School of Botany - Theses
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ItemDefining the roles of essential genes in the malaria parasite life cycle( 2017)The combination of drug resistance, lack of an effective vaccine and ongoing conflict and poverty mean that malaria remains a major global health crisis. Understanding metabolic pathways at all parasite life stages is important in prioritising and targeting novel anti-parasitic compounds. To overcome limitations of existing genetic tools to investigate all the parasite life stages, new approaches are vital. This project aimed to develop a novel genetic approach using post meiotic segregation to separate genes and bridge parasites through crucial life stages. The unusual heme synthesis pathway of the rodent malaria parasite, Plasmodium berghei, requires eight enzymes distributed across the mitochondrion, apicoplast and cytoplasm. Deletion of the ferrochelatase (FC) gene, the final enzyme in the pathway, confirms that heme synthesis is not essential in the red blood cell stages of the life cycle but is required to complete oocyst development in mosquitoes. The lethality of FC deletions in the mosquito stage makes it difficult to study the impact of these mutations in the subsequent liver stage. To overcome this, I combined locus-specific fluorophore expression with a genetic complementation approach to generate viable, heterozygous oocysts able to produce a mix of FC expressing and FC deficient sporozoites. In the liver stage, FC deficient parasites can be distinguished by fluorescence and phenotyped. Parasites lacking FC exhibited a severe growth defect from early to mid-stages of liver development in-vitro and could not infect naïve mice, confirming liver stage arrest. These results validate the heme pathway as a potential target for prophylactic drugs targeting liver stage parasites. Energy metabolism in malaria parasites varies remarkably over the parasite life cycle. Parasites depend solely on anaerobic glycolysis at blood stage but need Krebs cycle, the electron transport chain, and mitochondrial ATP synthase during mosquito stage development. Again, reverse genetic approaches to study the hepatic stage of Plasmodium have been thwarted because parasites with defects in energy pathways are unable to complete the mosquito stage. I used the genetic complementation approach established to study heme biosynthesis to bridge parasites lacking the β subunit of mitochondrial ATP synthase through mosquito stage and studied their development in the liver stage. ATPase knockouts were indistinguishable from wildtype in in-vitro liver stage assays of size, nuclear content, and merosome production. Robust progression to blood stage confirmed the dispensability of mitochondrial ATP synthesis in liver stages. I extended this approach to explore the essentiality of upstream mitochondrial electron transport and Krebs cycle during the liver stage. I speculate that energy metabolism in the liver stage resembles that in the blood stage, relying predominantly on glycolysis for ATP production. There are numerous genetic tools to manipulate the blood stage malaria parasite genome in general, but existing genetic tools to generate viable parasites with defects in blood stage essential genes are limited. To overcome this limitation, I have developed a novel strategy in which I first insert a complementary copy of the essential gene-of-interest, and then delete the endogenous gene, and then take advantage of meiosis and segregation during the mosquito stage to create haploid knockout sporozoites. I genotype the parasites along the way by fluorescence microscopy. As proof of principle, I created complemented knockouts of the blood stage essential 1-deoxy-D-xylulose-5- phosphate reductoisomerase (DXR) gene, crossed these with wildtype parasites, and then tracked the progeny through in-vitro and in-vivo liver development. Precomplementation proved difficult, perhaps due to inappropriate expression of important metabolic genes. Additionally, problems with apparent silencing of the fluorophore tags compromised my ability to genotype cross progeny preventing any firm conclusion on the function of isoprenoid precursor pathway of liver stage parasites. Nevertheless, my success in generating a blood stage essential gene knockout via precomplementation provides encouragement that this novel reverse genetic strategy can be implemented to investigate the role of blood stage-essential genes in sporozoite and liver stages of malaria parasites.
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ItemExploring how fatty acids synthesized by malaria parasites are incorporated into lipids: Characterization of the Plasmodium apicoplast glycerol-3-phosphate acyltransferase( 2015)The Plasmodium parasites responsible for malaria synthesize fatty acids in a reduced plastid organelle known as the apicoplast. There has been enormous interest in the apicoplast fatty acid synthesis pathway as a potential drug target, but comparatively little research into its role in parasite lipid metabolism. The fatty acid synthesis pathway is essential only at certain stages of the parasite life cycle, and appears to be required to support membrane lipid biosynthesis. However, the nature of the lipid species reliant on the apicoplast pathway and the intermediate steps in their synthesis have largely not been explored. The majority of parasite membrane lipid species can be synthesized from a phosphatidic acid precursor. Phosphatidic acid is composed of two fatty acids linked to glycerol-3-phosphate, and it is synthesized in two steps by a pair of acyltransferases. Plasmodium parasites were initially predicted to have two complete phosphatidic acid synthesis pathways located in the apicoplast and endoplasmic reticulum. Recent discoveries in the P. yoelii rodent model have demonstrated the apicoplast pathway is essential for parasites in the liver stage, consistent with the requirement for fatty acid synthesis in that species. However, it also indicated the apicoplast pathway may be incomplete, suggesting its function was instead to incorporate newly-made fatty acids into the intermediate lysophosphatidic acid. This thesis investigates the glycerol-3-phosphate acyltransferase of the P. falciparum and P. berghei apicoplast lysophosphatidic acid synthesis pathway to gain greater insight its role in lipid metabolism. The localization of the P. falciparum enzyme is confirmed and its activity demonstrated and explored through complementation, site-directed mutagenesis and structure homology modeling. The P. falciparum glycerol-3-phosphate acyltransferase is shown to be non-essential in the blood stage in standard culturing conditions, consistent with the dispensability of fatty acid synthesis at this stage. Unexpectedly, the P. falciparum enzyme is also found to be dispensable in lipid-depleted media conditions that induce fatty acid synthesis, suggesting the fatty acids made by parasites in this environment are used via other pathways. The P. berghei homologue is demonstrated to be essential in the liver stage, and its loss to closely mirror the phenotype of fatty acid synthesis knockouts in rodent models, implicating the pathway as a major route for newly-synthesized fatty acids to be incorporated into membrane lipid precursors. However, loss of the P. berghei enzyme does not impact expression of a key merozoite surface protein to the extent seen in the fatty acid synthesis knockouts, providing novel insight into how newly-made fatty acids might contribute to the synthesis of the lipid anchor of this protein. These findings provide information about the apicoplast lysophosphatidic acid synthesis pathway in two further Plasmodium species, and contribute towards understanding how newly-synthesized fatty acids are incorporated into precursors for membrane lipid synthesis.
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ItemEstablishing and elucidating conditional genetic manipulation of the malaria parasite, Plasmodium falciparum( 2013)Methods to alter gene function in Plasmodium are barely out of infancy, with much growth occurring within the past five years via adaptation of protocols established in other species. In this thesis, a method where the parasite’s innate ability to control gene expression in response to environmental stimuli, and a synthetic small molecule regulated protein stability system were applied to change the phenotypic expression of introduced transgenes. In chapter 2, we aim to control activity of a temperature sensitive mutant FLP recombinase protein. Our goal was to induce FLP activity and thus change the open reading frame of a GFP expressing plasmid to mCherry using single site recombination. Dual transgenic parasites were placed in a variety of conditions to favour FLP activity and to prevent negative selection. No evidence of conditional genetic recombination or phenotypic change from green to cherry was observed. Direct expression of FLP during a short period of drug selection produced a mixed population of GFP and mCherry genotypes. The activity of FLP is inefficient and was deemed unsuccessful for our goal of raising transgenic parasites poised for conditional genetic recombination. A homogenic population of mCherry genotypes by successful recombination of the GFP ORF was achieved after drug-cycled expression of evolved FLP (FLPe). Chapter 3 focuses on turning the Plasmodium specific phenomenon of altering rRNA expression profiles in response to fluctuations of temperature and glucose concentration into a tool suitable for conditional gene expression. We copied the promoter region of the S2-Type rRNA gene and measured its ability to express luciferase in altered environmental conditions. A low level of basal activity was observed during the intra-erythrocytic developmental cycle, with expression peaking at levels similar to the weak chloroquine transporter (CRT) promoter during the trophozoite stage. The S2-Type rRNA promoter region did not produce a notable increase of luciferase transcription or activity in response to environmental change. Use of the S2-Type rRNA promoter to express FLP at the enzymatic favourable temperature of 26°C did not result in genetic recombination. The tools developed are capable of expressing transgenes, however their ability to conditionally alter the gene product is limited.