetual. Here, we demonstrate that mice deficient in autophagy proteins are protected from the intestinal bacterial pathogen Citrobacter rodentium in a manner dependent on IFN-I signalling and nucleic acid sensing pathways. Enhanced IFN-stimulated gene expression in intestinal tissue of autophagy-deficient mice in the absence of infection was mediated by the gut microbiota. Additionally, monocytes infiltrating into the autophagy-deficient intestinal microenvironment displayed an enhanced inflammatory profile and were necessary for protection against C. rodentium. Finally, we demonstrate that the microbiota-dependent IFN-I production that occurs in the autophagy-deficient host also protects against chemical injury of the intestine. Thus, autophagy proteins prevent a spontaneous IFN-I response to microbiota that is beneficial in the presence of infectious and non-infectious intestinal hazards. These results identify a role for autophagy proteins in controlling the magnitude of IFN-I signalling at the intestinal barrier.

Linking Pathogen Virulence, Host Immunity and The Microbiota at the Intestinal Barrier.

The mechanisms that allow enteric pathogens to colonize the intestine and host immunity as well as the indigenous microbiota to inhibit pathogen colonization remain poorly understood. Our laboratory is using Citrobacter rodentium, a mouse pathogen that models human infections by enteropathogenic E. coli, to understand the mechanisms that regulate the colonization and clearance of the pathogen in the gut. These studies have revealed how the pathogen colonizes and replicates successfully early during infection and how host immunity and the indigenous microbiota cooperate to eradicate the pathogen in the later stage of the infection. The impairment of the immune system to control the barrier function of the intestine leads to pathogen invasion and the induction of a second layer of host protective immunity to limit the systemic spread of the pathogen.(Presented at the 1931st Meeting, January 17, 2017).

Citrobacter rodentium mouse model of bacterial infection.

Infection of mice with Citrobacter rodentium is a robust model to study bacterial pathogenesis, mucosal immunology, the health benefits of probiotics and the role of the microbiota during infection. C. rodentium was first isolated by Barthold from an outbreak of mouse diarrhea in Yale University in 1972 and was 'rediscovered' by Falkow and Schauer in 1993. Since then the use of the model has proliferated, and it is now the gold standard for studying virulence of the closely related human pathogens enteropathogenic and enterohemorrhagic Escherichia coli (EPEC and EHEC, respectively). Here we provide a detailed protocol for various applications of the model, including bacterial growth, site-directed mutagenesis, mouse inoculation (from cultured cells and after cohabitation), monitoring of bacterial colonization, tissue extraction and analysis, immune responses, probiotic treatment and microbiota analysis. The main protocol, from mouse infection to clearance and analysis of tissues and host responses, takes ∼5 weeks to complete.

Novel antimicrobial peptide prevents C. rodentium infection in C57BL/6 mice by enhancing acid-induced pathogen killing.

Citrobacter rodentium is a Gram-negative, murine-specific enteric pathogen that infects epithelial cells in the colon. It is closely related to the clinically relevant human pathogen, enterohemorrhagic Escherichia coli (EHEC), a leading cause of haemorrhagic colitis and haemolytic uremic syndrome. We have previously reported that a novel antimicrobial peptide, wrwycr, compromises bacterial DNA repair and significantly reduces the survival of acid-stressed EHEC, suggesting an antimicrobial strategy for targeting the survival of ingested EHEC. This study examines the impact of peptide pretreatment on survival of the closely related murine pathogen, C. rodentium, before and after acid stress, using both in vitro and in vivo investigations. Peptide pretreatment of C. rodentium significantly and dramatically increases acid-stress-induced killing in a peptide-dose-dependent and time-dependent manner. Reduction in survival rates after brief pretreatment with peptide (25-65 µM) followed by 1 h at pH 3.5 ranges from 6 to 8 log fold relative to untreated C. rodentium, with no detectable bacteria after 65 µM peptide-acid treatment. Using a C57BL/6 mouse model of infection, peptide pretreatment of C. rodentium with wrwycr prior to orogastric gavage eliminates evidence of infection based on C. rodentium colonization levels, faecal scores, colonic histology, faecal microbiome and visual observation of overall animal health. These findings provide compelling evidence for the role of the peptide wrwycr as a potential strategy to control the growth and colonization of enteric pathogens.

IL-36α expression is elevated in ulcerative colitis and promotes colonic inflammation.

A role for the IL-36 family of cytokines has been identified in the pathogenesis of psoriasis. Although significant mechanistic overlap can exist between psoriasis and inflammatory bowel disease (IBD), to date there have been no reports investigating the IL-36 family in gastrointestinal inflammation. Here we demonstrate that expression levels of IL-36α are specifically elevated in the colonic mucosa of ulcerative colitis patients. This elevated expression is mirrored in the inflamed colonic mucosa of mice, wherein IL-36 receptor deficiency confirmed this pathway as a mediator of mucosal inflammation. Il36r-/- mice exhibited reduced disease severity in an acute DSS-induced model of colitis in association with decreased innate inflammatory cell infiltration to the colon lamina propria. Consistent with these data, infection with the enteropathogenic bacteria Citrobacter rodentium, resulted in reduced innate inflammatory cell recruitment and increased bacterial colonization in the colons of il36r-/- mice. Il36r-/- mice also exhibited altered T helper cell responses in this model, with enhanced Th17 and reduced Th1 responses, demonstrating that IL-36R signaling also regulates intestinal mucosal T-cell responses. These data identify a novel role for IL-36 signaling in colonic inflammation and indicate that the IL-36R pathway may represent a novel target for therapeutic intervention in IBD.

Humoral Immunity in the Gut Selectively Targets Phenotypically Virulent Attaching-and-Effacing Bacteria for Intraluminal Elimination.

Virulence factors expressed by enteric bacteria are pivotal for pathogen colonization and induction of intestinal disease, but the mechanisms by which host immunity regulates pathogen virulence are largely unknown. Here we show that specific antibody responses are required for downregulation of virulence gene expression in Citrobacter rodentium, an enteric pathogen that models human infections with attaching-and-effacing bacteria. In the absence of antibodies against the pathogen, phenotypically virulent C. rodentium, accumulated and infected the epithelium and subsequently invaded the lamina propia, causing host lethality. IgG induced after infection recognized virulence factors and bound virulent bacteria within the intestinal lumen, leading to their engulfment by neutrophils, while phenotypically avirulent pathogens remained in the intestinal lumen and were eventually outcompeted by the microbiota. Thus, the interplay of the innate and adaptive immune system selectively targets virulent C. rodentium in the intestinal lumen to promote pathogen eradication and host survival.