Discovery of Bile Acid-Associated Molecular Changes in the Murine Gastrointestinal Tract During Clostridioides difficile Infection

Abstract

Clostridioides difficile is a nosocomial gastrointestinal pathogen with a major impact on the healthcare community and limited treatment methods. This pathogen undergoes germination from a dormant spore into a vegetative toxin-producing cell within the small intestine after encountering primary bile acids. This thesis is aimed to further elucidate the molecular environment of the gastrointestinal tract with imaging mass spectrometry (IMS) through the development a unique sample preparation workflow. Multimolecular IMS showed differential molecular localizations between tissue and luminal content in the small intestine. Successful application of this workflow to an infection model showed dramatic differences in bile acid abundances during C. difficile infection in the small intestinal lumen. To assess changes to overall bile acid pools, bile acid levels were quantified by liquid chromatography tandem MS, which showed that primary bile acids increase 10 to 100-fold within 24 hours of infection. This increase is partially a result of toxins produced by the pathogen, but is independent of inflammation. Using the bile acid-binding resin cholestyramine, it was shown that early access to bile acids is necessary for successful germination and colonization of C. difficile in the host. Analysis of pediatric patient samples showed that differential bile acid profiles existed in colonized patients based on their symptom status. Asymptomatic patients had higher levels of C. difficile toxin-binding bile acids in their stool compared to those with characteristic symptomology. Further studies aimed to elucidate if the enterohepatic circulation of bile acids was metabolically manipulated to cause the increased bile acid levels during infection. Utilizing a combined transcriptomic and proteomic approach, the production and reabsorption of bile acids were investigated. While transcriptomic data suggested that synthesis of bile acids was increased and absorption was decreased during infection, which would lead to increased bile acid abundance, protein abundances did not markedly change. Overall, this thesis highlights the mutual relationship between bile acids and C. difficile during infection, offering novel potential potentially novel targets for therapeutics.