Dissertation
The role of isocitrate dehydrogenase and assessment of intracellular ATP levels during the erythrocytic stages of Plasmodium falciparum
Doctor of Philosophy (Ph.D.), Drexel University
Mar 2017
DOI:
https://doi.org/10.17918/00000871
Abstract
Malaria remains one of the leading causes of morbidity and mortality worldwide. Despite many years of research on a global scale, our understanding of the biology of malaria parasites, while largely improved, is still incomplete. Plasmodium falciparum causes the most severe form of malaria in humans. In this dissertation, we studied two important biological aspects of this devastating pathogen: the role of the TCA enzyme, isocitrate dehydrogenase in the asexual and sexual blood-stages of the parasite; and a new method to assess intracellular ATP levels in live malaria parasites using a geneticallyencoded FRET-based ATP indicator. Our previous study suggested that the TCA cycle enzyme aconitase (Aco) alone, but not the complete TCA cycle, is essential for gametocyte development. Here, we described a defective phenotype of the parasites with genetic ablation of another TCA enzyme isocitrate dehydrogenase (IDH) in gametocyte development. Similar to the [delta]Aco, [delta]IDH gametocytes stall at stage III/IV and cannot form mature gametocytes or undergo exflagellation. Both Aco and IDH KO gametocytes exhibit fragmented mitochondrial morphology. Knockout of IDH also leads to a decrease in growth rate and fitness cost in erythrocytic stage. Two possible causes for the phenotype in [delta]Aco and [delta]IDH gametocytes were examined: (1) Accumulation of reactive oxygen species (ROS) within the parasite mitochondria due to lack of NADPH production, (2) and cytotoxicity of built up citrate. We concluded that IDH is required for gametocytogenesis most likely due to its role to generate mitochondrial NADPH, suggesting that IDH can be further explored as a target of transmission-blocking agents. We also reported the successful application of AT1.03, a genetically-encoded FRET-Based ATP indicator, in P. falciparum parasites. AT1.03 was stably expressed in the cytosol of the parasites, and FRET signals of live parasites were imaged by spinningdisk confocal microscopy. We demonstrated that cytosolic ATP levels are highly diverse between individual parasites, and drug-induced stress could be assessed by measuring the cytosolic ATP levels in the parasites. Our results established this method which could be developed into a powerful tool to study the mechanism of antimalarials as well as high throughput screens for new drug leads.
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Details
- Title
- The role of isocitrate dehydrogenase and assessment of intracellular ATP levels during the erythrocytic stages of Plasmodium falciparum
- Creators
- Ming Yang
- Contributors
- Akhil B. Vaidya (Advisor) - Drexel University, Microbiology and Immunology
- Awarding Institution
- Drexel University
- Degree Awarded
- Doctor of Philosophy (Ph.D.)
- Publisher
- Drexel University; Philadelphia, Pennsylvania
- Number of pages
- xii, 168 pages
- Resource Type
- Dissertation
- Language
- English
- Academic Unit
- Biochemistry and Molecular Biology; College of Medicine; Drexel University
- Other Identifier
- 991016814382704721