title: Identification of a novel virulence factor in Clostridium difficile that modulates toxin sensitivity of cultured epithelial cells.
abstract: Two glucosylating toxins named toxins A and B play a role in the pathogenesis of Clostridium Difficile infection. The interaction of the toxins with host cell factors proceeds to downstream stages of cytotoxic effects in cells, in which involvement of other C. difficile factors remains unknown. We utilized culture filtrate of C. difficile with a low dilution to characterize the influence of putative minor proteins on the organization of the actin cytoskeleton in cultured epithelial cells and found a previously uncharacterized F-actin aggregated structure, termed "actin aggregate," at the juxtanuclear region. We reasoned that formation of actin aggregate was due to an additional factor(s) in the culture filtrate rather than the glucosylating toxins, because treatment of purified toxins rarely caused actin aggregate in cells. We focused on a previously uncharacterized hypothetical protein harboring a KDEL-like sequence as a candidate. The product of the candidate gene was detected in culture filtrate of C. difficile ATCC 9689 and was renamed Srl. Purified glutathione S-transferase-tagged Srl triggered formation of actin aggregate in the cells in the presence of either toxin A or B and enhanced cytotoxicity of each of the two toxins, including decreases in both cell viability and transepithelial resistance of cultured epithelial monolayer, although the recombinant Srl alone did not show detectable cytotoxicity. Srl-neutralized culture filtrate partially inhibited morphological changes of the cells in parallel with decreased actin aggregate formation in the cells. Thus, Srl might contribute to the modulation of toxin sensitivity of intestinal epithelial cells by enhancing cytotoxicity of C. difficile toxins.
authors: Department of Foodborne Infection Research (SRL, Inc.), Kitasato University School of Medicine, 1-15-1 Sagamihara-shi, Minami-ku, Kanagawa 252-0374, Japan. miuram@med.kitasato-u.ac.jp
authors: Masashi
authors: M
authors: Miura
authors: Haru
authors: H
authors: Kato
authors: Osamu
authors: O
authors: Matsushita
mesh_terms: D000199:Actins / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins / Q000378:metabolism / Q000633:toxicity*; D001427:Bacterial Toxins / Q000378:metabolism / Q000633:toxicity*; D001483:Base Sequence; D018938:Caco-2 Cells; D002460:Cell Line; D016360:Clostridioides difficile / Q000235:genetics / Q000378:metabolism / Q000472:pathogenicity*; D017077:Culture Media, Conditioned; D004768:Enterotoxins / Q000378:metabolism / Q000633:toxicity*; D004847:Epithelial Cells / Q000187:drug effects* / Q000382:microbiology; D005982:Glutathione Transferase / Q000378:metabolism; D054884:Host-Pathogen Interactions; D006801:Humans; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D037521:Virulence Factors / Q000235:genetics / Q000633:toxicity*
chemical_list: D000199:Actins; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D017077:Culture Media, Conditioned; D004768:Enterotoxins; D037521:Virulence Factors; C041348:tcdA protein, Clostridium difficile; C031815:toxB protein, Clostridium difficile; D005982:Glutathione Transferase