Selective reciprocity in antimicrobial activity versus cytotoxicity of hBD-2 and crotamine

Abstract

Recent discoveries suggest cysteine-stabilized toxins and antimicrobial peptides have structure-activity parallels derived by common ancestry. Here, human antimicrobial peptide hBD-2 and rattlesnake venom-toxin crotamine were compared in phylogeny, 3D structure, target cell specificity, and mechanisms of action. Results indicate a striking degree of structural and phylogenetic congruence. Importantly, these polypeptides also exhibited functional reciprocity: (i) they exerted highly similar antimicrobial pH optima and spectra; (ii) both altered membrane potential consistent with ion channel-perturbing activities; and (iii) both peptides induced phosphatidylserine accessibility in eukaryotic cells. However, the Nav channel-inhibitor tetrodotoxin antagonized hBD-2 mechanisms, but not those of crotamine. As crotamine targets eukaryotic ion channels, computational docking was used to compare hBD-2 versus crotamine interactions with prototypic bacterial, fungal, or mammalian Kv channels. Models support direct interactions of each peptide with Kv channels. However, while crotamine localized to occlude Kv channels in eukaryotic but not prokaryotic cells, hBD-2 interacted with prokaryotic and eukaryotic Kv channels but did not occlude either. Together, these results support the hypothesis that antimicrobial and cytotoxic polypeptides have ancestral structurefunction homology, but evolved to preferentially target respective microbial versus mammalian ion channels via residue-specific interactions. These insights may accelerate development of antiinfective or therapeutic peptides that selectively target microbial or abnormal host cells.channel ͉ defensin ͉ host defense ͉ toxin

In vitro antibacterial and hemolytic activities of crotamine, a small basic myotoxin from rattlesnake Crotalus durissus

“…In our study, among five Gram-negative and two Gram-positive bacterial strains that we tested, crotamine showed an obvious bactericidal activity only against E. coli, with a weak or no activity against all others in the MIC assay, although the time-kill assay revealed a weak activity against S. aureus. Consistent with our results, Yount et al 22 reported the antibacterial activity of crotamine against E. coli, but not S. aureus, using a radial diffusion method. In the same study, the activity against a Gram-positive (Bacillus subtilis) and fungal (Candida albicans) pathogen was also observed with crotamine.…”

“…Following our comprehensive testing on the antibacterial and cytotoxic properties, we now can unequivocally conclude that crotamine is also a b-defensin-like AMP with a salt-sensitive antibacterial activity. To strengthen our conclusion, an independent study also demonstrated the antibacterial activity of crotamine 22 during the preparation of this manuscript.…”

“…Indeed, similar to crotamine, a b-defensin was shown to interact with K + channels, albeit with a different specificity. 22 Although hBD-2, a human b-defensin, interacted with both prokaryotic and eukaryotic voltage-sensitive K + channels, crotamine only interacted with eukaryotic K + channels. 22 The amino-acid sequence variation between crotamine and b-defensins may explain their difference in ion channel targeting.…”

“…22 Although hBD-2, a human b-defensin, interacted with both prokaryotic and eukaryotic voltage-sensitive K + channels, crotamine only interacted with eukaryotic K + channels. 22 The amino-acid sequence variation between crotamine and b-defensins may explain their difference in ion channel targeting. Taken together, our results showing the antibacterial activity of crotamine reinforce the notion that crotamine evolved from the duplication of the b-defensin gene lineage.…”

Construction of recombinant E. coli Nissle 1917 (EcN) strains for the expression and secretion of defensins

“…However, as other modes of defensin action are discussed, we cannot exclude other mechanisms of antimicrobial activity exhibited by HisMHBD2 (Yount et al 2009). …”