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Influence of environment in host cells on heterogeneity of virulence factors expression in Salmonella
Mathéová, Paulína ; Černý, Ondřej (advisor) ; Konopásek, Ivo (referee)
Bacteria of the genus Salmonella are one of the most important human pathogens. Salmonella expresses several virulence factors, including the type three secretion system encoded on the Salmonella pathogenicity island 2 (SPI-2 T3SS) and its effector proteins. SPI- 2 T3SS effector proteins allow the bacteria to colonize host cells, persist there and use them as a Trojan horse for migration throughout the host organism. Although the function of the SPI- 2 T3SS is essential for the bacterial population during systemic infection, some bacteria do not express this virulence factor and benefit from its expression by other members of the population. Similar heterogeneity in expression of other virulence factors has been shown to be crucial for their function. This work focuses on the heterogeneity of expression of selected SPI-2 T3SS effector proteins under the influence of host factors (pH, ROS, RNS) affecting bacteria during intracellular infection of macrophages. Using a fluorescent reporter system suitable for flow cytometry the expression of selected effector proteins was detected at the level of individual bacteria. The obtained data show, that individual bacteria respond differently to the decrease in pH in their vicinity. Part of the bacterial population starts to express the studied effector...
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Adenylate cyclase toxin of Bordetella pertussis, its conformation and ion balance in host cell.
Motlová, Lucia ; Konopásek, Ivo (advisor) ; Krůšek, Jan (referee)
Adenylate cyclase (CyaA, ACT) toxin is one of the major virulence factors of Bordetella pertussis. Although CyaA binds to many types of membranes, it is assumed that the integrin CD11b/CD18 is its receptor which is expressed on the surface of myeloid cells. CyaA belongs to the family of RTX toxin-hemolysins. CyaA acts on the host cells by two independent activities. One of them is the conversion of ATP to cyclic AMP, which is catalyzed by adenylate cyclase (AC) domain after its translocation into the cytosol of the host cell, which leads to the entry of calcium cations into the host cell. Translocation is probably initiated by interaction of CyaA monomer with the target membrane. The second activity is the formation of CyaA channel selective for cations, which probably causes colloid osmotic lysis of target cells. The channel forming activity is provided by RTX hemolysin domain which most probably forms oligomers, although it was found that CyaA as a monomer causes leakage of potassium cations from the host cell. It is also not clear whether the oligomerization of CyaA would occur in solution, or after interaction with the host membrane. The aim of this study was to examine the flow of sodium ions on the membrane of murine macrophages J774A.1, which express integrin CD11b/CD18 on their surface....
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Cold adaptation in stationary phase in Bacillus subtilis
Beranová, Anna ; Konopásek, Ivo (advisor) ; Svobodová, Jaroslava (referee)
Cold adaptation in stationary phase in Bacillus subtilis One of the most important abiotic factor which influences life of bacterial cells is the ambient temperature. A decrease of this temperature is usually accompanied usually with the loss of the fluidity of bacterial cytoplasmatic membrane. While the mechanisms of the responses to the cold shock during the exponential phase of growth are well known for Bacillus subtilis, the responses of stationary phase cells had not been studied yet (despite the stationary phase is the most common state of microorganism in the nature). There are two independent mechanisms which restores much needed fluidity in Bacillus subtilis - short-term adaptation and long-term adaptation. Short-term adaptation is based on the function of fatty acid desaturase coded by des gene. Long-term adaptation relies on the change in ratio of iso- and anteiso- branched fatty acids. In this work we examinated membrane adaptation during stationary phase under two different conditions, namely under cultivation at stable low temperature and after cold shock. The highest activity of Pdes was observed for cultivation at 25 řC and for the cold shock applied from cultivation in 37 řC to 25 řC. Anisotropy measurements and fatty acids analysis were also performed. Results indicated, that the...
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Differences in physiology between r and K bacterial strategists.
Moserová, Andrea ; Konopásek, Ivo (advisor) ; Krištůfek, Václav (referee)
Differences in physiology between r and K-bacterial strategists. The definition of bacterial r/K-strategists is currently based on the time interval they need to form a colony on agar plate. Also, their growth rate which is often used to identify r/K-position within a pair of bacterial strains. To date it was evidenced that also other physiological characteristics relate to bacterial r/K-status, for example their different ability to 1) adapt for changing conditions 2) utilize complex or very diluted substrates, 3) use secondary metabolites to cope with other strains and possibly also others. The intersection of macro- and microbiological r/K-conceptions lies in time distribution of r/K-strategists during succession. The aim of this study was to verify the basic r/K-characteristics on nine chosen strains and to correlate them with their physiological differences that are implicitly regarded as characteristic for r/K-groups. The study deals with growth rates measurements on both liquid and solid media, identification of fatty acid composition and membrane fluidity of strains cultivated at near-optimum and cold temperature in order to track the differences in cold adaptation. The study also deals with the description of possible new K-strategist characteristic: the lack of correlation between the colony...
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Bacterial RTX toxins and their calcium-binding sites
Lišková, Petra ; Konopásek, Ivo (advisor) ; Holoubek, Aleš (referee) ; Hof, Martin (referee)
FrpC protein produced by Neisseria meningitidis in a human host belongs to the family of bacterial RTX toxins due to the presence of RTX domain. FrpC possesses a calcium-dependent auto-catalytic cleavage activity which is localized within its 177 amino-acids long segment Self-Processing Module (SPM). As the SPM is naturally intrinsically disordered protein without bound Ca2+, the calcium binding is crucial for SPM folding which is followed by the auto-catalytic processing. The elucidation of the SPM structure may be the key step for understanding of enzymatic and biological function. The structure of folded SPM itself can be characterized only with difficulties due to the presence of flexible loop according to preliminary NMR data. The subject of this work is the description of SPM using fluorescence methods, characterization of ions binding to SPM and structural changes occurring during Ca2+ binding. In this work, the ion binding properties of SPM segment and its ion-induced folding was characterized. It was found that the dissociation constant kD of 17 μM coincided with the folding of SPM into the native calcium-bound state which occurs in the concentration range between 1 and 20 μM Ca2+. In the attempt to characterize the structure of ion binding site, the fully active single tryptophan mutants...
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