Nucleoside/Nucleobase Transporters in Leishmania major

大利什曼原虫中的核苷/核碱基转运蛋白

• 批准号: 6827962
• 负责人: Scott M Landfear
• 金额: $ 33.98万
• 依托单位: OREGON HEALTH & SCIENCE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-03-01 至 2009-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6827962
• 关键词: Leishmania donovani; Leishmania major; Saccharomyces cerevisiae; Xenopus; gene expression; gene targeting; genetic screening; host organism interaction; intracellular parasitism; intracellular transport; laboratory mouse; life cycle; membrane channels; membrane permeability; membrane transport proteins; molecular cloning; mutant; nucleic acid sequence; nucleobase; nucleosides; posttranslational modifications; protein quantitation /detection; protozoal genetics; purines; site directed mutagenesis; virulence

Amalgamating tools of molecular biology, genetics, biochemistry, and cell biology, this collaborative competing continuation application offers an interdisciplinary dissection of the nucleoside and nucleobase transporters from Leishmania. Because protozoan parasites are incapable of synthesizing purine nucleotides or nucleobases de novo, purine transporters provide an important, if not obligatory, nutritional function for the parasite and suggest several therapeutic paradigms. In the course of the previously funded investigations, we have cloned and functionally characterized two equilibrative nucleoside transporter (ENT) genes from L. donovani; LdNT1, the gene encoding the adenosine-pyrimidine nucleoside transporter, and LdNT2, the inosine-guanosine transporter gene. We have also created Aldntl and Aldnt2 knockouts by targeted gene replacement and characterized mutationally derived parasites deficient in either LdNT1 or LdNT2 activity. More recently, two additional ENT family members have been identified within the Leishmania major genome project database; LmaNT3, which recognizes purine nucleobases but not nucleosides, and a previously unidentified open reading frame, LmaNT4, which has recently been shown to possess nucleobase transport activity. These molecular and cellular reagents are the cornerstone of the three Specific Aims of this proposal. The first Specific Aim will examine the functional roles performed by these transporters in intact L. major parasites using targeted gene replacement strategies. We will test LmaNT1, LmaNT2, LmaNT3, and LmaNT4 function in L. major promastigotes, metacyclics, and infectious amastigotes by creating and characterizing deltalmant1, deltamant2, deltamant3, and deltalmant4 knockouts in various permutations. In Specific Aim II, we will implement an unbiased genetic screen for Imant2 loss-of-function mutants. This will enable us to identify in a nonintuitive manner key residues within LmaNT2 that are required for either permeation or ligand recognition. The final Specific Aim will be to functionally characterize the novel LmaNT4 nucleobase transporter in Xenopus laevis oocytes. We will determine the ligand specificity and affinities for LmaNT4 and evaluate LmaNT4 expression throughout the L. major life cycle.

结合分子生物学、遗传学、生物化学和细胞生物学的工具，这一合作竞争的延续应用提供了利什曼原虫核苷和核碱基转运体的跨学科解剖。由于原生动物寄生虫不能重新合成嘌呤核苷酸或核碱基，嘌呤转运体为寄生虫提供了重要的（如果不是强制性的）营养功能，并提出了几种治疗范例。在先前资助的研究过程中，我们已经克隆并功能表征了多诺瓦氏乳杆菌的两个平衡核苷转运蛋白（ENT）基因；LdNT1，编码腺苷-嘧啶核苷转运蛋白的基因，以及