Trypanosoma cruzi epimastigotes are able to store and mobilize high amounts of cholesterol in reservosome lipid inclusions

Background

Reservosomes 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 Findings

Using 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/Significance

Taken 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.     

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.     

Recently differentiated epimastigotes from Trypanosoma cruzi are infective to the mammalian host

Trypanosoma cruzi, the etiologic agent of Chagas disease, has a complex life cycle in which four distinct developmental forms alternate between the insect vector and the mammalian host. It is assumed that replicating epimastigotes present in the insect gut are not infective to mammalian host, a paradigm corroborated by the widely acknowledged fact that only this stage is susceptible to the complement system. In the present work, we establish a T. cruzi in vitro and in vivo epimastigogenesis model to analyze the biological aspects of recently differentiated epimastigotes (rdEpi). We show that both trypomastigote stages of T. cruzi (cell‐derived and metacyclic) are able to transform into epimastigotes (processes termed primary and secondary epimastigogenesis, respectively) and that rdEpi have striking properties in comparison to long‐term cultured epimastigotes: resistance to complement‐mediated lysis and both in vitro (cell culture) and in vivo (mouse) infectivity. Proteomics analysis of all T. cruzi stages reveled a cluster of proteins that were up‐regulated only in rdEpi (including ABC transporters and ERO1), suggesting a role for them in rdEpi virulence. The present work introduces a new experimental model for the study of host‐parasite interactions, showing that rdEpi can be infective to the mammalian host.     

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 carboxylesterases of Trypanosoma cruzi epimastigotes

Carboxylesterase activity in Trypanosoma cruzi was found mainly in the microsomal (40%) and the cytosolic fraction (26%). The Vmax for p-nitrophenyl acetate was 28.50 and 17.60 nmol per min and mg of protein for the microsomal and the cytosolic fractions, respectively. The Km was 0.78 mM for the microsomal activity and 0.55 mM for the cytosolic activity. The inhibition rate constant with N-ethylmaleimide were 38.10 M-1 min-1 and 2.56 M-1 min-1 for the cytosolic and the microsomal enzymes, respectively. The rate constants with Paraoxon were 8,360 M-1 X min-1 and 32,600 M-1 X min-1. Polyacrylamide gel electrophoresis under nondenaturing conditions showed three bands of microsomal activity with M.W. of 63, 136 and 153 kDA. Similarly, three bands of cytosolic activity with M.W. of 126, 294 and 479 kDA were identified.     

Isolation and characterization of a reservosome fraction from Trypanosoma cruzi

Resistance to different antibiotics was found in 26 of the 30 strains analyzed, more than 70% of the strains analyzed were resistant to carbenicillin and ampicillin and a significant correlation was found between the resistance to both antibiotics. Plasmids were found in 80% of the strains analyzed, and 11 different plasmid profiles were observed. The most common profile obtained had only a 21.2-kbp plasmid, a significant correlation was found between the presence of this plasmid and resistance to carbenicillin, although some exceptions could be detected. Plasmids were cured from a cephalothin resistant strain and reintroduced into the plasmid-free cell and into Escherichia coli DH5alpha, both strains gained resistance to this antibiotic.     

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.     

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.     

Differentiation of Trypanosoma cruzi epimastigotes: metacyclogenesis and adhesion to substrate are triggered by nutritional stress

Differentiation of Trypanosoma cruzi epimastigotes to metacyclic trypomastigotes occurs in the insect rectum, after adhesion of the epimastigotes to the intestinal wall. We investigated the effect of the nutritional stress on the metacyclogenesis process in vitro by incubating epimastigotes in the chemically defined TAU3AAG medium supplemented with different nutrients. Addition of fetal bovine serum induced epimastigote growth but inhibited metacyclogenesis. In this medium, few parasites attached to the substrate. Ultrastructural analysis demonstrated reservosomes at the posterior end of the epimastigotes. Incubation of the cells in TAU3AAG medium containing gold-labeled transferrin resulted in high endocytosis of the marker by both adhered and free-swimming epimastigotes. No intracellular gold particles could be detected in trypomastigotes. Addition of transferrin gold complexes to adhered epimastigotes cultivated for 4 days in TAU3AAG medium resulted in decrease of both metacyclogenesis and adhesion to the substrate, as compared with parasites maintained in transferrin-free medium. Adhesion to the substrate is triggered by nutritional stress, and proteins accumulated in reservosomes are used as energy source during the differentiation. A close relationship exists among nutritional stress, endocytosis of nutrients, adhesion to the substrate, and cell differentiation in T. cruzi epimastigotes.     

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.     

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.     

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.     

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.     

Trypanosoma cruzi: distribution of fluorescently labeled tubulin and actin in epimastigotes

Cytoskeletal components were visualized in epimastigote forms of Trypanosoma cruzi by double immunofluorescence microscopy using monospecific antibodies against tubulin and against actin. Intense staining of the flagellum and the edges of the cell body was observed when the cells were stained with anti-tubulin, reflecting the presence of the basal bodies, the flagellar axoneme and the subpellicular microtubules. A less intense staining was seen in the cell body of epimastigotes stained with anti-actin. However, an intense staining was observed with this antibody in the flagellum, in a pattern similar to that observed with anti-tubulin. It is suggested that the paraxial structure, which is formed by a complex array of 6-nm-thick microfilaments is composed, at least in part, of actin.     

Tryparedoxin peroxidases and superoxide dismutases expression as well as ROS release are related to Trypanosoma cruzi epimastigotes growth phases

Trypanosoma cruzi's antioxidant system is unique and relevant to the parasite. In this study, quantitative assays were performed to determine cytosolic and mitochondrial tryparedoxin peroxidases and superoxide dismutases expression (TcCPx, TcMPx, SODB and SODA) in correlation to H(2)O(2) release and O(2)(-) production. Differences were observed regarding H(2)O(2) release and O(2)(-) production between strains and along the growth curve. All of the enzymes studied exhibited varied expression as a function of time in culture. Although at lower levels, the Y strain exhibited the same pattern of Tulahuen 2 enzyme expression for all of the proteins studied, except SODA. In the stationary phase, the degree of expression of all of the enzymes in the Y strain returned to similar levels as those detected in the log phase with the exception of TcCPx and SODA. In Tulahuen 2, a higher expression of TcMPx, SODA and SODB was detected in the early stationary phase, and a slight decrease was observed in the late stationary phase for each enzyme, excluding TcMPx, which exhibited a marked decrease, and TcCPx, which increased its level. Because of the significance of ROS in redox signaling, these differences in enzyme expression underscore the importance of these parameters for epimastigote proliferation.     

Evidence for NADH- and NADPH-linked glutamate dehydrogenases in Trypanosoma cruzi epimastigotes.

Two glutamate dehydrogenases, NADH-linked (EC 1.2.1.2) and NADPH-linked (EC 1.2.1.4) were isolated from the epimastigote forms of Trypanosoma cruzi and purified. Both enzymes exist as hexamers. The molecular weights of the native NADH-and NADPH-linked glutamate dehydrogenases were estimated to be 360,000 and 265,000, respectively, and those of the subunits to be 58,000 and 43,000, respectively. The isoelectric point of the NADH-linked dehydrogenase is at pH 5.25 and that of the NADPH-linked enzyme at pH 5.1. The activities of both enzymes are regulated by product inhibition. In addition, purine nucleotides were shown to be potent inhibitors of the NADH-linked glutamate dehydrogenase.     

Pyruvate phosphate dikinase and pyrophosphate metabolism in the glycosome of Trypanosoma cruzi epimastigotes

Pyruvate phosphate dikinase (PPDK) was recently reported in trypanosomatids, but its metabolic function is not yet known. The present work deals with the cellular localization and the function of the Trypanosoma cruzi enzyme. First, we show by digitonin titration and cell fractionation that the enzyme was essentially present in the glycosome matrix of the epimastigote form. Second, we address the issue of the direction of the reaction inside the glycosome for one part, our bibliographic survey evidenced a quite exergonic ΔG°′ (at least −5.2 kcal/mol at neutral pH and physiologic ionic strength); for another part, no pyrophosphatase (PPase) could be detected in fractions corresponding to the glycosomes; therefore, glycosomal PPDK likely works in the direction of pyruvate production. Third, we address the issue of the origin of the glycosomal pyrophosphate (PPi): several synthetic pathways known to produce PPi are already considered to be glycosomal. This work also indicates the presence of an NADP⁺-dependent β-oxidation of palmitoyl-CoA in the glycosome. Several pyruvate-consuming activities, in particular alanine dehydrogenase (ADH) and pyruvate carboxylase (PC), were detected in the glycosomal fraction. PPDK appears therefore as a central enzyme in the metabolism of the glycosome of T. cruzi by providing a link between glycolysis, fatty acid oxidation and biosynthetic PPi-producing pathways. Indeed, PPDK seems to replace pyrophosphatase in its classical thermodynamic role of displacing the equilibrium of PPi-producing reactions, as well as in its role of eliminating the toxic PPi.