PROJECT SUMMARY/ABSTRACT Clostridioides difficile is an anaerobic spore-forming pathogen that causes around 500,000 infections, 13,000 deaths, and healthcare costs of around 1 billion dollars annually in the United States. C. difficile infections cause severe diarrhea and inflammation of the colon. Infection is transmitted by ingestion of C. difficile spores, which can survive oxygen, disinfectants, chemicals, and radiation. After ingestion, spores germinate into vegetative cells within the intestine. These vegetative cells colonize the gastrointestinal tract, generating toxins and forming more spores. Spore formation is triggered by nutritional deprivation of the pathogen in the gastrointestinal tract. Nutrient availability is are sensed in C. difficile by specific transcriptional regulators, such as CodY. CodY is a global transcription regulator that is present in low G + C Gram-positive bacteria and responds to intracellular concentrations of branched-chain amino acids and GTP. Although it is known that C. difficile codY mutants hyper-sporulate, the means by which CodY represses sporulation is not currently understood. Based on the sporulation repression by CodY and its role as a transcriptional regulator, I hypothesize that CodY regulates sporulation by controlling the expression of a sporulation factor(s). In preliminary studies, I performed transcriptomic analyses of codY and wild-type strains, in combination with mapping of CodY-binding sites and transcriptional knockdown. Through these experiments I identified direct CodY-regulated factors that impact sporulation. The top three candidate genes are CDR20291_1334, CDR20291_1851-1853, and CDR20291_2485-2486. To test the hypothesis, I propose to complete these Specific Aims: Aim 1, I will assess the roles of CDR20291_1334, CDR20291_1851-1853, and CDR20291_2485-2486 in sporulation by generating null mutants and subsequently analyzing their contribution to sporulation and transcriptional profiles. Also, I will characterize the basic functions of these genes in C. difficile using growth curves, fluorescent microscopy, and co-immunoprecipitation experiments. In Aim 2, I will compare CodY binding to these target genes on wild-type alleles and variants containing mutated CodY binding sites by electrophoretic mobility shift assay, followed by assessment of the impact of CodY-dependent regulation on sporulation frequency via complementation studies with these genes containing wild-type and mutated CodY boxes. The completion of these aims will identify CodY- regulated genes that control sporulation and uncover their role in sporulation regulation through CodY.