The role of glycerol kinase in plasmodium falciparum

A 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.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

Ecophysiological studies of the invasive weed Chromolaena odorata (L.) King and Robinson and its control in KwaZulu-Natal.

Ph.D. University of KwaZulu-Natal, Durban 2013.Despite increased interest in the control and spread of the alien weed, Chromolaena odorata, little is known of its photosynthetic characteristics under field conditions. The aim of the study was to obtain a better understanding of the ecophysiological attributes of C. odorata that contribute to its invasive success. Photosynthetic performance of C. odorata was evaluated by monitoring diurnal changes in gas exchange, chlorophyll a fluorescence and plant water relations. Gas exchange characteristics of plants growing in exposed and shaded environments, as well as seasonal patterns, were evaluated. The response of C. odorata to water stress was also determined. Chromolaena odorata exhibited high CO2 uptake rates with no light saturation. Shade plants had significantly larger leaf surface areas and greater concentrations of total chlorophyll, total carotenoids and chlorophylls a and b than sun plants. Relatively high photosynthetic uptake rates in C. odorata may allow for greater carbon gain in high light environments thus contributing to increased growth and spread of the species. Chromolaena odorata can successfully acclimatise to low photosynthetic photon flux density (PPFD), thus, outcompeting less tolerant species under low light conditions. Leaf conductance, CO2 uptake, transpiration and chlorophyll fluorescence parameters in winter were tightly coupled to summer. Plants had higher water use efficiency (WUE) in summer compared to winter, probably to maximise CO2 uptake and minimise water loss. There was a progressive decrease in leaf water potential with increase in water stress in water stressed (WS) plants. The leaves of WS plants showed signs of severe wilting 10 days after the onset of stress compared to well watered (WW) plants. Increased proline concentration and leaf wilting probably increase (WUE) and may be an adaptive strategy to protect against dehydration injury.The effects of the herbicide, glyphosate, on gas exchange and translocation were studied. Glyphosate treatment decreased leaf conductance leading to a reduction in CO2 uptake and transpiration. Glyphosate is a mobile herbicide that is transported from leaves to roots and caused death of plants within a week of treatment. The potential antimicrobial properties of the weed were evaluated using selected bacteria and fungi. Crude leaf extracts exhibited some antibacterial and antifungal activity. Extracts from the weed are unlikely to be useful antimicrobial sources due to low concentrations of active compounds. A co-ordinated strategy, taking into account the high plasticity of the weed, is needed to curtail the spread of C. odorata. The ecophysiological responses to environmental conditions should be considered when planning management and control strategies for C. odorata

Absence of Acanthocytosis in Huntington’s Disease-like 2: A Prospective Comparison with Huntington’s Disease

Background: Huntington’s Disease-like 2 (HDL2) is classified as a neuroacanthocytosis; however, this remains unverified. We aim to determine if acanthocytes are present in HDL2 and whether acanthocytes can differentiate HDL2 from Huntington’s disease (HD). Methods: We prospectively compared 13 HD and 12 HDL2 cases against 21 unaffected controls in Johannesburg. Blood smears were prepared using international standards and reviewed by at least two blinded reviewers. An acanthocytosis rate of greater than 1.2% in the dry smear or greater than 3.7% in the wet smear was designated a priori as the threshold for clinical significance based on previously established standards. Flow cytometry was performed on all but four of the cases. Red cell membrane protein analysis was performed on all participants. Results: There were 12 HDL2, 13 HD, and 21 controls enrolled. None of the HD or HDL2 participants had defined acanthocytosis or other morphological abnormalities. None of the HD or HDL2 cases had evidence of an abnormal band 3. Discussion: Acanthocytosis was not identified in either HDL2 or HD in our patient population. Our results, based on the first prospective study of acanthocytes in HDL2 or HD, suggest that screening for acanthocytes will not help establish the diagnosis of HD or HDL2, nor differentiate between the two disorders and raises the question if HDL2 should be placed within the neuroacanthocytosis syndromes

Evolutionary Patterning: A Novel Approach to the Identification of Potential Drug Target Sites in Plasmodium falciparum

Malaria continues to be the most lethal protozoan disease of humans. Drug development programs exhibit a high attrition rate and parasite resistance to chemotherapeutic drugs exacerbates the problem. Strategies that limit the development of resistance and minimize host side-effects are therefore of major importance. In this study, a novel approach, termed evolutionary patterning (EP), was used to identify suitable drug target sites that would minimize the emergence of parasite resistance. EP uses the ratio of non-synonymous to synonymous substitutions (ω) to assess the patterns of evolutionary change at individual codons in a gene and to identify codons under the most intense purifying selection (ω≤0.1). The extreme evolutionary pressure to maintain these residues implies that resistance mutations are highly unlikely to develop, which makes them attractive chemotherapeutic targets. Method validation included a demonstration that none of the residues providing pyrimethamine resistance in the Plasmodium falciparum dihydrofolate reductase enzyme were under extreme purifying selection. To illustrate the EP approach, the putative P. falciparum glycerol kinase (PfGK) was used as an example. The gene was cloned and the recombinant protein was active in vitro, verifying the database annotation. Parasite and human GK gene sequences were analyzed separately as part of protozoan and metazoan clades, respectively, and key differences in the evolutionary patterns of the two molecules were identified. Potential drug target sites containing residues under extreme evolutionary constraints were selected. Structural modeling was used to evaluate the functional importance and drug accessibility of these sites, which narrowed down the number of candidates. The strategy of evolutionary patterning and refinement with structural modeling addresses the problem of targeting sites to minimize the development of drug resistance. This represents a significant advance for drug discovery programs in malaria and other infectious diseases

Hepatitis B Virus Research in South Africa

Despite being vaccine-preventable, hepatitis B virus (HBV) infection remains the seventh leading cause of mortality in the world. In South Africa (SA), over 1.9 million people are chronically infected with HBV, and 70% of all Black chronic carriers are infected with HBV subgenotype A1. The virus remains a significant burden on public health in SA despite the introduction of an infant immunization program implemented in 1995 and the availability of effective treatment for chronic HBV infection. In addition, the high prevalence of HIV infection amplifies HBV replication, predisposes patients to chronicity, and complicates management of the infection. HBV research has made significant progress leading to better understanding of HBV epidemiology and management challenges in the SA context. This has led to recent revision of the national HBV infection management guidelines. Research on developing new vaccines and therapies is underway and progress has been made with designing potentially curative gene therapies against HBV. This review summarizes research carried out in SA on HBV molecular biology, epidemiology, treatment, and vaccination strategies

Absence of Acanthocytosis in Huntington's Disease-like 2: A Prospective Comparison with Huntington's Disease

Background:&nbsp;Huntington&rsquo;s Disease-like 2 (HDL2) is classified as a neuroacanthocytosis; however, this remains unverified. We aim to determine if acanthocytes are present in HDL2 and whether acanthocytes can differentiate HDL2 from Huntington&rsquo;s disease (HD).Methods:&nbsp;We prospectively compared 13 HD and 12 HDL2 cases against 21 unaffected controls in Johannesburg. Blood smears were prepared using international standards and reviewed by at least two blinded reviewers. An acanthocytosis rate of greater than 1.2% in the dry smear or greater than 3.7% in the wet smear was designated a priori as the threshold for clinical significance based on previously established standards. Flow cytometry was performed on all but four of the cases. Red cell membrane protein analysis was performed on all participants.Results:&nbsp;There were 12 HDL2, 13 HD, and 21 controls enrolled. None of the HD or HDL2 participants had defined acanthocytosis or other morphological abnormalities. None of the HD or HDL2 cases had evidence of an abnormal band 3.Discussion:&nbsp;Acanthocytosis was not identified in either HDL2 or HD in our patient population. Our results, based on the first prospective study of acanthocytes in HDL2 or HD, suggest that screening for acanthocytes will not help establish the diagnosis of HD or HDL2, nor differentiate between the two disorders and raises the question if HDL2 should be placed within the neuroacanthocytosis syndromes.</p