Characterizing the Early Events of Clostridium difficile Spore Germination

Abstract

Clostridium difficile is an anaerobic spore forming bacterium that is an important human pathogen and a leading cause of hospital acquired infectious diarrhea worldwide. C. difficile infections cause symptoms ranging from severe diarrhea, to pseudomembranous colitis, toxin megacolon, and even death. These symptoms are caused by the production of two large exotoxins (TcdA and TcdB), that attack the host intestinal epithelium. C. difficile infection is a toxin-mediated disease, however, they are dependent on the ability to produce spores. C. difficile spores are produced through a highly regulated process known as sporulation. Sporulation occurs when bacterial growth conditions are unfavorable which leads to the phosphorylation of the major transcription factor Spo0A. Spo0A phosphorylation leads to activation of a cascade of compartment specific transcription factors and the production of mature dormant spores. These spores allow the bacterium to survive in harsh environmental conditions such as oxygen, low pH, ethanol, dessication, etc. C. difficile spores are the infectious particles; when a susceptible patient ingests a spore, it becomes reactivated in the small intestine through a process known as germination. Germination is the first pathogenic event that occurs after ingestion of the spore and thus, is required for disease. Germination occurs after specific environmental cues, known as germinants, signal to the spore that conditions are favorable for vegetative cell growth. These germinants are typically amino acids that interact with specific receptors found within the spore (GerA). Much is known about bacterial spore germination in Bacillus spp., however, since the C. difficile genome does not encode any orthologs of these receptors, C. difficile spores germinate through a unique mechanism. Instead C. difficile germinates in response to the combination of amino acids and bile salts with a unique pseudo-protease receptor, CspC. The amino acid receptor has yet to be discovered. The goals of this thesis are to describe the earliest events of germination including the specific conditions that facilitate germination, the mechanism of bile salts amino acid induced germination, and characterize the role of calcium. In this work, I describe for the first time an additional germination pathway activated by bile salts and intestinal calcium. Data presented in this thesis indicate that germination of C. difficile spores is regulated within the gastrointestinal tract by pH and availability of specific germinants, limiting germination to the ileum. Bile salts, calcium, and amino acids synergize together to decrease the required concentrations to physiological levels. This synergy also allows C. difficile spores to overcome the effects of inhibitory bile salts facilitating germination in ileal contents from non-antibiotic treated mice. Overall, the data presented in this thesis contribute to the knowledge of C. difficile germination, and provide biological mechanisms that answer important questions regarding clinical correlates between patients with impaired calcium absorption and increased risk of contracting a C. difficile infection. The findings presented here should ultimately, lead to the development of novel therapeutics to prevent or improve the outcomes of C. difficile infections.