Nucleoside Transporters of Plasmodium falciparum

描述(由申请人提供)：融合了分子生物学、生物化学、遗传学和免疫细胞化学的工具，本提案对恶性疟原虫的核苷转运体进行了跨学科的剖析。由于原生动物寄生虫不能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(如果不是必需的)营养功能，并提供了几种治疗方案。两个核苷转运蛋白基因PfNT1和PfNT2已在现有的恶性疟原虫数据库中被鉴定，并在本实验室进行了克隆和测序。在将PfNT1cRNA注入非洲爪哇卵母细胞后，PfNT1的活性已初步确定，PfNT1在核苷转运缺陷的杜氏利什曼原虫中也有功能过表达。此外，还制备了针对PfNT1的兔多克隆抗血清，并用共聚焦显微镜和免疫电子显微镜将PfNT1定位于寄生虫的质膜。还产生了针对PfNT2的抗体。这些试剂是这项提案中三个具体目标的基石。多组分的特异性目标一将包括：i.关于PfNT1的配基特异性和对哺乳动物核苷转运抑制剂的亲和力和敏感性的全面生化表征；ii.评估PfNT2是否是一个功能性的核苷转运体，如果是，初步的分子和生化特征，包括该蛋白在感染恶性疟原虫的红细胞中的免疫定位；以及iii.利用非洲爪哇卵母细胞cRNA表达系统验证PfNT1和PfNT2是否是电性转运体。第二个具体目标是启动PfNTI的结构-功能分析。我们建议对功能缺失突变体进行遗传筛选，以无偏见的方式识别PfNT1中配体渗透和/或配体选择性所需的关键氨基酸。最后一个具体目标是通过转基因和基因打靶的方法探索PfNT1在寄生虫红细胞中的生理功能。具体地说，我们将尝试在野生型或遗传互补的恶性疟原虫中创建Apfnt1基因敲除，以测试PfNT1功能是否对完整的寄生虫是必要的。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.