IAI Accepts, published online ahead of print on 2 November 2009

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Infect. Immun. doi:10.1128/IAI.00778-09
Copyright (c) 2009, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Identification of a Claudin-4 residue important for mediating the host cell binding and action of Clostridium perfringens enterotoxin

Susan L. Robertson, James G. Smedley III, and Bruce A. McClane^*

Dept. of Microbiology & Molecular Genetics, University of Pittsburgh, Pittsburgh, PA, 15261

^* To whom correspondence should be addressed. Email: bamcc{at}pitt.edu.

The 24-member claudin protein family plays a key role in maintaining the normal structure and function of epithelial tight junctions. Previous studies with fibroblast transfectants and naturally-sensitive Caco-2 cells have also implicated certain claudins (e.g., Claudin-4) as receptors for Clostridium perfringens enterotoxin (CPE). The current study first provided evidence that the second extracellular loop (ECL-2) of claudins is specifically important for mediating the host cell binding and cytotoxicity of native CPE. Rat fibroblast transfectants expressing a Claudin-4 chimera, where the natural ECL-2 was replaced by ECL-2 from Claudin-2, exhibited no CPE-induced cytotoxicity. Conversely, CPE bound to, and killed, CPE-treated transfectants expressing a Claudin-2 chimera with a substituted ECL-2 from Claudin-4. Site-directed mutagenesis was then employed to alter an ECL-2 residue that invariably aligns as N in claudins known to bind native CPE, but as D or S in claudins that cannot bind CPE. Transfectants expressing a Claudin-4_N149D mutant lost the ability to bind or respond to CPE, while transfectants expressing a Claudin-1 mutant with the corresponding ECL-2 residue changed from D to N acquired CPE binding and sensitivity. Identifying carriage of this N residue in ECL-2 as being important for native CPE binding helps to explain why only certain claudins can serve as CPE receptors. Finally, preincubating CPE with soluble recombinant Claudin-4, or Claudin-4 fragments containing ECL-2, specifically blocked the cytotoxicity on Caco-2 cells. This result opens the possibility of using receptor claudins as therapeutic decoys to ameliorate CPE-mediated intestinal disease.