Trypanosomatid essential metabolic pathway: New approaches about heme fate in Trypanosoma cruzi

Trypanosoma cruzi, the causal agent of Chagas disease, has a complex life cycle and depends on hosts for its nutritional needs. Our group has investigated heme (Fe-protoporphyrin IX) internalization and the effects on parasite growth, following the fate of this porphyrin in the parasite. Here, we show that epimastigotes cultivated with heme yielded the compounds α-meso-hydroxyheme, verdoheme and biliverdin (as determined by HPLC), suggesting an active heme degradation pathway in this parasite. Furthermore, through immunoprecipitation and immunoblotting assays of epimastigote extracts, we observed recognition by an antibody against mammalian HO-1. We also detected the localization of the HO-1-like protein in the parasite using immunocytochemistry, with antibody staining primarily in the cytoplasm. Although HO has not been described in the parasite’s genome, our results offer new insights into heme metabolism in T. cruzi, revealing potential future therapeutic targets.     

Trypanosoma cruzi: Synergistic cytotoxicity of multiple amphipathic anti-microbial peptides to T. cruzi and potential bacterial hosts

The parasite Trypanasoma cruzi is responsible for Chagas disease and its triatomine vector, Rhodnius prolixus, has a symbiotic relationship with the soil bacterium, Rhodococcus rhodnii.R. rhodnii that was previously genetically engineered to produce the anti-microbial peptide, cecropin A was co-infected with T. cruzi into R. prolixus resulting in clearance of the infectious T. cruzi in 65% of the vectors. Similar anti-microbial peptides have been isolated elsewhere and were studied for differential toxicity against T. cruzi and R. rhodnii. Of the six anti-microbial peptides tested, apidaecin, magainin II, melittin, and cecropin A were deemed potential candidates for the Chagas paratransgenic system as they were capable of killing T.cruzi at concentrations that exhibit little or no toxic effects on R. rhodnii. Subsequent treatments of T. cruzi with these peptides in pair-wise combinations resulted in synergistic killing, indicating that improvement of the 65% parasite clearance seen in previous experiments may be possible utilizing combinations of different anti-microbial peptides.     

Genome size, karyotype polymorphism and chromosomal evolution in Trypanosoma cruzi

Background

The Trypanosoma cruzi genome was sequenced from a hybrid strain (CL Brener). However, high allelic variation and the repetitive nature of the genome have prevented the complete linear sequence of chromosomes being determined. Determining the full complement of chromosomes and establishing syntenic groups will be important in defining the structure of T. cruzi chromosomes. A large amount of information is now available for T. cruzi and Trypanosoma brucei, providing the opportunity to compare and describe the overall patterns of chromosomal evolution in these parasites.

Methodology/Principal Findings

The genome sizes, repetitive DNA contents, and the numbers and sizes of chromosomes of nine strains of T. cruzi from four lineages (TcI, TcII, TcV and TcVI) were determined. The genome of the TcI group was statistically smaller than other lineages, with the exception of the TcI isolate Tc1161 (José-IMT). Satellite DNA content was correlated with genome size for all isolates, but this was not accompanied by simultaneous amplification of retrotransposons. Regardless of chromosomal polymorphism, large syntenic groups are conserved among T. cruzi lineages. Duplicated chromosome-sized regions were identified and could be retained as paralogous loci, increasing the dosage of several genes. By comparing T. cruzi and T. brucei chromosomes, homologous chromosomal regions in T. brucei were identified. Chromosomes Tb9 and Tb11 of T. brucei share regions of syntenic homology with three and six T. cruzi chromosomal bands, respectively.

Conclusions

Despite genome size variation and karyotype polymorphism, T. cruzi lineages exhibit conservation of chromosome structure. Several syntenic groups are conserved among all isolates analyzed in this study. The syntenic regions are larger than expected if rearrangements occur randomly, suggesting that they are conserved owing to positive selection. Mapping of the syntenic regions on T. cruzi chromosomal bands provides evidence for the occurrence of fusion and split events involving T. brucei and T. cruzi chromosomes.     

Proteomic analysis of the Trypanosoma cruzi ribosomal proteins

Trypanosoma cruzi is a parasite responsible for Chagas disease. The identification of new targets for chemotherapy is a major challenge for the control of this disease. Several lines of evidences suggest that the translational system in trypanosomatids show important differences compared to other eukaryotes. However, there little is known information about this. We have performed a detailed data mining search for ribosomal protein genes in T. cruzi genome data base combined with mass spectrometry analysis of purified T. cruzi ribosomes. Our results show that T. cruzi ribosomal proteins have ∼50% sequence identity to yeast ones. Nevertheless, some parasite proteins are longer due to the presence of several N- or C-terminal extensions, which are exclusive of trypanosomatids. In particular, L19 and S21 show C-terminal extensions of 168 and 164 amino acids, respectively. In addition, we detected two 60S subunit proteins that had not been previously detected in the T. cruzi total proteome; namely, L22 and L42.     

Bottlenecks in domestic animal populations can facilitate the emergence of Trypanosoma cruzi,the aetiological agent of Chagas disease

Faeces-mediated transmission of Trypanosoma cruzi (the aetiological agent of Chagas disease) by triatomine insects is extremely inefficient. Still, the parasite emerges frequently, and has infected millions of people and domestic animals. We synthesize here the results of field and laboratory studies of T. cruzi transmission conducted in and around Arequipa, Peru. We document the repeated occurrence of large colonies of triatomine bugs (more than 1000) with very high infection prevalence (more than 85%). By inoculating guinea pigs, an important reservoir of T. cruzi in Peru, and feeding triatomine bugs on them weekly, we demonstrate that, while most animals quickly control parasitaemia, a subset of animals remains highly infectious to vectors for many months. However, we argue that the presence of these persistently infectious hosts is insufficient to explain the observed prevalence of T. cruzi in vector colonies. We posit that seasonal rains, leading to a fluctuation in the price of guinea pig food (alfalfa), leading to annual guinea pig roasts, leading to a concentration of vectors on a small subpopulation of animals maintained for reproduction, can propel T. cruzi through vector colonies and create a considerable force of infection for a pathogen whose transmission might otherwise fizzle out.     

Detection of matrix metallopeptidase-9-like proteins in Trypanosoma cruzi

In this study, the cell-associated and extracellular peptidases of Trypanosoma cruzi grown in modified Roitman’s complex (MRC) medium were analyzed by measuring peptidase activity in gelatin-containing zymograms. Our results showed that the cell-associated peptidases as well as peptidases extracellularly released by T. cruzi displayed two distinct proteolytic classes: cysteine and metallopeptidase activities. The major cysteine peptidase, cruzipain, synthesized by T. cruzi cells was detected in cellular parasite content, as a 50 kDa reactive polypeptide, after probing with anti-cruzipain antibody. In addition, metallo-type peptidases belonging to the matrix metallopeptidase-9 (MMP-9) family were revealed, after Western blotting, as a 97 kDa protein band in cellular extract and an 85 kDa polypeptide in both cellular and secreted parasite extracts. The MMP-9-like activity present in cells and spent culture medium was immunoprecipitated by an anti-MMP-9 polyclonal antibody. The surface location of MMP-9-like proteins in T. cruzi was also evidenced by means of flow cytometry analysis. Furthermore, doxycycline that has direct MMP-9 inhibiting properties in vitro, inhibited MMP-9-like activities in gel zymography, immunoprecipitation and flow cytometry analyses. This is the first report of the presence of MMP-9-like molecules in T. cruzi. The presence of a matrix extracellular-degrading enzyme may play a role in the T. cruzi-host cell interaction, making this enzyme a potential target for future drug development against this pathogenic trypanosomatid.     

Enzymes of the antioxidant network as novel determiners of Trypanosoma cruzi virulence

Virulence of Trypanosoma cruzi depends on a variety of genetic and biochemical factors. It has been proposed that components of the parasites’ antioxidant system may play a key part in this process by pre-adapting the pathogen to the oxidative environment encountered during host cell invasion. Using several isolates (10 strains) belonging to the two major phylogenetic lineages (T. cruzi-I and T. cruzi-II), we investigated whether there was an association between virulence (ranging from highly aggressive to attenuated isolates at the parasitemia and histopathological level) and the antioxidant enzyme content. Antibodies raised against trypanothione synthetase (TcTS), ascorbate peroxidase (TcAPX), mitochondrial and cytosolic tryparedoxin peroxidases (TcMPX and TcCPX) and trypanothione reductase (TcTR) were used to evaluate the antioxidant enzyme levels in epimastigote and metacyclic trypomastigote forms in the T. cruzi strains. Levels of TcCPX, TcMPX and TcTS were shown to increase during differentiation from the non-infective epimastigote to the infective metacyclic trypomastigote stage in all parasite strains examined. Peroxiredoxins were found to be present at higher levels in the metacyclic infective forms of the virulent isolates compared with the attenuated strains. Additionally, an increased resistance of epimastigotes from virulent T. cruzi populations to hydrogen peroxide and peroxynitrite challenge was observed. In mouse infection models, a direct correlation was found between protein levels of TcCPX, TcMPX and TcTS, and the parasitemia elicited by the different isolates studied (Pearson’s coefficient: 0.617, 0.771, 0.499; respectively, P < 0.01). No correlation with parasitemia was found for TcAPX and TcTR proteins in any of the strains analyzed. Our data support that enzymes of the parasite antioxidant armamentarium at the onset of infection represent new virulence factors involved in the establishment of disease.     

Identification and Functional Analysis of Trypanosoma cruzi Genes That Encode Proteins of the Glycosylphosphatidylinositol Biosynthetic Pathway

Background

Trypanosoma cruzi is a protist parasite that causes Chagas disease. Several proteins that are essential for parasite virulence and involved in host immune responses are anchored to the membrane through glycosylphosphatidylinositol (GPI) molecules. In addition, T. cruzi GPI anchors have immunostimulatory activities, including the ability to stimulate the synthesis of cytokines by innate immune cells. Therefore, T. cruzi genes related to GPI anchor biosynthesis constitute potential new targets for the development of better therapies against Chagas disease.

Methodology/Principal Findings

In silico analysis of the T. cruzi genome resulted in the identification of 18 genes encoding proteins of the GPI biosynthetic pathway as well as the inositolphosphorylceramide (IPC) synthase gene. Expression of GFP fusions of some of these proteins in T. cruzi epimastigotes showed that they localize in the endoplasmic reticulum (ER). Expression analyses of two genes indicated that they are constitutively expressed in all stages of the parasite life cycle. T. cruzi genes TcDPM1, TcGPI10 and TcGPI12 complement conditional yeast mutants in GPI biosynthesis. Attempts to generate T. cruzi knockouts for three genes were unsuccessful, suggesting that GPI may be an essential component of the parasite. Regarding TcGPI8, which encodes the catalytic subunit of the transamidase complex, although we were able to generate single allele knockout mutants, attempts to disrupt both alleles failed, resulting instead in parasites that have undergone genomic recombination and maintained at least one active copy of the gene.

Conclusions/Significance

Analyses of T. cruzi sequences encoding components of the GPI biosynthetic pathway indicated that they are essential genes involved in key aspects of host-parasite interactions. Complementation assays of yeast mutants with these T. cruzi genes resulted in yeast cell lines that can now be employed in high throughput screenings of drugs against this parasite.     

Trypanosoma cruzi: Genetic diversity influences the profile of immunoglobulins during experimental infection

The clonal evolution model postulated for Trypanosoma cruzi predicts a correlation between the phylogenetic divergence of T. cruzi clonal genotypes and their biological properties. In the present study, the linkage between phylogenetic divergence of the parasite and IgG, IgG1, IgG2a and IgG2b response has been evaluated during the acute and chronic phases of the experimental infection. Eight laboratory-cloned stocks representative of this phylogenetic diversity and including the lineages T. cruzi I (genotypes 19 and 20), T. cruzi II (genotype 32) and T. cruzi (genotype 39) have been studied. The results showed that the pattern of humoral immune response was correlated with T. cruzi genotype, and that stocks included in genotype 20 were responsible for the high IgG response in the acute and chronic phases. Moreover, T. cruzi I lineage was more efficient in over-expressing all subclasses of specific anti-parasite IgG than either T. cruzi II or T. cruzi lineages. Curiously, the alteration in the pattern of antibodies induced by Benznidazole treatment was related to the phase of the infection but not to the genotype of the parasite. The data suggest that genotypes of T. cruzi are able to drive levels/subclasses of specific IgG, hence giving rise to further concerns about the sensitivity of serological assays in the diagnosis of human Chagas disease.     

Crystal structure of Trypanosoma cruzi dihydroorotate dehydrogenase from Y strain

Trypanosoma cruzi is the etiological agent of Chagas’ disease, a pathogenesis that affects millions of people in Latin America. Here, we report the crystal structure of dihydroorotate dehydrogenase (DHODH) from T. cruzi strain Y solved at 2.2 Å resolution. DHODH is a flavin mononucleotide containing enzyme, which catalyses the oxidation of l-dihydroorotate to orotate, the fourth step and only redox reaction in the de novo biosynthesis of pyrimidine nucleotides. Genetic studies have shown that DHODH is essential for T. cruzi survival, validating the idea that this enzyme can be considered an attractive target for the development of antichagasic drugs. In our work, a detailed analysis of T. cruzi DHODH crystal structure has allowed us to suggest potential sites to be further exploited for the design of highly specific inhibitors through the technology of structure-based drug design.     

Trypanosoma cruzi: Multiplex PCR to detect and classify strains according to groups I and II

A multiplex PCR was developed for simultaneous detection of Trypanosoma cruzi DNA and classification of the parasite strain into groups I and II. As little as 10 fg of T. cruzi DNA could be detected by multiplex PCR. The technique was shown to be specific for T. cruzi DNA, since no PCR amplification products were obtained with DNA from other tripanosomatid species. Multiplex PCR was validated by assaying genomic DNA from 34 strains of T. cruzi that had been previously characterized; 24 blood samples from experimentally-infected mice and non-infected controls; 20 buffy coat samples from patients in the acute phase of Chagas disease and non-infected individuals, and 15 samples of feces from naturally-infected Triatoma infestans. T. cruzi samples from patients and from Y strain-infected mice were classified by multiplex PCR as T. cruzi II and samples from T. infestans and Colombiana strain-infected mice as T. cruzi I.     

Bioluminescent imaging of Trypanosoma cruzi infection

Chagas disease, caused by infection with the protozoan parasite Trypanosoma cruzi, is a major public health problem in Central and South America. The pathogenesis of Chagas disease is complex and the natural course of infection is not completely understood. The recent development of bioluminescence imaging technology has facilitated studies of a number of infectious and non-infectious diseases. We developed luminescent T. cruzi to facilitate similar studies of Chagas disease pathogenesis. Luminescent T. cruzi trypomastigotes and amastigotes were imaged in infections of rat myoblast cultures, which demonstrated a clear correlation of photon emission signal strength to the number of parasites used. This was also observed in mice infected with different numbers of luminescent parasites, where a stringent correlation of photon emission to parasite number was observed early at the site of inoculation, followed by dissemination of parasites to different sites over the course of a 25-day infection. Whole animal imaging from ventral, dorsal and lateral perspectives provided clear evidence of parasite dissemination. The tissue distribution of T. cruzi was further determined by imaging heart, spleen, skeletal muscle, lungs, kidneys, liver and intestines ex vivo. These results illustrate the natural dissemination of T. cruzi during infection and unveil a new tool for studying a number of aspects of Chagas disease, including rapid in vitro screening of potential therapeutical agents, roles of parasite and host factors in the outcome of infection, and analysis of differential tissue tropism in various parasite-host strain combinations.     

Molecular epidemiology of domestic and sylvatic Trypanosoma cruzi infection in rural northwestern Argentina

Genetic diversity of Trypanosoma cruzi populations and parasite transmission dynamics have been well documented throughout the Americas, but few studies have been conducted in the Gran Chaco ecoregion, one of the most highly endemic areas for Chagas disease, caused by T. cruzi. In this study, we assessed the distribution of T. cruzi lineages (identified by PCR strategies) in Triatoma infestans, domestic dogs, cats, humans and sylvatic mammals from two neighbouring rural areas with different histories of transmission and vector control in northern Argentina. Lineage II predominated amongst the 99 isolates characterised and lineage I amongst the six isolates obtained from sylvatic mammals. T. cruzi lineage IIe predominated in domestic habitats; it was found in 87% of 54 isolates from Tr. infestans, in 82% of 33 isolates from dogs, and in the four cats found infected. Domestic and sylvatic cycles overlapped in the study area in the late 1980s, when intense domestic transmission occurred, and still overlap marginally. The introduction of T. cruzi from sylvatic into domestic habitats is likely to occur very rarely in the current epidemiological context. The household distribution of T. cruzi lineages showed that Tr. infestans, dogs and cats from a given house compound shared the same parasite lineage in most cases. Based on molecular evidence, this result lends further support to the importance of dogs and cats as domestic reservoir hosts of T. cruzi. We believe that in Argentina, this is the first time that lineage IIc has been isolated from naturally infected domestic dogs and Tr. infestans.     

Construction of a genetic map for Theileria parva: identification of hotspots of recombination

The tick-borne protozoan parasite Theileria parva is the causal agent of East Coast Fever (ECF), a severe lymphoproliferative disease of cattle in eastern, central and southern Africa. The life cycle of T. parva is predominantly haploid, with a brief diploid stage occurring in the tick vector that involves meiotic recombination. Resolved genetic studies of T. parva are currently constrained by the lack of a genome-wide high-definition genetic map of the parasite. We undertook a genetic cross of two cloned isolates of T. parva to construct such a map from 35 recombinant progeny, using a genome-wide panel of 79 variable number of tandem repeat markers. Progeny were established by in vitro cloning of cattle lymphocytes after infection with sporozoites prepared from Rhipicephalus appendiculatus ticks fed on a calf undergoing a dual infection with the two clonal parental stocks. The genetic map was determined by assigning individual markers to the four chromosome genome, whose physical length is approximately 8309 kilobasepairs (Kb). Segregation analysis of the markers among the progeny revealed a total genetic size of 1683.8 centiMorgans (cM), covering a physical distance of 7737.62 Kb (∼93% of the genome). The average genome-wide recombination rate observed for T. parva was relatively high, at 0.22 cM Kb⁻¹ per meiotic generation. Recombination hot-spots and cold-spots were identified for each of the chromosomes. A panel of 27 loci encoding determinants previously identified as immunorelevant or likely to be under selection were positioned on the linkage map. We believe this to be the first genetic linkage map for T. parva. This resource, with the availability of the genome sequence of T. parva, will promote improved understanding of the pathogen by facilitating the use of genetic analysis for identification of loci responsible for variable phenotypic traits exhibited by individual parasite stocks.     

Trypanosoma cruzi: effect of the infection on the 20S proteasome in non-immune cells

Human infection with the protozoan Trypanosoma cruzi leads to Chagas disease. After 10-20 years of the normal acute phase, this disease develops to a chronic phase characterized mainly by dilated congestive cardiomyopathy. The mechanisms involved in the chronic phase are poorly understood, and it has been suggested that the parasite evades immune surveillance by down regulating the MHC class I antigen processing pathway. Here we analyzed whether composition or expression of the 20S proteasome, the major proteinase responsible for the generation of MHC class I ligands, were altered upon infection of HeLa cells by T. cruzi. Two-dimensional gel electrophoresis and RT-PCR experiments comparing non-infected and infected cells did not show differences between the composition of 20S proteasome or expression of its subunits. However, the proteasome’s trypsin- and chymotrypsin-like activities were 2.5 and 3.6 times higher in infected cells than in non-infected cells. Our results suggest that in vitroT. cruzi infection of human or rat cells do not alter the expression of 20S proteasomal subunits or particle composition, and fails to induce the formation of immunoproteasome. However, a significant increase in the trypsin- and chymotrypsin-like activities of the host proteasome was observed.     

Trypanosoma cruzi: In vitro effect of aspirin with nifurtimox and benznidazole

Nifurtimox and benznidazole are the only active drugs against Trypanosoma cruzi; however, they have limited efficacy and severe side effects. During primoinfection, T. cruzi infected macrophages mount an antiparasitic response, which the parasite evades through an increase of tumor growth factor β and PGE₂ activation as well as decreased iNOS activity. Thus, prostaglandin synthesis inhibition with aspirin might increase macrophage antiparasitic activity and increase nifurtimox and benznidazole effect.Aspirin alone demonstrated a low effect upon macrophage antiparasitic activity. However, isobolographic analysis of the combined effects of aspirin, nifurtimox and benznidazole indicated a synergistic effect on T. cruzi infection of RAW cells, with combinatory indexes of 0.71 and 0.61, respectively.The observed effect of aspirin upon T. cruzi infection was not related with the PGE₂ synthesis inhibition. Nevertheless, NO levels were restored by aspirin in T. cruzi-infected RAW cells, contributing to macrophage antiparasitic activity improvement.Thus, the synergy of aspirin with nifurtimox and benznidazole is due to the capability of aspirin to increase antiparasitic activity of macrophages.     

Trypanosoma cruzi: multiple nucleoside diphosphate kinase isoforms in a single cell

Nucleoside diphosphate kinases (NDPKs) are multifunctional enzymes involved mainly in the conservation of nucleotides and deoxynucleotides at intracellular levels. Here we report the characterization of two NDPKs from the protozoan parasite Trypanosoma cruzi, the etiological agent of Chagas disease. TcNDPK1 and TcNDPK2 were biochemically characterized presenting different kinetic parameters and regulation mechanisms. NDPK activity was mainly detected in soluble fractions according to the digitonin extraction technique; however 20% of the activity remains insoluble at digitonin concentrations up to 5 mg ml⁻¹. TcNDPK1 is a short enzyme isoform, whereas TcNDPK2 is a long one containing a DM10 motif. In addition, two other putative NDPK genes (TcNPDK3 and TcNDPK4) were detected by data mining at the T. cruzi genome database. The large number and diversity of NDPK isoforms are in agreement with those previously observed for other T. cruzi phosphotransferases, such as adenylate kinases.     

Natural sesquiterpene lactones induce programmed cell death in Trypanosoma cruzi: A new therapeutic target?

BackgroundChagas disease or American Trypanosomiasis is caused by the flagellated protozoan parasite Trypanosoma cruzi (T. cruzi) and is recognized by the WHO as one of the world's 17 neglected tropical diseases. Only two drugs (Benznidazol, Bz and Nifurtimox, Nx) are currently accepted for treatment, however they cause severe adverse effects and their efficacy is still controversial. It is then important to explore for new drugs.PurposeProgrammed cell death (PCD) in parasites offers interesting new therapeutic targets. The aim of this work was to evaluate the induction of PCD in T. cruzi by two natural sesquiterpene lactones (STLs), dehydroleucodine (DhL) and helenalin (Hln) as compared with the two conventional drugs, Bz and Nx.Material and MethodsHln and DhL were isolated from aerial parts of Gaillardia megapotamica and Artemisia douglassiana Besser, respectively. Purity of compounds (greater than 95%) was confirmed by ¹³C-nuclear magnetic resonance, melting point analysis, and optical rotation. Induction of PCD in T. cruzi epimastigotes and trypomastigotes by DhL, Hln, Bz and Nx was assayed by phosphatidylserine exposure at the parasite surface and by detection of DNA fragmentation using the TUNEL assay. Trypanocidal activity of natural and synthetic compounds was assayed by measuring parasite viability using the MTT method.ResultsThe two natural STLs, DhL and Hln, induce programmed cell death in both, the replicative epimastigote form and the infective trypomastigote form of T. cruzi. Interestingly, the two conventional antichagasic drugs (Bz and Nx) do not induce programmed cell death. A combination of DhL and either Bz or Nx showed an increased effect of natural compounds and synthetic drugs on the decrease of parasite viability.ConclusionDhL and Hln induce programmed cell death in T. cruzi replicative epimastigote and infective trypomastigote forms, which is a different mechanism of action than the conventional drugs to kill the parasite. Therefore DhL and Hln may offer an interesting option for the treatment of Chagas disease, alone or in combination with conventional drugs.     

Trypanosoma cruzi: in vitro activity of Epoxy-α-Lap, a derivative of α-lapachone, on trypomastigote and amastigote forms

Chagas disease is an endemic parasitic infection caused by Trypanosomacruzi that affects 18-20 million people in Central and South America. Recently we described the Epoxy-α-Lap, an oxyran derivative of α-lapachone, which presents a low toxicity profile and a high inhibitory activity against T.cruzi epimastigotes forms, the non-infective form of this parasite. In this work we described the trypanocidal effects of Epoxy-α-Lap on extracellular (trypomastigote) and intracellular (amastigote) infective forms of two T. cruzi strains (Y and Colombian) known by their different infective profile. Our results showed that Epoxy-α-Lap is lethal to trypomastigote Y and Colombian strains (97% and 84%, respectively). Interestingly, Epoxy-α-Lap also showed a trypanocidal effect in human macrophage infected with T. cruzi Y (85.6%) and Colombian (71.9%) strains amastigote forms. Similar effects were observed on T. cruzi amastigote infected Vero cells (96.4% and 95.0%, respectively). Our results pointed Epoxy-α-Lap as a potential candidate for Chagas disease chemotherapy since it presents trypanocidal activity on all T. cruzi forms with low) toxicity profile.