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Elucidation of the properties and structure of the pore-forming domain of colicin U produced by bacterium Shigella boydii.
Dolejšová, Tereza
Colicin U is a protein produced by bacterium Shigella boydii. It belongs to the group of pore-forming colicins. These colicins interact with receptors in the outer membrane of bacteria closely related to a producing colicinogenic strain. After interaction with the receptor, colicin is translocated across the outer membrane and periplasm to the cytoplasmic membrane where it forms pores. Consequently, the pore formation leads to membrane depolarization and cell death. In this thesis I decided to study the pore-forming properties of colicin U and its membrane topology. It is shown that colicin U pores are formed by only one colicin molecule and they are voltage dependent. Using measurements with nonelectrolytes we estimated a theoretical inner profile of the pore and its inner diameter to be between 0.7 and 1 nm. Above that, a membrane topology of colicin U pore-forming domain (PFD) is studied. BLM measurements with biotinylated colicin U showed that a significant part of colicin's PFD was translocated to the opposite side of the membrane after the pore opening. The segment between substituted amino acids F463 and D486 was evidenced to be on the trans side of the membrane after the pore opening. Additionally, properties of peptide H1, which reflects a significant part of the first α- helix of colicin...
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Conformation of the adenylate cyclase toxin of Bordetella pertussis.
Motlová, Lucia
This work is focused on the RTX (Repeats in ToXin) domains structure of selected RTX toxins and its impact on secretion and protein folding. The structural analysis included RTX domains of ApxI (Actinobacillus pleuropneumoniae-RTX-toxin I) from Actinobacillus pleuropneumoniae, HlyA (Alfa-hemolysin) from Escherichia coli and LtxA (Leukotoxin A) from Aggregatibacter actinomycetemcomitans and blocs 4 a 5 RTX domain CyaA (adenylate cyclase toxin) from Bordetella pertussis. The structures of LtxA RTX domain and CyaA RTX blocs 4 and 5 were obtained and characterized. Two models of CyaA RTX domain were built based on SAXS (Small Angle X-ray Scattering) model, previously solved RTX structures and RTX structures presented here.
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Elucidation of the properties and structure of the pore-forming domain of colicin U produced by bacterium Shigella boydii.
Dolejšová, Tereza ; Fišer, Radovan (advisor) ; Krůšek, Jan (referee) ; Osička, Radim (referee)
Colicin U is a protein produced by bacterium Shigella boydii. It belongs to the group of pore-forming colicins. These colicins interact with receptors in the outer membrane of bacteria closely related to a producing colicinogenic strain. After interaction with the receptor, colicin is translocated across the outer membrane and periplasm to the cytoplasmic membrane where it forms pores. Consequently, the pore formation leads to membrane depolarization and cell death. In this thesis I decided to study the pore-forming properties of colicin U and its membrane topology. It is shown that colicin U pores are formed by only one colicin molecule and they are voltage dependent. Using measurements with nonelectrolytes we estimated a theoretical inner profile of the pore and its inner diameter to be between 0.7 and 1 nm. Above that, a membrane topology of colicin U pore-forming domain (PFD) is studied. BLM measurements with biotinylated colicin U showed that a significant part of colicin's PFD was translocated to the opposite side of the membrane after the pore opening. The segment between substituted amino acids F463 and D486 was evidenced to be on the trans side of the membrane after the pore opening. Additionally, properties of peptide H1, which reflects a significant part of the first α- helix of colicin...
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The role of a conserved tyrosine residue of acylated domain in membrane insertion and penetration of RTX toxins
Lepesheva, Anna ; Mašín, Jiří (advisor) ; Petráčková, Denisa (referee)
Pore-forming RTX toxins are key virulence factors of many Gram-negative pathogens. These proteins share several common structural and functional features: (i) the presence of repetitive sequences rich in glycine and aspartate, which are important for calcium ion binding, (ii) transport from the bacterial cytosol through the type I secretion system (T1SS), (iii) modification by a fatty acid at specific lysines in the acylated domain by a specific acyltransferase, and (iv) the presence of an amphipathic region responsible for the formation of cation-selective pores in the target membrane. The aromatic side chain of the conserved tyrosine residue 940 in the acylated segment of the RTX adenylate cyclase toxin (CyaA, ACT or AC-Hly) of Bordetella pertussis plays a key role in the interaction of the toxin with the target cell membrane. The aim of this study was to determine whether the corresponding conserved residues Y940, Y642, Y643 and Y639 secreted by the homologous RTX toxin CyaA from Bordetella bronchiseptica, HlyA from Escherichia coli, ApxIA from Actinobacillus pleuropneumoniae and RtxA from Kingella kingae play the same critical role in membrane insertion and pore formation. The hemolytic and cytotoxic activities of these toxins were completely impaired only after replacement of the conserved...
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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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Conformation of the adenylate cyclase toxin of Bordetella pertussis.
Motlová, Lucia ; Fišer, Radovan (advisor) ; Obšil, Tomáš (referee) ; Holoubek, Aleš (referee)
This work is focused on the RTX (Repeats in ToXin) domains structure of selected RTX toxins and its impact on secretion and protein folding. The structural analysis included RTX domains of ApxI (Actinobacillus pleuropneumoniae-RTX-toxin I) from Actinobacillus pleuropneumoniae, HlyA (Alfa-hemolysin) from Escherichia coli and LtxA (Leukotoxin A) from Aggregatibacter actinomycetemcomitans and blocs 4 a 5 RTX domain CyaA (adenylate cyclase toxin) from Bordetella pertussis. The structures of LtxA RTX domain and CyaA RTX blocs 4 and 5 were obtained and characterized. Two models of CyaA RTX domain were built based on SAXS (Small Angle X-ray Scattering) model, previously solved RTX structures and RTX structures presented here.
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Effect of calcium ions and cholesterol on channel forming activity of Adenylate-cyclase toxin
Doktorová, Eliška ; Fišer, Radovan (advisor) ; Horák, Martin (referee)
1 Abstract Adenylate cyclase toxin (CyaA) is one of the major virulence factors of bacterium Bordetella pertussis, which is a causative agent of whooping cough. CyaA belongs to the family of RTX toxin-hemolysins. The toxin targets primarily cells expressing integrin receptor CD11b/CD18 but it can also penetrate cells lacking this receptor. CyaA acts on host cells by two independent activities. One is formation of small cation-selective channels, which can lead to colloid osmotic lysis of target cells. The second is disruption of cell signaling through the translocation of the adenylate cyclase (AC) domain to host cell cytosol, which leads to the conversion of ATP into cyclic AMP. It was recently shown that cholesterol affects endocytosis of CyaA. CyaA translocates it's AC domain after relocation of CyaA molecule to the cholesterol-rich lipid raft (Bumba et al. 2010). In this work I examined the effect of cholesterol on channel- forming activity and selectivity of ion channels created by CyaA. For measurements I used artificial membranes enriched with cholesterol. CyaA channels are voltage-dependent. The positive membrane potential on the side of toxin is rquired for incorporation of CyaA molecule into cell membrane. I tried to find out whether the value of voltage has effect on channels opening time....
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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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