Medical Biology - Theses
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ItemNovel membrane proteins of Plasmodium falciparum( 2011)Apicomplexan parasites such as Plasmodium spp. cause a multitude of illnesses through infection of both human and livestock hosts. Of the Plasmodium species that infect humans, P. falciparum is associated with a large proportion of symptomatic malarial disease, resulting in approximately 800,000 deaths and a further 250 million clinical cases annually (WHO World Malaria Report, 2010). Although there are many facets that contribute to the global health catastrophe caused by malaria, including social, geographical, economic and political; the biology of the parasite itself is a major contributing factor. P. falciparum has a complex lifecycle involving stages in both its mosquito vector and human host, within which it carries out its development in distinct phases in both the liver and blood stream. Symptomatic malaria is associated with blood-stage infection and it is investigations into this asexual life-stage that have formed the basis of my PhD research. In particular I have focused on investigating the functions of membrane proteins, particularly those residing in detergent resistant membranes. These membranes contain regions where the lipids are more ordered and enriched in proteins and are the sites of many important cellular activities such as signal transduction and membrane trafficking. Previous studies have shown that when detergent resistant membranes are recovered from the invasive merozoite stage of P. falciparum, many proteins from the parasite’s pellicle; a three-membrane layer that covers the parasite, are extracted also. Furthermore, proteins that reside in the parasitophorous vacuole, a membrane that envelops young intra-erythrocytic ring-stage parasites, also preferentially associate with the detergent resistant fraction. Within both the merozoite and ring stage parasites reside many proteins of unknown function and during my candidature I have studied several of these proteins. Initial studies presented here have focused on the characterisation of a novel family of Apicomplexan-specific proteins termed the GAPM family which have been identified within the detergent resistant membrane fraction of P. falciparum developing merozoites. It is shown here that the GAPM family of proteins is conserved across all sequenced Apicomplexa and associate into high molecular weight structures within the inner membrane complex of both P. falciparum and the related Toxoplasma gondii. The inner membrane complex forms the innermost pair of membranes of the parasite pellicle. The data presented here demonstrates that the GAPMs bind to both the actinmyosin host-cell invasion motor on the outside of the inner membrane complex and to the parasite cytoskeletal network on the inside of the complex, possibly linking the invasion motor and cytoskeleton together. It has long been recognized in the field that for the invasion motor to work and propel the merozoite into its red blood cell host that the motor must be attached to a solid scaffold such as the cytoskeleton. My functional characterization of the GAPMs has provided the first candidates for proteins that likely tether the parasite motor to the cytoskeleton and therefore shed light on important aspects of how the invasion machinery is assembled. The P. falciparum parasitophorous vacuole membrane is also enriched in detergent resistant membrane proteins and has been shown previously to contain the recently discovered PTEX complex that putatively helps export hundreds of proteins into the host human cell. Exported proteins help remodel the host and contribute greatly to parasite virulence and consequently there has been great interest in unraveling how the export process occurs. Investigations presented here have focused on the biochemical characterisation of the three major components of the PTEX complex, HSP101, PTEX150 and EXP2 in order to provide functional insight into PTEX’s trafficking role. Specifically these studies aimed to glean insight into the overall expression and formation of this important protein complex with particular focus on EXP2 and its putative role as a parasitophorous vacuole membrane-associated pore. Data presented here has revealed that the PTEX components are made in late schizonts/rings and prior to invasion are likely to reside within the dense granules prior to their secretion into the nascent parasitophorous vacuole during invasion. Within the parasitophorous vacuole, EXP2 is the most strongly membrane associated component and is present within this structure as a large oligomeric complex to which the remainder of the PTEX complex attaches. Overall, this data is consistent with EXP2 forming a membrane-associated oligomer, which anchors the remainder of the PTEX components, supporting the hypothesis that this protein is forming the putative membrane-associated pore. PTEX likely functions with several other proteins to establish an export pipeline starting within the parasite where exported proteins are first made and then cross the parasite into the vacuole, upon which time PTEX translocates the protein cargo into the red blood cell. To characterise other proteins possibly involved in export, in the last chapter of my thesis I have investigated a novel parasitophorous vacuole protein; PF14_0201, which was originally identified in the detergent resistant membrane proteome of P. falciparum rings. My studies provide a preliminary characterisation of this novel protein and investigate whether it is indeed a novel PTEX component as suggested by preliminary data. Although PF14_0201 is a resident parasitophorous vacuole protein that it is strongly membrane associated, likely via a GPI-anchor, no substantial interaction was detected between PF14_0201 and the PTEX components indicating that this protein is probably playing another role within the vacuole. Further possible functions of PF14_0201 were explored but proved uninformative. Overall my study of these three sets of detergent resistant membrane proteins, namely the GAPMs, PTEX and PF14_0201, have provided useful information about the formation and function of structures present within different phases of the P. falciparum asexual blood stage cell cycle.