Toxoplasma gondii is an obligate intracellular protozoan parasite with a worldwide prevalence. Together with the causative agent of malaria (Plasmodium falciparum) and other medically important pathogenic parasites it belongs to the phylum of the Apicomplexa. Besides identifiable eukaryotic organelles, apicomplexan parasites differ from other eukaryotic cells by an extra set of specialised secretory organelles (micronemes, rhoptries and dense granules), that are sequentially secreted during invasion of the host cell. Upon host cell contact the apically located micronemes are the first organelles to be released and contain crucial virulence factors that are secreted. In order to systematically analyse vesicular traffic with a special focus on the secretory pathway of rhoptry and microneme proteins the ddFKBP system was used to perform a systematic analysis of Rab proteins in Toxoplasma gondii. Rab proteins are small GTP- binding proteins that are involved in targeting and fusion of vesicles from a donor to an acceptor membrane. Whereas higher eukaryotes like human cells encode more than 60 different Rab proteins apicomplexan parasites possess only a reduced core set of Rab proteins. Performing co-localisation studies with generated parasite lines expressing ddFKBPmyc-tagged versions of Rab1A, 1B, 2, 4, 5A, 5C, 7, 18 and Rab5B-ddFKBPHA revealed, that all these Rabs localise to the early secretory pathway (Rab1B, 2 and 18), the Golgi (Rab4), or the late secretory pathway (Rab5A, Rab5B, Rab5C and Rab7). No exact localisation could be defined for Rab1A.Rab5A and Rab5C, normally involved in endocytic uptake, were identified as important regulators of traffic to micronemes and rhoptries in Toxoplasma gondii, using an overexpression screen of Rabs and the analysis of trans-dominant mutants of promising candidates.Intriguingly, some microneme proteins could be found to traffic independently on functional Rab5A and Rab5C, indicating the existence of independent transport routes to micronemes, which again indicates that apicomplexans have remodelled Rab5-mediated vesicular traffic into a secretory system that is essential for host cell invasion.By using two-colour super-resolution stimulated emission depletion (STED) microscopy, distinct localisations of independent microneme proteins could be verified. This demonstrated that micronemal organelles are organised in distinct subsets or subcompartments. Given these results, it can be assumed that apicomplexan parasites modify classic regulators of the endocytic system to carry out essential parasite-specific roles in the biogenesis of their unique secretory organelles.
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