PROJECT SUMMARY Clostridioides difficile is the leading cause of antibiotic-associated diarrhea, with over 223,000 infections, almost 13,000 deaths, and 1 billion dollars in healthcare cost in the United States every year. C. difficile infection starts when infectious spores are ingested and germinate in the gastrointestinal (GI) tract to produce the vegetative cells that cause the disease. In order to establish an infection, C. difficile must colonize the human colon, however, the factors that allow C. difficile to successfully colonize the intestine remain largely unknown. Hydrolytic enzymes such as collagenases and gelatinases have been known to help multiple pathogens to colonize distinct niches within the human body by facilitating penetration into host tissues, contributing to the dissemination of toxins, and increasing the availability of nutrients for the pathogen to use. These enzymes allow the degradation of collagen and its derivative gelatin, which are ubiquitous components of mammalian tissues (including gastrointestinal tissue). I hypothesize that the collagenase enzymes in C. difficile are colonization factors that allow this bacterium to degrade host collagen and gelatin, and that these hydrolytic activities improve C. difficile intestinal colonization and persistence in the gut to increase virulence. The overall objective of this application is to identify what enzyme(s) are responsible for the collagenolytic and gelatinolytic activities observed in C. difficile strains, and how these activities impact pathogenesis and the host. To test this hypothesis, and in pursuit of the overall goal, I am determined to complete the following Specific Aims: Aim 1. Determine the role of U32 peptidases CD0703 and CD1228 in collagen and gelatin degradation. Aim 2. Examine the role of the collagenase/gelatinase in C. difficile pathogenesis and colonization of the host intestine. The completion of these aims will identify the enzyme(s) responsible for the hydrolytic activities observed in C. difficile and determine the contribution of these hydrolytic activities during C. difficile infection. Elucidating these could provide novel targets for prevention or treatment of C. difficile infection.