Identification of an epithelial cell receptor responsible for Clostridium difficile TcdB-induced cytotoxicity

Abstract

Clostridium difficile is the leading cause of hospital-acquired diarrhea in the United States. The two main virulence factors of C. difficile are the large toxins, TcdA and TcdB, which enter colonic epithelial cells and cause fluid secretion, inflammation, and cell death. Using a gene-trap insertional mutagenesis screen, we identified poliovirus receptor-like 3 (PVRL3) as a cellular factor necessary for TcdB-mediated cytotoxicity. Disruption of PVRL3 expression by gene-trap mutagenesis, shRNA, or CRISPR/Cas9 mutagenesis resulted in resistance of cells to TcdB. Complementation of the gene-trap or CRISPR mutants with PVRL3 resulted in restoration of TcdB-mediated cell death. Purified PVRL3 ectodomain bound to TcdB by pull-down. Pretreatment of cells with a monoclonal antibody against PVRL3 or prebinding TcdB to PVRL3 ectodomain also inhibited cytotoxicity in cell culture. The receptor is highly expressed on the surface epithelium of the human colon and was observed to colocalize with TcdB in both an explant model and in tissue from a patient with pseudomembranous colitis. These data suggest PVRL3 is a physiologically relevant binding partner that can serve as a target for the prevention of TcdB-induced cytotoxicity in C. difficile infection.

Clostridium difficile Toxins TcdA and TcdB Cause Colonic Tissue Damage by Distinct Mechanisms

“…Rather, we think that the most likely reason is that the toxins engage different receptors. While the human receptor for TcdA has yet to be identified, two receptors for TcdB have been described: poliovirus receptor-like protein 3 (PVRL3) and chondroitin sulfate proteoglycan 4 (CSPG4) (47,48). PVRL3 seems to account for the cell death occurring at high concentrations in HeLa cells (where CSPG4 is also highly expressed), but it can mediate both necrotic and apoptotic mechanisms in Caco2 cells (47,48).…”

“…While the human receptor for TcdA has yet to be identified, two receptors for TcdB have been described: poliovirus receptor-like protein 3 (PVRL3) and chondroitin sulfate proteoglycan 4 (CSPG4) (47,48). PVRL3 seems to account for the cell death occurring at high concentrations in HeLa cells (where CSPG4 is also highly expressed), but it can mediate both necrotic and apoptotic mechanisms in Caco2 cells (47,48). We hypothesize that TcdB binding to PVRL3 initiates the assembly and activation of the NOX complex and that the lack of PVRL3 binding by TcdA accounts for the differences in the cell death responses of the two toxins.…”

Clostridium difficile Infection

“…Indeed, TcdA and TcdB lacking CROPS domains are still capable of intoxicating cells 113,114 , and the homologous toxin TpeL from Clostridium perfringens lacks a CROPS domain entirely 115 . Recently, two protein receptors have been reported for TcdB: poliovirus receptor-like protein 3 (PVRL3; also called nectin 3) 116 and chondroitin sulfate proteoglycan 4 (CSPG4) 117 . PVRL3 is highly expressed on the surface of human colon epithelial cells and co-localizes with TcdB in tissue resected from a C. difficile-infected individual 116 , suggesting that PVRL3 could serve as the initial receptor that TcdB encounters in the context of infection.…”

“…Recently, two protein receptors have been reported for TcdB: poliovirus receptor-like protein 3 (PVRL3; also called nectin 3) 116 and chondroitin sulfate proteoglycan 4 (CSPG4) 117 . PVRL3 is highly expressed on the surface of human colon epithelial cells and co-localizes with TcdB in tissue resected from a C. difficile-infected individual 116 , suggesting that PVRL3 could serve as the initial receptor that TcdB encounters in the context of infection. CSPG4 is highly expressed in the intestinal subepithelial myofibroblasts of mouse and human intestines 118 , suggesting that this receptor could be engaged after initial damage to the colonic epithelium 117 .…”

“…CSPG4 is highly expressed in the intestinal subepithelial myofibroblasts of mouse and human intestines 118 , suggesting that this receptor could be engaged after initial damage to the colonic epithelium 117 . Both CSPG4 and PVRL3 bind outside the CROPS domain 116,117 . It is conceivable that additional alternative receptors for TcdB exist.…”