Effect of epithelium association and toxin gene expression on Clostridioides difficile disease severity

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-oralroute which upon reaching the colon germinate into metabolically active vegetative cells that produce toxinsthat damage the intestinal epithelium. Despite toxins being essential for causing damage to the host toxin levelsand C. difficile biomass in fecal samples during murine infection do not correlate with disease severity. Thesefindings 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 onepithelial cells suggesting that penetration of the mucus layer would be advantageous to C. difficile. Since othershave also shown that C. difficile chemotaxes to the mucus layer and that its ability to utilize metabolites producedclose to the epithelium enhances colonization I hypothesize that the spatial distribution of C. difficile within thecolon is a critical factor in determining disease severity and may explain the lack of correlation between toxinlevels and C. difficile levels in feces and clinical outcomes. Furthermore toxin gene expression is heterogeneousin vitro suggesting that the sub-population of toxin gene-expressing cells generated during infection and theirproximity to the epithelium may further modulate disease severity. To address these hypotheses I haveconstructed and optimized various fluorescent reporter strains to visualize individual C. difficile cells and theirassociated toxin gene expression during murine infection. I am pairing these reporter strains with deletionmutants of genes hypothesized to drive C. difficile epithelium association to identify factors that drive C. difficilesassociation with the epithelium in vivo. I am also interrogating the impact of heterogeneity in toxin geneexpression during murine infection by expressing the dual constitutive and toxin gene-specific reporter systemin a strain that inducibly and uniformly expresses toxin genes. Collectively these analyses will advance ourunderstanding of the spatial distribution of C. difficile cells during infection and its impact on disease severity andhow heterogeneity in toxin gene expression impacts C. difficile localization in the colon and its ability to causedisease during murine infection. The information gained from these studies could lead to improved diagnosticsand methods for predicting disease severity which would greatly aid in the treatment and prevention of C. difficiledisease.