Zinc Trafficking Machinery in Malaria Infection

项目概述：所有活细胞都必须吸收一定数量的金属离子，而包括锌在内的许多必需金属的必要浓度在不同物种之间是相对保守的。然而，在被疟原虫恶性疟原虫寄生的人红细胞中，锌被发现在局部区域积聚，其浓度高达正常红细胞的20倍。有趣的是，其他必需金属（如铜和铁）的细胞内水平并没有因寄生虫的存在而显著改变。在本提案中，我们概述了一种研究疟疾寄生虫能够显著改变宿主细胞锌生理的机制的方法。我们相信，参与锌清除和囤积的蛋白质可以作为控制这种致命寄生虫的潜在药物靶点。该项目将提供关于这种传染病的基本知识，并允许开发新的治疗方法，这将有助于克服目前阻碍根除这种疾病的耐药性。我们的目的是研究候选锌进口和转运蛋白的金属生物化学，以深入了解细胞内疟原虫如何操纵锌配额以在宿主体内感染和增殖。具体来说，我们的目标是(1)通过研究恶性疟原虫在锌胁迫条件下的转录谱来鉴定候选蛋白，(2)研究这些蛋白和已知的转运蛋白同源物在亚细胞锌定位中的作用，(3)研究这些蛋白本身的亚细胞定位以及它们在体外结合和运输锌的能力。用于实现这些目标的方法将包括mRNA的DMA微阵列分析，使用x射线荧光和荧光锌传感器测定变异菌株的总锌和局部锌含量，以及通过免疫荧光法检测蛋白质位置。相关性：据估计，每年将有近10%的世界人口患有临床疟疾，每年造成150万至270万人死亡。将感染疟疾寄生虫的红细胞与健康红细胞区分开来的一个特征是锌含量升高。因此，我们相信，研究锌在疟疾扩散中的作用将有助于更好地了解这种疾病，并提出新的抗疟疾治疗方法。

DESCRIPTION (provided by applicant): Project Summary: All living cells must recruit metal ions to meet a certain quota, and the necessary concentration of many essential metals, including zinc, is relatively conserved among divergent species. However, in human erythrocytes parasitemized by the malaria parasite Plasmodium falciparum, zinc is found to accumulate in localized areas to a concentration of up to 20 times that of a normal erythrocyte. Interestingly, the intracellular levels of other essential metals such as copper and iron are not significantly altered by the presence of the parasite. In this proposal, we outline an approach to studying the mechanism by which the malaria parasite is able to dramatically alter the host cell zinc physiology. We believe that the proteins involved in zinc scavenging and hoarding serve as potential pharmaceutical targets in the effort to control this deadly parasite. This project will provide fundamental knowledge about this infectious disease and allow for the development of new treatments which will help to circumvent the drug resistances that currently hamper eradication of the disease. We aim to study the metallobiochemistry of candidate zinc import and transport proteins to develop an in- depth understanding of how the intracellular malaria parasite can manipulate the zinc quota in order to infect and proliferate in the host. Specifically, we aim to (1) identify candidate proteins by studying P. falciparum transcription profiles under zinc stress conditions, (2) examine the roles of these and known transporter homologs in subcellularzinc localization, and (3) examine the subcellular localization of the proteins themselves as well as their ability to bind and transport zinc in vitro. The methods used to accomplish these aims will include DMA microarray analysis of mRNA, determination of total and local zinc content in variant strains using X-ray fluorescence and fluorescent zinc sensors, and detection of protein location via immunofluorescent assays. Relevance: It is estimated that nearly 10% of the world's population will suffer from clinical malaria each year, resulting in between 1.5 and 2.7 million deaths annually. One characteristic that sets red blood cells infected with the malaria parasite apart from healthy red blood cells is the presence of elevated levels of zinc. Therefore we believe that studying the role of zinc in malaria proliferation will lead to a better understanding of this disease as well as suggesting new types of anti-malarial therapies.