Chelating agent inhibition of Trypanosoma cruzi epimastigotes In vitro

A number of chelating agents and some of their derivatives are as effective as, or superior to, benznidazole, the compound currently in clinical use, in the suppression of the reproduction of epimastigotes of Trypanosoma cruzi, the protozoa that causes Chagas' disease. All compounds were examined at a culture concentration of 5 μg/mL. The most effective compounds included N, N, N′, N′-tetrakis(2-pyridylmethyl)ethylenediamine, sodium diethylamine-N-carbodithioate, piperidine-N-carbodithioate and several of its analogs, a number of other carbodithioates with two nonpolar groups on the nitrogen, and tetraethylthiuram disulfide, a prodrug of sodium diethylamine-N-carbodithioate and widely used in the treatment of alcoholism. The introduction of additional ionic or nonionic polar groups on the chelating molecule generally results in a loss of tyrpanocidal activity. Common commercially available chelating agents which exhibited no activity included -penicillamine, meso-2,3-dimercaptosuccinic acid, and triethylenetetramine tetrahydrochloride. Dose-response data on the culture indicated that some of these compounds exhibited inhibition of Trypanosoma cruzi epimastigotes at concentrations as low as 0.625 μg/mL. It is proposed that the mechanism of action of these compounds is based on their ability to interface with the essential metal metabolism at intracellular sites of the epimastigote involving iron, copper, or zinc. The results also indicate that a certain degree of hydrophobicity may be necessary for the groups attached to the literal metal-bonding structure if the compounds are to successfully inhibit the epimastigotes of Trypanosoma cruzi. The development of antiprotozoal drugs which are chelating agents specifically designed to selectively disrupt the essential metal metabolism of Trypanosoma cruzi should furnish a new generation of drugs which can be used in the treatment of Chagas' disease.     

The in vivo distribution of Cr-labeled Trypanosoma cruzi in mice

The optimal conditions for labeling Trypanosoma cruzi culture forms with ⁵¹CrO₄²⁻ were determined. Labeled trypanosomes or labeled human red blood cells (RBCs) were injected intravenously into normal C3H(He) female mice and the rate of clearance and organ distribution of the isotope were observed over a 30 h period. It was found that trypanosomes and xenogeneic RBCs were cleared rapidly from the peripheral blood and accumulated primarily in the liver, spleen, lungs and kidneys. A difference was noted in accumulation of trypanosomes and RBCs in these mice.     

Trypanosoma cruzi: skin-penetration kinetics of vector-derived metacyclic trypomastigotes

In order to investigate the natural route of infection of nude and normal BALB/c mice with Trypanosoma cruzi via the skin, a drop of vector faeces/urine containing metacyclic trypomastigotes was placed onto the puncture site of a bite from Triatoma infestans. The periods of exposure, i.e. until removal of flagellates from the skin, and the time elapsed until surgical removal of the skin around the puncture were varied. After 15 min of exposure, T. cruzi developed in all nude mice without surgery, and in four of 10 mice if the puncture region of the skin was removed directly after exposure. In a shaved puncture region, 5 min of exposure were sufficient to infect all normal BALB/c mice without surgery and one of four mice with direct removal of the puncture region. Longer periods of exposure or time until removal of the skin only sometimes resulted in higher infection rates. Prepatent periods and the development of parasitaemia varied irrespective of the period of exposure or the period until skin removal at the puncture site. The importance of these findings is that they clearly prove that T. cruzi can rapidly invade the host via the puncture site of the bite of the vector and that at least some parasites are immediately transported away from this site.     

The lysis of Trypanosoma cruzi epimastigotes by eosinophils and neutrophils

López A.F., Bunn Moreno M.M. and Sanderson C.J. 1978. The lysis of Trypanosoma cruzi epimastigotes by eosinophils and neutrophils. International Journal for Parasitology8: 485–489. Antibody-dependent cell-mediated cytotoxicity of T. cruzi epimastigotes has been studied by means of an isotopic assay for parasite lysis, in which the release of labelled RNA is measured. It is shown that rat eosinophils and neutrophils have approximately equal activity against the parasite in the presence of antibody. Antibody enhances the activity of neutrophils about 8-fold, whereas eosinophils have no detectable activity in the absence of antibody.Attention is drawn to the tendency of eosinophils to be inactivated by in vitro manipulation, especially adherence techniques used for removing macrophages and neutrophils.     

Protein biosynthesis changes in Trypanosoma cruzi induced by supra-optimal temperature

Early during vertebrate infection, T. cruzi is exposed to the host blood at an elevated temperature. Bearing this in mind, the pattern of protein synthesis of two parasite forms was examined. SDS-PAGE of heated organisms showed an increase in at least four proteins (103, 92, 75 and 61 kD). The temperature effect is also manifested in cells whose RNA synthesis is reduced by actinomycin D treatment. The synthesis of the '29 degrees proteins' is inhibited at 40 degrees C in organisms growing in culture medium; when the organisms were maintained in serum, the inhibition was not observed. The inhibitory effect observed at 40 degrees C was reversed when the temperature was shifted to 29 degrees C. These proteins were synthesized for 180 min at 37 degrees C or 360 min at 40 degrees C. The increased protein synthesis manifested at 37 degrees C had decreased 45 min after the temperature was lowered to 29 degrees C. When the cells were pre-incubated at 40 degrees C and shifted to 29 degrees C, the synthesis of the heat-induced proteins proceeded for at least 180 min. This pattern of heat induction in epimastigotes and trypomastigotes is the same irrespective of whether the incubation medium is LIT (for epimastigotes), M-16 (for trypomastigotes), or when serum was used for both cell types.     

Function of the dense plaques during mitosis in Trypanosoma cruzi

During mitosis in Trypanosoma cruzi ten dense plaques originate from the single, large nucleolus present in interphase and the preliminary phase of mitosis. Each plaque becomes associated with two microtubular bundles which end at the poles of the dividing nucleus. After an equilibrium phase in which the plaques are located near the nuclear equator, each plaque divides into two half-plaques. Each half-plaque migrates to a pole in the next stage. Although the composition of the plaques is unknown, they are associated with chromatin fibers. It is concluded that plaques act as kinetochores of higher organisms and provide a mechanical device for equal distribution of chromatin to daughter nuclei.     

Genotype variation of Trypanosoma cruzi isolates from different Brazilian biomes

Chagas disease is an enzootic disease, in which the flagellate Trypanosoma cruzi infects a large variety of animals. Humans are accidentally infected due to the migration into wild environments. To identify T. cruzi discrete typing units (DTUs), 19 Brazilian isolates from different biomes and hosts were analyzed by PCR amplification of 24Sα rRNA, 18S rRNA and mini-exon gene sequences. The majority of the isolates was classified as TcIIb (TcII) but subtypes TcIIc (TcIII) and TcIId (TcV) were also identified. In addition, in monkeys TcI was detected.     

Colombian Trypanosoma cruzi major genotypes circulating in patients: Minicircle homologies by cross-hybridization analysis

Here we present compelling evidence of Trypanosoma cruzi genotypes infecting 77 human cases of Chagas disease in Santander Department of Colombia. The patients were clinically studied and classified according to the presence of cardiac symptoms. We describe the distribution of the major T. cruzi genotypes circulating in this area by means of direct PCR analysis of blood samples. PCR was directed to minicircles and amplified DNAs were hybridized using genotype-specific DNA probes. These samples were previously genotyped with miniexon, 24 α rRNA and cytochrome oxidase subunit II (COII) markers. Minicircle DNA analyses were more sensitive than miniexon, 24 α rRNA and CO II genes in detecting infective T. cruzi II (Tc II). Two Tc II genotypes were identified by hybridization using two complementary DNA probes in 27.3% of the patients, with 15.3% using all three markers. These corresponded to 10 cases genotyped only by hybridization. The lineage Tc I, determined by hybridization, was the most prevalent singly or combined with different genotypes (72.7%), and at least three different T. cruzi genotypes were identified. Attempts to find two T. cruzi genotypes Tc I and Tc II in other endemic areas of Colombia revealed that one similar to the most prevalent Tc I genotype was detected in distant geographical areas. A similar Tc II genotype was found in Bolivia and Chile, revealing the great distribution of some ancestral T. cruzi genotypes. We did not detect any association between infective Tc I and Tc II lineages and the severity of the patients’ cardiac symptoms.     

PGE2 involvement in experimental infection with Trypanosoma cruzi subpopulations

PGE₂ involvement in experimental Trypanosoma cruzi infection depends on the lethal capacity of the parasite subpopulation used. Mice acutely infected with non-lethal K98 displayed an enhancement in PGE₂ serum levels during the acute period, while those infected with lethal T. cruzi subpopulations (RA or K98-2) showed levels not different from normal mice. The enhancement detected in K98 group could be related both to an increased number of CD8+ T cell number and to enhanced PGE₂ release per cell by CD8+; values of PGE₂ release by adherent cells were not altered in this group. Treatment with cyclooxygenase inhibitors enhanced mortality rates of mice infected with K98, and administration of 16,16-dimethyl PGE₂ (dPGE) reversed this effect. However, mice infected with RA did not reduce their mortality rates by administration of diverse doses of dPGE. These findings suggest that PGE₂ could play a role in resistance in mice infected with K98.     

Characterization of messenger RNA from epimastigotes and metacyclic trypomastigotes of Trypanosoma cruzi

The cell-free translation products of polyribosomal and post-polyribosomal mRNAs from the non-infective epimastigotes and the infective metacyclic trypomastigotes of the parasitic protozoan Trypanosoma cruzi were compared by two-dimensional polyacrylamide gel electrophoresis. The result show that although many polypeptides are conserved, quantitative and qualitative differences are observed between both differentiation stages. The results also indicate the existence of post-polyribosomal mRNAs in equilibrium with polyribosomal counterparts. The immunoprecipitation of the in vitro synthesized polypeptides with chagasic human serum and the serum raised against an 85-kDa glycoprotein (P2-WGA), potentially involved in the process of T. cruzi penetration into mammalian cells, shows that while the chagasic serum recognizes the same 72-kDa, 68-kDa and 46-kDa polypeptides in both differentiation stages, the anti-P2-WGA serum immunoprecipitates a single 48-kDa polypeptide from in vitro translation products of metacyclic trypomastigotes.     

Trypanosoma cruzi: Insights into naphthoquinone effects on growth and proteinase activity

In this study we compared the effects of naphthoquinones (α-lapachone, β-lapachone, nor-β-lapachone and Epoxy-α-lap) on growth of Trypanosoma cruzi epimastigotes forms, and on viability of VERO cells. In addition we also experimentally analyzed the most active compounds inhibitory profile against T. cruzi serine- and cysteine-proteinases activity and theoretically evaluated them against cruzain, the major T. cruzi cysteine proteinase by using a molecular docking approach. Our results confirmed β-lapachone and Epoxy-α-lap with a high trypanocidal activity in contrast to α-lapachone and nor-β-lapachone whereas Epoxy-α-lap presented the safest toxicity profile against VERO cells. Interestingly the evaluation of the active compounds effects against T. cruzi cysteine- and serine-proteinases activities revealed different targets for these molecules. β-Lapachone is able to inhibit the cysteine-proteinase activity of T. cruzi proteic whole extract and of cruzain, similar to E-64, a classical cysteine-proteinase inhibitor. Differently, Epoxy-α-lap inhibited the T. cruzi serine-proteinase activity, similar to PMSF, a classical serine-proteinase inhibitor. In agreement to these biological profiles in the enzymatic assays, our theoretical analysis showed that E-64 and β-lapachone interact with the cruzain specific S2 pocket and active site whereas Epoxy-α-lap showed no important interactions. Overall, our results infer that β-lapachone and Epoxy-α-lap compounds may inhibit T. cruzi epimastigotes growth by affecting T. cruzi different proteinases. Thus the present data shows the potential of these compounds as prototype of protease inhibitors on drug design studies for developing new antichagasic compounds.     

Trypanosoma cruzi infection disturbs mitochondrial membrane potential and ROS production rate in cardiomyocytes

In this study, we investigated the role of Trypanosoma cruzi invasion and inflammatory processes in reactive oxygen species (ROS) production in a mouse atrial cardiomyocyte line (HL-1) and primary adult rat ventricular cardiomyocytes. Cardiomyocytes were incubated with T. cruzi (Tc) trypomastigotes, Tc lysate (TcTL), or Tc secreted proteins (TcSP) for 0–72 h, and ROS were measured by amplex red assay. Cardiomyocytes infected by T. cruzi (but not those incubated with TcTL or TcSP) exhibited a linear increase in ROS production for 2–48 h postinfection (max 18-fold increase), which was further enhanced by recombinant cytokines (IL-1β, TNF-α, and IFN-γ). We observed no increase in NADPH oxidase, xanthine oxidase, or myeloperoxidase activity, and specific inhibitors of these enzymes did not block the increased rate of ROS production in infected cardiomyocytes. Instead, the mitochondrial membrane potential was perturbed and resulted in inefficient electron transport chain (ETC) activity and enhanced electron leakage and ROS formation in infected cardiomyocytes. HL-1 rho (ρ) cardiomyocytes lacked a functional ETC and exhibited no increase in ROS formation in response to T. cruzi. Together, these results demonstrate that invasion by T. cruzi and an inflammatory milieu affect mitochondrial integrity and contribute to electron transport chain inefficiency and ROS production in cardiomyocytes.     

Trypanosoma cruzi: Multiple actin isovariants are observed along different developmental stages

The expression and biological role of actin during the Trypanosoma cruzi life cycle remains largely unknown. Polyclonal antibodies against a recombinant T. cruzi actin protein were used to confirm its expression in epimastigotes, trypomastigotes, and amastigotes. Although the overall levels of expression were similar, clear differences in the subcellular distribution of actin among the developmental stages were identified. The existence of five actin variants in each developmental stage with distinct patterns of expression were uncovered by immunoblotting of protein extracts separated 2D-SDS gels. The isoelectric points of the actin variants in epimastigotes ranged from 4.45 to 4.9, whereas they ranged from 4.9 to 5.24 in trypomastigotes and amastigotes. To determine if the actin variants found could represent previously unidentified actins, we performed a genomic survey of the T. cruzi GeneDB database and found 12 independent loci encoding for a diverse group of actins and actin-like proteins that are conserved among trypanosomatids.     

Kinetic properties and inhibition of Trypanosoma cruzi 3-hydroxy-3-methylglutaryl CoA reductase

A detailed kinetic analysis of the recombinant soluble enzyme 3-hydroxy-3-methylglutaryl CoA reductase (HMGR) from Trypanosoma cruzi has been performed. The enzyme catalyzes the normal anabolic reaction and the reductant is NADPH. It also catalyzes the oxidation of mevalonate but at a lower proportion compared to the anabolic reaction. We report that the catalytically active species of HMGR in solution is the tetrameric form. Fluvastatin inhibited competitively the enzyme while cerivastatin binds by a mechanism which is more accurately described by a biphasic process characteristic of a class of ‘slow, tight-binding’ inhibitors.     

Aromatic guanyl hydrazones: Synthesis, structural studies and in vitro activity against Trypanosoma cruzi

A series of 22 aromatic guanyl hydrazones, prepared by condensation of several aldehydes with aminoguanidine hydrochloride, were fully characterized by NMR techniques and tested in vitro against the trypomastigote form of Trypanosoma cruzi, the causative agent of Chagas disease. Most of the compounds, especially those without hydrogen bonding groups and possessing ortho-substitution, were significantly more active than crystal violet (ID₅₀ 536 μM). The most active compound has an ID₅₀ value of 17 μM (25 times more potent than gentian violet).     

Energetics of heart mitochondria during acute phase of Trypanosoma cruzi infection in rats

The energetics of heart mitochondria was studied in the acute phase of Trypanosoma cruzi infection in rats. Wistar rats were infected with 2 × 10⁵ trypomastigote forms of the Y strain of T. cruzi, and heart mitochondria and submitochondrial particles isolated after 7 and 25 days of infection. Ultrastructure of mitochondria seemed to be preserved, but cytochrome c levels were significantly depressed. Respiratory control ratios (RCR) were decreased for glutamate and succinate oxidations, as a consequence of inhibition of respiration in state 3 and/or of stimulation of respiration in state 4. Stimulation of hydrolytic activity of FoF1-ATPase by energization of mitochondria was approx. 2-fold higher in relation to controls. Mitochondrial ATP concentration remained constant. In conclusion, during the acute phase of T. cruzi infection in rats there is an energy impairment at the level of heart mitochondria, but their ultrastructure and ATP concentration seem to be preserved; the maintenance of ATP may be due to an adaptative mechanism of the cell which includes inhibition of the hydrolytic activity of FoF1-ATPase.     

Activity of juvenile hormone and juvenile hormone analogues on the growth of Trypanosoma cruzi

The effects of juvenile hormone-III (JH-III) and the juvenile hormone analogues (JHA) methoprene and fenoxycarb on the growth and macromolecular biosynthesis in Trypanosoma cruzi were studied in vitro. It was observed that JH-III and JHA blocked growth and ³H-thymidine incorporation without killing the cells within certain concentrations (≤1 × 10⁻⁴M), but they caused cellular death at concentrations over 1 × 10⁻³M. The inhibitory effect on growth was partially reversible. On the other hand, the inhibitory action of JH-III, methoprene and fenoxycarb was an unspecific effect according to the results obtained with Leishmania mexicana mexicana (promastigotes) and human peripheral blood lymphocytes. The JHA have a good possibility of being used in the control of trypanosomiasis.