Amphotericin B-induced damage of Trypanosoma cruzi epimastigotes

Amphotericin B (AmB) autoxidation resulted in oxygen consumption, superoxide anion formation and production of thiobarbituric acid (TBA)-reactive material (malondialdehyde). Malondialdehyde formation increased after incubation of the drug with ascorbate-ADP-FeCl3. Growth of Trypanosoma cruzi epimastigotes in the presence of AmB induced a decrease in the free fatty acid content of the cells (57% in control cells vs. 7% in AmB-treated cells), and in the proportion of unsaturated fatty acids as well as cell killing. No changes were detected on sterol content. No evidence was found for lipid peroxidation as a mechanism of cell injury by this antibiotic.     

The aerobic metabolism of metronidazole by Trypanosoma cruzi epimastigotes

Trypanosoma cruzi epimastigotes actively metabolize metronidazole under aerobic conditions to a polar compound tentatively identified as 2-methyl-5-nitroimidazole-1-yl-acetic acid. The rate of metabolite formation is increased by more than 50% by pretreatment with phenobarbital and inhibited by SKF-525A and metyrapone. The reaction is dramatically stimulated by the addition of flavone which suggests that the metabolite is produced via the cytochrome P-450 system. Apparently the nitro group in the metabolite is maintained intact. Detoxication reactions catalyzed by cytochrome P-450 appear to be more important than previously suspected as a basis to explain at least partially the resistance of these organisms to known antimicrobial agents. However, other factors such as the fate of nitro substituent in metronidazole require further evaluation.     

Non-histone protein methylation in Trypanosoma cruzi epimastigotes

Post-translational methylation of proteins, which occurs in arginines and lysines, modulates several biological processes at different levels of cell signaling. Recently, methylation has been demonstrated in the regulation beyond histones, for example, in the dynamics of protein-protein and protein-nucleic acid interactions. However, the presence and role of non-histone methylation in Trypanosoma cruzi, the etiologic agent of Chagas disease, has not yet been elucidated. Here, we applied mass spectrometry-based-proteomics (LC-MS/MS) to profile the methylproteome of T. cruzi epimastigotes, describing a total of 1252 methyl sites in 824 proteins. Functional enrichment and protein-protein interaction analysis show that protein methylation impacts important biological processes of the parasite, such as translation, RNA and DNA binding, amino acid, and carbohydrate metabolism. In addition, 171 of the methylated proteins were previously reported to bear phosphorylation sites in T. cruzi, including flagellar proteins and RNA binding proteins, indicating that there may be an interplay between these different modifications in non-histone proteins. Our results show that a broad spectrum of functions is affected by methylation in T. cruzi, indicating its potential to impact important processes in the biology of the parasite and other trypanosomes.     

Epoxide hydrase in Trypanosoma cruzi epimastigotes.

1. Microsomal fractions from Trypanosoma cruzi epimastigotes catalyze the hydration of styrene oxide to styrene glycol. The activity is linear up to 45 min of incubation, is proportional to microsomal protein concentration within certain range, and has an optimum pH of 8.5. 2. Double-reciprocal plots indicate a Km value of 5.3 . 10(-4) M for styrene oxide and a V of 29.6 pmol of styrene glycol formed/min per mg protein at 37 degrees C. 4-Chlorophenyl-2,3-epoxypropyl either (Ki = 2.08 . 10(-4) M) and juvenile hormone I (Ki = 2.7 . 10(-4) M) are competitive inhibitors; whereas, 1-chloro-2,3-epoxypropane is a non-competitive inhibitor. The enzyme is induced about three-fold by 5 mM phenobarbital in the growth medium. 3. The epoxide hydrase is not activated by detergents but rather inhibited by concentrations of Tween-80 and Lubrol as low as 0.025%. 4. Experiments with intact cells indicate that about 3% of [8-14C]styrene oxide penetrates after 90 min of incubation; whereas, over 30% of juvenile hormone I is found intracellularly after the same incubation period. Intracellular styrene oxide is hydrated to styrene glycol to a significant extent and the in vivo hydration is increased by pretreatment with phenobarbital and inhibited upon the addition of 4-chlorophenyl-2,3-epoxypropyl ether. Only a small amount of the intracellular juvenile hormone I is recovered as the corresponding diol ester.     

Effects of hydroquinones on intact Trypanosoma cruzi epimastigotes

1. Hydroquinones inhibited the culture growth of Trypanosoma cruzi epimastigotes at concentrations lower than 1 mM.2. Hydroquinones inhibited the oxygen consumption on the intact Trypanosoma cruzi cells. I₅₀ values for hydroquinone, terbutylhydroquinone and 2,5-di-t-butylhydroquinone were 24.87 mM, 0.88 mM and 0.26 mM, respectively. t-Butylhydroquinone and 2,5-di-t-butylhydroquinone had a Michaelian type kinetic inhibition; hydroquinone also showed a Michaelian type kinetic inhibition at low concentrations but at higher concentrations it showed a positive cooperativity.3. These hydroquinones changed the NAD(P) redox-state to a more reduced state and that of cytochrome b to a more oxidized state. The magnitude of the redox-state change was dependent of the hydrofobicity of the derivates.4. These results suggest that the growth and oxygen uptake inhibition by the hydroquinones is due to a blockage of the mitochondrial electron transport chain before cytochrome b.     

Lack of arginine decarboxylase in Trypanosoma cruzi epimastigotes

The presence of arginine decarboxylase (ADC) enzymatic activity in Trypanosoma cruzi epimastigotes is still a matter of controversy due to conflicting results published during the last few years. We have investigated whether arginine might indeed be a precursor of putrescine via agmatine in these parasites. We have shown that wild-type T. cruzi epimastigotes cultivated in a medium almost free of polyamines stopped their growth after several repeated passages of cultures in the same medium, and that neither arginine nor omithine were able to support or reinitiate parasite multiplication. In contrast, normal growth was quickly resumed after adding exogenous putrescine or spermidine. The in vivo labelling of parasites with radioactive arginine showed no conversion of this amino acid into agmatine, and attempts to detect ADC activity measured by the release of CO2 under different conditions in T. cruzi extracts gave negligible values for all strains assayed. The described data clearly indicate that wild-type T. cruzi epimastigotes lack ADC enzymatic activity.     

Impairment of cell division of Trypanosoma cruzi epimastigotes

The mechanisms that facilitate the adaptation of Trypanosoma cruzi to two distinct hosts, insect and vertebrate, are poorly understood, in part due to the limited ability to perform gene disruption studies by homologous recombination. This report describes a developmentally-defective phenotype that resulted from integration of a drug marker adjacent to the GAPDH gene in T. cruzi.     

Regulatory studies of L-glutamate dehydrogenase from Trypanosoma cruzi epimastigotes

Carboxylesterase activity corresponding to types A and B has been demonstrated in intact T. cruzi epimastigotes as shown by the hydrolysis of several esters of p-nitrophenol and the effect of suitable inhibitors. The in situ carboxylesterase activity was described by the Michaelis Menten kinetic approach. The apparent Vmax for the acetate and butyrate esters were 66.5 and 165.3 nmol hydrolysed per min and mg of protein respectively. An Arrhenius plot of the temperature dependent activity showed two sharp linear regions with a transition temperature of 31.6 degrees C. and energies of activation of 6.2 and 14.1 kcal/mol. The in situ carboxylesterase activity was inhibited 26% by paraoxon and 56% by N-ethylmaleimide, but not by p-chloromercuribenzoate.     

5-Aminolevulinic acid synthesis in epimastigotes of Trypanosoma cruzi

BACKGROUND AND AIMS: Trypanosoma cruzi is the causative agent of Chagas disease or American trypanosomiasis. The parasite manifests a nutritional requirement for heme compounds because of its biosynthesis deficiency. The aim of this study has been to investigate the presence of metabolites and enzymes of porphyrin pathway, as well as ALA formation in epimastigotes of T. cruzi, Tulahuén strain, Tul 2 stock. METHODS: Succinyl CoA synthetase, 5-aminolevulinic acid (ALA) synthetase, 4,5-dioxovaleric (DOVA) transaminase, ALA dehydratase and porphobilinogenase activities, as well as ALA, porphobilinogen (PBG), free porphyrins and heme content were measured in a parasite cells-free extract. Extracellular content of these metabolites was also determined. RESULTS: DOVA, PBG, porphyrins and heme were not detected in acellular extracts of T. cruzi. However ALA was detected both intra- and extracellularly This is the first time that the presence of ALA (98% of intracellularly formed ALA) is demonstrated in the extracellular medium of a parasite culture. Regarding the ALA synthesizing enzymes, DOVA transaminase levels found were low (7.13+/-0.49EU/mg protein), whilst ALA synthetase (ALA-S) activity was undetectable. A compound of non-protein nature, low molecular weight, heat unstable, inhibiting bacterial ALA-S activity was detected in an acellular extract of T. cruzi. This inhibitor could not be identified with either ALA, DOVA or heme. CONCLUSIONS: ALA synthesis is functional in the parasite and it would be regulated by the heme levels, both directly and through the inhibitor factor detected. ALA formed can not be metabolized further, because the necessary enzymes are not active, therefore it should be excreted to avoid intracellular cytotoxicity.     

Surface proteins in different isolates of Trypanosoma cruzi epimastigotes

Summary Epimastigotes from several Trypanosoma cruzi stocks were labeled by iodination with Chloramine T and their proteins detected by gel electrophoresis and autoradiography.The labeled proteins from the parasite surface were detected after immunoprecipitation with antisera against fixed trypanosomes or from infected rabbits. These antisera were able to recognize one or more proteins in all T. cruzi isolates analyzed, but the individual patterns differed from each other. Variations in the surface protein patterns were also observed in two Tulahuen stocks kept during several years under different conditions. Growth medium as well as the stage of growth at which the parasites were collected had also an effect upon the relative amount of the observed labeled proteins.     

Heme requirement and intracellular trafficking in Trypanosoma cruzi epimastigotes

Epimastigotes multiplies in the insect midgut by taking up nutrients present in the blood meal including heme bound to hemoglobin of red blood cell. During blood meal digestion by vector proteases in the posterior midgut, hemoglobin is clipped off into amino acids, peptides, and free heme. In this paper, we compared the heme and hemoglobin uptake kinetics and followed their intracellular trafficking. Addition of heme to culture medium increased epimastigote proliferation in a dose-dependent manner, while medium supplemented with hemoglobin enhanced growth after 3-day lag phase. Medium supplemented with globin-derived peptides stimulated cell proliferation in a dose-independent way. Using Palladium mesoporphyrin IX (Pd-mP) as a fluorescent heme-analog, we observed that heme internalization proceeded much faster than that observed by hemoglobin-rhodamine. Binding experiments showed that parasites accumulated the Pd-mP into the posterior region of the cell whereas hemoglobin-rhodamine stained the anterior region. Finally, using different specific inhibitors of ABC transporters we conclude that a P-glycoprotein homologue transporter is probably involved in heme transport through the plasma membrane.     

Acid and alkaline phosphatase activity in Trypanosoma cruzi epimastigotes

Phosphatase activity in intact Trypanosoma cruzi epimastigotes has been demonstrated. After subcellular fractionation three activities were characterized: (a) a membrane-bound microsomal acid activity with an optimum pH of 4.0 and a Km of 1.2 mM, strongly inhibited by tartrate and fluoride; (b) a soluble cytosolic acid activity with an optimum pH of 5.5 and a Km of 0.95 mM, strongly inhibited by p-hydroxymercuribenzoate, EDTA and copper ions and activated by cyanide, manganese and magnesium ions; and (c) a soluble cytosolic alkaline activity with an optimum pH of 8.0 and a Km of 3.8 mM, inhibited by p-hydroxymercuribenzoate, fluoride, EDTA, and copper, calcium and zinc ions. This activity was increased by magnesium and manganese ions.     

Ouabain-insensitive Na(+)-ATPase activity in Trypanosoma cruzi epimastigotes

In the present paper, the presence of a ouabain-insensitive Na(+)-stimulated, Mg(2+)-dependent ATPase activity in T. cruzi epimastigotes CL14 clone and Y strain was investigated. The increase in Na+ concentration (from 5 to 170 mM), in the presence of 2 mM ouabain, increases the ATPase activity in a saturable manner along a rectangular hyperbola. The Vmax was 18.0 +/- 1.0 and 21.1 +/- 1.1 nmoles Pi x mg-1 x min-1 and the half-activation value (K50) for Na+ was 34.3 +/- 5.8 mM and 37.7 +/- 5.3 in CL14 clone and in Y strain, respectively. The Na(+)-stimulated ATPase activity was inhibited by 5-[aminosulfonyl]-4-chloro-2-[(2-furanylmethyl)-amino] benzoic acid (furosemide) in a dose-dependent manner. The half-inhibition value (I50) was 0.22 +/- 0.03 and 0.24 +/- 0.07 mM, and the Hill number (n) was 0.99 +/- 0.2 and 2.16 +/- 0.29 for CL14 clone and Y strain, respectively. These data indicate that both cell types express the ouabain-insensitive Na(+)-ATPase activity, which might be considered the biochemical expression of the second Na+ pump.     

Stress response to high osmolarity in Trypanosoma cruzi epimastigotes

Trypanosoma cruzi undergoes differentiation in the rectum of triatomine, where increased osmolarity is caused mainly by elevated content of NaCl from urine. Early biochemical events in response to high osmolarity in this parasite have not been totally elucidated. In order to clarify the relationship between these events and developmental stages of T. cruzi, epimastigotes were subjected to hyperosmotic stress, which caused activation of Na(+)/H(+) exchanger from acidic vacuoles and accumulation of inositol trisphosphate (InsP(3)). Suppression of InsP(3) levels was observed in presence of intracellular Ca(2+) chelator or pre-treatment with 5-(N-ethyl-N-isopropyl)-amiloride (EIPA), which also inhibited the alkalinization of acidic vacuoles via a Na(+)/H(+) exchanger and the consequent increase in cytosolic calcium. These effects were activated and inhibited by PMA and Chelerythrine respectively, suggesting regulation by protein kinase C. The T. cruzi Na(+)/H(+) exchanger, TcNHE1, has 11 transmembrane domains and is localized in acidic vacuoles of epimastigotes. The analyzed biochemical changes were correlated with morphological changes, including an increase in the size of acidocalcisomes and subsequent differentiation to an intermediate form. Both processes were delayed when TcNHE1 was inhibited by EIPA, suggesting that these early biochemical events allow the parasite to adapt to conditions faced in the rectum of the insect vector.     

Effects of ketoconazole on sterol biosynthesis by Trypanosoma cruzi epimastigotes

Reproduction of Trypanosoma cruzi epimastigotes in a liver infusion-tryptose-serum medium was inhibited by the antimycotic agent ketoconazole. Effective drug concentrations also blocked the biosynthesis of the parasite's fungal-type sterols at the stage of C-14 demethylation, as demonstrated by radioisotopic, chromatographic and mass spectrometric methods. Coincidently, a 4,4,14 alpha-trimethyl sterol, 24-methylenedihydrolanosterol, accumulated and came to replace as much as three fourths of the free sterol content of the cells. Precedents from studies with fungi are invoked to suggest that the cytotoxicity of ketoconazole for T. cruzi is a consequence of the inability of 24-methylenedihydrolanosterol to perform the membrane lipid bilayer functions of the normal epimastigote sterols.     

The glycosome membrane of Trypanosoma cruzi epimastigotes: protein and lipid composition

Highly purified glycosomes from Trypanosoma cruzi epimastigotes were obtained by differential centrifugation and isopycnic ultracentrifugation. Glycosomal membranes, produced by carbonate treatment of purified glycosomes, exhibited about eight main protein bands and eight minor ones. Essentially the same protein pattern was observed in the detergent-rich fraction of a Triton X-114 fractionation of whole glycosomes, indicating that most of the membrane-bound polypeptides were highly hydrophobic. The orientation of these proteins was studied by in situ labelling followed by limited pronase hydrolysis of intact glycosomes. Three glycosome membrane proteins were characterized as peripheral by comparing the protein bands patterns of membrane fractions obtained by different treatments. Noteworthy membrane polypeptides were: (1) a peripheral 75k Da membrane protein, oriented towards the cytosol, which was the most abundant glycosomal membrane protein in exponentially growing epimastigotes but was essentially absent in stationary phase cells; (2) a pair of integral membrane proteins with molecular masses in the range of 85-100 kDa, which were only present in stationary phase cells; (3) a heme-containing 36k Da protein, strongly associated to the membrane, present in both growth phases; (4) a very immunogenic 41k Da integral membrane polypeptide, oriented towards the cytosol. The lipid composition of the glycosomal membranes was also investigated. The distribution of phospholipid species in glycosomes and glycosomal membranes was very similar to that of whole cells, with phosphatidyl-ethanolamine, phosphatidyl-choline, and phosphatidyl-serine as main components and smaller proportions of sphingomyelin and with phosphatidyl-inositol. On the other hand, glycosomes were enriched in endogenous sterols (ergosterol, 24-ethyl-5,7,22-cholesta-trien-3beta-ol), and precursors, when compared with whole cells, a finding consistent with the proposal that these organelles are involved in the de novo biosynthesis of sterols in trypanosomatids.     

The cytostome of Trypanosoma cruzi epimastigotes is associated with the flagellar complex.

Okuda, K., Esteva, M., Segura, E. L., and Bijovsky, A. T. 1999. The cytostome of Trypanosoma cruzi epimastigotes is associated with the flagellar complex. Experimental Parasitology 92, 223-231. Proliferative forms of Trypanosoma cruzi, amastigotes and epimastigotes, have a cytostome, a specialized structure formed by an invagination of the flagellar pocket's membrane surrounded by microtubules and frequently followed by a row of vesicles. All this assemblage penetrates deeply into the cytoplasm overpassing the nucleus. This structure, together with the flagellar pocket, appears to play an important role in the nutrition of the parasite. We demonstrated that the monoclonal antibody 2C4, made-up against isolated flagellar complex of T. cruzi epimastigotes, recognizes a protein doublet of 76 and 87 kDa in total epimastigotes homogenate. The 76-kDa polypeptide is enriched in the detergent-soluble fraction whereas the 87-kDa polypeptide is highly represented in the insoluble fractions and the purified flagella. Immuno-fluorescence assays show the antigen as a small spot at the flagellar pocket region. Immunogold labeling of ultrathin sections of epimastigote forms reveals gold particles at the opening of flagellar pocket, concentrated in the cytostome region. Immunocytochemistry of epimastigote whole-mount cytoskeletons reveals the labeling on an array of three to four microtubules that appears attached to flagellum, running in the direction of the nucleus. Ultrastructural observations have shown that the posterior region of isolated flagella, corresponding to the level of the flagellar pocket, possesses a microtubular structure compatible with that from the cytostome. The relationship between the cytostome, an endocytic organelle, and the flagellum is here described for the first time.     

Vaccination with epimastigotes of different strains of Trypanosoma rangeli protects mice against Trypanosoma cruzi infection

In our laboratory, we have developed a model of vaccination in mice with Trypanosoma rangeli, a non-pathogenic parasite that shares many antigens with Trypanosoma cruzi. The vaccinated mice were protected against infection with virulent T. cruzi. The goal of the present work was to study the protective activity of strains of T. rangeli of different origin, with the aim of analysing whether this protective capacity is a common feature of T. rangeli. BALB/c mice were vaccinated with live or fixed epimastigotes of two T. rangeli strains, Choachi and SC-58. Vaccinated (VM) and control mice (CM) were infected with virulent T. cruzi, Tulahuen strain. The results showed that the levels of parasitemia of VM, vaccinated with the two strains of T. rangeli were significantly lower than those developed in CM. The survival rate of VM was higher than that CM. Histological studies revealed many amastigote nests and severe inflammatory infiltrates in the heart and skeletal muscles of CM, whereas in the VM only moderate lymphomonocytic infiltrates were detected. Altogether, the results of the present work as well as previous studies show that the antigens involved in the protection induced by T. rangeli are expressed in different strains of this parasite. These findings could prove useful in vaccine preparation.