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.