title: Clostridium difficile toxins disrupt epithelial barrier function by altering membrane microdomain localization of tight junction proteins.
abstract: The anaerobic bacterium Clostridium difficile is the etiologic agent of pseudomembranous colitis. C. difficile toxins TcdA and TcdB are UDP-glucosyltransferases that monoglucosylate and thereby inactivate the Rho family of GTPases (W. P. Ciesla, Jr., and D. A. Bobak, J. Biol. Chem. 273:16021-16026, 1998). We utilized purified reference toxins of C. difficile, TcdA-10463 (TcdA) and TcdB-10463 (TcdB), and a model intestinal epithelial cell line to characterize their influence on tight-junction (TJ) organization and hence to analyze the mechanisms by which they contribute to the enhanced paracellular permeability and disease pathophysiology of pseudomembranous colitis. The increase in paracellular permeability induced by TcdA and TcdB was associated with disorganization of apical and basal F-actin. F-actin restructuring was paralleled by dissociation of occludin, ZO-1, and ZO-2 from the lateral TJ membrane without influencing the subjacent adherens junction protein, E-cadherin. In addition, we observed decreased association of actin with the TJ cytoplasmic plaque protein ZO-1. Differential detergent extraction and fractionation in sucrose density gradients revealed TcdB-induced redistribution of occludin and ZO-1 from detergent-insoluble fractions constituting "raft-like" membrane microdomains, suggesting an important role of Rho proteins in maintaining the association of TJ proteins with such microdomains. These toxin-mediated effects on actin and TJ structure provide a mechanism for early events in the pathophysiology of pseudomembranous colitis.
authors.affiliation: Epithelial Pathobiology Research Unit, Department of Pathology, Emory University School of Medicine, Atlanta, Georgia 30322, USA. anusrat@emory.edu
authors.forename: A
authors.initials: A
authors.lastname: Nusrat
authors.forename: C
authors.initials: C
authors.lastname: von Eichel-Streiber
authors.forename: J R
authors.initials: JR
authors.lastname: Turner
authors.forename: P
authors.initials: P
authors.lastname: Verkade
authors.forename: J L
authors.initials: JL
authors.lastname: Madara
authors.forename: C A
authors.initials: CA
authors.lastname: Parkos
mesh_terms: D000199:Actins / Q000378:metabolism; D001426:Bacterial Proteins; D001427:Bacterial Toxins / Q000633:toxicity*; D016764:Cell Polarity; D016360:Clostridioides difficile / Q000472:pathogenicity*; D004768:Enterotoxins / Q000633:toxicity; D005964:Glucosyltransferases / Q000494:pharmacology; D007413:Intestinal Mucosa / Q000187:drug effects*; D021962:Membrane Microdomains / Q000187:drug effects*; D008565:Membrane Proteins / Q000378:metabolism; D010539:Permeability / Q000187:drug effects; D010750:Phosphoproteins / Q000378:metabolism; D019108:Tight Junctions / Q000187:drug effects*; D062826:Zonula Occludens-1 Protein; D062827:Zonula Occludens-2 Protein
chemical_list: D000199:Actins; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D004768:Enterotoxins; D008565:Membrane Proteins; D010750:Phosphoproteins; D062826:Zonula Occludens-1 Protein; D062827:Zonula Occludens-2 Protein; C041348:tcdA protein, Clostridium difficile; C031815:toxB protein, Clostridium difficile; D005964:Glucosyltransferases