|
| |
|
|
Catalytic function of DNA-dependent RNA polymerases
Sýkora, Michal ; Vopálenský, Václav (advisor) ; Lichá, Irena (referee)
DNA-dependent RNA polymerase is a highly organised protein complex that is responsible for gene expression and its regulation. Multisubunit RNA polymerase with its several catalytic activities is responsible for transcription of genes to RNA copies in all cellular organisms. During transcription RNA polymerase undergoes substantial conformational changes depending on the conditions in a particular cell. RNA polymerase in a state designated as an elongation complex passes through repetitive cycles of adding a nucleotide to the growing RNA chain. The active center contains two magnesium ions which coordinate the reactive groups of substrates. Furthermore, the active center contains structural elements that participate in binding of substrate, propper orientation of substrate towards the template strand and translocation of the RNA polymerase. The most important of these mobile structural elements are the bridge helix and the trigger loop whose conformational changes accompanies nucleotide addition cycle. Advances in the structural and biochemical characterization of RNA polymerase open new possibilities in the understanding of the transcription mechanism, its fidelity and control.
|
|
|
Study of persistence mechanisms to antibiotics in Staphylococcus aureus
Balgová, Tamara ; Lichá, Irena (advisor) ; Ulrych, Aleš (referee)
In clinical practice, infections caused by persistent bacteria have become a worldwide problem. We are talking about a subpopulation of cells that are able to withstand lethal doses of antibiotics and after their elimination are capable of resuscitation and re-induction of the disease. The opportunistic pathogen Staphylococcus aureus is one of this bacteria and causes various serious chronic infections. During the long-term persistence in patients, persistent bacteria acquire adaptive mutations. The aim of this diploma thesis was to monitor the degree of persistence in selected clinical isolates, and at the same time to demonstrate the effect of adaptive mutations on the degree of persistence as well as to molecularly characterize the persistent state by gene expression. I had chronological isolates of S. aureus at my disposal, the initial one being the primoisolate, an isolate taken at the diagnostics of cystic fibrosis before the start of antibiotic treatment. Another was taken at a distance of one year and the last with a half-year interval from the previous one. Following whole genome sequencing, genes in which adaptive mutations occurred were identified. The first method determines the degree of persistence by calculating CFU (Colony Forming Units) after antibiotic treatment. I found that this...
|
|
|
Enterococcus spp. as a reservoir of resistance genes.
Zajíčková, Adéla ; Lichá, Irena (advisor) ; Balíková Novotná, Gabriela (referee)
Bacteria of the genus Enterococcus are categorised among common nosocomial pathogens. They are a significant reservoir of resistance genes to a majority of antibiotics and exhibit an intrinsic resistance to low levels of beta-lactams, glycopeptides, aminoglycosides, streptogramines and lincosamides. The aim of this paper is to review the main resistance genes and other mechanisms involved in the resistance of bacteria of this genus to antibiotics. The paper is mainly focused on the resistance to beta-lactam antibiotics, which is provided by the expression and mutations of low-affinity PBPs, the individual van resistance types mediating resistance to vancomycin, and the expression of enzymes capable of modifying the functional groups of aminoglycoside antibiotics. The paper also describes the resistance to newer antibiotics that are used to treat vancomycin-resistant isolates. The resistance to individual antibiotics can arise from the coding of their own chromosomal genes or entire signaling pathways leading to a reduction in the effect of antibiotics, the acquisition of genetic mutations, and especially the spread of new resistance genes by horizontal transfer.
|
|
|
Changes in the ability to form persisters in chronological isolates of Staphylococcus aureus
Kotková, Hana ; Lichá, Irena (advisor) ; Tkadlec, Jan (referee)
In immunodeficient patients, for example with cystic fibrosis (CF), the opportunistic pathogen Staphylococcus aureus causes chronic infections of respiratory tract that are treated with antibiotics (ATB) in the long term. However, exposure to antibiotics can lead to persistence, thereby result a recurrence of infection. The aim of this work was to examine in selected pairs of S. aureus chronological isolates from the respiratory tract of CF patients how their ability to form persisters is changing in time. I have found that the ability to persist within the clonal pair does not change significantly after two years of survival in the host, and that the ability to persist depends on the adaptative mutations of the isolates. Persister formation may depend on mutations in operon of the alternative sigma B factor (sigB) and the major virulence gene regulator (agr). By dual staining with DioC2(3) and To-pro-3, I was able to determine the changes in membrane potential and membrane permeability during the killing curve with ATBs. The distribution into subpopulations according to these parameters depends primarily on the antibiotic used. I conclude that various antibiotics can induce different mechanisms causing a persistent state. Futhermore, I have constructed plasmids with a labeled promoter to determine...
|
|
|
Heterogeneity of expression of virulence factors of Salmonella.
Mathéová, Paulína ; Černý, Ondřej (advisor) ; Lichá, Irena (referee)
The emergence of phenotypically heterogeneous individuals within an isogenic bacterial population is considered to be an important adaptation to the host environment. It allows survival of some bacterial subpopulations under diverse stress conditions caused by the host immune system, the emergence of a "division of labor" and cooperation between individual bacteria. Bacteria of the genus Salmonella are important pathogens in humans and livestock. Many of the key virulence factors of Salmonella are heterogeneously expressed. The phenotypic diversity of individual bacteria allows certain individuals to escape the host's immune system and ensure that the gene pool is preserved to future generations. In case when change in conditions causes complete extinction of part of the Salmonella population from the environment, the remaining individuals are able to restore the size of the population and phenotypic diversity, after overcoming unfavourable conditions. This work summarises the knowledge about heterogeneity of expression of virulence factors of Salmonella and the characteristics of individual subpopulations in different environmental conditions. Keywords: Salmonella, heterogeneity, virulence factors, bet-hedging, division-of-labor, bacterial subpopulations.
|
|
|
Molecular mechanisms of bacterial persistence to antibiotics
Jirsová, Anežka ; Lichá, Irena (advisor) ; Branny, Pavel (referee)
The ability to persist is inherent in the vast majority of bacterial species. Persisters represent a small heterogenous fraction of the bacterial population that can tolerate antibiotics. Unlike resistant cells, which are genetically distinct from the rest of the antibiotic-sensitive population, persister cells form a genetically indistinguishable subpopulation. Persisters arise either as a result of stress caused by adverse environmental conditions, or persister subpopulation arise as a result of stochastic induction independent of the presence of stressful conditions. There are various mechanisms by which bacterial cells have been shown to induce a persistent state. Toxin-antitoxin systems and their interaction with stringent response effectors play an important role during the development of persistence. The persistent state is also affected by changes in proton-motive force (PMF) and the fluctuations in the gene expression level of energy generating enzymes of the Krebs cycle (TCA). In this thesis, persister cells are characterized and the current knowledge about the molecular mechanisms leading to the induction of persistence in bacteria is summarized. Key words: bacteria, persistence, antibiotics, stringent response, PMF, TCA
|
|
|
Regulation of virulence factors of Staphylococcus aureus
Šaňková, Michaela ; Lichá, Irena (advisor) ; Černý, Ondřej (referee)
Staphylococcus aureus is a gram-positive pathogenic bacterium that regulates virulence factors production in response to changing environmental conditions. S. aureus cells evolved a complex regulatory network, including a number of regulatory proteins, transcriptional factors and two- component systems. One of the most important S. aureus regulatory systems is the Agr system (Accessory gene regulator) that perceives its own population density by sensing a "quorum-sensing" signal in a form of autoinducing peptid (AIP). Agr system encodes a global regulatory RNAIII that regulates the expression of target virulence factors, which includes surface proteins as well as extracellular toxins and enzymes. The family of global protein regulators SarA and transcriptional sigma factor B also play a significant role in the regulation of S. aureus virulence. The production of virulence factors is also regulated in response to specific signals from extracellular environment by two- component-systems, which includes the regulator of exoprotein production SaeRS, the regulator of autolysis ArlRS and the regulator of respiratory response SrrAB. Key words: Staphylococcus aureus, virulence factors, Agr, quorum-sensing, RNAIII, SarA, SigB
|
|
|
Evaluation of TD test for analysis of persistence or tolerance in clinical isolates of Staphylococcus aureus
Kotková, Hana ; Lichá, Irena (advisor) ; Fišer, Radovan (referee)
Persistence is the ability of bacteria to survive the impact of antibiotics even when the bacteria do not encode resistance genes. This is a very complex process, which is probably consequence of a reduction physiological process in subpopulation of bacteria. The aim of this study was to verify the suitability of the newly developed "Tolerance Disk Test" (TD test, Gefen et al, 2017) for detection of persistent or tolerant subpopulations of bacterial cells in clinical isolates of Staphylococcus aureus. We performed TD test for this kind of bacteria, which is a significant opportunistic pathogenic organism in humans, and compared its results with the killing curves. We have found that the ability to persist can be monitored semi-quantitatively also in this case and we consider this test suitable for introduction into clinical practice. In addition, we suggest that TD test could distinguish between persistent and tolerant subpopulations. Key words: Staphylococcus aureus, persistence, antibiotics, tolerance
|
|
|
Intracellular and intercellular regulation of gene expression in Gram-positive bacteria.
Pospíšil, Jiří ; Krásný, Libor (advisor) ; Lichá, Irena (referee) ; Malínský, Jan (referee)
Bacteria, the most dominant organisms on Earth, are an everyday presence in our lives. Symbiotic bacteria, which are present in the digestive tract of animals, usually have a beneficial effect on the body. On the opposite side of the spectrum are pathogenic species that cause more or less serious diseases around the world. In order to fight pathogens effectively, it is necessary to learn as much as possible about the molecular mechanisms by which bacteria respond to their environment, and also about the types of communication within bacterial populations that allow them to react to environmental changes as "multicellular" organisms. This Thesis consists of two main parts. In the first part, selected aspects of bacterial gene expression are characterized, using Bacillus subtilis and Mycobacterium smegmatis as model organisms. DNA-dependent RNA polymerase (RNAP) is the enzyme that is responsible for transcription of DNA into RNA, and thus plays a key role in gene expression. This Thesis deals with the structure of bacterial RNAP and important auxiliary factors (proteins and RNA) that associate with this enzyme and modulate its function. In the second part, the focus is on cell-to-cell communication, revealing which factors/mechanisms, including gene expression, affect this process in B. subtilis....
|
guest ::
