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Factors contributing to stress adaptation of human pathogen Bordetella pertussis
Hejnarová, Václava ; Večerek, Branislav (advisor) ; Černý, Ondřej (referee)
The role of bivalent metals as cofactors in various enzymes is essential for all domains of life. Their importance can be seen in interactions of hosts with patogens such as Bor- detella pertussis. This bacterium has several mechanisms that protect it from oxidative stress in phagosomes of immune cells. One example is Mn-dependent enzyme superoxide dismutase. Phagosome itself is an environment with limiting concentrations of several key bivalent metals. Thus, the transport of these metals must be controlled to ensure sufficent concentration for enzymatic activity and prevent from toxic cumulation and mismetallation. This study focuses on a particular locus BP3083-BP3077 that is highly expressed in B. pertussis surviving after internalization by THP-1 macrophages. The locus consists of two transporters, one of which shares homology with SitABCD Mn2` impor- ter of Salmonella enterica and is regulated by Fur family repressor. Bacterial cells were exposed to toxic concentrations of several bivalent metals and the level of gene expres- sion was studied by RT-qPCR. Deletion mutants were used for examination of bacterial phenotype. Results of the study show that the locus encodes Mn2` and Co2` transporters and becomes negatively regulated by Fur family repressor in toxic concentration of Co2` . The...
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Interaction of the human pathogen Bordetella pertussis with blood serum
Štipl, Daniel ; Večerek, Branislav (advisor) ; Kamanová, Jana (referee)
Bordetella pertussis is a Gram-negative strictly human pathogen and the major causative agent of whooping cough or pertussis. The incidence of this highly contagious respiratory disease in developed countries has increased in the last decades. One of the less characterized virulence factors of B. pertussis is the type three secretion system (TTSS) which is responsible for the secretion of the effector proteins into host eukaryotic cells. This diploma thesis sheds light onto factors influencing TTSS in vitro activity. Although TTSS of laboratory strain Tohama I was induced by biologically active compounds present in blood (e. g. complement proteins), TTSS of recent clinical isolate B1917 seems to be induced permanently. Furthermore, BB0302 encoding a GntR family transcription regulator in B. bronchiseptica RB50 (homologous to BP0209 of Tohama I) was studied, however, the deletion of this gene did not affect the TTSS functionality. Serum resistance is a factor that plays a key role in the pathogenesis of B pertussis. We show that Czech recent isolates (2008-2015) are significantly more resistant to serum killing in vitro than the original vaccine strains (1954-1965). This phenomenon seems to result from the adaptation of global B. pertussis population to its human host. In addition, this diploma...
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Virulence factors of Bordetella pertussis
Držmíšek, Jakub ; Večerek, Branislav (advisor) ; Vopálenská, Irena (referee)
Bordetella pertusis is a Gram-negative, aerobic, non-spore-forming coccobacillus. Although it's strictly human pathogen, it's possible to infect other mammals at laboratory conditions. Transmission among hosts is mediated via respiratory tract droplets. Infection could be direct, host to host, alternatively by contaminated environment. Bordetella colonizes upper respiratory tract, wherefrom descends into lungs and causes disease known as whooping cough or pertussis leading to 195 000 deaths of 16 mil. incidences per year (according to WHO report from 2010). More than twenty years before, respectively to found pertussis toxin, that time intensively under examination, pertussis was marked as toxin-mediated disease. In the course of time, more other virulence factors were revealed, that could be divided into groups of adhesins, toxins and others. Adhesins are filamentous haemagglutinin, pertactin and fimbriae. Toxins include pertussis toxin, adenylate cyclase, tracheal cytotoxin, dermonecrotic toxin and lipopolysaccharide. Most of virulence factors are regulated by two component system Bvg. However, it is needed lots of other factors for successful infection as for example autotransporters or so called siderophores serving as iron acquisition from environment. Secretion of virulence factors is mediated by its...
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The role of posttranslational modifications in action of bacterial toxins
Hudáčková, Kristýna ; Mašín, Jiří (advisor) ; Večerek, Branislav (referee)
Posttranslational modifications of proteins are a widespread mechanisms used by both prokaryotic and eukaryotic cells for increase the diversity of the proteome by the addition of functional groups, proteins, proteolytic cleavage of regulatory subunits, or degradation of entire proteins. These modifications include for example phosphorylation, glycosylation, acetylation, lipidation, ubiquitination or proteolysis and affect almost all aspects of cell biology and pathogenesis. Toxins produced by microorganisms are important virulence factors. Many of these bacterial toxins use posttranslational modification for their activation, as for example listeriolysin O, toxins of Bacillus anthracis or clostridial toxins. Large group of bacterial toxins activated by fatty acid are RTX (from Repeats-in-ToXin) toxins of Gram-negative pathogens, including Bordetella pertussis adenylate cyclase toxin or α-hemolysin secreted by uropathogenic Escherichia coli.
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Interaction of the human pathogen Bordetella pertussis with blood serum
Štipl, Daniel ; Večerek, Branislav (advisor) ; Kamanová, Jana (referee)
Bordetella pertussis is a Gram-negative strictly human pathogen and the major causative agent of whooping cough or pertussis. The incidence of this highly contagious respiratory disease in developed countries has increased in the last decades. One of the less characterized virulence factors of B. pertussis is the type three secretion system (TTSS) which is responsible for the secretion of the effector proteins into host eukaryotic cells. This diploma thesis sheds light onto factors influencing TTSS in vitro activity. Although TTSS of laboratory strain Tohama I was induced by biologically active compounds present in blood (e. g. complement proteins), TTSS of recent clinical isolate B1917 seems to be induced permanently. Furthermore, BB0302 encoding a GntR family transcription regulator in B. bronchiseptica RB50 (homologous to BP0209 of Tohama I) was studied, however, the deletion of this gene did not affect the TTSS functionality. Serum resistance is a factor that plays a key role in the pathogenesis of B pertussis. We show that Czech recent isolates (2008-2015) are significantly more resistant to serum killing in vitro than the original vaccine strains (1954-1965). This phenomenon seems to result from the adaptation of global B. pertussis population to its human host. In addition, this diploma...
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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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The effect of the environment on bacterial DNA topology and gene expression.
Mikesková, Klára ; Krásný, Libor (advisor) ; Večerek, Branislav (referee)
Biological processes in the cell are affected by DNA topology, i. e. by DNA structure and shape. An important topological parameter is the level of supercoiling - additional twisting of DNA is relieved by positive (twisting in the same direction as the helix turns) or negative (twisting in the opposite direction) supercoils. In this Thesis I review the role of supercoiling in gene expression regulation. I describe how supercoiling is involved in homeostatic mechanisms that control the transcriptional output from some genes. Environmental changes such as shifts in temperature, oxidative stress, extreme pH and antibiotics and other inhibitors affect the level of DNA supercoiling. DNA supercoiling then affects the expression of enzymes, which influence DNA topology, as well as some other genes/proteins. In summary, this Thesis describes how changes in the environment influence bacterial DNA topology and gene expression with a brief mention of this type of regulation in eukaryotes.
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BopN function in the Bordetella type III secretion system
Kincová, Veronika ; Kamanová, Jana (advisor) ; Večerek, Branislav (referee)
Species of the Bordetella genus cause the highly contagious whooping cough disease in humans (B. pertussis, B. parapertussis) and related respiratory diseases in other mammals (B. bronchiseptica, B. parapertussis). One of the virulence systems of Bordetellae is the type III secretion system (T3SS) employed for translocation of effector proteins directly from bacterial cytosol into the cytosol of host cells. The T3SS protein BopN protein has been categorized as a Bordetella effector protein. Nevertheless, the homologous proteins in other gram-negative bacteria function in establishing the secretion hierarchy through T3SS and some of them block T3SS secretion in high calcium environments before bacteria-host cell contact has been established. In this thesis I examined the function of the BopN protein and the role of calcium ions in T3SS activity of B. bronchiseptica. Two independent methods have been used for determination of T3SS secretion activity. Addition of 2 mM calcium ions into bacterial media decreased secretion of the T3SS reporter, while no such effect was observed in a B. bronchiseptica strain lacking the bopN gene. Mass spectrometry data confirmed the inhibition of T3SS activity in the presence of calcium ions. Enhanced calcium levels resulted in decreased mobilization and secretion of...
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The structure and role of type III secretion system and other virulence factors in pathogenesis of pertussis
Štipl, Daniel ; Večerek, Branislav (advisor) ; Pinkas, Dominik (referee)
Bordetella pertussis is a significant human pathogen which colonises a respiratory tract. The infection with B. pertussis results in serious and highly contagious disease called pertussis or whooping cough. B. pertussis produces wide range of virulence factors such as pertussis toxin, adenylate cyclase toxin, dermonecrotic toxin, tracheal cytotoxin, adhesins and type III secretion system (T3SS). The BvgAS is two-component signal transduction system that provides the complex regulation of B. pertussis virulence. The virulence factor T3SS is used by some Gram-negative bacteria to colonise the host and is responsible for pathogenesis of the infection. T3SS takes a role in virulence of mammalian pathogen B. bronchiseptica, closely related to B. pertussis. The importance of T3SS in virulence of B. pertussis remains to be investigated. Significant advance in structure, function and regulation of the most of virulence factors have been accomplished in last few decades. The causative agents of pathogenesis in that infection remain unknown. Key words: Bordetella, T3SS, gene expresssion regulation, virulence factor, pathogenesis
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Production and secretion of virulence factors in Bordetella pertussis
Držmíšek, Jakub ; Večerek, Branislav (advisor) ; Petráčková, Denisa (referee)
Bordetella pertussis is a strictly human pathogen and causative agent of infectious respiratory disease called whooping cough. In order to establish successful infection and colonization of the host, B. pertussis uses a broad spectrum of virulence factors such as adhesins (filamentous hemagglutinin, pertactin, and fimbriae) and toxins (adenylate cyclase and pertussis toxins). In addition, the type 3 secretion system (T3SS) was also found in the genus Bordetella. In connection to our previous characterisation of B. pertussis strain lacking the gene encoding RNA chaperone Hfq (Δhfq), which proved that Hfq is required for T3SS functionality, the recombinant T3SS proteins BopB, BopD, BopC and BopN were purified to homogeneity. Next, the specific antibodies were obtained using purified recombinant proteins in order to study the production of the T3SS components in B. pertussis. Using refined anti- BopC antibodies it was for the first time shown that laboratory-adapted B. pertussis strain secretes BopC protein into medium. The recombinant translocators BopB and BopD were also used to examine their pore-forming activity using planar black lipid membranes. Based on the characterisation of hfq deletion mutant, having impaired production of membrane proteins when compared to the wild type, mass spectrometry...
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