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/首席研究员（姓、名、中）：Ullman, Buddy 2R01 AI041622-11A1 这一竞争性的更新应用融合了分子生物学、遗传学和药理学的工具和技术，提供了一个实验范例来验证杜氏利什曼原虫多胺途径的关键成分，并实施针对治疗利什曼病的药物发现的策略。该提案将主要关注杜氏乳杆菌鸟氨酸脱羧酶 (LdODC)，但也会影响多胺途径的其他成分，包括精氨酸酶 (LdARG)、腺苷甲硫氨酸脱羧酶 (LdADOMETDC) 和亚精胺合酶 (LdSPDSYN)。在我们对利什曼原虫多胺代谢的研究过程中，我们得到了：1）从杜诺瓦尼利什曼原虫或墨西哥利什曼原虫中分离出编码所有四种蛋白质的基因； 2) 在 L. donovani 中创建并表征了 ?ldodc、?ldadometdc 和 ?ldspdsyn 敲除型，以及在 L. mexicana 中创建了 ?lmarg 敲除型； 3) 产生针对 LdODC、LdADOMETDC 和 LdSPDSYN 的单特异性抗血清。迄今为止，我们的大部分研究都是以寄生虫的昆虫载体形式进行的，该应用现在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。这项提议是基于我们的关键发现，即 ldodc L. donovani 严重丧失了感染小鼠的能力，并启动了从药理学角度利用经过基因验证的 LdODC 酶的研究。正在考虑获得 2009 年美国复苏和再投资法案支持的提案有一个具体目标 I，由四个部分组成：1) 我们将确定 LdODC 的产品腐胺是否可以逆转 ?ldodc 寄生虫受损的感染性表型老鼠； 2) 我们将尝试通过在饮用水中施用LdODC抑制剂α-二氟甲基鸟氨酸（DFMO）来在药理学上模拟LDODC损伤对小鼠寄生虫感染性的有害影响； 3) 我们将扩大对叙利亚金仓鼠的 ?ldodc 敲除感染性研究，叙利亚金仓鼠是一种内脏利什曼病的啮齿动物模型，与人类疾病更相似； 4) 我们将通过评估 ldarg、ldadometdc 和 ldspdsyn 基因缺失突变体感染小鼠后的寄生虫负荷，确定 ldodc 无效突变体的毒力缺陷是否延伸至 LdARG、LdADOMETDC 和 LdSPDSYN 缺陷。该目的的实验将证实 LdODC 作为治疗内脏利什曼病的药物靶点的有效性，并将扩展我们对 LdARG、LdADOMETDC 和 LdSPDSYN 作为寄生虫感染形式的潜在药物靶点的表征。