Trypanosoma cruzi: Fatty acid metabolism in vitro

Trypanosoma cruzi populations, composed primarily of trypomastigote forms, readily converted palmitic acid, linoleic acid, oleic acid, and stearic acid to CO₂. Appreciable amounts of carbon from these four fatty acids were also incorporated into neutral and phospholipid lipids by these parasites. Palmitic acid, a 16 carbon saturated fatty acid, was converted at rates greater than those of the other three fatty acids.     

Development and interactions of Trypanosoma cruzi within the insect vector

Transmission of Chagas disease or American trypanosomiasis depends on Trypanosoma cruzi development and differentiation within its triatomine insect vector. In this review, Eloi Garcia and Patricia de Azambuja aim to outline the current areas of research that may explain aspects of the parasite-vector interaction.     

Trypanosoma cruzi: high ribosomal resistance to trichosanthin inactivation

Trypanosoma cruzi is the parasite causing Chagas Disease. Several results already published suggest that T. cruzi ribosomes have remarkable differences with their mammalian counterparts. In the present work, we showed that trypanosomatid (T. cruzi and Crithidia fasciculata) ribosomes are highly resistant to inactivation by trichosanthin (TCS), which is active against mammalian ribosomes. Differential resistance is an intrinsic feature of the ribosomal particles, as demonstrated by using assays where the only variable was the ribosomes source. Because we have recently described that TCS interacts with the acidic C-terminal end of mammalian ribosomal P proteins, we assayed the effect of a TCS variant, which is unable to interact with P proteins, on trypanosomatid ribosomes. This mutant showed similar shifting of IC(50) values on rat, T. cruzi and C. fasciculata ribosomes, suggesting that the resistance mechanism might involve other ribosomal components rather than the C-terminal end of P proteins.     

Lipid composition of three morphological stages of Trypanosoma cruzi

The neutral and phospholipid content at each of the three morphological stages of the parasite, Trypanosoma cruzi, was analyzed by thin-layer chromatography. Total lipid fatty acid composition at each stage was analyzed by gas-liquid chromatography and the results were compared. Change in lipid composition at each stage was observed.     

Some factors inducing formation of metacyclic stages of Trypanosoma cruzi

Epimastigotes of Trypanosoma cruzi, Peru strain, incubated in Contreras' artificial triatomine urine transformed into metacyclic trypomastigotes within 48 h at 28 degrees C when 10 mM L-proline or L-glutamate was added to the medium. Smaller numbers of metacyclic stages were induced in the presence of glucose or L-alanine. The L-leucine and L-isoleucine, 2 amino acids known to inhibit proline catabolism, inhibited proline-induced metacyclogenesis. Cells gassed with 5% CO2 showed significantly faster rates and higher levels of transformation than those not gassed, therefore indicating the additional importance of CO2 for transformation.     

Acetate oxidation by bloodstream forms of Trypanosoma cruzi.

Bloodstream forms of Trypanosoma cruzi had a substantial increase in respiration in the presence of acetate. Oxidation of acetate took place via the tricarboxylic acid cycle and involved an antimycin A-sensitive respiratory pathway. Oxygen uptake in the presence of acetate was a sensitive to antimycin A inhibition as was CO2 production. There was a 6--7% residual O2 uptake which was not inhibited by high antimycin concentrations. Human anti-T. cruzi sera had no effect on oxygen uptake.     

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.     

A sialidase activity in the midgut of the insect Triatoma infestans is responsible for the low levels of sialic acid in Trypanosoma cruzi growing in the insect vector

Trypanosoma cruzi expresses a unique trans-sialidase that isresponsible for the transfer of sialic acid from host glycoproteinsand glycolipids to mucin-like glycoprotein acceptors on theparasite surface. The enzyme and the sialic acid acceptors arepresent in the mammalian forms of the parasite and in the parasiteforms that grow in axenic cultures, which correspond to thedevelopmental stages found in the insect vectors. Here we showthat parasite forms growing in the vector Triatoma infestansexpress trans-sialidase in the hind gut portions of the insectHowever, the sialic acid acceptors are poorly sialylated dueto the low concentration of sialic acid donors in the gut lumenof T.infestans, which feeds exclusively on blood that is richin sialic acid donors. These low levels of sialic acid donorsare due to a novel sialidase activity present mainly in theanterior midgut with high specificity for     

CTLA-4 blockage increases resistance to infection with the intracellular protozoan Trypanosoma cruzi

Recent studies have revealed an important role for CTLA-4 as a negative regulator of T cell activation. In the present study, we evaluated the importance of CTLA-4 to the immune response against the intracellular protozoan, Trypanosoma cruzi, the causative agent of Chagas' disease. We observed that the expression of CTLA-4 in spleen cells from naive mice cultured in the presence of live trypomastigote forms of T. cruzi increases over time of exposure. Furthermore, spleen cells harvested from recently infected mice showed a significant increase in the expression of CTLA-4 when compared with spleen cells from noninfected mice. Blockage of CTLA-4 in vitro and/or in vivo did not restore the lymphoproliferative response decreased during the acute phase of infection, but it resulted in a significant increase of NO production in vivo and in vitro. Moreover, the production of IFN-gamma in response to parasite Ags was significantly increased in spleen cells from anti-CTLA-4-treated infected mice when compared with the production found in cells from IgG-treated infected mice. CTLA-4 blockade in vivo also resulted in increased resistance to infection with the Y and Colombian strains of T. cruzi. Taken together these results indicate that CTLA-4 engagement is implicated in the modulation of the immune response against T. cruzi by acting in the mechanisms that control IFN-gamma and NO production during the acute phase of the infection.     

Endogenous IFN-gamma is required for resistance to acute Trypanosoma cruzi infection in mice

In order to study the role of endogenous IFN-gamma in Trypanosoma cruzi infection in mice, a potent murine IFN-gamma-specific mAb was injected i.p. on days -1, 7, and 14, relative to infection. Irrespective of the parasite inocula (100 or 25,000), groups of antibody-treated mice had significantly greater cumulative mortality rates than did appropriate controls. In antibody-treated mice, mean survival times were also significantly shorter, and maximum mean parasitemia levels were significantly higher, than in controls. Moreover, the number of amastigote nests in tissues was higher than in control mice and attained a maximum at the same time as parasitemia. As evident from kinetic studies of neutralizing activity, injected mAb were rapidly consumed in infected, but not in noninfected, mice, which is suggestive of massive IFN-gamma production during the early parasitemic phase of the disease. Nevertheless, IFN-gamma remained undetectable in the sera of infected but untreated mice. Unexpectedly, however, a peak of IFN-like antiviral activity, characterizable as a mixture of IFN-gamma and IFN-beta, appeared in mAb-treated mice that survived to infection at a time when neutralizing activity of injected mAb had drastically decreased in the circulation. We hypothesize that this high level of artificially induced endogenous IFN-gamma, not neutralized by the amounts of injected mAb, was due to the more intense parasite multiplication occurring in mAb-treated mice, which in turn may have induced an increased amount of various cytokines. TNF-alpha was not found in the serum of our mice. The humoral immune response entered its exponential phase at a time point later than that when protection by endogenous IFN-gamma was evident. Treatment with IFN-gamma-specific antibody, as applied in our study, failed to affect the level of different Ig isotypes or of T. cruzi-specific antibodies. Our study clearly indicates that IFN-gamma is produced early in acute T. cruzi infection and exerts a protective effect that is probably independent from the humoral immune response.     

Differences in the nuclear chromatin among various stages of the life cycle of Trypanosoma cruzi

Trypanosoma cruzi is the etiological agent of Chagas. Although the nuclear chromatin of this parasite is organized in the form of nucleosome filaments, its chromatin is physically and enzymatically fragile, and no condensation into chromosomes occurs during mitosis. All previous investigations have been carried out with epimastigote form in its proliferate stage. It is not known whether these differences in chromatin structure are also found in the non-proliferate stationary epimastigote forms and in tissue derived trypomastigotes. Our results confirm that chromatin of logarithmic epimastigotes presents limited compaction when increasing salt concentrations from 1 to 100 mM NaCl, and no 30-nm fibers were formed. Contrary to these results, non-proliferative forms of the parasites showed a pattern of compactation similar to that observed in rat liver chromatin, where solenoids of 30-nm fibers are formed at 100-mM NaCl. In accordance with these results, digestion of the nuclear chromatin with DNase I revealed that the chromatin of logarithmic phase epimastigotes was more accessible to the enzyme. We conclude from these results that structural differences in the chromatin exist not only between T. cruzi and higher eukaryotes but also among various forms of the parasite. The functional significance of these differences are currently under investigation.     

Enhanced multiplication of intracellular (amastigote) stages of Trypanosoma cruzi in vitro

Amastigotes of different strains of Trypanosoma cruzi responded to stimulation with concanavalin A in an axenic medium by increased DNA synthesis and cell multiplication. These effects were inhibited by alpha-methyl mannoside. Other mitogens, i.e. phytohemagglutinin P, castor bean ricin Type II isolated from Ricinus communis, and a bacterial lipopolysaccharide, had no effect on amastigote growth. Amastigote stimulation by concanavalin A lends itself to studies on the biochemistry and cell cycle of this human pathogen.     

Energy generation in insect stages of Trypanosoma brucei: metabolism in flux

The generation of energy in African trypanosomes is a subject of undoubted importance. In bloodstream-form organisms, substrate-level phosphorylation of glucose is sufficient to provide the energy needs of the parasite. The situation in procyclic-form trypanosomes is more complex. For many years, it was accepted that glucose metabolism followed a conventional scheme involving glycolysis, the tricarboxylic acid cycle and ATP-producing oxidative phosphorylation linked to the electron-transport chain. However, progress in sequencing the Trypanosoma brucei genome and the development of gene-knockout and RNA interference technology has provided novel insight. Coupling these new technologies with classical approaches, including NMR and mass spectrometry to analyse glycolytic intermediates and end products, has yielded several surprises. In this article, we summarize how these recent data have helped to change the view of metabolism in procyclic-form T. brucei.