Cellular analysis of host cell infection by different developmental stages of Trypanosoma cruzi

Trypanosoma cruzi is an obligate intracellular parasite that infects phagocytic and non-phagocytic mammalian cells by a complex process that appears to involve several discrete steps. Even though the infection process was described many years ago, the molecular mechanisms involved remain poorly understood. As fluorescent proteins have proven to be excellent tools for live-cell imaging, we used EGFP- and DsRed1-1-transfected trypomastigotes, amastigotes and epimastigotes to study the infection process in living cells. Contrary to what has been reported, our results showed that epimastigotes are as infective as trypomastigotes and amastigotes. Besides, differences in replication, differentiation and parasite release times were observed among the stages. Our results suggest that the different developmental stages use distinct attachment and invasion mechanisms. We propose that fluorescent-based plasmid expression systems are good models for studying the infection process of intracellular microorganisms and could offers insights about the molecular mechanisms involved.     

Biochemical detection of adenosine deaminase in Trypanosoma evansi

Biochemical and molecular research on parasites has increased considerably in trypanosomes in the recent years. Many of them have the purpose of identify areas, proteins and structures of the parasite which are vulnerable and could be used in therapy against the protozoan. Based on this hypothesis this study aimed to detect biochemically the enzyme adenosine deaminase (ADA) in Trypanosoma evansi, and to adapt an assay to the measurement of its activity in trypomastigotes. Firstly, the parasites were separated from the blood of mice experimentally infected with a DEAE-cellulose column. The ADA activity in trypomastigotes was evaluated at concentrations of 0.1, 0.2, 0.5, 0.6 and 0.8 mg of protein by spectrophotometry. ADA activity was observed in the parasites at all concentrations tested and its activity was proportional to the concentration of protein, ranging between 0.64 and 2.24 U/L in the lowest and highest concentration of protein, respectively. Therefore, it is possible to detect biochemically ADA in T. evansi, an enzyme that may be associated with vital functions of the parasite, similar to what occurs in mammals. This knowledge may be useful in the association of the chemotherapic treatment with specific inhibitors of the enzyme, in future studies.     

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.     

All <Emphasis Type="Italic">Trypanosoma cruzi</Emphasis> developmental forms present lysosome-related organelles

Trypanosoma cruzi epimastigote forms concentrate their major protease, cruzipain, in the same compartment where these parasites store macromolecules obtained from medium and for this ability these organelles were named as reservosomes. Intracellular digestion occurs mainly inside reservosomes and seems to be modulated by cruzipain and its natural inhibitor chagasin that also concentrates in reservosomes. T. cruzi mammalian forms, trypomastigotes and amastigotes, are unable to capture macromolecules by endocytosis, but also express cruzipain and chagasin, whose role in infectivity has been described. In this paper, we demonstrate that trypomastigotes and amastigotes also concentrate cruzipain, chagasin as well as serine carboxypeptidase in hydrolase-rich compartments of acidic nature. The presence of P-type proton ATPase indicates that this compartment is acidified by the same enzyme as epimastigote endocytic compartments. Electron microscopy analyzes showed that these organelles are placed at the posterior region of the parasite body, are single membrane bound and possess an electron-dense matrix with electronlucent inclusions. Three-dimensional reconstruction showed that these compartments have different size and shape in trypomastigotes and amastigotes. Based on these evidences, we suggest that all T. cruzi developmental stages present lysosome-related organelles that in epimastigotes have the additional and unique ability of storing cargo.     

Trypanosoma cruzi: New foci of enzootic Chagas' disease in California

Trypanosoma cruzi was discovered in 2–3rd instar nymphs of Triatoma protracta protracta from Paicines, 57 of 257 adults from Mulholland Overcrossing, and 2 males of 27 bugs from Juniper Hills, California. Thirty experimentally infected albino Mus musculus revealed low grade parasitaemias. Of 785 laboratory-raised Triatoma fed on 11 mice with Paicines isolate, 675 T. p. navajoensis and 1 T. p. protracta became infected; of 856 T. p. navajoensis fed on 13 mice with Mulholland isolate, 792 were positive; and of 137 T. p. navajoensis fed on 6 mice with the Juniper Hills isolate, 32 were infected with Trypanosoma cruzi. Examination of 7599 mm² area of tissue films for Paicines isolate revealed 31 type b and 15 type c sphaeromastigotes; 5650 mm² for the Mulholland isolate revealed 9 a-, 149 sphaero-, 8 epi-, and 7 trypo-mastigotes; and 15,209 mm² for the Juniper Hills isolate revealed 245 a-, 2865 sphaero-, 42 epi-, and 21 trypo-mastigotes. Field xenodiagnosis of 228 rodents in Juniper Hills revealed 11 Peromyscus truei montipinoris and 1 Neotoma fuscipes macrotis with Chagas' trypanosome.     

Trypanosoma cruzi: Comparative fatty acid metabolism of the epimastigotes and trypomastigotes in vitro

In vitro studies on the fatty acid metabolism of the epimastigotes and trypomastigotes of Trypanosoma cruzi showed the following: (1) Trypomastigotes demonstrated the ability to convert labeled palmitic acid to CO₂. Epimastigotes either did not convert this fatty acid to CO₂ or did so at a very low rate. (2) Trypomastigotes incorporated palmitic acid into neutral lipids, but, at a rate less than that of the epimastigotes. (3) While epimastigotes readily incorporated palmitic acid into phospholipid lipids, trypomastigote forms seemed to lack this ability.