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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI069057-05
Application #
8064395
Study Section
Pathogenic Eukaryotes Study Section (PTHE)
Program Officer
Rogers, Martin J
Project Start
2007-05-01
Project End
2012-12-30
Budget Start
2011-05-01
Budget End
2012-12-30
Support Year
5
Fiscal Year
2011
Total Cost
$367,285
Indirect Cost
Name
Seattle Biomedical Research Institute
Department
Type
DUNS #
070967955
City
Seattle
State
WA
Country
United States
Zip Code
98109
Surve, Sachin V; Jensen, Bryan C; Heestand, Meredith et al. (2017) NADH dehydrogenase of Trypanosoma brucei is important for efficient acetate production in bloodstream forms. Mol Biochem Parasitol 211:57-61
Carnes, Jason; Anupama, Atashi; Balmer, Oliver et al. (2015) Genome and phylogenetic analyses of Trypanosoma evansi reveal extensive similarity to T. brucei and multiple independent origins for dyskinetoplasty. PLoS Negl Trop Dis 9:e3404
Dean, Samuel; Gould, Matthew K; Dewar, Caroline E et al. (2013) Single point mutations in ATP synthase compensate for mitochondrial genome loss in trypanosomes. Proc Natl Acad Sci U S A 110:14741-6
Surve, Sachin; Heestand, Meredith; Panicucci, Brian et al. (2012) Enigmatic presence of mitochondrial complex I in Trypanosoma brucei bloodstream forms. Eukaryot Cell 11:183-93
Schnaufer, Achim (2010) Evolution of dyskinetoplastic trypanosomes: how, and how often? Trends Parasitol 26:557-8
Zikova, Alena; Schnaufer, Achim; Dalley, Rachel A et al. (2009) The F(0)F(1)-ATP synthase complex contains novel subunits and is essential for procyclic Trypanosoma brucei. PLoS Pathog 5:e1000436
Amaro, Rommie E; Schnaufer, Achim; Interthal, Heidrun et al. (2008) Discovery of drug-like inhibitors of an essential RNA-editing ligase in Trypanosoma brucei. Proc Natl Acad Sci U S A 105:17278-83