Chagas' disease is a parasitic disease that affects more than 20 million individuals in Latin America (12). As yet, prophylaxis of the disease has been limited because of the lack of a vaccine or safe chemotherapy. Trypanosoma cruzi, the causative agent of this disease (4), displays a complex life cycle that involves two intermediary hosts (triatomines and mammals) and three differentiation stages: epimastigotes, which replicate in the insect vector; amastigotes, which replicate in mammalian cells; and the infective and nonreplicative trypomastigotes (11).The transformation of replicative forms into infective forms involves important morphogenetic changes. One of the most striking modifications occurs at the level of kinetoplast ultrastructure. The kinetoplast is that portion of the unique mitochondrion from members of the order Kinetoplastida which contains the mitochondrial DNA (kinetoplast DNA [K-DNA]). During cell proliferation, the K-DNA must replicate, while on transformation of amastigotes or epimastigotes into trypomastigotes, the K-DNA changes from a compact (bar) to a more relaxed (basket) form (11). This suggests that topoisomerases may play an important role in both T. cruzi proliferation and differentiation.Our recent work on T. cruzi metacyclogenesis (i.e., the transformation of epimastigotes into metacyclic trypomastigotes) resulted in the development of chemically defined in vitro differentiation conditions (1,8). In addition to allowing the production of metacyclic trypomastigotes and the study of the metacyclogenesis process, these conditions are very useful for testing the effects of drugs on T. cruzi differentiation.In this study we tested the effect of topoisomerase II inhibitors on T. cruzi metacyclogenesis. In addition, we also tested the effects of these inhibitors on T. cruzi proliferation and during the intracellular cycle of the parasite inside Parasites. The T. cruzi Dm28c clone was obtained and was kept in the laboratory as described previously (6). Epimastigotes were grown in liver infusion tryptose (LIT) medium (2). In vitro metacyclogenesis was performed as described elsewhere (8). Briefly, epimastigotes were harvested from LIT medium and incubated for 2 h in artificial triatomine urine (TAU) (190 mM NaCl, 17 mM KCl, 2 mM MgCl2, 2 mM CaCl2, 8 mM phosphate buffer [pH 6.0]) and were then incubated in TAU supplemented with 10 mM L-proline (TAUP) for different times up to 96 h in the presence or absence of the drugs being tested. T. cruzi trypomastigotes of the Y strain (21) were obtained from the blood of albino mice at the peak of parasitemia and were isolated by differential centrifugation. Amastigote forms were obtained from the supernatant of a J774G-8 macrophage cell line, after 6 to 7 days after infection with bloodstream trypomastigotes (3). The proliferation of amastigotes in axenic culture (5 x 106 cells ml-') was performed in Warren medium at 29°C (10). In all cases, cells were counted in a Neubauer chamber.Treatment of heart muscle cell culture. Cultures that were 3 to 5 d...
Highly purified lectins with specificities for receptor molecules containing sialic acid, N-acetylglucosamine (DGlcNAc), N-acetylgalactosamine (D-GalNAc), galactose (D-Gal), mannose-like residues (D-Man) or L-fucose (LFuc), were used to determine changes in cell-surface carbohydrates of the protozoal parasite Trypanosomu 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-GIcNAc 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-Gabspecific), 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 cufinaris lectins (specific for D-Man-like residues); however, they were unreactive with the L-fucose-binding lectins from Lotus tetrugonolobos and Ufex europueus. These agglutination assays were further confirmed by binding studies using I z51-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.
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.
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