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Title: Molecular and biochemical pharmacology of mitochondrial enzymes in the malaria parasite Plasmodium falciparum
Author: Abd Majid, Roslaini
ISNI:       0000 0004 2737 1792
Awarding Body: University of Liverpool
Current Institution: University of Liverpool
Date of Award: 2011
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The mitochondria of malaria have big potential to be explored as a drug targeting site. This is due to the differences in the composition of the Plasmodium respiratory complex compared with the human host. The Plasmodium respiratory chain consists of 2 unique dehydrogenases PfNDH2 and MQO which are only encoded in prokaryotic cells. Apart from these two, thy Plasmodium bc1 complex has low amino acid similarity when compared to the bc1 cQmplex of other eukaryotes. Currently the only approved antimalarial drug targeting Plasmodium mitochondria is atovaquone. This drug is used in combination with proguanil (Malarone™) and is widely used for the curative and prophylactic treatment of malaria. Atovaquone, a 2-hydroxynaphthoquinone, is a competitive inhibitor of the quinol oxidation (Qo) site of the mitochondrial cytochrome bc] complex. Inhibition of this enzyme results in the collapse of the mitochondrial membrane potential and subsequent parasite death. However, atovaquone resistance developed very soon after its implementation as malaria chemotherapy. Previous studies have established that the resistance of the parasite is the result of mutations in the bc1 Q0. To date we are the first group to elucidate the genotype and biochemical characterisation of the atovaquone resistant P. Jalciparum TM90C2B. The GeXP multiplex quantitative PCR (qPCR) revealed that a number of genes encoding energy metabolism proteins and genes associated with redox control are upregulated in the resistant parasite compared to the atovaquone sensitive strain 3 D7. This has been supported by automated sequencing results which indicate the presence of a single point mutation in the DNA sequence of TM90C2B cytochrome b which resulted in the substitution of tyrosine by serine at position 268. The drug sensitivity assays and kinetic studies were conducted in order to understand the phenotypic consequences of this mutation. As expected, TM90C2B strain was resistant to almost all electron transport chain inhibitors. The enzymological characterisation of TM90-C2B bc] complex (steady-state decylubiquinol:cytochrome oxidoreductase assay) showed that enzyme turnover was approximately 50% of the atovaquone-sensitive strain with a threefold increase in Krn for decylubiquinol (3D7 : Vrnax = 97.4 ± 5.1 nmol cyt c reduced/min/mg protein, Krn = 5.5± 1.1 mM dQH2, ICSD 6 nM, K; = 0.6 nM; TM90C2B : Vrnax = 60.2 ± 3.2 nmol cyt C reduced/min/mg protein, Krn = 18.5± 2.6 mM dQH2, leSD 600 nM, K; = 162 nM). The other mitochondrial respiratory complexes' specific activities were also measured using samples prepared from Percol® fractionation. Apart from Complex II, we also managed to demonstrate the specific activities of the other complexes'. The PfNDH2 and bCI complex proteins ofTM90C2B strain had 50% less activity than the 3D7 strain. This was supported by data from western blot analysis showing a decrease in PfNDH2 and ISP proteins content of the TM90C2B compared to the 3D7 strain. In addition to this study, we also characterised the Plasmodium Jalciparum mitochondrial malate quinone oxidoreductase (MQO). MQO is involved in the electron transport chain, oxidising malate to oxaloacetate in the oxidative arm of Plasmodium tricarboxylic acid cycle (TCA cycle). This enzyme contains a flavin cofactor which donates electrons to cytochrome bCI complexvia reduction ofubiquinone to ubiquinoL The bioinformatic analysis data indicates that PfMQO is a membrane-bound mitochondrial protein with only one transmembrane domain. The PfMQO gene was successfully amplified from genomic DNA of 3D7 P. falciparum and cloned into 2 expression vectors pET-15b and pUCI9. The presence and orientation of the gene in the respective vector were confirmed by restriction enzyme analysis and automated sequencing. The putative PfMQO gene (1566 bp) was successfully amplified and predicted to produce an approximately 60-kDa protein. Only when the PfMQO gene was cloned into pET15b was overexpression of the recombinant protein achieved. The recombinant PfMQO was purifiable under denaturing conditions due to its insolubility and formation of inclusion bodies. This protein is inactive and appears to be improperly folded thus producing the inconsistent results of the kinetic analyses. In conclusion, our study provides new insights into the understanding of the moleCular and biochemical regulation of atovaquone resistant parasites through a mutation in the bc1 complex. The partial characterisation of PfMQO is a starting point for the development of a new drug target in Plasmodium mitochondria by taking into account the uniqueness of this enzyme which is not found in the human host.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral