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Evolution of hydrogenosomes: adaptation of free living protists Mastagamoeba balamuthi and Naegleria gruberi to oxygen-poor environment
Nývltová, Eva ; Tachezy, Jan (advisor) ; TIELENS, Aloysius Gerard Marie (referee) ; Markoš, Anton (referee)
Various protists from different eukaryotic groups are able to live in the oxygen-poor niches. Their metabolic adaptation to anaerobiosis is usually associated with loss of the typical mitochondrial functions, including the tricarboxylic acid cycle and oxidative phosphorylation. Anaerobic forms of mitochondria generate ATP exclusively by the substrate level phosphorylation in the hydrogen-producing hydrogenosomes, or the ATP synthesis is completely lost as observed in mitosomes. Consequently, the proteomes of such organelles are considerably reduced. It is a question of debate whether the anaerobic forms of mitochondria evolved directly from premitochondrial organelles that might be present in ancient anaerobic eukaryotes or during the secondary adaptation of aerobic eukaryotes to anaerobic niches. The protist from super group Amoebozoa, Mastigamoeba balamuthi, is very attractive for study of mitochondria evolution, because it is closely related with two very different organisms: (i) the aerobic, free-living slime molds such as Dictyostelium that possesses classical aerobic mitochondria, as well as (ii) the anaerobic parasite Entamoeba histolytica that contains mitosomes, the most reduced form of mitochondria. The mitochondria derived organelles in anaerobic, free-living M. balamuthi could represent...
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FeS cluster assembly in cytosol of Trichomonas vaginalis
Stojanovová, Darja ; Tachezy, Jan (advisor) ; Šuťák, Róbert (referee)
Iron-sulfur (FeS) cluster assembly is extensively studied in model organisms, e.g. Saccharomyces cerevisiae, Homo sapiens, and more recently in Trypanosoma brucei. However, little is known about FeS assembly in divergent anaerobic organisms such as Trichomonas vaginalis, which parasites in the human urogenital tract. This parasitic protist possesses anaerobic form of mitochondria, the hydrogenosome, in which some component of FeS cluster assembly machinery (ISC) has been identified, whereas the cytosolic CIA pathway has not been studied so far. Our work deals mainly with TvIscU, a component of ISC pathway, and T. vaginalis CIA pathway. We suggest that both hydrogenosomal and cytosolic FeS cluster assembly pathways of this parasite differ from typical models. We examined possible ISC-CIA relationship. Next, we found homologues for several key components involved CIA machinery, namely Nbp35, Cfd1, Nar1, Cia1 and Cia2. However, we did not identify any homologous proteins to Tah18, Dre2 and Mms19. We expressed identified proteins with HA-tag and localized them by cell fractionation and immunofluorescence microscopy in T. vaginalis. Finally, we immunoprecipitated two Cfd1 paralogues, TvCfd1A and TvCfd1B to search for their interacting partners. The results suggest that these two paralogues interact with...
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Pathogenic trichomonads in cats and dogs
Vobořilová, Pavlína ; Tachezy, Jan (advisor) ; Hampl, Vladimír (referee)
Trichomonads are anaerobic flagellated protists that are either parasites or commensals. They frequently inhabit digestive, respiratory, and urogenital tracts of vertebrates, including domestic cats and dogs. In these hosts, four trichomonad species has been described: Tetratrichomonas canistomae and Tetratrichomonas felistomae that are commensals of the host oral cavity; Pentatrichomonas hominis, a commensal of intestinal tract that could be found in dogs and cats but also in other mammals including humans; and pathogenic Tritrichomonas foetus that causes, in addition to cattle infection, feline intestinal trichomonosis. Although, trichomonads in dogs and cats are probably of cosmopolitan distribution we have no information about their presence in Czech Republic. The first aim of this study was to distinguish types of trichomonads present in the oral cavity of dogs and cats and to get preliminary epidemiological data. The second aim was to demonstrate the presence of Tritrichomonas foetus in cats and dogs in the Czech Republic and to identify potential risk factors for the disease. Cultivation and nested PCR were used to determine the presence of trichomonads in dogs and cats. Sequencing and phylogenetic analysis based on ITS1-5.8rRNA-ITS2 gene sequence was used to identify species of isolated...
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Reverse genetics in anaerobic protists
Stojanovová, Darja ; Tachezy, Jan (advisor) ; Doležal, Pavel (referee)
This work is focused on reverese genetics of anaerobic protists, mainly T. vaginalis, G. intestinalis and E. histolytica and deals with techniques and experimental procedures of genome manipulation in these parasites. Both DNA and RNA can be manipulated and the gene function can be disclosed using methods of reverse genetics. The knowledge gained is useful in many ways. For example, using these techniques crucial aspects of biology of parasitic prostist are studied, providing basis for potential development of new drugs. Utilization of such methods also helps to understand the cellular and metabolic pathways and mechanisms, that could be very diverse or reduced in protists. The methods of reverse genetics that result in permanent and inheritable changes in DNA are, for instance, homologous recombination or DNA integration. There is also a transcriptional gene silencing (TGS) technique to stop gene expression even though the coding DNA remains unchanged. TGS could be realized by several mechanisms, for example by RNA interference. RNA interference pathway, commonly known as posttranscriptional gene silencing mechanism, causes the breakage of mRNA or stops its translation. Other techniques of gene silencing involve, e.g., the expression of antisense RNA, oligonucleotudes and ribozymes.
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Protein translocation into hydrogenosomes of "Trichomonas vaginalis"
Radhakrishna Makki, Abhijith ; Tachezy, Jan (advisor) ; Hashimi, Hassan (referee) ; JACKSON, Catherine Lynn (referee)
Mitochondria carry out several important functions in eukaryotic cells such as energy metabolism, iron-sulfur cluster assembly, apoptosis, signaling pathways, protein quality control etc. Most mitochondrial proteins are synthesized on the cytosolic ribosomes and transported to the organelles by the cytosolic chaperones and mitochondrial protein import machinery based on specific targeting signals. Although, the basic principles of protein import have been explained, many questions remain unanswered, particularly for highly modified mitochondria such as hydrogenosomes. The aim of the study was to investigate protein translocation into hydrogenosomes of a human parasite, Trichomonas vaginalis (Tv) with a focus on the composition, function and structure of protein translocases and the role of targeting signals. The translocase of the outer membrane (TOM) is responsible for the import of most proteins into the organelle. Even though, the presence of a TOM complex in trichomonad hydrogenosomes was predicted, its components were not known. Moreover, the generic structure of the mitochondrial TOM complex was not resolved. This study showed that the TvTOM complex is highly divergent consisting of two modified core subunits - channel- forming TvTom40 isoforms and a Tom22-like protein, and two...
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