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Expression and purification of N-terminal fragment of filamentous hemagglutinin from Bordetella Pertussis in E. Coli
Jurnečka, David ; Stiborová, Marie (advisor) ; Kavan, Daniel (referee)
: Whooping cough is highly contagious disease caused by gram-negative bacteria Bordetella pertussis. During infection the bacteria produces many types of toxins and adhesive molecules including a filamentous hemagglutinin(FHA). FHA is 220 kDa surface-exposed and secreted protein, which plays a key role in host-cell interactions. The project aims at construction of heterologous expression system for production of the N-terminal part of B. pertussis FHA (FHA1-862) in E. coli. The expression vector is composed of system of two independent T7/Lac promoters and enables secretion of FHA1-862 into the culture media. Downstream of the first promoter is fhaB gene encoding FHA1-862 and the letter is followed by fhaC gen encoding the FhaC transport protein, which allows translocation of FHA from periplasmic space to extracellular milieu. FHA1-862 was successfully secreted in E. coli strain BL21 carrying plasmid pMM100 (Laclq) at 30 řC and purified by affinity chromatography on Cellufine resin. These results indicate that FHA1-862 protein can be produced in E. coli, however, the system is inefficient and the yield of the protein is very low. (In Czech)
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Structural mass spectrometry of Bordetella virulence factors
Jurnečka, David
The Bordetellae are aerobic Gram-negative coccobacilli colonizing the upper respiratory tract of mammals and thereby causing diseases with similar symptoms but different host specificity. The bacteria produce a variety of adhesins and toxins that facilitate their ability to promote infection and evade the innate immune system. Among them, the filamentous hemagglutinin (FHA) and the adenylate cyclase toxin (CyaA) are the major virulence factors providing the adherence to the host epithelial cells and the protection against bactericidal activity of phagocytic cells, respectively. Moreover, CyaA along with the Escherichia coli α-hemolysin (HlyA) and the Kingella kingae cytotoxin (RtxA) represent a prominent group of Repeats in ToXin (RTX) cytotoxins/hemolysins that undergo post-translational acylation on conserved lysine residues. Here, different mass spectrometry approaches were employed to analyze the structural features of FHA and to characterize the acylation status of the RTX toxins and their various hybrid molecules. First, the differential 16O/18O labeling revealed that the mature FHA proteins of B. pertussis (Bp-FHA) and the B. bronchiseptica (Bb-FHA) are processed at different sites, after Ala2348 and Lys2479 of the FhaB precursor, respectively. Second, the bottom-up proteomics of the...
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Structural mass spectrometry of Bordetella virulence factors
Jurnečka, David
The Bordetellae are aerobic Gram-negative coccobacilli colonizing the upper respiratory tract of mammals and thereby causing diseases with similar symptoms but different host specificity. The bacteria produce a variety of adhesins and toxins that facilitate their ability to promote infection and evade the innate immune system. Among them, the filamentous hemagglutinin (FHA) and the adenylate cyclase toxin (CyaA) are the major virulence factors providing the adherence to the host epithelial cells and the protection against bactericidal activity of phagocytic cells, respectively. Moreover, CyaA along with the Escherichia coli α-hemolysin (HlyA) and the Kingella kingae cytotoxin (RtxA) represent a prominent group of Repeats in ToXin (RTX) cytotoxins/hemolysins that undergo post-translational acylation on conserved lysine residues. Here, different mass spectrometry approaches were employed to analyze the structural features of FHA and to characterize the acylation status of the RTX toxins and their various hybrid molecules. First, the differential 16O/18O labeling revealed that the mature FHA proteins of B. pertussis (Bp-FHA) and the B. bronchiseptica (Bb-FHA) are processed at different sites, after Ala2348 and Lys2479 of the FhaB precursor, respectively. Second, the bottom-up proteomics of the...
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Structural mass spectrometry of Bordetella virulence factors
Jurnečka, David ; Bumba, Ladislav (advisor) ; Novák, Petr (referee) ; Řehulka, Pavel (referee)
The Bordetellae are aerobic Gram-negative coccobacilli colonizing the upper respiratory tract of mammals and thereby causing diseases with similar symptoms but different host specificity. The bacteria produce a variety of adhesins and toxins that facilitate their ability to promote infection and evade the innate immune system. Among them, the filamentous hemagglutinin (FHA) and the adenylate cyclase toxin (CyaA) are the major virulence factors providing the adherence to the host epithelial cells and the protection against bactericidal activity of phagocytic cells, respectively. Moreover, CyaA along with the Escherichia coli α-hemolysin (HlyA) and the Kingella kingae cytotoxin (RtxA) represent a prominent group of Repeats in ToXin (RTX) cytotoxins/hemolysins that undergo post-translational acylation on conserved lysine residues. Here, different mass spectrometry approaches were employed to analyze the structural features of FHA and to characterize the acylation status of the RTX toxins and their various hybrid molecules. First, the differential 16O/18O labeling revealed that the mature FHA proteins of B. pertussis (Bp-FHA) and the B. bronchiseptica (Bb-FHA) are processed at different sites, after Ala2348 and Lys2479 of the FhaB precursor, respectively. Second, the bottom-up proteomics of the...
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Regulatory mechanisms governing the virulence of the human pathogen Bordetella pertussis
Hejnarová, Václava ; Večerek, Branislav (advisor) ; Jurnečka, David (referee)
Bordetella pertussis is human pathogen, which causes severe respiratory disease called per- tussis or whooping cough. Pathogenicity of B. pertussis is mediated by a wide variety of vi- rulence factors including pertussis toxin, adenylate cyclase toxin, pertactin and filamentous haemagglutinin. Successful infection and colonization of the host depend on the precise timing of virulence factors production. For this purpose bacteria developed miscellaneous mechanisms of gene regulation. Two-component phosphotransferase systems, such as BvgAS, RisAK and PlrSR are involved in response to external stimuli. These systems of signal transduction modu- late bacterial gene expression profiles and establish consecutive phases of infection. Non-coding RNAs, particularly sRNAs and RNA chaperone Hfq provide additional level of regulation. Hfq is a post-transcriptional regulator, which mediates interaction of sRNA with target mRNA and thereby modulates their translation. Hfq affects approximately 10% of all B. pertussis genes including virulence factors such as type III secretion system, adenylate cyclase toxin, pertussis toxin and filamentous haemagglutinin. Knowledge of these regulatory mechanisms plays a key role in understanding of the pathogenesis of whooping cough and can lead to improved control over the spread...
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Preparation of Oryzasin 1 for protein digestion in hydrogen/deuterium exchange.
Šintáková, Kristýna ; Man, Petr (advisor) ; Jurnečka, David (referee)
Hydrogen/deuterium exchange coupled to mass spectrometry (HXMS) is an increasingly popular technique in structural biology. Its spatial resolution strongly depends on the efficiency of the fragmentation or proteolytic cleavage of the studied protein. Therefore, it is desired to search for new proteases that would not only be able to digest the protein of interest under the HXMS conditions, but also to provide the best possible coverage of the protein sequence. Finding optimal conditions for production of Oryzasin 1 aspartate protease for its potential use in HXMS experiments was done in this thesis. Selected production clones were selected from available plasmids, the identity of the produced protein was verified by peptide mapping, and optimal production conditions were found. Based on these results, large-scale protein production and inclusion body isolation were undertaken. (In Czech) Keywords: Oryzasin 1, proteases, hydrogen/deuterium exchange coupled to mass spectrometry (HXMS)
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Structural analysis of filamentous hemagglutinin (FhaB) from Bordetella pertusis
Jurnečka, David ; Kavan, Daniel (advisor) ; Man, Petr (referee)
: Filamentous hemagglutinin (FHA) is adhesive protein molecule that is secreted by Gram- negative bacterium Bordetella pertusis, the causative agent of whooping cough (pertussis). The C-terminal segment of FHA plays a crucial role in host-pathogen interaction, however, the structural features are still unknown. Here, we identified the C-terminal residue of FHA and processed form of FHA (FHA*) as alanine residues in position 2304 and 2228, respectively. Circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy demonstrated that the C-terminal segment of FHA(FHA 1995-2228) is characterized by alpha-helical contribution without any compact protein fold. Moreover, suppression of transcription of small regulatory RNA pairing to the 5'-end of fhaB transcript resulted in two- fold increase of FHA production. These data suggested that the C-terminal segment of FHA appear to be an unstructured protein and FHA secretion is negatively regulated by small regulatory RNA. (In Czech) Keywords: Bordetella pertussis, filamentous hemagglutinin, small RNA
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Expression and purification of N-terminal fragment of filamentous hemagglutinin from Bordetella Pertussis in E. Coli
Jurnečka, David ; Stiborová, Marie (advisor) ; Kavan, Daniel (referee)
: Whooping cough is highly contagious disease caused by gram-negative bacteria Bordetella pertussis. During infection the bacteria produces many types of toxins and adhesive molecules including a filamentous hemagglutinin(FHA). FHA is 220 kDa surface-exposed and secreted protein, which plays a key role in host-cell interactions. The project aims at construction of heterologous expression system for production of the N-terminal part of B. pertussis FHA (FHA1-862) in E. coli. The expression vector is composed of system of two independent T7/Lac promoters and enables secretion of FHA1-862 into the culture media. Downstream of the first promoter is fhaB gene encoding FHA1-862 and the letter is followed by fhaC gen encoding the FhaC transport protein, which allows translocation of FHA from periplasmic space to extracellular milieu. FHA1-862 was successfully secreted in E. coli strain BL21 carrying plasmid pMM100 (Laclq) at 30 řC and purified by affinity chromatography on Cellufine resin. These results indicate that FHA1-862 protein can be produced in E. coli, however, the system is inefficient and the yield of the protein is very low. (In Czech)
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