Phosphatidylcholine biosynthesis and miltefosine mode of action in Leishmania

描述（由申请人提供）：利什曼原虫物种引起被称为利什曼病的重要人类疾病，影响全世界超过1200万患者，因此代表了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，对于它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，因为这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，其中磷脂酰胆碱是通过磷脂酰乙醇胺的三倍甲基化合成的，以及从头开始的途径，通过吸收宿主衍生的胆碱开始。我们确定利什曼原虫的新生途径是不可缺少的，因此，甲基化途径可能代表了磷脂酰胆碱合成的生理相关途径。胆碱类似物米替福辛，目前用于治疗几种利什曼病，抑制磷脂酰乙醇胺转化为磷脂酰胆碱，这一事实进一步支持了这一观点。本申请的目的是鉴定与磷脂酰胆碱前体乙醇胺生物合成有关的酶/基因，并确定抗利什曼病药物米特福辛如何干扰这一代谢途径。这项拟议研究的基本原理是，更好地了解米替福辛的作用方式可能有助于预测耐药性机制，这对寄生虫病的治疗和预防具有重要意义。我们已经在L. donovani基因组中鉴定了两个推定的磷脂酰乙醇胺甲基转移酶基因，LdPEM1和LdPEM2。我们的假设是，由LdPEM1和/或LdPEM2编码的甲基转移酶与磷脂酰胆碱的生物合成有关，其中至少有一个是米特福辛的靶标。我们将通过追求以下三个目标来验证我们的假设：在Specific Aim 1中，我们将确定L. donovani的潜在LdPEM1和LdPEM2类似物，因为其完整的基因组序列尚未获得；在Specific Aim 2中，我们将培养针对LdPEM1和LdPEM2的抗体，研究它们在利什曼原虫生命周期中的表达谱；在Specific Aim 3中，我们将确定LdPEM1和LdPEM2底物的特异性、酶活性和在米替福辛存在下的抑制谱。提出的研究意义重大，因为它有望促进我们对利什曼原虫中磷脂酰胆碱的生物发生和米特福辛的作用模式的理解。

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.