Functional Characterization of Leishmania Transporters

描述（由申请人提供）：本提案的目的是研究墨西哥利什曼原虫中葡萄糖转运蛋白的生物学功能。利什曼原虫在全世界造成约1200万人患病，是一个重大的公共卫生问题。鉴定为寄生虫提供基本功能的基因和蛋白质对于确定开发新药的靶点至关重要。先前的研究已经证明，L. mexicana的葡萄糖转运体，介导葡萄糖和相关己糖摄取的整体膜蛋白，在感染脊椎动物并导致疾病的生命周期的无尾线虫阶段是必不可少的。因此，本研究将重点关注寄生虫葡萄糖转运体的生化功能，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。第一个目标是检查细胞中葡萄糖的三种主要命运，并确定当葡萄糖转运蛋白被靶向基因删除时，它们中的哪一种受到损害。葡萄糖的这些命运包括：1)结合到糖缀合物和复杂的碳水化合物中，2)通过戊糖磷酸途径代谢产生NADPH和磷酸核糖，3)通过糖酵解代谢。这些实验将确定这三种途径中的哪一种会受到葡萄糖转运蛋白零突变体无法输入己糖的影响，以及这些缺陷中的哪一种可能导致该零突变体作为无纺丝体无法生存。第二个目的是研究3种葡萄糖转运蛋白异构体GT1、GT2和GT3的具体功能。将制备这些基因的零突变体，并在生命周期的promastigote和amastigote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种葡萄糖转运蛋白中的每一种的不同功能。总之，这一提议将提供葡萄糖转运蛋白功能的详细生化和遗传学解剖，以及这些渗透物在寄生虫生命周期中的重要作用。

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.