Mitochondrial Function in Bloodstream Trypanosoma brucei

描述(申请人提供)：锥虫是原生动物病原体，可引起人和牲畜的疾病。由于毒性和耐药性的发展，大多数药物越来越无效。线粒体基因的表达和功能对寄生虫的生存至关重要--尽管细长血流(LS)形式中没有氧化磷酸化--并可能成为新药的强大靶点。然而，LS阶段的线粒体生物学知之甚少，细胞器的基本功能也不清楚。该项目将检验一种假设，即呼吸复合体I(NADH：泛醌氧化还原酶)和/或V(三磷酸腺苷合成酶)是LS寄生虫生存所必需的，并且这些复合体中的一个或两个的基本成分是线粒体表达的。这一假说是基于这些复合体以LS形式活跃的证据，以及对编码这些复合体假定亚单位的线粒体基因的电子鉴定。(1)通过沉默必需的核编码亚基的表达，将使复合体I和V失活，并将检测对细胞生长和对线粒体功能参数的影响，如ATP水平、呼吸作用、膜电位和蛋白质输入。(2)通过确定线粒体基因产物在体内的相互作用伙伴，验证线粒体基因编码呼吸复合体I和V亚基的假设。蛋白质的纯化和鉴定将通过亲和标记和质谱分析来实现。(3)对(1)中确定的RNA编辑和活性的要求将在运动发育障碍(DK)锥体中确定，这些锥体已经失去了线粒体DNA，因此可能表明潜在的耐药途径。(4)通过克隆和测序关键的核编码亚基，将确定DK锥体潜在的补偿性突变。这些研究将为锥虫线粒体功能提供新的见解，验证新的药物靶点，并评估针对线粒体功能的药物耐药性的可能性。相关：锥虫是单细胞寄生虫，是非洲昏睡病和利什曼病等具有破坏性健康和经济后果的疾病的病原体。这个项目将描述线粒体--一种细胞器--在寄生虫致病阶段的作用。这项研究将有助于确定新的药物靶点，并了解现有药物的作用。

DESCRIPTION (provided by applicant): Trypanosomatids are protozoan pathogens that cause disease in man and livestock. Most drugs are increasingly ineffective due to toxicity and development of resistance. Mitochondrial gene expression and function are essential for survival of the parasite - despite the absence of oxidative phosphorylation in the long slender bloodstream (LS) form - and may represent powerful targets for new drugs. However, mitochondrial biology in the LS stage is poorly understood and the essential function of the organelle for the parasite is not known. This project will test the hypothesis that respiratory complexes I (NADH:ubiquinone oxidoreductase) and/or V (ATP synthase) are required for viability of LS parasites and that essential components of one or both of these complexes are mitochondrially expressed. This hypothesis is based on evidence that these complexes are active in the LS form and on the in silico identification of mitochondrial genes encoding putative subunits of these complexes. (1) Complexes I and V will be inactivated by silencing the expression of essential nuclearly encoded subunits and the effects on cell growth and on parameters of mitochondrial function such as ATP levels, respiration, membrane potential, and protein import will be examined. (2) The hypothesis will be tested that mitochondrial genes encode subunits of respiratory complexes I and V by identifying the interaction partners of the gene products in vivo. Protein purification and identification will be achieved through affinity tagging and mass spectrometry. (3) The requirement for RNA editing and activities identified in (1) will be determined in dyskinetoplastic (dk) trypanosomes, which have lost their mitochondrial DNA and may thus indicate potential routes for drug resistance. (4) Potential compensatory mutations in dk trypanosomes will be identified by cloning and sequencing of key nuclearly encoded subunits. These studies will provide novel insights into mitochondrial function in trypanosomatids, validate new drug targets, and assess the potential for resistance to drugs targeting mitochondrial function. Relevance: Trypanosomatids are unicellular parasites and the causative agents of diseases with devastating health and economic consequences, such as African sleeping sickness and Leishmaniasis. This project will characterize the role of the mitochondrion - a cell organelle - in the disease-causing stage of the parasite. This research will help to identify new drug targets and to understand the action of existing drugs.