Impact of Nutrition on Sporulation and Pathogenesis in Clostridium difficile Open Access

Nawrocki, Kathryn (2016)

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Clostridium difficile is an antibiotic-associated nosocomial pathogen that causes severe gastrointestinal disease. C. difficile is a Gram-positive obligate anaerobe and is transmitted via the fecal-oral route as a dormant spore. Spores are ingested and germinate into vegetative cells in the presence of bile salts in the gastrointestinal tract. As vegetative C. difficile traverses the large intestine and colon, it begins to undergo sporulation so it can survive in the aerobic environment after exiting the host. The regulation of sporulation in C. difficile is poorly understood, but prior work suggested a link between sporulation and the nutritional state of the cell. We decided to investigate the role of CodY in sporulation. CodY is a global transcriptional repressor, but only binds its target sites when guanosine triphosphate and branched chain amino acids are readily available. CodY is a direct link between the metabolite pool and transcription in the cell. We concluded that CodY is a negative regulator of C. difficile sporulation. In conjunction with this work, we also examined the role of the readily available gastrointestinal metabolite, ethanolamine. Many pathogenic bacteria utilize ethanolamine to gain an advantage over the microbiota. We verified that C. difficile utilizes ethanolamine, but ethanolamine does not appear to have an effect on sporulation in vitro. We investigated ethanolamine metabolism in the hamster model of infection. While ethanolamine did not impact sporulation post-mortem, it does influence the progression of disease in infected hamsters. When we infected hamsters with mutants that were unable utilize ethanolamine, they developed symptoms and succumbed to infection at an earlier time than hamsters infected with wild-type. While the inability to utilize ethanolamine causes disease at an earlier time, it does not appear to have any direct effect on virulence factor production. This suggests that ethanolamine utilization may play a role in delaying the onset of disease. This work determined that the initiation of sporulation and nutritional state are linked, but that the metabolite ethanolamine does not appear to modulate sporulation, but instead alters disease progression. Overall, this work has shed light onto sporulation and pathogenesis of C. difficile.

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List of Tables and Figures

Chapter 1: Introduction. 1

Chapter 2: CodY-dependent Regulation of Sporulation in Clostridium difficile. 18

Chapter 3: The Impact of Ethanolamine Utilization on Pathogenesis of Clostridium difficile. 77

Chapter 4: Discussion. 121

Chapter 5: Appendix I - Supplemental Material for Chapter 2. 132

Chapter 6: Appendix II - Supplemental Material for Chapter 3. 142

Chapter 7: Appendix III - Conserved Oligopeptide Permeases Modulate Sporulation Initiation in Clostridium difficile. 150

Chapter 8: Appendix IV - An Alkaline Phosphatase Reporter for use in Clostridium difficile. 203

Chapter 9: Appendix V - The Clostridium difficile Dlt pathway is controlled by the ECF sigma factor, sV, in response to lysozyme. 230

Chapter 10: Appendix VI - The Phosphotransfer Protein CD1492 Represses Sporulation Initiation in Clostridium difficile. 282

Chapter 11: Appendix VII - Antimicrobial Peptide Resistance Mechanisms of Gram-Positive Bacteria. 322

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