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Effect of surfactin on the lipid moiety of Bacillus subtilis cytoplasmic membrane
Sklenářová, Petra ; Seydlová, Gabriela (advisor) ; Lichá, Irena (referee)
Surfactin, a secondary metabolite produced by Bacillus subtilis, is a surface active compound and antibiotic permeabilizing membrane bilayer. The aim of this study was to reveal the self-resistance strategy at the level of the lipid moiety of cytoplasmic membrane, which B. subtilis employs to combat surfactin in concentrations that are lethal for other bacterial species. Non-producing strain B. subtilis 168 was cultivated in the presence of two different sublethal concentrations of surfactin (350 a 650 µg/ml), which was isolated from the culture broth of B. subtilis ATCC 21332. Presence of surfactin in the medium resulted in a concentration dependent lag phase, which took 40 min (350 µg/ml) and 3 h (650 µg/ml), respectively. Afterwards, the culture grew with the altered doubling time of 44 min (350 µg/ml) and 126 min (650 µg/ml), respectively. Surfactin induced substantial changes in the phospholipid composition of the cytoplasmic membrane. The proportion of the major phospholipid component phosphatidylglycerol decreased and inversely, the level of phosphatidylethanolamine increased. Interestingly, the content of phosphatidic acid rose considerably in the presence of surfactin concentration causing stimulation of B. subtilis growth (350 µg/ml). Liposome leakage assay using phospholipids mimicking...
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Effect of membrane lipid composition on resistance against surfactin
Pinkas, Dominik ; Seydlová, Gabriela (advisor) ; Sýkora, Jan (referee)
Surfactin is an antibiotic produced by several strains of B. subtilis. Its broad range of biological activities is interesting from perspective of medicine, food industry and bioremediation and is based on its surface-active properties and interaction with biological membranes. The latter means mainly forming ion channels, conductive pores and with increasing concentration eventually disrupting membrane structure in detergent-like manner. Mechanism of resistance of producing strain against its own toxic product is not yet fully understood. This work shows that it could be based on surfactin target modification - which means altering membrane lipid composition. We were able to recognize surfactin-formed ion channels or pores with a broad range of conductivities spanning from 2 pS to 2 nS using BLM method. Liposome leakage assay with carboxyfluorescein revealed few distinct mechanisms of lysis, differing in amplitude, rate of lysis and cooperativity. Increased content of anionic lipids with conical shape, namely cardiolipin and phosphatidic acid led to substantial increased membrane resistance to surfactin-induced permeabilization. Key words: membrane, surfactin, Bacillus subtilis, cardiolipin, black lipid membranes, liposomes
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Cell wall integrity signalling pathway and yeast colony morphology
Reslová, Gabriela ; Schierová, Michaela (advisor) ; Seydlová, Gabriela (referee)
In the yeast Saccharomyces cerevisiae, stress on the cell wall is caused by various external influences (e.g. exposure to chemicals, oxidative stress, osmotic changes, pH changes or heat shock) which trigger the cell wall integrity signalling pathway (CWI). The aim of my work was to investigate the effect of the CWI pathway on yeast colony morphogenesis. Using strains with deletions in genes of the CWI pathway derived from two parental strains BR-F-Flo11p-GFP and PORT, I have found that differences in genetic background influences the process and activation of this pathway. Among the strains derived from BR-F-Flo11p-GFP, only the strain with the deletion of MID2 affects the appearance of colonies. MID2 encodes a cell-surface sensor of CWI pathway. In all deletion strains derived from PORT, the disruption of the CWI pathway causes a slower development of colonies growing on glycerol medium supplemented with 0,05 mM selenate inducing fluffy colony morphology. The largest effect has deletion of gene MTL1 which also encodes a cell-surface sensor with homology to Mid2. I have confirmed that strains with deletions in genes of CWI pathway have altered sensitivity to inhibitors disrupting cell wall integrity (Calcofluor white, Congo red, zymolyase). By means of zymolyase assay, I have confirmed the...
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Bordetella pertussis and whooping cough: Bacterium and its virulence factors, epidemiology of disease and vaccination strategy.
Bočková, Barbora ; Holubová, Jana (advisor) ; Seydlová, Gabriela (referee)
Bordetella pertussis, a gram-negative bacterium, is a human pathogen which affects the upper respiratory tract. It is the causative agent of whooping cough or pertussis. B. pertussis produces several virulence factors consisting of toxins and adhesins. Whole cell vaccine and subsequent acellular vaccine were developed against pertussis in the past. However, a gradual increase of pertussis incidences has been reported in the last twenty years. This thesis provides basic information about B. pertussis and whooping cough. The main aim of the herein presented work is to summarize the contemporary epidemiologic situation along with determining reasons for increased pertussis cases. In addition, possible solutions for the present situation are proposed.
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Study of biofilm formation of bacterial isolates from milk and dairy products
Nováková, Veronika ; Demnerová, Kateřina (advisor) ; Seydlová, Gabriela (referee)
Bacterial biofilm is a complex community of microbial cells, which are embed into the extracellular pollysacharide matrix. Typical ability of the biofilm is the ability to adhere to either abiotic, or biotic surfaces. The formation of biofilm is a dynamic process, which finally formes a 3D multicellular complex. The initial phase is called the adhesion. After that process is done, the extracellular polymeric matrix is produced to create living conditions of bacteria in biofilm. Biofilm has hight level of antimicrobial resistance. This resistance consists of physical and chemical barriers, which effectively block diffusion and penetration of antimicrobial substances inside the biofilm. Biofilms cause problems in food industry, where it could be origin of food contaminations. Thefore it is necessary to understand the relationship between microorganisms and materials used in food industry. Key words: biofilm, milk and dairy products, milk and dairy industry, disifectanc, Escherichia coli, Staphylococcus spp.
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Construction of a genetically detoxified Bordetella pertussis strain to develope a new generation of whole-cell vaccine
Bočková, Barbora ; Holubová, Jana (advisor) ; Seydlová, Gabriela (referee)
Bordetella pertussis is a strictly human pathogen colonizing the upper respiratory tract, causing a respiratory disease known as whooping cough or pertussis. The introduction of whole-cell vaccines and acellular vaccines, resulted in a significant reduction in the incidence of disease and reduce the fatalities associated with infection. However, epidemiological data show a significant increase in the incidence of the disease in recent decades. The increasing incidence is mainly attributed to the transition from the whole- cell vaccine to an acellular vaccine. Based on research from recent years has shown that acellular vaccines have many drawbacks, and it is therefore necessary to change the vaccination strategy. One possible solution to the situation is the development of a new generation of whole-cell vaccines with reduced reactogenicity. The new whole-cell vaccine was prepared by a genetically modified B. pertussis strain. B. pertussis was modified using allelic exchange to develop strain encoding enzymatically inactive pertussis toxin, modified lipid A and lacking dermonecrotic toxin. This combination of genetic modifications in mice led to a decrease in reactogenicity test vaccine in vivo. In case of intranasal infection whole-cell vaccine containing genetically modified strain is providing...
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Bacterial role in silicate mineral alteration in near-neutral pH conditions
Duchoslav, Vojtěch ; Falteisek, Lukáš (advisor) ; Seydlová, Gabriela (referee)
It is well established that bacteria are able to catalyze dissolution of silicate minerals. Bacteria may dissolve silicates for two different purposes. They may use certain elements that can undergo redox transitions as substrates for their energetic metabolism or they can leach nutrients, that are otherwise unaccessible in their habitat. The main mechanisms of bacterially mediated silicate leaching are acidic or basic catalysis and surface complexation. The main nutrients extracted are K, Mg, P and Fe. The only element significantly exploited as substrate for dissimilative energetic metabolism is iron. In order to leach iron as a nutrient, even extremely strong complexants (i.e. siderophores) may be employed. However, only moderate complexing agents can serve to obtain iron as terminal electron acceptor. The second possibility is to reduce iron directly in the crystal grid by means of the conductive nanofibres. The oxidative dissolution of silicates by chemoautotrophs is rare, in contrast to that of sulfides. Bacterial dissolution of silicates leaves morphological and geochemical signatures, but it is still problematic to recognize and interpret them. Although it is well-known that bacteria can dissolve most of the rock-forming minerals in diverse environments we are unable to quantify their contribution...
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Mode of action and nature of different susceptibility of bacteria to antibacterial compounds lipophosphonoxins
Havlová, Noemi ; Seydlová, Gabriela (advisor) ; Krásný, Libor (referee)
Lipophosphonoxins (LPPO) are small synthetic antibacterial compounds targeting the cytoplasmic membrane. 1st generation of LPPO (LPPO I) displays an antimicrobial activity against Gram positive bacteria, however they do not show any activity against Gram negatives. After the modification of the iminosugar module (bearing the positive charge) the 2nd generation of LPPO (LPPO II) were synthetized. LPPO II exhibit broadened activity against Gram positive bacteria and also kill Gram negatives, including multiresistant strains. This work focuses on the mode of action of LPPO - the pore-forming activity of these substances is investigated on model membranes as well as in vivo. It also deals with the nature of different activity against Gram positive and Gram negative bacteria using model bacteria Bacillus subtilis and Escherichia coli. The results show that the insensitivity of Gram negative bacteria against LPPO I is probably caused by the different cell wall structure and the presence of the outer membrane that LPPO are almost unable to overcome. Also, the composition of phospholipids in the target membrane influences the antimicrobial activity of LPPO. Higher proportion of phospholipids with neutral charge reduces the LPPO pore-forming activity but is also responsible for low cytotoxicity in...
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