Medical Biology - Theses
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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.