Polyamine Metabolism in Leishmania

描述（由申请人提供）：结合分子生物学、遗传学、生物化学和药理学的工具和技术，这一竞争性更新申请提供了一个实验范例，以进一步验证多诺瓦利什曼原虫多胺生物合成途径的关键成分，内脏利什曼病的病原体。该应用侧重于从宿主获得多胺在建立寄生虫感染中的作用，并实现了一种用于治疗内脏和其他形式利什曼病的药物发现策略。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）四种酶组成。寄生虫也可以通过摄取机制挽救宿主多胺及其前体，其中一种是POT1腐胺-亚精胺转运体。该应用程序将解决宿主和寄生虫多胺途径之间复杂且基本上未表征的关系，并且是基本观察的逻辑延伸，即ldarg， ldodc， ldadometdc和lldspdsyn病变，所有这些病变都导致多胺肌萎缩，在很大程度上影响了L. donovani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)编码每个多胺酶和[POT1渗透酶]的基因；2) L. donvani的‘ ldarg, ’ ldodc, ' ldadometdc, ‘ ldadspdsyn ’零突变体；3) Arg1 flox/flox；巨噬细胞中缺乏ARG1的Tie2cre小鼠及其Tie2cre Cre缺失对照。[具体目的1]有三个组成部分：1)通过比较对照和Arg1 flox/flox的肝脏和脾脏中的寄生虫负荷，确定多胺或多胺前体从宿主中回收的作用；两种接种过的小鼠

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.