Phosphatidylcholine biosynthesis and miltefosine mode of action in Leishmania

描述（由申请人提供）：利什曼原虫属物种引起重要的人类疾病，称为利什曼病，影响全球超过1200万患者，因此是一个主要的健康问题。甘油脂的生物合成对于寄生虫的生长是必不可少的，因此，对于其引起疾病的能力也是必不可少的。我们的长期目标是确定参与寄生虫脂质代谢的必需蛋白质/基因，因为它对于更好地理解基于脂质的抗寄生虫药物的作用模式至关重要，因此，对于开发更有效的治疗方法至关重要。磷脂酰胆碱是利什曼原虫的一种必需脂质，通过两种途径产生：甲基化途径，其中磷脂酰胆碱通过磷脂酰乙醇胺的三倍甲基化合成;以及从头途径，其起始于宿主来源的胆碱的摄取。我们建立了从头途径是利什曼原虫，因此，甲基化途径可能代表磷脂酰胆碱合成的生理相关途径。目前用于治疗几种形式的利什曼病的胆碱类似物米替福新抑制磷脂酰乙醇胺转化为磷脂酰胆碱的事实进一步支持了这一观点。本申请的目的是鉴定参与由前体磷脂酰乙醇胺生物合成磷脂酰胆碱的酶/基因，并确定抗利什曼原虫药物米替福新如何干扰该代谢途径。拟议研究的基本原理是，更好地了解米替福新的作用模式可能有助于预测耐药性机制，这对治疗和预防寄生虫病具有重要意义。我们已经确定了两个假定的磷脂酰乙醇胺甲基转移酶基因在L。donovani基因组，LdPEM 1和LdPEM 2。我们的假设是LdPEM 1和/或LdPEM 2编码的甲基转移酶参与磷脂酰胆碱的生物合成，并且其中至少一种是米替福新的靶点。我们将通过追求以下三个目标来测试我们的假设：在具体目标1中，我们将识别L的潜在LdPEM 1和LdPEM 2旁系同源物。在特定目标2中，我们将产生LdPEM 1和LdPEM 2特异性抗体，以研究利什曼原虫生命周期期间的表达谱;在特定目标3中，我们将确定LdPEM 1和LdPEM 2底物特异性、酶活性和存在米替福新时的抑制谱。这项研究意义重大，因为它有望促进我们对利什曼原虫磷脂酰胆碱生物合成和米替福新作用模式的理解。

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.