RESUMO
The study of Trypanosoma cruzi type II DNA-topoisomerase should provide new clues for the rational development of new drugs for the chemotherapy of Chagas' disease. This enzyme is very likely involved in the processes leading to T. cruzi replication and differentiation since both processes are blocked by bacterial type II DNA topoisomerase inhibitors. In this article, we review and discuss our recent data related to the cloning, sequencing, and expression of T. cruzi type II topoisomerase.
Assuntos
DNA Topoisomerases Tipo II/metabolismo , Genes de Protozoários , Trypanosoma cruzi/enzimologia , Sequência de Aminoácidos , Animais , Doença de Chagas/tratamento farmacológico , Clonagem Molecular , Crithidia fasciculata/enzimologia , Crithidia fasciculata/genética , DNA Topoisomerases Tipo II/genética , Dados de Sequência Molecular , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Trypanosoma brucei brucei/enzimologia , Trypanosoma brucei brucei/genética , Trypanosoma cruzi/genéticaRESUMO
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.
Assuntos
Antígenos de Protozoários/metabolismo , Antígenos de Superfície/metabolismo , Metabolismo dos Carboidratos , Trypanosoma cruzi/metabolismo , Testes de Aglutinação , Animais , Antígenos de Protozoários/imunologia , Antígenos de Superfície/imunologia , Lectinas/metabolismo , Neuraminidase/metabolismo , Trypanosoma cruzi/crescimento & desenvolvimento , Trypanosoma cruzi/imunologiaRESUMO
Bacterial topoisomerase II inhibitors (ofloxacin and its commercial derivative Tarivid, nalidixic acid, and novobiocin) were tested as blockers of Trypanosoma cruzi differentiation and proliferation. The transformation of either epimastigotes into metacyclic trypomastigotes or amastigotes into trypomastigotes was inhibited by the drugs in a dose-dependent manner. The inhibition of epimastigote differentiation was also dependent on the time of drug addition to the medium. Proliferation of T. cruzi was also blocked in a dose-dependent manner by the drugs, with the exception of novobiocin, which did not inhibit epimastigote replication and resulted in cell lysis when it was used at high concentrations. On the other hand, the transformation of amastigotes into epimastigotes in axenic culture was not inhibited; this process did not require either kinetoplast (mitochondrial) DNA replication or changes in the DNA network organization. Electron microscopy of cells treated with Tarivid (ofloxacin) showed damage to the kinetoplast, suggesting that this organelle might be the target of the drug. These results indicate that a bacterial-like topoisomerase II plays an important role in T. cruzi proliferation and differentiation.
Assuntos
Inibidores da Topoisomerase II , Trypanosoma cruzi/crescimento & desenvolvimento , Animais , Coração/efeitos dos fármacos , Interações Hospedeiro-Parasita/efeitos dos fármacos , Músculos/citologia , Músculos/efeitos dos fármacos , Músculos/fisiologia , Miocárdio/citologia , Ofloxacino/farmacologia , Trypanosoma cruzi/efeitos dos fármacos , Trypanosoma cruzi/enzimologiaRESUMO
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.
Assuntos
Ácidos Graxos/análise , Trypanosoma cruzi/análise , Animais , Diferenciação Celular , Trypanosoma cruzi/fisiologiaRESUMO
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.