Ethanolaminephosphate cytidyltransferase is essential for survival, lipid homeostasis and stress tolerance in Leishmania major

Abstract

Glycerophospholipids including phosphatidylethanolamine (PE) and phosphatidylcholine (PC) are vital components of biological membranes. Trypanosomatid parasites of the genus Leishmania can acquire PE and PC via de novo synthesis and the uptake/remodeling of host lipids. In this study, we investigated the ethanolaminephosphate cytidyltransferase (EPCT) in Leishmania major, which is the causative agent for cutaneous leishmaniasis. EPCT is a key enzyme in the ethanolamine branch of the Kennedy pathway which is responsible for the de novo synthesis of PE. Our results demonstrate that L. major EPCT is a cytosolic protein capable of catalyzing the formation of CDP-ethanolamine from ethanolamine-phosphate and cytidine triphosphate. Genetic manipulation experiments indicate that EPCT is essential in both the promastigote and amastigote stages of L. major as the chromosomal null mutants cannot survive without the episomal expression of EPCT. This differs from our previous findings on the choline branch of the Kennedy pathway (responsible for PC synthesis) which is required only in promastigotes but not amastigotes. While episomal EPCT expression does not affect promastigote proliferation under normal conditions, it leads to reduced production of ethanolamine plasmalogen or plasmenylethanolamine, the dominant PE subtype in Leishmania. In addition, parasites with epsiomal EPCT exhibit heightened sensitivity to acidic pH and starvation stress, and significant reduction in virulence. In summary, our investigation demonstrates that proper regulation of EPCT expression is crucial for PE synthesis, stress response, and survival of Leishmania parasites throughout their life cycle. AUTHOR SUMMARYIn nature, Leishmania parasites alternate between fast replicating, extracellular promastigotes in sand fly gut and slow growing, intracellular amastigotes in macrophages. Previous studies suggest that promastigotes acquire most of their lipids via de novo synthesis whereas amastigotes rely on the uptake and remodeling of host lipids. Here we investigated the function of ethanolaminephosphate cytidyltransferase (EPCT) which catalyzes a key step in the de novo synthesis of phosphatidylethanolamine (PE) in Leishmania major. Results showed that EPCT is indispensable for both promastigotes and amastigotes, indicating that de novo PE synthesis is still needed at certain capacity for the intracellular form of Leishmania parasites. In addition, elevated EPCT expression alters overall PE synthesis and compromises parasites tolerance to adverse conditions and is deleterious to the growth of intracellular amastigotes. These findings provide new insight into how Leishmania acquire essential phospholipids and how disturbance of lipid metabolism can impact parasite fitness.