| dc.description.abstract | Clostridioides difficile is a Gram-positive, spore-forming bacterium and the leading cause of hospital-acquired diarrhea in the United States. Upon colonizing the colon of susceptible hosts, C. difficile causes disease through two secreted protein toxins, toxin A (TcdA) and toxin B (TcdB). The glucosyltransferase activity of the toxins inactivates small Rho-family GTPases involved in cell signaling to promote inflammation, fluid secretion, and cell death. While multiple proteins have been identified to bind each toxin in vitro, the significance of these interactions remains unclear for mediating disease. In this dissertation, I investigated the roles of chondroitin sulfate proteoglycan 4 (CSPG4), Frizzled (FZD), and Nectin-3, three receptors proposed to mediate the endocytosis of TcdB. Using a rectal instillation mouse model of C. difficile toxins, I show that FZD interactions with TcdB are not essential for mediating pathologies. Furthermore, I show that ribotype 027 C. difficile, an epidemic C. difficile ribotype, produces a TcdB that is unable to interact with FZD proteins but is still capable of promoting fulminant disease in hosts. Mechanistically, I propose that TcdB promotes injuries to the colonic epithelium through interactions with Nectin-3 and CSPG4. By developing a method to visualize TcdB on cells using fluorescent microscopy, I demonstrate that both Nectin-3 and CSPG4 colocalize with TcdB on 18Co cells. On host colonic tissue, I observed novel localization of Nectin-3 within the brush border of epithelial cells and CSPG4 localization along the epithelial cell junction. As colonic epithelial cells do not express CSPG4, I propose that this CSPG4 signal is likely mediated by shed forms of CSPG4 produced by stromal cells along the crypt-surface axis. In support of this model, I show that shed forms of CSPG4 can be endocytosed by epithelial cells, a concept that TcdB can exploit to potentiate its activity. Collectively, my study provides new insights into how TcdB utilizes receptor interactions to intoxicate colonic epithelial cells to cause disease. | |
