Abstract
A bottle neck in malaria research is the investigation of Plasmodium falciparum liver stage parasites because of technical issues in the infection of Anopheles mosquitoes with these parasites and subsequent generation of infectious sporozoites. Therefore I, in close collaboration with a colleague, established a combined in vitro/in vivo P. falciparum life-cycle in our lab. For that a protocol was established that included the generation of sexual P. falciparum stages in cell culture that were subsequently transmitted to Anopheles mosquitoes utilizing a special membrane feeding system. Later in the life-cycle sporozoites were extracted from the mosquito salivary glands to nally infect liver cells for further studies. We were able to establish a constant mosquito-infection rate for several months to perform experiments on P. falciparum sporozoites and exo-erythrocytic forms. To help decipher the in apicomplexans so far mostly uncharacterized cellular process endocytosis I investigated the function and localization of an EH-domain containing dynamin-like protein in Toxoplasma and Plasmodium. It belongs to a family of eukaryotic Eps15-homology domain containing proteins (EHDs) that have been characterized in higher eukaryotes and especially vertebrates to be part of endocytic events such as vesicular tracking and endocytic recycling. I was able to show by an in silico analysis that in contrast to vertebrates (four dierent EHDs) there is only one protein member of this familiy existing in each apicomplexan. Nevertheless, the apicomplexan EHD-protein has similar to all other EHD-proteins a predicted characteristic ATPase-domain (dynamin-like G-domain) and the Eps15-homology domain (EH). Through a uorescent tagging approach I was able to show a dynamic localization of the Toxoplasma EHD-protein member TgRME-1 (named after its ortholog in C. elegans receptor-mediated endocytosis protein 1) within the parasites. It localized to a vesicular compartment within the parasites that did not colocalize with known organelles so far. The compartment fragmented upon cellular division and is most likely involved in vesicular tracking of supply vesicles that transport lipids or other nutrients to the newly forming daughter-cells. From the data obtained in this thesis it can be hypothesized that the TgRME-1 labelled compartment represents a storage compartment that is filled up during the non-replicative phase and during endodyogeny helps to form daughter-cells. Structural analysis of the protein by deletion of either the G-domain or the EH-domain revealed a similar architecture of the protein compared to published data on mammalian EHDs. Investigation of the Plasmodium berghei EHD (PbEHD) with an antibody generated against the protein revealed a dfferent localization in different parasite stages. Whereas the protein localized to several vesicular compartments in the sporozoite stage it concentrated to a single organelle-like compartment in liver-stages 24 hours after invasion. This compartment later (48 hours after invasion) also fragmented and was distributed to the newly forming merozoites during schizogony, similar to TgRME-1. This subcellular localization indicated that both proteins might share a similar function in tachyzoites of Toxoplasma and Plasmodium liver stage parasites. A phenotypical analysis of PbEHD via generation of a pbehd (-) parasite revealed a putative function for the protein during intrahepatic development. The pbehd (-) liver stage parasite showed a reduced growth rate in vivo and in vitro but was still able to complete the life-cycle. In vivo, C57BL/6 mice infected with pbehd (-) parasites showed a prolonged prepatency period and did not develop experimental cerebral malaria in contrast to wildtype-infected mice. I was able to narrow down this protective effect solely to both the prolonged liver-stage phase and the involvement of the immunemodulator cytokine IL-10. Even though a defined role for the EHD-protein in the apicomplexan parasites could not be determined in this thesis I was able to characterize its architecure and localization in Toxoplasma gondii and Plasmodium berghei. I was able to identify a so far uncharacterized compartment in these parasites that is most likely involved in endocytic-recycling and storage of nutrients such as lipids for the parasites. In addition, my studies showed that the apicomplexan EHD-protein is involved in processes of the cellular division. A better understanding of these and other mechanisms of endocytosis will lead to anti-parasitic strategies that may reduce the burden caused by apicomplexan parasites.
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