Clostridium difficile toxins disrupt epithelial barrier function by altering membrane microdomain localization of tight junction proteins

The anaerobic bacterium Clostridium difficile is the etiologic agent of pse udomembranous colitis. C. difficile toxins TcdA and TcdB are UDP-glucosyltr ansferases that monoglucosylate and thereby inactivate the Rho family of GT Pases (W.P. Ciesla, Jr., and D. A. Bobak, J. Biol. Chem. 273:16021-16026, 1 998). We utilized purified reference toxins of C. difficile, TcdA-10463 (Tc dA) and TcdB-10463 (TcdB), and a model intestinal epithelial cell line to c haracterize their influence on tight-junction (TJ) organization and hence t o analyze the mechanisms by which they contribute to the enhanced paracellu lar permeability and disease pathophysiology of pseudomembranous colitis. T he increase in paracellular permeability induced by TcdA and TcdB was assoc iated with disorganization of apical and basal F-actin. F-actin restructuri ng was paralleled by dissociation of occludin, ZO-1, and ZO-2 from the late ral 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 a nd fractionation in sucrose density gradients revealed TcdB-induced redistr ibution of occludin and ZO-1 from detergent-insoluble fractions constitutin g "raft-like" membrane microdomains, suggesting an important role of Rho pr oteins in maintaining the association of TJ proteins with such microdomains . These toxin-mediated effects on actin and TJ structure provide a mechanis m for early events in the pathophysiology of pseudomembranous colitis.