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.

Overview of the Effect of Citrobacter rodentium Infection on Host Metabolism and the Microbiota.

Citrobacter rodentium is a natural enteric mouse pathogen that models human intestinal diseases, such as pathogenic E. coli infections, ulcerative colitis, and colon cancer. Upon reaching the monolayer of intestinal epithelial cells (IECs) lining the gut, a complex web of interactions between the host, the pathogen, and the microbiota ensues. A number of studies revealed surprisingly rapid changes in IEC bioenergetics upon infection, involving a switch from oxidative phosphorylation to aerobic glycolysis, leading to mucosal oxygenation and subsequent changes in microbiota composition. Microbiome studies have revealed a bloom in Enterobacteriaceae during C. rodentium infection in both resistant (i.e., C57BL/6) and susceptible (i.e., C3H/HeN) strains of mice concomitant with a depletion of butyrate-producing Clostridia. The emerging understanding that dysbiosis of cholesterol metabolism is induced by enteric infection further confirms the pivotal role immunometabolism plays in disease outcome. Inversely, the host and microbiota also impact upon the progression of infection, from the susceptibility of the distal colon to C. rodentium colonization to clearance of the pathogen, both via opsonization from the host adaptive immune system and out competition by the resident microbiota. Further complicating this compendium of interactions, C. rodentium exploits microbiota metabolites to fine-tune virulence gene expression and promote colonization. This chapter summarizes the current knowledge of the myriad of pathogen-host-microbiota interactions that occur during the progression of C. rodentium infection in mice and the broader implications of these findings on our understanding of enteric disease.

Citrobacter rodentium Infection Induces Persistent Molecular Changes and Interferon Gamma-Dependent Major Histocompatibility Complex Class II Expression in the Colonic Epithelium.

Most studies of infections at mucosal surfaces have focused on the acute phase of the disease. Consequently, little is known about the molecular processes that underpin tissue recovery and the long-term consequences postinfection. Here, we conducted temporal deep quantitative proteomic analysis of colonic intestinal epithelial cells (cIECs) from mice infected with the natural mouse pathogen Citrobacter rodentium over time points corresponding to the late steady-state phase (10 days postinfection [DPI]), the clearance phase (13 to 20 DPI), and 4 weeks after the pathogen has been cleared (48 DPI). C. rodentium, which relies on a type III secretion system to infect, is used to model infections with enteropathogenic and enterohemorrhagic Escherichia coli. We observe a strong upregulation of inflammatory signaling and nutritional immunity responses during the clearance phase of the infection. Despite morphological tissue recovery, chromogranin B (ChgB)-positive endocrine cells remained significantly below baseline levels at 48 DPI. In contrast, we observed an increased abundance of proteins involved in antigen processing and presentation 4 weeks after pathogen clearance. In particular, long-term changes were characterized by a persistent interferon gamma (IFN-γ) response and the expression of major histocompatibility complex class II (MHCII) molecules in 60% of the EpCAM+ cIECs, which were not seen in Ifnγ-/- mice. Nonetheless, both wild-type and Ifnγ-/- mice mounted similar systemic and colonic IgG responses to C. rodentium and were equally protected from rechallenge, suggesting that cIEC MHCII is not necessary for protective immunity against C. rodentium. IMPORTANCE Mucosal surfaces respond to infection by mounting an array of metabolic, inflammatory, and tissue repair responses. While these have been well studied during acute infection, less is known about tissue recovery after pathogen clearance. We employ the mouse pathogen Citrobacter rodentium, which binds colonic intestinal epithelial cells (cIECs), to investigate the long-term effects of bacterial infection on gut physiology. Using global proteomic analysis, we study cIEC temporal responses during and after the clearance phase of infection. While the overall tissue morphology recovered, cIECs showed persistent signs of infection 4 weeks after pathogen clearance. These were characterized by a strong IFN-γ signature, including the upregulation of major histocompatibility complex class II (MHCII) antigen presentation proteins, suggesting that the tissue remains on "high alert" for weeks after the acute insult is resolved. However, we demonstrate that cIEC MHCII expression, which is induced by IFN-γ, is not required for protective IgG-mediated immunity against C. rodentium; instead, it may play a role in mucosal recovery.

Citrobacter rodentium induces rapid and unique metabolic and inflammatory responses in mice suffering from severe disease.

The mouse pathogen Citrobacter rodentium is used to model infections with enterohaemorrhagic and enteropathogenic Escherichia coli (EHEC and EPEC). Pathogenesis is commonly modelled in mice developing mild disease (e.g., C57BL/6). However, little is known about host responses in mice exhibiting severe colitis (e.g., C3H/HeN), which arguably provide a more clinically relevant model for human paediatric enteric infection. Infection of C3H/HeN mice with C. rodentium results in rapid colonic colonisation, coinciding with induction of key inflammatory signatures and colonic crypt hyperplasia. Infection also induces dramatic changes to bioenergetics in intestinal epithelial cells, with transition from oxidative phosphorylation (OXPHOS) to aerobic glycolysis and higher abundance of SGLT4, LDHA, and MCT4. Concomitantly, mitochondrial proteins involved in the TCA cycle and OXPHOS were in lower abundance. Similar to observations in C57BL/6 mice, we detected simultaneous activation of cholesterol biogenesis, import, and efflux. Distinctly, however, the pattern recognition receptors NLRP3 and ALPK1 were specifically induced in C3H/HeN. Using cell-based assays revealed that C. rodentium activates the ALPK1/TIFA axis, which is dependent on the ADP-heptose biosynthesis pathway but independent of the Type III secretion system. This study reveals for the first time the unfolding intestinal epithelial cells' responses during severe infectious colitis, which resemble EPEC human infections.

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.