The Insect Peptide Coprisin Prevents Clostridium difficile-Mediated Acute Inflammation and Mucosal Damage through Selective Antimicrobial Activity

Abstract

Clostridium difficile-associated diarrhea and pseudomembranous colitis are typically treated with vancomycin or metronidazole, but recent increases in relapse incidence and the emergence of drug-resistant strains of C. difficile indicate the need for new antibiotics. We previously isolated coprisin, an antibacterial peptide from Copris tripartitus, a Korean dung beetle, and identified a nine-amino-acid peptide in the ␣-helical region of it (LLCIALRKK) that had antimicrobial activity (J. Here, we examined whether treatment with a coprisin analogue (a disulfide dimer of the nine peptides) prevented inflammation and mucosal damage in a mouse model of acute gut inflammation established by administration of antibiotics followed by C. difficile infection. In this model, coprisin treatment significantly ameliorated body weight decreases, improved the survival rate, and decreased mucosal damage and proinflammatory cytokine production. In contrast, the coprisin analogue had no apparent antibiotic activity against commensal bacteria, including Lactobacillus and Bifidobacterium, which are known to inhibit the colonization of C. difficile. The exposure of C. difficile to the coprisin analogue caused a marked increase in nuclear propidium iodide (PI) staining, indicating membrane damage; the staining levels were similar to those seen with bacteria treated with a positive control for membrane disruption (EDTA). In contrast, coprisin analogue treatment did not trigger increases in the nuclear PI staining of Bifidobacterium thermophilum. This observation suggests that the antibiotic activity of the coprisin analogue may occur through specific membrane disruption of C. difficile. Thus, these results indicate that the coprisin analogue may prove useful as a therapeutic agent for C. difficile infection-associated inflammatory diarrhea and pseudomembranous colitis.-

The Insect Peptide CopA3 Increases Colonic Epithelial Cell Proliferation and Mucosal Barrier Function to Prevent Inflammatory Responses in the Gut

“…difficile Toxin A Preparation-Toxin A was purified from C. difficile strain VPI 10463 (American Type Culture Collection, Manassas, VA) as described previously. The purity of native toxin A was assessed by gel electrophoresis, which confirmed a single protein at the expected molecular mass of 307 kDa (16).…”

“…Peptide Synthesis-The CopA3 peptide (from Dung beetles, Copris tripartitus) and P4 peptide (RLLLAIGRG-NH 2 , from the white-spotted flower chafer, Protaetia brevitarsis) were synthesized by AnyGen (Gwang-ju, South Korea) (15,16). The peptides were purified by reverse phase HPLC using a Capcell Pak C18 column (Shiseido, Japan) and eluted with a linear gradient of water-acetonitrile (0 -80%) containing 0.1% trifluoroacetic acid (45% recovery).…”

“…We recently found that CopA3, a 9-mer disulfide dimer peptide (LLCIALRKK-NH 2 , D-form) isolated from the Korean dung beetle, exhibited neurotropic effects in neuronal cell lines and mouse brain stem cells (15) and also showed antimicrobial activity (16). Although the biological activity of CopA3 has been examined in a variety of cellular systems (15,16), no previous study has examined its actions on mucosal epithelial cells and gut inflammation.…”

“…Although the biological activity of CopA3 has been examined in a variety of cellular systems (15,16), no previous study has examined its actions on mucosal epithelial cells and gut inflammation. Here, we show for the first time that CopA3 strongly increases cell proliferation in colonic epithelial cells, enhances the mucosal barrier, and inhibits both C. difficile toxin A-induced enteritis and DSS-induced colitis in mice.…”

Acquisition and retention of Clostridium difficile by Musca domestica larvae and pupae during metamorphosis

“…22 It is possible that the presence of antibacterial peptides in M. domestica larvae could be influential in the lack of retention of C. difficile through metamorphosis and although yet to be discovered in M. domestica, some insects (the Korean dung beetle, Copris tripartitus) do possess antimicrobial peptides (coprisin) with activity against vegetative cells of C. difficile. 23 …”