Structural Mechanisms of Clostridioides difficile pathogenesis

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Summary Clostridioides difficile is a Gram-positive, spore-forming anaerobe that infects the colon, causing a range of human disease including diarrhea, pseudomembranous colitis, and toxic megacolon. The United States Centers for Disease Control reports that, in 2017, there were 223,900 estimated cases of C. difficile infection (CDI) in hospitalized patients in the United States with an estimated 12,800 deaths. The incidence of community- acquired CDI is also common making C. difficile a significant public health concern. The bacterium makes a toxin, TcdB, which is responsible for the majority of CDI symptoms. The goal of the proposed project is to define the structural basis and physiologic consequences of TcdB binding to receptor proteins on the host cell surface. In Aim 1, we will define the structures and key residues involved in TcdB-receptor interactions using a combination of structural biology, mutagenesis, and quantitative binding approaches. We will use this information to generate C. difficile strains with defined mutations in TcdB receptor binding sites. In Aim 2, we will evaluate the cellular tropism of the toxin in the context of a human explant intoxication model, taking advantage of significant technological advances in high resolution light microscopy imaging. These studies will be paired with mechanistic studies in a novel cellular model of TcdB intoxication as well as the mouse model of CDI. Collectively, these studies will define the physiologic consequences associated with defined TcdB-receptor interactions and are expected to provide a mechanistic framework for advancing novel therapeutic and CDI prevention strategies.