title: 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.
authors.affiliation: Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, TN 37232;
authors.forename: Michelle E
authors.initials: ME
authors.lastname: LaFrance
authors.affiliation: Department of Pathology, Microbiology and Immunology, Vanderbilt University School of Medicine, Nashville, TN 37232;
authors.forename: Melissa A
authors.initials: MA
authors.lastname: Farrow
authors.affiliation: Department of Pathology, Microbiology and Immunology, Vanderbilt University School of Medicine, Nashville, TN 37232;
authors.forename: Ramyavardhanee
authors.initials: R
authors.lastname: Chandrasekaran
authors.affiliation: Department of Medicine, Vanderbilt University School of Medicine, Nashville, TN 37232;
authors.forename: Jinsong
authors.initials: J
authors.lastname: Sheng
authors.affiliation: Department of Pathology, Microbiology and Immunology, Vanderbilt University School of Medicine, Nashville, TN 37232; Department of Medicine, Vanderbilt University School of Medicine, Nashville, TN 37232; Department of Research Medicine, Veterans Affairs Tennessee Valley Healthcare System, Nashville, TN 37212.
authors.forename: Donald H
authors.initials: DH
authors.lastname: Rubin
authors.affiliation: Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, TN 37232; Department of Pathology, Microbiology and Immunology, Vanderbilt University School of Medicine, Nashville, TN 37232; borden.lacy@vanderbilt.edu.
authors.forename: D Borden
authors.initials: DB
authors.lastname: Lacy
mesh_terms: D000704:Analysis of Variance; D000911:Antibodies, Monoclonal / Q000378:metabolism; D001426:Bacterial Proteins / Q000378:metabolism / Q000633:toxicity*; D001427:Bacterial Toxins / Q000378:metabolism / Q000633:toxicity*; D018938:Caco-2 Cells; D015815:Cell Adhesion Molecules / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D016360:Clostridioides difficile / Q000737:chemistry*; D003106:Colon / Q000378:metabolism; D004768:Enterotoxins / Q000378:metabolism / Q000633:toxicity*; D004847:Epithelial Cells / Q000378:metabolism*; D005816:Genetic Complementation Test; D006367:HeLa Cells; D006801:Humans; D016254:Mutagenesis, Insertional; D000075983:Nectins
chemical_list: D000911:Antibodies, Monoclonal; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D015815:Cell Adhesion Molecules; D004768:Enterotoxins; C000619014:NECTIN3 protein, human; D000075983:Nectins; C031815:toxB protein, Clostridium difficile
keywords: PVRL3; nectin-3; toxin