Understanding the crucial roles of lipid synthesis in Leishmania proliferation and stress response



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Leishmaniasis are a group of neglected tropical diseases transmitted through the bite of female phlebotomine sandflies. The causative agents are protozoan parasites of the genus Leishmania which alternate between flagellated promastigotes colonizing the midgut of sandflies and non-flagellated amastigotes residing in the macrophages of mammals. The 3 forms of Leishmaniasis are cutaneous, mucocutaneous and visceral caused by L. major, L. mexicana and L. donovani. Without a safe vaccine, disease management primarily depends on vector control and drugs like pentavalent antimonials, itraconazole, myriocin administered in high doses, having adverse side effects and increased drug resistance Discoveries that reveal fundamental insights into Leishmania biology can lead to new drug targets, better treatments, and improved vector control strategies. Therefore, research involving the identification and characterization of metabolic pathways that could provide new drug targets is of considerable value. Understanding biology behind lipid synthesis and acquisition in Leishmania is important because lipids play a crucial role in cell homeostasis. Plasma membrane and other organelle membrane contain lipids as important constituents, so understanding how important lipids like PC, sterol and sphingolipid are synthesized de novo and utilized for both life stages of Leishmania in sandfly and mammalian hosts, respectively is crucial. The long-term goal can be utilizing this new biological knowledge about lipid synthesis and acquisition by applying them to identify possible novel drug targets against the parasite. Potentially, we can generate novel inhibitors or use this knowledge to create combinatorial treatment with existing chemotherapeutics helping to cure Leishmaniasis (second-deadliest parasitic disease after malaria). Phosphatidylcholine (PC) is the most abundant group of phospholipids in eukaryotes constituting 30–35% of total lipids in Leishmania. PC synthesis mainly occurs via the choline branch of the Kennedy pathway (choline  choline-phosphate  CDP-choline  PC) and the N-methylation of phosphatidylethanolamine (PE). In addition, Leishmania parasites can also acquire lipids from the host or culture medium. Our previous study on the choline-phosphate cytidylyltransferase (CPCT) demonstrates that the formation of CDP-choline from choline-phosphate and CTP is dispensable for the promastigotes and amastigotes of Leishmania major. Thus, these parasites may bypass CPCT through an alternative route of CDP-choline production, PE N-methylation or lipid salvage. In this study, we assessed the function and essentiality of choline ethanolamine phosphotransferase (CEPT) which is directly responsible for the de novo synthesis of both PC and PE. This is important because in addition to being principle membrane components, PC and PE are precursors for a number of vital intermediates including diacylglycerol, lysophospholipid and phosphatidic acid. Understanding how Leishmania generate PC and PE may reveal new ways to block their growth. Our data indicate that L. major CEPT is localized in the ER and possesses the activity to synthesize PC from CDP-choline and diacylglycerol. Targeted deletion of CEPT is only possible in the presence of an episomal CEPT in the promastigote stage of L. major. These chromosomal null parasites require the episomal expression of CEPT for survival in culture, confirming its essentiality during the promastigote stage. In contrast, during in vivo infection of BALB/c mice, these chromosomal null parasites appeared to lose the episomal copy of CEPT while maintaining a normal level of virulence, replication and cellular PC. Therefore, while the de novo synthesis of PC/PE is indispensable for proliferation of promastigotes, intracellular amastigotes could acquire the majority of their lipids from the host. Within the mid gut of sand fly, Leishmania promastigotes need to colonize, proliferate and differentiate while facing a number of challenges including sand fly immunity, nutrient competition, potential microbial toxins and osmolality changes associated with sugar and blood meal digestion. Sterols and sphingolipids are key components of the plasma membrane and regulate diverse processes including membrane stability, ligand-receptor interaction, and vesicular trafficking. Previous findings show, sphingolipids and sterol biosynthetic pathway enzymes are crucial for stress response and virulence. To decipher the protective role of these lipids in Leishmania, we examined the sensitivity of ergosterol and sphingolipid mutants to membrane perturbation agents, bacterial toxins and osmotic stress. My results show sterol and sphingolipid inhibition result in mitochondrial abnormalities, making Leishmania hypersensitive to temperature, osmostress and Pentamidine (mitochondrion targeting drug), thereby indicating sterols and sphingolipids crucially maintain Leishmania homeostasis during temperature and osmotic stress encountered in its lifecycle. Alteration of lipid synthesis can make Leishmania extremely vulnerable to membrane stress and nutrient deprivation. My work also tries to improve current drug efficacy by combined usage of lower doses of existing drugs like Pentamidine and Antimony tartrate respectively with sterol inhibitor Itraconazole and sphingolipid inhibitor Myriocin respectively against Leishmania spp ( L.major, L. mexicana and L. donovani) in vitro with least to no side effects. My results show various Leishmania spp. (L. major, L. mexicana, L. donovani) on sterol inhibition with sublytic Itraconazole doses, and sphingolipid inhibition with sublytic Myriocin doses will show hypersensitivity to sublytic doses of Pentamidine and Antimony tartrate when used in combination. The significance of this proposed work is to find better combinatorial treatment strategies against Leishmaniasis with existing drugs and inhibitors with least possible doses, having least to no side effects. Future work will elucidate: 1) how sterol/sphingolipid synthesis affects the composition and physiology of plasma membrane, and 2) how lipids in plasma membrane affect the transmission of Leishmania parasites in the sand fly.

Embargo status: Restricted until 01/2027. To request the author grant access, click on the PDF link to the left.



Leishmania, Phospholipid, Sterol, Sphingolipid, Stress Response