2013 DIPLOMOVÁ PRÁCE Lukáš Novák Abstract Free-living microaerophilic protist Trimastix pyriformis is closely related to oxymonads which are the largest eukaryotic group without any known mitochondrion. In contrast to oxymonads, an enigmatic reduced mitochondrion has been found in the cell of T. pyriformis. In EST data of T. pyriformis, a number of genes has been identified whose products are putatively localized in the mitochondrion. Among these are genes for all the components of the glycine cleavage system, [FeFe]hydrogenases and the mitochondrial marker Cpn60. We performed experiments in order to determine the cellular localization of these proteins. Our results show that the glycine cleavage system is localized in the mitochondrion. Results of the experiments carried out in order to localize two hydrogenases suggest also the mitochondrial localization but are not fully convincing. The attempt to localize Cpn60 has failed. We have also identified a set of new genes in transcripts of T. pyriformis and Monocercomonoides sp. (Oxymonadida). These genes code for some components of the SUF system of FeS cluster synthesis and a peroxidase rubrerythrin. Key words: Trimastix, Monocercomonoides, mitochondrion, hydrogenosome, mitosome, hydrogenase, glycine cleavage system, SUF system.

The diplomonads are a diverse group of eukaryotic microbes found in oxygen limited environments such as the intestine of animals were they may cause severe disease. Among them, the prominent human parasite Giardia intestinalis non-invasively colonizes the small intestine of humans and animals where it induces the gastrointestinal disease giardiasis. Two of the eight genetic groups of G. intestinalis, assemblage A and B, are known to infect humans and have zoonotic potential. At the start of project, genome scale data from assemblage B-H was either sparse or entirely missing. In this thesis, genome sequencing was performed on the assemblage B isolate GS (Paper I) and the P15 isolate (Paper III) of the hoofed-animals specific assemblage E to investigate the underlying components of phenotypic diversity in Giardia. Comparisons to assemblage A isolate WB revealed large genomic differences; entirely different repertoires of surface antigens, genome rearrangements and isolate specific coding sequences of potential bacterial origin. We established that genomic differences are also manifested at the transcriptome level (Paper VIII). In a follow up analysis (Paper IV) we concluded that the Giardia assemblages are largely reproductively isolated. The large genomic differences observed between Giardia isolates can explain the phenotypic diversity of giardiasis. The adaptation of diplomonads was further studied in Spironucleus barkhanus (Paper II), a fish commensal of grayling, that is closely related to the fish pathogen Spironucleus salmonicida, causative agent of systemic spironucleosis in salmonid fish. We identified substantial genomic differences in the form of divergent genome size, primary sequence divergence and evidence of allelic sequence heterozygosity, a feature not seen in S. salmonicida. We devised a transfection system for S. salmonicida (Paper VI) and applied it to the study of the mitochondrial remnant organelle (Paper VII). Our analyses showed that S. salmonicida harbor a hydrogenosome, an organelle with more metabolic capabilities than the mitosome of Giardia. Phylogenetic reconstructions of key hydrogenosomal enzymes showed an ancient origin, indicating a common origin to the hydrogenosome in parabasilids and diplomonads. In conclusion, the thesis has provided important insights into the adaptation of diplomonads in the present and the distant past, revealing hidden diversity.