RESUMO
A comparative study of proteolytic enzymes and cell-surface protein composition in virulent and avirulent Leishmania (Leishmania) amazonensis promastigote forms was carried out using one- and two-dimensional dodecyl sulfate sodium-polyacrylamide gel electrophoresis (SDS-PAGE). The surface iodinated protein profiles showed two major polypeptides of 65-60 and 50-47 kDa that were expressed in both virulent and avirulent promastigote forms. However, minor quantitative differences were observed in the cell-surface profile between the avirulent and virulent promastigotes. These included polypeptides of 115, 52, 45, 32, and 25 kDa that were preferentially expressed in the virulent forms. Two-dimensional SDS-PAGE showed an accentuated expression of acidic polypeptides; some of them differentially expressed in the promastigote forms analyzed. Live parasites treated with glycosylphosphatidylinositol (GPI)-specific phospholipase C (PLC) from Trypanosoma brucei and immunoprecipitated with the cross-reacting determinant (CRD) antibody recognized three major polypeptides of 65-60, 52, and 50-47 kDa, hence suggesting that these peptides were anchored to the plasma membrane domains through GPI anchor. Moreover, the polypeptides of 65-60 and 52 kDa were also recognized by the gp63 antiserum. Several metalloproteinase activities were similar in both virulent and avirulent promastigote forms, whereas cysteine proteinase activities, sensitive to E-64, were preferentially expressed in virulent promastigotes. These results suggest that cell-surface polypeptides and intracellular cysteine proteinases might play an important role in the virulence of L. (L.) amazonensis.
Assuntos
Endopeptidases/biossíntese , Leishmania mexicana/patogenicidade , Proteínas de Membrana/biossíntese , Proteínas de Protozoários/biossíntese , Animais , Cricetinae , Eletroforese em Gel Bidimensional , Eletroforese em Gel de Poliacrilamida , Endopeptidases/análise , Humanos , Leishmania mexicana/enzimologia , Leishmania mexicana/metabolismo , Proteínas de Membrana/análise , Camundongos , Testes de Precipitina , Proteínas de Protozoários/análise , VirulênciaRESUMO
In this study, we analyzed the influence of proteinase expression on the cellular differentiation of Herpetomonas samuelpessoai. Along cellular differentiation, which was induced by dimethylsulfoxide (DMSO), the trypanosomatids secreted several molecules with variable proteolytic activity. All of them were inhibited by 10 m M 1,10-phenanthroline, suggesting that they are zinc-metalloproteinases. Analysis of parasite extracts revealed the occurrence of a 63-kDa metalloproteinase and a 45-kDa cysteine proteinase. After extraction with Triton X-114 followed by water-detergent partition, the 63-kDa component was present in both aqueous and detergent phases, which indicated that this enzyme may be distributed over different cellular compartments including membrane domains. The 45-kDa component, however, presented hydrophilic properties and was predominantly expressed by DMSO non-treated parasites, suggesting that proteinases may be involved in the process of cellular differentiation in H. samuelpessoai. This was confirmed by the fact that a cysteine proteinase inhibitor abrogated parasite differentiation. The role of proteinases and their relevance in the differentiation of H. samuelpessoai are discussed.
Assuntos
Dimetil Sulfóxido/farmacologia , Metaloendopeptidases/metabolismo , Trypanosomatina/enzimologia , Animais , Diferenciação Celular , Concentração de Íons de Hidrogênio , Metaloendopeptidases/análise , Trypanosomatina/efeitos dos fármacos , Trypanosomatina/crescimento & desenvolvimentoRESUMO
The surface anionogenic groups and sialoglycoconjugate structures of Paracoccidioides brasiliensis yeast forms were analysed by cell microelectrophoresis, binding assays with lectins and viral particles, ultrastructural cytochemistry, enzymic digestion and flow cytofluorimetry. P. brasiliensis yeast forms, particularly the budding primordia, reacted strongly with cationized ferritin. Binding assays showed that the reaction with sialic-acid-specific Limax flavus lectin (LFA) was distributed over the entire P. brasiliensis cell wall. Treatment of yeast forms with neuraminidase significantly reduced their negative surface charge and LFA labelling, which suggests that sialic acid residues are major anionogenic groups exposed on the P. brasiliensis surface. Furthermore, after neuraminidase treatment, labelling with Arachis hypogaea (peanut) agglutinin increased due to unmasking of subterminal beta-D-galactopyranosyl residues. The sialic acid linkages to galactose are alpha 2,6 and alpha 2,3 as assessed, respectively, by fungal attachment to M1/5 and M1/5 HS8 strains of influenza A virus and binding of Sambucus niger and Maackia amurensis agglutinins. The alpha 2,6 linkage clearly predominated in both experiments. Flow cytofluorimetry analysis revealed the heterogenicity of P. brasiliensis yeast cell populations, which comprised young and mature budding yeasts. Both express binding sites to LFA and Limulus polyphemus agglutinin.
Assuntos
Ácido N-Acetilneuramínico/análise , Ácido N-Acetilneuramínico/metabolismo , Paracoccidioides/química , Paracoccidioides/metabolismo , Aglutininas/metabolismo , Parede Celular/química , Parede Celular/metabolismo , Ferritinas/farmacocinética , Citometria de Fluxo , Proteínas Fúngicas/metabolismo , Galactose/metabolismo , Vírus da Influenza A/metabolismo , Lectinas/metabolismo , Microscopia Eletrônica , Neuraminidase/farmacologia , Paracoccidioides/ultraestrutura , Ligação Proteica , Receptores de Superfície Celular/metabolismoRESUMO
The influence og growth conditions, as well as of propranolol on Fonsecaea pedrosoi morphogenesis was eatablished using the chemically defined media of Czapeck-Dox (CD) and Butterfield (BF). Mycelial growth of F. pedrosoi in both media was obtained at room temperature (25°C) for 14 days, without shaking, whereas conidia formed ar 37°C, for days, in shaken cultures and could be isolated free from the mycelium by filtration in gauze. At low pH (2.5-3.0), there appeared sclerotic calls attached to normal hyphae. When propranolol was added to the CD medium moniliform hyphae were observed, whereas this drug in the BF medium induced fromation of sclerotic cells. Ultrastructural examination revealed that the propranolol-induced sclerotic cell were very similar to those observed in infected tissues