Medical Biology - Theses
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ItemStudies of Plasmodium-Iron Interactions( 2023-09)Background Iron deficiency anaemia and malaria co-exist across sub-Saharan Africa where they disproportionately affect young children and pregnant women. Iron supplementation is recommended to treat anaemia, but there are concerns regarding its safety and the potential protection afforded by iron deficiency. An interaction between host iron status and malaria risk has been hypothesised for decades, but confounding factors mean that conclusions are difficult to draw from field studies alone. In this thesis the complex interaction between Plasmodium and iron is investigated using clinical, pre-clinical and in vitro studies. Methods The effect of host iron deficiency on the risk of Plasmodium falciparum parasitaemia by PCR was assessed in a cohort of 711 anaemic Malawian pregnant women, and the risk of intravenous iron supplementation on subsequent risk of parasitaemia explored. These findings were dissected using Plasmodium berghei infection in C57Bl/6 mouse models of iron deficiency (Tmprss6 knockout (Tmprss6-KO)) and iron overload (inducible hepcidin knockout (iHamp-KO)). Tmprss6 knockdown (Tmprss6-KD) was used to assess the phenotype in Tmprss6-KO mice and to explore Tmprss6 as a druggable target; achieved through treatment of wild-type C57Bl/6 mice with siTMP, a GalNAc conjugate targeting Tmprss6. The effect of iron restriction on the parasite was investigated through iron chelation of in vitro P. falciparum cultures, followed by transcriptomic and proteomic analysis. A role of post-transcriptional regulation in the response to iron chelation was further explored. In the setting of known post transcriptional regulation of cellular iron in other organisms via the iron responsive element (IRE)/Iron regulatory protein (IRP) system, and with an IRP-like protein described in P. falciparum, the role of PfIRP was explored through the generation and characterisation of PfIRP-KO parasites. Results Among anaemic Malawian pregnant women iron deficiency was associated with a 53% reduced risk of P. falciparum parasitaemia (Adjusted risk ratio 0.47, 95% confidence interval (0.34, 0.60), p<0.0001), with this finding robust to varied definitions of iron deficiency. Intravenous iron did not increase the subsequent risk of P. falciparum parasitaemia. These findings were supported by the mouse models. Tmprss6-KO mice had improved survival when infected with P. berghei. Conversely, iHamp-KO mice exhibited decreased liver stage infection but an unaltered course in the blood stage of infection. Tmprss6-KD did not replicate the KO phenotype; the disease course was not changed in siTMP treated mice. In vitro, iron chelation inhibited parasite growth and induced substantial changes in the transcriptome and proteome of P. falciparum. Differential expression of genes and proteins involved in key processes in the parasite’s lifecycle, and with plausible links to iron were identified, as was a possible role for post-transcriptional regulation. Investigating this showed PfIRP-KO parasites were more susceptible to iron chelation. Transcriptomic and proteomic analysis identified proteins that might be regulated in an IRE/IRP-like manner. Conclusions This work adds to the current understanding of the complex interaction between Plasmodium and iron. Taken together, these data support iron deficiency being protective against P. falciparum infection. In vitro studies highlighted genes of potential further interest in P. falciparum iron homeostasis and support an iron related role for PfIRP.
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ItemNo Preview AvailableSignaling pathways in apicomplexan parasites( 2022)The phylum Apicomplexa contains several parasitic species of medical and agricultural importance, including Plasmodium falciparum and Toxoplasma gondii. Apicomplexan parasites have complex life cycles involving several morphological stages across multiple hosts, and rely on extensive signaling networks to sense their environment, transduce external signals and coordinate appropriate responses. Currently, there are few specific and effective therapeutics to treat parasitic infections and great efforts have been made to understand the signaling pathways and downstream post-translational modifications that regulate crucial parasite life cycle transitions. In the current literature (reviewed in Chapter 1), phosphorylation and the upstream pathways that mediate this post-translational modification is well described in apicomplexan parasites, while ubiquitination is less well understood due to a lack of extensive characterisation of the ubiquitination machinery. Intracellular Ca2+ signaling and the downstream activity of Ca2+-dependent protein kinases (CDPKs) is paramount to parasite motility, and therefore facilitates host cell invasion and egress. In recent years the function of cyclic nucleotide signaling in regulation of Ca2+ mobilisation and motility has come to light. cGMP signaling and its downstream effector kinase, protein kinase G (PKG) is crucial for activating parasite motility, while cAMP signaling via protein kinase A (PKA) has been shown to be a negative regulator of motility in Toxoplasma tachyzoites. PKA has been implicated in Plasmodium merozoite invasion as the kinase responsible for phosphorylation of the key invasion ligand apical membrane antigen 1 (AMA1), however it is unclear how Plasmodium PKA functions in motility. Chapter 3 explores the function of PKA in Plasmodium falciparum through conditional knockdown of the PKA catalytic subunit (PfPKAc) in asexual stage parasites. I show this kinase to be essential in merozoite invasion of erythrocytes, and provide further evidence that PfPKAc is responsible for phosphorylation of AMA1. In Chapter 4, I perform a comprehensive characterisation of the OTU deubiquitinase (DUB) complement in Toxoplasma. OTU DUBs play important regulatory functions in eukaryotic cells, and through bioinformatic analysis I show that OTU DUBs are expanded in Toxoplasma which suggests a functional significance of OTU DUB function in these parasites. I perform a comprehensive biochemical characterisation of OTU DUBs in Toxoplasma. I identify activities against both ubiquitin and NEDD8-based substrates, and reveal ubiquitin linkage preferences Lys6, Lys11, Lys48 and Lys63-linked chain types. I also show that accessory domains of Toxoplasma OTU DUBs are important for regulating OTU domain function and impose linkage preferences. Utilising the auxin-inducible degron (AID) to generate knockdown parasite lines, I also identify TgOTUD6B is important for Toxoplasma growth. This dissertation explores several aspects of post-translational modifications in apicomplexan parasites and provides new insights into PKA function in Plasmodium, and the OTU deubiquitinase family in Toxoplasma.
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ItemIdentifying and Characterising Exported Hepatic Effector Proteins of the Human Malaria Parasite Plasmodium falciparum( 2022)The parasitic disease malaria is caused by symptomatic infection of Plasmodium parasites during their infection in erythrocytes. Plasmodium falciparum, the most lethal parasite that infects humans, must first undergo replication during the asymptomatic liver stage, which makes it a primary target for prophylactic intervention. Our current understanding of P. falciparum liver stage biology is limited, particularly around the parasite’s ability to subvert host innate defences and exploit host nutrients. This is due to the limited models for liver stage research as P. falciparum replicates in the hepatocyte for 7 days forming tens of thousands of merozoites in an enclosed parasitophorous vacuole. Merozoites in turn infect erythrocytes upon liver stage egress and subsequent release into the blood stream. By infecting an erythrocyte, the parasite forms a parasitophorous vacuole and exports proteins across this vacuole membrane into the host, leading to molecular changes causing malaria-associated pathology. Many parasitic proteins are targeted for export by a pentameric amino-acid motif known as the PEXEL (RxLxE/D/Q), that is proteolytically cleaved in the ER by the aspartyl protease plasmepsin V. Matured effector proteins, as well as PEXEL-negative exported proteins, are translocated into the host-cytoplasm by the PTEX complex. The process of protein export has been widely studied during blood stage infection, but little is known about this process during liver stage. Important questions are whether the export pathway is active and important during the liver stage of the parasite life cycle and whether effector proteins can be identified. In this PhD. we are aiming to address these questions by attempting to identify and characterise novel liver stage exported proteins using two approaches. First, an agnostic approach involving proximity ligation proteomics has been hypothesised to identify liver-stage proteins in subcellular compartments of the parasite and host cell. Second, PEXEL-searching has identified 20 effector candidates of which some have been visualised utilising epitope tagged parasites and polyclonal antibodies. Some effectors have been functionally characterised using rapamycin-mediated conditional gene excision to disrupt expression of respective genes during the P. falciparum life cycle. In vitro sporozoite invasion and traversal assays as well as in vivo liver stage experiments utilising chimeric mice with engrafted human hepatocytes were used to demonstrate the role of these proteins during liver stage. Future identification of novel P. falciparum liver stage exported proteins may provide new drug and vaccine targets for preventive therapeutics targeting the pre-erythrocytic stage of infection by malaria parasites that infect humans.
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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.