Defining the roles of essential genes in the malaria parasite life cycle

© 2017 Dr. Gallallalage Upeksha Lakmini RathnapalaThe 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