Changes in fatty acid composition associated with differentiation of Trypanosoma cruzi

The fatty acid composition during the transformation of Trypanosoma cruzi epimastigotes into metacyclic trypomastigotes (metacyclogenesis) was analysed by gas-liquid chromatography and mass spectrometry. Significant qualitative and quantitative changes in the fatty acid composition occurred during incubation of epimastigotes derived from LIT medium in the triatomine artificial urine (TAU). Metacyclogenesis was also followed by alterations in the fatty acid pattern but these were considerably less pronounced when compared to the pattern obtained for TAU-incubated epimastigotes. These results suggest that changes in the lipid composition precede the morphological transformation of epimastigotes into metacyclic trypomastigotes.     

Cultivation of Trypanosoma cruzi epimastigotes in low glucose axenic media shifts its competence to differentiate at metacyclic trypomastigotes

This study offers an insight into why Trypanosoma cruzi epimastigotes lose their capacity to differentiate into metacyclic forms, if maintained in culture media long-term through serial passages. The biological and metabolic behaviour of two T. cruzi strains isolated from various origins (human, opossum), and maintained under two schedules (alternate triatomine/mouse passages and serial culture media) were compared. To determine the effect of the environment on the parasites, the epimastigotes were grown under extreme conditions (high and low glucose concentrations), and the glucose consumption, ammonia production and changes in pH, either in one compartment (along the growth curve) or two compartments (induced metacyclogenesis) were compared. The glucose effect on the stages involved in metacyclogenesis at antigenic level was also evaluated. The results indicate that T. cruzi adapts to various environmental conditions and also that the ability of epimastigotes to undergo metacyclogenesis are influenced by the maintenance schedule. Antigenic profile analysis supports the idea that epimastigotes adapted to culture media do not complete their molecular differentiation into the trypomastigote metacyclic stage. These transition forms conserve some degree of gene expression of the epimastigote stage.     

Characterization and expression of proteases during Trypanosoma cruzi metacyclogenesis

Investigation of protease activities during the transformation of Trypanosoma cruzi epimastigotes into metacyclic trypomastigoes (metacyclo-genesis) revealed three major components with apparent molecular weights of 65, 52, and 40 kDa. The 65-kDa protease is a metacyclic trypomastigote stage-specific protease with an isoelectric point of 5.2 whose activity is inhibited by 1,10-phenanthroline, suggesting that it might be a metalloprotease. The 52-kDa component is also a metalloprotease which is constitutively expressed in epimastigotes and metacyclic trypomastigoes. On the other hand, the 40-kDa component is apparently made up of several isoforms of a cysteine protease which is expressed in much higher levels in epimastigotes than in metacyclic trypomastigote forms. The fact that the 65- and 40-kDa proteases are developmentally regulated suggests that proteases might be important for T. cruzi differentiation. Accordingly, T. cruzi metacyclogenesis is blocked by metallo- and cysteine-protease inhibitors.     

Antagonic activities of Trypanosoma cruzi metacaspases affect the balance between cell proliferation, death and differentiation

Metacaspases are distant relatives of animal caspases present in plants, fungi and protozoa. At variance with caspases, metacaspases exhibit stringent specificity for basic amino-acid residues and are absolutely dependent on millimolar concentrations of calcium. In the protozoan parasite Trypanosoma cruzi, metacaspases have been suggested to be involved in an apoptosis-like phenomenon upon exposure of the parasite to fresh human serum (FHS). Nuclear relocalization of metacaspases was observed after FHS treatment and overexpression of metacaspase-5 led to enhanced sensitivity to this stimulus. Here we report some biochemical properties of T. cruzi metacaspases. Performing fluorescent-activated cell sorting (FACS) analysis of epimastigotes inducibly overexpressing metacaspase-3, we demonstrate a role for this metacaspase in cell cycle progression, protection of epimastigotes from naturally occurring cell death and differentiation to infective metacyclic trypomastigotes. We also show that regulation of metacaspase-3 activity is important for cell cycle completion inside the mammalian host. On the other hand, inducible overexpression of metacaspase-5 lacking its C-terminal domain caused an apoptotic-like response. These results suggest that the two T. cruzi metacaspases could play an important role in the life cycle and bring to light the close relationship between cell division, death and differentiation in this ancient unicellular eukaryote.     

Cyclic AMP and adenylate cyclase activators stimulate Trypanosoma cruzi differentiation

A chemically defined in vitro differentiating condition was used to study the potential role of cyclic AMP (cAMP) and adenylate cyclase activators on the transformation of Trypanosoma cruzi epimastigotes to the infective metacyclic trypomastigotes (metacyclogenesis). It was observed that both addition of cAMP analogs or adenylate cyclase activators to the differentiating medium stimulated the transformation of epimastigotes to metacyclic trypomastigotes. These results were further corroborated by showing that inhibitors of cAMP phosphodiesterase were stimulatory while activators of this enzyme inhibited the metacyclogenesis process. On the other hand, inhibitors of calmodulin inhibited the transformation of epimastigotes to metacyclic trypomastigotes, suggesting that T. cruzi adenylate cyclase might be activated by calmodulin. In addition, the results strongly suggest that guanine nucleotide binding proteins are involved in T. cruzi adenylate cyclase activation. This system may be useful for studying cell differentiation mechanisms in eukaryotes.     

Protein synthesis attenuation by phosphorylation of eIF2α is required for the differentiation of Trypanosoma cruzi into infective forms

Chagas' disease is a potentially life-threatening illness caused by the unicellular protozoan parasite Trypanosoma cruzi. It is transmitted to humans by triatomine bugs where T. cruzi multiplies and differentiates in the digestive tract. The differentiation of proliferative and non-infective epimastigotes into infective metacyclic trypomastigotes (metacyclogenesis) can be correlated to nutrient exhaustion in the gut of the insect vector. In vitro, metacyclic-trypomastigotes can be obtained when epimastigotes are submitted to nutritional stress suggesting that metacyclogenesis is triggered by nutrient starvation. The molecular mechanism underlying such event is not understood. Here, we investigated the role of one of the key signaling responses elicited by nutritional stress in all other eukaryotes, the inhibition of translation initiation by the phosphorylation of the eukaryotic initiation factor 2α (eIF2α), during the in vitro differentiation of T. cruzi. Monospecific antibodies that recognize the phosphorylated Tc-eIF2α form were generated and used to demonstrate that parasites subjected to nutritional stress show increased levels of Tc-eIF2α phosphorylation. This was accompanied by a drastic inhibition of global translation initiation, as determined by polysomal profiles. A strain of T. cruzi overexpressing a mutant Tc-eIF2α, incapable of being phosphorylated, showed a block on translation initiation, indicating that such a nutritional stress in trypanosomatids induces the conserved translation inhibition response. In addition, Tc-eIF2α phosphorylation is critical for parasite differentiation since the overexpression of the mutant eIF2α in epimastigotes abolished metacyclogenesis. This work defines the role of eIF2α phosphorylation as a key step in T. cruzi differentiation.     

Cell-substrate adhesion during Trypanosoma cruzi differentiation

The transformation of Trypanosoma cruzi epimastigotes to the mammal infective metacyclic trypomastigotes (metacyclogenesis) can be performed in vitro under chemically defined conditions. Under these conditions, differentiating epimastigotes adhere to a surface before their transformation into metacyclic trypomastigotes. Scanning and transmission electron microscopy of adhered and non-adhered parasites during the metacyclogenesis process show that only epimastigotes and few transition forms are found in the first population, whereas metacyclic trypomastigotes are exclusively found in the cell culture supernatant. PAGE analysis of the [35S]methionine metabolic labeling products of adhered and non-adhered parasites shows that although most of the polypeptides are conserved, adhered parasites express specifically four polypeptides in the range of 45-50 kD with an isoelectric point of 4.8. These proteins might be involved in the adhesion process and are recognized by an antiserum against total adhered parasite proteins. This antiserum also recognized a group of 45-50 kD in the iodine-radiolabeled surface proteins of differentiating cells, providing direct evidence that these components are indeed surface antigens. The results suggest that epimastigotes must adhere to a substrate before their transformation to metacyclic trypomastigotes, being released to the medium as the metacyclogenesis process is accomplished. This could correspond to the process naturally occurring within the triatomine invertebrate host.     

Differential energetic metabolism during Trypanosoma cruzi differentiation. I. Citrate synthase, NADP-isocitrate dehydrogenase, and succinate dehydrogenase

The activities of the mitochondrial enzymes citrate synthase (citrate oxaloacetatelyase, EC 4.1.3.7), NADP-linked isocitrate dehydrogenase (threo-Ds-isocitrate:NADP+ oxidoreductase (decarboxylating), EC 1.1.1.42), and succinate dehydrogenase (succinate: FAD oxidoreductase, EC 1.3.99.1) as well as their kinetic behavior in the two developmental forms of Trypanosoma cruzi at insect vector stage, epimastigotes and infective metacyclic trypomastigotes, were studied. The results presented in this work clearly demonstrate a higher mitochondrial metabolism in the metacyclic forms as is shown by the extraordinary enhanced activities of metacyclic citrate synthase, isocitrate dehydrogenase, and succinate dehydrogenase. In epimastigotes, the specific activities of citrate synthase at variable concentrations of oxalacetate and acetyl-CoA were 24.6 and 26.6 mU/mg of protein, respectively, and the Michaelis constants were 7.88 and 6.84 microM for both substrates. The metacyclic enzyme exhibited the following kinetic parameters: a specific activity of 228.4 mU/mg and Km of 3.18 microM for oxalacetate and 248.5 mU/mg and 2.75 microM, respectively, for acetyl-CoA. NADP-linked isocitrate dehydrogenase specific activities for epimastigotes and metacyclics were 110.2 and 210.3 mU/mg, whereas the apparent Km's were 47.9 and 12.5 microM, respectively. No activity for the NAD-dependent isozyme was found in any form of T. cruzi differentiation. The particulated succinate dehydrogenase showed specific activities of 8.2 and 39.1 mU/mg for epimastigotes and metacyclic trypomastigotes, respectively, although no significant changes in the Km (0.46 and 0.48 mM) were found. The cellular role and the molecular mechanism that probably take place during this significant shift in the mitochondrial metabolism during the T. cruzi differentiation have been discussed.     

Trypanosoma cruzi: differentiation after interaction of epimastigotes and Triatoma infestans intestinal homogenate

Incubation of Trypanosoma cruzi epimastigotes with Triatoma infestans intestinal homogenate leads to differentiation to the metacyclic trypomastigote. Features of this interaction are presented. The morphogenetic mechanism was triggered almost at once; for the minimum interaction period assayed (15 min), the degree of differentiation achieved in Grace medium by Day 6 was 70.0 +/- 9.0%. Longer interaction periods failed to improve differentiation. The morphogenesis became irreversible at 4 hr after interaction. Epimastigotes incubated for 4 hr with T. infestans intestinal homogenate and then washed reached significant differentiation, while those washed before this time failed to do so. Treatment of epimastigotes with albumin improved the experimental conditions thereby hastening morphogenesis, the same percentage of metacyclics occurring in only 4 days. The factors capable of triggering differentiation were adsorbed by T. cruzi epimastigotes, as expected, but also by Leishmania mexicana and, to a lesser degree, by sheep red blood cells. Once the morphogenetic mechanism had been triggered following interaction of epimastigotes with intestinal homogenate for 15 min, metacyclic forms developed when parasites were transferred to Grace but not to other media. Treatment of epimastigotes with trypsin abolished their capacity to differentiate, which was completely reversed following a 5 hr incubation in LIT medium.     

Flagellar elongation induced by glucose limitation is preadaptive for Trypanosoma cruzi differentiation

Trypanosomes must sense and respond to environmental change in order to progress through their life cycles. The American trypanosome, Trypanosoma cruzi, differentiates from the noninfective epimastigote form to the infective metacyclic form spontaneously in axenic culture. Here, we investigate the initial stimulus for that change and demonstrate that T. cruzi epimastigotes sense limitation of glucose in the medium and respond by undergoing significant morphological and biochemical change. As part of this change, the mean flagellar length of the population triples, which is correlated with an increased ability to maintain interactions with hydrophobic substrates, a requirement for differentiation to the next life cycle stage.     

Mechanism of resistance to lysis by the alternative complement pathway in Trypanosoma cruzi trypomastigotes: effect of specific monoclonal antibody

Trypanosoma cruzi G strain epimastigotes were lysed by normal human serum (NHS) through activation of the alternative complement pathway (ACP), whereas metacyclic trypomastigotes were resistant to lysis. Epimastigotes and metacyclics with equivalent amounts of C3b deposited on their surface bound factor B with similar affinities. In contrast, factor H bound with higher affinity to metacyclics than to epimastigotes. Both T. cruzi forms with bound C3b were extensively (60 to 80%) lysed after formation of surface C3-convertase and the addition of a C3-C9 complement source. In the presence of factors H and I, or incubation with NHS with EDTA, the percentage of lysis of metacyclics decreased faster than that of epimastigotes with increasing incubation times. These data suggest, as a possible mechanism of resistance to lysis in metacyclic trypomastigotes, the higher binding affinity of factor H to C3b and the inactivation of the latter by serum regulatory proteins. Metacyclics were lysed by NHS, through ACP, in the presence of human immune serum to T. cruzi or anti-T. cruzi monoclonal antibody, but not with the Fab fragment of the latter, which recognizes a 90,000 m.w. antigen from T. cruzi metacyclics. Protection of parasite-bound C3b from serum control proteins was observed when parasites were incubated, before C3 deposition, with the lytic monoclonal antibody but not with its Fab fragment or a nonrelated IgG control. When C3b was deposited on metacyclics before antibody binding, C3b inactivation occurred. In the lysis of metacyclics, through ACP activation, binding of antibody apparently creates new acceptor sites which prevent the activity of serum regulatory proteins.     

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.     

Chromosomal size conservation through the cell cycle supports karyotype stability in Trypanosoma cruzi

The Trypanosoma cruzi karyotype shows an extensive chromosomal size polymorphism. Absence of condensed mitotic chromosomes and chromatin fragility are characteristic features of T. cruzi which would allow DNA breaks and chromosomal rearrangements during cell proliferation. We have investigated by pulsed field gel electrophoresis (PFGE) eventual changes in chromosomal size during exponential and stationary phases of T. cruzi epimastigotes in culture, in G0 trypomastigotes and throughout the cell cycle in synchronized epimastigotes. T. cruzi molecular karyotype was stable throughout the cell cycle and during differentiation. Thus, the chromosomal size polymorphism previously reported in T. cruzi contrasts with the stability of the molecular karyotype observed here and suggests that chromosomal rearrangements leading to changes in chromosomal size are scarce events during the clonal propagation of this parasite.     

Changes in cell-surface carbohydrates of Trypanosoma cruzi during metacyclogenesis under chemically defined conditions.

Highly purified lectins with specificities for receptor molecules containing sialic acid, N-acetylglucosamine (D-GlcNAc), N-acetylgalactosamine (D-GalNAc), galactose (D-Gal), mannose-like residues (D-Man) or L-fucose (L-Fuc), were used to determine changes in cell-surface carbohydrates of the protozoal parasite Trypanosoma cruzi during metacyclogenesis under chemically defined conditions. Of the D-GalNAc-binding lectins, BS-I selectively agglutinated metacyclic trypomastigotes, MPL was selective for replicating epimastigotes, whereas SBA strongly agglutinated all developmental stages of T. cruzi. WGA (sialic acid and/or D-GlcNAc specific) was also reactive with differentiating epimastigotes and metacyclic trypomastigotes but displayed a higher reactivity with replicating epimastigote forms. A progressive decrease in agglutinating activity was observed for jacaline (specific for D-Gal) during the metacyclogenesis process; conversely, a progressive increase in affinity was observed for RCA-I (D-Gal-specific), although the reactivity of other D-Gal-specific lectins (PNA and AxP) was strong at all developmental stages. All developmental stages of T. cruzi were agglutinated by Con A and Lens culinaris lectins (specific for D-Man-like residues); however, they were unreactive with the L-fucose-binding lectins from Lotus tetragonolobos and Ulex europaeus. These agglutination assays were further confirmed by binding studies using 125I-labelled lectins. Neuraminidase activity was detected in supernatants of cell-free differentiation medium using the PNA hemagglutination test with human A erythrocytes. The most pronounced differences in lectin agglutination activity were observed between replicating and differentiating epimastigotes, suggesting that changes in the composition of accessible cell-surface carbohydrates precede the morphological transformation of epimastigotes into metacyclic trypomastigotes.     

Characterization of host cell-derived membrane proteins of the vacuole surrounding different intracellular forms of Trypanosoma cruzi in J774 cells. Evidence for phagocyte receptor sorting during the early stages of parasite entry.

Trypanosoma cruzi, an obligate intracellular protozoan parasite, exhibits developmental regulation of virulence. Although both noninfective epimastigote and infective trypomastigote stages of T. cruzi enter phagocytic cells via the formation of a parasitophorous vacuole (PV), only the latter developmental stages survive ingestion and perpetuate the infection. To determine whether the membrane composition of PV surrounding these different stages might contribute to differences in the outcome of infection, we identified selected membrane constituents by immunofluorescence and intracellular radioiodination, and studied their incorporation into PV. Complement receptors (CR3) are incorporated preferentially into the PV membrane surrounding serum-opsonized epimastigotes but not culture-derived metacyclic trypomastigotes. FcR are not preferentially incorporated into PV membranes unless epimastigotes or culture-derived metacyclic trypomastigotes are opsonized with anti-T. cruzi antibody. PV surrounding either parasite stage contain beta 1 integrins and lysosomal membrane glycoproteins (lgp). These results indicate that the plasma membrane glycoproteins incorporated into the surrounding PV membrane differ depending upon the stage of parasite being internalized, and that these differences reflect, at least in part, selective ligation of cell surface receptors mediating uptake. Furthermore, they imply that although virulent trypomastigote stages may avoid host cell uptake by conventional phagocytic receptors, i.e., CR3 or FcR, they do not escape fusion with an lgp-containing vacuole where they could still be exposed to lysosomal antimicrobial mechanisms.     

Biological characterization of Trypanosoma cruzi strains

Biological parameters of five Trypanosoma cruzi strains from different sources were determined in order to know the laboratory behaviour of natural populations. The parameters evaluated were growth kinetics of epimastigotes, differentiation into metacyclic forms, infectivity in mammalian cells grown in vitro and parasite susceptibility to nifurtimox, benznidazole and gentian violet. Differences in transformation to metacyclic, in the percentage of infected cells as well as in the number of amastigotes per cell were observed among the strains. Regarding to pharmacological assays, Y strain was the most sensitive to the three assayed compounds. These data demonstrate the heterogeneity of natural populations of T. cruzi, the only responsible of infection in humans.     

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.     

Differential gene expression during Trypanosoma cruzi metacyclogenesis

The transformation of epimastigotes into metacyclic trypomastigotes involves changes in the pattern of expressed genes, resulting in important morphological and functional differences between these developmental forms of Trypanosoma cruzi. In order to identify and characterize genes involved in triggering the metacyclogenesis process and in conferring to metacyclic trypomastigotes their stage specific biological properties, we have developed a method allowing the isolation of genes specifically expressed when comparing two close related cell populations (representation of differential expression or RDE). The method is based on the PCR amplification of gene sequences selected by hybridizing and subtracting the populations in such a way that after some cycles of hybridization-amplification genes specific to a given population are highly enriched. The use of this method in the analysis of differential gene expression during T. cruzi metacyclogenesis (6 hr and 24 hr of differentiation and metacyclic trypomastigotes) resulted in the isolation of several clones from each time point. Northern blot analysis showed that some genes are transiently expressed (6 hr and 24 hr differentiating cells), while others are present in differentiating cells and in metacyclic trypomastigotes. Nucleotide sequencing of six clones characterized so far showed that they do not display any homology to gene sequences available in the GeneBank.