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.
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