Camila M. Adade

Melittin peptide kills Trypanosoma cruzi parasites by inducing different cell death pathways

Antimicrobial peptides (AMPs) are components of the innate immune response that represent desirable alternatives to conventional pharmaceuticals, as they have a fast mode of action, a low likelihood of resistance development and can act in conjunction with existing drug regimens. AMPs exhibit strong inhibitory activity against both Gram-positive and Gram-negative bacteria, fungi, viruses, metazoans and other parasites, such as the protozoan Leishmania. Melittin is a naturally occurring AMP, which comprises 40-50% of the dry weight of Apis mellifera venom. Our group has recently shown that crude A. mellifera venom is lethal to Trypanosoma cruzi, the Chagas disease etiologic agent, and generates a variety of cell death phenotypes among treated parasites. Here, we demonstrate that the melittin affected all of T. cruzi developmental forms, including the intracellular amastigotes. The ultrastructural changes induced by melittin suggested the occurrence of different programmed cell death pathways, as was observed in A. mellifera-treated parasites. Autophagic cell death appeared to be the main death mechanism in epimastigotes. In contrast, melittin-treated trypomastigotes appeared to be dying via an apoptotic mechanism. Our findings confirm the great potential of AMPs, including melittin, as a potential source of new drugs for the treatment of neglected diseases, such as Chagas disease.

Crovirin, a snake venom cysteine-rich secretory protein (CRISP) with promising activity against Trypanosomes and Leishmania.

Background The neglected human diseases caused by trypanosomatids are currently treated with toxic therapy with limited efficacy. In search for novel anti-trypanosomatid agents, we showed previously that the Crotalus viridis viridis (Cvv) snake venom was active against infective forms of Trypanosoma cruzi. Here, we describe the purification of crovirin, a cysteine-rich secretory protein (CRISP) from Cvv venom with promising activity against trypanosomes and Leishmania. Methodology/Principal Findings Crude venom extract was loaded onto a reverse phase analytical (C8) column using a high performance liquid chromatographer. A linear gradient of water/acetonitrile with 0.1% trifluoroacetic acid was used. The peak containing the isolated protein (confirmed by SDS-PAGE and mass spectrometry) was collected and its protein content was measured. T. cruzi trypomastigotes and amastigotes, L. amazonensis promastigotes and amastigotes and T. brucei rhodesiense procyclic and bloodstream trypomastigotes were challenged with crovirin, whose toxicity was tested against LLC-MK2 cells, peritoneal macrophages and isolated murine extensor digitorum longus muscle. We purified a single protein from Cvv venom corresponding, according to Nano-LC MS/MS sequencing, to a CRISP of 24,893.64 Da, henceforth referred to as crovirin. Human infective trypanosomatid forms, including intracellular amastigotes, were sensitive to crovirin, with low IC50 or LD50 values (1.10–2.38 µg/ml). A considerably higher concentration (20 µg/ml) of crovirin was required to elicit only limited toxicity on mammalian cells. Conclusions This is the first report of CRISP anti-protozoal activity, and suggests that other members of this family might have potential as drugs or drug leads for the development of novel agents against trypanosomatid-borne neglected diseases.

Apis mellifera venom induces different cell death pathways in Trypanosoma cruzi.

Chagas disease chemotherapy is based on drugs that exhibit toxic effects and have limited efficacy, such as Benznidazole. Therefore, research into new chemotherapeutic agents from natural sources needs to be exploited. Apis mellifera venom consists of many biologically active molecules and has been reported to exhibit remarkable anti-cancer effects, often promoting an apoptosis-like death phenotype. This study demonstrates that A. mellifera venom can affect the growth, viability and ultrastructure of all Trypanosoma cruzi developmental forms, including intracellular amastigotes, at concentrations 15- to 100-fold lower than those required to cause toxic effects in mammalian cells. The ultrastructural changes induced by the venom in the different developmental forms led us to hypothesize the occurrence of different programmed cell death pathways. Autophagic cell death, characterized by the presence of autophagosomes-like organelles and a strong monodansyl cadaverine labelling, appears to be the main death mechanism in epimastigotes. In contrast, increased TUNEL staining, abnormal nuclear chromatin condensation and kDNA disorganization was observed in venom-treated trypomastigotes, suggesting cell death by an apoptotic mechanism. On the other hand, intracellular amastigotes presented a heterogeneous cell death phenotype profile, where apoptosis-like death seemed to be predominant. Our findings confirm the great potential of A. mellifera venom as a source for the development of new drugs for the treatment of neglected diseases such as Chagas disease.

In vitro effects of suramin on Trypanosoma cruzi

Suramin has been previously reported to inhibit distinct cellular enzymes and to affect the synthesis and distribution of cytoskeleton proteins, cell differentiation and proliferation. The present study indicates that prolonged incubation of Trypanosoma cruzi-infected cells in the presence of 500 microM suramin during intracellular development of the parasite causes morphological changes in amastigote and trypomastigote forms related to the cell division and differentiation process. Our results also show that trypomastigotes obtained from suramin-treated host cells were significantly less infective than control parasites and that amastigotes derived from those trypomastigote forms were less proliferative.

Antiproliferative and ultrastructural effects of phenethylamine derivatives on promastigotes and amastigotes of Leishmania (Leishmania) infantum chagasi.

Leishmania (Leishmania) infantum chagasi is one of the agents that cause visceral leishmaniasis. This disease occurs more frequently in third world countries, such as Brazil. The treatment is arduous, and is dependent on just a few drugs like the antimonial derivatives and amphotericin B. Moreover, these drugs are not only expensive, but they can also cause severe side effects and require long-term treatment. Therefore, it is very important to find new compounds that are effective against leishmaniasis. In the present work we evaluated a new group of synthetic amides against the promastigote and amastigote forms of L. infantum chagasi. The results showed that one of these amides in particular, presented very effective activity against the promastigotes and amastigotes of L. infantum chagasi at low concentrations and it also presented low toxicity for mammal cells, which makes this synthetic amide a promising drug for combating leishmaniasis.