Identification and evaluation of Galleria mellonella peptides with antileishmanial activity

Leishmaniasis is a very serious group of diseases, they are caused by more than 20 species of protozoan parasites belonging to the genus of Leishmania. Epidemiological surveillance reveals that more than 350 million people in 98 countries are in potential risk of infection [1], which makes Leishmaniasis one of the six endemic diseases considered as high priorities worldwide [2]. The main clinical manifestations in humans are cutaneous (CL) affecting the skin, mucocutaneous (MCL) able to induce severe consequences in the mucous membranes; and visceral leishmaniasis (VL) considered life threatening when it is not treated. CL is considered by the World Health Organization (WHO) as an emerging and uncontrolled disease [3], with more than 1 million cases reported yearly worldwide [1,[4], [5], [6], [7], [8]]. It is mainly caused by the species Leishmania mexicana, Leishmania braziliensis and Leishmania panamensis in America. Although CL it is considered the most common and non-lethal clinical form of the disease and the consequences are severe. It is characterized for single or multiples ulcerative skin lesions, which result from infection of macrophages in the dermis. Ulcerations can persist for months and sometimes years and eventually cause disfiguring scars [9].

WHO recommendations for cutaneous leishmaniasis treatment rely primarily on chemotherapeutic agents. First-line treatment is pentavalent antimonial (SbV) compounds such as sodium stibogluconate (Pentostam^®) and meglumine antimoniate (Glucantime^®), they have been used since the 1940s [[10], [11], [12]]. Despite these drugs are effective in approximately 90% of the cases, there is a growing evidence of variable efficacy depending on Leishmania species, geographic region, presence of resistant strains and therapeutic schemes employed [[13], [14], [15]]. Substitute treatments are Amphotericin B, liposomal Amphotericin B (AmBisome^®) [16], Pentamidine isethionate and Miltefosine approved by FDA in march of 2014 [17]. Unfortunately, the use of first or second-line treatments is commonly associated with serious side effects, high toxicity (hepatic and cardio) and severe adverse reactions (musculoskeletal pains and renal failure). Additionally, infected patients that stay under medication for long periods; due to the drug side effects decide to abandon the medication and therefore fail to complete the full course of the treatment [3].

There is a considerable interest in identifying novel antileishmanial drugs with minimum adverse effects and low resistance generation. Based on these characteristics, a growing number of researchers have centered their attention in the evaluation of antimicrobial peptides (AMPs) as potential drugs. AMPs are considered part of the chemical defense system of most organisms; they play a very important role in the non-specific response against pathogen invasion [18]. Most of the AMPs contain 8 to 50 amino acid residues, are amphipathic and have a positively charge at physiological pH [19]. These characteristics are important because the AMPs mechanism of action is based on the interaction between the peptide and the lipids of microorganism cell membrane, resulting in the destabilization of the lipid bilayer and therefore cell death [20,21]. These molecules have been isolated from bacteria, fungi, plants and animals, showing a broad spectrum of antimicrobial activity, including the capability to kill cancer cells [22,23]. One of the most promising characteristics of AMPs is the membrane disturbing mechanism of action whereby they exert the biological activity and minimize the potential development of resistance from microorganisms.

The greater wax moth Galleria mellonella (G. mellonella) has been successfully used as a biological model to study the insect immune response [24,25]. Different studies of this biological model revealed a different response depending on the kind of challenge [[26], [27], [28]]. AMPs are synthesized into fat body and hemocytes of insects and they are secreted into hemolymph as part of the systemic response against pathogens [29,30]. At least eighteen defense peptides with different biochemical and antimicrobial properties have been identified in G. mellonella so far [29,31,32]. Among these peptides, Moricins, Cecropin-D an homologous from Cecropin-A (H. cecropia), that belongs to the chemical arsenal in Lepidoptera insects presented a recognized antifungal activity [33,34]. The evaluation of Cecropin-D has demonstrated a broad antimicrobial activity against filamentous fungi (A. niger), Gram-negative (E. coli D31) and Gram-positive bacteria (M. luteus, L. monocytogenes, and S. lutea) [32]. Regardless of the well-recognized antimicrobial activity of G. mellonella peptides, there are no studies about their activity against parasites. For this reason, the main goal of this study was to investigate the differential expression of peptides on G. mellonella hemolymph due to the immunization with Leishmania (Viannia) panamensis (L. (V) panamensis) promastigotes and their potential antileishmanial activity. The immunological response of G. mellonella larvae was followed by using Tris-tricine sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), reversed-phase HPLC (RP-HPLC) and two-dimensional gel electrophoresis. The identification of the peptides was performed by liquid chromatography-mass spectroscopy (LC/MS). The results described the changes observed in the peptide profiles when the non-challenged and challenged hemolymph was evaluated by using the analytical techniques previously mentioned. Additionally, this paper reported the antileishmanial activity of hemolymph fractions from G. mellonella larvae and identification of four antimicrobial peptides in these anti-parasitic fractions, of which two of these antimicrobial peptides showing anti-parasitic activity and their potential as antileishmanial drugs.