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.