Citólise mediada por lipídeos de Leishmania (Leishmania) amazonensis: indícios da formação de poros mediada por lisofosfatidilcolinas e seu envolvimento na invasão celular por promastigotas

Abstract

Previous work by our group have described and characterized a cytolytic activity, whose mechanism is by pore formation in the membrane of target cells, the molecule responsible for this activity being called leishporin. Although several features of leishporin have already been determined, the molecular identity of this cytolysin is still unknown. Previous work, also from our group, has shown that lysophosphatidylcholines (LPCs) purified from extracts of promastigotes are lytic. In the present work, we have studied the cytolytic activity of lipids from Leishmania amazonensis promastigotes, in an attempt to contribute to the identification of leishporin and the confirmation of this mechanism in lipid-mediated lysis. We have thus demonstrated that lipid extracts (ext-lip) of L. amazonensis promastigotes, totally devoid of proteins, are cytolytic, lysing human red blood cells. We found that ext-lip retain all the hemolytic activity present in the total extract (ext-t) and in the membrane extract (ext-m) of promastigotes, suggesting that lipids are responsible for the formation of pores and that the protein fraction lacks hemolytic activity. These results were corroborated by the fact that the profile of hemolytic activity of ext-lip during the days of cultivation of promastigotes in vitro follows the same profile of the activity of ext-t and ext-m. Confirming previous results, we have verified that ext-t and ext-m are thermolabile, suggesting the participation of proteins in hemolytic activity or in its regulation, while hemolytic lipids are, as expected, thermoresistant. This made us hypothesize that leishporin is a lipoprotein complex, the lipid part of which is responsible for the pore-forming activity or that some ext-t or ext-m protein is important for the formation of pores when in the presence of lytic lipid. An important finding was the demonstration that ext-m-mediated hemolysis of promastigotes is colloid-osmotic, strongly suggesting that the mechanism of hemolysis by the total lipids of the parasite is by pore formation, and corroborating previous findings that purified LPCs-mediated hemolysis is colloid-osmotic. Knowing that type A phospholipases are the enzymes responsible for the synthesis of LPCs, we have used the Rosenthal‘s inhibitor, which inhibits phospholipases A1 and A2, in the promastigotes culture. We have demonstrated that ext-t of treated promastigotes entirely lose its hemolytic activity. These results thus suggest that LPCs are, in fact, leishporin. Using promastigotes treated or not with Rosenthal inhibitor, we have also found that parasites without hemolytic activity are less invasive for macrophages or fibroblasts than hemolytic parasites, suggesting that leishporin is important in the process of phagocytic and non-phagocytic cell invasion by the parasites. Obtaining this phenotype of promastigotes, completely devoid of cytolytic activity, was an important step for future studies of leishporin function.