DataSheet_7_De Novo Synthesis of Phosphatidylcholine Is Essential for the Promastigote But Not Amastigote Stage in Leishmania major.pdf

Phosphatidylcholine (PC) is the most abundant type of phospholipids in eukaryotes constituting ~30% 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 acquire PC and other lipids from the host or culture medium. In this study, we assessed the function and essentiality of choline ethanolamine phosphotransferase (CEPT) in Leishmania major which is responsible for the final step of the de novo synthesis of PC and PE. Our data indicate that CEPT is localized in the endoplasmic reticulum and possesses the activity to generate PC from CDP-choline and diacylglycerol. Targeted deletion of CEPT is only possible in the presence of an episomal CEPT gene in the promastigote stage of L. major. These chromosomal null parasites require the episomal expression of CEPT to survive 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 normal levels of virulence, replication and cellular PC. Therefore, while the de novo synthesis of PC/PE is indispensable for the proliferation of promastigotes, intracellular amastigotes appear to acquire most of their lipids through salvage and remodeling.

磷脂酰胆碱（Phosphatidylcholine, PC）是真核生物中含量最丰富的磷脂类型，在利什曼原虫内约占总脂质的30%。PC的合成主要通过肯尼迪途径（Kennedy pathway）的胆碱分支（胆碱→磷酸胆碱→CDP-胆碱→PC）以及磷脂酰乙醇胺（Phosphatidylethanolamine, PE）的N-甲基化途径完成。此外，利什曼原虫还可从宿主或培养基中获取PC及其他脂质。本研究评估了硕大利什曼原虫（Leishmania major, L. major）中胆碱乙醇胺磷酸转移酶（choline ethanolamine phosphotransferase, CEPT）的功能与必需性，该酶负责PC与PE从头合成的最后一步。研究数据显示，CEPT定位于内质网，具备以CDP-胆碱与二酰甘油为底物合成PC的酶活性。在硕大利什曼原虫的前鞭毛体阶段，仅当存在游离型CEPT基因时，方可实现CEPT的靶向敲除。这类染色体缺陷型原虫需依赖游离型CEPT的表达才能在培养基中存活，证实了其在前鞭毛体阶段的必需性。与之相反，在BALB/c小鼠体内感染实验中，这类染色体缺陷型原虫会丢失游离型CEPT拷贝，但仍维持正常的毒力、增殖能力与细胞内PC水平。由此可见，尽管PC/PE的从头合成对前鞭毛体的增殖不可或缺，但细胞内无鞭毛体似乎主要通过获取与重塑途径获取自身所需的大部分脂质。