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Infection and Immunity, March 2001, p. 1329-1336, Vol. 69, No. 3
0019-9567/01/$04.00+0   DOI: 10.1128/IAI.69.3.1329-1336.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Clostridium difficile Toxins Disrupt Epithelial Barrier Function by Altering Membrane Microdomain Localization of Tight Junction Proteins

A. Nusrat,1,* C. von Eichel-Streiber,2 J. R. Turner,3,4 P. Verkade,5 J. L. Madara,1 and C. A. Parkos1

Epithelial Pathobiology Research Unit, Department of Pathology, Emory University School of Medicine, Atlanta, Georgia1; Institut fur Medizinische Mikrobiologie und Hygiene, Universität Mainz, Mainz,2 Cell Biology and Biophysics Program, EMBL, Heidelberg,3 and Max Planck Institute of Molecular Cell Biology and Genetics, Dresden,4 Germany; and Department of Pathology, Wayne State University, Detroit, Michigan5

Received 28 July 2000/Returned for modification 14 September 2000/Accepted 20 November 2000

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.

* Corresponding author. Mailing address: Department of Pathology and Laboratory Medicine, Emory University, WMRB 2335, 1639 Pierce Dr., Atlanta, GA 30322. Phone: (404) 727-8543. Fax: (404) 727-3321. E-mail: anusrat{at}emory.edu.

Infection and Immunity, March 2001, p. 1329-1336, Vol. 69, No. 3
0019-9567/01/$04.00+0   DOI: 10.1128/IAI.69.3.1329-1336.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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