Science Collected Works - Theses
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ItemAn investigation of transporters of the relict plastid in the malaria parasite Plasmodium falciparum(University of Melbourne, 2009)The malaria parasite, Plasmodium falciparum, harbours a relict plastid known as the �apicoplast�. The organelle is essential and has emerged as a promising target for new antimalarials. The predicted organelle proteome identified complete pathways for fatty acid and isoprenoid biosynthesis plus a partial set of haem synthesis enzymes. Biochemical validation of the pathway components and the identification of inhibitors are ongoing. Although the apicoplast is capable of making several substances, the nature and fates of these substances remain uninvestigated to date. As such, the exact reason to why the apicoplast is indispensable is unknown. This thesis explores the apicoplast�s �permeome�, transporter proteins embedded in the apicoplast membrane that regulate exchange between the organelle and the surrounding cytosol. The first chapter provides an overview of the origin, metabolism and the functions of the apicoplast and outlines its probable intracellular role(s). Potential membrane components of the apicoplast are discussed in relation to what is known for plant plastids. A bioinformatic search for the central carbon transport and metabolizing enzymes in apicomplexan genomes compares our models for the P. falciparum apicoplast metabolism with related apicoplasts from coccidians and piroplasms. Although apicoplasts have relinquished the ability to photosynthesise, there is still a need to somehow procure carbon and energy to fuel their metabolism. Subsequent chapters address these questions. At the commencement of this thesis, only two apicoplast membrane proteins (PfoTPT and PfiTPT) had been identified. PfoTPT and PfiTPT are plant-like transporters that reside in the outermost and innermost apicoplast membranes and are postulated to transport reduced carbon compounds into, and perhaps out of, the apicoplast. In Chapter Two I describe the first characterization of the transport activities of PfoTPT and PfiTPT using a novel cell-free assay not previously applied to parasite transporters. PfoTPT and PfiTPT transport reduced 3-carbon compounds (phosphoenolpyruvate [PEP], triose phosphate [DHAP] and 3-phosphoglycerate) but not the 6-carbon compound (glucose-6-phosphate). I present a metabolic model in which the apicoplast imports PEP and DHAP to drive its isopentenyl diphosphate and fatty acid biosynthesis pathways. I also discuss how apicoplast might export 3-phosphoglycerate in exchange for DHAP as a means of generating ATP and reducing power in the organelle. Chapter Three investigates the targeting requirements of PfoTPT, the only known protein in the outermost membrane of the P. falciparum apicoplast. Nuclear- encoded proteins in the apicoplast stroma and the inner membranes utilize an N- terminal bipartite leader sequence to enter the organelle but no such leader occurs on PfoTPT, which presumably uses an alternative route to traffic to the apicoplast. I made a series of deletion constructs of the ten transmembrane domain-containing PfoTPT to identify elements essential for targeting to the outermost apicoplast membrane. I show that transmembrane domain 1 of PfoTPT functions as a recessed signal anchor and mediates insertion of PfoTPT into the endomembrane. However, despite making a range of deletions across the protein, no specific apicoplast membrane targeting element could be identified leaving the question of how transporters, or indeed other proteins, are specifically inserted into this membrane. Lastly, in Chapter Four I explored the presence of other transporters in the apicoplast. Bioinformatics was utilized to develop lists of candidates for more in depth in vivo analysis. Putative apicoplast transporter genes were modified with epitope tags by 3� replacement to localize the gene products. A new apicoplast membrane transporter belonging to the ABC (ATP-binding cassette) transporter family is shown to localize to the periphery of the apicoplast and its possible function is discussed.
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ItemDissecting apicoplast targeting in plasmodium falciparum and toxoplasma gondii(University of Melbourne, 2004)The phylum Apicomplexa is a group of obligate intracellular parasites, responsible for a wide range of diseases of humans and livestock. Two notable species are Toxoplasma gondii and Plasmodium falciparum. T. gondii is the causative agent of toxoplasmosis and causes congenital birth defects and medical complications in AIDS patients. Plasmodium falciparum, on the other hand, is responsible for the most deadly of all human diseases � cerebral malaria. The plastid (termed apicoplast) of apicomplexan parasites contains many plant-like features and is indispensable. It therefore provides a new avenue of treatment for tackling the huge medical and economic burden this group of parasites cause. Like other plastids the apicoplast contains its own genome, but only encodes a fraction of the proteins that it requires to meet all its biological functions. Instead, most of the proteins that function within this organelle are encoded in the nucleus and are post-translationally targeted across the apicoplast�s four membranes. In chapter one I review the field of plastid targeting, including the targeting across two, three and four membrane plastids. I then extensively review data on the rapidly expanding field of apicoplast protein targeting. The study of protein targeting has been greatly enhanced by the ability to genetically modify organisms and P. falciparum is no exception. However, creating transgenic P. falciparum is challenging due to the parasites low transfection efficiency and the troubles associated with creating the transfection vectors. In chapter two I describe two new series of P. falciparum transfection vectors based around Gateway� recombinatorial cloning that I have used to create the transfection vectors described throughout this thesis. These new plasmids are high copy number in E. coli and circumvent many of the problems associated with making P. falciparum transfection vectors to express fluorescent protein chimeras. Indeed, the second set of vectors based on Gateway�s multisite cloning system completely eliminates the need for restriction enzyme mediated cloning methods. In chapter two I also describe a new parasite fixation method for light microscopy and together with the new vectors I show their application in the study of P. falciparum cell biology. By creating different transgenic cell lines I perform localization studies on several important organellar proteins and also investigate the morphology of the endoplasmic reticulum (ER) and mitochondrion throughout the intraerythrocytic lifecycle. By creating double transgenic parasites expressing two fluorescent proteins I also preliminarily characterize the relationship between the apicoplast and mitochondria throughout the intraerythrocytic lifecycle. Targeting nuclear encoded proteins across the apicoplast�s four membranes requires a bipartite N-terminal extension. The first domain resembles a eukaryotic signal peptide, which is then followed by a domain resembling a plant transit peptide. In chapter three I investigate the nature of the first targeting domain - the apicoplast signal peptide. By swapping signal peptides with other secretory proteins, I show that apicoplast signal peptides contain no novel information and show that apicoplast protein targeting initiates via the general secretory pathway. I also show that the apicoplast shares an intimate relationship with the ER and show, using brefeldin A as an effector molecule, that nuclear-encoded apicoplast-targeted proteins, most likely do not pass through the Golgi apparatus. In chapter four I investigate the properties of the second targeting domain � the apicoplast transit peptide. Like plant plastid transit peptides, apicoplast transit peptides are enriched in hydrophilic and basic amino acids and depleted in acidic residues. Other lab members used these characteristics to create an algorithm to predict apicoplast proteins (termed �PlasmoAP�). I demonstrate the biological importance of rules embedded within this algorithm by creating a series of point mutations in model apicoplast transit peptides in both P. falciparum and T. gondii. I also show that the exact position of N-terminal positive charge is not important but that N-terminal transit peptide positive charge is more important than C-terminal positive charge. This set of experiments agrees strongly with the rules embedded in PlasmoAP and provides great confidence in this algorithm�s prediction of the apicoplast proteome. I also show using a point mutagenesis approach, that binding of the chaperone Hsp70 is most likely important in apicoplast transit peptide fidelity and that phenylalanine at +1 (relative to the signal peptide cleavage site) and hydroxylated amino acids may also be important apicoplast transit peptide features. In chapter five I further my analysis of apicoplast transit peptides. I demonstrate the evolutionary conservation of the apicoplast transit peptide by showing the functional equivalency of three plant transit peptides. Furthermore, with aid of PlasmoAP I show that both randomly generated sequence and exons from non-apicoplast genes can behave like transit peptides in vivo, illustrating that apicoplast transit peptides are just a simple collection of amino acids with certain properties. This also suggests that these loose parameters of transit peptides would allow these targeting signals to be easily acquired throughout the process of organelle evolution. In summary, this thesis dissects the mechanisms and pathway adopted by apicomplexan parasites to target proteins to the apicoplast. The work presented in this thesis outlines the biological parameters governing apicoplast targeting, and taken in a broader sense answers many questions relating to targeting to plastids with four membranes. This is also the first study to illustrate the simplicity of plastid transit peptides. This work has also led to the robust prediction of the apicoplast proteome, which in turn can be used as an avenue to explore new antiparasitic drugs.
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ItemThe Function of the apicoplast in the malaria parasite, plasmodium falciparum(University of Melbourne, 2003)Apicomplexan parasites such as Plasmodium falciparum and Toxoplasma gondii contain a relict plastid (the apicoplast) homologous to chloroplasts of plant and algal cells. The apicoplast is essential to parasite survival, but its function is unknown. Numerous nuclear-encoded proteins are translocated into the apicoplast courtesy of a bipartite N- terminal extension. This extension consists of a signal peptide and a transit peptide, but transit peptides are only poorly understood. I identified more than one hundred apicoplast proteins from Plasmodium falciparum and other apicomplexan parasites to learn more about their transit peptides. I found that although apicoplast transit peptides share some properties with plant chloroplast transit peptides, some features are distinctive to apicoplast proteins. I also extracted amino acid features from P. falciparum apicoplast transit peptides that distinguish them from other non-apicoplast P. falciparum proteins. This allowed the development of automated tools to recognise apicoplast proteins from raw sequence. I used a combination of pre-existing and custom designed bioinformatic tools to identify 545 putative apicoplast proteins from the Plasmodium falciparum genome. Expression of all genes that encoded predicted apicoplast proteins was analysed using oligonucleotide microarrays, revealing some groups of differentially expressed genes. The putative apicoplast proteome brings to light enzymes responsible for almost complete pathways for fatty acid, isoprenoid and haem biosynthesis. The apicoplast pathways, like those of chloroplasts, are bacterial in nature and offer excellent targets for antimalarials.