Transcriptomic response to different heme sources in Trypanosoma cruzi epimastigotes

Unstimulated mouse peritoneal exudate cells were cultured on coverslips in Medium 199 containing 10 per cent (v/v) calf serum. Cytochalasin B dissolved in dimethyl sulphoxide (DMSO) and diluted in Medium 199 was added to cultures to give final concentrations of 1, 5 and 10 mug/ml. Equal numbers of Leishmania mexicana promastigotes, Trypanosoma cruzi epimastigotes and sheep red cells were added to 24 hr cultures incubated at 37 C. The macrophage monolayers were fixed and stained at various time intervals. L. mexicana promastigotes sheep red blood cells were found to attach to macrophages in the presence of the drug but did not enter the cells. When the medium containing the cytochalasin was replaced with normal medium phagocytosis of the adherent parasites and red cells followed rapidly. T. cruzi epimastigotes were found inside macrophages in both drug-treated and drug-free cultures although the number found to be intracellular in the latter was significantly greater. This study suggest that L. mexicana promastigotes enter macrophages by being phagocytosed, whereas T. cruzi epimastigotes can actively penetrate these cells.

Although Balamuthia mandrillaris was identified more than two decades ago as an agent of fatal granulomatous encephalitis in humans and other animals, little is known about its ecological niche, biological behavior in the environment, food preferences and predators, if any. When infecting humans or other animals, Balamuthia feeds on tissues; and in vitro culture, it feeds on mammalian cells (monkey kidney cells, human lung fibroblasts, and human microvascular endothelial cells). According to recent reports, it is believed that Balamuthia feeds on small amebae, for example, Acanthamoeba that are present in its ecological niche. To test this hypothesis, we associated Balamuthia on a one-on-one basis with selected protozoa and algae. We videotaped the behavior of Balamuthia in the presence of a potential prey, its ability to hunt and attack its food, and the time required to eat and cause damage to the target cell by direct contact. We found that B. mandrillaris ingested trophozoites of Naegleria fowleri, Naegleria gruberi, Acanthamoeba spp., Trypanosoma cruzi epimastigotes, Toxoplasma gondii tachyzoites, and Giardia. However, it did not feed on Acanthamoeba cysts or algae. Balamuthia caused cytolysis of T. cruzi epimastigotes and T. gondii tachyzoites by direct contact. Balamuthia trophozoites and cysts were, however, eaten by Paramecium sp.

Chagas disease affects about 7 million people worldwide. Only two drugs are currently available for the treatment for this parasite disease, namely, benznidazol (Bzn) and nifurtimox (Nfx). Both drugs have limited curative power in the chronic phase of the disease. Therefore, continuous research is an urgent need so as to discover novel therapeutic alternatives. The development of safer and more efficient therapeutic anti-T. cruzi drugs continues to be a major goal in trypanocidal chemotherapy. Synthesis, 2D-QSAR and drug-like physicochemical properties of a set of quinazolinone and quinazoline derivatives were studied as trypanocidal agents. All compounds were screened in vitro against Trypanosoma cruzi (Tulahuen strain, Tul 2 stock) epimastigotes and bloodstream trypomastigotes. Out of 34 compounds synthesized and tested, six compounds (5a, 5b, 9b, 9h, 13f and 13p) displayed significant activity against both epimastigotes and tripomastigotes, without exerting toxicity on Vero cells. The antiprotozoal activity of these quinazolinone and quinazoline derivatives represents an interesting starting point for a medicinal chemistry program aiming at the development of novel chemotherapies for Chagas disease.

Our laboratories have been investigating biological effects of a sesquiterpenoid polygodial and its natural and synthetic analogues. Herein, we report the evaluation of these compounds against the three forms of Trypanosoma cruzi, amastigotes, trypomastigotes and epimastigotes. Although polygodial was found to be poorly active, its natural congener epipolygodial and synthetic Wittig-derived analogues showed low micromolar potency against all three forms of the parasite. Synthetic α,β-unsaturated phosphonate 9 compared favorably with clinically approved drugs benznidazole and nifurtimox, and was effective against trypomastigotes, toward which benznidazole showed no activity.

Background: Parasitic diseases, particularly Chagas disease caused by Trypanosoma cruzi, primarily affect developing countries but are now spreading to wealthier nations due to changing migration patterns. With approximately 8 to 9 million cases annually and a rise in drug resistance and side effects, there is an urgent need for new therapeutic approaches. Objectives: This study aimed to identify potential pharmacological compounds to target the TATA Binding Protein (TBP) of T. cruzi. Methods: Over eleven thousand FDA-approved pharmacological compounds were analyzed using in silico methods, including homology modeling, molecular docking, and molecular dynamics simulations. In addition, in vitro assays were conducted to assess the trypanocidal activity of promising candidates against T. cruzi epimastigotes and their selectivity toward macrophage J774.2. Results: Two compounds, DB00890 and DB07635, emerged as promising candidates, demonstrating significant potential against T. cruzi TBP. Compound DB00890 had trypanocidal activity against T. cruzi epimastigotes, with IC50 values of 70.4 µM (SI 2.84) and 37.3 µM (SI 5.36) for the NINOA and A1 strains, respectively. Conclusions: Our findings suggest DB00890 is a promising candidate for the development of new agents against Chagas disease, with the potential for targeted therapies that minimize side effects. These results provide a strong foundation for further research into novel treatments for parasitic diseases caused by T. cruzi.

Understanding the energy-transduction pathways employed by Trypanosoma cruzi, the etiological agent of Chagas disease, may lead to the identification of new targets for development of a more effective therapy. Herein, the contribution of different substrates for O(2) consumption rates along T. cruzi epimastigotes (Tulahuen 2 and Y strains) growth curve was evaluated. O(2) consumption rates were higher at the late stationary phase not due to an increase on succinate-dehydrogenase activity. Antimycin A and cyanide did not totally inhibit the mitochondrial respiratory chain (MRC). Malonate at 10 or 25 mM was not a potent inhibitor of complex II. Comparing complex II and III, the former appears to be the primary site of H(2)O(2) release. An update on T. cruzi MRC is presented that together with our results bring important data towards the understanding of the parasite's MRC. The findings mainly at the stationary phase could be relevant for epimastigotes transformation into the metacyclic form, and in this sense deserves further attention.

Poly(ADP-ribosylation) is a post-translational covalent modification of proteins catalyzed by a family of enzymes termed poly(ADP-ribose) polymerases (PARPs). In the human genome, 17 different genes have been identified that encode members of the PARP superfamily. Poly (ADP-ribose) metabolism plays a role in a wide range of biological processes. In Trypanosoma cruzi, PARP enzyme appears to play a role in DNA repair mechanisms and may also be involved in controlling the different phases of cell growth. Here we describe the identification of potent inhibitors for T. cruzi PARP with a fluorescence-based activity assay. The inhibitors were also tested on T. cruzi epimastigotes, showing that they reduced ADP-ribose polymer formation in vivo. Notably, the identified inhibitors are able to reduce the growth rate of T. cruzi epimastigotes. The best inhibitor, Olaparib, is effective at nanomolar concentrations, making it an efficient chemical tool for chacterization of ADP-ribose metabolism in T. cruzi. PARP inhibition also decreases drastically the amount of amastigotes but interestingly has no effect on the amount of trypomastigotes in the cell culture. Knocking down human PARP-1 decreases both the amount of amastigotes and trypomastigotes in cell culture, indicating that the effect would be mainly due to inhibition of human PARP-1. The result suggests that the inhibition of PARP could be a potential way to interfere with T. cruzi infection.

3-Hydroxy-2-methylene-3-(4-nitrophenylpropanenitrile): A new highly active compound against epimastigote and trypomastigote form of Trypanosoma cruzi

BackgroundReservosomes are lysosome-related organelles found in Trypanosoma cruzi epimastigotes. They represent the last step in epimastigote endocytic route, accumulating a set of proteins and enzymes related to protein digestion and lipid metabolism. The reservosome matrix contains planar membranes, vesicles and lipid inclusions. Some of the latter may assume rectangular or sword-shaped crystalloid forms surrounded by a phospholipid monolayer, resembling the cholesterol crystals in foam cells.Methodology/Principal FindingsUsing Nile Red fluorimetry and fluorescence microscopy, as well as electron microscopy, we have established a direct correlation between serum concentration in culture medium and the presence of crystalloid lipid inclusions. Starting from a reservosome purified fraction, we have developed a fractionation protocol to isolate lipid inclusions. Gas-chromatography mass-spectrometry (GC-MS) analysis revealed that lipid inclusions are composed mainly by cholesterol and cholesterol esters. Moreover, when the parasites with crystalloid lipid-loaded reservosomes were maintained in serum free medium for 48 hours the inclusions disappeared almost completely, including the sword shaped ones.Conclusions/SignificanceTaken together, our results suggest that epimastigote forms of T. cruzi store high amounts of neutral lipids from extracellular medium, mostly cholesterol or cholesterol esters inside reservosomes. Interestingly, the parasites are able to disassemble the reservosome cholesterol crystalloid inclusions when submitted to serum starvation.

Three enzyme immunoassays (EIAs) for diagnosis of Chagas' disease were developed with fixed forms of Trypanosoma cruzi using a panel of 435 sera from the following groups: Venezuelan subjects positive by immunofluorescence (n = 70), Venezuelan healthy controls (n = 85), healthy Canadians (n = 166), and subjects with other parasitic diseases (n = 114). All assays achieved 100% sensitivity and reasonable specificity for amastigotes (97.6%), epimastigotes (98.3%), and trypomastigotes (99.3%). The fixed-trypomastigote assay was stable over 4 months at 4 degrees C and room temperature. These data suggest that a fixed-trypomastigote EIA may be a suitable candidate for blood bank screening.

The mouse macrophage cell line J774 was easily infected by T. cruzi epimastigotes which were transformed to amastigotes that multiplied inside the cells. Spleen-T-cells from T. cruzi immune mice stimulated with Concanavalin A or T. cruzi, but not with unrelated antigens, released lymphokines into the supernatants that when added to J774 cells were unable to induce complete trypanocidal activity, although they were able to delay the rate of infection by protecting the cells from being infected. Addition of bacterial lipopolysaccharide (LPS), although inactive by itself, acted synergistically with the supernatants in inducing complete trypanocidal activity without affecting the susceptibility of J774 cells to infection. Gamma-interferon (gamma-IFN) activity was detected in the supernatants, however, but was not solely responsible for the trypanocidal inducing activities, since: there was no correlation between the levels of gamma-IFN and macrophage activation; gamma-IFN alone was less effective than the supernatants alone; and two active fractions of 100,000-150,000 mol. wt and 30,000 mol. wt were separated by gel filtration chromatography of the lymphokine preparations. The latter, which showed the characteristics of gamma-IFN with respect to size, pH 2 sensitivity and antiviral activity, had some trypanocidal activity alone. However, the 100,000-150,000 mol. wt fraction was active only in the presence of LPS. Finally, this trypanocidal inducing activity of the supernatants was not due to the induction of synthesis of gamma-IFN by the J774 cells.

The existence of a GTP-binding protein of the Ns type in Trypanosoma cruzi was explored. Epimastigote membranes were labelled by cholera toxin in the presence of [adenine-14C]NAD+. After SDS/polyacrylamide-gel electrophoresis of extracted membrane proteins, a single labelled polypeptide band of apparent Mr approx. 45,000 was detected. Epimastigote cells were treated with N-ethylmaleimide and electrofused to lymphoma S49 cells lacking the Ns protein. Evidence indicates that in such electrofusion-generated cell hybrids a heterologous adenylate cyclase system was reconstituted with the Ns protein provided by T. cruzi epimastigotes.

Background: Worldwide, the number of cases of parasitic diseases has been increasing; however, available treatments have variable adverse effects and low efficacy, mainly in Neglected Tropical Diseases such as Chagas disease and Leishmaniasis. Therefore, the development of new and more effective antiparasitic drugs is important. Natural products are the source of secondary metabolites with different biological activities, such as antibacterial, anticancer, anti-inflammatory, and antiparasitic. Objectives: In this work, secondary metabolites (phenols and terpenes) from natural products were selected to be evaluated against the epimastigotes of NINOA and A1 strains of Trypanosoma cruzi and the promastigotes of M379 strain and FCQEPS native isolate of Leishmania mexicana. Additionally, their cytotoxicity and selectivity index were determined. Methods: Eighteen secondary metabolites were evaluated in vitro against T. cruzi epimastigotes and L. mexicana promastigotes; additionally, their cytotoxicity on the J774.2 macrophage cell line was determined. Results: The compounds l-(-)-menthol (14, IC50 = 24.52 µM) and β-citronellol (11, IC50 = 21.54 µM) had higher trypanocidal activity than the reference drug (benznidazole) against NINOA and A1 strains of T. cruzi, respectively. On the other hand, para-anisyl alcohol (4, IC50 = 34.89 µM) had higher leishmanicidal activity than the reference drug (glucantime®) against M379 and the FCQEPS native isolate of L. mexicana. Finally, in silico, the determination of their pharmacokinetic and toxicological properties showed that they are promising candidates for oral and topical uses. Conclusions: This study opens the possibility of using secondary metabolites as scaffolds for access to the development of new molecules for the treatment of parasite diseases.

Infection with the protozoan parasite Trypanosoma cruzi causes human Chagas disease. Benznidazole (BNZ) and nifurtimox are the current drugs for the treatment; however, they induce severe adverse side effects in patients; therefore, there is a need to improve the treatment effectiveness and efficiency of these drugs for its safer use. Background/Objective: Glyburide, glipizide, and gliquidone, hypoglycemic drugs for diabetes treatment, were previously predicted to bind to dihydrofolate reductase-thymidylate synthase from T. cruzi by in silico docking analysis; they also showed antiproliferative effects against T. cruzi epimastigotes, the stage of the insect vector. In the present study, the potential parasiticidal effect of these antidiabetic drugs was tested in monotherapy and bi-therapy with BNZ in human cells in vitro and in animals. Methods: Evaluation was performed in (a) a model of in vitro infection of T. cruzi trypomastigotes using human fibroblasts as host cells and (b) in mice infected with T. cruzi. Results: The antidiabetic drugs in monotherapy showed antiparasitic effects in preventing infection progression (trypomastigotes release), with an IC50 of 8.4-14.3 µM in comparison to that of BNZ (0.26 µM) in vitro. However, in bi-therapy, the presence of just 0.5 or 1 µM of the antidiabetics decreased the BNZ IC50 by 5-10 times to 0.03-0.05 µM. Remarkably, the antidiabetic drugs in monotherapy decreased the infection in mice by 40-60% in a similar extent to BNZ (80%). In addition, the combination of BNZ plus antidiabetics perturbed the antioxidant metabolites in epimastigotes. Conclusions: These results identified antidiabetics as potential drugs in combination therapy with BNZ to treat T. cruzi infection.

The transcription promoter of the spliced leader gene from Trypanosoma cruzi