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 α-helical region of it (LLCIALRKK) that had antimicrobial activity (J.-S. Hwang et al., Int. J. Pept., 2009, doi:10.1155/2009/136284). 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.

Fig. 1.

Coprisin analogue (coprisin) has antibiotic activity against C. difficile. (A) Disc diffusion tests. Sterile discs containing coprisin (10 to 100 μg/ml in water) were placed on BHI agar plates inoculated with C. difficile (10⁸ CFU/ml), anaerobically incubated for 48 h at 37°C. Data are representative of the results obtained with three independent samples. (B) Comparison of the antibiotic activity of coprisin analogue (100 μg/ml) with that of vancomycin (100 μg/ml). (C) Water (control), l-type or d-type coprisin analogue (10 μg/ml), or vancomycin (10 μg/ml) was added to anaerobically growing cultures of C. difficile (2 × 10⁵ CFU/ml) in BHI broth at 37°C. After 2 days at a plateau of growth, suitable dilutions were plated onto BHI agar and the residual numbers of C. difficile bacteria were counted. Data are representative of the results obtained with three independent samples. *, P < 0.05 versus water-treated control culture.

Fig. 2.

Coprisin analogue (coprisin) did not have antibiotic activity against the tested commensal bacteria. (A) Kanamycin, coprisin analogue, vancomycin, or clindamycin (0 to 30 μg/ml) was added to growing cultures of L. casei (2 × 10⁵ CFU/ml) at 37°C, and the cultures were incubated under anaerobic conditions. Samples were diluted after 48 h at a plateau of growth and plated onto MRS agar to score the residual numbers of CFU. The survival percentage values represent the numbers of colonies in treated samples relative to the numbers of colonies in control cultures (water treated). *, P < 0.01 versus water-treated control culture. (B) The indicated antibiotics (0 to 30 μg/ml) were added to growing cultures of L. delbrueckii subsp. lactis (2 × 10⁵ CFU/ml), and the cultures were incubated anaerobically. After 36 h at a plateau of growth, suitable dilutions were plated onto M17 agar and the residual numbers of CFU were counted. *, P < 0.01 versus water-treated control culture. (C) The indicated antibiotics were added to growing cultures of B. thermophilum (2 × 10⁵ CFU/ml), and the cultures were incubated anaerobically for 36 h. *, P < 0.01 versus water-treated control culture; #, P < 0.05 versus water-treated control culture.

Fig. 3.

Coprisin analogue (coprisin) causes membrane damage to C. difficile. (A) Bacteria were incubated with PI (100 ng/ml) and coprisin analogue (1 μg/ml) for 1 h and analyzed using a confocal scanning laser microscope (magnification, ×1,000). (B) The fluorescence in each bacterial suspension. The bars represent the means ± SEM of the results of three independent experiments, each with triplicate determinations. *, P < 0.005 versus medium-treated control (con) bacteria. (C) Coprisin analogue had no effect on cell membranes of B. thermophilum (magnification, ×1,000). (D) *, P < 0.005 versus medium-treated control bacteria.

Fig. 4.

Coprisin analogue (coprisin) prevents mucosal damage and inflammatory responses in mice infected with C. difficile. (A to F) Mice were treated with the antibiotic mixture for 3 days, followed by administration of clindamycin. After 1 day, C. difficile (5 × 10⁸ CFU) was added by gavage in the presence (CD plus coprisin analogue) and absence (CD) of coprisin analogue (1 μg/ml) administered orally. Control mice treated with the antibiotic mixture were administered water alone after clindamycin treatment. (A) Changes in body weight are presented as percentages of initial body weight. Data represent means ± SEM (n = 8 mice per group, with each experiment performed with triplicate determinations). (B) Kaplan-Meier survival plots. (C) Representative hematoxylin- and eosin-stained histological sections (magnification, ×200). (D) Concentrations of IL-6 in ileal tissues (n = 8). *, P < 0.001 versus C. difficile-infected mice. (E) Caspase activation by proteolytic processing. Tissue lysates were resolved on 15% polyacrylamide gels and probed with antibodies against caspase-3, caspase-8, and β-actin. Shorter fragments (lower bands) represent active forms of caspase-3 or caspase-8. (F) Ileal tissue extracts were plated on C. difficile-selective agar and incubated anaerobically for 48 h. (G) Colonocytes were exposed to tissue extracts (50 μl of extract/ml of culture medium) for 12 h, and cell rounding was measured (magnification, ×100).