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Anaerobic fungi and herbivores
Pristašová, Paulína ; Tachezy, Jan (advisor) ; Hrdý, Ivan (referee)
Anaerobic fungi Neocallimastigomycotina participate in rumen symbiotic microbiome. This highly specific group of fungi are a part of the basal lineages of the Fungi kingdom, differing from their aerobic counterparts in many aspects, including the possession of hydrogenosomes and the production of cellulosomes. Their entire life cycle occurs in the rumen, comprising of motile zoospores, which after colonizing plant biomass, germinate into vegetative thallus, forming a sporangium. In the rumen ecosystem, anaerobic fungi are primary colonizers of ingested plant biomass, where with the help of extracelullar multienzyme complexes- cellulosomes- degrade recalcitrant plant cell wall. The ability to form cellulosomes, until now observed in prokaryots only, was acquired by horizontal gene transfer from rumen symbiotic bacteria. Their long coevolution also led to many syntrophic interactions with methanogens, which utilise H2 produced by fungal hydrogenosomes. Their potential use in biotechnology includes biogas production and increased digestibility of agricultural feed. Key words: anaerobic fungi, rumen, hydrogenosome, cellulosome
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Molecular characterization of the factors participating in the formation of Giardia intestinalis cyst wall.
Pastyříková, Aneta ; Doležal, Pavel (advisor) ; Hrdý, Ivan (referee)
1 Abstract The surface of Giardia intestinalis cysts is covered by a thick fibrillar wall providing the parasite with the necessary protection against the external environment. The cyst wall consists of proteins and carbohydrates, among which is a unique β-1,3-N-acetylgalactosamine homopolymer (known as Giardan). This carbohydrate is synthesized from glucose by cytosolic enzymes induced during encystation. This thesis focuses on the biosynthesis of Giardan and the functional characterization of UDP-N-acetylglucosamine 4'-epimerase (UA4E), one of the enzymes involved in the synthetic pathway. For these purposes, the corresponding gene was targeted using the CRISPR/Cas9 system. A stable cell line was obtained in which all four copies of the UA4E gene were deleted. The success of gene knock-out was confirmed on the basis of proteomic analysis. Using fluorescence microscopy, we observed that ΔUA4E cells induced to encystation are able to initiate encystation processes and cell differentiation. The presence of ESV vesicles and typical morphological changes corresponding to cysts were observed in the cells. However, these stages had a thin surface layer and were unable to form typical thick wall with fibrillar appearance at their surface leading to the subsequent production of non-viable cysts. Key words Giardia...
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Current methods in protein-protein interactions research
Křivková, Jana ; Hrdý, Ivan (advisor) ; Kučerová, Jitka (referee)
Protein-protein interactions (PPI) have a crucial role in all processes in living cells. Understanding the interactions between proteins allows us to describe cell processes in more detail and their study opens new possibilities for drug design. The importance of the question of studying PPI is shown in the recent development of various methods for their identification and description. The aim of this thesis is to give an overview of new and improved experimental methods of identification and characterization of protein-protein interactions. Methods described in this thesis are divided in four chapters - proximity-dependent labelling methods (BioID, BioID2, APEX, TurboID, MiniTurbo, PUP-IT, AirID, SPPLAT, EMARS), cross-linking methods (XS-MS), fluorescence methods for identification and visualization (BiFC, FRET, BRET) and biophysical methods for description of kinetics and thermodynamics parameters of interaction (SPR, ITC, MT).
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Novel protease in the reduced mitochondria of Giardia intestinalis.
Vondráčková, Pavlína ; Doležal, Pavel (advisor) ; Hrdý, Ivan (referee)
The proteom of Giardia mitosomes is represented by twenty proteins, which are imported from the cytoplasm, as mitochondrial genome is missing. The N-terminal sequences, which target proteins to mitosomes are removed upon translocation and the remaining protein is folded into its native comformation. Out of three types of mitochondrial processing peptidases only functional half of dominant matrix peptidase is present in the mitosomes of G. intestinalis - feature unique among all eukaryotes. Based on the proteomic analyses, we identified novel putative mitochondrial peptidase (peptidase X) in G. intestinalis mitosomes. Overexpressing the protein with hemagglutinin tag we confirmed its mitosomal localization. Using specific polyclonal antibody combined with carbonate extraction and protease protection assay we have specified the distribution of the protein in the mitosomal subcompartments. Based on these results, peptidase X is anchored in the inner mitosomal mambrane via its C-terminus facing the mitosomal matrix. Peptidase X has mitochondrial targeting sekvence on the N-terminus, which is also capable of targeting protein into yeast mitochondria. By using bioinformatic tools, we are trying to detect structural similarities of peptidase X to other proteins in order to identify its substrate specifity and thus...
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Pyruvate:ferredoxin oxidoreductase homologues from Trichomonas vaginalis hydrogenosomes
Zedníková, Věra ; Hrdý, Ivan (advisor) ; Doležal, Pavel (referee)
Oxidative decarboxylation of pyruvate is a fundamental reaction of living organisms in general, leading to energy conservation. In some anaerobic or microaerophilic eukaryotic or prokaryotic organisms pyruvate decarboxylation is carried out by a single enzyme, pyruvate:ferredoxin oxidoreductase (PFO). PFO contains Fe-S clusters and thiamin pyrophosphate cofactor (TPP). In the reaction catalyzed by PFO, from pyruvate and Co-A arise acetyl-CoA, CO2, and two electrons are released. Those electrons are accepted by low molecular carrier proteins. Most frequently these proteins are ferredoxins or flavodoxins such as in nitrogen fixating bacteria. PFO can perform a reversible reaction. Trichomonads are mostly parasitic or endosymbiotic organisms with mitochondria-like organelles, hydrogenosomes. These organelles possess PFO which is one of the key enzymes in the metabolism of Trichomonas vaginalis hydrogenosomes. PFO of T. vaginalis, a sexually transmitted pathogen of man, plays also a role in a term of medical importance. PFO is, by a universally accepted concept, one of the key proteins acting in the activation of antimicrobial drugs against trichomoniasis 5-nitroimidazoles, including metronidazole. In the genome of T. vaginalis seven PFO genes were identified. They were named PFO A, B1, B2, C, D, E and...
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Anthelmintic and other xenobiotic biotransformation in helminths and its contribution to resistance development
Prchal, Lukáš ; Szotáková, Barbora (advisor) ; Hrdý, Ivan (referee) ; Kučera, Radim (referee)
Charles University, Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate Mgr. Lukáš Prchal Supervisor prof. Ing. Barbora Szotáková, Ph.D. Title of Doctoral Thesis Anthelmintic and other xenobiotic biotransformation in helminths and its contribution to resistance development Parasitic helminths such as tapeworms, flukes or nematodes pose a threat for domestic, farm and wild living animals. Helminths cause significant health problems in animals. Moreover, they cause economical losses to farmers due to production decrease and treatment costs. The treatment with veterinary anthelmintics is still a basic method to fight off helminth infections. However, the long-term use of anthelmintics caused an emergence of resistance to anthelmintics. The increasing incidence of multiresistant strains of many helminth species is the greatest problem. Therefore, research of drug resistance mechanisms became an actual field of study. Increased biotransformation of anthelmintics, transport out of parasite bodies and contact of lower developmental stages with anthelmintics in the environment are some of possible mechanisms that lead to decrease of anthelmintic therapy effectivity and resistance development. The presented thesis focuses on the study of these mechanisms in three helminth...
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Mechanisms of metronidazole resistance in bacteria and anaerobic eukaryotes
Pilařová, Kateřina ; Hrdý, Ivan (advisor) ; Kulda, Jaroslav (referee)
This bachelor thesis tries to describe mechanisms of resistance in different groups of organisms, where metronidazole is used for treatment. These organisms are bacteria (genus Bacteroides, Helicobacter, Clostridium) as well as anaerobic eukaryota (genus Trichomonas, Tritrichomonas, Entamoeba, Giardia). It is important, that metronidazole acts selectively only on cells living in anaerobic or microaerophilic environments. Metronidazole enters the cell by passive diffusion and is subsequently reduced to cytotoxic intermediate which damages the cell at different levels. Metronidazole can also enter the cell with aerobic metabolism, also the reduction can occur, but the eventual radical is then thanks to oxygen transformed back to metronidazole in the process called futile cycling. Oxygen then forms superoxide, O2-. Organisms with aerobic or microaerophilic metabolism cannot detoxify nitroradical with oxygen, so they developed other ways how they protect themselves against the harmful effects of nitroradicals. Sensitivity of these patogens to metronidazole correlates with the changes of their enzymatic constitution. Perhaps the most basic mechanism how the patogens combat metronidazole is the prevention of reduction of the drug. The way how the organisms prevent reduction differs between species. There is a...
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Purification of recombinant proteins by affinity chromatography
Zemek, Ondřej ; Mácha, Jaroslav (advisor) ; Hrdý, Ivan (referee)
The isolation and purification of recombinant proteins is essential for further study of their structural and functional properties. The affinity chromatography is usually the method of choice for this task. In this paper the most used affinity tags are reviewed for their properties and experience with their application. The tags covered here include CBP, MBP, GST, polyhistidine and polyarginine tags, FLAG-tag, Strep-tag II and SpA. The origin and properties of the tags, their influence on form and localization of fusion protein as well as binding, elution and removal are discussed. Keywords: affinity chromatography, recombinant protein, purification, affinity tag
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The role of Toxoplasma gondii on the expression of Major Urinary Proteins in the house mouse
Hladovcová, Denisa ; Stopka, Pavel (advisor) ; Hrdý, Ivan (referee)
Major Urinary Proteins (MUP) are pheromonal transmitters involved in chemical communication in rodents. Complexes of MUPs and ligands mediate information about genetical background of an individual and co-create individual scent profile. They play a significant role in kinship determination, the crucial factor in the choice of a mating partner. It is assumed that the MUP production is energetically demanding due to the pressure of sexual selection, and the transmitted signal is thus supposed to be honest. This theory hasn't yet been experimentally tested, neither was proven the effect of another intense selection pressure- a parasitic infection. In my thesis, I describe the effect of an infection of a parasite Toxoplasma gondii on MUP production. The results suggest that the production is altered in both sexes, the production decreasing in males to the level of male castrates (or the female level). Considering the results of more detailed analysis of the infection we assume that the energetical demands of the MUP production doesn't allow the production of ordinary amounts if MUPs during the first phase of the infection as the energy is primarily devoted to the infection control. The increased production in latter phase can be attributed to the importance of scent communication. Decreased levels of...
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