Clostridium difficile is the leading cause of antibiotic-associated diarrhea in the hospital and long term health care settings. In addition to the patient toll, the treatment-associated costs of C. difficile infections to the United States healthcare system have been estimated at $5 billion. Although the rate of C. difficile infection in the United States is rising, surprisingly little is known about the mechanisms of C. difficile pathogenesis. C. difficile is believed to be acquired by the host in the form of a dormant spore. To cause disease, the spore must respond in the gastrointestinal tract to signals that trigger germination, thereby allowing growth as a vegetative bacterium, toxin production and subsequent spore formation before excretion into the environment. Taurocholic acid, a bile acid normally found in the GI tract, and glycine are co- germinants for C. difficile spores. Another bile acid, chenodeoxycholic acid, inhibits taurocholic acid-mediated germination and is toxic for C. difficile vegetative growth. In prior work, the molecular target of bile acids on the C. difficile spore was identified - identifying the first C. difficile spore germinant receptor. This led to the finding that C. difficile spore germination proceeds through a novel, ?outside ? in? germination pathway. More recently, the target of the amino acid co-germinants on the C. difficile spore was identified. In this proposal, the investigator proposes to: (1) characterize how the C. difficile ?germinosome? proteins interact; (2) define the mechanism of localization of these proteins in the C. difficile spore; (3) globally identify YabG protease targets; and (4) characterize the impact of these targets on C. difficile pathogenesis. Successful completion of the experiments outlined herein will extend the understanding of the mechanisms of C. difficile germination, open new avenues in the study of C. difficile spore formation and spore germination.
Successful completion of the experiments listed in this proposal will characterize, in detail, the Clostridium difficile spore ?germinosome? complex ? a complex consisting of the CspB, CspA, CspC and SleC proteins ? and how this complex is formed. Moreover, this application will identify and characterize YabG protease substrates (other than CspBA and preproSleC). The knowledge gained from these experiments could lead to the design of novel therapeutics to combat C. difficile spore germination.
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