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

克氏锥虫生物学和发病机制中的核苷-核碱基转运蛋白

基本信息

批准号：
8897847
负责人：
BUDDY ULLMAN
金额：
$ 19.25万
依托单位：
OREGON HEALTH & SCIENCE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2015
资助国家：
美国
起止时间：
2015-01-01 至 2016-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8897847
关键词：
Address Affinity Back Biology Cardiac Myocytes Cardiovascular system Cell Survival Cell membrane Cells Central America Chagas Disease Communities Cosmetics Coupling Development Disease Drug Targeting Environment Exhibits Family Family member Feedback Foundations Future Gene Targeting Genetic Goals Growth Health Immigration In Vitro Infection Instruction Investigation Knock-out Knowledge Lead Life Cycle Stages Ligands Location Mediating Metabolic Metabolic Pathway Metabolism Mexico Modeling Mus Nucleoside Transporter Nucleosides Nutrient Nutritional Nutritional Requirements Outcome Parasites Parasitic Diseases Pathogenesis Pathway interactions Pharmaceutical Preparations Phenotype Plague Play Positioning Attribute Process Proliferating Public Health Purine Nucleosides Purines Pyrimidine Pyrimidine Nucleosides Pyrimidines RNA Interference Research Research Project Grants Role South America Specific qualifier value Specificity System Testing Text Therapeutic Trypanosoma cruzi United States Validation Virulence chemotherapy drug development drug discovery enzyme pathway gastrointestinal system gene replacement genome-wide member mutant novel nucleobase permease prospective public health relevance purine purine/pyrimidine metabolism research study response therapeutic target trait uptake

项目摘要

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.

描述（由申请人提供）：查加斯病是一种破坏性的心血管和消化系统疾病，由原生动物寄生虫克氏锥虫引起，该疾病是墨西哥、中美洲和南美洲的一个主要公共卫生问题，约有 300,000 人。据估计，美国的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，尚无可靠有效的治疗恰加斯病的化疗方法，因此需要验证新药。靶向并发现新药物，特别是那些针对独特寄生虫特征的药物，势在必行。[该应用的总体目标是通过功能表征和治疗验证来检验寄生虫和宿主代谢密切相关的假设。 4] 平衡核苷转运蛋白 (NT) 家族成员 TcNT1、TcNT2、TcNT3 和 TcNT4，启动嘌呤和嘧啶的易位最近，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克氏锥虫细胞内复制的关键决定因素，这是一个至关重要的过程。此外，已知克氏锥虫的嘌呤清除是一种不可或缺的营养功能，因为与哺乳动物宿主相比，该寄生虫是嘌呤营养缺陷，因此必须强制清除宿主嘌呤才能生存和增殖。我将重点关注两个具体目标。我们将确定 TcNT1、TcNT2、TcNT3 和 TcNT4 的配体特异性和亲和力，评估这四种 NT 中的每一种是否在哺乳动物形式的寄生虫中表达，并验证其完整的亚细胞位置。 Specific Aim II 提供了四种 NT 作为潜在治疗靶点的遗传验证，我们将创建 TcNT1、TcNT2 缺陷的无效突变体。通过靶向基因替换的 TcNT3 和 TcNT4 及其相应的“回加”，评估无效系的营养需求和运输表型，测试无效突变体在体外感染心肌细胞的能力，并确定 T 是否cruzi 基因敲除在 Balb/c 小鼠中表现出毒力表型受损。这些实验的完成将验证细胞内 T. cruzi 和 T. cruzi 的嘌呤和嘧啶途径的假设。哺乳动物宿主是相互关联的，并验证这些途径不仅在寄生虫中，而且在哺乳动物宿主中作为潜在的药物靶标，这些信息将为未来治疗心血管和胃肠系统这种破坏性感染的药物发现工作提供信息。