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Molecular and Biochemical Characterisation of the Electron Transport Chain of Plasmodium Falciparum

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Molecular and Biochemical Characterisation of the Electron Transport Chain of Plasmodium Falciparum Molecular and biochemical characterisation of the electron transport chain of Plasmodium falciparum Thomas ANTOINE Ph.D. February 2012 Molecular and biochemical characterisation of the electron transport chain of Plasmodium falciparum Thesis is submitted in accordance with the requirements of the University of Liverpool for the degree of Doctor of Philosophy By Thomas ANTOINE February 2012 Declaration This thesis is the result of my own work. The material contained in the thesis has not been presented, nor is currently being presented, either wholly or as a part, for any other degree or other qualification. The research work was carried out in the Liverpool School of Tropical Medicine, University of Liverpool, United Kingdom. ……………………………………….. Thomas ANTOINE (2012) i Acknowledgments First of all, I would like to express my gratitude to my supervisors Pr. Steve Ward, Dr. Giancarlo Biagini and Dr. Nick Fisher for their supervision, continued support and constant encouragement throughout my PhD. A special thank goes to the InterMal PhD program and the Marie Curie Fellowship for providing the generous funding which allowed me to undertake this research, but also for giving me the opportunity to attend conferences and workshops as well as meet so many interesting people. I am grateful to Prof. Alister Craig and Prof. Sylke Muller for accepting to be the two members of my oral defense committee. The members of the Ward's research group have contributed immensely to my personal and professional time at Liverpool. I am very grateful to Ashley Warman for insightful comments in my work, for reviewing my thesis and providing valuable feedback as well as for many motivating discussions. I would like also to thank David Waterhouse for his friendship, enthusiasm and support in mass spectrometry analysis. I have greatly enjoyed the opportunity to work with Enrique Salcedo and Jill Davies, whose expertise and technical excellence in malaria research have been particularly helpful. Other past and present group members that I have had the pleasure to work with are Gavin Laing, Alison Ardrey, Ally Shone and Gemma Nixon. Thanks to Alasdair Hill, Alison Mbekiani, Doss, Murad Ali Mubaraki, Taghreed Abdulaziz Hafiz, Francesca Tamoranezzi, Alice Halliday, Susana Barbosa, Darren Cook, Archana Kaniti, Upali Goonetilleke and others who made the lab a friendly environment for working. I would also like to acknowledge my French comrades Rodolphe and Mathieu for being great friends throughout my last year in Liverpool. I would like to thank Natalia for her love, encouragement and support when I have needed it the most. Thank you with all my heart! Finally, I dedicate this thesis to my mom, dad and sister for always believing in me, for their continuous love and their supports in my decisions. Merci pour tout! ii Abstract The single mitochondrion of Plasmodium falciparum has some unusual functional features and unconventional biochemical properties. The electron transport chain (ETC) has critical roles in generating the mitochondrial membrane potential (Δψm) and driving pyrimidine biosynthesis. Enzymes of the respiratory chain became attractive targets for antimalarial drugs such as atovaquone, an inhibitor of the bc1 complex and lethal to malaria parasites. Although much progress has been made, there are a number of knowledge gaps in our understanding of the underlying biochemical mechanisms of the respiratory chain and some major components are still not completely characterised. In addition, recent studies have generated conflicting data and hypotheses with regards to mitochondrial function. This thesis, using a multidisciplinary approach, was undertaken to improve our understanding of the mitochondrion and its function and to clarify some of the confusing and contradictory data in this area. Due to high sequence divergence, two ETC complexes remain to be clearly annotated in Plasmodium species: the succinate dehydrogenase (or complex II) and ATP synthase (or complex V). A pyramid-shaped bioinformatic strategy based on structural fingerprints and motif patterns was applied to identify candidate genes encoding the membrane anchors of complex II (SdhC and SdhD) and the ATP synthase F0 sector (subunits a and b). In order to validate the genes predicted, 2D (non-)gradient BNE/SDS-Page approaches were established to separate respiratory chain complexes from solubilised P. falciparum membranes and identify their subunits via NanoLC- MS/MS analysis. Although the proteomic strategy was validated with bovine mitochondrial preparation, no positive identification of the genes of interest could be obtained with P. falciparum extract due to low-expression of target proteins and difficulties in the preparation of isolated mitochondria of high purity. For the first time, the direct activities (or ubiquinone reduction activity) of the five dehydrogenases delivering ubiquinol to the bc1 complex were compared. Due to a higher rate of turnover, the type II NADH dehydrogenase (PfNdh2) appears to be the main enzyme for feeding the electron transport chain with activity two to three times greater than the other dehydrogenases (dihydroorotate dehydrogenase, malate quinone oxidoreductase, glycerol-3- phosphate dehydrogenase). Additionally, spatiotemporal confocal imaging of parasite mitochondria revealed that loss of PfNDH2 function provoked a collapse of the mitochondrial transmembrane potential (Δψm). This observation reinforced the interest of targeting PfNdh2 as a chemotherapeutic strategy for drug development. The catalytic properties of the P. falciparum complex II were also examined using various electron donors and acceptors. Although complex II has been previously considered as a succinate dehydrogenase, results obtained indicate that complex II functions as a ubiquinol-fumarate reductase (QFR) forming succinate from fumarate in the asexual stages of P. falciparum. The capacity of menaquinone, an alternative electron carrier of anaerobic species and recently detected in malaria parasites was also evaluated to replace iii Abstract ubiquinone within the respiratory chain in anaerobic conditions. Data obtained demonstrated that the menaquinone pool is not involved in the ETC and only the ubiquinone pool interacts with the different enzymes. Artemisinin and its derivatives are frontline drugs employed in the treatment of uncomplicated malaria usually in the form of combination therapies. The ETC has been previously implicated in the mode of action of artemisinin and its derivatives. In this chapter, this hypothesis was tested using a single-cell imaging and enzyme-assay based approach. Data presented reveal that endoperoxide drugs provoke a rapid collapse of mitochondrial and plasma membrane potentials, both essential for parasite survival. Addition of the iron chelator desferrioxamine or the superoxide scavenger Tiron drastically reduces the depolarization highlighting the role of ferrous ions and oxidant stress in the artemisinins activation process and membrane damaging activity. Deoxyartemisinin, which lacks the endoperoxide bridge, has no effect on membrane potential indicating that this peroxide functionality is the key pharmacophore responsible for the pharmacological activity of this class of compound. Thus, the results presented, suggest that artemisinin and its derivatives act as a generator of additional reactive oxygen species that overcome the oxidative defenses of the malaria parasite and cause a widespread and rapid membrane potential depolarization leading to mitochondrial dysfunction and parasite death. TM Atovaquone is a bc1 inhibitor used in combination with proguanil (e.g. Malarone ) for the curative and prophylactic treatment of malaria. However, resistance to atovaquone associated with point mutations have been detected in the field. In this thesis, the first description of the effect of the Y268S mutation (harbored by the atovaquone-resistant field isolate TM902CB) on parasite bc1 catalytic turnover and stability has been reported. This mutation was shown to confer a 270-fold shift of the inhibitory constant (Ki) for atovaquone with a concomitant reduction in the Vmax of the bc1 complex of 40% and a 3-fold increase in the observed Km for ubiquinol. Western blotting analyses revealed a reduced iron-sulfur protein content in Y268S bc1 suggestive of a weakened interaction between this subunit and the cytochrome b. It was concluded that the reduced enzyme activity affects protein stability and should incur a fitness penalty to the parasite, features that were not fully discernable using the yeast model alone. Due to a small mitochondrial genome (mtDNA), the great majority of mitochondrial proteins, including those composing the ETC, are imported post-translationally from the cytosol into the organelle. Thus, it is essential to understand the import and processing machinery of mitochondrial proteins in malaria parasites. However, the description of the apicomplexan model including P. falciparum is fragmented across different studies and no general overview, including recent insights, has been proposed. An updated picture of the whole protein import and processing machinery in apicomplexa is presented. Novel putative components are revealed by comparison with five other apicomplexan species (Toxoplasma gondii, Cryptosporidium muris, Theileria parva, Babesia bovis and Neospora caninum). Aspects of the apicomplexan model are highly divergent from that seen in yeast, mammalians or plants. iv Publications and presentations Publications: 1. Fisher N*,
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