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14th International Bordetella Symposium
Šebo, Peter
We shall discuss the latest research achievements in the Bordatella/pertussis filed and how to tackle the challenge of current pertussis resurgence. We will assess what needs to be done to overcome the limitations of current acellular pertussis vaccines in order to curb the resurgence and transmission of whoopong cough.
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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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Preparation and characterization of protein constructs for structural analysis of acylate domain of adenylate cyclase toxin from bacteria B. pertussis
Ryvola, Vojtěch ; Ječmen, Tomáš (advisor) ; Chmelík, Josef (referee)
Whooping cough or pertussis is a contagious bacterial airways diasease caused by B. pertussis. Despite the high vaccination coverage of the population in developed countries, whooping cough is one of the most common diseases that can be prevented by vaccination. Bacteria B. pertussis produces a variety of toxins that facilitate the colonization and proliferation of bacterial cells in the host ciliary epithelium of the upper respiratory tract. One of these toxins is adenylate cyclase toxin, which is released from bacterial cells by a type I secretory apparatus and, after it's calcium-induced packaging, is able to transport its N-terminal adenylate cyclase domain across the cytoplasmic membrane of host cells. This domain able to catalyse conversion of adenosine triphosphate (ATP) to a cyclic adenosine monophosphate (cAMP) after the interaction with cytoplasmatic calmodulin. High levels of cAMP subsequently lead to impaired cell signaling and to a fundamental reduction in the bactericidal functions of phagocytic cells. The aim of this bachelor thesis was to prepare and characterize 'hybrid' protein constructs, which consist of a C-terminal part of the acylation domain and an N-terminal part of the RTX domain (at positions 881-1038 and 881-1047) linked to the C-terminal part of the RTX domain (at...
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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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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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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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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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Interaction of pathogenic Bordetella species with host cells
Čurnová, Ivana ; Petráčková, Denisa (advisor) ; Mašín, Jiří (referee)
Most of the members of the Gram-negative genus Bordetella cause severe infections of the respiratory tract in their hosts. B. pertussis and specific lineages of B. parapertussis infect humans and cause the disease known as whooping cough, a highly contagious respiratory disease that is currently on the rise even in highly vaccinated populations. Therefore, a more detailed understanding of the B. pertussis interactions with the host is crucial. B. pertussis produces a great variety of virulence factors, majority of which is regulated by the two- component BvgAS system. These factors assist the pathogen in the colonization of the host and evasion of the host immune system. The studies on host-pathogen interactions use both in vitro and in vivo infection models, which complement each other appropriately. Recently, it was demonstrated that B. pertussis escape killing and persists in macrophages, suggesting that B. pertussis can be considered as a facultative intracellular pathogen. This ability may allow the pathogen cells to persist within the host and potentially spread to the new host. The aim of this bachelor thesis was to summarize the knowledge on the host-pathogen interactions between B. pertussis and its host with focus on in vitro and in vivo infection models. The attention is paid especially...
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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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Fluorescence studies of bacterial membrane proteins and cell signalling.
Fišer, Radovan ; Konopásek, Ivo (advisor) ; Hof, Martin (referee) ; Forstová, Jitka (referee)
(English) This work is based on five publications studying mostly adenylate cyclase toxin (CyaA) from Bordetella pertussis and its interaction with biological membranes. CyaA permeabilizes cell membranes by forming small cationselective pores and subverts cellular signaling by delivering an adenylate cyclase (AC) enzyme that converts ATP to cAMP into host cells. First study clarifies the membrane disruption mechanisms of CyaA and another bacterial RTX toxin; αhemolysin (HlyA) from Escherichia coli. For this purpose, we employed a fluorescence requenching method using liposomes as target membranes. We showed that both toxins induced a graded leakage of liposome content with different ion selectivities (Fišer a Konopásek 2009). Both AC delivery and pore formation were previously shown to involve a predicted amphipathic αhelix(502522). In the second publication we investigated another predicted transmembrane αhelix(565591) that comprises a Glu(570) and Glu(581) pair. We examined the roles of these glutamates in the activity of CyaA, mostly on planar lipid membranes end erythrocytes. Negative charge at position 570, but not at position 581, was found to be essential for cation selectivity of the pore, suggesting a role of Glu(570) in...
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