Metabolic Phenotypes in <i>Clostridioides difficile</i> and the Gut Microbiome

Clostridioides difficile infection (CDI) is a major healthcare-acquired infection caused by dysbiosis of the gut microbiome, typically due to broad-spectrum antibiotics. C. difficile pathogenesis is mediated by its two toxins, TcdA and TcdB which damage the gut epithelial barrier. Toxin production in C. difficile is metabolically sensitive, responding to a variety of external metabolites. However, the intracellular metabolic phenotypes of C. difficile connecting these external variables to a specific toxin state are unclear. In this dissertation, I focus on elucidating the metabolic phenotypes associated with low and high toxin states in C. difficile. By using publicly available RNAseq data from C. difficile grown in 16 unique conditions in conjunction with genome-scale metabolic models, I discovered metabolic differences between toxin states and strains of C. difficile. Furthermore, I found reactions that transformed C. difficile from a metabolic state associated with a high toxin state to a low toxin state in the model. To further explore metabolic phenotypes in C. difficile, I next focused on metabolic interactions between C. difficile and the environment, using C. difficile strain type, diet, and gut commensals as the variables. By creating 105 strain-specific metabolic models, I was able to identify metabolic phenotypes that varied by both diet and strain. Notably, I found a subset of C. difficile strains that have increased flux through energy-generating redox pathways. I also found that simulations of C. difficile-commensal interactions were not affected by the C. difficile strain type, however they were affected by diet. This suggests a hierarchical relationship between diet, commensals, and C. difficile genetics. Together, this dissertation provides a wholistic understanding of metabolic phenotypes in C. difficile related to toxin production, diet, genetics, and commensal interactions. I used disparate datasets and variables to investigate how they affect metabolism both in isolation and all together. These unique analyses have improved our understanding of the metabolic and ecological processes that are important to CDI pathogenesis, and they are a critical stepping stone towards improving treatment and management of disease.