Effect of epithelium association and toxin gene expression on Clostridioides difficile disease severity - ABSTRACT Clostridioides difficile is an anaerobic, spore-forming, bacterial pathogen that is the most common cause of healthcare-associated infections in the United States. C. difficile is transmitted by spores through the fecal-oral route, which upon reaching the colon, germinate into metabolically active vegetative cells that produce toxins that damage the intestinal epithelium. Despite toxins being essential for causing damage to the host, toxin levels and C. difficile biomass in fecal samples during murine infection do not correlate with disease severity. These findings indicate that our understanding of the factors determining disease severity during C. difficile infection (CDI) is limited. Recent work has shown that the mucus layer impairs toxin binding to receptors found on epithelial cells suggesting that penetration of the mucus layer would be advantageous to C. difficile. Since others have also shown that C. difficile chemotaxes to the mucus layer and that its ability to utilize metabolites produced close to the epithelium enhances colonization, I hypothesize that the spatial distribution of C. difficile within the colon is a critical factor in determining disease severity and may explain the lack of correlation between toxin levels and C. difficile levels in feces and clinical outcomes. Furthermore, toxin gene expression is heterogeneous in vitro, suggesting that the sub-population of toxin gene-expressing cells generated during infection and their proximity to the epithelium may further modulate disease severity. To address these hypotheses, I have constructed and optimized various fluorescent reporter strains to visualize individual C. difficile cells and their associated toxin gene expression during murine infection. I am pairing these reporter strains with deletion mutants of genes hypothesized to drive C. difficile epithelium association to identify factors that drive C. difficile’s association with the epithelium in vivo. I am also interrogating the impact of heterogeneity in toxin gene expression during murine infection by expressing the dual constitutive and toxin gene-specific reporter system in a strain that inducibly and uniformly expresses toxin genes. Collectively, these analyses will advance our understanding of the spatial distribution of C. difficile cells during infection and its impact on disease severity and how heterogeneity in toxin gene expression impacts C. difficile localization in the colon and its ability to cause disease during murine infection. The information gained from these studies could lead to improved diagnostics and methods for predicting disease severity, which would greatly aid in the treatment and prevention of C. difficile disease.
