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Abstract

Malaria, a life-threatening disease caused by Plasmodium parasites, poses a significant global health challenge. Despite efforts to combat malaria through prevention, diagnosis, and treatment, it continues to claim a high number of lives, particularly among vulnerable populations such as young children and pregnant women. The motility of Plasmodium ookinetes and sporozoites in the mosquito vector as well as in the mammalian host is a crucial aspect of the parasite life cycle. Ookinetes play a critical role in the establishment of an infection in the mosquito. Motility enables these parasite forms to traverse the midgut epithelium, a key step during parasite development. Sporozoites need to be motile to egress from oocysts, to enter salivary glands and during their transmission to the mammalian host. During an infectious mosquito bite, sporozoites exhibit remarkable motility, allowing them to navigate through the skin, invade blood vessels and reach the liver, where they establish an infection. Understanding the mechanisms underlying parasite motility is essential for developing effective strategies to target and disrupt their life cycle, ultimately aiding in the control and eradication of the disease. Parasite motility relies on the secretion of specialized secretory vesicles at the apical side of the cell, to present proteins on the cell surface needed to interact with host cell surface receptors. In my thesis, I first focused on identifying novel proteins residing in the secretory vesicles of ookinetes and sporozoites using two different proteomics approaches. For ookinetes, I employed Apex2-based proximity labeling to determine the protein content of micronemes. However, a similar approach in sporozoites proved unsuccessful due to a high background signal on the cell surface. Therefore, I chose to collect and analyze secreted proteins in the supernatant of activated cells. Both screens identified both known microneme resident and new proteins. I then selected the most promising novel candidates (akratin and concavin) for further characterization. Concavin is localized on the cell surface of ookinetes and sporozoites and gene deletion of this protein resulted in deformed sporozoites. During maturation of concavin(-) sporozoites they round up from the posterior end of the cell due to detachment of the inner membrane complex from the parasite membrane. This structural aberration limits the efficiency of parasite transmission from the mosquito to the host during an infectious bite. Therefore, the role of concavin emerged as pivotal in ensuring the proper form and function of sporozoites, ultimately impacting the successful conclusion of the malaria parasite's life cycle. In-depth analyses of parasites lacking akratin revealed a dual function of the protein, both in microgametes and ookinetes. Specifically, a mutation of a putative tyrosine-containing motif within the C-terminus showed an exclusive function during ookinete to oocyst transition, distinct from its role during gametogenesis. I also discovered two similar tyrosine containing motifs in the C- terminus of the previously characterized pantothenic acid transporter PAT which was shown to play a role in microgametes and sporozoites. Remarkably, both motifs in the C-terminus of PAT are indispensable for ookinetes but not important for gametocytes, mirroring the findings observed with akratin. Stage-specific expression of the respective mutants further underscores the significance of the C-terminus in sporozoites. Interestingly, in the case of sporozoites, only one of the two motifs is important, presenting a nuanced difference compared to ookinetes. High resolution imaging is important for investigating the small malaria parasite. Yet STED imaging was restricted to non-hemozoin containing life cycle stages due to the high light absorbing capacity of the hemozoin crystals leading to cell damage. I developed a novel method to facilitate Plasmodium blood stage whole cell STED nanoscopy. Using CUBIC-P I was able to elute hemozoin from the parasites enabling STED of whole cells without restrictions. This opens up new possibilities for more detailed investigations at the nanoscale level within Plasmodium-infected red blood cells and potentially enables to a deeper understanding of the cellular dynamics during Plasmodium blood stage development.