Phosphatidylcholine biosynthesis and miltefosine mode of action in Leishmania

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DESCRIPTION (provided by applicant): Leishmania species cause important human diseases termed leishmaniasis that affects more than twelve million patients worldwide, and thus, represents a major health problem. Glycerolipid biosynthesis is essential for parasite growth, and thus, for its ability to cause disease. Our long-term goals are to identify essential proteins/genes involved in parasite lipid metabolism, as it is critical for a better understanding of the mode of action of lipid-based anti-parasitic drugs, and hence, for the development of more efficacious therapeutics. Phosphatidylcholine, an essential lipid in Leishmania, is produced via two pathways: the methylation pathway in which phosphatidylcholine is synthesized by a three-fold methylation of phosphatidylethanolamine, and the de novo pathway that initiates with the uptake of host derived choline. We established that the de novo pathway is dispensable for Leishmania, and thus, the methylation pathway may represent the physiologically relevant route for phosphatidylcholine synthesis. The fact that the choline analog miltefosine, currently used to treat several forms of leishmaniasis, inhibits the conversion of phosphatidylethanolamine into phosphatidylcholine, further supports this idea. The objective of this application is to identify the enzymes/genes implicated in phosphatidylcholine biosynthesis from the precursor phosphatidylethanolamine, and establish how the anti-leishmanial drug miltefosine interferes with this metabolic route. The rationale for the proposed research is that a better understanding of miltefosine's mode of action may help anticipate drug resistance mechanisms, which has critical implications for the treatment and prevention of parasitic diseases. We have identified two putative phosphatidylethanolamine methyltransferase genes in the L. donovani genome, LdPEM1 and LdPEM2. Our hypothesis is that the methyltransferases encoded by LdPEM1 and/or LdPEM2 are implicated in phosphatidylcholine biosynthesis, and at least one of them is the target of miltefosine. We will test our hypothesis by pursuing the three following aims: in Specific Aim 1, we will identify potential LdPEM1 and LdPEM2 paralogs of L. donovani, as its complete genome sequence is not available yet; in Specific Aim 2, we will raise antibodies specific to LdPEM1 and LdPEM2 to investigate their expression profiles of during the life cycle of Leishmania; and in Specific Aim 3, we will determine LdPEM1 and LdPEM2 substrate specificities, enzymatic activities, and inhibition profiles in the presence of miltefosine. The proposed research is significant, because it is expected to advance our understanding of phosphatidylcholine biogenesis in Leishmania and of miltefosine's mode of action.