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. 2022 Oct 10:13:1023114.
doi: 10.3389/fphar.2022.1023114. eCollection 2022.

HAS 1: A natural product from soil-isolated Streptomyces species with potent activity against cutaneous leishmaniasis caused by Leishmania tropica

Bassel Awada  1   2 , Maguy Hamie  1   2 , Rana El Hajj  3 , Ghada Derbaj  1   2 , Rania Najm  4 , Perla Makhoul  1   2 , Dima Hajj Ali  1 , Antoine G Abou Fayad  1   2 , Hiba El Hajj  1   2
Affiliations

HAS 1: A natural product from soil-isolated Streptomyces species with potent activity against cutaneous leishmaniasis caused by Leishmania tropica

Bassel Awada et al. Front Pharmacol. .

Abstract

Cutaneous Leishmaniasis (CL) is a neglected tropical disease, classified by the World Health Organization (WHO) as one of the most unrestrained diseases. The Syrian war and the significant displacement of refugees aggravated the spread of this ailment into several neighboring countries in the Eastern Mediterranean Region (EMR). In Syria, Leishmania tropica is identified as one of the most aggressive and endemic identified species, causing localized or generalized lesions, often chronic or relapsing. Pentavalent antimonial drugs are currently used as first line treatment against CL. Nonetheless, these drugs exhibit several limitations, including the repetitive painful injections, high cost, poor availability, and mainly systemic toxicity. Besides, the emergence of acquired parasitic resistance hinders their potency, stressing the need for new therapies to combat CL. Natural products (NPs) epitomize a valuable source in drug discovery. NPs are secondary metabolites (SMs) produced by plants, sponges, or a wide variety of organisms, including environmental microorganisms. The EMR is characterized by its immense biodiversity, yet it remains a relatively untapped area in drug discovery. NPs of the region were explored over the last 2 decades, but their discoveries lack biogeographical diversity and are limited to the Red Sea. Here, we isolated previously uncultured environmental soil-dwelling Streptomyces sp. HAS1, from Hasbaya region in southeast Lebanon. When fermented in one of our production media named INA, HAS1 produced a crude extract with significant potency against a clinical Leishmania tropica isolate. Using bio-guided fractionation, the bioactive compound was purified and the structure was elucidated by NMR and LC-HRMS. Our findings establish NPs as strong candidates for treating Leishmania tropica and further dwells on the importance of these natural sources to combat microbial infections.

Keywords: Leishmania tropica; bio-guided fractionation; cutaneous leishmaniasis; natural products; soil extract; streptomyces.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
Crude extracts produced by environmental bacteria derived from Lebanese soil inhibit L. tropica amastigotes replication. (A). Pipeline of secondary metabolite production by environmental bacteria isolated from soils collected from selected region across Lebanon. Bacteria from Hasbaya (HAS), Koura (KR) and Bchamoun (BCH) were fermented in a collection of 14 different production media. XAD resins were added 14 days post bacterial inoculation to adsorb the SMs, which were then extracted via organic solvents. The overall SMs collected from an isolate in a specific medium were named after the isolate followed by the medium name, for example HAS1 INA. (B). Real-time quantitative PCR detection of THP-1 derived macrophages infected with patient-derived L. tropica amastigotes treated with 0.01 μg/ml of crude extracts HAS1 C, BCH8 NL2, HAS1 M8, KR24 V6, and HAS1 INA for 24 h. Briefly, THP-1 monocytes were differentiated into macrophages, activated with LPS, and infected with L. tropica with a ratio of one parasite/three cells. Treatment with 0.01 μg/ml of the crude extracts or 1 μM of EABP0503 was then performed for 24 h. The results are shown as percentage of untreated infected macrophages. Amastigote transcripts were evaluated by Syber green RT-PCR using kinetoplast specific primers and their percentage of expression was normalized to GAPDH. Results are expressed as percentage of the untreated control (±) SEM and are representative of at least three independent experiments. To validate statistical significance, t-tests were used with *** representing p-values < 0.001.
FIGURE 2
FIGURE 2
Morphological, physiological, and biochemical characterization of the bacterial isolate HAS1. (A). Growth, shape, color of the mycelium of the isolate HAS1 and spore formation on ISP3 agar 10 days after bacterial inoculation with an incubation at 28°C. (B). Physiological characterization of HAS1 isolate using bacterial growth on the 5,339 agar with different NaCl concentrations (0, 2.5, 5, 7.5 and 10%) after incubation for 10 days at 28°C. (C). Growth of HAS1 on ISP2 agar with different pH (3, 4, 5, 6, 7, 8, 9, and 10) after a 10 days at 28°C. (D). Biochemical testing of HAS1 using the API 20E kit.
FIGURE 3
FIGURE 3
Bio-guided fractionation of the antileishmanial compounds of HAS1 INA. (A). An upscaled fermentation of the HAS1 INA crude was first subjected to liquid-liquid partition generating four fractions: HAS1 INA Hexane, HAS1 INA chloroform, HAS1 INA Ethyl Acetate, and HAS1 INA Water. HAS1 INA chloroform was further fractionated into four pure compounds using HPLC: HAS1-F1, HAS1-F2, HAS-F3, and HAS1-F4. (B,C). Real-time quantitative PCR detection of THP-1 derived macrophages infected with patient-derived L. tropica amastigotes treated with 0.01 μg/ml of HAS1 INA fractions and the crude extract (B) or with 1 μg/ml of the pure compounds for 24 h or with 1 μM of EABP0503 for 24 h (C). Amastigote transcripts were evaluated by Syber green RT-PCR using kinetoplast specific primers and their percentage of expression was normalized to GAPDH. Results are expressed as percentage of the untreated control (±) SEM and are representative of at least three independent experiments (C) and one representative experiment for (B). To validate statistical significance, t-tests were used with ** representing p-values < 0.01, and *** representing p-values < 0.001. (D). H&E staining of THP-1 derived macrophages infected with patient-derived L. tropica amastigotes treated with 1 μg/ml of HAS1-F1 or HAS1-F2 for 24 h. (E). Immunofluorescence microscopy of THP-1 derived macrophages infected with patient-derived L. tropica amastigotes treated with 1 μg/ml of HAS1-F1 or HAS-F2. The Gp63 surface parasite was stained with an anti-Gp63 antibody (green), and nuclei were stained with Hoechst 33,342 (blue). The graph shows the quantification of GP63 as averages from 50 different cells which were then expressed as percentage of the untreated control (±) SEM. To validate statistical significance, t-tests were used with * representing p-values < 0.05, and ** representing p-values < 0.01. (F). Blind count of motile promastigotes of L. tropica treated with 0.01 μg/ml of HAS1 INA, 1 μg/ml of HAS1-F1 or HAS1-F2, or 1 μM of EABP0503 for 24 h. Results are expressed as percentage of the untreated control (±) SEM and are representative of at least three independent experiments. To validate statistical significance, t-tests were used with *** representing p-values < 0.001.
FIGURE 4
FIGURE 4
Structure of HAS1-F1.
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