PROJECT SUMMARY Clostridium difficile is a major public health threat, causing disease ranging from mild diarrhea to potentially fatal pseudomembranous colitis. C. difficile disease symptoms are largely mediated by the secreted cytotoxins, TcdA and TcdB. Many aspects of the pathogenicity of this bacterium remains poorly understood, including how C. difficile adapts to the host intestinal environment. The components of the bacterial cell surface play critical roles in physiology and virulence and are commonly immunogenic antigens and potential antibiotic targets. In C. difficile the signaling molecule cyclic diguanylate (c-di-GMP) controls the production of flagella, type IV pili, and multiple additional cell surface proteins, indicating a key role for c-di-GMP in reorganizing the C. difficile cell surface in response to the host intestinal environment. Phase variation is a means by which many bacterial species introduce phenotypic heterogeneity into the population as a strategy to ensure survival of the population in the face of changing selective pressures. We recently showed that flagella and toxins phase vary in C. difficile, and sequencing analyses identified several other putative phase variable loci including two that encode c-di-GMP hydrolases. Our central hypothesis is that C. difficile combines c-di-GMP signaling and phase variation to coordinate global changes to the cell surface, enabling adaptation to extracellular pressures encountered during colonization of the intestinal tract. The objective of this proposal is to define the mechanisms of phase variation that control c-di-GMP signaling, the phenotypic responses to changes in c-di- GMP, and the impact of these regulatory mechanisms on C. difficile physiology and virulence. To accomplish this goal, we employ molecular genetics, biochemical techniques, and animal models of C. difficile disease to examine phase variation and c-di-GMP signaling at the population and single cell levels. Our discovery that C. difficile links phase variation with c-di-GMP signaling reveals a previously unknown mechanism for coordinated modification of the bacterial cell surface. Completion of these aims may expose new targets for attenuating C. difficile fitness in the host, facilitating efforts to combat this increasingly problematic pathogen.