Oxidative stress in plants and the malarial parasite Plasmodium falciparum

Author: Thi Thu Suong Cu

Cu, Thi Thu Suong, 2011 Oxidative stress in plants and the malarial parasite Plasmodium falciparum, Flinders University, School of Biological Sciences

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This thesis is divided into three main parts. Part A of the project aimed to investigate the effects of the mixed-culture of wheat (Triticum aestivum L.) with white lupin (Lupinus albus L.) on the availability of soil-P to the wheat and to study the mechanisms by which white lupin mobilises P from unavailable soil-P pools making it more available to the wheat crop partner. P limitation is a major problem for many agricultural systems, particularly in the tropics and subtropics. White lupin is well-known for its ability to mobilise P from a P pool that is normally unavailable to plants. In this study, white lupin was grown in a mixed culture with wheat and the effects of the mixed culture on the availability of P to the wheat was investigated using a novel leaching system. The results show that while lupin was capable of mobilising the P that was locked up in the soil, as the level of the immobile P pool was significantly lower in the presence of white lupin compared to the wheat monoculture. This ability of white lupin to mobilise the unavailable P was demonstrated to have beneficial effects on the growth and P uptake of the wheat when they were grown in a mixed-culture. Part B and C of this study focuses on the 2-Cys peroxiredoxin proteins of malarial parasite Plasmodium falciparum; PfPrx-1 and PfPrx-2 respectively. Malaria parasites are highly exposed to oxidative burden due to a large amount of reactive oxygen and nitrogen species produced by haemoglobin degradation in the food vacuole of the parasite and the immune effects of the host in response to parasite infection. Therefore, inside erythrocytes, the ability of the parasite to defend itself against oxidative damage is of vital importance for parasite survival. Such defences are thus expected to be potential targets for malaria control strategies. This study focuses on the 2-Cys peroxiredoxin (Prx) system of P. falciparum; these enzymes are mainly responsible for the detoxification of hydrogen peroxide and other organic peroxides in the parasite, thereby protecting the parasite against oxidative stress. Part B of the project aimed to study the factors influencing the oligomeric states of the P. falciparum PfPrx-1 protein recombinantly expressed in E. coli and to investigate its three dimensional structure using X-ray crystallography. Firstly, the PfPrx-1 gene was successfully expressed in E.coli for protein production. Following high-level expression, the PfPrx-1 protein was purified to homogeneity. The purified PfPrx-1 protein was studied for its Prx activity and its oligomeric structure. The PfPrx-1 protein exists in both decameric and dimeric forms. High pH (8.5) and high concentrations of urea (2.8 M) seem to favour the formation of dimers. Attempts at crystallization of the protein yielded crystals, of which one diffracted to around 6-7 A. Eventhough these data were not enough to sovle the structure of the protein, they could serve as a ground for further studies in order to obtain the full details of PfPrx-1 3-D structure enabling inhibitor studies for structure-based drug design. Part C of this study aimed to develop a Pichia pastoris expression system for the P. falciparum PfPrx-2 protein and to investigate the oligomeric states and the subcellular localization of the recombinant PfPrx-2 protein. The P. pastoris expression system was demonstrated to be able to translate this P. falciparum A+T rich sequence and recognise the mitochondrial targeting sequence of the protein. This production system could be up-scaled to obtain sufficient quantities of recombinant PfPrx-2 protein to enable structural and functional studies for structure-based drug design. Evidence for the production by P. pastoris of the PfPrx-2 protein as an oligomer (decamer and larger) was obtained by gel filtration chromatography, SDS-PAGE and Western blotting. It was found that purification of the functionally active PfPrx-2, by Ni-affinity chromatography, resulted in the protein being 'locked' in the dimeric state and it could not be converted to the monomer by a variety of reducing agents as indicated by SDS-PAGE.

Keywords: oxidative stress,antioxidant,malaria,Plasmodium falciparum

Subject: Biological sciences thesis

Thesis type: Doctor of Philosophy
Completed: 2011
School: School of Biological Sciences
Supervisor: Kathy Schuller