Repurposing the Antibacterial Agents Peptide 19-4LF and Peptide 19-2.5 for Treatment of Cutaneous Leishmaniasis

Abstract

The lack of safe and cost-effective treatments against leishmaniasis highlights the urgent need to develop improved leishmanicidal agents. Antimicrobial peptides (AMPs) are an emerging category of therapeutics exerting a wide range of biological activities such as anti-bacterial, anti-fungal, anti-parasitic and anti-tumoral. In the present study, the approach of repurposing AMPs as antileishmanial drugs was applied. The leishmanicidal activity of two synthetic anti-lipopolysaccharide peptides (SALPs), so-called 19-2.5 and 19-4LF was characterized in Leishmania major. In vitro, both peptides were highly active against intracellular Leishmania major in mouse macrophages without exerting toxicity in host cells. Then, q-PCR-based gene profiling, revealed that this activity was related to the downregulation of several genes involved in drug resistance (yip1), virulence (gp63) and parasite proliferation (Cyclin 1 and Cyclin 6). Importantly, the treatment of BALB/c mice with any of the two AMPs caused a significant reduction in L. major infective burden. This effect was associated with an increase in Th1 cytokine levels (IL-12p35, TNF-α, and iNOS) in the skin lesion and spleen of the L. major infected mice while the Th2-associated genes were downregulated (IL-4 and IL-6). Lastly, we investigated the effect of both peptides in the gene expression profile of the P2X7 purinergic receptor, which has been reported as a therapeutic target in several diseases. The results showed significant repression of P2X7R by both peptides in the skin lesion of L. major infected mice to an extent comparable to that of a common anti-leishmanial drug, Paromomycin. Our in vitro and in vivo studies suggest that the synthetic AMPs 19-2.5 and 19-4LF are promising candidates for leishmaniasis treatment and present P2X7R as a potential therapeutic target in cutaneous leishmaniasis (CL).

Keywords: antimicrobial peptides (AMPs); cytokines; drug repurposing; drug resistance; leishmaniasis; peptide 19-2.5; peptide 19-4LF; proliferation.

Figure 1

Leishmanicidal activity of 19-2.5 (1 µg/mL) and 19-4 LF (1 µg/mL) against intramacrophage amastigotes (A). Bars represent the mean ± SD values from three independent experiments. Significant reductions in the number of amastigotes per infected macrophage were detected when compared to untreated controls (***, p < 0.001). Representative images of untreated infected cells and infected macrophages treated with 1 µg/mL of 19-2.5 or 19-4LF peptides (B).

Figure 2

The effect of 19-2.5 and 19-4 LF on L. major amastigotes gene expression levels. The graphs show the expression levels of genes related to drug resistance, [yip 1 (A), ABCC6 (B)], virulence [gp63 (C)], and Leishmania proliferation [cyclin 1 (D), and cyclin 6 (E)] after 24 h of treatment with 19-2.5 (1 µg/mL) and 19-4 LF (1 µg/mL). GAPDH was used as a housekeeping gene to normalize L. major gene expression. The amount of each transcript was expressed by the formula 2^{ct(GAPDH)−ct(gene)}. Bars represent the mean ± SD from three independent experiments. Significant differences compared to control (untreated cells) were indicated (*, p < 0.05; **, p < 0.01).

Figure 3

Effect of peptides 19-2.5 and 19-4 LF on parasite burden in the skin lesions (A) and the spleen (B) of L. major infected mice after treatment compared to controls (untreated mice). The parasite burden was evaluated by quantifying the Lm18S mRNA gene expression levels using qPCR method. Bars represent the mean ± the SD. Significant difference was indicated (**, p < 0.01 compared with the untreated control).

Figure 4

Gene expression levels of Th1 cytokines (IL 12 p35 and TNFα), iNOS gene, Th2 cytokines (IL 4 and IL 6), cytokine receptors (TNFR1, IL4 Rα and IL6 R) and CDKN1A gene from the skin lesions of BALB/c mice treated with 19-2.5 and 19-4 LF peptides or untreated. β-actin was used as a reference gene to normalize mouse gene expression. The amount of each transcript was expressed by the formula 2^{ct(actin)−ct(gene)}. Bars represent the means (±SD) (*, p < 0.05; **, p < 0.01).

Figure 5

Gene expression levels of Th1 cytokines (IL 12 p35 and TNFα), iNOS gene, Th2 cytokines (IL 4 and IL 6), cytokine receptors (TNFR1, IL4 Rα and IL6 R) and CDKN1A gene from the spleen of BALB/c mice treated with 19-2.5 and 19-4 LF peptides or untreated. β-actin was used as a housekeeping gene to normalize mouse gene expression. The amount of each transcript was expressed by the formula 2^{ct(actin)−ct(gene)}. Bars represent the means (±SD) (*, p < 0.05; **, p < 0.01).

Figure 6

Effect of PM and the two peptides (19-2.5 and 19-4LF) on the expression levels of P2X7 receptor gene in the skin lesion and the spleen of treated BALB/c mice compared to untreated mice (controls). The bars represent the means ± SD. Significant differences were indicated (*, p < 0.05; **, p < 0.01 compared to controls).

Figure 7

Leishmanicidal effect of PM (50 µM) (A), Ampho B (0.025 µM) (B) and their combinations with peptides Pep19-2.5 (1 µg/mL) and Pep19-4 LF (1 µg/mL). Bars represent the means (±SD) from three independent experiments (*, p < 0.05; ***, p < 0.001).