Hastings, CH; (2012) Novel malaria parasite proteins involved in erythrocyte invasion. Doctoral thesis , UCL (University College London).

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Abstract

Erythrocyte invasion is a key step in the Plasmodium life cycle. This process is tightly regulated, involving the sequential release of specialised apical secretory organelles – the micronemes, rhoptries and dense granules. These organelles contain proteins required for invasion and establishment of the parasitophorous vacuole, but most of the proteins remain uncharacterised. The aim of this project was to uncover novel proteins with a role in invasion by the human malaria parasite Plasmodium falciparum merozoites. I identified proteins using the following selection criteria: a) expression in the schizont/merozoite form of the parasite; b) conservation across the genus; c) the presence of a signal peptide and d) one or more transmembrane (TM) domains. A list of 64 proteins was identified, and filtered further based on novelty, presence in the merozoite proteome, expression in other life cycle stages, and difficulty of study. Five proteins were selected, and I produced recombinant protein and raised antibodies against three, which I used to identify the sub-cellular location of the protein within the parasite. The proteins appear to reside in either the rhoptries or the endoplasmic reticulum of the merozoite. Attempts were made to epitope-tag and delete all 3 genes, with a focus on one protein, the type IV Hsp40, PF11_0443. This protein contains two TM domains and is expressed during schizogony. By immunofluorescence it is present in the ER of early schizonts, before accumulating at the apex of merozoites in a rhoptry location. Immunoprecipitation experiments indicated that the protein binds known rhoptry proteins and other chaperones. The protein has been epitope-tagged but attempts to delete the gene by genetic recombination were unsuccessful. The gene is conserved in Plasmodium spp. and there are orthologues in higher eukaryotes, but it is absent from other Apicomplexa. Current studies are focused on the role of this protein in erythrocyte invasion.

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