Progress Towards Understanding the Role of Clostridioides difficile Transferase Toxin in C. difficile Induced Inflammation and Pathogenesis

Abstract

Clostridioides difficile is an anaerobic pathogen and the most common cause of healthcare-associated diarrhea and colitis in the United States. The symptoms of the infection arise due to production of toxin A and toxin B. These toxins disrupt the intestinal barrier and cause an acute host inflammatory response, a major hallmark of pathophysiology in C. difficile infection. Epidemic C. difficile strains additionally produce the C. difficile transferase toxin (CDT, a binary toxin consisting of enzymatic CDTa subunit and pore forming CDTb subunit) suggesting that it may be important for the severity of CDI. However, the role of CDT during C. difficile pathogenesis is unclear because of limited understanding of its contribution to host inflammation and limited research tools for knowing how much CDT is produced during infection. In this dissertation, I showed that the pore forming subunit, CDTb, activates the NLRP3 inflammasome in myeloid-derived murine cells. The activation was attenuated in the presence of CDTa. While I found that CDT contributes to weight loss and edema in an acute C. difficile mouse infection model, this was independent of the inflammasome function. As a result, I discussed why translating toxin-specific cellular responses into a physiological context may present several challenges. In addition, I utilized a CDTb-specific nanobody clone library and chose to express and purify five with promising CDTb-binding properties. I discovered that selected nanobodies have high affinity binding interactions and potent neutralizing properties in vitro. Finally, I developed a nanobody-based sandwich ELISA assay and quantified CDTb levels produced over the time course of an in vivo murine infection. In summary, my dissertation work presented a significant advance toward appreciating the complexity of the role of CDT in host inflammation and reported physiological toxin concentrations beneficial for designing in vitro experiments and toxin monitoring strategies.