IAI Accepts, published online ahead of print on 27 May 2008

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Infect. Immun. doi:10.1128/IAI.00460-08
Copyright (c) 2008, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Non-cytotoxic Clostridium perfringens Enterotoxin (CPE) Variants Localize CPE Intestinal Binding and Demonstrate a Relationship Between CPE-Induced Cytotoxicity and Enterotoxicity

James G. Smedley III, Juliann Saputo, Jacqueline C. Parker, Mariano E. Fernandez-Miyakawa, Bruce A. McClane, Susan Robertson, and Francisco A. Uzal^*

Department of Microbiology and Molecular Genetics, and Molecular Virology and Microbiology Graduate Program, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261 USA; California Animal Health and Food Safety Laboratory System, School of Veterinary Medicine, University of California, Davis, San Bernardino, CA 92408, USA

^* To whom correspondence should be addressed. Email: fuzal{at}cahfs.ucdavis.edu.

Clostridium perfringens enterotoxin (CPE) causes the symptoms of a very common food poisoning. To assess whether CPE-induced cytotoxicity is necessary for enterotoxicity, a rabbit ileal loop model was used to compare the in vivo effects of native CPE or recombinant CPE (rCPE), both of which are cytotoxic, versus the non-cytotoxic rCPE variants rCPE D48A and rCPE_168-319. Both CPE and rCPE elicited significant fluid accumulation in rabbit ileal loops, along with severe mucosal damage that starts at villus tips and then progressively affects the entire villus, including necrosis of epithelium and lamina propria, villus blunting and fusion, and transmural edema and hemorrhage. Similar treatment of ileal loops with either of the non-cytotoxic rCPE variants produced no visible histologic damage or fluid transport changes. Immunohistochemistry revealed strong CPE or rCPE_168-319 binding to villus tips, which correlated with the abundant presence of claudin-4, a known CPE receptor, in this villus region. These results support, i) cytotoxicity as being necessary for CPE-induced enterotoxicity, ii) the CPE sensitivity of villus tips as being at least partially attributable to the abundant presence of receptors in this villus region and iii) claudin-4 as being an important intestinal receptor for CPE. Finally, rCPE_168-319 was able to partially inhibit CPE-induced histologic damage, suggesting that non-cytotoxic rCPE variants might be useful for protecting against some intestinal effects of CPE.