Medical Biology - Theses
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ItemMerozoite surface protein 1: insights into complex formation and function in erythrocyte invasion( 2016)The invasion of Plasmodium falciparum into host erythrocytes during the parasites’ asexual blood stage is a critical step in the perpetuation of symptomatic infection of malaria in the human host. Merozoites, the invasive form of the parasite, express a glycosylphosphatidylinositol-anchored 190 kDa Merozoite Surface Protein 1 (MSP1) on the surface. This abundant, essential protein exists in a large complex that includes other peripheral Merozoite Surface Proteins (MSPs). Together, these large macromolecular complexes are thought to mediate the initial stages of invasion. MSPs are of great interest to the field as they are exposed to the host immune system and also contribute directly to the invasion process. Therefore, there is a strong consideration for considering them as therapeutic targets. The majority of the work assessing these molecules as potential vaccine candidates has been performed with single MSP antigens and vaccine trials on these MSPs have shown variable results. A main concern arising from these trials is the fact that these antigens are often found in complex with other antigens and therefore have regions that are masked when found on the parasite surface. In order to address this, the work presented in this thesis utilises parasite-derived complexes to understand how peripheral MSPs: MSP3, MSP6, MSPDBL1, MSPDBL2 and MSP7 utilise MSP1 as a platform to be presented on the merozoite surface to form an array of complexes with different functional roles. In addition, multiple forms of erythrocyte binding complexes were found to have overlapping functions in invasion. Complexes that are involved in erythrocyte binding were characterised, where two components, MSPDBL1 and MSPDBL2 were shown to mediate erythrocyte binding directly. Overall, this study has identified and validated the presence of multiple Merozoite Surface Protein 1 complexes that are involved in mediating the interaction of the merozoite to receptors on the red blood cell surface, which is a vital process for successful invasion of parasites into host erythrocytes. Together, these findings have provided valuable insights into the complexity of MSP1 complexes and have contributed to the most complete model for the molecular arrangements that occur on the parasite surface to date.
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ItemAnalysis of 6-cys proteins and calcium fluxes during erythrocyte invasion by Plasmodium falciparum parasites( 2015)Plasmodium parasites amplify their population within the human host by invading, growing and replicating within the body’s erythrocytes. When the population becomes high enough, the damage caused produces symptomatic malaria disease. To develop new drugs and vaccines against malaria it is therefore important to know as much possible about how parasites grow within the human host and particularly about how the extracellular merozoite stage invades erythrocytes, since this short-lived stage is highly vulnerable. This thesis provides new information from the most deadly human malaria pathogen P. falciparum, on the biochemical characteristics of a little known family of merozoite surface proteins which were thought to facilitate erythrocyte invasion as well revealing with unprecedented resolution, new details about how merozoites enter erythrocytes. P12, P38, P41, and P92 comprise a group of blood-stage merozoite surface proteins that belong to the 6-cys family and all except P41 are predicted to have membrane anchors. To functionally characterize the proteins, specific antibodies were made and were then employed to block merozoite invasion by interfering with the binding of 6-cys to erythrocytes. The effect of the antibodies was very weak and therefore not indicative of a major role for 6-cys in invasion. The antibodies were then used as localization probes and indicated that P12 and P41 were at the merozoite periphery with some concentrated towards the apex. In addition, the non-anchored P41 was held on the merozoite surface through heterodimerization with the membrane anchored P12. Despite the P12/P41 heterodimer being in prime position to bind erythrocytes during invasion no evidence for binding could be established. Characterisation of P92 was next conducted and revealed that like the P12/P41 heterodimer, it was tightly associated with the parasite membrane and later cleaved off possibly during invasion. On the other hand, P38 did not shed from the merozoite surface, and it was carried into the erythrocyte. P92 was strictly localised to the apical end of the merozoite while P38 displayed both apical and surface localisation. Similar to the P12/P41 heterodimer, P92 does not appear to bind erythrocytes. In a final attempt to derive a function for the blood stage 6-cys, their genes were individually knocked out but none of the mutants produced any defective growth or invasion phenotypes suggestive of function. To further study invasion, the morphology and kinetics of this process in P. falciparum merozoites was examined with high-speed live-cell microscopy. With greater temporal resolution, novel cellular actions of the merozoites were observed. For example, during the 7.5 s pre-invasion phase the merozoite deforms the erythrocyte plasma membrane multiple times whilst re-orientating. After a brief rest, the merozoite invaded over a ~17 s period forming a vacuole mainly from wrapping the erythrocyte’s membrane around itself. About 18.5 s after entry, the merozoite began spinning in a clockwise direction to possibly to help disconnect itself from the erythrocyte membrane. After spinning had commenced the host erythrocyte began to develop a spiculated appearance called echinocytosis. Suspecting that calcium influx into the erythrocyte during invasion might be responsible for the echinocytosis, the appearance of these fluxes was monitored during invasion by live cell imaging. These observations confirmed for the first time, that a calcium flux originated as an intense spot emanating from the area of contact between the merozoite and erythrocyte suggestive of pore formation between the cells. Further experiments with modified levels of calcium indicated the ion is required for efficient invasion and may play role in causing echinocytosis. Other work using the calcium flux as a visual marker indicated that pore formation coincided with the deployment of tight adhesive proteins from the merozoite that commit it to invasion. The live cell imaging work presented therefore sheds considerable light on many details of merozoite invasion that could inform future drug and vaccine development. Supplementary Videos: Video 1. High-speed time-lapse acquisition of 3D7 merozoite invading the erythrocyte (40 fps). Video 2. The 3D7 merozoite invading the BODIPY FL C12-sphingomyelin labelled erythrocyte (2 fps, 2× real speed). Video 3. The 3D7 merozoite invading the erythrocyte in the presence of Fluo-4 AM showing the punctate apical calcium and calcium influx in the infected erythrocyte (3 fps, 8× real speed) Video 4. Fluo-4-stained 3D7 parasite culture showing merozoites attempting to invade in the presence of R1 peptide (3 fps, 8× real speed). The punctate apical calcium and influx in the attached erythrocyte were detectable. Video 5. Fluo-4-stained 3D7 parasite culture showing merozoites attempting to invade in the presence of R1 peptide (variable speed). The echinocytotic erythrocyte had not recovered after ~20 min of recording. Video 6. CytD-treated 3D7 merozoite attempting to invade the Fluo-4 labelled erythrocyte (variable speed). The punctate apical calcium was visible but the calcium influx was difficult to observe. The echinocytotic erythrocyte had not recovered after ~20 min of recording.
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ItemPlasmodium falciparum merozoite invasion mechanisms and inhibitors of invasion( 2012)Malaria threatens 40% of the global population resulting in approximately 225 million cases of disease and 800 000 deaths per year. Recently marked improvements in the implementation of control measures and increased use of artemisinin combination therapies (ACTs) have contributed to a reduction in malaria mortality and morbidity (World Health Organization, Global Malaria Programme, 2010). Furthermore the licensing and roll-out of the first malaria vaccine, RTS,S, is hoped to occur by 2015 (White, 2011; Agnandji et al., 2011). However, a sustained reduction in malaria burden and eradication of malaria seems unlikely with current control strategies alone. The largest malaria burden is caused by infection with P. falciparum parasites. All symptomatic illness occurs during asexual replication in the blood that is initiated when the merozoite form of the parasite invades red blood cells (RBCs). A limited understanding of merozoite invasion and immune mechanisms inhibiting invasion has hampered rational vaccine and drug development targeting this stage of the parasite life cycle. This thesis is based on the study of merozoite invasion of the RBC with a particular focus on the role of merozoite surface proteins in invasion and mechanisms of inhibition targeting these proteins. Part of the difficulty in studying P. falciparum merozoite invasion is due to the lack of efficient techniques to isolate merozoites that maintain invasive capacity. Chapter 3 describes the development of methods to isolate merozoites that maintain viability. Importantly, the method only requires basic laboratory equipment, therefore is accessible to both resource rich and developing laboratories. Highly synchronized cultures were treated with a cysteine protease inhibitor to block schizont rupture and then merozoites isolated via membrane filtration (Boyle et al., 2010a). Approximately 15% of isolated merozoites maintained viability and were able to successfully invade when incubated with RBCs. This allowed for the development of methods to fix merozoites during invasion for microscopy and invasion inhibition assays. Invasion of isolated merozoites was independent of serum components and the invasive half-life of merozoites was approximately 8 minutes. Merozoite isolation and invasion assays are now being used by a number of research groups and are a powerful technique to study merozoite invasion mechanisms (Riglar et al., 2011) and inhibitors of invasion. In Chapter 4, microscopy of invading merozoites is used to investigate the shedding of merozoite surface antigens during invasion. The initial steps of merozoite invasion are hypothesized to be mediated by merozoite surface proteins that contact with the RBC via weak receptor-ligand interactions. During invasion it is thought that merozoite surface proteins are cleaved and then shed from the merozoite to allow invasion to occur. This has been most clearly demonstrated for MSP1, with compounds that inhibit MSP1 cleavage and/or shedding also inhibiting invasion (Blackman et al., 1994; Singh et al., 2006; Woehlbier et al., 2010; Fleck et al., 2003; Blackman and Holder, 1992). Contrary to the current paradigm, it was found that merozoite surface proteins MSP2 and MSP4 were not shed from the merozoite surface during invasion and were instead carried into the RBC without apparent cleavage. Post invasion, MSP2 was rapidly degraded within a few minutes, whereas MSP4 was maintained for a number of hours. Interestingly, during invasion some MSP2 antibodies were found to be internalized into the RBC. Internalized antibodies were maintained for approximately 24 hours post invasion. This work establishes that there is differential cleavage and shedding of merozoite surface proteins during invasion and suggests that some merozoite surface proteins may have roles outside initial contact events. Chapter 5 investigates the mechanisms by which antibodies inhibit merozoite invasion in the presence of physiological relevant concentrations of complement-active serum. This work was possible due to capacity of isolated merozoites to invade in both the absence of serum and high serum concentrations. While the importance of IgG in mediating parasite clearance is well established (Sabchareon et al., 1991; McGregor, 1964b), the mechanisms of antibody function remain poorly understood. Naturally acquired antibodies from malaria-exposed individuals, as well as antibodies from vaccinated rabbits and humans had complement-dependent inhibition activity targeting merozoite invasion. The complement component C1q was required and appeared to be sufficient for complement-dependent inhibition. MSP1 and MSP2 were identified as targets of complement-dependent antibody mediated inhibition. Antibody mediated complement-dependent inhibition of invasion is a novel mechanism targeting merozoites that may be important in understanding protective immunity and for evaluating candidate merozoite vaccines. Finally, Chapter 6 explores the interaction of heparin with merozoite surface proteins and the potential of heparin-like-molecules (HLMs) as the basis for novel drug development. Heparin is a known inhibitor of merozoite invasion, and appears to act by inhibiting early contact events. Utilizing a heparin-binding assay with native merozoite proteins, heparin was shown to bind the processed fragment of MSP1, known as MSP1- 42. A panel of novel HLMs were screened for growth/invasion inhibition activity and a number of highly inhibitory compounds were identified. This work will be a basis for further studies to identify novel invasion inhibitors that may be used as the basis for drug development. The development of a method to isolate viable merozoites has allowed this thesis to explore a number of aspects of merozoite invasion mechanisms and inhibition of invasion. As well as increasing our understanding of P. falciparum merozoite biology and immunity to malaria, it is hoped that this work will contribute to the development of tools to combat malaria disease.