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Dissertation
Investigation of the unusual tricarboxylic acid metabolism and mitochondrial electron transport chain functions in Plasmodium falciparum
Doctor of Philosophy (Ph.D.), Drexel University
Apr 2011
DOI:
https://doi.org/10.17918/00009659
Abstract
Tricarboxylic acid (TCA) metabolism in malaria parasites is poorly understood. We knocked out [alpha]-ketoglutarate dehydrogenase (KDH), succinyl-CoA synthetase (SCS), succinate dehydrogenase (SDH) and isocitrate dehydrogenase (IDH) genes individually. KDH/SCS and SCS/SDH were also knocked out simultaneously. Remarkably, none of the knockout lines exhibited growth defects, suggesting these enzymes are nonessential. The KDH/SCS double-knockout line indicated an unusual mode of heme biosynthesis. Fumarate hydratase could not be deleted, suggesting it is essential. We fed the knockout lines 13C-5N-glutamine and examined changes of TCA metabolites by mass-spectrometry. No significant changes in the TCA metabolites measured were found in the [delta]SCS line. [delta]KDH knockout lines contained lower levels of +4-13C-succinate and +4-13C-malate. [delta]SDH knockout lines produced higher succinate and lower +4-13C-malate levels. The [delta]IDH knockout line had lower levels of malate (unlabeled, +3-13C-labeled, +4- 13C-labeled), citrate (+5-13C-labeled), and succinate (unlabeled, +4-13C-labeled). These data suggest such changes in TCA metabolites do not affect parasite survival, indicating a high degree of metabolic redundancy in malaria parasites. Previous work has shown that the most important function of the mtETC (mitochondrial electron transport chain) in P. falciparum (D10 strain) is to provide an electron sink for pyrimidine biosynthesis. To investigate general applicability of this finding, we integrated a single copy the yeast DHODH gene into the genomes of D10, 3D7, Dd2 and HB3. The gene was equally expressed in all strains. All transgenic strains were highly resistant to atovaquone and DSM1 (P. falciparum DHODH inhibitor) in short-term assays. When exposed to atovaquone for longer periods, D10 and 3D7 transgenic parasites were able to survive, while Dd2 and HB3 transgenic parasites died after 3-4 days of exposure. No differences were found in the responses to DSM1. The ubiquinone analog, decylubiquinone, substantially rescued Dd2 and HB3 transgenic parasites during extended growth under atovaquone. These data suggest there are strain specific differences in the requirement for mtETC. It's possible that dehydrogenases in the mtETC other than DHODH are more essential in some strains than in others. Overall, our studies have discovered unique aspects of TCA metabolism and mtETC functions in blood-stage P. falciparum with the potential to inform drug discovery efforts.
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Details
- Title
- Investigation of the unusual tricarboxylic acid metabolism and mitochondrial electron transport chain functions in Plasmodium falciparum
- Creators
- Hangjun Ke
- Contributors
- Akhil B. Vaidya (Advisor) - Drexel University, Drexel University (1970-)
- Awarding Institution
- Drexel University
- Degree Awarded
- Doctor of Philosophy (Ph.D.)
- Publisher
- Drexel University; Philadelphia, Pennsylvania
- Number of pages
- xix, 160 pages
- Resource Type
- Dissertation
- Language
- English
- Academic Unit
- Microbiology and Immunology; College of Medicine; Drexel University
- Other Identifier
- 991021888744804721