Chagas disease and leishmaniasis are major human vector-borne neglected tropical diseases caused by the related hemoflagellate parasites Trypanosoma cruzi and Leishmania spp. In 2019, approximately 6 to 8 million people were estimated to be infected with T. cruzi, and an additional 70 million were estimated to be at risk of infection (Lidani et al., 2019). The World Health Organization considers leishmaniasis to be an emerging and uncontrolled disease complex (Anon, 2024), with an estimated 1 million new cases annually in 2012 while 12 million people were considered to be infected (Alvar et al., 2012). Since then, the number of cases of cutaneous leishmaniasis, which is the most common and transient form of the disease, peaked in 2019 with 280,679 reported cases (Anon, 2024).

Leishmaniasis is transmitted by sand flies, which egest the infectious metacyclic stage of the parasite into the vertebrate host upon taking a blood meal (Serafim et al., 2021). The disease has a broad clinical spectrum, causing fatal visceral, cutaneous, and mucocutaneous leishmaniasis. Cutaneous leishmaniasis is the most common clinical manifestation that is caused by L. major in the Old World and may lead to disfiguring ulcerative lesion that occurs at the site of infection in humans (Desjeux, 2004). The life cycle of Leishmania parasites includes the extracellular promastigote stages within the sand fly vector and an intracellular amastigote form which inhabits the macrophages of the vertebrate host (Mann et al., 2021; Serafim et al., 2021).

The life cycle of T. cruzi, the vector for Chagas disease, starts when the trypomastigote (blood form) is ingested by blood-sucking triatomine bugs during a blood meal. Then, the parasites reproduce and transition from epimastigotes to the infectious metacyclic trypomastigotes in the insects’ gut. Upon taking a blood meal, the infectious metacyclic trypomastigotes are transmitted to the vertebrate host from infectious insect feces by either rubbing them at the bite site of the triatomine bug or by ingestion of the infected vector by the mammal. In the vertebrate host, the pathological human life stages of T. cruzi are the intracellular amastigotes (Cucunubá et al., 2024).

Current treatment options for leishmaniasis are limited to a small number of drugs, including arsenicals, amphotericin B, pentavalent antimonials, and anticancer alkyl-lysophospholipids that are expensive, toxic, and not fully effective (Croft et al., 1996; Mitropoulos et al., 2010). Nifurtimox and Benznidazole are the two most common drugs currently used to treat Chagas disease, however, these drugs are often associated with toxicity problems (e.g. nephrotoxicity and cardiotoxicity) and emergence of resistance (Berhe et al., 2024). In an effort to address the need for new and efficacious drugs, research studies have evaluated small peptide antimicrobial compounds for killing both trypomastigotes and amastigotes parasite stages (Vizioli and Salzet, 2002). Antimicrobial peptides (AMPs) are cationic proteins that are common components of the innate immune systems of many organisms, and studies have shown that host defense lytic peptides of both insects and vertebrates have cytotoxic activity toward Leishmania promastigotes and amastigotes (Perez-Cordero et al., 2011; Lynn et al., 2011). Lytic peptides are part of the nonspecific immune system of eukaryotes that destroy the membranes of microorganisms (Leuschner and Hansel, 2004; Guaní-Guerra et al., 2010) but are not likely to harm higher eukaryotes because they do not affect the electrically neutral cholesterol-containing cell membranes of higher eukaryotes (Javadpour et al., 1996; Bell, 2011).

McGwire and Kulkarni (2010) reviewed the interactions of AMPs found in aquatic animals, insects, plants and mammals with pathogenic Leishmania and Trypanosoma species. Multiple studies on insect AMPs used different forms of cecropin-like lytic peptides; the lytic peptide cecropin was first described from silkworm. Barr et al. (1995) used three peptides (DC-1, DC-2, and DC-2R) that were synthesized and had similar amphipathic and hydrophobic properties of cecropin B. The authors showed that all three peptides were effective in killing 100% of T. cruzi trypomastigotes in vitro at a concentration of 10 μM. In addition, a significant reduction in amastigote numbers in infected Vero cells was observed for all three peptides at 2.5 μM, with no toxicity at that concentration. The peptide DC-1 (Hecate) was tested intravenously for toxicity in mice with no observed effects at 50 micrograms daily for 10 days, and a significant reduction in T. cruzi parasitemia in infected mice was observed for mice treated 5 times over 10 days with 25 micrograms IV. DC-2 was effective in vitro at less than 1 μM and ranked at 4+ on a 1-4+ scale, placing it among the most potent AMPs in the insect category along with attacin and cecropin-melittin hybrids (McGwire and Kulkarni, 2010).

Lytic peptide action can be targeted to specific cell types by the addition of a ligand. For example, Hansel et al. (2007) reported that lytic peptides conjugated with cancer cell membrane receptor ligands could be used to destroy breast cancer cells, while lytic peptides alone or conjugated with non-specific peptides were not effective. Lytic peptides also have been conjugated with human hormones that bind to receptors on tumor cells for targeted destruction of prostate and testicular cancer cells (Leuschner and Hansel, 2004). Phage display technique was used to identify peptides that attach to the vital lignocellulose-digesting flagellate protozoa (phylum Parabasalia) in the guts of workers of the Formosan subterranean termite, Coptotermes formosanus (Husseneder et al., 2010; Sethi et al., 2014). The authors selected and synthesized two of 19 candidates, attached the fluorophore EDANS, and confirmed binding to the protozoa in vitro. Then they fused the ligand that was associated with the variant surface glycoprotein of T. brucei with Hecate (Ligand-Hecate) and demonstrated that the fusion peptide killed the protozoa in the termite gut more effectively than Hecate alone in vitro and in vivo. In addition, the authors confirmed that ligands designed against Parabasalia protozoa also bind to at least 4 other phyla and are thus likely conserved enough to target trypanosomatid life stages (Husseneder et al., 2010; Sethi et al., 2014).

The purpose of this study is to determine if the concept of targeting extracellular life stages of T. cruzi and L. major and intracellular L. major using the ligands developed in prior studies (Husseneder et al., 2010; Sethi et al., 2014) with the synthetic lytic peptide Hecate (Barr et al., 1995) can be applied to provide safe and efficient parasite treatment. In the current study, we compared the antiparasitic activity and cytotoxicity of Hecate and Ligand-Hecate constructs against L. major promastigotes and T. cruzi epimastigotes and assessed the inhibition of L. major amastigote development in macrophages after treatment with these lytic peptides.

As a first step towards efficient targeting of lytic peptides to trypanosomatids, we tested two ligands for their capacity to bind L. major promastigotes. These ligands were originally designed via phage display of linear random heptapeptides to identify termite protozoa-recognizing peptides (Husseneder et al., 2010; Sethi et al., 2014). Two out of 19 peptides were selected for ligand development and shown using fluorophore EDANS to bind with the symbiotic termite gut protozoa but not the gut wall in vivo (Husseneder et al., 2010). Initially, both ligands were tested and shown to attach across the cell membrane of L. major promastigotes. Since both ligands showed similar binding patterns, only Ligand 1 was chosen to synthesize fusion proteins with the lytic peptide Hecate (Ligand-Hecate) and testing for parasiticidal activity. Ligand 1 was selected because it showed homology to epitopes present on the variant surface glycoprotein of Trypanosoma brucei. Additionally, we had prior evidence that a Ligand 1-Hecate fusion protein successfully kills protozoan symbionts in termites and is evolutionary conserved enough to target membrane receptors across a variety of protozoan phyla (Husseneder et al., 2010; Sethi et al., 2014).

Herein, we showed that the Hecate and Ligand-Hecate lytic peptides were most effective in killing both T. cruzi epimastigotes and L. major promastigotes at 48 h. Importantly, both lytic peptides showed negligible cytotoxicity to intraperitoneal murine macrophages even at high concentrations across all treatment durations. Similarly, Barr et al. (1995) showed that Hecate had no toxicity effect on Vero cell cultures at 2.5 μM but did cause a significant reduction in the number of T. cruzi amastigotes in infected Vero cells when administered at a double exposure of 2.5 μM at 24 h and 48 h post-incubation. The therapeutic indices of Hecate and Ligand-Hecate were both higher against L. major promastigotes than T. cruzi epimastigotes.

The proliferation of intracellular L. major amastigotes propagated inside murine macrophages was significantly reduced after treatment with Hecate or Ligand-Hecate and both lytic peptides were equally successful in reducing the percentage of infected cells as 5 μM of the commercial trypanomiasis treatment Amphotericin B. However, the Ligand-Hecate treatment outperformed treatment with Hecate alone at lower concentrations. The addition of the ligand increased the efficiency of Hecate in inhibiting the proliferation of the L. major intracellular amastigotes at a 4X lower concentration (0.41 μM) when compared to Hecate alone (1.62 μM) and thus Ligand-Hecate ranks highest among AMPs previously tested (McGwire and Kulkarni, 2010). Similarly, Sethi et al. (2014) showed that Ligand-Hecate had increased efficiency over Hecate alone killing the symbiotic protozoa of termites faster than Hecate alone.

In the present study we only tested the lytic peptides against T. cruzi in vitro. However, Barr et al. (1995) did provide proof that Hecate reduced the development of parasitemia of T. cruzi in mice and was not toxic to mice. Furthermore, the authors showed that 100% of mice infected with T. cruzi died by day 14 post-infection, while mice treated with Hecate after infection showed no weight loss or mortality by this timepoint. Studies like the one of Barr et al. (1995) but adding ligands to Hecate would be the next step towards development of AMP therapy against trypanosomatids. Importantly, the performance of the Ligand-Hecate treatment tested in our study warrants future studies using available in vivo models for pathogenic species of Trypanosoma and Leishmania.

Our study indicates the therapeutic action of AMPs against both extracellular and intracellular protozoa parasites with no significant toxicity to host cells. However, the mechanisms of cytolysis by AMPs are not well understood. The AMPs are small cationic proteins of the innate immune system of organisms which contain high percentages of basic amino acids that form either α-helical of β-pleated sheets. The AMPs have been promoted as alternatives for conventional antiprotozoal drugs because of low toxicity and less likely occurrence of resistance (Giovati et al., 2018). The amphipathicity of AMPs enables them to interact with negatively charged membranes and destabilize surface-membranes through a variety of mechanisms (McGwire and Kulkarni, 2010). Mutwiri et al. (2000) studied the potential mechanisms for membrane disruption of Trichomonas spp. caused by treatment with Hecate. The authors used scanning electron microscopy to demonstrate the extensive damage to plasma membranes of the trichomonads caused by 10 ppm D-Hecate in just 10 min. Mutwiri et al. (2000) concluded that time dependent accumulation of high concentrations of peptides was more likely to contribute to membrane disruption and cell disintegration rather than changes osmotic pressure.

The need for and approaches to developing conjugates to increase specificity and efficacy of AMPs varies by targeted cell membranes, hosts, and desired outcomes. Studies have shown that lytic peptides conjugated with cancer cell membrane receptor ligands or human hormones destroy tumor cells while lytic peptides alone or conjugated with non-specific peptides were not effective (Leuschner and Hansel, 2004; Hansel et al., 2007). Similarly, the ligand designed via phage display to attach to termite protozoa, protected non-targets like bacteria and termite gut tissue from lytic peptide action when fused to Hecate (Husseneder et al., 2010; Sethi et al., 2014), while efficiently killing cellulose digesting symbiotic protozoa of Formosan subterranean termites in vitro and in vivo. The same Ligand-Hecate construct was used in the present study to target L. major and T. cruzi, indicating conserved membrane receptors across distant phyla of protozoa. While we did not compare specificity of Hecate vs. Ligand-Hecate in this study, Sethi et al.'s (2014) results did provide evidence for the protective effect of adding a ligand to the lytic peptide to increase specificity and reduce off target effects against bacteria and insect tissue. Many studies showed that AMPs can be used in vitro to kill protozoa, but development of ligands can provide the flexibility and specificity to address in vivo success to kill protozoa with small peptide chemistries. The use of AMPs for vector borne pathogens is not limited to developing safe and effective treatments for trypomastigote parasites affecting humans. Moreira et al. (2007) showed that the in vitro growth of intracellular forms of Plasmodium falciparum, the causative agent of Malaria, was inhibited when treated with the spider AMP gomesin. Furthermore, Anopheles stephensi mosquitoes infected with P. falciparum or P. berghei that fed on mice treated with gomesin failed to produce the usual or expected number of oocysts (Moreira et al., 2007). Therefore, the area-wide use of AMPs for curative therapy of malaria in humans also could lower the infection rates of mosquitoes in communities.

The use of AMPs for control of transmission of T. cruzi in human domiciles has been promoted. Beard et al. (2002) conducted studies using a transgenic bacterial symbiont (Rhodococcus rhodnii) of Rhodnius prolixus to express Cecropin A resulting in the death of T. cruzi trypomastigotes in the gut of the vector. The genetically modified symbionts were delivered in a synthetic paste to simulate feces of R. prolixus, which is a source of symbiotic bacteria for first instar bugs via coprophagy. Sethi et al. (2014) employed a similar paratransgenesis approach for termite control by targeting and killing cellulose-digesting gut protozoa of Formosan subterranean termites. The authors genetically engineered yeast to express Ligand-Hecate and incorporated freeze-dried transgenic yeast into a cellulose bait. Termite foragers ingested the yeast, transferred it among colony member workers via coprophagy and trophallaxis, and the loss of protozoa killed the termite colony within weeks. The use of a transgenic yeast rather than a bacterium likely would add environmental stability for baits and facilitate scaled-up drug production for targeting reservoir vertebrates or invertebrates aiming to break transmission cycles.

In conclusion, most of the drugs currently used for treatment of Chagas disease and leishmaniasis have high vertebrate toxicity and low specificity for the parasites. The ligand-based drug design approach has been reported for improving or identifying new chemotypes (Berhe et al., 2024). Our study was an in vitro demonstration of efficacy and safety of Ligand-Hecate for killing intracellular and extracellular life stages of Leishmania and extracellular stages of Trypanosoma parasites. The addition of the ligand increased the lytic peptide action at lower concentrations. We also showed that lytic peptide (Hecate) treatment alone is safe but gains efficiency for curative L. major amastigote therapy with a ligand-based design. The efficacy of Hecate was increased by 4X (from 1.62 μM to 0.41 μM) by addition of a ligand known to bind with symbiotic protozoa of termites that showed homology to epitopes present on the variant surface glycoprotein of Trypanosoma brucei (Husseneder et al., 2010; Sethi et al., 2014). The efficacy at treatment levels less than 1μM rank highest among AMPs previously tested (McGwire and Kulkarni, 2010). Furthermore, this protozoacidal construct can be produced and delivered by transgenic yeast (Sethi et al., 2014). Therefore, the potential of using transgenic yeast or bacteria in the production of ligand-lytic peptides for therapy or even as delivery systems for humans, reservoir vertebrate hosts, and invertebrate hosts is an innovative approach to drug development.