Polyamine Metabolism in Leishmania

描述(由申请人提供)：融合了分子生物学、遗传学、生物化学和药理学的工具和技术，这项竞争性的更新申请提供了一个实验范例，进一步验证内脏利什曼病的病原体杜氏利什曼原虫多胺生物合成途径的关键成分。申请的重点是从宿主获取多胺在确定寄生虫感染的过程中的作用]，并实施一项旨在发现治疗内脏利什曼病和可能其他形式的利什曼病的药物的战略。利什曼原虫多胺生物合成途径由精氨酸酶(LdARG)、鸟氨酸脱羧酶(LdODC)、S腺苷蛋氨酸脱羧酶(LdADOMETDC)和亚精胺合成酶(LdSPDSYN)4种酶组成。[寄生虫也可以通过摄取机制挽救宿主多胺及其前体，其中之一是POT1腐胺-亚精胺转运蛋白。该应用程序将解决宿主和寄生虫多胺途径之间这种复杂且基本上未描述的关系)，并是基本观察结果的逻辑延伸，即�ldarg、�ldodc、�ldadometdc和�ldspdsyn损伤，所有这些损伤都导致多胺轴营养不良，在极大不同的程度上影响杜诺瓦尼乳杆菌感染哺乳动物宿主的能力。可用于这些研究的关键试剂包括：1)编码每种多胺酶和[POT1通透酶]的基因；2)杜氏乳杆菌的�ldarg、�ldodc、�ldadometdc和�ldspdsyn零突变体；以及3)Arg1Flox/Flox；Tie2cre小鼠及其Tie2cre Cre Deleter对照。[特定目标I]有三个组成部分：1)通过比较对照和Arg1 Flox/Flox的肝脏和脾中的寄生虫负载，确定从宿主中抢救多胺或多胺前体的作用；Tie2cre小鼠 目的：1)研究野生型、�ldarg、�ldodc或�ldspdsyn L.donovani野生型、�ldarg、�ldodc或�ldspdsyn L.donovani在适当营养补充方案下的致病机制；2)对来源于Arg1 flx/flx的野生型和突变株的腹膜巨噬细胞中的寄生虫进行互补的感染性研究；3)通过检测pot1的过表达是否增加了野生型小鼠感染�ldodc或�ldspdsyn L.donovani时的寄生虫负担，并通过对�ldpot1基因敲除的表型评估来确定LdPOT1是否是捐赠者体内唯一的多胺渗透机制，以确定POT1在调节寄生虫感染中的作用。特异性目标2是药物开发中合乎逻辑的一步，为发现杜氏乳杆菌多胺途径的小分子抑制剂提供了蓝图。将对5,000-6,000种已证实的抗利什曼类化合物进行简单、成本效益和包容性的反向化学遗传筛选，这些化合物是从结构和化学上不同的小分子库的两个全面的高通量正向化学遗传筛选中出现的，以识别针对杜诺瓦尼乳杆菌多胺途径的四种酶中的任何一种的化学物质。

DESCRIPTION (provided by applicant): Amalgamating the tools and techniques of molecular biology, genetics, biochemistry, and pharmacology, this competing renewal application offers an experimental paradigm to further validate key components of the polyamine biosynthetic pathway of Leishmania donovani, the causative agent of visceral leishmaniasis. The application focuses on the role of polyamine acquisition from the host in the establishment of parasite infection] and implements a strategy directed toward drug discovery for the treatment of visceral and perhaps other forms of leishmaniasis. The polyamine biosynthetic pathway of Leishmania consists of four enzymes: arginase (LdARG), ornithine decarboxylase (LdODC), S-adenosylmethionine decarboxylase (LdADOMETDC), and spermidine synthase (LdSPDSYN). [The parasite can also salvage host polyamines and their precursors via uptake mechanisms, one of which is the POT1 putrescine-spermidine transporter. The application will address this intricate and largely uncharacterized relationship between the host and parasite polyamine pathways] and is a logical extension of the fundamental observations that �ldarg, �ldodc, �ldadometdc, and �ldspdsyn lesions, all of which confer polyamine axotrophy, impact the capacity of L. donovani to infect the mammalian host to dramatically different extents. Key reagents available for these investigations include: 1) genes encoding each of the polyamine enzymes and [the POT1 permease]; 2) �ldarg, �ldodc, �ldadometdc, and �ldspdsyn null mutants of L. donovani; and 3) Arg1 flox/flox; Tie2cre mice that lack ARG1 in macrophages and their Tie2cre Cre deleter controls. [Specific Aim I has three components: 1) to ascertain the role of polyamine or polyamine precursor salvage from the host by comparing parasite loads in livers and spleens of both control and Arg1 flox/flox ; Tie2cre mice that have been inoculated with either wild type, �ldarg, �ldodc, or �ldspdsyn L. donovani followed by appropriate nutritional supplementation protocols to test mechanism; 2) to perform complementary infectivity studies with the wild type and mutant parasites in peritoneal macrophages derived from the Arg1flox/flox; Tie2cre and Tie2cre strains; 3) to establish the role of POT1 in modulating parasite infection by testing whether POT1 overexpression boosts parasite burdens when wild type mice are inoculated with either �ldodc or �ldspdsyn L. donovani and by ascertaining whether LdPOT1 is the sole polyamine permeation mechanism in L. donovani via the creation and phenotypic assessment of a �ldpot1 knockout.] Specific Aim 2 is a logical step in drug development and offers a blueprint to discover small molecule inhibitors of the genetically validated polyamine pathway of L. donovani. A simple, cost-effective, and inclusive reverse chemical genetic screen of 5,000 - 6,000 proven anti-leishmanial compounds that have emerged from two comprehensive high throughput forward chemical genetic screens of structurally and chemically diverse small molecule libraries will be performed in order to identify chemicals that target any o the four enzymes of the L. donovani polyamine pathway.