Clostridioides difficile infection (CDI) is the leading cause of hospital-acquired diarrhea. Toxins, especially TcdB, cause epithelial cell apoptosis, but the underlying cell death mechanism is less clear. ABSTRACT Clostridioides difficile… Click to show full abstract
Clostridioides difficile infection (CDI) is the leading cause of hospital-acquired diarrhea. Toxins, especially TcdB, cause epithelial cell apoptosis, but the underlying cell death mechanism is less clear. ABSTRACT Clostridioides difficile infection (CDI) is the leading cause of antibiotic-associated intestinal disease, resulting in severe diarrhea and fatal pseudomembranous colitis. TcdB, one of the essential virulence factors secreted by this bacterium, induces host cell apoptosis through a poorly understood mechanism. Here, we performed an RNA interference (RNAi) screen customized to Caco-2 cells, a cell line model of the intestinal epithelium, to discover host factors involved in TcdB-induced apoptosis. We identified plakoglobin, also known as junction plakoglobin (JUP) or γ-catenin, a member of the catenin family, as a novel host factor and a previously known cell death-related chromatin factor, high-mobility group box 1 (HMGB1). Disruption of those host factors by RNAi and CRISPR resulted in resistance of cells to TcdB-mediated and mitochondrion-dependent apoptosis. JUP was redistributed from adherens junctions to the mitochondria and colocalized with the antiapoptotic factor Bcl-XL. JUP proteins could permeabilize the mitochondrial membrane, resulting in the release of cytochrome c. Our results reveal a novel role of JUP in targeting the mitochondria to promote the mitochondrial apoptotic pathway. Treatment with glycyrrhizin, an HMGB1 inhibitor, resulted in significantly increased resistance to TcdB-induced epithelial damage in cultured cells and a mouse ligated colon loop model. These findings demonstrate the critical roles of JUP and HMGB1 in TcdB-induced epithelial cell apoptosis. IMPORTANCE Clostridioides difficile infection (CDI) is the leading cause of hospital-acquired diarrhea. Toxins, especially TcdB, cause epithelial cell apoptosis, but the underlying cell death mechanism is less clear. Through an apoptosis-focused RNAi screen using a bacterium-made small interfering (siRNA) library customized to a human colonic epithelial cell model, we found a novel host factor, plakoglobin (γ-catenin), as a key factor required for cell apoptosis induced by TcdB. Plakoglobin targets and permeabilizes mitochondria after stimulation by TcdB, demonstrating a hitherto underappreciated role of this catenin family member in the apoptosis of intestinal epithelial cells. We also found a previously known cell death-related chromatin factor, HMGB1, and explored the inhibition of HMGB1 for CDI therapy in vivo.
               
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