Project Summary Clostridiodies difficile is an opportunistic pathogen that can colonize a patient’s gastrointestinal tract following antibiotic perturbation of the intestinal microbiome. The quality of the host immune response to infection is an important factor in determining disease outcome. Immunodeficiencies leave the host acutely susceptible to infection, while unregulated host inflammation can drive disease (28). Interestingly, numerous clinical and animal studies have found the host immune response limits C. difficile-mediated tissue damage but does not directly drive pathogen clearance (4-12). However, the potential for host immune factors to shape C. difficile biology beyond total pathogen burden remains largely undefined. Previous studies have demonstrated that the biogeography and transcriptional activity of other pathogenic intestinal bacteria within the intestinal tract can be shaped by the mucosal immune system (13-16). Whether immunologic pressures shape C. difficile spatial and transcriptional profile has not been explored. This proposal utilizes distinct immunodeficient mice previously reported to exhibit a spectrum from mild to severe disease upon C. difficile infection despite indistinguishable C. difficile burden or toxin production. The aims of this proposal will first, visualize and quantify the C. difficile vegetative and spore burden in the mucus layer compared to the central lumen of the intestine under distinct immunologic conditions. Second, in complementary studies, the in vivo transcriptional profile of C. difficile will be assessed in mice harboring distinct immunologic deficiencies. Transcriptional pathways that promote sporulation, virulence factors, nutrient uptake, antimicrobial detoxification, and metabolic activity will be examined to understand how C. difficile responds to immune pressure to promote persistence and transmission. These aims will reveal novel immune-C. difficile interactions that determine disease severity and provide a template for how the immune response can be modulated to support conventional antibiotic treatment in treating C. difficile associated disease. 1