Nationwide, Clostridioides (formerly Clostridium) difficile infection is the most common cause of antibiotic- associated diarrhea and the most frequent hospital-acquired infection. Recent epidemiological studies have shown an alarming increase in incidence of C. difficile infection in disabled Veterans. Hospital-based infection control programs and careful choice of antibiotic treatments have been beneficial, but recurrent and drug- resistant C. difficile infections persistently burden the VA healthcare system. Therapeutically, fecal microbiota transplant has shown promising success but lacks specificity and standardization. To identify new therapeutic targets or strategies for disease prevention, a better fundamental understanding of how C. difficile toxins damage colonic tissue, and how the human host responds, is essential. Pathogenic strains of C. difficile produce toxin A (TcdA) and/or toxin B (TcdB), which directly kill host cells and induce an inflammatory response in the colonic mucosa. Clinical isolates of C. difficile encoding TcdB alone are sufficient to produce the whole spectrum of pathology in patients with C. difficile infection. Bezlotoxumab is a neutralizing antibody against TcdB and a therapeutic drug for preventing recurrent C. difficile infection. Therefore, TcdB is considered a prominent driver of human disease and will be the specific focus of this proposal. Recently, significant progress has been made to identify host cell surface receptors for TcdB, specifically Nectin- 3, CSPG4, and the Frizzled family of Wnt receptor proteins. The expression of these receptors in human colonic tissue and their respective contribution to TcdB function is not clearly understood. Furthermore, the epithelial cell subtypes targeted by TcdB and how interactions with these cells contribute to pathogenesis during the course of C. difficile infection is understudied. Emerging data suggest TcdB specifically targets the stem and progenitor cells in the base of colonic crypts potentially with the effect of delaying epithelial reconstitution. The Frizzled family of receptors are critical for regulating colonic stem cell function. Additionally, TcdB transactivates the epidermal growth factor receptor (EGFR), a mediator of colonic epithelial cell stem cell behavior. This proposal is based on preliminary data suggesting EGFR, ErbB2, and ErbB3 receptors facilitate TcdB-induced cell death. The effect of TcdB on ErbB signaling has not been studied in vivo, and the impact of this pathway on C. difficile pathogenesis is not known. Given the relevance of ErbB signaling to colonic stem cell behavior in homeostasis and injury repair, it may be that targeting stem or progenitor cells is a critical mechanism of TcdB virulence. The main hypothesis of this proposal is TcdB induces ErbB signaling in colonic stem/progenitor cells as an important component of the pathogenesis of C. difficile infection. The proposed experiments are aimed at using recombinant TcdB and...