Roles of P-glycoprotein genes in drug resistance and intracellular parasitism in Leishmania

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We isolated and characterized two different P-glycoprotein genes in a parasitic protozoan, Leishmania amazonensis. The LaMDR1 gene encodes 1341 amino acids for a protein consisting of two similar halves, each containing one ATP-binding and six transmembrane domains. Transfection and over-expression of the LaMDR1 gene in wild-type promastigotes of L. amazonensis conferred resistance to vinblastine, doxorubicin and actinomycin D, indicating that the LaMDR1 gene is a homologue of human multidrung resistance MDR1 gene. The LaMDR2 gene encodes 1267 amino acids, revealing only 47% amino acid identity to the LaMDR1 protein. Transfectants with the LaMDR2 gene showed resistance to 5-fluorouracil (5-FU), a fluoropyrimidine used for cancer chemotherapy, but not to other anti-cancer drugs described above. A slightly higher intracellular accumulation of [ィイD13ィエD1H] vinblastine was observed in the LaMDR1-transfected cells compared with control transfectant cells. In transfectants with the LaMDR2 gene, an increased accumulation of [ィイD13ィエD1H]5-FU was measured in the first 30 min after drug exposure, but the drug accumulation was gradually decreased and reached to 20-30% of the control level at 120 min after the exposure. This 5-FU accumulation kinetics in transfectants with the LaMDR2 gene suggests that the LaMDR2 protein may locate on the membrane of cytoplasmic vesicles and transport 5-FU inside the vesicles during the initial phase, and then drugs were secreted by exocytosis during the later phase. These results revealed that the LaMDR2 gene is a new member of the ATP-binding cassette (ABC) family of protein with the P-glycoprotein structure and play a novel role in survival of the parasites in the host cells. Subcellular localization of the LaMDR2 protein using anti-LaMDR2 antibodies is under study. Functional analysis of the LaMDR2 gene by gene-disruption technology is also in process.