Nucleoside-Nucleobase Transporters in the Biology and Pathogenesis of T. cruzi

 描述（由申请人提供）：恰加斯病是一种由原生动物寄生虫克氏锥虫引起的心血管和消化系统的毁灭性疾病。这种疾病是墨西哥、中美洲和南美洲的一个主要公共卫生问题，据估计，美国约有30万人感染了这种寄生虫，这一数字随着移民的增加而增加。目前，对恰加斯病没有依赖性有效的化疗，并且需要验证新的药物靶点并发现新的药物，特别是那些针对独特寄生虫特征的药物，这是必要的。我们的应用程序解决了这一紧急情况。[The本申请的总体目的是通过功能表征和治疗验证平衡核苷转运蛋白（NT）家族的四个成员TcNT 1、TcNT 2、TcNT 3和TcNT 4来检验寄生虫和宿主代谢密切交织的假设，所述四个成员启动嘌呤和嘧啶核苷/核碱基从宿主易位到寄生虫中。最近，针对哺乳动物宿主细胞中基因的全基因组RNA干扰筛选证实了宿主对嘌呤和嘧啶的摄取是T. cruzi，这是疾病发病机理的基础过程。此外，T.已知Cruzi是不可缺少的营养功能，因为与哺乳动物宿主相反，寄生虫是嘌呤营养缺陷型的，因此必须强制性地依赖宿主嘌呤以存活和增殖。有两个具体目标。具体目标我会集中 关于四种NT的功能特征。我们将确定TcNT 1，TcNT 2，TcNT 3和TcNT 4的配体特异性和亲和力，评估四种NT中的每一种是否在哺乳动物形式的寄生虫中表达，并验证它们在完整寄生虫中的亚细胞位置。特异性目的II提供了四个NT作为潜在治疗靶点的遗传验证。我们将通过靶向基因替换以及相应的“添加”来创建TcNT 1、TcNT 2、TcNT 3和TcNT 4缺陷的无效突变体，评估无效系的营养需求和转运表型，测试无效突变体体外感染心肌母细胞的能力，并确定T. Cruzi敲除在Balb/c小鼠中表现出受损的毒力表型。这些实验的完成将验证细胞内T细胞的嘌呤和嘧啶途径可能与细胞内T。cruzi和哺乳动物宿主是相互关联的，并且不仅在寄生虫中，而且在哺乳动物宿主中验证这些途径作为预期的药物靶标。这些信息将为未来的药物发现工作提供信息，以治疗这种破坏性的心血管和胃肠道系统感染。

 DESCRIPTION (provided by applicant): Chagas disease is a devastating disease of the cardiovascular and digestive systems that is caused by the protozoan parasite Trypanosoma cruzi. The disease is a major public health problem in Mexico, Central America, and South America, and ~300,000 people in the United States are estimated to be infected with the parasite, a figure that is increasing with immigration. Presently, there are no dependably effective chemotherapies for Chagas disease, and the need to validate novel drug targets and discover new drugs, particularly those that target unique parasite traits, is imperative. Our application addresses this exigency. [The overall objective of this application is to test the hypothesis that parasite and host metabolism are intimately intertwined by functionally characterizing and therapeutically validating the four] members of the equilibrative nucleoside transporter (NT) family, TcNT1, TcNT2, TcNT3, and TcNT4, that initiate the translocation of purine and pyrimidine nucleosides/nucleobases from the host into the parasite. Recently, a genome-wide RNA interference screen targeting genes in mammalian host cells authenticated purine and pyrimidine uptake from the host as critical determinants to the intracellular replicatio of T. cruzi, a process that is fundamental to disease pathogenesis. Moreover, purine scavenge by T. cruzi is known to be an indispensable nutritional function since, in contrast to the mammalian host, the parasite is auxotrophic for purines and thus, must obligatorily scavenge host purines for survival and proliferation. There are two Specific Aims. Specific Aim I will focus on the functional characterization of the four NTs. We will determine the ligand specificities and affinities for TcNT1, TcNT2, TcNT3, and TcNT4, evaluate whether each of the four NTs is expressed in mammalian forms of the parasite, and verify their subcellular locations in intact parasites. Specific Aim II affords a genetic validation of the four NTs as potential therapeutic targets. We will create null mutants deficient in TcNT1, TcNT2, TcNT3, and TcNT4 by targeted gene replacement, as well as their corresponding "add-backs," assess the nutritional requirements and transport phenotypes of the null lines, test the capacity of the null mutants to infect cardiomyoblasts in vitro, and determine whether the T. cruzi knockouts exhibit a compromised virulence phenotype in Balb/c mice. Accomplishment of these experiments will verify the hypothesis that the purine and pyrimidine pathways of intracellular T. cruzi and the mammalian host are interconnected and validate these pathways, not only in the parasite but, as well, in the mammalian host, as prospective drug targets. This information will inform future drug discovery efforts to treat this devastating infection of the cardiovascular and gastrointestinal systems.