Trypomastigote Forms Research Articles

Trypanosoma cruzi: A kinetoplast-associated protein of the photolyase/cryptochrome family

A photolyase-like protein gene found in the Trypanosoma cruzi genome database was cloned and expressed in Escherichia coli resulting in the formation of inclusion bodies. Antibodies against this protein were used to determine expression of the protein in the different forms of the parasite. It was visualized in the epimastigote form but not in amastigote or trypomastigote forms obtained from culture in Vero cells. In epimastigotes, this protein is located at the level of the mitochondrion associated to both sides of the kinetoplast. Sequence analyses indicated that this protein, as well as other photolyases from Leishmania spp. and Trypanosoma brucei are related to single-stranded photolyases or cryptochromes DASH.

Enalapril prevents cardiac immune-mediated damage and exerts anti-Trypanosoma cruziactivity during acute phase of experimental Chagas disease

Chagas heart disease (CHD), caused by Trypanosoma cruzi infection, is a significant cause of morbidity and mortality in South and Central America. Enalapril, an angiotensin converting enzyme (ACE) inhibitor, is an important drug used to ameliorate heart functional capacity and its remodelling in individuals presenting CHD. In this study, we evaluated the effects of enalapril on systemic and cardiac immune response during experimental acute CHD. C57BL/6 mice infected with 50 trypomastigote forms of T. cruzi (Colombian strain) were treated daily with enalapril (25 mg/kg) and, after 30 days, a reduction in seric levels of IFN-gamma, TNF-alpha, CCL5/RANTES and nitric oxide, but not in that of IL-10, was detected. This imbalance of cytokines reflects in a reduction of heart mononuclear infiltration and in an increasing of cardiac mast cells. Enalapril also presents a new and interesting in vitro and in vivo anti-T. cruzi activity probably acting on parasite oxidative pathway via cytochrome-P450. Our data show that enalapril exerts an important anti-T. cruzi and anti-inflammatory activity during acute CHD reducing inflammatory cells and, possibly, preventing fibrotic process in the chronic phase. Nevertheless, further studies are still necessary to clarify the mechanisms by which this drug is acting on the parasites and on the immune pathways.

Primeiro registro de Trypanosoma vivax em bovinos no Estado do Rio Grande do Sul, Brasil

Este estudo teve o objetivo de relatar a ocorrência de Trypanosoma vivax no Sul do Brasil. O protozoário foi diagnosticado em esfregaço sanguíneo de um bovino e a identificação baseada na morfologia das formas tripomastigotas e confirmada pela técnica de reação em cadeia de polimerases (PCR). O animal infectado apresentou sintomatologia compatível com a forma nervosa da infecção por T. vivax. Outros bovinos que compartilhavam o mesmo ambiente apresentaram resultados negativos para T. vivax por PCR. Este é o primeiro registro de T. vivax no Estado do Rio Grande do Sul e na região Sul do Brasil.

Influence of Trypanosoma evansi in blood, plasma, and brain cholinesterase of experimentally infected cats

Changes in blood, plasma and brain cholinesterase activities in Trypanosoma evansi-infected cats were investigated. Seven animals were infected with 10 8 trypomastigote forms each and six were used as control. Animals were monitored for 56 days by examining daily blood smears. Blood samples were collected at days 28 and 56 post-inoculation to determine the activity of acetylcholinesterase (AChE) in blood and the activity of butyrylcholinesterase (BChE) in plasma. AChE was also evaluated in total brain. The activity of AChE in blood and brain, and the activity of BChE in plasma significantly reduced in the infected cats. Therefore, the infection by T. evansi influenced cholinesterases of felines indicating changes in the responses of the cholinergic system.

Melatonin and dehydroepiandrosterone combination: does this treatment exert a synergistic effect during experimental Trypanosoma cruzi infection?

Previous studies showed that melatonin or dehydroepiandrosterone (DHEA) enhances the immune response against parasitic pathogens. The present study investigated the in vitro activity of melatonin combined with DHEA in a period of 24 hr during the course of in vivo T. cruzi infection. The in vitro activity of melatonin or DHEA alone, as well as together, were tested for the trypomastigote forms (doses ranging from 0.5 to 128 microm). In vitro, neither melatonin nor DHEA alone had any activity against trypomastigote forms, although when the highest concentration of combined melatonin and DHEA was used, it was active against the trypomastigote forms of the parasite. However, for this concentration, a quite toxicity on peritoneal macrophages was observed. For in vivo evaluation, male Wistar rats were infected with the Y strain of T. cruzi. They were orally treated with 10 mg/kg body weight/day of melatonin and subcutaneously with 40 mg/kg body weight/day of DHEA. Treatment with melatonin, DHEA and the association showed a significant reduction in the number of blood trypomastigotes during the acute phase of infection as compared to untreated animals (P < 0.05). A significant increase in the number of macrophages and nitric oxide (NO) concentrations were observed during the peak of parasitaemia with melatonin alone or combined with DHEA. However, with DHEA alone the highest concentration of NO was observed (P < 0.05). Moreover, DHEA treatment increased TNF-alpha levels during the infection (P < 0.05). These results show that melatonin, DHEA or the combination of both reduces parasitemia during the acute phase of infection. The combined action of both molecules did not exert a synergic action on the host's ability to fight infection, and it seems that among all treatments DHEA induces a more efficient immune response.

In vitro analyses of the effect of aromatic diamidines upon Trypanosoma cruzi

Aromatic diamidines (ADs) have been recognized as promising antiparasitic agents. Therefore, in the present work, the in vitro trypanocidal effect of 11 ADs upon the relevant clinical forms of Trypanosoma cruzi was evaluated, as well as determining their toxicity to mammalian cells and their subcellular localization. The trypanocidal effect upon trypomastigotes and amastigotes was evaluated by light microscopy through the determination of the IC(50) values. The cytotoxicity was determined by the MTT colorimetric assay against mouse cardiomyocytes. For the subcellular localization, transmission electron microscopy and fluorescence approaches were used. The fluorescence intensity within the kinetoplast DNA (kDNA) and nuclear DNA (nDNA) of treated parasites was determined using the Image J program. Compounds 2, 5 and 7 showed the lowest IC(50) values (micromolar range) against intracellular amastigotes and trypomastigotes. In the presence of blood, all the tested ADs exhibited a reduction of their activity. The compounds did not exhibit toxicity to cardiac cells and the highest selectivity index (SI) was achieved by compound 5 with an SI of >137 for trypomastigotes and compound 7 with an SI of >107 for intracellular parasites. The subcellular effects upon bloodstream forms treated with compounds 5 and 7 were mainly on kDNA, leading to its disorganization. The higher accumulation in the kDNA observed for all tested ADs was not directly related to their efficacy. Our results show the high activity of this new series of ADs against both trypomastigote and amastigote forms, with excellent SIs, especially compound 7, which merits further in vivo evaluation.

Design, synthesis and cruzain docking of 3-(4-substituted-aryl)-1,2,4-oxadiazole- N-acylhydrazones as anti- Trypanosoma cruzi agents

Research in recent years has demonstrated that the Trypanosoma cruzi cysteine protease cruzain (TCC) is a valid chemotherapeutic target, since inhibitors of this protease affect the pathology appropriately. By exploring the N-acylhydrazones (NAH) as privileged structures usually present in antiparasitic agents, we investigated a library of 16 NAH bearing the 3-(4-substituted-aryl)-1,2,4-oxadiazole scaffold (NAH 3a– h, 4a– h). The in vitro bioactivity against epimastigote and trypomastigote forms of T. cruzi was evaluated, and some NAH under study exhibited antitrypanosomal activity at concentrations that are not toxic to mammalian cells. The series of compounds based on the 3-(4-substituted-aryl)-1,2,4-oxadiazole scaffold revealed the remarkable importance of each substituent at the phenyl’s 4-position for the inhibitory activity. Non-nitrated compounds 3a and 4e were found to be as potent as the reference drug, Benznidazole. In addition, the molecular origin of the antitrypanosomal properties for these series was investigated using docking studies of the TCC structure.

Identification of the mechanism of action of eupomatenoid-5 isolated from Piper regnellii var. pallescens on Trypanosoma cruzi

Parasitic protozoa cause several diseases, affecting hundreds of millions, particularly in underdeveloped countries [1]. Chagas disease is one of these clinical conditions triggered by infection with the protozoan Trypanosoma cruzi. The currently drugs available for treatment of this infection are unsatisfactory due to limited efficacy and toxic side effects, making the search for more specific pharmacological agents a priority [2]. In previous work, eupomatenoid-5 isolated from Piper regnellii var. pallescens showed activity against the proliferative stages of T. cruzi [3]. Here, we decided to investigate the mechanism of action of the eupomatenoid-5 on T. cruzi. The components of the trypanothione-dependent antioxidant system from T. cruzi have been pointed out as potential chemotherapeutic targets [2] by presenting differences with mammalian host. Thus, epimastigotes were pre-treated with eupomatenoid-5 and challenged with doses of H2O2 in IB medium. Then, the cells were resuspended in LIT medium and the growth index (GI) determined after 5 days. Control of the eupomatenoid-5 and H2O2 were also performed. For lipoperoxidation assay, the same treatment was performed and determined as the amount of thiobarbituric acid-reactive substances in terms of malondialdehyde [4]. The results showed a decrease of GI after pre-treatment and challenged with non-toxic dose (20mM) (35.4%) and sub-lethal dose of H2O2 (100mM) (17.9%), while the control eupomatenoid-5 GI was 61.5%. Additionally, the same treatment increased lipoperoxidation compared with the controls H2O2 and eupomatenoid-5. These results have indicated that eupomatenoid-5 can act in the detoxification system of T. cruzi making these cells more susceptive to H2O2. Studies in trypomastigote and amastigote intracellular forms are being performed.

Trypanocidal, leishmanicidal and cytotoxic effects of anthecotulide type linear sesquiterpene lactones from Anthemis auriculata

Trypanosomiasis and leishmaniasis pose major public health threats for many countries, particularly those in sub-Saharan Africa and South America. In the present study, we evaluated the in vitro antiprotozoal activity of three irregular, linear sesquiterpene lactones recently isolated from Greek Anthemis auriculata, namely anthecotulide (1), 4-hydroxyanthecotulide (2) and 4-acetoxyanthecotulide (3). Trypomastigote forms of Trypanosoma brucei rhodesiense and T. cruzi as well as axenic amastigotes of Leishmania donovani were used for testing. The cytotoxic potential of the compounds was also assessed against mammalian (rat) skeletal myoblasts (L6 cells). All compounds showed potent trypanocidal and leishmanicidal activity. 4-Hydroxyanthecotulide (2) appeared to be the most active compound against all parasites, particularly towards T. b. rhodesiense (IC(50) 0.56 microg/ml), whereas 4-acetoxyanthecotulide (3) was the least active. All three metabolites possessed toxicity on mammalian cells, which might limit their use as antiprotozoal agents.

Diminazene aceturate in the control of Trypanosoma evansi infection in cats

The aim of this study was to investigate the efficacy of diminazene aceturate in the control of the infection by Trypanosoma evansi in cats. Fourteen animals were infected with 10 8 trypomastigote forms each and six were used as negative control (group A). Seven of the infected cats were used as positive control (group B) and seven were treated with diminazene aceturate (3.5 mg kg −1) for 5 consecutive days (group C). Biochemical and hematological parameters were evaluated during the experiment. Blood with anticoagulant was collected at day 49 post-inoculation and preserved in ethanol for DNA extraction. Samples were analyzed using PCR T. evansi-specific to assess the effectiveness of treatment. The treatment with diminazene aceturate had an efficacy of 85.7%. Alanine aminotransferase, gamma-glutamyltransferase, urea, and creatinine values remained within the normal physiological range in the treated cats. Hemogram was normalized in all the cured animals. Therefore, the therapy used is effective in controlling T. evansi in cats.

Ruthenium complexes endowed with potent anti- Trypanosoma cruzi activity: Synthesis, biological characterization and structure–activity relationships

Although effective against epimastigotes (proliferative form) and of low cytotoxicity in mammals, the aryl-4-oxothiazolylhydrazones (ATZ) display only limited activity against trypomastigotes (bloodstream form) of Trypanosoma cruzi. Considering the metal complexation approach with bioactive ligands as one possible strategy for improving the biological efficacy of ATZ, a set of eight new ruthenium–ATZ complexes (RuCl 2ATZCOD, COD is 1,5-cyclooctadiene) were prepared, chemically and biologically characterized, including in vitro assays against epimastigotes and trypomastigote forms of the parasite and also assessment of cytotoxicity in mammals. Two of these complexes presented antitrypanosomal activity at non-cytotoxic concentrations on mammalian cells and of higher potency than its metal–free ligands, while the metallic precursor [RuCl 2COD(MeCN) 2] showed only moderate antitrypanosomal activity. Comparative analysis between the ruthenium complexes and metal–free ligands demonstrated the usefulness of this approach, with the establishment of new SAR data. Additional pharmacological tests, including a DNA bond assay, gave rise to the proposal of a single preliminary explanation for the molecular origin of the bioactivity.

Proteomic Analysis of Detergent-Solubilized Membrane Proteins from Insect-Developmental Forms of Trypanosoma cruzi

The cell surface of Trypanosoma cruzi, the etiologic agent of Chagas disease, is covered by a dense layer of glycosylphosphatidylinositol (GPI)-anchored molecules. These molecules are involved in a variety of interactions between this parasite and its mammalian and insect hosts. Here, using the neutral detergent Triton X-114, we obtained fractions rich in GPI-anchored and other membrane proteins from insect developmental stages of T. cruzi. These fractions were analyzed by two-dimensional liquid chromatography coupled to tandem mass spectrometry (2D-LC-MS/MS), resulting in the identification of 98 proteins of metacyclic trypomastigotes and 280 of epimastigotes. Of those, approximately 65% (n=245) had predicted lipid post-translational modification sites (i.e., GPI-anchor, myristoylation, or prenylation), signal-anchor sequence, or transmembrane domains that could explain their solubility in detergent solution. The identification of some of these modified proteins was also validated by immunoblotting. We also present evidence that, in contrast to the noninfective proliferative epimastigote forms, the infective nonproliferative metacyclic trypomastigote forms express a large repertoire of surface glycoproteins, such as GP90 and GP82, which are involved in adhesion and invasion of host cells. Taken together, our results unequivocally show stage-specific protein profiles that appear to be related to the biology of each T. cruzi insect-derived developmental form.

Use of l -Proline and ATP Production by Trypanosoma cruzi Metacyclic Forms as Requirements for Host Cell Invasion

The process of host cell invasion by Trypanosoma cruzi depends on parasite energy. What source of energy is used for that event is not known. To address this and other questions related to T. cruzi energy requirements and cell invasion, we analyzed metacyclic trypomastigote forms of the phylogenetically distant CL and G strains. For both strains, the nutritional stress experienced by cells starved for 24, 36, or 48 h in phosphate-buffered saline reduced the ATP content and the ability of the parasite to invade HeLa cells proportionally to the starvation time. Inhibition of ATP production by treating parasites with rotenone plus antimycin A also diminished the infectivity. Nutrient depletion did not alter the expression of gp82, the surface molecule that mediates CL strain internalization, but increased the expression of gp90, the negative regulator of cell invasion, in the G strain. When L-proline was given to metacyclic forms starved for 36 h, the ATP levels were restored to those of nonstarved controls for both strains. Glucose had no such effect, although this carbohydrate and L-proline were transported in similar fashions. Recovery of infectivity promoted by L-proline treatment of starved parasites was restricted to the CL strain. The profile of restoration of ATP content and gp82-mediated invasion capacity by L-proline treatment of starved Y-strain parasites was similar to that of the CL strain, whereas the Dm28 and Dm30 strains, whose infectivity is downregulated by gp90, behaved like the G strain. L-Proline was also found to increase the ability of the CL strain to traverse a gastric mucin layer, a property important for the establishment of T. cruzi infection by the oral route. Efficient translocation of parasites through gastric mucin toward the target epithelial cells in the stomach mucosa is an essential requirement for subsequent cell invasion. By relying on these closely associated ATP-driven processes, the metacyclic trypomastigotes effectively accomplish their internalization.

Clotting disturbances in Trypanosoma evansi-infected cats

Coagulopathy in cats (Felis catus) parasitized by Trypanosoma evansi in both the initial and chronic phases of the disease has been investigated. To address this issue, seven animals were infected with 108 trypomastigote forms each, and six were used as control. Animals were monitored for 56 days by examining daily blood smears. Blood samples for hematocrit, platelet counting, and fibrinogen levels were collect at 15-day intervals. Prothrombin time, activated partial thromboplastin time, and number of megakaryocytes were analyzed at days 14 and 42 post-inoculation. A decrease in hematocrit values and platelet counts and an increase in plasmatic fibrinogen concentration were observed in the infected cats (P < 0.05). Coagulation time did not differ between the infected and non-infected groups. The reduction in platelet count increased the number of megakaryocytes in the infected group (P < 0.05). These results indicate that the infection by T. evansi in cats increased the number of megakaryocytes in response to primary clotting disorders.

The antitumoral, trypanocidal and antileishmanial activities of extract and alkaloids isolated from Duguetia furfuracea

The alkaloid extract and five alkaloids isolated from subterranean stem bark of Duguetia furfuracea (Annonaceae) were investigated for the following activities: antitumoral, trypanocidal and leishmanicidal. Dicentrinone showed weak cytotoxicity, but it had the strongest leishmanicidal activity (IC(50) 0.01 microM). Duguetine and duguetine beta-N-oxide caused considerable antitumoral activity in every cell lines evaluated, although duguetine was more active against trypomastigote forms (IC(50) 9.32 microM) than other alkaloids tested.

2,3-Diphenyl-1,4-Naphthoquinone: A Potential Chemotherapeutic Agent Against Trypanosoma cruzi

Chagas disease, caused by Trypanosoma cruzi, is a widespread infection in Latin America. Currently, only 2 partially effective and highly toxic drugs, i.e., benznidazole and nifurtimox, are available for the treatment of this disease, and several efforts are underway in the search for better chemotherapeutic agents. Here, we have determined the trypanocidal activity of 2,3-diphenyl-1 ,4-naphthoquinone (DPNQ), a novel quinone derivative. In vitro, DPNQ was highly cytotoxic at a low, micromolar concentration (LD50 = 2.5 microM) against epimastigote, cell-derived trypomastigote, and intracellular amastigote forms of T. cruzi, but not against mammalian cells (LD50 = 130 microM). In vivo studies on the murine model of Chagas disease revealed that DPNQ-treated animals (3 doses of 10 mg/kg/day) showed a significant delay in parasitemia peak and higher (up to 60%) survival rate 70 days post-infection, when compared with the control group (infected, untreated). We also observed a 2-fold decrease in parasitemia between the control group (infected, untreated) and the treated group (infected, treated). No apparent drug toxicity effects were noticed in the control group (uninfected, treated). In addition, we determined that DPNQ is the first competitive inhibitor of T. cruzi lipoamide dehydrogenase (TcLipDH) thus far described. Our results indicate that DPNQ is a promising chemotherapeutic agent against T. cruzi.

A single pair of CD4+ and CD8+ T cell epitopes can induce protective T. cruzi immunity (39.56)

Abstract Both CD4+ and CD8+ T cells are required for immunity protective against the protozoan parasite, Trypanosoma cruzi. The unique T. cruzi trans-sialidase (TS) antigen can induce both mucosal and systemic protection. BALB/c mice develop CD8 responses directed against the H2-Kd-restricted TS epitope IYNVGQVSI (aa 359-367). We have used overlapping peptides (18mers with 15 aa overlaps) spanning the TS amino-terminus (aa 33-275) to identify an H2-IAd-restricted CD4 epitope that induces potent TS-specific Th1 responses (KVTERVVHSFRLPALVNV; aa 58-75). Here we demonstrate that dendritic cells (DC) pulsed only with KVTERVVHSFRLPALVNV and IYNVGQVSI can induce potent protective immunity. DC generated in vivo with flt3-ligand were pulsed with 50 ug/ml of each peptide and injected intravenously into BALB/c mice (1 million/mouse) 3 times at 2 week intervals. One month later these mice were challenged with 5000 Tulahuén blood form trypomastigotes. Controls included unvaccinated mice and mice given DC only. Both parasitemia and mortality were significantly reduced (p&amp;lt;0.05) in mice receiving DC pulsed with the TS peptides. These results indicate that monospecific CD4+ and CD8+ T cells can protect against a complex parasitic pathogen. We are preparing transgenic mice expressing TCR specific for these two epitopes with the goal of assessing in detail the mechanisms involved in the development of protective T. cruzi immunity. This work was funded by NIH RO1 #AI040196 awarded to DFH.

Perspectives on the Trypanosoma cruzi-host cell receptor interactions.

Chagas disease is caused by the parasite Trypanosoma cruzi. The critical initial event is the interaction of the trypomastigote form of the parasite with host receptors. This review highlights recent observations concerning these interactions. Some of the key receptors considered are those for thromboxane, bradykinin, and for the nerve growth factor TrKA. Other important receptors such as galectin-3, thrombospondin, and laminin are also discussed. Investigation into the molecular biology and cell biology of host receptors for T. cruzi may provide novel therapeutic targets.

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 Trypanosoma cruzi 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 .

Trypanosoma caninum n. sp. (Protozoa: Kinetoplastida) isolated from intact skin of a domestic dog (Canis familiaris) captured in Rio de Janeiro, Brazil

An unknown Trypanosoma species was isolated from an axenic culture of intact skin from a domestic dog captured in Rio de Janeiro, Brazil, which was co-infected with Leishmania (Viannia) braziliensis. Giemsa-stained smears of cultures grown in different media revealed the presence of epimastigotes, trypomastigotes, spheromastigotes, transitional stages, and dividing forms (epimastigotes or spheromastigotes). The highest frequency of trypomastigotes was observed in RPMI (15.2%) and DMEM (9.2%) media containing 5% FCS, with a mean length of these forms of 43.0 and 36.0 mum, respectively. Molecular analysis by sequential application of PCR assays indicated that this trypanosome differs from Trypanosoma cruzi and T. rangeli when specific primers were applied. On the other hand, a PCR strategy targeted to the D7 domain of 24salpha rDNA, using primers D75/D76, amplified products of about 250 bp in that isolate (stock A-27), different from the amplification products obtained with T. cruzi and T. rangeli. This organism differs from T. cruzi mainly by the size of its trypomastigote forms and kinetoplasts and the absence of infectivity for macrophages and triatomine bugs. It is also morphologically distinct from salivarian trypanosomes reported in Brazil. Isoenzyme analysis at 8 loci demonstrated a very peculiar banding pattern clearly distinct from those of T. rangeli and T. cruzi. We conclude that this isolate is a new Trypanosoma species. The name T. caninum is suggested.