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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ItemDissection of the coordinated events during Plasmodium falciparum infection of the human erythrocyte( 2013)Malaria disease continues to place significant social and economic burdens on the developing world. Of the Plasmodium parasites responsible for the disease, P. falciparum causes the most severe form and thus kills up to 1 million people each year. Unfortunately, recent years have seen rising signs of resistance to even our most successful drug-based therapies and a continued underperformance of promising vaccine prospects during clinical trials. This signals a need for continued research, particularly that focussed on providing new targets for therapy and on the development of methods to more effectively understand new and existing therapeutic approaches during their early stages of development. Invasion and subsequent remodelling of the erythrocyte by the merozoite form of the parasite mark two areas of particular interest. Indeed, both are critical for the establishment of symptomatic infection. Despite their importance and interest as therapeutic targets, study of the P. falciparum merozoite, erythrocyte invasion and early remodelling events have all been hampered by shortfalls in methodology. This has left much to be understood about this period of the lifecycle. Using recent advances in our ability to isolate free, viable, P. falciparum merozoites, I therefore develop methods to fix parasites at each step of, and in the minutes following, erythrocyte invasion. For the first time, this allows detailed imaging of these processes on a molecular level using various imaging platforms, including widefield deconvolution, ‘super-resolution’ three-dimensional structured illumination, and transmission electron microscopies, along with electron tomography. In particular, the application of cutting edge microscopy combined with sophisticated quantitative imaging analysis makes for a powerful investigative approach. Initially, these techniques are developed and used to investigate a number of processes that are critical for merozoite invasion: attachment, tight junction formation, surface protein shedding, actomyosin motor activation and organelle secretion. This study identifies interactions mediated by merozoite surface adhesins as the important initiator of subsequent invasion processes, which all follow without further checkpoints. It also points to the tight junction as a nexus that organises and directs these processes. I then dissect aspects of erythrocyte remodelling, providing previously lacking cellular evidence for the role of the Plasmodium translocon of exported proteins (PTEX) complex during protein export from the parasite. In particular, this study identifies key events that occur in the latter parts of invasion which are critical for subsequent protein export. This points to an important level of coordination between invasion and remodelling events that may be occurring up to 24 hours later. Together the contributions made during this PhD provide the most complete model for invasion and early parasite remodelling to date. The methods developed also provide an important platform from which others can develop our understanding of these critical events in the future.