Biochemistry and Pharmacology - Theses
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ItemAlternative splicing and stage differentiation in apicomplexan parasites( 2017)Alternative splicing is the phenomenon by which single genes code for multiple mRNA isoforms. This is common in metazoans, with alternative splicing observed in over 90% of human genes (Wang et al., 2008). However, the full extent of alternative splicing in apicomplexans has been previously under-reported. Here, I address this deficiency by transcriptomic analysis of two apicomplexan parasites: Toxoplasma gondii, which causes toxoplasmosis; and Plasmodium berghei, which is a murine model for human malaria. I identified apicomplexan homologues to SR (serine-arginine–rich) proteins, which are alternative-splicing factors in humans. I then localised a homologue, which I named TgSR3, to a subnuclear compartment in T. gondii. Conditional overexpression of TgSR3 was deleterious to growth. I detected perturbation of alternative splicing by qRT-PCR. Parasites were sequenced with RNA-seq, and 2000 genes were identified as constitutively alternatively spliced. Overexpression of TgSR3 perturbed alternative splicing in over 1000 genes. Previously, computational tools were poorly suited to compacted parasite genomes, making these analyses difficult. I alleviated this by writing a program, GeneGuillotine, which deconvolutes RNA-seq reads mapped to these genomes. I wrote another program, JunctionJuror, which estimates the amount of constitutive alternative splicing in single samples. Most alternative splicing in humans is tissue specific (Wang et al., 2008; Pan et al., 2008). However, unicellular parasites including Apicomplexa lack tissue. Nevertheless, I have shown that alternative splicing can still be common. I hypothesised that the tissue-specific alternative splicing of metazoans is analogous to stage-specific alternative splicing in unicellular organisms. I purified female and male gametocytes of P. berghei and sequenced these stages, with the aim of investigating alternative splicing and its relationship to stage differentiation. As a reference point, I first established the wild-type differences between female and male gametocytes. I detected a trend towards downregulation of transcripts in gametocytes compared to asexual erythrocytic stages, with this phenomenon more marked in female gametocytes. I was also able to identify many female- and male-specific genes, some previously-characterised, and some novel. My findings were further placed in an evolutionary context. Sex-specific genes were well conserved within the Plasmodium genus, but relatively poorly conserved outside this clade, suggesting that many Plasmodium sex-related genes evolved within this genus. This trend is least pronounced in male-specific genes, which suggests that sexual development of male gametocytes may have preferentially evolved from genes already present in organisms outside this genus. I then analysed these transcriptomes, now focusing on changes in alternative splicing. While non-gendered gametocyte differentiation is modulated by known transcription factors such as AP2-G (Sinha et al., 2014), I provide evidence that alternative splicing adds another level of regulation, which is required for differentiation into specific genders. I ablated a Plasmodium SR-protein homologue, which I named PbSR-MG. By transcriptomic analysis, I show that it regulates alternative splicing, predominantly in male gametocytes. Ablation was also associated with a drastic reduction in the viability of male gametocytes. Hence, I have shown that alternative splicing is common in apicomplexan parasites, is regulated by specific genes, and acts on specific targets. Alternative splicing is important for parasite viability and fundamental to stage differentiation in Plasmodium.
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ItemProtein localisation in a divergent eukaryotic parasite( 2013)Malaria infects 200 million people every year, with more than half a million of those cases resulting in death. Understanding the cell biology of Plasmodium falciparum remains largely an unsolved and unexplored problem. This doctoral assertion furthers understanding by investigating the sub-cellular localisation of Plasmodium falciparum proteins. A comprehensive literature review was undertaken, and the sub-cellular localisations of hundreds of proteins has been recorded in a transparent, traceable, publicly accessible and tactile fashion, and this serves as the basis for the further studies undertaken. It has been named ApiLoc. Using this curated set of protein localisations, the first Plasmodium falciparum-specific algorithm to predict sub-cellular localisation globally was created. Amino-acid based protein features were useful for prediction, but the predictor leveraged other predictive data types, such as microarray information and evolutionary conservation of the protein. Predictions were rigorously validated using both computational and epitope tagging approaches. ApiLoc also served as the basis for studies into the evolutionary conservation of protein localisation itself. This showed a remarkable lack of conservation, with only ~50% of protein localisations conserved across Apicomplexa, and ~20% throughout Eukaryota. The nucleus was studied in particular detail, given my involvement in a project to analyse isolated nuclei using a proteomic approach. Biochemical and bioinformatic studies were undertaken, providing further evidence that a classical nuclear import system involving basic-rich nuclear localisation signals is functional in Plasmodium falciparum and the evolutionarily related parasite Toxoplasma gondii.
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ItemAnalysis of architectural rearrangements in Plasmodium falciparum gametocytes( 2013)Transmission of the most virulent human malaria parasite, Plasmodium falciparum, is dependent on the parasite’s ability to produce viable gametocytes. Over two weeks the parasite prepares itself for transmission into mosquitoes by undergoing a series of cellular rearrangements. Once the metamorphosis is completed, mature gametocytes release from their sequestration sites, enter the circulation and become accessible to feeding mosquitoes. Whilst the formation of mature gametocytes represents a bottleneck in the parasite’s lifecycle, and thus an attractive target for transmission blocking strategies, very little of the basic biology of this lifecycle stage has been described. As the intraerythrocytic asexual stage parasite develops, it modifies its host red blood cell (RBC) by forming an exomembrane network of parasite-derived protein sorting organelles that facilitate the delivery of proteins to the RBC cytoplasm and membrane. Despite early ultrastructural descriptions of the gametocyte exomembrane network, no molecular characterisation of this system has been performed. In this thesis, modern high-resolution microscopy and immuno labelling techniques were used to re-evaluate the fine structure and molecular identity of several key components of the exomembrane system in the gametocyte. Early ultrastructural studies identified a sub-pellicular membrane complex in gametocytes. This structure consists of a flattened cisternal membrane beneath the parasite plasma membrane, which is supported by a network of microtubules. We have further described the molecular composition and origin of the sub-pellicular membrane complex, by identifying the presence of glideosome-associated proteins in gametocytes. We show that the gametocyte pellicle is analogous to the inner membrane complex (IMC), an organelle with structural and motor functions that is conserved across the Apicomplexan phylum. Thus we have proposed that the sub-pellicular membrane complex be renamed the gametocyte IMC (gIMC). We have also shown that the coordinated assembly of the microtubule network alongside the gIMC is responsible for driving shape change in the P. falciparum gametocyte. Interestingly, changes in cellular deformability correspond with gametocyte shape shifting. An increase in deformability coincides with the time the banana-shaped stage V gametocytes reappear in circulation. It has therefore been postulated that modifying the deformability of the host cell enables the gametocyte to circulate in the blood stream without being detected and removed by the mechanical filtering mechanisms in the host's spleen. Further investigation of how these changes occur show that the loss of cellular deformability is not dependent of the tubulin cytoskeleton of the parasite. Instead, it is associated with an altered molecular organization of the host cell membrane, as indicated by a loss of lateral mobility of the major RBC membrane protein, Band 3. Our studies have shown that several parasite proteins, which modulate rigidity of the RBC membrane in asexual stage parasites, are not exported in gametocytes. It appears that changes in gametocyte deformability are due to the rearrangement of cytoskeletal components, including host cell actin. The work presented in this thesis demonstrates that changes in gametocyte morphology and cellular deformability are crucial for the development of the P. falciparum gametocyte and discusses their likely contribution to the survival and transmission of the parasite.