title: Clostridium difficile toxin B-induced necrosis is mediated by the host epithelial cell NADPH oxidase complex.
abstract: Clostridium difficile infection (CDI) is a leading cause of health care-associated diarrhea and has increased in incidence and severity over the last decade. Pathogenesis is mediated by two toxins, TcdA and TcdB, which cause fluid secretion, inflammation, and necrosis of the colonic mucosa. TcdB is a potent cytotoxin capable of inducing enzyme-independent necrosis in both cells and tissue. In this study, we show that TcdB-induced cell death depends on assembly of the host epithelial cell NADPH oxidase (NOX) complex and the production of reactive oxygen species (ROS). Treating cells with siRNAs directed against key components of the NOX complex, chemical inhibitors of NOX function, or molecules that scavenge superoxide or ROS confers protection against toxin challenge. To test the hypothesis that chemical inhibition of TcdB-induced cytotoxicity can protect against TcdB-induced tissue damage, we treated colonic explants with diphenyleneiodonium (DPI), a flavoenzyme inhibitor, or N-acetylcysteine (NAC), an antioxidant. TcdB-induced ROS production in colonic tissue was inhibited with DPI, and both DPI and NAC conferred protection against TcdB-induced tissue damage. The efficacy of DPI and NAC provides proof of concept that chemical attenuation of ROS could serve as a viable strategy for protecting the colonic mucosa of patients with CDI.
authors.affiliation: Department of Pathology, Microbiology, and Immunology, Chemical and Physical Biology Program, Department of Surgery, Epithelial Biology Center, Department of Medicine, and Department of Cell and Developmental Biology, Vanderbilt University School of Medicine, Nashville, TN 37232.
authors.forename: Melissa A
authors.initials: MA
authors.lastname: Farrow
authors.forename: Nicole M
authors.initials: NM
authors.lastname: Chumbler
authors.forename: Lynne A
authors.initials: LA
authors.lastname: Lapierre
authors.forename: Jeffrey L
authors.initials: JL
authors.lastname: Franklin
authors.forename: Stacey A
authors.initials: SA
authors.lastname: Rutherford
authors.forename: James R
authors.initials: JR
authors.lastname: Goldenring
authors.forename: D Borden
authors.initials: DB
authors.lastname: Lacy
mesh_terms: D001426:Bacterial Proteins / Q000633:toxicity*; D001427:Bacterial Toxins / Q000378:metabolism / Q000633:toxicity*; D015153:Blotting, Western; D018938:Caco-2 Cells; D004768:Enterotoxins / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D018613:Microscopy, Confocal; D046912:Multiprotein Complexes / Q000378:metabolism*; D019255:NADPH Oxidases / Q000378:metabolism*; D009336:Necrosis / Q000378:metabolism*; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection; D037521:Virulence Factors / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D001427:Bacterial Toxins; D004768:Enterotoxins; D046912:Multiprotein Complexes; D034741:RNA, Small Interfering; D037521:Virulence Factors; C041348:tcdA protein, Clostridium difficile; C031815:toxB protein, Clostridium difficile; D019255:NADPH Oxidases