Toxin B receptor mediated events in Clostridioides difficile disease

PROJECT SUMMARY Clostridioides difficile is a toxigenic, anaerobic bacterium that is a leading cause of antibiotic associated diarrhea. Patients with a dysbiotic gut microbiome due to antibiotic therapy, proton pump inhibitors, inflammatory bowel disease, or hospitalization are particularly susceptible to C. difficile infections. Following colonization, C. difficile secretes two toxins, TcdA and TcdB, which contribute to disease symptoms by entering and damaging host cells in the colon. TcdB isotypes from various clinically relevant C. difficile strains exhibit differences in cell tropism and receptor binding capabilities. Specifically, the TcdB2 isotype produced by the hypervirulent, endemic C. difficile strain has evolved to rely on chondroitin sulfate proteoglycan 4 (CSPG4) as its primary cell surface receptor. During injury, CSPG4 functions as a potent signaling molecule when cleaved from the cell surface to promote wound healing. However, since CSPG4 is cleaved from the cell surface, it remains unknown how TcdB2 is internalized by this proteoglycan and whether or not these interactions disrupt CSPG4 function. The overarching goal of this proposal is to understand how CSPG4 is used by TcdB2 to promote the pathologies observed during C. difficile disease. Preliminary evidence suggests TcdB2 residues 1769-1787 facilitate interactions with the chondroitin sulfate (CS) chains of CSPG4 in a divalent cation-dependent fashion to mediate cell entry. Thus, I hypothesize that TcdB2 interacts with the CS chains of CSPG4 to facilitate cell entry and interfere with CSPG4 signaling networks known to be critical for wound healing. In this research training plan, investigations in Aim 1 will determine how interactions between TcdB2 and the CS chains of CSPG4 drive toxin internalization, while Aim 2 will examine how TcdB2 binding impacts known functions regulated by CSPG4. The results of these experiments will provide important insight into the mechanism of TcdB2 uptake by the cell surface proteoglycan CSPG4, and how these interactions contribute to C. difficile disease.