Metabolic phenotyping of Plasmodium falciparum mutants with impaired pyruvate dehydrogenase complex activity

Biddau, Marco (2017) Metabolic phenotyping of Plasmodium falciparum mutants with impaired pyruvate dehydrogenase complex activity. PhD thesis, University of Glasgow.

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Abstract

Malaria remains one of the most important infectious diseases in the world, counting 200 million cases and killing 300,000 people every year, mostly represented by children under the age of 5. Apicomplexan parasites of the genus Plasmodium cause malaria and P. falciparum is responsible for the most severe form of the disease. The development of drug resistance is one of the most important challenges that eradication programs face and first line therapies have started to become ineffective. A better understanding of Plasmodium biology and metabolic functions is essential for the identification of new drug targets and the interpretation of the drug resistance mechanisms to help and fight this devastating disease. The pyruvate dehydrogenase complex (PDC) is a member of the -ketoacid dehydrogenase complexes (KADH), representing essential elements of the intermediary metabolism. PDC has the important role in decarboxylating pyruvate generating NADH+H+ and acetyl-CoA, which are essential for many metabolic processes including energy and lipid metabolism. Three enzymes catalyse PDC reaction represented by pyruvate dehydrogenase (E1), dihydrolipoamide acetyltransferase (E2) and dihydrolipoamide dehydrogenase (E3). Specific cofactors such as thiamine pyrophosphate, FAD and NAD+ are required for the activity of the enzymes and lipoic acid is essential for E2 catalysis. Usually, the main role of PDC is to link glycolysis to the tricarboxylic acid cycle and downstream oxidative phosphorylation. This dogma does not apply to Plasmodium PDC, which is exclusively found in the apicoplast and provides acetyl-CoA and NADH+H+ for fatty acid biosynthesis. Both, PDC and fatty acid biosynthesis are dispensable during the intraerythrocytic development of P. falciparum but are essential for sexual development. Therefore, the complex may be exploitable for potential transmission-blocking agents. Given that the genes encoding the PDC subunits are actively expressed during the intraerythrocytic life of P. falciparum, it was postulated that they might have additional functions independent of PDC as has been reported from other organisms. To test this hypothesis, P. falciparum mutants with a deletion of apicoplast e3 (ae3) and with impaired E2 activity, through interruption of the apicoplast located lipoic acid biosynthesis pathway by deletion of the lipoic acid protein ligase B gene (lipB), were investigated using targeted and untargeted metabolomics approaches. Metabolic fluxes in the parasite metabolism were characterised after stable isotopic labelling using 13C-U-D-glucose and 13C, 15N-U-L-glutamine. Further growth phenotypes of the mutants were detailed and quantitative western blotting was used to assess the relative expression levels of several antioxidant and redox proteins. The analysis of the growth phenotypes confirmed that disruption of lipb accelerates parasite lifecycle, while deletion of ae3 produces parasites with a persistent synchronicity. The targeted metabolomics analysis of the LipB mutants showed an up-regulation of the TCA cycle activity characterised by an increased flux of isotopic carbons from 13C-U-D-glucose to 13C-2-citrate. These results imply an incomplete cyclic activity of the TCA cycle and the transferring of citrate outside of the mitochondrion, possibly contributing to the cytosolic acetyl-CoA pool. Results obtained from the untargeted analysis sustained this hypothesis showing increased levels for CoA biosynthesis intermediates and different acetylated metabolites, suggesting adaptations in the parasite acetylome. Additionally, levels of ATP were significantly reduced in this mutant line and carbon flux into NAD(P)+ was increased suggesting the instalment of starvation and redox stress. The e3 mutants hardly showed any significant metabolic change, despite its peculiar synchronous growth phenotype. These results suggest that the two different PDC subunits may have independent roles affecting specific metabolic functions in the parasite. On the other hand, the analysis of expression levels of antioxidant and redox active proteins in both parasite lines showed that they have increased the relative expression of peroxiredoxins and glutathione S-transferase, possibly to compensate a redox-regulating role of E2 and E3. In addition, the expression levels of the mitochondrial branched-chain -ketoacid dehydrogenase were significantly up-regulated, possibly pointing towards compensatory activity providing acetyl-CoA. In a more direct approach to assess the role of P. falciparum E2, a DiCre based conditional knockout of the gene was attempted. Preliminary data suggest that conditional deletion of e2 might affect parasite growth during intraerythrocytic development. However, isolation of a clonal e2 mutant line for detailed analyses was unsuccessful due to time limitations. The data provided in this thesis suggest that the E2 and E3 subunits of PDC possibly have independent roles during P. falciparum intraerythrocytic development that affect the parasites’ energy and redox metabolisms. They also provide some evidence that the mutant parasites have adapted to the metabolic changes by up-regulating proteins involved in antioxidant and redox activities and by increasing the carbon flux from glucose into the generation on NAD(P)+ to allow for increased activities of their antioxidant machinery. In addition, it appears that mitochondrial citrate synthesis increases in response to the loss of E2 activity and that citrate may be a conduit for the transport of acetyl-CoA into the cytosol and other cellular compartments where acetylation reactions take place, in order to maintain general cellular functions and regulatory processes. The metabolic changes may affect regulation of cell cycle progression as is evidenced by the growth phenotypes of the two parasites lines.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Additional Information: Prof Sylke Müller has retired a few months before the end of my PhD and I concluded my work under Prof Michael Barrett supervision.
Keywords: Plasmodium falciparum, malaria, pyruvate dehydrogenase complex, metabolism, apicoplast, metabolomics, dihydrolypoyl transacetylase, dihydrolypoyl dehydrogenase, lipoic acid protein ligase B, TCA cycle, citrate, DiCre conditional knockout system, redox, antioxidant, targeted approach metabolomics, mzMatch-ISO, growth curve.
Subjects: Q Science > QR Microbiology
Colleges/Schools: College of Medical Veterinary and Life Sciences > Institute of Infection Immunity and Inflammation > Parasitology
Funder's Name: European Commission (EC), Wellcome Trust (WELLCOTR)
Supervisor's Name: Müller, Prof. Sylke and Barrett, Prof. Michael
Date of Award: April 2017
Embargo Date: 6 April 2020
Depositing User: Mr Marco Biddau
Unique ID: glathesis:2017-8089
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 19 Apr 2017 14:55
Last Modified: 03 Jul 2017 12:23
URI: http://theses.gla.ac.uk/id/eprint/8089

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