Genetic Mechanisms of Sporulation Induction in C. difficile

Project Summary/Abstract Clostridioides difficile (formerly Clostridium) is a major nosocomial pathogen that causes severe diarrheal disease that is highly infectious and difficult to treat. C. difficile is easily transmitted due to the formation and expulsion of contagious spores from infected hosts. The spore form of C. difficile is resistant to most disinfectants and is critical for the survival of the bacterium outside of the host intestine. The gastrointestinal tract is the only natural environment known to support C. difficile spore formation, but we understand little about how spore formation is initiated by the bacterium. The long-term goal of this project is to uncover the molecular mechanisms that control the initiation of C. difficile sporulation. Based on our data, we hypothesize that several early sporulation proteins function in independent pathways to regulate the initiation of sporulation by controlling activation of the master transcriptional regulator, Spo0A. The specific objectives of this application are to define the direct regulatory mechanisms that act upon Spo0A and to delineate the molecular pathways that control sporulation initiation. Capitalizing on our previous experiences in C. difficile molecular genetics, gene regulation, and Gram-positive intestinal pathogenesis, we will meet the objectives through the experiments detailed in two specific aims. In Aim 1, we will extend our current studies to uncover direct interacting partners of Spo0A using a combination of genetic and biochemical approaches. In parallel, Aim 2 expands epistatic analyses to construct Spo0A regulatory pathways, followed by functional analyses of the proteins within the initiation cascade. This research is innovative because it combines biomolecular and genetic approaches to resolve a fundamental question about this important and complex biological process. Completion of these aims is expected to expose potential vulnerabilities in spore initiation. This work is an essential step in the development of rational strategies to impede C. difficile transmission by preventing the formation of infectious spores in the host.