McAllister, Kathleen Nicole (2019-08). The Study of the Bacterial Pathogen Clostridioides difficile from Genetic Tools to In Vitro and In Vivo Physiology. Doctoral Dissertation.
Thesis
C. difficile is a Gram-positive, anaerobic gut pathogen which infects hundreds of thousands of individuals each year and is a significant concern as a nosocomial and community-acquired pathogen. Genetic tools are important when analyzing the physiology of such organisms so that the underlying physiology / pathogenesis of the organisms can be studied. We find that Stickland metabolism, a metabolic process that is used by only a small fraction of the microbiota, is important for C. difficile physiology. We first used TargeTron mutagenesis to investigate the role of selenoproteins in C. difficile Stickland metabolism. In this study, we found that a TargeTron insertion into selD, encoding the selenophosphate synthetase that is essential for the specific incorporation of selenium into selenoproteins, results in a significant growth defect and a global loss of selenium incorporation. However, because of potential polar effects of the TargeTron insertion as well as other drawbacks of other available genetic tools, we developed a CRISPR-Cas9 mutagenesis system for C. difficile. We then built upon our initial characterization of the CRISPR-Cas9-generated selD mutant by creating a CRISPR-Cas9-mediated restoration of the selD gene at the native locus and used these strains to analyze the importance of selenium-containing proteins on C. difficile physiology. Our findings support the hypothesis that selenium-containing proteins are important for several aspects of C. difficile physiology (e.g., vegetative growth, spore formation, and outgrowth post-germination). Using RNAseq, we identified multiple candidate genes that likely aid the cell in overcoming the global loss of selenoproteins to grow in medium which is favorable for using Stickland metabolism. Lastly, we analyzed samples from hospitalized patients for their bile acid content and abundances in order to study the effects of antibiotic treatment, diarrheal symptoms, C. difficile infection, and recurrence of infection. We found that groups of bile acids are associated with all health statuses of patients. These studies collectively give insight into the importance of C. difficile physiology and the development of genetic tools in an attempt to analyze pathways that will stop the C. difficile life cycle so that targeted therapeutics can be developed.
