SUMMARY Clostridioides difficile (formerly named Clostridium difficile) is a Gram-positive, spore-forming pathogen, and the leading cause of nosocomial and antibiotic-associated intestinal infections. Susceptibility to C. difficile infection (CDI) often follows antibiotic treatment and subsequent disruption of the resident intestinal microbiota, however the rise of infections in healthy young adults suggests that there are additional factors that contribute to CDI. To colonize the gastrointestinal tract, C. difficile must compete with both the host and members of the gut microbiota for critical nutrients. Access to nutrient metals can profoundly impact the outcome of CDI as metals are required cofactors for approximately 30% of all proteins. This fact is exploited by host metal binding proteins which sequester nutrient metals to restrict microbial growth in a process termed nutritional immunity. A hallmark of CDI is the secretion of potent toxins that cause severe damage to the gastrointestinal epithelium and trigger the production of pro-inflammatory cytokines and chemokines. These events initiate the immune-mediated recruitment of inflammatory factors to the site of infection. One of the most abundant inflammatory proteins that accumulates at the site of CDI is calprotectin. Calprotectin is the most abundant protein in neutrophils and is a component of nutritional immunity that directly inhibits microbial growth through nutrient metal sequestration. Calprotectin is also a potent immunomodulatory protein, and a common clinical inflammatory biomarker whose abundance correlates with CDI severity. It is unknown how the massive infiltration of calprotectin affects metal availability in the gastrointestinal tract and shapes competition between C. difficile and members of the gut microbiota. In addition, how C. difficile adapts to calprotectin-dependent metal limitation and resists nutritional immunity during CDI remains unclear. We propose a model whereby nutrient metals make a critical contribution to the outcome of CDI. Toxin driven inflammation drives the recruitment of immune cells into the gut which leads to the accumulation of large amounts of CP. CP chelates available nutrient metals and exerts potent pro- inflammatory activities. This massive inflammatory response and redistribution of nutrients alters C. difficile gene expression and affects the interaction between C. difficile and members of the microbiota. Finally, we hypothesize that C. difficile encodes multiple gene products that compete with both CP and the microbiota for nutrient metals and this competition has a profound effect on the outcome of CDI. Experiments described in this proposal will test this model and define the contribution of nutritional immunity to the pathogenesis of C. difficile, determine the role of metal binding and immune cell recruitment in the protective properties of calprotectin, and identify C. difficile genes required to compete with the microbiome for nutr...