title: Essential role of toxin A in C. difficile 027 and reference strain supernatant-mediated disruption of Caco-2 intestinal epithelial barrier function.
abstract: Clostridium difficile induces mucosal inflammation via secreted toxins A and B and initial interactions between the toxins and intestinal epithelial cells (which lead to loss of barrier function) are believed to be important in disease pathogenesis. Secreted toxin-specific antibodies may inhibit such interactions. Using the Caco-2 epithelial cell line, we have investigated the use of an anti-toxin A monoclonal antibody (ATAA) in providing protection against toxin A-mediated disruption of epithelial barrier function (assessed by measurement of transepithelial electrical resistance and luminal to basolateral flux of labelled dextran). In contrast to free antibody, ATAA conjugated to sepharose beads was more effective in neutralizing the activity of purified toxin A. Sepharose bead-conjugated ATAA was subsequently used to investigate the contribution of toxin A in epithelial injury mediated by C. difficile supernatant samples (containing toxins A, B and other products). Loss of barrier function mediated by apical application of supernatant samples of reference and epidemic 027 strains of C. difficile was abrogated by neutralization of toxin A. However, this was not the case when the supernatant samples were applied to the basal surface of epithelial monolayers. In conclusion, our studies have shown that (i) sepharose bead-conjugated ATAA is more effective in neutralizing toxin A than free antibody and (ii) when the apical (luminal) surface of epithelial monolayers is exposed to the secretory products of reference and 027 strains of C. difficile, toxin A is required for the initial injury that leads to loss of barrier function.
authors.affiliation: Institute of Infection, Immunity and Inflammation, University of Nottingham, Nottingham, UK.
authors.forename: P A
authors.initials: PA
authors.lastname: Sutton
authors.forename: S
authors.initials: S
authors.lastname: Li
authors.forename: J
authors.initials: J
authors.lastname: Webb
authors.forename: K
authors.initials: K
authors.lastname: Solomon
authors.forename: J
authors.initials: J
authors.lastname: Brazier
authors.forename: Y R
authors.initials: YR
authors.lastname: Mahida
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000494:pharmacology*; D001427:Bacterial Toxins; D018938:Caco-2 Cells; D002522:Chlorocebus aethiops; D016360:Clostridioides difficile / Q000472:pathogenicity*; D004768:Enterotoxins / Q000633:toxicity*; D004847:Epithelial Cells / Q000187:drug effects*; D006801:Humans; D000922:Immunotoxins / Q000494:pharmacology*; D007413:Intestinal Mucosa / Q000187:drug effects; D007422:Intestines / Q000187:drug effects*; D012685:Sepharose / Q000494:pharmacology; D014709:Vero Cells / Q000276:immunology
chemical_list: D000911:Antibodies, Monoclonal; D001427:Bacterial Toxins; D004768:Enterotoxins; D000922:Immunotoxins; C041348:tcdA protein, Clostridium difficile; D012685:Sepharose