title: Clostridium difficile Toxin B causes epithelial cell necrosis through an autoprocessing-independent mechanism.
abstract: Clostridium difficile is the most common cause of antibiotic-associated nosocomial infection in the United States. C. difficile secretes two homologous toxins, TcdA and TcdB, which are responsible for the symptoms of C. difficile associated disease. The mechanism of toxin action includes an autoprocessing event where a cysteine protease domain (CPD) releases a glucosyltransferase domain (GTD) into the cytosol. The GTD acts to modify and inactivate Rho-family GTPases. The presumed importance of autoprocessing in toxicity, and the apparent specificity of the CPD active site make it, potentially, an attractive target for small molecule drug discovery. In the course of exploring this potential, we have discovered that both wild-type TcdB and TcdB mutants with impaired autoprocessing or glucosyltransferase activities are able to induce rapid, necrotic cell death in HeLa and Caco-2 epithelial cell lines. The concentrations required to induce this phenotype correlate with pathology in a porcine colonic explant model of epithelial damage. We conclude that autoprocessing and GTD release is not required for epithelial cell necrosis and that targeting the autoprocessing activity of TcdB for the development of novel therapeutics will not prevent the colonic tissue damage that occurs in C. difficile - associated disease.
authors.affiliation: Chemical and Physical Biology Program, Vanderbilt University School of Medicine, Nashville, Tennessee, United States of America.
authors.forename: Nicole M
authors.initials: NM
authors.lastname: Chumbler
authors.forename: Melissa A
authors.initials: MA
authors.lastname: Farrow
authors.forename: Lynne A
authors.initials: LA
authors.lastname: Lapierre
authors.forename: Jeffrey L
authors.initials: JL
authors.lastname: Franklin
authors.forename: David B
authors.initials: DB
authors.lastname: Haslam
authors.forename: David
authors.initials: D
authors.lastname: Haslam
authors.forename: James R
authors.initials: JR
authors.lastname: Goldenring
authors.forename: D Borden
authors.initials: DB
authors.lastname: Lacy
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001427:Bacterial Toxins / Q000235:genetics / Q000378:metabolism*; D018938:Caco-2 Cells; D016360:Clostridioides difficile / Q000235:genetics / Q000378:metabolism*; D004761:Enterocolitis, Pseudomembranous / Q000235:genetics / Q000378:metabolism* / Q000382:microbiology / Q000473:pathology; D004847:Epithelial Cells / Q000378:metabolism* / Q000382:microbiology / Q000502:physiology; D005260:Female; D006367:HeLa Cells; D006801:Humans; D008297:Male; D009154:Mutation; D009336:Necrosis; D013552:Swine
chemical_list: D001426:Bacterial Proteins; D001427:Bacterial Toxins; C031815:toxB protein, Clostridium difficile