Nucleoside/Nucleobase Transporters in Leishmania major

融合了分子生物学、遗传学、生物化学和细胞生物学的工具，这种协作竞争的延续应用程序提供了对利什曼原虫核苷和核碱基转运体的跨学科剖析。由于原生动物寄生虫不能合成嘌呤核苷酸或碱基，嘌呤转运蛋白为寄生虫提供了重要的营养功能，并提出了几种治疗方案。在之前资助的研究过程中，我们克隆了杜诺氏乳杆菌的两个平衡核苷转运蛋白(ENT)基因，编码腺苷-嘧啶核苷转运蛋白的基因LdNT1，并对其进行了功能鉴定。 LdNT2，肌苷-鸟苷转运蛋白基因。我们还通过有针对性的基因替换创建了Aldnt1和Aldnt2基因敲除，并鉴定了突变衍生的寄生虫缺乏LdNT1或LdNT2活性。最近，在利什曼原虫主要基因组计划数据库中又发现了两个ENT家族成员：LmaNT3，它识别嘌呤核苷酸碱基，但不识别核苷；以及以前未确定的开放阅读框架LmaNT4，它最近被证明具有核苷酸碱基运输活性。这些分子和细胞试剂是这项提案的三个具体目标的基石。第一个特定的目的是利用有针对性的基因替换策略来研究这些转运蛋白在完整的主要寄生虫中所发挥的功能作用。我们将测试 LmaNT1、LmaNT2、LmaNT3和LmaNT4通过在不同的排列中创建和表征deltalmant1、delTamant2、delTamant3和deltalmant4基因敲除而在主要乳杆菌前鞭毛体、后循环和感染性无鞭毛体中发挥作用。在特定目标II中，我们将对Imant2功能缺失突变体实施无偏见的遗传筛选。这将使我们能够以非直观的方式识别LmaNT2中渗透或配体识别所需的关键残基。最终的具体目标将是在非洲爪哇卵母细胞中鉴定新的LmaNT4碱基转运蛋白的功能。我们将确定LmaNT4的配基特异性和亲和力，并评估LmaNT4在主要生活周期中的表达。

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.