Modification and analysis of proteins involved in gliding motility and invasion of Plasmodium

Abstract

Motility is an important feature of cells that is required for the development of multicellular organisms (Trepat et al. 2012), to heal tissue and fight invading pathogens. But also pathogens itself depend on motility to migrate through tissue barriers and infect hosts. Especially parasites of the genus Plasmodium that are the causative agents of malaria in humans require active movement in order to complete their life cycles. Motility of these parasites is not dependent on alterations of their cell shape, as it is usually observed in mammalian cells, and is therefore called gliding motility. While the mechanism of gliding motility is still not fully understood it is known that specific surface proteins called adhesins are essential for the parasite to perform active movement. In order to better understand the function of adhesins in gliding motility this thesis investigates sporozoite-specific adhesins by using genetic approaches like gene knockout, mutation of single amino acids as well as deletion or exchange of whole domains. A particular focus is put on the thrombospondin related anonymous protein (TRAP) that is specifically expressed at the sporozoite stage. Previous research showed that the deletion of TRAP abrogates directed movement of sporozoites as well as the invasion of the salivary glands of the mosquito. In addition sporozoites lacking TRAP are not infectious to mice if intravenously injected (Sultan et al. 1997). However, while the functions of TRAP are well characterised the mode of action of TRAP is still unknown. To gain further insight into the functions of this adhesin this thesis investigates the Von Willebrandt factor like A-domain as well as the thrombospondin type-I repeat in TRAP s extracellular portion and their implications in gliding motility and invasion of sporozoites. To analyse the phenotype of transgenic sporozoites in vitro and in vivo the rodent malaria parasite Plasmodium berghei was used. Utilizing this strategy it is possible to investigate the transmission potential of generated parasite lines by infecting mice via intravenous injection of sporozoites or via bites of infected mosquitos which are experiments that are difficult to perform with the human malaria parasite Plasmodium falciparum. Taken together this thesis aims to gain more insight into ligand recognition by TRAP and Plasmodium transmission in general.