Biochemistry and Pharmacology - Theses

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Organellar translation and inhibition in Plasmodium falciparum

Bulloch, Michaela Susan ( 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.
Reaction hijacking tyrosyl-tRNA synthetase as a new anti-infectives strategy

Tai, Chia-Wei ( 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.
Ubiquitination in the malaria parasite Plasmodium falciparum

Tutor, Madel Verra ( 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.
Understanding virulence protein trafficking in the P. falciparum infected red blood cell

Carmo, Olivia Maria Silva ( 2022)

After invading the red blood cell (RBC), the malaria-causative parasite P. falciparum traffics an adhesin, erythrocyte membrane protein 1 (here referred to as EMP1), to the host cell surface. EMP1 is essential for parasite survival in vivo as it prevents splenic clearance. Moreover, variants of EMP1 that confer cytoadherence to cerebral or placental tissue can lead to fatal complications of the disease. In addition to EMP1, ~500 other parasite proteins are exported into the host cell compartment, localizing to parasite- induced membrane-bound and membraneless structures and knob-like protrusions at the RBC surface. These exported proteins do not resemble canonical trafficking machinery (e.g., ESCRT, SNARE, and Rab machineries), and there is no remnant secretory machinery for the parasite to co-opt, so how EMP1 is transported through the host cell cytoplasm remains unclear. Here we present functional characterization of two exported proteins with potential roles in EMP1 transport. First focusing on the gametocyte exported protein 7 (GEXP07), we found that GEXP07 localizes to the Maurer’s clefts, an intermediate compartment for EMP1 trafficking. In the absence of GEXP07, the clefts segment into smaller membrane- bound structures, the knobs are larger and clustered, and EMP1 transport to the host cell surface is reduced. The work confirms the critical role of Maurer’s clefts in EMP1 trafficking and reveals a previously unappreciated link between EMP1 transport and host cell remodeling. We also characterized the PfEMP1 trafficking protein 7 (PTP7). We found that PTP7 localizes to the Maurer’s cleft and associated structures, including vesicles and membraneless structures called J-dots. In the absence of PTP7, the clefts become decorated with budding vesicles - seemingly stalled in the process of fission. The knobs morphology is altered and forward trafficking of EMP1 from the cleft to the infected RBC surface is ablated. We show that the poly-asparagine repeat-containing C-terminal domain of PTP7 is essential for its function. The work leads to the intriguing suggestion that low complexity domains might be important for the function of these non-canonical trafficking proteins. We sought to further understand the physical processes that might drive trafficking of exported proteins and explored the role of intrinsically unstructured protein domains, which have been shown to drive some trafficking events in other organisms. As mobile, membrane-less structures, the J-dots resemble the phase separated biomolecular condensates observed in other eukaryotes. Here, we used an optogenetic technique in a heterologous mammalian cell system to explore the condensate-forming potential of full-length J-dot proteins and protein domains. We identified the central region of a protein called 0801 as having a propensity to phase separate. We generated sequence variants of this protein region and determined the molecular grammar responsible for condensate formation. This work points to the possibility that intrinsically unstructured protein domains could play a previously unrecognized role in protein trafficking and/or protein sequestration in P. falciparum. Overall, the work presented in this thesis adds new insights to our understanding of EMP1 trafficking. Also, in the context of the RBC cytoplasm which lacks canonical trafficking machinery, our preliminary findings regarding the phase separation capacity of some exported proteins may inform the wider field of protein transport. If validated in P. falciparum, our findings prompt a re-evaluation of the molecular requirements for coordinated protein transport in eukaryotes more broadly.
Alternative splicing and stage differentiation in apicomplexan parasites

Yeoh, Lee Ming ( 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.
High-content screening of antimalarial drugs using metabolomic approaches

Chua, Hwa Huat ( 2016)

Malaria continues to have an appalling health and economic impact on many of the poorest countries in the world, with more than 214 million cases and 438,000 deaths a year. While considerable progress has been made in reducing the overall impact of malaria, the emergence of clinical resistance to current first-line antimalarial drugs threatens to roll back these advances. The reliance on drug treatments is further exacerbated by the limited efficacy of recent vaccine trials. Malaria is caused by mosquito-transmitted protists belonging to the genus Plasmodium. Of these, P. falciparum is the cause of the most serious form of malaria. In order to identify new lead compounds, a number of high-throughput live cell-based screens using P. falciparum have been undertaken with millions of compounds derived from the GlaxoSmithKline (GSK), St. Jude Children's Research Hospital, and Novartis compound libraries. These screens identified thousands of new drug-like compounds which selectively kill asexual intraerythrocytic stages of P. falciparum. In order to promote research on the modes of action of these compounds the Medicines for Malaria Venture (MMV), assembled a subset of 400 compounds into the Malaria Box which has been made freely available to the research community for further characterisation. The major goal of this study was to develop a generic metabolomic approach for investigating the mode of action of the Malaria Box compound library and other lead compounds identified in live-cell screens. A high content metabolomics screen was developed to allow measurement of several hundred intracellular metabolites using gas chromatography-mass spectrometry (GC-MS). GC-MS was chosen as it allows highly reproducible coverage of many metabolites in parasite central carbon metabolism. Initial studies demonstrated that GC-MS profiling could readily differentiate between uninfected red blood cell (uRBC) cultures and P. falciparum infected RBC (iRBC) cultures (at 10% parasitaemia) without fractionation of the infected cultures (i.e. separation of infected RBC from 90% uninfected RBC). A screen was subsequently developed in which synchronised P. falciparum-infected RBC cultures in a 24-well plate format was treated with drugs of interest (1 µM concentration) for 12 hours when they reached mid-trophozoite stage (32-hour post-invasion). Extensive method development resulted in an optimised protocol for rapid quenching of parasite/RBC metabolism and metabolite extraction. This screen was validated using metabolic inhibitors with known modes of action, as well as several front-line antimalarial drugs. Atovaquone, an antimalarial that is thought to primarily target the cytochrome bc1 complex in P. falciparum asexual erythrocytic stages was found to induce changes in a limited number of mitochondrial metabolites including fumarate and γ-aminobutyric acid (GABA). In contrast, drugs such as artemisinin and chloroquine that kills the parasites induced changes in a large number of metabolites across many metabolic pathways. This screen was subsequently used to profile the impact of the 80 most potent Malaria Box compounds on the metabolome of mid-trophozoite stage (1 µM concentration, 12 hours of exposure). Hierarchical cluster analysis (HCA) was used to triage these compounds into groups with putatively similar modes of action. Strikingly, this analysis allowed clustering to many of the 80 compounds into two major groups: In particular, 31 Malaria Box compounds, as well as chloroquine, resulted in characteristic changes in a range of metabolites, including a signature decrease (> 1.5-fold) in the metabolite pipecolate (a putative intermediate in lysine degradation). Another 22 Malaria Box compounds clustered together, and resembled atovaquone-induced metabolic phenotype, that included a > 1.5-fold accumulation of fumarate. Three additional metabolic phenotypes were also identified in this initial screen, highlighting its utility in broadly grouping drugs with known antimalarial activity and potentially similar modes of action. To further characterise the mode of action for the 22 Malaria Box compounds that induced atovaquone-like metabolic perturbations, the effect of these compounds on the mitochondrial electron transport chain and pyrimidine biosynthesis were investigated. These processes are linked as one of the key enzymes in pyrimidine biosynthesis, dihydroorotate dehydrogenase (DHODH), is dependent on the respiratory chain for activity. The impact of the Malaria Box compounds on pyrimidine biosynthesis was assessed by measuring changes in early intermediates in this pathway using liquid chromatography-mass spectrometry (LC-MS) and by metabolic labelling studies with H13CO3- which is incorporated into these intermediates. Eight of the 22 Malaria Box compounds inhibited pyrimidine biosynthesis in the same way as atovaquone, leading to the accumulation of N-carbamoyl aspartate and depletion of downstream metabolite uridine monophosphate (UMP). Further differences in the mode of action of these Malaria Box compounds and atovaquone were indicated by the finding that episomal-expression of the yeast DHODH, which by-passes the need for a mitochondrial respiratory activity in asexual erythrocytic stages, conferred resistance to fourteen of these compounds. In contrast, expression of yeast DHODH leads to effective resistant to atovaquone. Finally, none of the Malaria Box compounds exhibited synergistic activity with proguanil, indicating that they either target different sites on the cytochrome bc1 complex or other proteins altogether. Studies were also undertaken to investigate the function of pipecolate, which appears to be part of a parasite specific metabolic pathway, as it was only found in P. falciparum infected RBC and was selectively depleted when infected RBC were treated with several first-line antimalarials (artemisinin and chloroquine) and 31 Malaria Box compounds. Several lines of evidence suggested that pipecolate is generated during catabolism of lysine, which in turn is produced during haemoglobin degradation in the parasite food vacuole. In particular, inhibition of haemoglobin degradation led to concomitant decreases in both lysine and pipecolate, while metabolic labelling studies with 13C-lysine demonstrated conversion to 13C-pipecolate in vivo. Further, in vitro studies using positionally labelled lysine suggested the operation of an unanticipated lysine catabolic pathway involving the initial conversion of lysine to saccharopine. Pipecolate, but not lysine, is actively secreted by P. falciparum-infected RBC, and its synthesis may constitute an important mechanism for removing and/or detoxifying excess lysine generated by constitutive haemoglobin degradation. Further studies are needed to determine whether lysine degradation is essential in P. falciparum, which could be further explored as a potential novel drug target. Finally, the 24-well plate screen was further extended to measure macromolecule biosynthesis using heavy water, 2H2O labelling. Incubation of cells in the presence of low concentrations of 2H2O leads to the incorporation of deuterium (2H) into a wide range of metabolic intermediates that are subsequently used for macromolecule biosynthesis. A workflow was developed for measuring the turnover of DNA, RNA, proteins and lipids in P. falciparum-infected RBC following labelling of cells in 5% 2H2O and GC-MS analysis of constituent components of these macromolecules (i.e. deoxyribose, ribose, amino acids and fatty acids). Incorporation of 2H enrichment in DNA (deoxyribose) and RNA (ribose) at different stages of intraerythrocytic parasite development indicated stage-specific expression of DNA and RNA throughout the 48-hour cycle. As expected, incorporation into these molecules was inhibited by aphidicolin (DNA synthesis inhibitor) or actinomycin (RNA synthesis inhibitor), respectively. Interestingly 2H-enrichment in protein and lipids was minimal or not detected indicating that precursors for these molecules are largely derived from the host cell. Screening of late ring stage parasites (18-hour post-invasion) with 240 of the Malaria Box compounds (1 µM concentration, 12 hours of exposure) led to the identification of 47 compounds that inhibited DNA biosynthesis by > 50% DNA synthesis, with concomitant partial or strong inhibition of RNA biosynthesis. Detailed stage-specific analysis of these compounds provided further insights into different modes of DNA/RNA biosynthesis. Collectively, these studies have led to the development of two screens for investigating the mode of action of compounds that have been shown to have antimalarial activity in live-cell screens. The high content GC-MS based metabolomic screen demonstrated that many of the Malaria Box compounds can be grouped based on the metabolic responses that they generated. This raises the prospect that many of the top hits identified in these screens may target relatively few processes in the parasite (or the host cell). It will be of interest to extend these studies to the next tier of compounds which may exhibit lower malarial activity but allow identification of other drug targets. These studies have also highlighted unanticipated metabolic pathways in P. falciparum, such as lysine degradation, which may be important for virulence and potential drug targets.
Tryptophanyl-tRNA synthetases as drug targets in the malaria parasite Plasmodium falciparum

Pasaje, Charisse Flerida ( 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.
Mechanism of action of artemisinin antimalarials and implications for drug resistance in Plasmodium falciparum

Xie, Stanley Cheng ( 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.
Synthesis and investigation of activities and modes of action of antimalarial compounds

TEGUH, SILVIA ( 2015)

Syntheses, structure-activity relationships and mode of action investigations of a novel compound series of conjugated quinoline-indole, revealed that they are active against blood stages of multiple strains of Plasmodium falciparum, and appear to dissipate mitochondrial potential. To aid in the study of current antimalarial drug, artemisinin, components of a “bait-and-probe” strategy were synthesised (using click chemistry). The work in this thesis has shown that the azide-linked drug-“bait” can be covalently incorporated into a protein, which acts as a potential drug target, prior to being coupled with its identifier “probe” (e.g. biotin).
Dissecting the molecular basis of malaria parasite movement and host cell traversal in the mosquito midgut

Angrisano, Fiona ( 2015)

Understanding the processes by which vector-borne pathogens colonise their invertebrate host is a fundamental question both in terms of the co-evolutionary biology of host-pathogen interactions and in dissecting the molecular basis of disease transmission. For the malaria parasites in particular, from the genus Plasmodium, the process by which the parasite targets and traverses the mosquito midgut epithelium is a critical bottleneck in lifecycle progression. Motility is a fundamental part of cellular life and survival for Plasmodium parasites. The motile life cycle forms achieve motility, called gliding, via the activity of an internal actomyosin motor. Although gliding is based on the well-studied system of actin and myosin, its core biomechanics are not completely understood. Although decades of research have revealed several key molecules involved in parasite traversal there is still little real understanding of the stepwise events that govern the journey of a parasite from the blood bolus to its destination under the basal lamina of the midgut. Despite the recent gains in reducing the burden of malaria disease in human populations there is still a pressing need to generate new therapeutics and strategies targeting this global pathogen. It has been recognised that any successful programme aiming towards disease eradication or elimination cannot rely on preventative and therapeutic treatments alone, but must also incorporate strategies to block parasite transmission through the mosquito vector. Much of the current literature surrounding Plasmodium transmission focuses on other lifecycle stages, neglecting the insect stage ookinete, which in itself presents a natural lifecycle bottleneck during progression through the mosquito midgut. Our findings suggest that ookinetes require dynamic actin in order to move and that this movement occurs in a left-handed helical fashion due to parasite shape. Extending our knowledge of key traversal proteins and their function, work on the vaccine candidate Cell Traversal protein for Ookinetes and Sporozoites, show that this protein is secreted in a Calcium stimulated, cGMP dependent protein kinase supported manner. Taken together, this body of work sheds light into the major interactions between the parasite and the mosquito in order to help support the broader goal to identify targets for transmission- blocking vaccine therapies against malaria disease.

Biochemistry and Pharmacology - Theses

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