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Subcellular localization of resistant proteins Vga(A)LC and Msr(A) using fluorescence microscopy
Nguyen Thi Ngoc, Bich ; Balíková Novotná, Gabriela (advisor) ; Lichá, Irena (referee)
Vga(A)LC and Msr(A) are clinically significant resistant proteins in staphylococci that confer resistance to translational inhibitors. They belong to ARE ABC-F protein subfamily, which is part of ABC transporters. Unlike typical ABC transporters, ABC-F proteins do not have transmembrane domains that are responsible for the transport of substances through the membrane. Therefore, they do not have characteristic transport function but regulatory or resistance function. Their mechanism of action on the ribosome has been described only recently, where these proteins displace the antibiotic from the ribosome. However, some aspects of their function are still unclear. For example, what is the function of the Vga(A) location on a membrane that has been detected in the membrane fraction but not in the ribosomal. In this work, using fluorescence microscopy, I observed subcellular localization of the Vga(A)LC-mEos2, Vga(A)LC-GFP and Msr(A)-eqFP650 resistant fusion proteins in live cells of S. aureus under different culture conditions . It has been shown that Vga(A)LC-GFP and Msr(A)-eqFP650 occur in a foci near the membrane. Depending on ATPase activity or the presence of an antibiotic, the localization of Msr(A)-eqFP650 in the cell changes from focal to diffuse, presumably on ribosomes, suggesting a...
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Comparison of specific expression in bacterial and yeast biofilms
Kicko, Peter ; Palková, Zdena (advisor) ; Lichá, Irena (referee)
The development and maintenance of biofilm is a complex process that is based on a change in genetic expression. The biofilm formation is observed in some prokaryotic and eukaryotic cells. During its formation, cell aggregation occurs, extracellular matrix is created and we observe the formation of metabolically differentiable cells, often with increased resistance to antimicrobial drugs. This work focuses on important steps leading to biofilm formation associated with specific gene expression and highlights the similar and different processes between bacterial and yeast cells. The work begins by comparison of cell signalling, it continues by comparing the expression of the adhesive proteins and extracellular enzymes, synthesis of exopolysaccharides, formation of extracellular nucleic acid, and in the last chapter we focused on the formation of persistors. The aim of this work is to connect the acquired information and to contribute to the understanding the complexity of this process. Key words: biofilm, signalling, adhesins, exopolysaccharides, extracellular nucleic acid, persistor
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Mode of daptomycin action and mechanism of resistance
Helusová, Michaela ; Mikušová, Gabriela (advisor) ; Lichá, Irena (referee)
Daptomycin is one of antimicrobial peptides. These molecules are part of immune system of all organisms. Daptomycin consists of a cyclic peptide core and a lipophilic tail. Daptomycin is produced by Streptomyces roseosporus and is used to treat serious gram-positive infections. Daptomycin is active also against methicillin-resistant Staphylococcus aureus. Its antimicrobial effect depends on the presence of calcium ions and phosphatidylglycerol. Daptomycin targets bacterial cytoplasmic membrane, where it forms oligomers. Mode of action of daptomycin probably includes pore-forming effect leading to membrane damage. This disturbance causes ion efflux from cytoplasm which leads to membrane potential disruption, which results in inhibition of macromolecular synthesis and cell death. Daptomycin also causes changes in cell morphology. Despite its unusual mode of action, several mechanisms of resistance have emerged in some pathogenic strains. These are for example decrease in the amount of phosphatidylglycerol in the membrane, increase in the amount of lysylphosphatidylglycerol, release of membrane phospholipids or mutations in genes which control peptidoglycan synthesis.
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Oxidative stress in bacteria - with an emphasis on the model organism of Escherichia coli
Moravcová, Andrea ; Lichá, Irena (advisor) ; Čáp, Michal (referee)
Most bacterial species encounter aerobic conditions during their life, which can be toxic. This leads to a state of oxidative stress. Toxicity of aerobic environment results from the chemical properties of molecular oxygen and its reactive species (ROS). Bacteria had to adapt to these conditions in the past to ensure preservation and prosperity. This thesis is preferably focused on oxidative stress adaptations in the most elaborated bacterial model - Escherichia coli. Regulation of adaptations at the regulation of transcription, translation and metabolism level are described with emphasis on molecular mechanisms. The main adaptation mechanism against oxidative stress is the deactivation of ROS, as well as the repair of damaged cell structures (macromolecules). These enzyme activities are regulated by several transcriptional regulators. The transcriptional regulators OxyR and SoxRS have been well studied in E. coli. Even though these regulators are conserved across the bacterial spectrum, they may not have the same function in all organisms. For this reason, also other, more or less studied bacterial species - Bacillus subtilis, Streptomyces coelicolor, Pseudomonas putida, Pseudomonas aeruginosa - were included in this thesis. The various strategies of how these bacteria use not only OxyR and SoxRS...
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The use of animal models in the study of pathogenesis of Bordetella pertussis
Traganová, Veronika ; Holubová, Jana (advisor) ; Lichá, Irena (referee)
Bordetella pertussis is a strictly human pathogen and a causative agent of whooping cough. The study of bacterial transmission, virulence factors and vaccine efficacy testing became a very relevant topic, due to the disease resurgence in well-vaccinated populations during the past decades. Detailed investigation of bacterial interactions in vivo requires a suitable animal model. The most common used animal for B. pertussis testing is a laboratory mouse, however mice are unable to develop the typical B. pertussis infection symptomps. In the field of vaccine research and testing, the newborn pig has also proved to be a suitable model. By far the best nonhuman host of B. pertussis is the recently discovered baboon model which can perfectly imitate the human respiratory tract conditions, immune response and also the host-to-host transmission. This thesis summarizes basic knowledge of model organisms used in the past and in the present for the study of B. pertussis. At the same time, it shows the comparison of the advantages and disadvantages of the discussed animal models.
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