| dc.description.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. | |
