Functional cloning of the miltefosine transporter -: A novel P-type phospholipid translocase from Leishmania involved in drug resistance

The antitumor drug miltefosine (hexadecylphosphocholine, MIL) has recently been approved as the first oral agent for the treatment of visceral leishmaniasis. Little is known about the mechanisms of action and uptake of MIL in either parasites or tumor cell lines. We have cloned a putative MIL transporter (LdMT) by functional rescue, using a Leishmania donovani-resistant line defective in the inward-directed translocation of both MIL and glycerophospholipids. LdMT is a novel P-type ATPase belonging to the partially characterized aminophospholipid translocase subfamily. Resistant parasites transfected with LdMT regain their sensitivity to MIL and edelfosine and the ability to normally take up [C-14]MIL and fluorescent-labeled glycerophospholipids. Moreover, LdMT localizes to the plasma membrane, and its overexpression in Leishmania tarentolae, a species non-sensitive to MIL, significantly increases the uptake of [C-14]MIL, strongly suggesting that this protein behaves as a true translocase. Finally, both LdMT-resistant alleles encompass single but distinct point mutations, each of which impairs transport function, explaining the resistant phenotype. These results demonstrate biochemically and genetically the direct involvement of LdMT in MIL and phospholipids translocation in Leishmania and describe for the first time a P-type ATPase involved in MIL uptake and potency in eukaryotic cells.