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Dissertation
Investigation of mitochondrially targeted proteins in blood stage malaria parasites
Doctor of Philosophy (Ph.D.), Drexel University
Apr 2011
DOI:
https://doi.org/10.17918/00000738
Abstract
Malaria parasites contain reduced-function mitochondria. Nevertheless, their mitochondrion is a validated target for antimalarials, such as atovaquone, which selectively inhibits the parasite mitochondrial electron transport chain (mtETC). We previously demonstrated that a critical function of the mtETC in blood stages of Plasmodium falciparum is to regenerate ubiquinone as co-substrate of dihydroorotate dehydrogenase (DHODH), and that transgenic P. falciparum expressing type I DHODH from yeast (yDHODH) are resistant to all mtETC inhibitors. In this study, we constructed a transfection vector, pUF1 that uses yDHODH as a selectable marker. As a proof of concept, we disrupted the type II vacuolar proton-pumping pyrophosphatase gene in P. falciparum. This is the third selectable marker for P. falciparum transfections. Five dehydrogenases supply electrons to the mtETC: malate: quinone oxidoreductase (MQO), succinate dehydrogenase, DHODH, glycerol-3-phosphate dehydrogenase and alternative NADH dehydrogenase (NDH2). Among these, we aimed to identify the roles of MQO and NDH2 in malaria parasites. Gene disruption and protein knock-down experiments of MQO and NDH2 were unsuccessful, suggesting that they may be essential in blood stages of Plasmodium. We also determined that these proteins are localized in the parasite mitochondrion. Examination of HA-tagged MQO by blue native polyacrylamide gel electrophoresis suggested that MQO might be present in larger complexes, possibly in association with other mitochondrial proteins. Combined with previous results, these findings suggest that ubiquinone-dependent mitochondrial dehydrogenases may have roles beyond their known enzymatic activities in blood stage parasites. In Plasmodium, the glycine cleavage complex (GCV) was proposed to be involved in folate metabolism within the mitochondrion. GCV is made up of 4 proteins; glycine decarboxylase, aminomethyltransferase (AMT), carrier protein and lipoamide dehydrogenase. Here, we investigated the putative AMT (AMT1). We knocked out AMT1 both in vitro and in vivo. P. falciparum AMT1 knockout (Pf[delta]AMT1) parasites exhibited no growth deficiency in vitro. Surprisingly, Pf[delta]AMT1 parasites were found to be deficient in lipoylation of the apicoplast-localized pyruvate dehydrogenase (PDH) E2 subunit. Complementation of Pf[delta]AMT1 appeared to rescue PDH lipoylation. Localization studies revealed that AMT1 is found mainly in the cytoplasm. Currently, we are investigating P. berghei[delta]AMT1 parasites in mosquito and liver stages.
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Details
- Title
- Investigation of mitochondrially targeted proteins in blood stage malaria parasites
- Creators
- Suresh Maddur Ganesan
- Contributors
- Akhil B. Vaidya (Advisor)
- Awarding Institution
- Drexel University
- Degree Awarded
- Doctor of Philosophy (Ph.D.)
- Publisher
- Drexel University; Philadelphia, Pennsylvania
- Number of pages
- xv, 211 pages
- Resource Type
- Dissertation
- Language
- English
- Academic Unit
- Microbiology and Immunology; College of Medicine; Drexel University
- Other Identifier
- 991014970202304721