Characterization of ABC transporters in parasitic nematodes

ABC转运蛋白广泛存在于所有生物体中，是最大的蛋白质家族之一。生活在不同环境中并必须适应不同外部条件的生物拥有大量的ABC转运蛋白(例如，秀丽线虫染色体编码60个ABC转运蛋白)，而占据较有限生长生态位的生物拥有相当少的ABC转运蛋白(例如，布氏锥虫染色体编码21个ABC转运蛋白)。ABC转运蛋白在许多细胞过程中是不可或缺的，但对寄生线虫中的这些蛋白知之甚少。在农场动物和人类中，线虫会导致疾病、死亡和经济损失。对它们的生存至关重要的是运输机制，其中包括ABC转运蛋白。目前用于治疗线虫的所有驱虫剂都以转运蛋白为目标。在寄生线虫中已鉴定出一些ABC转运蛋白基因序列，但目前尚无功能研究。这项研究的长期目标是了解ABC转运蛋白介导的线虫耐药性。在此之前，必须扩展寄生线虫中ABC转运蛋白的基本知识。为了做到这一点，人类淋巴系统中的一种线虫--马来丝虫将被用作模式生物。本研究的具体目标是：(1)利用生物信息学和基因组分析对布鲁吉亚ABC转运蛋白超家族进行鉴定和分类；(2)通过将寄生基因导入线虫来建立布鲁贾转运蛋白的空间蛋白质组；(3)利用定量RT-PCR技术确定布鲁贾转运蛋白在所有性周期和生命周期阶段的表达模式；以及(4)利用定点突变和结合分析来确定布鲁贾转运蛋白的底物特异性和转运动力学。这项研究对扩展寄生线虫ABC转运蛋白的基本知识和深入了解其细胞功能具有重要意义。由于许多ABC转运蛋白是驱虫药的靶标，本研究将提出新的或替代的控制策略，并扩大现有的对驱虫药抗性机制的知识。

ABC transporters have been found in all living organisms and form one of the largest characterized protein families. Organisms that live in diverse environments and that must adapt to various external conditions have a large repertoire of ABC transporters (e.g., Caenorhabditis elegans chromosomes encode 60 ABC transporters) while organisms that occupy a more restricted growth niche have considerably less (e.g., Trypanosoma brucei chromosomes encode 21 ABC transporters). ABC transporters are integral to many cellular processes, yet little is known of these proteins in parasitic nematodes. In farm animals and humans, nematodes cause disease, death and economic loss. Essential to their survival are transport mechanisms, which include ABC transporters. All anthelmintics currently used to treat nematodes target transporters. Some ABC transporter gene sequences have been identified in parasitic nematodes however, functional studies do not exist. The long-term goal of the research is to understand ABC transporter mediated drug resistance in nematodes. Before this can be accomplished, the basic knowledge of ABC transporters in parasitic nematodes must be expanded. To do this Brugia malayi, a nematode that resides in the lymphatic system of humans, will be used as the model organism. The specific goals of the research are to (1) use bioinformatic and genomic analyses to identify and classify the Brugia ABC transporter superfamily; (2) create a spatial proteome of Brugia transporters by transfection of C. elegans with parasite genes; (3) determine the expression patterns in all sexual and life cycle stages using quantitative RT-PCR; and (4) determine the substrate specificity and transport kinetics of Brugia transporters using site-directed mutagenesis and binding assays. The research has implications for expanding the basic knowledge of ABC transporters in parasitic nematodes and providing insight into their cellular functions. As many ABC transporters are targets for anthelmintics, this research will suggest new or alternative control strategies and also expand the existing knowledge of the mechanism of resistance to anthelmintics.