Nucleoside Transporters of Plasmodium falciparum

描述（由申请人提供）：结合分子生物学，生物化学，遗传学和免疫细胞化学的工具，本提案提供了恶性疟原虫核苷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，因此核苷转运体为寄生虫提供了重要的营养功能，并提供了几种治疗范例。在现有的恶性疟原虫数据库中已鉴定出两个核苷转运基因PfNT1和PfNT2，并在该实验室对两者进行了克隆和测序。在将PfNT1 cRNA注射到非洲爪蟾卵母细胞后，PfNT1活性已被初步表征，PfNT1也在核苷转运缺陷的多诺瓦利什曼原虫中功能性过表达。此外，在家兔中培养了PfNT1特异性的多克隆抗血清，并通过共聚焦和免疫电镜将PfNT1定位到寄生虫的质膜上。针对PfNT2的抗体也已产生。这些试剂是本提案中三个具体目标的基石。多组分特异性目标I将包括：I . PfNT1在配体特异性、对哺乳动物核苷转运抑制剂的亲和力和敏感性方面的全面生化表征；2。评估PfNT2是否是功能性核苷转运体，如果是，则进行初步的分子和生化表征，包括该蛋白在恶性疟原虫感染的红细胞中的免疫定位；ⅲ。，利用爪蟾卵母细胞cRNA表达系统验证PfNT1和PfNT2是否为电致转运体。第二个特定目标启动PfNTI的结构-功能分析。我们建议对功能丧失突变体进行遗传筛选，以无偏方式鉴定PfNT1中配体渗透和/或配体选择性所需的关键氨基酸。最后一篇文章将通过转染和基因靶向的方法探讨PfNT1在被寄生红细胞中的生理功能。具体来说，我们将尝试在野生型或基因互补型恶性疟原虫中创建Apfntl敲除，以测试PfNT1功能是否对完整的寄生虫至关重要。然后，我们将描述由此产生的运输和生长表型。

DESCRIPTION (provided by the applicant): Amalgamating tools of molecular biology, biochemistry, genetics, and immunocytochemistry, this proposal offers an interdisciplinary dissection of the nucleoside transporters of Plasmodium falciparum. As protozoan parasites are incapable of synthesizing purine nucleotides de novo, nucleoside transporters provide an important, if not obligatory, nutritional function for the parasite and present several therapeutic paradigms. Two nucleoside transporter genes, PfNT1 and PfNT2, have been identified within available P. falciparum databases, and both have been cloned and sequenced in this laboratory. PfNT1 activity has been characterized in a preliminary fashion after PfNT1 cRNA injection into Xenopus laevis oocytes, and PfNT1has also been functionally over-expressed in nucleoside transport-deficient Leishmania donovani. In addition polyclonal antisera specific for PfNT1 have been raised in rabbits and used to localize PfNT1 to the parasite plasma membrane by confocal and immuno-electron microscopy. Antibodies against PfNT2 have also been generated. These reagents are the cornerstones of the three specific aims in this proposal. The multi-component Specific Aim I will encompass: i., a thorough biochemical characterization of PfNT1 with respect to ligand specificity and affinities and sensitivities to inhibitors of mammalian nucleoside transport; ii., an assessment of whether PfNT2 is a functional nucleoside transporter, and if so, a preliminary molecular and biochemical characterization, including immuno-locatization of the protein in P. falciparum-infected erythrocytes; and iii., a verification of whether PfNT1 and PfNT2 are electrogenic transporters using the Xenopus oocyte cRNA expression system. The second Specific Aim initiates a structure-function analysis of PfNTI. We propose to implement a genetic screen for loss-of-function mutants to identify in an unbiased fashion key amino acids in PfNT1 that are required for ligand permeation and/or ligand selectivity. The last Specific Aim will explore the physiological function of PfNT1 within the parasitized erythrocyte using transfection and gene targeting approaches. Specifically, we will attempt to create Apfntl knockouts in either wild type or genetically complemented P. falciparum in order to test whether PfNT1 function is essential to the intact parasite. We will then characterize the resultant transport and growth phenotypes.