Caracterização de microdomínios de membrana resitentes a detergente não iônico em promastigotas de Leishmania (Viannia) braziliensis. Papel dos microdomínios na infectividade. / Characterization of non-ionic detergent resistant membrane microdomains in Leishmania (Viannia) braziliensis promastigotes. Role of these microdmains in the infectivity

Kelly Aparecida Geraldo Yoneyama Tudini

Glycoconjugates present in Leishmania surface are an important class of molecules involved in parasite-macrophage interaction. The structural elucidation of Leishmania (Viannia) braziliensis promastigote glycolipids is very relevant because in this specie the glycolipids represent the major promastigote surface glycoconjugate. Six glycolipid fractions, termed B1 to B6, were purified from L. (V.) braziliensis promastigotes by high performance liquid chromatography. All fractions were sensitive to mild alkaline hydrolysis and gas chromatography/mass spectrometry (GC/MS) analysis showed the presence of mannose, galactose, glucose, glucosamine and myo-inositol suggesting that glycolipids are glycoinositolphospholipids (GIPLs). The partially methylated alditol acetates analysis showed the presence of 2,4-di-O-methyl-mannitol tetracetate and 2,3,4,6- tetra-O-methyl-galactitol diacetate suggesting a branched oligosaccharide structure containing terminal galactose residue. These results indicate that L. (V.) braziliensis promastigotes express GIPLs which could be included in the hybrid GIPLs class, although present new glycan structures. The concept of lipid rafts, and membrane microdomains, which are non-ionic detergent-resistant at 4C, enriched by cholesterol and sphingolipid, have being developed in the last years and have being involved in many processes such as adhesion, signal transduction, endocytosis and polarized traffic of proteins. Recently it was demonstrated the presence of these microdomains in T. brucei and L. (L.) major, suggesting a broad distribution throughout the Kinetoplastida. Membrane microdomains present in L. (V.) braziliensis promastigotes were analyzed using two protocols. The first was based on insolubility in non-ionic detergent at 4C and the second was based on the low density of these membranes. Detergent-resistant membranes (DRMs) were purified by ultracentrifugation on sucrose gradient. About 85% of GIPLs, 85% of sterols, 65% of inositol phosphorylceramide and 35% of phospholipids were detected in detergent-insoluble fraction at 4C. The presence of GIPLs, sterols and IPC were also verified on DRMs fractions obtained after ultracentrifugation. By GC-MS it was verified that GIPLs and IPC are composed mainly by saturated fatty acids. On the other hand, the detergentsoluble fraction present 65% of phospholipids, which contain higher percentage of unsaturated fatty acids. Analysis by SDS-PAGE demonstrated the presence of 46 kDa (glyco)protein that could correspond to the gp46 anchored in membrane by GPI, and related with protective immunity in mice immunized with this purified glycoprotein. Parasites depleted of sterols after treatment with ketoconazole and methyl-β-cyclodextrin (MβCD) were used in macrophage infectivity. Analysis of infectivity index showed that MβCD at 20 mM and 40 mM inhibited 53% and 50% of L. (V.) braziliensis infectivity, respectively. Parasites treated with MβCD at 40 mM showed a 70% sterol depletion and their GIPLs were present mainly in the fractions containing detergentsoluble components, indicating the this drug disrupted the membrane microdomains. Parasites treated with ketoconazole presented an inhibition of 92% of macrophage infectivity. The high inhibition of macrophage infectivity by parasites treated with ketoconazole could be related with low membrane microdomains disruption and with morphological changes observed in promastigotes mainly in their flagellar structure, suggesting the involvement of flagella in macrophage infectivity by L. (V.) braziliensis promastigotes. The results presented in these work demonstrate that the intact membrane microdomains are essential for L. (V.) braziliensis infectivity.

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