The role of glycerol kinase in plasmodium falciparum

Simple item page
dc.contributor.authorNaidoo, Kubendran
dc.date.accessioned2013-03-26T06:13:27Z
dc.date.available2013-03-26T06:13:27Z
dc.date.issued2013-03-26
dc.descriptionA thesis submitted to the Faculty of Science, University of the Witwatersrand, Johannesburg, in fulfilment of the requirements for the degree of Doctor of Philosophy. Johannesburg, November 2012.en_ZA
dc.description.abstractMalaria continues to be a devastating disease. Plasmodium falciparum is the most lethal human malaria parasite, responsible for the majority of the hundreds of millions of cases of malaria and approximately 665,000 thousand deaths in 2010. Understanding the biology of the parasite is vital in identifying potential drug targets necessary to develop novel treatments to combat the disease. During every 48-hour asexual intra-erythrocytic replication cycle, a single parasite can produce up to 32 progeny. This extensive proliferation implies that parasites require substantial amounts of lipid precursors. Glycerol kinase (GK) is a highly conserved enzyme that functions at the interface of lipid synthesis and carbohydrate metabolism. GK catalyzes the ATP-dependent phosphorylation of glycerol to glycerol-3-phosphate, a major phospholipid precursor. In this study, the full length 1,506bp P. falciparum glycerol kinase (PfGK) gene was cloned and expressed as a glutathione S-transferase (GST) fusion protein in E. coli. The recombinant PfGK (rPfGK) enzyme was predominantly expressed as an insoluble aggregate, however, ~3μg soluble rPfGK was purified from an 800ml induced culture. SDS-PAGE analysis showed that the protein migrated at ~73kDa and its enzyme activity was verified using an ADP-coupled spectrophotometric assay. The kinetic parameters for rPfGK were Km = 15.7μM for glycerol and Km = 15.9μM for ATP. To evaluate the role of the enzyme in asexual blood-stage development, PfGK was disrupted using double crossover homologous DNA recombination to generate a glycerol kinase knockout parasite line (3D7ΔPfGK). Southern hybridization and PfGK mRNA expression analysis verified that the gene had been disrupted. 3D7ΔPfGK growth rates were evaluated using thiazole orange, a DNA staining dye, coupled to flow cytometry analysis for improved sensitivity. Highly synchronized ring stage parasites were monitored over one 48-hour developmental cycle and results showed that 3D7ΔPfGK growth was significantly reduced to 56.5 ± 1.8% when compared to wild type parasites. This reduced proliferation of 3D7ΔPfGK knockout parasites suggests that PfGK is required for optimal proliferation during the blood stages but is not essential for viability and therefore, not a potential drug target. However, PfGK mRNA expression is markedly elevated in gametocytes and sporozoites. This suggests that PfGK may play a significant role in the mosquito- and liver-stage parasites, with implications for a potential transmission-blocking target. Thus, using a novel bioinformatics method, Evolutionary Patterning, in combination with structural modelling, three potential drug target sites that were different to the human GK orthologue and less likely to develop resistance to compounds were identified. Further studies in the mosquito stages will provide insight into the role of PfGK in the lifecycle of P. falciparum parasites.en_ZA
dc.identifier.urihttp://hdl.handle.net/10539/12599
dc.language.isoenen_ZA
dc.subject.lcshGlycerin.
dc.subject.lcshPlasmodium falciparum.
dc.titleThe role of glycerol kinase in plasmodium falciparumen_ZA
dc.typeThesisen_ZA
Files
Original bundle
Now showing 1 - 1 of 1
No Thumbnail Available
Name:
PhD Thesis - Kubendran Naidoo.pdf
Size:
7.58 MB
Format:
Adobe Portable Document Format
Description:
Main article
Download
License bundle
Now showing 1 - 1 of 1
No Thumbnail Available
Name:
license.txt
Size:
1.71 KB
Format:
Item-specific license agreed upon to submission
Description:
Download
Collections
ETD Collection