We previously demonstrated that coprisin analogue has antibacterial activity against various pathogenic bacterial species (13
). Here, we assessed whether this peptide had antibiotic activity against C. difficile
, the primary etiologic agent of antibiotic-associated pseudomembranous colitis and severe diarrhea in humans and animals (4
). Our results revealed that coprisin analogue treatment significantly inhibited the growth rate of C. difficile
() but did not alter the growth rates of Lactobacillus
(). Given that normal microbiota (46
), along with probiotics, have inhibitory activities against pathogenic bacteria (3
) and that effective antibiotics should have specific antimicrobial activity against pathogenic but not nonpathogenic microbes, coprisin analogue may be a good candidate for use as a potential therapeutic reagent for C. difficile
-associated acute colitis.
Although antibiotic-associated diarrhea has been linked to numerous antibiotics, including the beta-lactam antibiotics (26
), clindamycin, which is usually used to treat anaerobic bacterial infections, is considered to be a primary cause of C. difficile
-associated diarrhea and pseudomembranous colitis (16
). Here, we found that clindamycin treatment markedly inhibited the growth of the tested Lactobacillus
species (L. casei
and L. delbrueckii
), as well as Bifidobacterium
and C. difficile
. This nonselective antibiotic activity against normal microbiota, which can inhibit the growth of pathogenic bacteria, may facilitate C. difficile
colonization and subsequent damage. Compared to vancomycin, which has antimicrobial activity against Bifidobacterium
but not Lactobacillus
species (), coprisin analogue is more selective and does not appear to have antibiotic activity against the tested normal gut microorganisms.
The amino acid sequence of coprisin is very similar to that of the ~40-amino-acid defensin and defensin-like peptides, which confer antibacterial activity by disrupting the membrane or suppressing cell cycle signaling (48
). In the current study, we found that coprisin analogue treatment damaged the plasma membrane of C. difficile
but not that of Bifidobacterium
sp. ( and D). Since both of those bacterial species are Gram positive and since they have biologically similar membranes (3
), future work would be necessary to determine the basis for the selective antimicrobial activity of coprisin analogue. For example, a specific receptor for coprisin analogue may exist on the plasma membrane of C. difficile
, or negatively charged components of the lipid membrane, such as teichoic acids (30
) and peptidoglycan (30
), may play a role in the interaction with coprisin analogue. Alternatively, the selectivity of coprisin analogue may arise as a result of the presence of three positively charged amino acids (NH2
-RKK-COOH) at the C terminus of the peptide. The defensin family peptides can interact with a wide variety of membrane components, including lipopolysaccharides (41
), cardiolipin (11
), and sphingolipids (44
); beyond those interactions, structural features of the peptides further determine their specificity for binding to the surface of a given microorganism (e.g., via disulfide cross-linking through a cysteine) (7
). Since coprisin analogue contains a cysteine in the middle position, potentially allowing it to dimerize, the peptide structure itself may affect its microbial binding capacity or selectivity.
In a mouse model of acute gut inflammation following C. difficile infection, the presence of coprisin analogue markedly ameliorated inflammatory responses and weight loss and improved survival rates. The sharp decreases in body weight on days 2 and 3 following C. difficile infection were only partially reversed by coprisin analogue treatment; it was not until day 4 that coprisin analogue-treated mice returned to control-level body weights. However, coprisin analogue did not prevent inflammation resulting from injection of purified toxin A into the ileal lumen (data not shown), suggesting that the apparent anti-inflammatory activity of coprisin analogue is associated with its antimicrobial activity rather than with inhibition of the activity of the toxins.
In summary, we report that coprisin analogue, a disulfide dimer and insect-derived peptide, has selective antibiotic activity against C. difficile but not Lactobacillus and Bifidobacterium, members of the normal bowel flora. Furthermore, coprisin treatment has a strong beneficial effect on C. difficile infection-induced mouse gut inflammation. These novel findings suggest that coprisin could be a useful candidate for therapeutic use against C. difficile-associated diarrhea and pseudomembranous colitis.