Medical Biology - Theses
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ItemCell-cell interactions during malaria parasite invasion of the human erythrocyte( 2015)Red blood cells are remarkably resilient, flexible and dynamic structures. These properties are required for their passage through small capillaries and are imparted by the cytoskeleton, a network of proteins that underlies and links to the cell membrane. To successfully invade the blood stage malaria parasite, called a merozoite, must induce rapid and drastic changes to the structure of the target erythrocyte, including the formation of a tight junction and a new cellular compartment, the parasitophorous vacuole. These key modifications involve the infolding of the red blood cell membrane, membrane fusion and fission events and the secretion of parasite proteins into the host. Although detailed cellular descriptions of merozoite invasion have been achieved over the past few decades, comparatively little is known about the molecular basis of how the host cell responds to parasite entry. In fact, in contrast to what is known about the invasion strategies of most other intracellular pathogens, the prevailing model of Apocomplexan invasion imagines a largely binary system within which an active parasite, driven by its acto-‐ myosin motor, invades a passive host cell. There is a growing body of evidence, however, that suggests that Apicomplexan host cells may not be as inactive as initially thought. Nonetheless, to date, there is no direct evidence for the notion that erythrocytes contribute actively to merozoite invasion. This PhD took at its starting point the hypothesise that to invade, merozoites interface with endogenous erythrocyte pathways that regulate membrane and cytoskeletal remodelling, and that the tight junction is a key structure that coordinates the this host-‐pathogen interaction during the brief moment of entry. To address this proposition, this PhD studied P. falciparum merozoite invasion using a combination of in silico bioinformatic screening, high-‐definition imaging, quantitative and high-‐throughput invasion inhibition assays and quantitative phospho-‐proteomics. Work presented in this thesis further elaborates the molecular architecture of the P. falciparum merozoite tight junction, outlines a model for the secretion of virulence factors by the parasite during entry, establishes that an active erythrocyte is a prerequisite for successful merozoite invasion and demonstrates, for the first time, that the red blood cell responds to early invasion events through the phosphorylation of components of its membrane and cytoskeleton. Taken together, these findings provide strong support for a shift in how we conceptualise invasion, from paradigm that focuses almost exclusively on the activity of the parasite towards one in which both the merozoite and the erythrocyte act cooperatively to achieve the requisite remodelling events that lead to successful intracellular infection. By further expounding the way in which the malaria merozoite orchestrates its interaction with its target red blood cell during invasion, and in particular shedding light on the potential host-‐cell contribution to this process, this work informs future endeavours aimed at the development of novel chemotherapeutic targets to stop invasion and hence prevent or treat malaria disease.
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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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ItemB cell responses during severe malaria: the impact of inflammation on T follicular helper cell and germinal centre responses( 2015)Despite many advances in malaria control and elimination, infection by Plasmodium remains a significantly widespread cause of morbidity and mortality worldwide. Naturally acquired immunity to the parasite plays an important role in protection against malaria infection and the development of symptomatic disease. However, no evidence exists of sterile immunity to the disease and the development of sustained clinically protective antibody responses has been shown to require repeated infections. While many studies have focused on the complex nature of these responses against the antigenically diverse parasite, few have addressed the effect of malaria infection on the generation of memory B cell responses. A study of children in areas of high seasonal malaria transmission revealed a delay in malaria-specific MBC generation despite continual exposure to the parasite. In contrast, in a low transmission setting, lasting memory B cell responses were detected in adults following a single exposure to the parasite. These data indicate clinical malaria infections may hinder the generation and maintenance of malaria-specific memory B cell populations. Long-lived populations of B cells, including memory B cells and long-lived plasma cells, are generated during the germinal centre (GC) reaction in secondary lymphoid organs, such as the spleen. In support of the notion that clinical malaria episodes hinder the induction of humoral memory, histological studies revealed that human fatal malaria infections are accompanied by dramatic changes in splenic architecture, including impaired GC formation. The bulk of studies examining the induction of GC responses following malaria infection have made use of self-resolving infection models in mice. To specifically address the impact of severe malaria infections on these processes, the development of GC responses was assessed using the P. berghei ANKA model of severe malaria in comparison to immunisation with an equivalent antigenic load of attenuated parasites. This model permitted the uncoupling of the effects of severe malaria infection and parasite exposure, and demonstrated that severe malaria infections profoundly impede the correct generation of GC structures. Further, compared to immunised control animals, infected animals had reduced numbers of GC B cells. Critically, the excessive inflammatory processes caused by severe malaria infection directly impaired T follicular helper cell differentiation and lead to the preferential accumulation of Tfh precursors. As a consequence of impaired GC induction, memory responses were not efficiently generated following severe malaria. Collectively, the data presented in this thesis demonstrate a novel role for inflammation in the control of Tfh and GC responses and provide valuable insight into the mechanisms underlying inefficient B cell responses following clinical malaria infections in humans.