ETD Collection

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Subject: search.filters.subject.Plasmodium falciparum.

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Now showing 1 - 4 of 4
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    The effect of iron and iron chelators on the growth of an in vitro plasmodium falciparum culture.
    (1991) Jairam, Karuna Thaker
    The influence of iron on the outcome of various infections have been extensively reviewed. Clinical observations suggests that iron deficiency may be protective against malaria. Various researchers have shown that certain iron chelators blocked the proliferation of plasmodium falciparum in vitro and in vivo. (Abbreviation abstract)
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    The adaptor protein 1 medium subunit of plasmodium falciparum
    (2014-03-04) Bezuidenhout, Belinda Catherine
    Malaria is a tropical disease affecting millions of people worldwide. Plasmodium falciparum is the causative agent of the most severe form of malaria, and therefore insights into the molecular mechanisms by which it functions are critical. The intraerythrocytic stage of the life cycle is responsible for the clinical manifestations of the disease. Numerous proteins are required for the invasion and remodelling of host erythrocytes, and need to be transported to the highly specialized organelles from which they are secreted (invasion proteins), or to the erythrocyte cytoplasm or membrane (exported proteins). It is postulated that newly synthesized proteins are transported from the Golgi network to their target destinations by specific interactions of target sequences of the proteins with the medium subunit (μ) of an adaptor protein (AP1) complex. Bioinformatic analysis of the putative P. falciparum AP1μ subunit, encoded by Pf13_0062, revealed a cargo-binding domain. Three regions, one of which encompassed the putative binding domain, while the other two interrupted this domain, were cloned into the pGEX-4T-2 expression vector. These recombinant proteins were expressed in E. coli with a GST tag, purified and immobilized on glutathione magnetic beads and used to biopan P. falciparum phage display libraries to identify interacting proteins. No binding was observed with the truncated domains, but several specific interactions were identified with the binding domain. One of these peptides was 13 amino acids long and contained a Yxx motif, indicating that PfAP1, like its homologues in higher eukaryotes, binds specifically to this motif in cargo proteins. Other sequences identified included a RRNIFLFINRKKE peptide; exported protein PHISTa; and conserved protein PFL0675c. In the C-terminal region of PFL0675c an armadillo repeat structure was predicted, just downstream of the binding domain identified by biopanning. This region of PFL0675c was therefore cloned into the pET-15b expression vector and expressed as a recombinant His-tagged protein. Slot overlays and far western blotting confirmed the specificity of the interaction with PfAP1. Since PFL0675c does not display the characteristics typical of AP1 cargo, it is postulated to be an accessory protein to the complex. Localization studies performed by transfection V of P. falciparum parasites with pARL2AP1GFP showed that in vivo, PfAP1 localized to distinct foci around the nucleus. Co-localization studies confirmed that PfAP1 localizes to the cis-Golgi in P. falciparum. PfAP1 may therefore be involved in trafficking proteins from the Golgi network to specific subcellular compartments within the parasite. This is the first study identifying interacting partners of PfAP1, and demonstrating its localization in P. falciparum 3D7 parasites.
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    Characterization of a plasmodium falciparum protein kinase
    (2014-02-07) Roets, Sasha
    Malaria is caused by Plasmodium parasites and is the world’s most devastating tropical infectious disease. The need for identifying novel drug targets is fuelled by an increased resistance of these parasites against available drugs. The human host red cell membrane plays an important role during invasion and subsequent development of the parasite within the red cell and undergoes several structural, functional and biochemical changes triggered by various protein-protein interactions between the parasite and the host cells. These interactions form a fundamental part of malaria research, since the parasite spends the pathogenic stage of its life cycle in the human erythrocyte. The Plasmodium kinome is complex and the exact role of protein phosphorylation in malaria parasites is not yet fully understood. This study aims to characterise the kinase domain of Plasmodium falciparum (3D7) Protein Kinase 8 (PfPK8), described as a putative protein on the Plasmodium falciparum database. PfPK8 is encoded by the PfB0150c gene (recently renamed as PF3D7_0203100) situated on chromosome 2 of the parasite genome. A 1 507bp section of the PfB0150c gene, containing a 822bp centrally located kinase domain was cloned into a pTriEx-3 expression vector. A soluble recombinant octa-histidine-tagged PfPK8 was expressed in Escherichia coli Rosetta 2 (DE3) cells, but with relatively low yield and purity.To improve the expression, a recombinant PfB0150c-baculovirus infected Spodoptera frugiperda (Sf9) insect cell system was attempted, but without success. A different tag was employed and glutathione-S-transferase-PfPK8 was successfully expressed in Escherichia coli Rosetta 2 (DE3) cells, with a higher yield and purity. Recombinant GST-PfPK8 was used in non-radioactive coupled spectrophotometric kinase assays in the presence of known kinase substrates casein, MBP and H1 to determine kinetic parameters of the enzyme. It phosphorylated all three substrates at a temperature of 37ºC and pH of 7.4. Recombinant GST-PfPK8 was inactive at a pH below 6 and most active at pH 7.4. The relative activity of the enzyme was highest at a temperature synonymous to a fever spike in a Plasmodium falciparum infected individual. Secondary structural analysis of PfPK8 revealed the position of a conserved substrate binding domain containing an ATP-binding site and binding loop within the kinase domain. The kinase domain of rPfPK8 was modelled using available crystal structures of its identified homologues. The gene is expressed throughout the intraerythrocytic stages of the parasite life cycle, as well as in gametocytes. Protein-protein binding studies revealed that host-parasite protein-protein interactions exist between rPfPK8 and erythrocyte membrane protein, band 3. Plasmodium falciparum PK8 could therefore play a role during invasion of host erythrocytes and during the intraerythrocytic development of the parasite, by phosphorylating red blood cell membrane proteins. This study provides the groundwork for future X-ray crystallographic studies to elucidate the structure of the enzyme, and for additional gene manipulation experiments to ascertain whether it is essential for parasite survival in all the intraerythrocytic stages and therefore a potential new drug target candidate.
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    The role of glycerol kinase in plasmodium falciparum
    (2013-03-26) Naidoo, Kubendran
    Malaria 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.