Project Summary Clostridium difficile toxin B (TcdB) is a major virulent factor responsible for diseases associated with C. difficile infection. Our understanding of TcdB pathogenesis remains limited, owing in large part to a lack of established receptors through which to understand the in vivo targeting and disease progression in colonic tissues. We recently carried out genome-wide screens using the CRISPR-Cas9 approach and identified two distinct candidate receptors: Frizzled family proteins (FZDs) and chondroitin sulfate proteoglycan 4 (CSPG4). We further demonstrated that both FZDs and CSPG4 are functional receptors for TcdB, and each can mediate binding and entry of TcdB independently. However, how FZDs and CSPG4 contribute to TcdB pathogenesis in colonic tissues during C. difficile infection remains to be established. Here we propose three aims to address this key knowledge barrier. First, we found that FZDs and CSPG4 are differentially expressed in colonic tissues, with FZDs mainly in the colonic epithelium and CSPG4 largely expressed in sub-epithelial myofibroblasts. We thus propose a novel “two-receptor, two-stage” hypothesis to explain TcdB pathogenesis: TcdB first targets and enter the colonic epithelium via binding to FZDs. Disruption of the epithelium allows the toxin to gain access to sub-epithelial myofibroblasts, where TcdB utilizes CSPG4 as an alternative receptor. Aims 1 and 2 will examine this hypothesis, with Aim 1 focusing on the role of FZDs for TcdB binding and entry into the colonic epithelium during C. difficile infection in animal models, and Aim 2 focusing on the role of CSPG4 and myofibroblasts in TcdB pathogenesis during C. difficile infection. Furthermore, we also found that binding of TcdB to FZDs inhibits Wnt signaling, which is essential for colonic stem cells. We thus hypothesize a novel mechanism contributing to TcdB pathogenesis: TcdB may disrupt colonic stem cells through toxin-receptor engagement, independent of its enzymatic activity inside cells. Aim 3 will test this hypothesis by examining the biological consequences and pathological relevance of Wnt signaling inhibition utilizing colonic organoid models and by infection experiments in animal models with engineered C. difficile that expressing the receptor-binding domain of TcdB. We will also explore the therapeutic benefit of GSK-3 inhibitors, which modulate Wnt signaling, for reducing the damage to the colonic epithelium by TcdB during C. difficile infection. Together, these studies will provide a molecular understanding of disease progression during C. difficile infection and potentially lead to novel therapeutic interventions.