We report the feasibility of enterocin AS-48, a circular cationic peptide produced by Enterococcus faecalis , as a new leishmanicidal agent. AS-48 is lethal to Leishmania promastigotes as well as to axenic and intracellular amastigotes at low micromolar concentrations, with scarce cytotoxicity to macrophages. AS-48 induced a fast bioenergetic collapse of L. donovani promastigotes but only a partial permeation of their plasma membrane with limited entrance of vital dyes, even at concentrations beyond its full lethality. Fluoresceinated AS-48 was visualized inside parasites by confocal microscopy and seen to cause mitochondrial depolarization and reactive oxygen species production. Altogether, AS-48 appeared to have a mixed leishmanicidal mechanism that includes both plasma membrane permeabilization and additional intracellular targets, with mitochondrial dysfunctionality being of special relevance. This complex leishmanicidal mechanism of AS-48 persisted even for the killing of intracellular amastigotes, as evidenced by transmission electron microscopy. We demonstrated the potentiality of AS-48 as a new and safe leishmanicidal agent, expanding the growing repertoire of eukaryotic targets for bacteriocins, and our results provide a proof of mechanism for the search of new leishmanicidal bacteriocins, whose diversity constitutes an almost endless source for new structures at moderate production cost and whose safe use on food preservation is well established.

The in vitro antimicrobial potency of the bacteriocin AS-48 is well documented, but its clinical application requires investigation, as its toxicity could be different in in vitro (haemolytic and antibacterial activity in blood and cytotoxicity towards normal human cell lines) and in vivo (e.g. mice and zebrafish embryos) models. Overall, the results obtained are promising. They reveal the negligible propensity of AS-48 to cause cell death or impede cell growth at therapeutic concentrations (up to 27 μM) and support the suitability of this peptide as a potential therapeutic agent against several microbial infections, due to its selectivity and potency at low concentrations (in the range of 0.3-8.9 μM). In addition, AS-48 exhibits low haemolytic activity in whole blood and does not induce nitrite accumulation in non-stimulated RAW macrophages, indicating a lack of pro-inflammatory effects. The unexpected heightened sensitivity of zebrafish embryos to AS-48 could be due to the low differentiation state of these cells. The low cytotoxicity of AS-48, the absence of lymphocyte proliferation in vivo after skin sensitization in mice, and the lack of toxicity in a murine model support the consideration of the broad spectrum antimicrobial peptide AS-48 as a promising therapeutic agent for the control of a vast array of microbial infections, in particular, those involved in skin and soft tissue diseases.