Biochemistry and Pharmacology - Theses
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ItemOrganellar translation and inhibition in Plasmodium falciparum( 2023-09)The malaria parasite Plasmodium falciparum has two prokaryote-derived organelles: the mitochondrion and a relic plastid known as the apicoplast. These contain their own distinct, reduced genomes which must be transcribed and translated to maintain parasite viability. The bacterial-like proteins and metabolic functions of these organelles make malaria parasites susceptible to many anti-bacterials. This study aims to investigate organellar translation in P. falciparum, including the expression of apicoplast-targeted translation enzymes, tracking the cellular consequences of apicoplast translation inhibition, and measuring active organellar protein synthesis. Aminoacyl tRNA synthetases are a family of essential enzymes required for protein translation in the cytosol, apicoplast and mitochondrion. Several of these enzymes are encoded by single genes, from which two protein isoforms are proposed to be generated by alternative translation initiation. One isoform contains an N-terminal apicoplast localisation sequence, while the other lacks this and is cytosolic. In this study we investigate the significance of the nucleotides surrounding canonical and proposed translation start sites and show that these are important for their recognition by translation machinery. Additionally, we verify one of these dual-localised enzymes - threonine aminoacyl tRNA synthetase - as the target of the potent anti-microbial agent borrelidin in P. falciparum. Most organelle translation inhibitors have a lethal, but slow phenotype, killing parasites in the cycle following their administration. This has been attributed to disruption of apicoplast translation, with parasite death due to the inability to continue synthesis of essential apicoplast-derived isoprenoid metabolites. The consequences of isoprenoid starvation has been partially characterised, implicating lipophilic prenyl and isoprene chains as important, however not all essential isoprenoid products have been identified. We therefore aimed to investigate other downstream consequences of apicoplast translation inhibitors in Plasmodium. We found that apicoplast isoprenoids are required for synthesis of the major parasite sugar anchor glycophosphatidylinositol. Following inhibition of apicoplast translation, proteins typically anchored via this glycoconjugate became untethered, resulting in parasite segmentation, egress, and invasion defects. Difficulty in detecting proteins derived from organellar genomes had made the verification of organellar translation inhibitors challenging. Here, we use a mass spectrometry approach to directly detect and measure organellar translation in P. falciparum. This has facilitated the confirmation of the anti-apicoplast mechanism of action for the clinically used anti-malarials doxycycline and clindamycin. In addition, doxycycline was determined to inhibit mitochondrial translation, which was found to affect the activity of the electron transport chain. Together, this work has confirmed both the direct mechanism of action and indirect cellular consequences of organellar translation inhibitors on P. falciparum. In verifying the essentiality of glycophosphatidylinositols for multiple processes during the asexual stages, we have highlighted the potential for designing therapies that directly target aspects of glycophosphatidylinositol maturation or their protein attachment. Furthermore, determining the secondary target of doxycycline to be the mitochondrion has important clinical implications and may influence which drugs can be safely recommended for combination treatments.
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ItemUbiquitination in the malaria parasite Plasmodium falciparum( 2022)Ubiquitin is a post-translational modification that plays a role in many cellular processes, including protein degradation, trafficking, and signaling. The ubiquitination machinery includes E1 ubiquitin-activating enzymes, E2 ubiquitin-conjugating enzymes, E3 ubiquitin ligases, ubiquitin-binding domain-containing proteins, and deubiquitinases. In the malaria parasite P. falciparum, only a few ubiquitination proteins have been characterised and <10 more have been implicated in drug resistance. Post-translational mechanisms are known to be important in sexual development in Plasmodium, and so we investigated the role of selected ubiquitination proteins in differentiation into sexual forms called gametocytes. Using a CRISPR/Cas9 knockout strategy, we initiated the characterisation of selected ubiquitination genes that are upregulated in gametocytes compared to asexual parasites. We found two ubiquitination genes, encoding for a polyubiquitin binding protein and an E2 ubiquitin-conjugating enzyme, that play an important role on the regulation of sex-specific differentiation and stage development. Loss of the polyubiquitin binding protein produced gametocytes that reached late stages but lack a defined sex. Loss of the E2 ubiquitin-conjugating enzyme produced gametocytes with a morphological defect in the late stages and lack a defined sex. We also investigated the role of Kelch 13 (K13), a protein mutated in artemisinin-resistant parasites and hypothesised to be a ubiquitination protein and demonstrate that it is required for normal parasite uptake of haemoglobin. This work furthers our knowledge on the role of ubiquitination and of K13 in P. falciparum.
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ItemNo Preview AvailableInvestigation of alternative splicing in apicomplexan parasites( 2021)Alternative splicing is the phenomenon by which coding and non-coding regions of pre-mRNA molecules can be differentially spliced to yield multiple mRNA isoforms from a single gene. In metazoans, alternative splicing occurs to a substantial degree, contributing to protein diversity and the post-transcriptional regulation of gene expression. However, to what extent this occurs in apicomplexan parasites is much less understood. This thesis examines the landscape, regulators and function of alternative splicing in two apicomplexan parasites, T. gondii and P. falciparum. Technological advances in the short read sequencing of nucleic acids at unprecedented depths have enabled deep profiling of the transcriptome. However, the short reads present a limitation in the analysis of complex splicing events that span beyond the length of the reads. We evaluated the capability of a third generation long read sequencing technology, Oxford Nanopore Technologies (ONT) sequencing, in sequencing full-length native mRNA from T. gondii and P. falciparum, and established a method to analyse the alternative splicing landscape from the long reads. We successfully identified full-length transcripts spanning annotated and non-annotated junctions, implying a suitability in exploring complex splicing events. The analysis reveals an unusually high level of intron retained transcripts with premature terminating codons (PTCs). This suggests that most alternative splicing events in T. gondii and P. falciparum are unlikely to be productive. Alternative splicing in metazoans is modulated by alterative splicing factors, most notably the SR (serine-arginine–rich) protein family. We characterised the suite of SR proteins and two putative kinases/regulators of SR proteins in T. gondii. The proteins were found localised to sub-nuclear compartments characteristic of splicing factors. We demonstrated through genetic ablation and whole-transcriptome sequencing that the SR proteins modulate distinct but overlapping subsets of mostly non-productive alternative splicing events, as well as impacting transcript abundance. Alternatively spliced junctions were also enriched in characteristic SR binding motifs. The putative kinases of SR proteins were found to be essential to parasite survival and modulate extensive splicing events, but the events poorly mirrored that modulated by the SR proteins. This suggests a complex system of splicing regulation that do not conform to other eukaryotic models. The targeting of non-productive alternatively spliced transcripts for degradation through the nonsense mediated decay (NMD) pathway is one mechanism by which metazoans post-transcriptionally regulate gene expression. To explore if this was the case for T. gondii, we characterised the three core NDM proteins- UPF1, UPF2 and UPF3. The three proteins were found to co-immunoprecipitate with one another, implying a conservation of the core NMD complex. However, when we conditionally ablated the UPF proteins, parasite growth and survival was not impacted. We sequenced the parasite mRNA and found that only UPF2 impacted global intron retention rates. Moreover, a link between intron retention and gene expression regulation could not be established. Our results show that the fitness cost of mis- splicing determines intron retention rates, rather than targeted regulation. Hence, this thesis has shown that although non-productive alternative splicing is widespread and regulated in T. gondii, it is not a mechanism for post-transcriptional regulation of gene expression through the NMD pathway.