Clostridium difficile is a major nosocomial pathogen that causes severe diarrheal disease that is highly infectious and difficult to treat. C. difficileis easily transmitted due to its ability to form dormant spores. The spore form of C. difficile is resistant to most disinfectants and is critical for the survival of the bacterium outside of the hot. The only natural environment known to support C. difficile spore formation is the gastrointestinal tract, yet we know very little about how spore formation occurs. We hypothesize that sporulation of C. difficile is initiated in response to signals found within the host intestine, through an unknown and unique genetic mechanism. The long-term goal of this project is to uncover how C. difficile forms spores in the host so that molecular targets can be identified to disrupt the sporulation process. The specific objectives of this application are to identify the genetic mechanisms required for spore initiation and determine where sporulation is initiated in the host. Capitalizing on our previous experience with Bacillus subtilis sporulation, Gram-positive intestinal pathogenesis and C. difficile molecular genetics, we will meet these objectives through the experiments detailed in two specific aims. First, we will reveal the genetic mechanisms of spore initiation in C. difficile through genetic analysis of sporulation-defective mutants and newl identified early sporulation regulators. In parallel, we will define the progression of spore formation and explore the activation of sporulation regulators within the intestinal environment. The research proposed in this application is innovative because it represents a departure from the model sporulation paradigm as the archetypal standard for spore initiation by C. difficile and seeks to uncover the unique mechanisms that regulate sporulation this pathogen. The expected contribution of the proposed research is a detailed understanding of the genes required for spore initiation and the development of spores within the host. This contribution is significant because it is the first step in the development of strategies to prevent spore formation by C. difficile.