Nucleoside Transporters of Plasmodium falciparum

描述（由申请人提供）：融合分子生物学、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蛋白的跨学科解剖。由于原生动物寄生虫无法从头合成嘌呤核苷酸，核苷转运蛋白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式。两个核苷转运蛋白基因，PfNT 1和PfNT 2，已被确定在恶性疟原虫数据库中，并已在本实验室克隆和测序。PfNT 1活性的特点是在一个初步的方式后，PfNT 1 cRNA注射到非洲爪蟾卵母细胞，PfNT 1也已在功能上过表达的核苷转运缺陷型杜氏利什曼原虫。此外，已在兔中提出了特异性针对PfNT 1的多克隆抗血清，并通过共聚焦和免疫电子显微镜将PfNT 1定位于寄生虫质膜。还产生了针对PfNT 2的抗体。这些试剂是本提案中三个具体目标的基石。多组成部分的具体目标一将包括：PfNT 1在配体特异性和对哺乳动物核苷转运抑制剂的亲和力和敏感性方面的彻底生物化学表征; ii.，评估PfNT 2是否是功能性核苷转运蛋白，如果是，初步的分子和生物化学表征，包括在恶性疟原虫感染的红细胞中蛋白质的免疫定位;和iii.，使用非洲爪蟾卵母细胞cRNA表达系统验证PfNT 1和PfNT 2是否是产电转运蛋白。第二个特异性目的启动PfNTI的结构-功能分析。我们建议实施一个遗传筛选功能丧失突变体，以确定在一个公正的方式关键氨基酸PfNT 1所需的配体渗透和/或配体的选择性。最后一个具体目标将探讨PfNT 1的生理功能，在寄生红细胞内使用转染和基因打靶方法。具体地，我们将尝试在野生型或遗传互补的恶性疟原虫中产生ApfNTl敲除，以测试PfNTl功能是否对完整的寄生虫是必需的。然后，我们将表征所得的运输和生长表型。

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.