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Application of human monocytic cell line THP-1 for study of pathogenesis in whooping cough agent Bordetella pertussis
Čurnová, Ivana ; Petráčková, Denisa (advisor) ; Mašín, Jiří (referee)
Bordetella pertussis is strictly human pathogen that causes severe infection of the respiratory tract known as whooping cough, which is currently on the rise. B. pertussis was considered as an extracellular pathogen for a very long time. Recently it was shown the ability of B. pertussis to survive inside early endosomes of macrophages. This ability is studied in the human monocytic cell line THP-1 and also in primary macrophages from human donors. This diploma thesis is focused on THP-1 infectious model and mainly for the early phase of infection. A previously performed transcriptomic study showed significantly affected genes of B. pertussis during intracellular survival in THP-1 macrophages. In this study, we selected genes that are in some way related to intracellular survival inside human macrophages or have significantly effect for intracellular survival. The effect of the mutation in these genes was tested both on the level of cytotoxicity to THP-1 cells and the related number of surviving bacteria inside the macrophages. The deletion strain in two genes for cysteine dioxygenase (BP2871 and BP3011) and the mutant strain allocated in the Bvg+ phase were less cytotoxic than the control strain. Monitoring the effect of opsonization to intracellular survival have not such clear results. The effect...
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The role of a conserved tyrosine residue of acylated domain in membrane insertion and penetration of RTX toxins
Lepesheva, Anna ; Mašín, Jiří (advisor) ; Petráčková, Denisa (referee)
Pore-forming RTX toxins are key virulence factors of many Gram-negative pathogens. These proteins share several common structural and functional features: (i) the presence of repetitive sequences rich in glycine and aspartate, which are important for calcium ion binding, (ii) transport from the bacterial cytosol through the type I secretion system (T1SS), (iii) modification by a fatty acid at specific lysines in the acylated domain by a specific acyltransferase, and (iv) the presence of an amphipathic region responsible for the formation of cation-selective pores in the target membrane. The aromatic side chain of the conserved tyrosine residue 940 in the acylated segment of the RTX adenylate cyclase toxin (CyaA, ACT or AC-Hly) of Bordetella pertussis plays a key role in the interaction of the toxin with the target cell membrane. The aim of this study was to determine whether the corresponding conserved residues Y940, Y642, Y643 and Y639 secreted by the homologous RTX toxin CyaA from Bordetella bronchiseptica, HlyA from Escherichia coli, ApxIA from Actinobacillus pleuropneumoniae and RtxA from Kingella kingae play the same critical role in membrane insertion and pore formation. The hemolytic and cytotoxic activities of these toxins were completely impaired only after replacement of the conserved...
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Biofilm formation in Streptococcus pneumoniae
Jarošová, Václava ; Petráčková, Denisa (advisor) ; Matyska Lišková, Petra (referee)
Biofilm is a structured community of cells adhered to the surface or to each other and surrounded by extracellular matrix. Biofilm is fomed in several steps starting from single cells adhered to the surface up to microcolonies linked by channels. Because of a higher resistance to antibiotics the current hot topics in the biofilm research are formation of biofilms on medical materials and treatment of bacterial infections associated with biofilms. This work is focused on a biofilm forming bacteria Streptococcus pneumoniae. It is a potentially pathogenic bacterium which colonizes the upper respiratory tract and causes a number of diseases. Biofilms formed by S. pneumoniae exhibit increased resistance to antibiotics, therefore, alternative antimicrobial agents have been recently studied. For biofilm formation studies both open and closed systems are used. The flow cell and biofilm reactors represent commonly used open systems. Closed cultivation systems are for example a Calgary biofilm device and a micro titer plate-system developed by Christensen et al.
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Study of the interaction between fungus Pleurotus ostreatus and bacterial cultures on the abiotic surfaces - morphological, biochemical and proteomic analysis
Kozická, Barbora ; Petráčková, Denisa (advisor) ; Konopásek, Ivo (referee)
Ligninolytic fungi are well known for their ability to degrade a wide range of xenobiotics contaminating the environment, including synthetic industrial dyes. In this work Pleurotus ostreatus was used for decolorization of a synthetic textile dye Remazol Brilliant Blue R (RBBR). To set up a model fungal "fixed-bed" bioreactor the fungus was immobilized on a polyurethane foam and artificially contaminated with a model bacterium Rhodococcus erythropolis. The development of bacterial contamination can be expected during a real application of fungal bio filters in wastewater treatment. The main aim of the work was to study interspecies interactions in the model bioreactors during the dye decolorization. Ligninolytic enzyme activities were followed in the bioreactor cultures as markers of fungal biodegradation ability. In contrast to the controls, no bacterial growth was observed in the P. ostreatus bioreactor culture liquid. The results showed that fungal laccase, pH of the culture liquid, and glucose consumption by the fungus had no effect on the bacterial growth. However, 4*105 - 1,3*106 CFU/ml of R. erythropolis was detected to be associated with the fungal solid support. The presence of these bacteria had no effect on the decolorization performance of the bioreactors. Dye decolorization efficiency...
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Antibiotic resistance in clinically important strains of Enterococcus spp.
Kozická, Barbora ; Petráčková, Denisa (advisor) ; Motlová, Lucia (referee)
The Enterococcus spp. is a common part of microflora in the digestive tract; it is used in the food industry and added to probiotics. However, in the last few decades it comes to the fore particularly as a cause of nosocomial diseases. Its importance grows with its increasing resistance to antibiotics. The Enterococcus is intrinsically resistant to many types of antibiotics. In addition to that it may acquire additional resistance determinants by mutations or horizontal gene transfer. This work focuses on the Enterococcus faecium and the Enterococcus faecalis intrinsic and acquired resistances, as these two strains have the major clinical importance. In this work, the most attention is dedicated to the antibiotics vancomycin and linezolid. For several decades, vancomycin was the last treatment option in the case of a failure of commonly used antibiotics. The fact that the resistance to this antibiotic was spreading rapidly became a significant problem in these cases of treatment. Hence the antibiotic linezolid was developed as a response to the growing resistance of gram-positive bacteria to available antibiotics. It is also proved to be effective against the vancomycin- resistant strains E. faecium and E. faecalis.
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The role of fatty acylation in activity of proteins
Grobarčíková, Michaela ; Mašín, Jiří (advisor) ; Petráčková, Denisa (referee)
Post-translational modifications are covalent and generally enzyme-mediated modifications of proteins. These modifications can serve to create active forms of proteins and they can also expand the cellular repertoire of proteins derived from standard amino acids. Known post-translational modifications include for example phosphorylation, glycosylation, ubiquitination, proteolysis and also acylation discussed in more detail in this thesis. Acylation of proteins, the covalent attachment of an acyl group, is a very frequent protein modification. This reaction is typically mediated by specific acyl transferases and involves transport of an acyl group from a donor to an amino acid residue. A diverse spectrum of cellular proteins is post-translationally acylated and therefore become biologically active. This phenomenon occurs in bacterial toxins, which are important factors of the virulence of pathogenic bacteria. Protein lipidation regulates numerous biological pathways such as membrane targeting, protein secretion, cell signaling, and apoptosis. Posttranslational acylation is also required for Ras activity and many other cancer-causing proteins. Therefore, inhibitors of acyltransferases of these proteins are being tested as targets for antitumor drugs. In this work, findings about individual types of...
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Interaction of pathogenic Bordetella species with host cells
Čurnová, Ivana ; Petráčková, Denisa (advisor) ; Mašín, Jiří (referee)
Most of the members of the Gram-negative genus Bordetella cause severe infections of the respiratory tract in their hosts. B. pertussis and specific lineages of B. parapertussis infect humans and cause the disease known as whooping cough, a highly contagious respiratory disease that is currently on the rise even in highly vaccinated populations. Therefore, a more detailed understanding of the B. pertussis interactions with the host is crucial. B. pertussis produces a great variety of virulence factors, majority of which is regulated by the two- component BvgAS system. These factors assist the pathogen in the colonization of the host and evasion of the host immune system. The studies on host-pathogen interactions use both in vitro and in vivo infection models, which complement each other appropriately. Recently, it was demonstrated that B. pertussis escape killing and persists in macrophages, suggesting that B. pertussis can be considered as a facultative intracellular pathogen. This ability may allow the pathogen cells to persist within the host and potentially spread to the new host. The aim of this bachelor thesis was to summarize the knowledge on the host-pathogen interactions between B. pertussis and its host with focus on in vitro and in vivo infection models. The attention is paid especially...
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Mechanism of action of bacterial toxins elevating the cAMP in host cells
Lepesheva, Anna ; Mašín, Jiří (advisor) ; Petráčková, Denisa (referee)
Cyclic adenosine monophosphate (cAMP) is an universal second messenger that regulates a large number of molecular mechanisms inside the eukaryotic cell. The level of synthesized cAMP is tightly regulated by endogenous adenylatecyclase (AC), and therefore this enzyme is often a target for various bacterial toxins. To manipulate intracellular cAMP levels in a target cell, bacteria have developed two different strategies for their toxins. Bordetella pertussis adenylate cyclase toxin (CyaA), Bacillus anthracis edema factor (EF) and Pseudomonas aeruginosa exotoxinY have in their structure an enzymatic AC domain which is activated by an intracellular cofactor and has several times higher activity than the eukaryotic AC enzyme itself. Other toxins, such as Bordetella pertussis pertussis toxin (PT), Vibrio cholerae cholera toxin (CT), and Escherichia coli heat labile toxin use ADP-ribosylation reaction of AC-coupled heterotrimeric G proteins to increase its activity and uncontrolled cAMP production. This work presents a literature search with accent on the molecular mechanism of interaction of these toxins with the target cell. Keywords: bacterial pathogens, virulence factors, intracellular cAMP elevation, bacterial toxins, adenylatecyclase (adenylylcyclase), Bordetella pertussis, Vibrio cholerae,...
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The Occurence Of Bla(Tem) And Tet(W) Genes In Czech Wastewaters And Their Fate In Fungal-Based Wastewater Treatment Processes
Svobodová, Kateřina ; Adámek, M. ; Petráčková, Denisa
Antibiotic resistant bacteria and genes (ARB&Gs) have presently emerged as serious environmental pollutants with a high health risk. This study analyzed the occurrence of ARGs tet(W), bla(TEM) and intl1 in microbiomes from 4 Czech wastewater treatment plants showing that bla(TEM) displayed slightly higher gene abundance compared to tet(W). Next, simulated wastewater seeded with activated sludge bacteria was treated with Pleurotus ostreatus-based bioreactor to evaluate the effect of the fungal filter on bacterial community structure and bacterial resistome in wastewaters. The results showed that functional diversity of bacterial communities was significantly changed by their contact with fungal biofilm as revealed by EcoPlate assays and analyses of bacterial 16S rDNA by denaturing gradient gel electrophoresis. A significant decrease in tet(W) abundance (48 times) was observed in fungal-treated wastewater compared to untreated one. The abundance of bla(TEM) genes increased 4 times during the adaptation of bacteria to nutrient conditions and remained unaffected by the fungal treatment. To our knowledge this work is first to study the fate of ARGs in wastewaters during their advanced biological treatment. It documented that P. ostreatus has a potential to lower the abundance of some ARGs in wastewaters.
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