Functional Characterization of Leishmania Transporters

描述(申请人提供)：本提案的目的是研究墨西哥利什曼原虫葡萄糖转运蛋白的生物学功能。据估计，全世界有1200万人感染利什曼原虫，这是一个重大的公共卫生问题。识别为寄生虫提供基本功能的基因和蛋白质对于识别新药开发的靶标至关重要。前人的工作表明，墨西哥乳杆菌的葡萄糖转运蛋白是介导葡萄糖和相关己糖吸收的完整膜蛋白，在感染脊椎动物并导致疾病的生活史的无鞭毛体阶段是必不可少的。因此，这项建议将侧重于寄生虫葡萄糖转运蛋白的生化功能，并试图解释为什么它们在寄生虫生命周期的致病阶段是必不可少的。第一个目标是研究葡萄糖在细胞中的三种主要命运，并确定当葡萄糖转运蛋白被靶基因缺失从基因上去除时，它们中的哪一种受到损害。这些葡萄糖的命运包括：i)与糖共轭和复合碳水化合物结合；ii)通过磷酸戊糖途径代谢生成NADPH和磷酸核糖；以及iii)通过糖酵解代谢。这些实验将确定这3条途径中的哪一条受到葡萄糖转运蛋白缺失突变体不能输入己糖的影响，以及这些缺失中的哪一条可能导致该缺失突变体作为无鞭毛体而丧失生存能力。第二个目的是研究3种葡萄糖转运蛋白亚型GT1、GT2和GT3的特殊功能。将准备这些基因中每一个的零突变体，并在生命周期的前鞭毛体和无鞭毛体阶段评估它们的表型。这些实验将有助于阐明这种寄生虫表达的3种葡萄糖转运蛋白的不同功能。总体而言，这项提案将对葡萄糖转运蛋白的功能和这些通透性在寄生虫生命周期中的基本作用提供详细的生化和遗传剖析。

DESCRIPTION (provided by applicant): The objective of this proposal is to study the biological function of glucose transporters in the parasite Leishmania mexicana. Leishmania species cause disease in an estimated 12 million individuals worldwide and are a major public health problem. Identification of genes and proteins that serve essential functions for the parasite is critical for the identification of targets for development of novel drugs. Previous work has demonstrated that the glucose transporters of L. mexicana, integral membrane proteins that mediate the uptake of glucose and related hexose sugars, are essential in the amastigote stage of the life cycle that infects vertebrates and causes disease. Hence, this proposal will focus upon the biochemical functions of parasite glucose transporters and attempt to explain why they are essential for the disease causing stage of the parasite life cycle. The first aim will examine each of the 3 principal fates of glucose in the cell and determine which of them is/are impaired by when glucose transporters are genetically ablated by targeted gene deletion. These fates of glucose include: i) incorporation into glycoconjugates and complex carbohydrates, ii) metabolism via the pentose phosphate pathway to generate NADPH and ribose phosphate, and iii) metabolism via glycolysis. These experiments will establish which of these 3 pathways are affected by the inability of a glucose transporter null mutant to import hexoses and which of these deficiencies may contribute to the non-viability of this null mutant as amastigotes. The second aim will investigate the specific functions of the 3 glucose transporter isoforms GT1, GT2, and GT3. Null mutants in each of these genes will be prepared and their phenotypes assessed in both the promastigote and amastigote stages of the life cycle. These experiments will help elucidate distinct functions for each of the 3 glucose transporters expressed by this parasite. Overall, this proposal will provide a detailed biochemical and genetic dissection of glucose transporter function and the essential role of these permeases in the parasite life cycle.