Harris, P.K.; (2006) Functional analysis of PfSUB2: A malaria merozoite serine protease. Doctoral thesis , University of London.

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Abstract

Malaria is caused by members of the apicomplexan parasite genus Plasmodium. Of the four species that infect humans, P. falciparum is associated with the most severe forms of the disease. P. falciparum has a complicated life cycle involving two hosts. The disease in humans is associated with the red blood cell (RBC) cycle. Merozoites invade a RBC, replicate asexually within it, and cause the RBC to rupture releasing daughter merozoites into the blood stream to repeat the cycle. Invasion relies on an actinomyosin motor within the parasite and is also associated with the proteolytic shedding of a number of merozoite surface proteins, including the essential proteins AMA1 and MSP1. Both are shed by a merozoite surface 'sheddase' called MESH. The leading candidate for MESH is PfSUB2, an essential membrane-bound subtilisin-like protease (subtilase). In this project I have used transfection of the parasite to explore the role of PfSUB2 within the asexual blood stage life cycle of P. falciparum. PfSUB2 has been epitope tagged by targeted homologous recombination, and shown to initially accumulate in apical organelles called micronemes. Following merozoite release PfSUB2 is capped to the rear of the parasite in an actin-dependent manner and following invasion is detected distributed around the plasma membrane of the intracellular parasite. This pattern of trafficking is similar to that predicted for MESH. The tagged PfSUB2 has been purified from parasite extracts and the activity assessed. I have also attempted to over-express PfSUB2 from an episome within the parasite. As part of this work I have begun to characterise the pfsub2 promoter. A region at the 5' region of the gene has been shown to display promoter activity. This work would not have been possible without the help of many people. Firstly, I would like to thank my supervisor Mike Blackman for his excellent guidance. I would also like to thank the rest of the lab for their support, in particular Fiona Hackett for help in the early days, Rebecca O'Donnell for transfection and parasite advice and Sharon Yeoh for the PfSUB2 alignment. Peter Fletcher was responsible for manufacturing all the peptides used in this work and performed the mass spectroscopy. On a personal note I would like to thank my family for their continuing support. Last but not least I would like to thank Tom for putting up with me and for his useful suggestions on practical techniques.

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