Polyamine Metabolism in Leishmania

利什曼原虫的多胺代谢

基本信息

批准号：
7849950
负责人：
BUDDY ULLMAN
金额：
$ 38.48万
依托单位：
OREGON HEALTH & SCIENCE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
1997
资助国家：
美国
起止时间：
1997-07-01 至 2012-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7849950
关键词：
Ablation Address Adenosylmethionine Decarboxylase Affect African Trypanosomiasis Alleles American Amines Anabolism Animal Experiments Back Biochemical Biological Assay Carboxy-Lyases Cells Clinical Complement Control Animal DL-alpha-Difluoromethylornithine Defect Disease Drug Delivery Systems Drug resistance Enzyme Gene Enzymes Escherichia coli Funding Gene Deletion Genes Genetic Grant Growth Human Immune Sera In Vitro Infection Insect Vectors Investigation Knock-in Mouse Knock-out Leishmania Leishmania donovani Leishmaniasis Lesion Libraries Mesocricetus auratus Metabolism Molecular Molecular Biology Techniques Molecular Genetics Mus Nature Oral Administration Ornithine Decarboxylase Parasites Parasitic Diseases Pathogenicity Pathway interactions Pharmaceutical Preparations Pharmacology Phenotype Play Polyamines Principal Investigator Proteins Putrescine Reagent Recombinant Proteins Recombinants Recovery Rodent Model Role Saccharomyces cerevisiae Scheme Simulate Spermidine Synthase Staging Technology Trypanosoma brucei brucei Vaccines Virulence Visceral Leishmaniasis arginase base biological systems chemotherapy drinking water drug discovery gene function gene replacement high throughput screening human disease inhibitor/antagonist killings macrophage mouse model mutant novel overexpression programs research study small molecule libraries tool

项目摘要

Program Director/Principal Investigator (Last, First, Middle): Ullman, Buddy 2R01 AI041622-11A1 Amalgamating the tools and techniques of molecular biology, genetics, and pharmacology, this competing renewal application offers an experimental paradigm to validate key components of the polyamine pathway of Leishmania donovani and to implement a strategy directed toward drug discovery for the treatment of leishmaniasis. The proposal will focus primarily on the L. donovani ornithine decarboxylase (LdODC) enzyme but will also impact upon other components of the polyamine pathway as well, including arginase (LdARG), Sadenosylmethionine decarboxylase (LdADOMETDC), and spermidine synthase (LdSPDSYN). During the course of our investigations on polyamine metabolism in Leishmania we have: 1) isolated genes encoding all four proteins from either L. donovani or L. mexicana; 2) created and characterized ?ldodc, ?ldadometdc, and ?ldspdsyn knockouts in L. donovani and a ?lmarg knockout in L. mexicana; and 3) generated monospecific antisera against LdODC, LdADOMETDC, and LdSPDSYN. The majority of our studies thus far have been performed with the insect vector form of the parasite, and this application will now extend our characterization of the polyamine pathway to the mammalian stage of the parasite, primarily in rodent models. This proposal follows up upon our pivotal finding that ?ldodc L. donovani are profoundly incapacitated in their ability to infect mice and initiates studies that will pharmacologically exploit the genetically validated LdODC enzyme. The proposal that is under consideration for support from the American Recovery and Reinvestment Act of 2009 has one Specific Aim I with four components: 1) we will determine whether putrescine, the product of LdODC, can reverse the compromised infectivity phenotype of ?ldodc parasites in mice; 2) we will attempt to pharmacologically simulate the deleterious effects of a ?ldodc lesion on parasite infectivity in mice by administration of a-difluoromethylornithine (DFMO), an inhibitor of LdODC, in the drinking water; 3) we will expand our infectivity studies on the ?ldodc knockout to Syrian golden hamsters, a rodent model of visceral leishmaniasis that more closely resembles the human disease; and 4) we will establish whether the virulence defect of the ?ldodc null mutant extends to LdARG, LdADOMETDC, and LdSPDSYN deficiencies by assessing parasite burdens after infection of mice with ?ldarg, ?ldadometdc and ?ldspdsyn gene deletion mutants. The experiments in this aim will confirm the validity of LdODC as a drug target for the treatment of visceral leishmaniasis and will extend our characterization of LdARG, LdADOMETDC, and LdSPDSYN as potential drug targets to the infectious form of the parasite.

项目总监/首席调查员（最后一个，中间）：Ullman，好友2R01 AI041622-11A1 这种竞争性更新应用提供了分子生物学，遗传学和药理学的工具和技术，提供了一个实验性范式，以验证利什曼尼亚多诺瓦尼（Leishmania Donovani）多胺途径的关键组成部分，并实施针对药物发现的策略，以治疗利什曼病的治疗。该提案将主要集中于多诺瓦氏梭菌脱羧酶（LDODC）酶，但也将影响多胺途径的其他成分，包括精氨酸酶（LDARG），sadenosylmethionshionine decarebosine decarboseline decarbosyline dearboxylase（ldadadometdc）（ldadodometdc），以及Espermidine consnthasenase（ldspdsysnastase）。在利什曼尼亚多胺代谢的调查过程中，我们有：1）孤立的基因，编码了来自多诺瓦氏乳杆菌或墨西哥乳杆菌的所有四种蛋白质； 2）在L. Donovani中创建并描述了？ 3）对LDODC，LDADOMETDC和LDSPDSYN产生单特异性抗血清。迄今为止，我们的大多数研究都是用寄生虫的昆虫载体形式进行的，现在，该应用将将我们对多胺途径的表征扩展到寄生虫的哺乳动物阶段，主要是在啮齿动物模型中。这项提议遵循了我们关键的发现，即Donovani ldodc L. donovani在感染小鼠的能力方面非常无能为力，并启动了将在药理上利用遗传学验证的LDODC酶的研究。在2009年《美国恢复和再投资法案》中正在考虑的支持的提议中，有一个特定的目标I具有四个组成部分：1）我们将确定LDODC的putrescine是否能否逆转小鼠中的lDODC寄生虫的受损感染表型； 2）我们将尝试通过在饮用水中的抑制剂A-二氟甲基氨酸氨酸（DFMO）a-二氟甲基氨酸氨酸（DFMO）来模拟药理学对小鼠寄生虫感染性的有害作用； 3）我们将向叙利亚金仓鼠扩大有关LDODC淘汰的感染性研究，叙利亚金仓鼠是内脏利什曼病的啮齿动物模型，与人类疾病更相似。 4）我们将确定ldoDC无效突变体的毒力缺陷是否延伸到LDARG，LDADOMETDC和LDSPDSYN缺陷，通过评估用？ldarg感染小鼠后寄生虫负担，？ldadometdc和？ldadometdc和？ldspdsyn Genen Genen gene deleten deletion突变体。此目标的实验将证实LDODC作为治疗内脏利什曼病的药物的有效性，并将扩展我们对LDARG，LDADOMETDC和LDSPDSYN作为潜在药物靶标的表征，以对寄生虫的传染性形式。