Medical Biology - Theses
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ItemAspartic proteases and their potential for transmission blocking strategies( 2019)Sexual stage development in Plasmodium spp. is essential for transmission through the mosquito and to the human host. It represents objects to study a broad range of biological processes, including stage conversion and parasite/host co-adaptation. After the bloodmeal, male and female gametes emerge from intracellular gametocytes and zygote formation follows fertilization. Ookinetes develop from the zygote and traverse through the midgut epithelial cell layer to the basal lamina side of outer wall and develop into oocysts, the only parasite developmental stage that grows extracellularly and this growth and development creates thousands of sporozoites. Once fully developed and egressed, these sporozoites are released into the mosquito hemocoel and they migrate to the salivary gland ready to infect next mammalian host and continue their life cycle. This sexual stage also represents a major bottleneck during the life cycle of Plasmodium as, in mosquito midgut, parasites have to persevere for up to 24 hours outside host cell, exposed themselves to various risk factors such as components of human immune system included within bloodmeal, natural midgut microbial flora in mosquito midgut, and mosquito innate immune system. This exposure can lead up to an approximate 300-fold decrease in parasite survivability during the transmission to mosquito. Due to this unique feature, sexual stage is prime target for transmission blocking intervention strategies aimed to inhibit spread of the disease by the mosquito. Protease enzymes are essential during many steps of malaria parasite development in the blood and transmission stages and an important group of these enzymes are the plasmepsins, of which there are 10 in Plasmodium acting at various points through the life cycle. So far, only 4 plasmepsins are identified to be involved in critical processes and required for transmission. Firstly, plasmepsin VI is highly expressed during sexual stages and was previously shown to be involved in sporozoite development in P. berghei. Secondly, plasmepsin VIII is expressed in mature sporozoite and responsible for sporozoite motility in P. berghei. Finally, PMIX and X are found to be essential in both blood and mosquito stages, making them stand out as promising drug targets. In this study, we attempted to determine the biological functions of plasmepsin VI, IX, and X during transmission of malaria parasites. We found that plasmepsin VI is required for transmission of P. falciparum and might plays an important role in sporozoite egress process instead of sporozoite development as observed in P. berghei. We also found that our dual inhibitor that target both plasmepsin IX and X is able to block the transmission of P. falciparum to mosquito while another antimalaria compound that target only plasmepsin X is enough to block transmission of P. berghei from mouse to mosquito suggesting that both plasmepsin IX and X are essential for transmission. Taken together, our data has identified 3 plasmepsins that play important roles in sexual stage of malaria parasites and more works are needed in order to determine the mechanism of action of these 3 proteases.
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ItemCharacterization of plasmepsin X as a cross-species antimalarial target( 2019)The emergence and spread of drug resistance have hindered the campaign for malaria eradication. The development of new drug targets is critical for our anti-malarial arsenal of interventions. Plasmepsins, which are aspartic proteases expressed by malaria parasites, serve important functions for parasite survival. Among the 10 members of this enzyme family, plasmepsin X (PMX) is essential for P. falciparum growth and has been shown to be involved in the egress of merozoites from infected red blood cells and the invasion of merozoites into red blood cells. Several aspartic protease inhibitors have anti-malarial activity on P. falciparum and are proposed to target PfPMX. The aim of this project was to investigate if these compounds affect P. knowlesi growth and whether PMX is a cross-species target for antimalarial development. This work showed that two aspartic protease inhibitors, 49c and 1SR, caused inhibition of P. knowlesi parasite growth. In further studies, live cell imaging demonstrated that these compounds inhibit P. knowlesi parasite growth by blocking parasite egress. Next, the optimal condition for protease activity was characterised after the expression and purification of a functional recombinant P. knowlesi plasmepsin X (rPkPMX). Using a fluorogenic protease assay, both 49c and 1SR were shown to inhibit the activity of rPkPMX. Furthermore, rPkPMX was able to cleave synthetic substrates, which were based on the predicted cleavage sites of PfSUB1, PfRAP1, PfRh2, TgROP1 and TgMIC6 predicted cleavage sites. By screening a panel of aspartic protease inhibitors, the BACE1 inhibitor, LY2886721, was identified as an inhibitor of rPkPMX activity as well as P. knowlesi and P. falciparum parasite growth. Therefore, PMX can be used as a cross-species target for antimalarial drug development.
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ItemCharacterisation of the Plasmodium aspartyl proteases DNA-damage inducible protein 1 (DDI1) and Plasmepsin VII (PMVII)( 2019)Plasmodium falciparum resistance to artemisinin-(ART) based combination therapies (ACTs) and other antimalarials poses a major threat to malaria control and elimination. Current efforts are aimed towards identifying potent antimalarials which inhibit multiple stages of the parasite lifecycle or discovering novel drug targets which may help overcome ART-resistance. This work aimed to characterise two aspartyl proteases of P. falciparum which may hold promise as antimalarial targets. One strategy recently proposed to overcome ART-resistance is the synergistic use of a parasite-selective proteasome inhibitor to sensitise ART-resistant parasites to artemisinin. Therefore, development of an inhibitor targeting a parasite-specific protein involved in the P. falciparum ubiquitin-proteasome system (UPS) could yield a combination therapy to tackle ART-resistance. DNA-damage inducible protein 1 (DDI1) is a previously uncharacterised essential aspartyl protease in P. falciparum. Recent studies have shown that the catalytic domain of human DDI2 upregulates the UPS in mammalian cells. In other organisms, DDI1 plays a role in shuttling proteins to the proteasome for degradation via its ubiquitin-like domain. We hypothesise PfDDI1 is an active aspartyl protease and plays a role in the parasite’s UPS. To investigate the role of DDI1 in the UPS and parasite survival, we identified a DDI1 orthologue in P. falciparum and characterised this using several strategies. We utilised CRISPR-Cas9 to knock out, tag and inducibly knock down DDI1 across the asexual lifecycle of P. falciparum, and study the effect of this on parasites. Expression of recombinant DDI1 proteins provided insight into the protease activity and substrate repertoire of PfDDI1. Together these studies provide insight into the domain architecture, essentiality and function of PfDDI1 and clues into its potential as an antimalarial target. Development of an antimalarial to block parasite transmission between humans and mosquitos is also a viable strategy to reduce malaria burden. In this study, we also explore a potential transmission-blocking target, Plasmepsin VII (PMVII) and create tools to enable further study of this aspartyl protease in sexually reproductive gametocytes. These tools are vital to determine the function and substrate repertoire of PMVII and elucidate its potential as an antimalarial target.
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ItemMolecular mechanism of cell traversal by Plasmodium falciparum( 2016)Malaria is an infectious mosquito-borne disease caused by apicomplexan parasites of the genus Plasmodium. Each year, malaria affects over 200 million people, causing considerable morbidity and mortality. A central feature of the virulence of malaria parasites is the ability of the liver-infective form of the parasite, known as sporozoites, to migrate from the mosquito bite site in the skin through host tissues to the target organ, the liver. The ability of sporozoites to traverse through different host cell types is crucial for the establishment and development of parasites within the mammalian host. Over the past decade, our understanding of traversal has become clearer through important studies using rodent models of malaria, such as P. berghei and P. yoelii. However, it remains unclear how these findings apply to malaria parasite species that infect humans, such as P. falciparum and P. vivax. Furthermore, proteins involved in the process, as well as a step-wise molecular model of it, remain unknown. In order to address these questions, the work presented in this thesis utilises molecular genetics and cellular biology to investigate the role of proteins in the traversal mechanism. Overall, this study has identified a novel role for two well-known proteins, Apical Membrane Antigen 1 (AMA1) and Merozoite Apical Erythrocyte Binding Ligand (MAEBL), in the traversal process. Furthermore, this study has validated the role Sporozoite Protein Essential for Cell Traversal (SPECT) and Perforin-Like Protein 1 (PLP1) in P. falciparum sporozoites, which are two proteins that previously have been identified as playing a crucial role in traversal using rodent models of malaria. Using mice engrafted with human hepatocytes, this study also demonstrated the importance of traversal for P. falciparum sporozoites to establish infection of human hepatocytes in vivo. Together, these findings provide the first molecular understanding of cell traversal by P. falciparum and give valuable insights into the complexity of traversal and allowed the formation of a basic molecular model for this process.
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ItemNaturally acquired humoral responses to Plasmodium vivax and Plasmodium falciparum: identification of antigenic targets to inform rational biomarker and vaccine development( 2016)Malaria is an infectious disease caused by Plasmodium spp. parasites, transmitted by the bite of infected Anopheles mosquitoes. Among the five species that can cause disease in humans, P. falciparum and P. vivax are responsible for the majority of the cases and deaths. Due to increased political commitment and funding, the last decades have experienced a dramatic reduction in the burden of malaria, with several countries now attempting to permanently eliminate this disease. Achieving the goal of malaria elimination would be greatly facilitated by the development of biomarkers that can identify the remaining populations at-risk, as well as an effective vaccine. However, while it is clear that individuals living in endemic areas become gradually protected against malaria disease, the targets and mechanisms underlying the acquisition of natural immunity are complex and still poorly understood, hindering the development of such tools. This thesis aimed to investigate comprehensive panel of P. vivax and P. falciparum proteins as targets of natural immunity in Asia Pacific populations, and how this information can be used to inform rational vaccine and biomarker development. Strong associations of antibody responses to both novel and known P. vivax antigens with protection against clinical malaria were identified, as well as optimal antigenic combinations with predicted protective efficacy above 90%. By comparing humoral responses to P. vivax and P. falciparum, this thesis shows that early immune responses are markers of exposure and thus increased risk, whereas prolonged exposure and higher antibody titers are required to achieved clinical protection. The findings of this study support the development of a highly efficacious multicomponent malaria vaccine, and the use of serology as a surveillance tool.
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ItemMerozoite antigens of Plasmodium falciparum elicit strain transcending opsonising immunity( 2015)Despite progress towards reducing the global burden, malaria continues to cause approximately 200 million cases and 600,000 deaths annually (World Health Organization, 2014). Although several malaria vaccines are currently in clinical trials, no advanced vaccine candidate has yet demonstrated sufficient efficacy to be a stand-alone vaccine against the highly variant Plasmodium falciparum parasite. Development of effective vaccine strategies requires knowledge of the essential mechanisms for protective immunity and robust assays to serve as correlates of protective immunity. However, exactly which antibody functions are necessary to control parasitemia and clinical symptoms during natural infection remains unclear. The merozoite represents an attractive vaccine target, as antibodies to numerous merozoite antigens have been associated with protective immunity in human cohort studies. This thesis aimed to investigate the importance of merozoite opsonising antibodies for immunity to malaria. Opsonising antibodies, and the Fc Receptor-mediated functions these antibodies elicit, have been poorly studied in malaria partly due to limitations of in vitro assays. Therefore, in this thesis a merozoite phagocytosis assay was developed and validated (Chapter 3), and robust and reproducible phagocytosis responses from THP-1 cells were observed. This assay was then used to measure merozoite opsonisation in a longitudinal study of semi-children from Papua New Guinea (PNG), and phagocytosis responses were demonstrated to correlate with protection from clinical disease and high-density parasitemia (Chapter 4). Due to the highly diverse nature of P. falciparum merozoites, it was important to assess whether merozoite opsonisation involved strain-specific or strain-transcending specificities (Chapter 5). Highly consistent opsonisation and associations with immunity were observed across a panel of common laboratory strains and PNG parasites adapted to growth in vitro. Through use of transgenic parasite lines, the absence of MSP3, MSP6, MSPDBL1 or MSP1-19 was not observed to impact the overall level of merozoite phagocytosis. By depleting antibody reactivity to 3D7 merozoites, opsonisation of merozoites from PNG strains also declined, suggestive of conserved antigenic targets across parasite strains. The findings in this thesis have demonstrated the importance of opsonising antibodies and their associated phagocytic responses for protective immunity to malaria. Robust, reproducible and well-validated assays are a priority to aid pre-clinical and clinical malaria vaccine development. The consistent responses and protective associations provide strong support for merozoite opsonisation as a robust correlate of protective immunity in malaria endemic populations. As the majority of merozoite opsonising antibodies were strain-transcending, uncovering these conserved domains within merozoite surface antigens may yield important novel vaccine candidates with which to tackle this deadly disease.
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ItemActin regulation in Plasmodium falciparum: towards understanding the elusive nature of malarial actin filaments( 2015)Malaria disease, caused by the unicellular parasites from the genus Plasmodium, is a major cause of morbidity and mortality in many developing countries throughout the world. While there have been many improvements in intervention strategies in recent years, parasite resistance to front-line therapeutics is on the rise, highlighting the need for new and improved treatments and vaccines. To this end, a greater understanding of the biological mechanisms underpinning the disease will be crucial in the push towards malaria eradication. Across the malaria life cycle the parasite must traverse tissues and invade host cells in order to establish an infection and replicate. A conserved acto-myosin motor, anchored at the parasite periphery, generates the requisite force to drive the parasite forward, facilitating both invasion and motility. The actin at the heart of this motor is extremely divergent, forming filaments that are highly dynamic and unstable. Tightly controlled regulation of malaria actin is therefore necessary to direct the formation and disassembly of filaments in an appropriate spatio-temporal manner. However, malaria parasites possess a markedly reduced repertoire of actin regulators, of which coronin is one of the only predicted filament regulators. Much of the current literature surrounding Plasmodium actin biology relies on the production of actin from recombinant sources. In this study I investigate the various published methods for purifying recombinant malaria actin, and determine that the unusual characteristics previously reported for this actin are likely artifacts driven by incomplete protein folding in heterologous expression systems. This finding lead to the identification of the key actin folding chaperonin CCT in the Plasmodium genome, an essential protein complex required for producing native, functional actin in the cell. In parallel, characterization of the filament regulator, coronin, revealed its critical role in the organization of actin filaments. Using in vitro observations from recombinant Plasmodium falciparum coronin (PfCoronin), I have demonstrated that PfCoronin binds to actin filaments and bundles them together in parallel arrays. Furthermore, in vivo observations revealed PfCoronin to be located at the periphery of the parasite, consistent with the pellicular space in which the actin-myosin motor is housed. This localization is likely mediated by peripheral interactions with PI(4,5)P2 at the plasma membrane. These data identify PfCoronin as a potentially key regulator of actin filament recruitment and bundling at the cell cortex of motile Plasmodium parasites. Taken together, the identification of Plasmodium CCT and the characterization of PfCoronin have opened up new avenues for further development of these as potential drug targets, with the eventual aim of potentially crippling the motile malaria parasite and halting the progression of disease.