PROJECT SUMMARY/ABSTRACT Significance: Clostridium difficile is the most common pathogen to cause healthcare-associated infections and has led to diarrheal diseases being the only cause of infectious diseases mortality in the United States to increase from 2000 to 2014. Not only are adults aged 65 or older more likely to be infected, they make up 90% of deaths from C. difficile infection (CDI). Research to look into mechanisms that increase the risk of deaths and severe outcome from CDI in the aged host is now more important than ever. Approach: Our preliminary studies show that aged mice have lower early innate immune responses and higher mortality from CDI, but these differences in mortality and immune responses with aging can be overcome by fecal microbiota transplant from young to aged mice. Using a novel aged mouse model of CDI we have developed, I will identify factors that mediate the protection in aged mice on the intestinal microbiome/metabolome side (Aim 1) and on the host response side (Aim 2). Aim 1 will analyze the microbiome and metabolome to identify protective bacteria and metabolites to test for protection against CDI in aged mice. Aim 2 will utilize RNA sequencing and flow cytometry to identify immune cells and pathways that mediate the microbiome effect on host response and test them by reversing the pathway during fecal transplant experiment. Combining findings from both aims will allow us to more completely characterize the pathogenesis and find targets for developing novel therapeutics. Career development: This proposal leverages the institutional commitment and training environment at University of Virginia, resources of the Warren/Guerrant Lab, mentorship from world experts on C. difficile and mucosal immunology, and expert collaborations in the field of bioinformatics. My career development plan includes structured oversight and guidance from my mentorship team, targeted coursework to acquire novel skills in key areas of the project (immunology, bioinformatics), focused workshops to enhance faculty development skills, publication benchmarks and plans to apply for independent funding, all of which will help establish me as an independent investigator in the field of infections in the aging host.