Project Summary/Abstract Clostridioides difficile (formerly Clostridium) is a major nosocomial pathogen that causes severe diarrheal disease that is highly infectious and difficult to treat. C. difficile is easily transmitted due to the formation and expulsion of up to 108 spores/ml of stool from infected hosts. The spore form of C. difficile is resistant to most disinfectants and is critical for the survival of the bacterium outside of the host intestine. Unfortunately, little is known about the intestinal signals that lead to spore formation. The long-term goal of this investigation is to uncover how nutrition drives C. difficile pathogenesis and transmission. The specific objectives of this application are to determine which distinct nutritional pathways and effectors have the greatest impact on spore production, and how these effects are regulated. Based on our data, we hypothesize that specific intestinal metabolites promote toxin expression and spore formation, while other metabolites impede these processes. The rationale underlying these studies is that uncovering the nutritional cues that drive the production of spores will help us understand how disease and transmission occur. In our preliminary studies, we identified specific nutrients that have dramatic effects on C. difficile spore formation. Capitalizing on our previous experiences in C. difficile molecular genetics, metabolic gene regulation, and Gram-positive intestinal pathogenesis, we will meet the objectives through the experiments detailed in two specific aims. To begin, we will define the specific pathways affected by these compounds and then evaluate the impact of influential nutrients on C. difficile spore formation and transmission. In parallel, we will dissect the regulatory mechanisms that link specific nutrients to spore formation. This research is innovative because it takes advantage of the latest information about the intestinal environment that supports CDI, and combines biochemical and genetic approaches to answer fundamental questions about this important and complex biological process. The expected contribution of this research is a detailed understanding of the relevant bacterial pathways that enable C. difficile sporulation in response to specific nutrients. Moreover, this work is an essential step in the development of rational strategies to impede C. difficile transmission by preventing the formation of infectious spores in the host.