Phosphatidylcholine biosynthesis and miltefosine mode of action in Leishmania

描述(申请人提供)：利什曼原虫会引起重要的人类疾病，称为利什曼病，影响全世界1200多万患者，因此是一个主要的健康问题。甘油脂的生物合成对寄生虫的生长至关重要，因此对其致病能力也是必不可少的。我们的长期目标是确定涉及寄生虫脂质代谢的必要蛋白质/基因，因为这对于更好地了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是至关重要的。磷脂酰胆碱是利什曼原虫体内的一种必需脂质，通过两条途径产生：甲基化途径，即磷脂酰乙醇胺三次甲基化合成磷脂酰胆碱；从头途径，起始于摄取宿主来源的胆碱。我们证实从头途径对于利什曼原虫来说是必不可少的，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形式的利什曼病，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。这项应用的目的是确定与磷脂酰胆碱生物合成有关的酶/基因，并确定抗利什曼药物米替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模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关键意义。我们在杜氏乳杆菌基因组中发现了两个可能的磷脂酰乙醇胺甲基转移酶基因，LdPEM1和LdPEM2。我们的假设是，LdPEM1和/或LdPEM2编码的甲基转移酶参与了磷脂酰胆碱的生物合成，其中至少有一个是米替福辛的靶标。我们将通过追求以下三个目标来验证我们的假设：在特定目标1，我们将确定潜在的LdPEM1和LdPEM2平行基因，因为其完整的基因组序列尚未获得；在特定目标2，我们将产生针对LdPEM1和LdPEM2的抗体，以研究它们在利什曼原虫生活史中的表达谱；在特定目标3，我们将确定LdPEM1和LdPEM2底物的特异性，酶活性，以及在米替福辛存在的情况下的抑制谱。这项拟议的研究意义重大，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以及米替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.