Medical Biology - Theses
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ItemThe Parasite Genetic and Host Immunological Determinants of Immune Escape in Plasmodium falciparum Malaria( 2022-12)Abstract Human malaria remains a major global public health problem with an estimated 241 million clinical cases and 627,000 deaths in 2020, expected to increase in future years. Highly effective vaccines are urgently needed to progress the control and elimination of the disease. There are dozens of candidates in development, however only one vaccine (RTS, S) targeting the most virulent human malaria parasite, Plasmodium falciparum, has reached Phase 4 implementation trials with 50% efficacy that is short-lived and strain specific. As WHO has outlined a goal for malaria vaccines with a 75% efficacy against clinical malaria in all malaria-endemic countries by 2030, novel approaches are needed to increase efficacy. The limited efficacy of malaria vaccines to date has been in part attributed to the extreme diversity of parasite antigens being developed as ‘subunit’ vaccines, with only one or two randomly selected allelic variants as the basis for inducing immune responses. Antigen diversity has evolved as a means for malaria parasites to evade host immune responses - a process known as an immune escape. Pinpointing specific antigen polymorphisms that drive immune escape would help to prioritise antigens and alleles for inclusion in vaccine formulations. In my Ph.D. project, I investigated the hypothesis that specific polymorphisms in leading P. falciparum vaccine candidates are associated with immune escape. To test this hypothesis, I first analysed the publicly available MalariaGEN genome sequence data to catalogue the global genetic diversity of the genes encoding 25 leading P. falciparum vaccine candidate antigens. Predicted regions of immune selection were identified on both the linear gene sequence and the 3-dimensional protein structure. We then focused on two cohorts of children from malaria endemic regions of PNG conducted during moderate and high transmission periods. We analysed samples from 758 children, conducting multiplexed high-throughput assays on serum samples to measure IgG responses against 27 antigens, and targeted amplicon sequencing of 38 parasite antigen genes in sequentially collected samples from each child to measure the rate of allelic turnover for each antigen. The analysis identified critical immune escape genes and their specific polymorphisms that contribute to immune escape. The relationship between measures of genetic diversity and immune selection in the global data, and the antibody response in the children identifies antigens driving immune escape and those where diversity did not appear to contribute to immune escape. This research provides a vital framework for the prioritization of vaccine candidate antigens and a ‘serotype classification system’ to identify immune escape polymorphisms and for evaluating strain specific efficacy during vaccine trials.
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ItemMolecular mechanisms of liver infection by the human malaria parasite Plasmodium falciparum( 2021)Malaria is the disease caused by Plasmodium parasites. The parasite infects the red blood cells giving rise to symptoms but it musts first infect the liver to reach the blood. Blocking liver infection would prevent both malaria disease and onward transmission as well as stimulate immunity. However, little is known about parasite-host interactions during liver infection of Plasmodium falciparum, the species responsible for the most lethal form of malaria in humans, as its pre-erythrocytic stages are challenging to study. Plasmodium sporozoites are injected in the dermis by the bite of an infected mosquito. They make their way from the skin to the bloodstream and finally the liver, where they invade and replicate within a hepatocyte. The sporozoite’s journey from the skin to the host liver is enabled by a remarkable process called cell traversal that allows parasites to migrate and penetrate deeper into host tissues by entering and then rupturing host cells. Little is known about the key molecular interactions involved in this mechanism especially with respect to the host cell. There is a lack of knowledge about the importance of host factors and proteins involved in sporozoite infectivity. A deeper understanding of cell traversal and hepatocyte invasion could lead to novel interventions. This work aimed to identify key proteins involved in cell traversal and hepatocyte invasion by P. falciparum. A robust sporozoite production protocol was initially established to ensure the feasibility of the project. Host factors involved in cell traversal were systematically investigated using a whole genome CRISPR/Cas9 knock-out screen. The unbiased screen was enabled by the design of a new positive selection cell traversal assay that kills traversed hepatocytes, permitting the enrichment of traversal-resistant cells. Validation of more than one hundred curated hits identified several human genes involved in infection by other pathogens that are putative proteins involved in P. falciparum cell traversal. Finally, antibodies targeting different regions of the most abundant P. falciparum sporozoite surface protein — the circumsporozoite protein (CSP) — were characterised for their inhibition potential. To do so, an improved method allowing both cell traversal and hepatocyte invasion by P. falciparum sporozoite to be quantified by flow cytometry was established before inhibition assays were performed. Different inhibition profiles were identified, highlighting a role for the N-terminus of CSP in hepatocyte invasion. Identifying essential factors and parasite-host interactions during this first step of the malaria parasite lifecycle will provide more insight into support of a prophylactic treatment for malaria.
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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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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.