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Most of the studies in cell biology primarily focus on models from the opisthokont group of eukaryotes. However, opisthokonts do not encompass the full diversity of eukaryotes. Thus, it is necessary to broaden the research focus to other organisms to gain a comprehensive understanding of basic cellular processes shared across the tree of life. In this sense, Trypanosoma brucei, a unicellular eukaryote, emerges as a viable alternative. The collaborative efforts in genome sequencing and protein tagging over the past two decades have significantly expanded our knowledge on this organism and have provided valuable tools to facilitate a more detailed analysis of this parasite. Nevertheless, numerous questions still remain.
The survival of T. brucei within the mammalian host is intricately linked to the endo-lysosomal system, which plays a critical role in surface glycoprotein recycling, antibody clearance, and plasma membrane homeostasis. However, the dynamics of the duplication of the endo-lysosomal system during T. brucei proliferation and its potential relationship with plasma membrane growth remain poorly understood. Thus, as the primary objective, this thesis explores the endo-lysosomal system of T. brucei in the context of the cell cycle, providing insights on cell surface growth, endosome duplication, and clathrin recruitment. In addition, the study revisits ferritin endocytosis to provide quantitative data on the involvement of TbRab proteins (TbRab5A, TbRab7, and TbRab11) and the different endosomal subpopulations (early, late, and recycling endosomes, respectively) in the transport of this fluid-phase marker. Notably, while these subpopulations function as distinct compartments, different TbRabs can be found within the same region or structure, suggesting a potential physical connection between the endosomal subpopulations. The potential physical connection of endosomes is further explored within the context of the cell cycle and, finally, the duplication and morphological plasticity of the lysosome are also investigated. Overall, these findings provide insights into the dynamics of plasma membrane growth and the coordinated duplication of the endo-lysosomal system during T. brucei proliferation. The early duplication of endosomes suggests their potential involvement in plasma membrane growth, while the late duplication of the lysosome indicates a reduced role in this process. The recruitment of clathrin and TbRab GTPases to the site of endosome formation supports the assumption that the newly formed endosomal system is active during cell division and, consequently, indicates its potential role in plasma membrane homeostasis.
Furthermore, considering the vast diversity within the Trypanosoma genus, which includes ~500 described species, the macroevolution of the group was investigated using the combined information of the 18S rRNA gene sequence and structure. The sequence-structure analysis of T. brucei and other 42 trypanosome species was conducted in the context of the diversity of Trypanosomatida, the order in which trypanosomes are placed. An additional analysis focused on Trypanosoma highlighted key aspects of the group’s macroevolution. To explore these aspects further, additional trypanosome species were included, and the changes in the Trypanosoma tree topology were analyzed. The sequence-structure phylogeny confirmed the independent evolutionary history of the human pathogens T. brucei and Trypanosoma cruzi, while also providing insights into the evolution of the Aquatic clade, paraphyly of groups, and species classification into subgenera.
During the past years, the internal transcribed spacer 2 (ITS2) was established as a commonly used molecular phylogenetic marker for the eukaryotes. Its fast evolving sequence is predestinated for the use in low-level phylogenetics. However, the ITS2 also consists of a very conserved secondary structure. This enables the discrimination between more distantly related species. The combination of both in a sequence-structure based analysis increases the resolution of the marker and enables even more robust tree reconstructions on a broader taxonomic range. But, performing such an analysis required the application of different programs and databases making the use of the ITS2 non trivial for the typical biologist. To overcome this hindrance, I have developed the ITS2 Workbench, a completely web-based tool for automated phylogenetic sequence-structure analyses using the ITS2 (http://its2.bioapps.biozentrum.uni-wuerzburg.de). The development started with an optimization of length modelling topologies for Hidden Markov Models (HMMs), which were successfully applied on a secondary structure prediction model of the ITS2 marker. Here, structure is predicted by considering the sequences' composition in combination with the length distribution of different helical regions. Next, I integrated HMMs into the sequence-structure generation process for the delineation of the ITS2 within a given sequence. This re-implemented pipeline could more than double the number of structure predictions and reduce the runtime to a few days. Together with further optimizations of the homology modelling process I can now exhaustively predict secondary structures in several iterations. These modifications currently provide 380,000 annotated sequences including 288,000 structure predictions. To include these structures in the calculation of alignments and phylogenetic trees, I developed the R-package "treeforge". It generates sequence-structure alignments on up to four different coding alphabets. For the first time also structural bonds were considered in alignments, which required the estimation of new scoring matrices. Now, the reconstruction of Maximum Parsimony, Maximum Likelihood as well as Neighbour Joining trees on all four alphabets requires just a few lines of code. The package was used to resolve the controversial chlorophyceaen dataset and could be integrated into future versions of the ITS2 workbench. The platform is based on a modern, feature-rich Web 2.0 user interface equipped with the latest AJAX and Web-service technologies. It performs HMM-based sequence annotation, structure prediction by energy minimization or homology modelling, alignment calculation and tree reconstruction on a flexible data pool that repeats calculations according to data changes. Further, it provides sequence motif detection to control annotation and structure prediction and a sequence-structure based BLAST search, which facilitates the taxon sampling process. All features and the usage of the ITS2 workbench are explained in a video tutorial. However, the workbench bears some limitations regarding the size of datasets. This is caused mainly due to the immense computational power needed for such extensive calculations. To demonstrate the validity of the approach also for large-scale analyses, a fully automated reconstruction of the Chlorophyta (Green Algal) Tree of Life was performed. The successful application of the marker even on large datasets underlines the capabilities of ITS2 sequence-structure analysis and suggests its utilization on further datasets. The ITS2 workbench provides an excellent starting point for such endeavours.