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Adenylate cyclase toxin of bacteria Bordetella pertussis: mechanism of potassium efflux from macrophages
Pospíšilová, Eva ; Konopásek, Ivo (referee) ; Mašín, Jiří (advisor)
The adenylate cyclase toxin-hemolysin (ACT or CyaA) is a key virulence factor of Bordetella pertussis, the agent of the human respiratory disease whooping cough. CyaA penetrates phagocytes expressing the CD11b/CD18 integrin and exhibits two different activities. One toxin conformer oligomerizes in cell membrane and permeabilizes it by forming small cation-selective pores. Another toxin conformer appears to act as monomer. It forms a calcium influx path in the membrane, concomitantly with translocating into cells the adenylate cyclase enzyme domain that binds calmodulin and catalyzes unregulated conversion of cytosolic ATP into cAMP. We show here that CyaA causes efflux of K+ from CD11b+ cells by a mechanism that requires binding of CyaA to integrin CD11b/CD18 and permeabilization of the cellular membrane by pore-forming conformer of CyaA. Intact CyaA and the enzymatically inactive CyaA-AC- toxoid unable to generate cAMP produced the same kinetics of K+ efflux showing that elevation or signaling of cAMP had no role in this activity. The truncated CyaA variant (CyaA-AC) devoid of its invasive AC domain failed to promote K+ efflux despite a normal pore forming activity on erythrocyte and artificial membranes. However, binding of the MAb 3D1, which recognizes the distal segment of the AC domain...
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Effect of environmental stresses on mutability of Bacillus subtilis - role of mismatch-repair system
Nunvář, Jaroslav ; Lichá, Irena (advisor) ; Konopásek, Ivo (referee)
The everchanging nature of bacterial environment requires adaptation to emerging novel conditions. One proposed way of adaptation involves increased generation of genetic variability in response to harmful conditions - a phenomenon called adaptive mutagenesis. However, the details of mechanisms of adaptive mutagenesis, and even its very existence, are far from clear. Our goal was to subject the Gram-positive model bacterium Bacillus subtilis to variety of environmental stresses, examine the rate of mutagenesis occuring and compare it to unstressed conditions. Next we wondered if there was a role for mismatch-repair system (MMR), the major pathway for mutation avoidance, in these processes. To accomplish this, we constructed systems to monitor the expression of MMR components both on transcription and translation level. We also developed a mathematical model for precise mutation rate determination in order to quantify the intensity of mutagenic processes. The monitoring of MMR proteins translation failed due to high background endogenous fluorescence present in B. subtilis cells. However, we found out, using transcription reporter system, that the expression of MMR is not influenced by imposition of harsh hyperosmotic shock upon cells. The expression of MMR was also barely influenced by nutrient limitation...
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Growth of Escherichia coli on nanocrystalline diamond
Jurková, Blanka ; Kozak, Halyna ; Artemenko, Anna ; Ukraintsev, Egor ; Beranová, J. ; Konopásek, I. ; Kromka, Alexander
In this contribution, we compared the attachment of gram-negative model bacterium Escherichia coli to\nuncoated glass and glass coated by hydrogenated and oxidized NCD films. For attachment experiments,\ncontinuous cultivation in commercially available CDC Bioreactor was used. Antibacterial tests indicated\nhigher attachment of gram-negative model bacterium Escherichia coli to NCD surface compared to uncoated\nglass. We assign this effect to higher roughness of NCD surface compared to glass. Bacterial cells preferred\nthe hydrophobic surface of hydrogenated NCD surface to hydrophilic oxidized NCD for their attachment.
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