A Synthetic Peptide Corresponding to the Extracellular Loop 2 Region of Claudin-4 Protects against Clostridium perfringens Enterotoxin In Vitro and In Vivo

Abstract

bClostridium 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 ( r claudin-4), which is a CPE receptor, but not with recombinant nonreceptor claudins, such as r claudin-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 r claudin-4 or the claudin-4 ECL-2 peptide in both a preincubation assay and a coincubation assay. This inhibitory effect was specific, since r claudin-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.

Claudin-4 is required for modulation of paracellular permeability by muscarinic acetylcholine receptor in epithelial cells

“…The extracellular loops of claudins interact with each other to seal the cellular sheet and regulate paracellular transport between luminal and basolateral spaces (Lal-Nag and Morin, 2009). Claudin-4 has been detected in diverse epithelia, such as salivary, renal, lung, intestinal and epidermal cells (Akazawa et al, 2013;Cong et al, 2013;Gong et al, 2014;Kage et al, 2014;Shrestha et al, 2014). In rat submandibular SMIE cells, overexpression of claudin-4 increases TER and decreases the epithelial permeability of 70 kDa dextran (Michikawa et al, 2008).…”

Suppressive Effects of Hot-water Extract of Magnolia obovata on Clostridium perfringens Enterotoxin-induced Cytotoxicity in Human Intestinal Caco-2 Cells

“…CPE mainly uses the tight junction proteins claudin-3 and claudin-4 as receptors, which are constitutively expressed in the intestinal epithelial cells and Caco-2 cells [22,23]. CPE binds to the extracellular second loop region of claudin-3 and claudin-4 [24,25]. The C-terminal region of CPE contributes to the binding and facilitates the formation of the membrane attack complex in the plasma membrane of target cells by the N-terminal region [26][27][28].…”

The Potential Therapeutic Agent Mepacrine Protects Caco-2 Cells against Clostridium perfringens Enterotoxin Action

“…A peptide corresponding to the 30 C-terminal amino acids of CPE was shown to successfully block CPE binding to rabbit small intestinal brush border membranes by competing against the native enterotoxin for claudin binding ( 21 ). Similarly, a peptide corresponding to the second extracellular loop sequence of claudin-4 was successfully used as a receptor decoy to protect Caco-2 cells or rabbit small intestine from CPE effects ( 22 , 23 ). However, those peptide-based approaches required the use of high concentrations of expensive synthetic peptides that are susceptible to inactivation in the gastrointestinal tract, limiting their practicality as potential clinical therapeutics.…”