title: A synthetic peptide corresponding to the extracellular loop 2 region of claudin-4 protects against Clostridium perfringens enterotoxin in vitro and in vivo.
abstract: Clostridium perfringens enterotoxin (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 as rclaudin-1. The current study evaluated whether a synthetic peptide corresponding to the claudin-4 ECL-2 sequence can similarly inhibit CPE action in vitro and in vivo. Significant protection of Caco-2 cells was also observed using either rclaudin-4 or the claudin-4 ECL-2 peptide in both a preincubation assay and a coincubation assay. This inhibitory effect was specific, since rclaudin-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 repeated in vivo using 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 similar in vivo protection 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.
authors.affiliation: Department of Microbiology and Molecular Genetics, University of Pittsburgh, School of Medicine, Pittsburgh, Pennsylvania, USA.
authors.forename: Archana
authors.initials: A
authors.lastname: Shrestha
authors.affiliation: Department of Microbiology and Molecular Genetics, University of Pittsburgh, School of Medicine, Pittsburgh, Pennsylvania, USA.
authors.forename: Susan L
authors.initials: SL
authors.lastname: Robertson
authors.affiliation: California Animal Health and Food Safety Laboratory System, School of Veterinary Medicine, University of California, Davis, San Bernardino, California, USA.
authors.forename: Jorge
authors.initials: J
authors.lastname: Garcia
authors.affiliation: California Animal Health and Food Safety Laboratory System, School of Veterinary Medicine, University of California, Davis, San Bernardino, California, USA.
authors.forename: Juliann
authors.initials: J
authors.lastname: Beingasser
authors.affiliation: Department of Microbiology and Molecular Genetics, University of Pittsburgh, School of Medicine, Pittsburgh, Pennsylvania, USA.
authors.forename: Bruce A
authors.initials: BA
authors.lastname: McClane
authors.affiliation: California Animal Health and Food Safety Laboratory System, School of Veterinary Medicine, University of California, Davis, San Bernardino, California, USA fuzal@cahfs.ucdavis.edu.
authors.forename: Francisco A
authors.initials: FA
authors.lastname: Uzal
mesh_terms: D000818:Animals; D001681:Biological Assay; D018938:Caco-2 Cells; D062506:Claudin-4 / Q000737:chemistry*; D003016:Clostridium perfringens / Q000235:genetics / Q000378:metabolism*; D004768:Enterotoxins / Q000633:toxicity*; D006801:Humans; D007421:Intestine, Small / Q000382:microbiology; D010455:Peptides / Q000737:chemistry / Q000494:pharmacology*; D011485:Protein Binding; D011817:Rabbits
chemical_list: C568157:CLDN4 protein, human; D062506:Claudin-4; D004768:Enterotoxins; D010455:Peptides; C035598:enterotoxin, Clostridium