Roles of Plasmenylethanolamine Biosynthesis in Leishmania virulence

描述(申请人提供)：利什曼原虫在人类身上引起一系列破坏性疾病，称为利什曼病。目前的药物治疗不充分，往往是有毒的，而且没有安全的疫苗可用。开发新疗法的一个主要障碍是缺乏对利什曼原虫如何在哺乳动物宿主中建立感染的了解。以前对利什曼原虫表面糖偶联物和鞘磷脂的研究已经强调了它们在毒力中的重要性。相比之下，在包括利什曼原虫在内的寄生原生动物中，丰富的膜脂如磷脂酰乙醇胺(PE)、磷脂酰胆碱和磷脂酰肌醇的功能仍然研究得很少，尽管我们最近的数据表明它们的重要性远远超出了作为基本的膜成分。长期目标是阐明膜脂及其代谢物在利什曼原虫感染中的作用。有趣的是，利什曼原虫中的磷脂与哺乳动物宿主中的磷脂有很大不同。特别是，利什曼原虫中绝大多数的PE属于血浆乙醇胺(PLE)，这是一组鲜为人知的磷脂。这项应用的总体目标是评估PLE对利什曼原虫毒力的贡献。核心假设是PLE的生物合成对于利什曼原虫感染的建立是必不可少的。这一假说是基于以下观察得出的：1)随着寄生虫从复制性的、无毒的前循环向非复制的、高毒力的元环分化，PLE的丰度增加了2-4倍；2)在合成PLE所需的ETN的产生中存在缺陷的突变株，在固定相的PLE水平大大降低，并且未能形成有感染性的元环；以及，3)初步数据表明，在鞘脂降解方面存在缺陷的突变株毒力严重减弱。综上所述，这些结果表明PLE的生物合成对利什曼原虫的感染至关重要。为了验证中心假说，这一建议关注与PLE合成有关的两个候选基因：LmEPCT和LmISC。具体目标包括：1)通过靶向基因置换获得利什曼原虫LmEPCT和LmISC的零突变；2)评估LmEPCT和LmISC对利什曼原虫生长和感染性的影响；3)确定LmEPCT和LmISC的功能，揭示PLE生物合成与利什曼原虫毒力的相关性。拟议研究的成功完成将阐明PLE生物合成对利什曼原虫毒力的贡献，从而完成实现长期目标的重要一步。了解磷脂在利什曼原虫感染中的作用具有重要意义，因为它不仅可以从根本上深入了解利什曼原虫的毒力机制，还可以揭示新的治疗靶点，以帮助控制这些危险的寄生虫。 公共卫生相关性：这项建议旨在阐明以前研究不足的一组磷脂在利什曼原虫发病机制中的作用，利什曼原虫会导致人类一系列毁灭性的疾病，称为利什曼病。拟议研究的成功完成将为利什曼原虫的毒力机制提供新的见解。这些发现最终将被用来改善和保护健康。

DESCRIPTION (provided by applicant): Leishmania parasites cause a spectrum of devastating diseases in humans known as leishmaniasis. Current drug treatments are inadequate, often toxic, and no safe vaccine is available. A major obstacle in the development of new therapeutics is the lack of understanding on how Leishmania parasites establish infection in mammalian hosts. Previous studies on the surface glycoconjugates and sphingolipids in Leishmania have highlighted their importance in virulence. In contrast, functions of abundant membrane lipids such as phosphatidyl- ethanolamine (PE), phosphatidylcholine, and phosphatidylinositol remain extremely understudied in parasitic protozoans including Leishmania, although our recent data suggest their importance extends far beyond being basic membrane components. The long term goal is to elucidate the roles of membrane lipids and their metabolites in Leishmania infection. Interestingly, phospholipids in Leishmania parasites are drastically different from those in mammalian hosts. In particular, the vast majority of PE in Leishmania parasites belongs to plasmenylethanolamine (PLE), a group of phospholipids that is poorly understood. The overall objective of this application is to evaluate the contribution of PLE to the virulence of Leishmania parasites. The central hypothesis is that PLE biosynthesis is essential for the establishment of Leishmania infection. This hypothesis is formulated based on the following observations: 1) the abundance of PLE increases 2-4 fold as parasites differentiate from replicative, non-virulent procyclics to non-replicative, highly virulent metacyclics; 2) mutants defective in the production of EtN, which is required for the synthesis of PLE, showed greatly reduced level of PLE in stationary phase and failed to form infective metacyclics; and, 3) preliminary data indicate mutants defective in the degradation of sphingolipids, which could be the first step towards the synthesis of PLE, were severely attenuated in virulence. Together, these results suggest PLE biosynthesis is crucial for Leishmania infection. To test the central hypothesis, this proposal focuses on two candidate genes involved in the synthesis of PLE: LmEPCT and LmISC. Specific aims include: 1) to generate null mutants of LmEPCT and LmISC in L. major through targeted gene replacement; 2) to evaluate the impacts of LmEPCT and LmISC on Leishmania growth and infectivity; 3) to confirm the functions of LmEPCT and LmISC, and reveal the correlation between PLE biosynthesis and Leishmania virulence. Successful completion of the proposed studies will elucidate the contribution of PLE biosynthesis to Leishmania virulence; therefore complete an important step towards achieving the long term goal. Understanding the roles of phospholipids in Leishmania infection is highly significant, because it will not only provide fundamental insight into the mechanism of virulence, but also could reveal new therapeutic targets to help control these dangerous parasites. PUBLIC HEALTH RELEVANCE: This proposal aims to elucidate the roles of a previously understudied group of phospholipids in the pathogenesis of Leishmania parasites, which cause a spectrum of devastating diseases in humans known as leishmaniasis. Successful completion of the proposed studies will provide new insight into the mechanism of virulence in Leishmania. Such findings will, ultimately, be used to improve and protect health.