Studies on genetic and pharmacological perturbations on carbon and energy metabolism in toxoplasma gondii and plasmodium falciparum

Abstract

The causative agents for malaria and toxoplasmosis in humans are the apicomplexan parasites Plasmodium falciparum and Toxoplasma gondii. These infectious diseases are a huge health and economic burden, especially in poor countries. Understanding the nutritional needs, metabolic capabilities, and response to anti-parasitic molecules are key aspects for developing new therapeutic intervention against these diseases. In the first part of this study, the focus was on applying mass spectrometry based metabolomics in combination with genetics, pharmacology and biochemical techniques to comprehensively dissect central carbon and energy metabolism in T. gondii. In the second part of this study, the focus is on characterizing atovaquone resistance in P. falciparum, studying the effect on mtETC efficiency due to mutations in Pfcytb, and identification of novel atovaquone like compounds targeting Pfcytb enzyme. In addition, we have used untargeted metabolomics to capture inhibitor specific metabolic response in P. falciparum, to understand primary and secondary effects of the treatment, and to pursue mode of action and target identification studies. Toxoplasma gondii is a promiscuous parasite and serves as a model organism for studies and apicomplexan parasite biology and drug discovery. The ability of T. gondii to adapt to different environmental conditions, by modulating various cellular processes, including cellular metabolism, makes it one of the most successful pathogen. Previous studies have shown that glucose is a dispensable nutrient in the presence of glutamine as an alternate source of carbon during asexual development. To further confirm the dispensability of glycolysis and its effect on virulence and tissue cyst formation by the parasite, we generated a hexokinase gene null mutant (∆hk) in two strains of T. gondii; RH (Type-1; virulent and cannot form tissue cyst in mice) and Prugniad (Type-2; moderately virulent and can form tissue cyst in mice). The knockout parasite showed minor growth defect in comparison to wild type parasites in the presence of glutamine. Interestingly, in the absence of glutamine, the parasite die not die, but continued to grow with a severe fitness defect. This growth defect could not be compensated with non-essential amino acid (NEAA) or acetate. RH ∆hk mutants remained virulent in mice while Prugniad ∆hk mutants were compromised in virulence. Prugniad ∆hk mutants were capable of differentiating into bradyzoites in vitro, but were unable to form mature tissue cysts in mice. 13C-labeled metabolic flux analysis revealed that in the absence of glucose, glutamine is utilized as a major carbon source and metabolic intermediates of glycolysis and pentose phosphate pathway incorporated 13C derived from glutamine, thus providing evidence that gluconeogenesis is operational. To further validate the essentiality of gluconeogenesis for asexual stages of the parasite, we generated mutant parasites lacking a functional phosphoenolpyruvate carboxykinase (PEPCK) gene in RH strain background. PEPCK was found to be conditionally essential; dispensable in presence of glucose but essential in glucose depleted media. Our study highlights the metabolic flexibility in T. gondii, which confers the ability to either use glucose or glutamine as the sole nutrient for asexual propagation. These studies also revealed that the parasite is capable of obtaining bulk cellular ATP efficiently from either substrate level phosphorylation via glycolysis or from mitochondrial oxidative phosphorylation. P. falciparum and T. gondii can perform de novo biosynthesis of pyrimidine nucleotides, which can be blocked by atovaquone, a potent and lethal inhibitor of mtETC complex III protein CYTb. P. falciparum readily develops resistance to atovaquone by mutating the Pfcytb gene to abolish atovaquone binding. We generated atovaquone resistant P. falciparum parasites to study the effect of Pfcytb gene mutation on pyrimidine biosynthesis and mtETC. Several clonal lines of Pfcytb mutants were tested, and one of them, with >1000 fold decreased sensitivity to atovaquone, was selected for further studies. We mapped mutation as a P177L change in the Pfcytb gene. Further biochemical and metabolic studies showed that the mutant parasites have intact pyrimidine biosynthesis and mtETC despite the mutation in Pfcytb gene. We then identified novel atovaquone like molecules with similar nano molar potency against both wild type and Pfcytb mutant P. falciparum. Thus, our study validates mtETC, particularly Pfcytb, as an important target for development of novel antimalarial drugs. Untargeted metabolomics is a useful approach for studying drug MOA. In fact, this has already been used for characterizing a variety of antimalarial molecules, including some frontline antimalarial drugs such as chloroquine and pyrimethamine. In this study, we have characterized the early (4 h) and late (8 h) metabolic response to two potent P. falciparum aminoacyl tRNA synthetase inhibitors; cladosporine and halofuginone. These two compounds showed distinct metabolic effects in comparison to the antimalarial drugs chloroquine and pyrimethamine. Interestingly, although both cladosporine and halofuginone act by inhibiting protein sythesis in P. falciparum, we found that their metabolic response was very different. Our findings will be helpful in further characterizing the effects of these two novel antimalarial compounds.