SUMMARY - PROJECT 2 (ANTIGEN DISCOVERY) Clostridium difficile is a spore-forming pathogen that causes a wide range of gastrointestinal (GI) disorders varying in severity from mild diarrhea to fulminant colitis and death. Difficulties in treating infections with conventional antibiotics and increasing rates of recurrent infection underscore the need for the development of new therapeutic strategies to combat this urgent public health threat. To date vaccines for C. difficile have not fully met this need, thus future strategies will require innovative approaches that limit disease and promote clearance of this pathogen. Our objective is to generate a pipeline to identify novel vaccine antigens on the C. difficile cell and spore surface and develop these as the next generation of multi-valent mRNA vaccines against C. difficile. Antigen discovery strategy will incorporate aspects of the C. difficile life cycle, antigen variation on the cell surface, and microbial ecology. We hypothesize that detailed characterization of C. difficile proteins and gene systems important for fitness in the infected gut and competition with the resident microbiota will lead to validation of highly effective new vaccine targets. Our approach will use whole genome sequence (WGS) and surface proteome analyses on a wide array of hospital and community-associated strains and in-depth investigation into metabolic correlates during infection. We will combine advanced imaging mass spectrometry (IMS), mouse models of infection, and genetics to define nutritional requirements for C. difficile during infection and target these surface exposed proteins for mRNA-LNP vaccination. Together, this work will generate a translational workflow that leverages microbial ecology for discovery of novel vaccine targets for eradication of C. difficile.