A Synthetic Peptide Corresponding to the Extracellular Loop 2 Region of Claudin-4 Protects against Clostridium perfringens EnterotoxinIn VitroandIn Vivo

Abstract

ABSTRACTClostridium perfringensenterotoxin (CPE) action starts when the toxin binds to claudin receptors. Claudins contain two extracellular loop domains, with the second loop (ECL-2) being slightly smaller than the first. CPE has been shown to bind to ECL-2 in receptor claudins. We recently demonstrated that Caco-2 cells (a naturally CPE-sensitive enterocyte-like cell line) can be protected from CPE-induced cytotoxicity by preincubating the enterotoxin with soluble full-length recombinant claudin-4 (rclaudin-4), which is a CPE receptor, but not with recombinant nonreceptor claudins, such asrclaudin-1. The current study evaluated whether a synthetic peptide corresponding to the claudin-4 ECL-2 sequence can similarly inhibit CPE actionin vitroandin vivo. Significant protection of Caco-2 cells was also observed using eitherrclaudin-4 or the claudin-4 ECL-2 peptide in both a preincubation assay and a coincubation assay. This inhibitory effect was specific, sincerclaudin-1 and a synthetic peptide based on the claudin-1 ECL-2 offered no protection to Caco-2 cells. However, the claudin-4 ECL-2 peptide was unable to neutralize cytotoxicity if CPE had already bound to Caco-2 cells. When the study was repeatedin vivousing a rabbit small intestinal loop assay, preincubation or coincubation of CPE with the claudin-4 ECL-2 peptide significantly and specifically inhibited the development of CPE-induced luminal fluid accumulation and histologic lesions in rabbit small intestinal loops. No similarin vivoprotection from CPE was afforded by the claudin-1 ECL-2 peptide. These results suggest that claudin-4 ECL-2 peptides should be further investigated for their potential therapeutic application against CPE-associated disease.