RESUMO
The present study demonstrates that the Leishmania (Viannia) braziliensis strain MCAN/BR/1998/R619 is composed of multiple subpopulations with measurable distinctions. Single parasites were separated from a culture of promastigotes in stationary phase by cell sorting and then cultivated as subpopulations. Subsequently, these subpopulations were evaluated for features of in vitro growth, infectivity to murine macrophages and proteinase gene expression. The first evidence of distinct characteristics was observed during the in vitro cultivation of isolated subpopulations, as distinct clusters of patterns were formed among the cultures, indicating the existence of quantifiable fluctuations in metrics. Further, when infecting murine macrophages, the subpopulations induced distinct patterns of production of immune response mediators. While some subpopulations mainly induced the production of IL-1ß, IL-6 and TNF-α, others induced the production of IL-12p70 and nitric oxide. Finally, amastigotes of these subpopulations had higher expression of proteinase genes than promastigotes. Additionally, cysteine proteinase, serine proteinase, metalloproteinase and aspartic proteinases were differentially expressed in promastigote and amastigote forms. These data suggest the existence of distinct profiles for the L. (V.) braziliensis MCAN/BR/1998/R619 strain and subpopulations that could drive the success of parasite adaptation to the environments that they inhabit.
Assuntos
Leishmania braziliensis/crescimento & desenvolvimento , Animais , Humanos , Interleucina-1beta/imunologia , Interleucina-6/imunologia , Leishmania braziliensis/classificação , Leishmania braziliensis/genética , Leishmania braziliensis/isolamento & purificação , Leishmaniose Cutânea/imunologia , Leishmaniose Cutânea/parasitologia , Macrófagos/imunologia , Macrófagos/parasitologia , Camundongos , Peptídeo Hidrolases/genética , Peptídeo Hidrolases/metabolismo , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Fator de Necrose Tumoral alfa/imunologiaRESUMO
Leishmaniasis is a vector-borne disease and an important public health issue. Glycosaminoglycan ligands in Leishmania parasites are potential targets for new strategies to control this disease. We report the subcellular distribution of heparin-binding proteins (HBPs) in Leishmania (Viannia) braziliensis and specific biochemical characteristics of L. (V.) braziliensis HBPs. Promastigotes were fractionated, and flagella and membrane samples were applied to HiTrap Heparin affinity chromatography columns. Heparin-bound fractions from flagella and membrane samples were designated HBP Ff and HBP Mf, respectively. Fraction HBP Ff presented a higher concentration of HBPs relative to HBP Mf, and SDS-PAGE analyses showed 2 major protein bands in both fractions (65 and 55 kDa). The 65 kDa band showed gelatinolytic activity and was sensitive to inhibition by 1,10-phenanthroline. The localization of HBPs on the promastigote surfaces was confirmed using surface plasmon resonance (SPR) biosensor analysis by binding the parasites to a heparin-coated sensor chip; that was inhibited in a dose-dependent manner by pre-incubating the parasites with variable concentrations of heparin, thus indicating distinct heparin-binding capacities for the two fractions. In conclusion, protein fractions isolated from either the flagella or membranes of L. (V.) braziliensis promastigotes have characteristics of metallo-proteinases and are able to bind to glycosaminoglycans.