Leishmania braziliensis: Phospholipase A activity in the subcellular fractions of the different parasite stages.

BELAUNZARÁN ML,; BOTT E,; GIMENEZ G.,; LAMMEL EM; ISOLA ELD.

Congreso; XVIII International Congress for Tropical Medicine and Malaria and XLVIII Congress of the Brazilian Society for Tropical Medicine.; 2012

Leishmaniasis is a diverse and complex disease caused by a protozoa of the genus Leishmania (Trypanosomatidae family) transmitted to humans by sandflies. In Argentina, L. braziliensis, L guyanensis and L. amazonensis are the main species related to human disease. Phospholipase A1 (PLA1) specifically hydrolyses acyl groups from phospholiplids (PL) at the sn-1 position, being considered virulence factor for many pathogens. In Trypanosoma cruzi, protozoa of the Trypanosomatidae family, we demonstrated the presence PLA1, involved in parasite invasion and pathogenesis of Chagas disease. In L. braziliensis and L. mexicana promastigotes we previously determined the presence of a phosphatidylcholine PLA1 activity. We here study the subcellular distribution of PLA1 in L. braziliensis amastigotes (AMA) and promastigotes (PRO). Material and Methods Parasites: L. braziliensis, clon MHOM/BR/75M2904, AMA and PRO were axenically cultured in LIT media at 24ºC or 37ºC, respectively. Subcellular fractions: AMA or PRO were disrupted by freezing/thawing and sequentially centrifuged to obtain membrane-enriched, organelle-enriched and soluble fractions. PLA activity: Samples were incubated with 1-palmitoyl-2-[1-14C]oleoyl-phosphatidylcholine (PC) or 1-palmitoyl-2-[1-14C] araquidonyl-phosphatidylethanolamine (PE) 1h, 37ºC. Lipids were extracted by Bligh&Dyer, separated by thin layer chromatography and evaluated by autoradiography. PLA1 expression: Samples were analyzed by Immunoblot with specific antibodies against T. cruzi and T. brucei PLA1, prepared in our laboratory. Results The presence of lysoPC was indicative of PLA1 activity and was significantly higher in AMA with respect to PRO, when using PC as substrate. The major PLA1 activity in both stages was associated to the soluble fractions, followed by the organelle- and membrane- enriched fractions. As regards PE, it was also hydrolyzed by both stages, confirming the presence of a PLA activity acting on zwitterionic phospholipids. Immunoblot analyses with anti-PLA1 specific antibodies, showed in AMA soluble fractions two bands of ~50-60 kDa and a band of ~100 kDa and in membrane- enriched fractions a band of 35 kDa. Whereas, in PRO only in membrane enriched fraction a band of ~100 kDa was detected. Conclusions Herein, we demonstrated that L. brazilienses PLA1 activity was mainly associated to the infective stages, as we already reported for T. cruzi, thus suggesting its possible implications in the pathogenesis of Leishmaniosis. On the other hand, cytosolic PLA1 localization was similar to T. brucei, in contrast to our previous results in T. cruzi where it was localized in membranes. Supported by FONCYT/CONICET.