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Abstract

Malaria is caused by a unicellular parasite of the genus Plasmodium which has a complex life cycle involving an Anopheles mosquito vector and a vertebrate host. The development in the mosquito vector is crucial for the parasite to be transmitted to new hosts and to ensure genetic diversity. This requires parasite motility, crossing of barriers and persistence for long periods of time. Especially the sporozoite stage which develops in the mosquito gut has to overcome all these challenges to assure transmission back to the vertebrate host. Among others, this demands a very stable but flexible composition of the cytoskeleton. Besides actin filaments and intermediate filaments, microtubules (MTs) are one of the building blocks of the eukaryotic cytoskeleton. MTs are hollow cylinders composed of α- and β-tubulin heterodimers that are essential for many cellular processes, including intracellular transport, chromosome segregation, cell polarity, and migration. In most eukaryotes, the number and length of microtubules is variable whereas the highly polarized and crescent shaped Plasmodium sporozoites contain a special subset of cytosolic microtubules called subpellicular microtubules (sMTs) that are well defined in number and length. However, the significance of both parameters has not been studied to date. Little is known about the two α-tubulin isotypes existing in Plasmodium that are required for sMT polymerization in sporozoites as well as the influence of sMT parameters (e.g. number, length, arrangement) on sporozoite development and infectivity. In addition, other microtubule subsets such as hemispindle microtubules (hMTs), required for chromosome segregation during sporozoite budding, have been sparsely investigated due to difficulties in labelling MTs within insect stages. In this study, I quantitatively examine the impact of MT number and length in Plasmodium berghei by using state of the art gene manipulation strategies and imaging techniques. I could show, that α1- tubulin is essential for hMT and sMT formation during sporozoite budding, while replacement of α1-tubulin with α2-tubulin partially rescued microtubule polymerization. My data show, that microtubules are not essential for nuclear division and onset of sporozoite budding during sporogenesis. However, deletion of α1-tubulin led to severely deformed sporozoites unable to detach of the sporoblast after budding. Modifications of α1-tubulin regulatory elements resulted in reduced tubulin expression levels that highly correlated with the number of sMTs found in sporozoites. Reduced sMT numbers impaired sporozoite formation, motility and infectivity. Only sporozoites with 10 or more sMTs were infectious to mice. Replacement of α1-tubulin with α2-tubulin resulted in shortened sMTs leading to shorter sporozoites with increased curvature. Parasite lines with reduced MT length and/or number showed reduced transmission efficiencies when mice where bitten by infected mosquitoes while parasites containing shortened sMTs displayed also delayed prepatencies when mice where infected by intravenous injections indicating also a reduced efficacy in liver infection. Taken together, my findings emphasize the importance of defined microtubule numbers and length in a unicellular organism to ensure efficient transmission as well as functional and transcriptional differences of the two α-tubulin isotypes found in Plasmodium berghei.