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（由适用提供）：Chagas疾病是由原生动物寄生虫锥虫引起的心血管和消化系统的毁灭性疾病。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，据估计，在美国，约有30万人感染了寄生虫，这一数字随着移民而增加。目前，尚无针对Chagas疾病的可靠化学疗法，并且需要验证新型药物靶标并发现新药，尤其是那些针对独特的寄生虫特征的药物。我们的应用程序解决了这一外科基础。 [该应用的总体目的是检验以下假说：寄生虫和宿主代谢是通过功能表征和治疗验证这四个的表征和治疗验证的两个]等效核外侧转运蛋白（NT）家族的成员，TCNT1，TCNT2，TCNT2，TCNT3，TCNT3，TCNT3和TCNT4启动了纯粹的核心和核心核心的核定核心/核定的核心。最近，哺乳动物宿主细胞中的全基因组RNA干扰筛查靶向基因从宿主身份验证的嘌呤和嘧啶的摄取作为关键的决定剂对T. cruzi的细胞内复制品，这是疾病病原体基础的过程。此外，众所周知，克鲁兹（T. cruzi）的嘌呤清除是一种必不可少的营养功能，因为与哺乳动物宿主相反，寄生虫是购买的可达寄生虫，因此，必须为生存和增殖而必须进行强制性的清除宿主购买。有两个具体的目标。我将集中精力的具体目标关于四个NT的功能表征。我们将确定TCNT1，TCNT2和TCNT3和TCNT4的配体规范和亲和力，评估四个NTs中的每一个是否以寄生虫的哺乳动物形式表达，并验证其在完整寄生虫中的亚细胞位置。特定的目标II提供了四个NT作为潜在治疗靶标的遗传验证。我们将通过靶向基因更换及其相应的“添加后卫”来创建缺乏TCNT1，TCNT2，TCNT3和TCNT4缺陷的无效突变体，评估了无效线的营养需求和运输表型，测试了零突变体对感染的cardie虫的表现。 BALB/C小鼠。这些实验的完成将验证以下假设：克鲁齐细胞内t.和哺乳动物宿主的纯和嘧啶途径相互联系并验证这些途径，不仅在寄生虫中，而且在哺乳动物宿主中也是前瞻性药物靶标。这些信息将为未来的药物发现努力提供治疗心血管和胃肠道系统的毁灭性感染的努力。