The unicellular parasite Giardia intestinalis is one of the organisms carrying mitochondrion-related organelle known as mitosome, which is adapted to the microaerobic lifestyle. The only known fuction of the mitosome is the synthesis of the iron-sulphur clusters. The research of the mitosomal proteome provides new information on the biogenesis and function of this unusual organelle. One of the means of the mitosome research is the analysis of the interactome of the known mitosomal proteins. The state-of- the-art method of the interactome approach is the use of the chemical crosslinking and the subsequent immunoaffinity isolation of the complexes, containing the protein of interest. In this thesis, the interactomes of GiTom40 and GiMOMP35 were characterized with the bioinformatic tools. The cellular localization of four of the chosen proteins was determined by the fluorescent microscopy. One of the proteins, the predicted dynein intermediate chain DIC6939, was phylogenetically classified as an axonemal dynein. The superresolution microsopy was utilized to observe the possible colocalization of DIC6939 with the mitosomes and blue native PAGE led to the visualization of its native complexes. In this work, the optimal conditions for DIC6939 interactome isolation were succesfully determined. The outcome...

Charles University in Prague, Faculty of Science Department of Parasitology Ph.D. study program: Parasitology Abstract of the Ph.D. Thesis Reductive Evolution of Mitochondria - Related Organelles in Anaerobic Protist Petr Rada Supervisor: Prof. RNDr. Jan Tachezy,Ph.D. Advisor: Doc. RNDr. Ivan Hrdý, Ph.D. Praha, 2011 1 ABSTRACT Trichomonas vaginalis and Giardia intestinalis are parasitic protists of the Excavata group. Both contain anaerobic forms of mitochondria called hydrogenosomes (Trichomonas) and mitosomes (Giardia). Hydrogenosomes produce hydrogen and ATP by substarte level phosphorylation and mitosomes represent the highly-reduced form of mitochondria that do not participate in cellular energy metabolism and ATP generation. Both types of organelles lost the majority of mitochondrial pathways and their genomes during the mitochondrion to hydrogenosome transition. Consequently, hydrogenosomes and mitosomes facilitate translocation of nuclearly encoded proteins into the matrix of the organelle as well as exchange of metabolites and ions across their membranes. Little is known about the membrane machineries required for the biogenesis of the organelle and metabolite exchange and the limited knowledge of mitosomal proteomes has been mostly gained from genomic analysis and localization studies of a few...

Advances in DNA sequencing led to a technological breakthrough, that allowed analyzis of complete genomes including those of parasitic protists Trichomonas vaginalis and Giardia intestinalis . These organisms are studied not only for their clinical importance, but also from the evolutionary point of view for their adaptation to anaerobic environment. Genome sequencing and annotations of predicted proteins alone did not bring detail view into functioning of their mitochondrion related organelles ­ in G. intestinalis mitosomes, not­participating in energetic metabolism, in T. vaginalis hydrogenosomes, producing molecular hydrogen and ATP by means of substrate phosphorylation. Traditional methods based on a fractionation by ultracentrifuging in density gradient and subsequent biochemical and enzymological analyzes were extended by one­ and two­dimensional electrophoresis with subsequent identification of proteins by mass spectrometry. Methods of multidimensional separation of peptides produced by specific proteolysis of a complex mixture...

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.