Leishmania parasites alternate between extracellular promastigotes in sandfly midgut and intracellular amastigotes in mammals. These protozoan parasites cause significant morbidity and mortality due to the lack of a vaccine, limitations of vector control, and inadequacy of drugs. The long-term goal of this project is to elucidate the roles of lipid metabolism in Leishmania survival and infection. Understanding how parasites acquire essential lipids from the mammalian host will shed new light into Leishmania-host interaction and the findings may lead to novel drug targets and drug candidates. The previous funding period focused on how Leishmania parasites generate phosphatidylethanolamine (PE) including plasmalogen PE and diacyl PE. A key finding is that the enzyme ethanolamine-phosphate cytidylyltransferase (EPCT) is essential for both the promastigotes and amastigotes of Leishmania major. EPCT is required for the de novo synthesis of PE via the Kennedy pathway. The essentiality of EPCT in the pathogenic amastigote stage is likely due to the high demand of plasmalogen PE in amastigotes, and the lack of plasmalogen PE scavenging from the mammalian host. Another important finding from the previous funding period is that intracellular amastigotes contain an unusually high level of lysophosphatidylcholine (lyso PC) which has only one fatty acyl chain on the glycerol backbone. In most eukaryotes, lyso PC is a minor lipid component with signaling roles in several processes including intracellular calcium homeostasis, cell migration, inflammation, and apoptosis. Lyso PC also possesses membrane permeabilization activity and promotes positive membrane curvature due to its inverted cone shape. The objective of the next funding period is to determine how Leishmania amastigotes acquire lyso PC and elucidate its functions. Based on our understanding of Leishmania lipid metabolism, we hypothesize that amastigotes acquire lyso PC by hydrolyzing host PC with a parasite-derived phospholipase A2 (PLA2) and efficient lyso PC production is required for their intracellular proliferation. Two specific aims will be pursued concurrently to test this hypothesis. Aim 1 will characterize the molecular machinery involved in lyso PC metabolism in L. major amastigote using molecular biology approaches. Aim 2 will explore the origin and impact of amastigote lyso PC on Leishmania-macrophage interactions. These proposed studies are designed based on published and unpublished work from the previous funding period and are well within the research team’s area of expertise. Successful completion of these aims will lead to new insight into the interaction between Leishmania amastigotes and mammalian host. The findings will also help identify novel drug targets on lyso PC metabolism and understand the mechanism of action for miltefosine, a non-hydrolysable lyso PC analog and the only oral drug to treat leishmaniasis. Finally, the renewal application will allow us to continue expanding...