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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ItemReaction hijacking tyrosyl-tRNA synthetase as a new anti-infectives strategy( 2022)Malaria is a deadly disease of humans, with Plasmodium falciparum responsible for the most cases. Disappointingly, drug resistance is observed against current front-line therapies; thus, new drugs with novel mechanisms are urgently needed. ML901, a nucleoside sulfamate derivative, has been shown to possess good antimalarial efficacy and to specifically target P. falciparum tyrosyl-tRNA synthetase (PfYRS). PfYRS is a pivotal enzyme that participates in the protein synthesis pathway, in which tyrosine-charged tRNA is formed. ML901 appears to target PfYRS via a novel reaction hijacking mechanism in which PfYRS catalyzes the synthesis of a Tyr-ML901 adduct, which in turn poisons the enzyme. Human YRS is not susceptible to the reaction hijacking mechanism. This project sought to understand the molecular basis for the potency and specificity of ML901 and to determine if reaction hijacking could be exploited more widely. All YRS sequences harbor a conserved motif, referred to as “KMSKS”, in a loop that is reported to change conformation to facilitate ATP binding and the aminoacylation reaction. Sequence alignment across species reveals that most pathogenic parasites, including P. falciparum, possess a KMSKS motif, whereas higher eukaryotes possess an equivalent KMSSS motif. Structural analysis revealed that the motif in human YRS is part of a flexible (unstructured) loop while the equivalent loop is structured in PfYRS. Here we examined the role of the second lysine (K250) in determining loop flexibility and activity of PfYRS as well as the susceptibility of the mutant enzyme to reaction hijacking. Surprisingly, the X-ray crystal structure of recombinant PfYRS harboring the K250S mutation (PfYRSK250S) showed that the KMSSS loop is even more stable than the wildtype KMSKS loop. PfYRSK250S was found to consume substantively less ATP in the initial activation step. However, the weakly active PfYRSK250S is still susceptible to reaction hijacking by ML901. This study shows that the flexibility of the loop is not determined simply by the K250. Moreover, it shows that K250 plays an important role in enzymatic mechanism. Further investigations are required to understand the important factors that contribute to the particular susceptibility of PfYRS to reaction hijacking by ML901. Broad specificity nucleoside sulfamates, such as adenosine sulfamate (AMS), have previously been shown to have inhibitory activity against Gram-positive and Gram-negative bacteria. The equivalent of the KMSKS motif in the Escherichia coli YRS sequence is KFGKT. Here we explored the possibility that bacterial YRS might also be susceptible to inhibition via a reaction hijacking mechanism. A screen of a range of bacterial species revealed that AMS inhibits growth of E. coli and Enterococcus faecium. Targeted mass spectrometry confirmed the production of a range of amino acids adducts upon treatment in E. coli with AMS. Recombinant EcYRS was purified and expressed and shown to be inhibited via the reaction hijacking mechanism by AMS. These data suggest that bacterial amino acyl tRNA synthetases may be exciting new targets for reaction-hijacking nucleoside sulfamates.
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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.
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ItemUnderstanding the role of Maurer’s clefts in virulence protein trafficking( 2017)The malaria parasite Plasmodium falciparum modifies the host red blood cell to establish virulence protein-trafficking pathways. The major virulence protein, P. falciparum erythrocyte membrane protein 1 (PfEMP1) is exported from the parasite to the red blood cell surface, where it mediates attachment of the infected cell to ligands on the host vascular endothelium. This process of sequestration enables infected red blood cells to avoid immune detection in the spleen and contributes to the development of severe malaria. The Maurer’s clefts are organelles formed by the parasite and are present in the red blood cell cytoplasm. The primary function of Maurer’s clefts is thought to be the transport of PfEMP1 to the red blood cell membrane. We investigate a Maurer’s clefts protein, ring-exported protein-1 (REX1) and its role in PfEMP1 trafficking. We show that Maurer’s clefts morphology is disrupted by knocking down REX1 and that PfEMP1 surface display is decreased. Using transfectant parasites expressing truncated forms of the protein, we identify a repeat region of REX1 that mediates Maurer’s clefts morphology and is required for efficient PfEMP1 trafficking. We have developed a method to enrich the Maurer’s clefts from infected red blood cells and define their protein composition by tandem mass spectrometry. We epitope-tag a number of putative and established Maurer’s clefts proteins and confirm the location of several novel Maurer’s clefts proteins. Using super-resolution microscopy, we localise Maurer’s clefts proteins to subcompartments within these organelles. Finally, we use co- precipitation to describe a protein interaction network at the Maurer’s clefts.
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ItemTryptophanyl-tRNA synthetases as drug targets in the malaria parasite Plasmodium falciparum( 2016)Increasing resistance to first-line antimalarials has a strong impact on the health and economic burden of the disease, highlighting the need for new drugs with novel modes of action. The malaria parasite Plasmodium falciparum relies on efficient protein translation, so the loss of function of factors involved in protein biosynthesis could be detrimental to the parasites. Aminoacyl-tRNA synthetases (aaRS) are enzymes that are key to the production of substrates for protein translation, an event that occurs in three cellular compartments of Plasmodium: the cytosol, the mitochondrion, and a remnant chloroplast called the apicoplast. This work explores the tryptophanyl-tRNA synthetase (TrpRS), which charges tRNATrp, as a promising antimalarial target owing to its specificity within the parasite and its essential role in protein production. This work identified two isoforms of TrpRS in Plasmodium; one eukaryotic type that localises to the cytosol and a bacterial type that localises to the apicoplast. Using in vitro biochemical assays, the cytosolic TrpRS was found to preferentially aminoacylate tRNATrp from a eukaryotic source while the apicoplast TrpRS efficiently charges tRNATrp from a bacterial source. A structural analogue of tryptophan and an inhibitor of bacterial TrpRSs, indolmycin, specifically inhibits aminoacylation by the apicoplast TrpRS in vitro, and inhibits intraerythrocytic stage Plasmodium parasite growth, killing parasites with a delayed death effect characteristic of apicoplast inhibitors. Indolmycin treatment inhibits apicoplast inheritance and is rescuable by addition of the apicoplast metabolite isopentenyl pyrophosphate (IPP). These data establish that inhibition of an apicoplast housekeeping enzyme leads to loss of the apicoplast and this is sufficient for delayed death. Furthermore, repression of protein translation through the control of translation initiation, which is activated in response to various stressors, was explored as the mechanism of resistance not only to indolmycin but also to another potent aaRS inhibitor, borrelidin. Taken together, these findings identified the apicoplast TrpRS as an essential component of protein translation and a promising antimalarial target.
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ItemA network of protein interactions involved in the trafficking of PfEMP1 in Plasmodium falciparum-infected erythrocytes( 2016)Plasmodium falciparum is the most virulent human malaria parasite. Parasites invade red blood cells (RBCs) and extensively modify the structure and morphology of their host cell. As part of these modifications, the parasite establishes multi-protein virulence complexes that assemble within knob-like structures under the surface of the RBC plasma membrane. These structures allow infected RBCs to cytoadhere and sequester within the microvasculature of the host, effectively bypassing human clearance mechanisms that occur in the spleen. The most prominent members of these virulence complexes are protein members of the P. falciparum Erythrocyte Membrane Protein 1 (PfEMP1) family. The trafficking of PfEMP1 from the parasite to the host cell and its correct display on the RBC surface involves a complement of both host and parasite-derived trafficking machineries that are generated de novo during the approximate 48-hour lifecycle of the parasite inside its host RBC. In this work we examine the physical organisation of PfEMP1 trafficking intermediates in infected RBCs and determine interacting protein partners using an epitope-tagged minimal construct (PfEMP1B). Known and novel interacting-proteins were identified across multiple parasite and host compartments, consistent with our current knowledge of the PfEMP1 trafficking pathway. We show that PV-located PfEMP1B interacts directly with components of the Plasmodium Translocon of EXported proteins (PTEX) as well as a novel protein complex we refer to as the Exported Protein-Interacting Complex (EPIC). We define the EPIC interactome and using an inducible knockdown approach, show that depletion of one of its components, the parasitophorous vacuolar protein-1 (PV1), results in attenuation of infected RBC cytoadherence to endothelial cell ligands. Within the RBC cytoplasm, we show that PfEMP1B interacts with components of the Maurer’s clefts, and suggest a role for membranous tether structures in the transfer of PfEMP1 to the infected RBC surface. We also demonstrate a direct interaction between PfEMP1B and the RBC chaperonin complex, TCP-1 Ring Complex (TRiC), which may represent a novel mechanism for parasite recruitment of host factors in the export of parasite proteins such as PfEMP1. Ultimately, understanding and targeting the export of parasite virulence proteins may ultimately allow us to ablate parasite virulence in vivo.
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ItemMechanism of action of artemisinin antimalarials and implications for drug resistance in Plasmodium falciparum( 2015)The most deadly cases of malaria in humans are caused by Plasmodium falciparum. Artemisinin-based combination therapy is the current first-line treatment against this disease. However, artemisinin resistance has recently emerged in Southeast Asia, manifesting as delayed parasite clearance times in patients. Efforts to monitor and contain artemisinin resistance were initially frustrated by a lack of correlation between in vitro parasite sensitivity to artemisinins and in vivo drug efficacy in patients. Even though artemisinins have short in vivo half-lives, standard in vitro assays have traditionally employed extended drug exposure formats to assess parasite sensitivity to the drugs. We hypothesized that short drug pulses would better mimic in vivo conditions and be more clinically relevant for artemisinins. Using novel pulsed drug exposure assays, we demonstrate that laboratory and field (Pailin, Cambodia) parasite strains exhibit stage- and strain-dependent differences in drug sensitivities. Three stages with distinct drug sensitivities are identified in laboratory strains, namely hypersensitive early rings, insensitive mid-rings and sensitive trophozoites. Moreover, using this assay format, we are able to clearly distinguish the in vitro response of sensitive and resistant Pailin strains. We find that resistant field strains exhibit the highest levels of resistance at the very early ring stage and the late schizont stage. Our detailed analysis of field strains reveals that exposure to short pulses of artemisinins induces growth retardation in both sensitive and resistant parasites. Following this growth retardation, resistant strains survive, while sensitive parasites succumb. The data suggest that artemisinins are activated, and cause cellular damage, in both strains, but resistant parasites are better able to withstand the damage. As arrest in growth is often part of a cellular stress response, we postulated that artemisinin resistance is caused by an up-regulated parasite cellular defense mechanism. Consistent with this, we demonstrate that proteasome inhibitors effectively synergize artemisinin activity against both sensitive and resistance strains, with particularly strong synergism evident during the most resistant stage of the resistant strains. This suggests that the parasite proteasome system could be targeted to enhance drug action, offering a way to overcome artemisinin-resistant malaria. The mechanism of drug action is also investigated. Trophozoites take up and digest large amounts of host hemoglobin, and previous reports showed that the products of hemoglobin digestion can act as artemisinin activators in the trophozoite stage. However, the relevant activator or mechanism of action in rings is not clear. We show that chelating the labile iron pool has little effect on artemisinin activity against early rings. By contrast, hemoglobinases inhibitors strongly antagonize artemisinin action. We show for the first time that the hemoglobinases, falcipain-2 and -3, are expressed in rings. Moreover knockout of falcipain-2 and knockdown of falcipain-3, render the very early ring stage insensitive to artemisinins. These data lead to the surprising conclusion that hemoglobin digestion is active at the early ring stage and is involved in artemisinin activation.
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ItemThe Plasmodium food vacuole: protein targeting of transmembrane proteins( 2011)The causative agent of severe malaria in humans is Plasmodium falciparum – a parasite whose complex life cycle includes an asexual proliferation within human erythrocytes. Adaptations to the intra-erythrocytic lifestyle have created new and in some cases unique organelles, such as an endosomal/ lysosomal-like organelle, the food vacuole (FV). The only verified function of the FV is haemoglobin degradation and detoxification of the breakdown byproduct, haem. Several anti-malarial drugs interfere with this detoxification and thus kill the parasite. Two multi-spanning membrane proteins in the bounding membrane of the FV, Multidrug Resistant 1 (PfMDR1) and the Chloroquine Resistance Transporter (PfCRT), are associated with resistance against anti-malarial drugs. Protein targeting has been investigated for PfCRT, yet insights from this study are not readily transferable to PfMDR1. In this thesis I investigate the functions of the FV and underlying targeting processes by analysing its membrane proteome. I have expressed PfCRT as a green fluorescence protein (GFP)-tagged fusion and performed further studies on underlying targeting events, and on the biogenesis and development of the FV. I demonstrate that PfCRT targeting is dynamin-dependent and show the hitherto uncharacterised dynamic nature of the FV during intra-erythrocytic life stages. We are confronted with a limited knowledge about the functions and targeting events of membrane proteins to the FV. I have addressed these questions by applying bioinformatic approaches and thereby predicted 74 candidate FV membrane proteins. I have successfully expressed PfMDR1 and four candidate FV membrane proteins as epitope- or GFP-tagged proteins, as well as identifying a sixth likely FV membrane protein using a generated anti-peptide antibody. The subcellular localisation of four candidate proteins confirmed their FV association. Bioinformatic analysis of the putative functions of these revealed two transporter proteins, a pore-forming protein and a subunit of a targeting complex that is involved in retrograde transport from endosomes to the Golgi apparatus in other organisms. Further bioinformatic analysis of the remaining 70 candidate FV membrane proteins revealed eight putative transporter proteins involved in transport of ions, metabolites and amino acids in other organisms (two of which are localised to endosomes and lysosomes). To study the targeting processes of these known and novel FV membrane proteins, I investigated FV membrane trafficking routes using treatment of parasites with the fungal metabolite Brefeldin A, a compound inhibiting early secretory pathway processes. Finally, I have studied the contribution of the adaptin protein (AP) complexes to FV membrane protein targeting. The AP complexes mediate targeting of membrane proteins to endosomes and lysosomes in other organisms. The bioinformatic identification of four distinct AP complexes is described, as well as their subcellular localisation. I show that all four AP complexes are partially associated with the FV, and that AP-1 and AP-2 are strongly linked with the FV membrane. This suggests that AP complexes are involved in targeting of FV membrane proteins. Additionally, I have characterised the four P. falciparum AP complexes in regard to their sensitivity to Brefeldin A.