Enterocin AS-48 as Evidence for the Use of Bacteriocins as New Leishmanicidal Agents

Abstract

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.

Keywords: antimicrobial peptide; bioenergetics; enterocin AS-48; intracellular parasite.

FIG 1

In vivo monitoring of intracellular ATP levels of L. donovani 3-Luc promastigotes after AS-48 addition. Promastigotes were resuspended at 2 × 10⁷ cells/ml. DMNPE-luciferin was added at 25 μM. Once the readout became stable, AS-48 was added (t = 0), and variation in luminescence was expressed as the percentage of luminescence with respect to the luminescence of control, untreated parasites. AS-48 micromolar concentration is indicated by symbols: ○, 0.8; ■, 1.6; □, 3.1; ▼, 6.2; △, 12.5; ▲, 25.0. These results are from an experiment representative of three other experiments performed independently.

FIG 2

Permeabilization of the plasma membrane of L. donovani promastigotes by AS-48. Parasites were resuspended at 2 × 10⁷ cells/ml in HBSS-Glc with the corresponding probe. AS-48 was added (t = 0), and fluorescence is noted as a percentage relative to that of fully permeabilized parasites. (A) Kinetics of intracellular entrance of the vital dye Sytox green. Promastigotes were resuspended in HBSS-Glc–1 μM Sytox green. Fluorescence settings: λ_EXC = 485 nm, λ_EM = 520 nm. The arrow shows the time of the addition of TX-100 (final concentration, 0.1%), taken as fully permeabilized parasites. (B) Kinetics of plasma membrane depolarization. Parasites were resuspended in HBSS-Glc–0.1 μM bisoxonol. Fluorescence settings: λ_EXC = 544 nm, λ_EM = 584 nm. Fully depolarized parasites (100% fluorescence) were considered to be those treated with 10 μM CA(1-8)M(1-18). AS-48 micromolar concentration is indicated by symbols: ●, 0.78; ■, 1.6; □, 3.1; ▼, 6.2; △, 12.5; ▲, 25.0. (C) Endpoint cytofluorometric determination for both parameters after 4 h of incubation. Propidium iodide (PI; 1 μg/ml) and bisoxonol (0.1 μM) were added to the parasites 5 min before the cytofluorometric analysis. Fluorescence settings: for bisoxonol, λ_EXC = 488 nm, λ_EM = 525 nm; for PI, λ_EXC = 488 nm, λ_EM = 620 nm. Positive controls for permeabilization were 0.1% TX-100 (PI) and 10 μM CA(1-8)M(1-18) (bisoxonol). The percentage of the fully permeabilized population is shown inside the corresponding histogram. a.u., arbitrary units.

FIG 3

Confocal microscopy of L. donovani promastigotes treated with fluoresceinated AS-48. Promastigotes were incubated with fluoresceinated AS-48 (Fl-AS-48) at different concentrations. Incubation was carried out with 2 × 10⁷ cells/ml for 4 h in HBSS-Glc. Promastigotes were stained with DAPI (10 μg/ml, 5 min) prior to their observation as unfixed parasites in confocal microscopy. Nomarski, differential interference contrast microscopy. Fluorescence settings: Fl-AS-48, λ_EXC = 488 nm, λ_EM = 519 nm; DAPI, λ_EXC = 358 nm, λ_EM = 461 nm. Bar, 10 μm.

FIG 4

Assessment of mitochondrial damage to L. donovani promastigotes by AS-48. (A) Inhibition of ΔΨ_m and respiration (inset). Parasites were incubated with AS-48 for 4 h (HBSS-Glc, 2 × 10⁷ cells/ml), loaded with rhodamine 123, and analyzed by cytofluorometry. Parasites incubated with 10 mM KCN were used for positive control of mitochondrial depolarization. (A) (Inset) Oxygen consumption rate of promastigotes treated with AS-48. Respiration was measured at 10⁸ cells/ml in a Clark oxygen electrode, and values were expressed as percentages with respect to respiration in untreated cells. Lanes 1, 2, and 3, parasites treated with AS-48 at 12.5, 25, and 50 μM, respectively; lane 4, parasites treated with 10 μM CA(1-8)M(1-18) as a positive internal control. (B) Production of mitochondrial ROS induced by AS-48. Parasites were loaded with 0.5 μM Mitosox Red. Control parasites (black [left] bars). Parasites treated either with 3 μg/ml antimycin A (light-gray [middle] bars) as a positive control or with 5 μM AS-48 (dark-gray [right] bars). a.u., arbitrary units. Samples were taken at different times and analyzed by cytofluorometry (λ_EXC = 488 nm, λ_EM = 520 nm); *, P < 0.05; **, P < 0.01.

FIG 5

Electron microscopy of RAW 264.7 murine macrophage cells infected with L. pifanoi and treated with AS-48. Raw 264.7 cells were infected with L. pifanoi axenic amastigotes as described in Materials and Methods. Once the infection was established, infected macrophages were left untreated or were treated with 7 μM AS-48 for 24 h and processed for electron microscopy. The white arrow and star indicate the cellular debris from killed amastigotes inside the parasitophorous vacuole of AS-48-treated macrophages. Upper row, control parasites; lower row, AS-48-treated parasites. Bar, 10 μm.