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Anaerobic peroxisomes in Archamoebae
Le, Tien ; Tachezy, Jan (advisor) ; Michels, Paul A. M. (referee) ; Yurchenko, Vyacheslav (referee)
Peroxisomes and mitochondria play a key role in oxygen metabolism. It was widely accepted that the evolutionary adaptation of eukaryotes to anaerobiosis is reflected by the metabolic reduction of mitochondria, and concomitant loss of peroxisomes. The anaerobic protists Mastigamoeba balamuthi (Mb), Pelomyxa schiedti (Ps), and Entamoeba histolytica (Eh) contradict this paradigm. They possess anaerobic types of mitochondria (hydrogenosomes, mitosomes) but also host "anaerobic peroxisomes". Mb/Ps peroxisomes contain a common set of 13 peroxins (Pexs) that retain the core members of each functional category including components of both PTS1 and PTS2 machinery for matrix protein import. However, Eh peroxisomes harbour a reduced set of 7 known Pexs and lacks several components that are highly conserved among most eukaryotic lineages, including components of PTS2 machinery (Pex7), the RING complex (Pex2/10/12), docking complex (Pex13), and peroxisomal membrane protein import receptor (Pex3). Concerning the functional annotation, no clear biochemical context has been found in these anaerobic peroxisomes. They are diverse in enzymatic contents and are involved in various metabolic reactions, while catalase and typical peroxisomal enzymes of fatty acid beta-oxidation are absent. Mb peroxisomes appear to be involved in...
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Analysis of lysosomes of Trichomonas vaginalis
Zimmann, Nadine ; Tachezy, Jan (advisor) ; Walochnik, Julia (referee) ; Field, Mark (referee)
Lysosomes represent the central degradative compartment of eukaryote cells. Harboring a variety of acid hydrolases at acidic pH, this organelle is designed for the degradation and recycling of material for cellular homeostasis and sustenance. Studies on mammalian lysosomes have been extensive and revealed a long list of lysosomal proteins. While the function of most of these remains elusive, it is not surprising that a large subset have been found to be hydrolases. However, little is known about the biogenesis and function of this organelle in parasitic protists, and even less about its role in secretion. This work aimed to shed light on the (phago-)lysosomal proteome of the human parasite Trichomonas vaginalis, its protein targeting, and involvement in hydrolase secretion. Our studies revealed a lysosomal proteome of 462 proteins in 21 functional classes. Hydrolases represented the largest functional class and included proteases, lipases, phosphatases, and glycosidases. The identification of a large set of proteins involved in vesicular trafficking and cytoskeleton rearrangement indicates a dynamic phagolysosomal compartment. Our research, as well as the research of others, have identified several hydrolases also in the secretome, including the cysteine protease TvCP2. However, previously the mode...
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Resistance of Trichomonas vaginalis and other anaerobic pathogens to metronidazole
Havelka, Michal ; Tachezy, Jan (advisor) ; Rada, Petr (referee)
The aim of this bachelor thesis is to desbribe mechanisms of resistance to metronidazole in three anaerobic eukaryotic pathogens (T. vaginalis, G. intestinalis, E. histolytica). Diseases caused by these pathogens belong to the list of important but currently neglected diseases. Metronidazole acts only on microbes with an anaerobic metabolism. The drug enters the cell by passive diffusion and needs to be activated by reduction of the nitro group. Mechanisms of activation are different for every pathogen. Enzymes bound with energetic metabolism in hydrogenosomes, cytosolic thioredoxin reductase and nitroreductases play a major role in the activation. The drug damages cells in three ways - DNA damage, formation of covalent bonds with proteins, and covalent bonds with thiols. Pathogens have specific mechanisms to defend themselves against the drug. They can either down-regulate enzymes that activate metronidazole, reduce it to non-reactive aminoimidazole or they can increase the intracellular concentration of oxygen which leads to deactivation of the drug by futile cycling. These mechanisms are bound with physiological changes and subsequently with lowered viability of these pathogens. Furazolidone and benzimidazole derivatives are the best candidates to become an alternative to metronidazole for the...
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Role of exosomes and ectosomes in Trichomonas vaginalis virulence
Göblová, Rebeka ; Tachezy, Jan (advisor) ; Ditrich, Oleg (referee)
Trichomonas vaginalis is a causative agent of the most common non-viral sexually transmitted disease with approximately 275 mil new cases annually. Virulence of this parasitic depends on at least four factors: cell shape transformation, cytoadherence, secretion of cysteine proteases, and presence of endosymbionts. Over the past decades, extracellular vesicles appeared being another important player in the host-parasite interaction. It was discovered that T. vaginalis is one of the protists that can shed the extracellular vesicles such as exosomes and ectosomes. These vesicles are possibly involved in host-parasite communications, however limited information is available about their function. To investigate a possible role of exosomes in T. vaginalis virulence, we first selected suitable strain, which is free of endosymbionts (TV 17-2MI). Next we prepared six clones of TV 17-2MI strain to test whether the strain is homogenous concerning the virulence, or there are differences in virulence among individual cells. Mouse intraperitoneal virulence tests revealed that the clones displayed significant differences in virulence level, particularly in abscess formation and mortality of infected animals. Thus, for the first time we demonstrated heterogeneity of cells derived from a single T. vaginalis strain...
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Analysis of the genome of a free-living amoeba Mastigamoeba balamuthi and its comparison with pathogenic Entamoeba histolytica
Žárský, Vojtěch ; Tachezy, Jan (advisor) ; Bruchhaus, Iris (referee) ; Beneš, Vladimír (referee)
Charles University, Faculty of Science Department of parasitology Doctoral study programme: Parasitology Abstract (en) Analysis of the genome of a free-living amoeba Mastigamoeba balamuthi and its comparison with pathogenic Entamoeba histolytica Mgr. Vojtěch Žárský Supervisor: prof. RNDr. Jan Tachezy, Ph.D. Praha, 2020 Abstract Examination and comparison of organisms have been tremendously important for the study of life's history on earth. The progress of our understanding of the genetic basis of heredity and the recent boom of sequencing technologies allows us to continue in this exciting field of research from the perspective of genes and genomes. In this work, I focus on the study of an anaerobic amoeba Mastigamoeba balamuthi, which is related to an important human pathogen Entamoeba histolytica. Comparative analysis allows us to draw some conclusions about the nature of the common ancestor of Mastigamoeba and E. histolytica, how it adapted to the anaerobic lifestyle, and about the way the Entamoeba lineage evolved to become a successful parasite. Surprisingly we also noticed that besides hydrogenosomes (hydrogen-producing organelles related to mitochondria), M. balamuthi also harbors peroxisomes - organelles thought to be absent in anaerobic organisms. This finding motivated us to inquire more about...
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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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Interaction between hydrogenosomes and endoplasmic reticulum in Trichomonas vaginalis
Kučerová, Jitka ; Tachezy, Jan (advisor) ; Černý, Jan (referee)
Endoplasmic reticulum-mitochondria encounter structure (ERMES) is a protein complex tethering ER and mitochondria. ERMES consists of four core subunits - Mmm1, Mmm2 (Mdm34), Mdm10 and Mdm12. It was first discovered in Saccharomyces cerevisiae and most functional information is based on studies of this organism. ERMES affects mitochondrial distribution and morphology, participates in lipid trafficking and is important for homeostasis of the cell. In Trichomonas vaginalis, the human urogenital parasite, three genes for putative, highly divergent components of ERMES complex were predicted. However, the cell localization of these proteins and their function is unknown. This thesis is focused on investigation of ERMES components in T. vaginalis, their cellular localization, interactions between components and identification of their possible interacting partners.
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Virulence factors of Entamoeba histolytica
Novotná, Monika ; Tachezy, Jan (advisor) ; Tůmová, Pavla (referee)
The parasitic protist Entamoeba histolytica causes intestinal disease called amoebiasis (amoebic dysentery), which is one of the most significant diseases worldwide, mainly in developing countries. The goal of this bachelor thesis is to summarize current knowledge about virulence factors of E. histolytica. It is primarly focused on adhesive lectin Gal/GalNAc, cysteine proteases, phagocytosis of apoptotic cells, amoebapore forming pores in the membranes of the target cells and trogocytosis. Keywords: virulence factors, Entamoeba histolytica, parasite, protist, amoebiasis, lectin Gal/GalNAc, cysteine proteinase, phagocytosis, trogocytosis, amoebapore
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Exosomes in parasitic protists
Horáčková, Jana ; Tachezy, Jan (advisor) ; Štáfková, Jitka (referee)
Most cells release extracellular vesicles. These vesicles are exosomes, ectosomes and apoptotic bodies. The main function of ectosomes and exosomes is the intracellular communication and transport of molecules between cells. The goal of this bachelor thesis is to review the current knowledge about exosomes and ectosomes and how these vesicles contribute to the pathogenicity of parasitic protists. The parasitic protists release exosomes and ectosomes that contribute to their development in the host and play a key role in their pathogenicity. Exosomes and ectosomes can also affect the immune system of the host and facilitate the invasion of parasitic protists into the host cells.
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Mitochondrion-related organelles in diplomonads
Stoklasa, Michal ; Tachezy, Jan (advisor) ; Kolísko, Martin (referee)
Order Diplomonadida includes parasitic and free-living species that adapted to the oxygen-poor environment. They possess reduced form of mitochondria (hydrogenosome or mitosome). These organelles lack Krebs cycle and membrane electron-transport chain. ATP synthesis by oxidative phosphorylation and other mitochondrial metabolic pathways are modified or entirely absent. Main difference between hydrogenosome and mitosome is synthesis of hydrogen using the enzyme hydrogenase and ATP synthesis by substrate level phosphorylation in hydrogenosomes that are absent in mitosomes. The most studied diplomonads are a human pathogen Giardia intestinalis possessing the mitosomes and a salmon parasite Spironucleus salmonicida with hydrogenosomes. This thesis was focused on determining the type of mitochondrial organelles in angelfish parasite Spironucleus vortens and free living Hexamita sp. It has not been described whether they possess the hydrogenosomes or the mitosomes so far. In both protists transmission electron microscopy revealed presence of double membrane vesicles, possibly their mitochondrial organelles. Homologous S. vortens anti- hydrogenase and anti-HydE antibodies were produced and tested in order to determine their cellular localization. Using the western blot analysis and immunofluorescence...
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