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Identification of type III secretion effectors in Aeromonas spp.
Jirsová, Anežka ; Kamanová, Jana (advisor) ; Lišková, Petra (referee)
Bacteria of the genus Aeromonas are Gram-negative, rod-shaped bacteria that are ubiquitous in the aquatic environment. The mesophilic species of this genus, represented by A. hydrophila, A. dhakensis, A. veronii and A. schubertii, are not only important pathogens in fish but also emerging pathogens in humans. They are associated with various gastrointestinal, systemic and wound infections. Their virulence has been described as multifactorial and associated with the presence of a type 3 secretion system. However, not much is known about the effector proteins that are introduced into the host cell by this system. The aim of this work was to characterize the two different type 3 secretion systems API-1 and API-2 of A. schubertii with respect to their cellular cytotoxicity and to identify the effector proteins that they transport into the host cell. To achieve these goals, a variety of methods were used, including microbiological techniques, molecular cloning, tissue culture techniques, SDS polyacrylamide gel electrophoresis, and mass spectrometry. Successful construction of mutant strains of A. schubertii with inactivated API-1 or API-2 and a strain with deletion of the gene encoding the negative regulator of API-1 enabled me to identify potential type 3 effector proteins of this species. Among 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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Signalization of adenylate cyclase toxin of Bordetella pertussis in macrophages.
Černý, Ondřej ; Kamanová, Jana (advisor) ; Kuthan, Martin (referee)
Adenylate cyclase toxin (CyaA) is a key virulence factor of Bordetella pertussis, the causative agent of whooping cough. The toxin targets primarily myeloid phagocytes expressing CD11b/CD18 (αMβ2, CR3, Mac-1) and by elevation of cytosolic cAMP levels it paralyses their macropinocytic and opsono-phagocytic functions. Here, we dissected the cAMP-regulated pathway responsible for the block of macrophage macropinocytosis and characterized the capacity of CyaA-treated macrophages to shut- down Akt (protein kinase B, PKB) signaling; that controls nitric oxide (NO) production by macrophages. By using specific activators of protein kinase A (PKA) and for the exchange protein activated by cAMP (Epac), we show that activation of the cAMP effector Epac inhibits macropinocytosis in macrophages. Moreover, upon transfection of macrophages by the constitutively active and dominant negative variants of a downstream effector of Epac, the small GTPase Rap1, inhibition or upregulation of macrophage macropinocytosis was observed, respectively. It was reported previously that the Epac/Rap1 pathway regulates activity of tyrosin phosphatase SHP-1 as well as of protein phosphatase 2 A (PP2A). We show that inhibition of both tyrosin phosphatases and PP2A interferes with CyaA-mediated block of macropinocytosis. These...
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Role of cAMP signaling in phagocyte migration
Dáňová, Klára ; Kamanová, Jana (advisor) ; Paňková, Daniela (referee)
Cell migration plays a key role in a wide diversity of biological processes. Migration enables phagocytic cells to localize into the site of inflammation and to lymph nodes, thereby leading to initiation of innate and adaptive immune responses, respectively. The signal transduction that coordinates phagocyte migration consist of diverse signaling proteins, being often under control of 3'-5'-cyclic adenosine monophosphate (cAMP) and its two effectors, protein kinase A (PKA) and Epac (exchange protein activated by cAMP). Small GTPase Rap is activated by Epac and controls phagocyte migration via activation of RAPL and RIAM proteins. On the other hand, PKA regulates cell migration via modulation of activity of other proteins, which comprise actin, integrins, small GTPases Rho, Rac, Cdc42 as well as protein VASP. A prominent feature of cAMP signalization is its spatio-temporal organization. Therefore, besides description of cAMP-regulated signaling cascades in cell migration, this bachelor thesis also depicts how changes of activity of cAMP effectors in time and place are involved in regulation of cell movement.
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Role of bacteria and mucosal immune system and their interaction in the pathogenesis of inflammatory bowel disease
Du, Zhengyu ; Hudcovic, Tomáš (advisor) ; Prokešová, Ludmila (referee) ; Kamanová, Jana (referee)
Although the etiology and pathogenesis of inflammatory bowel disease (IBD) is not fully understood, it is generally accepted that the inflammation results from aberrant immune responses to antigens of gut microbiota in genetically susceptible individuals (Sartor et al., 2006). Alteration in intestinal microbiota has been found in IBD patients with increased abundance of certain bacteria and decreased abundance of others. Due to the complexity of the disease, multifaceted interactions between genetic factors, host immune response, gut microbiota and environment factors need to be taken into account. In this thesis, the pathogenesis of IBD was first reviewed in respect with the four factors mentioned above. Then we concentrated on the interaction between IBD-associated bacteria and mucosal immune system. We investigated the ability of mucosal-associated bacteria (MAB) from IBD patients to induce spontaneous colitis in germ-free (GF) mice and the impact of those bacteria on the development of dextran sulfate sodium (DSS)-colitis. Together with the analysis of the composition of gut microbiota of MAB colonized mice, we demonstrated the potential deleterious microbes were able to increase the susceptibility to DSS-colitis once they found a suitable niche. We revealed the mechanism of an E.coli strain...
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Bordetella Adenylate Cyclase: Molecular Mechanism of Action and Its Use for Antigen Delivery
Kamanová, Jana
Dissertation title: Bordetella Adenylate Cyclase: Molecular Mechanism of Action and Its Use for Antigen Delivery Author: Jana Kamanová The first part of this PhD. thesis deals with molecular mechanism of action of the adenylate cyclase toxin (CyaA), a key virulence factor of the whooping cough agent Bordetella pertussis. CyaA belongs to the family of RTX (Repeat-in-ToXin) proteins secreted by Gram-negative bacteria and primarily targets myeloid phagocytes, expressing the CD11b/CD18 integrin receptor (also known as αMβ2, CR3 or Mac-1). Upon binding, CyaA permeabilizes cell membranes by forming small cation-selective pores, and subverts cellular signaling by delivering into host cells an adenylate cyclase (AC) enzyme that converts ATP to cAMP. Elevation of the cytosolic cAMP levels by CyaA then knocks down bactericidal functions of host innate immunity. CyaA is unique among other enzymatically active toxins in its capacity to penetrate cells directly from cell surface across the cytoplasmic membrane, without the need for endocytosis. Penetrating activity of CyaA depends on plasma membrane potential and on an intact, acylated and calcium-loaded RTX cytolysin moiety. By examining a set of 18 CyaA constructs that bear overlapping deletions within AC domain and a CD8+ OVA T-cell epitope tag, we showed that 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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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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Bordetella Adenylate Cyclase: Molecular mechanism of Action and Its Use for Antigen Delivery
Kamanová, Jana ; Šebo, Peter (advisor) ; Dráber, Petr (referee) ; Černý, Jan (referee)
(English) 4 SUMMARY (English) The first part of this PhD. thesis deals with molecular mechanism of action of the adenylate cyclase toxin (CyaA), a key virulence factor of the whooping cough agent Bordetella pertussis. CyaA belongs to the family of RTX (Repeat-in-ToXin) proteins secreted by Gram-negative bacteria and primarily targets myeloid phagocytes, expressing the CD11b/CD18 integrin receptor (also known as αMβ2, CR3 or Mac-1). Upon binding, CyaA permeabilizes cell membranes by forming small cation-selective pores, and subverts cellular signaling by delivering into host cells an adenylate cyclase (AC) enzyme that converts ATP to cAMP. Elevation of the cytosolic cAMP levels by CyaA then knocks down bactericidal functions of host innate immunity. CyaA is unique among other enzymatically active toxins in its capacity to penetrate cells directly from cell surface across the cytoplasmic membrane, without the need for endocytosis. Penetrating activity of CyaA depends on plasma membrane potential and on an intact, acylated and calcium-loaded RTX cytolysin moiety. By examining a set of 18 CyaA constructs that bear overlapping deletions within AC domain and a CD8+ OVA T-cell epitope tag, we showed that the first 371 amino-terminal residues are dispensable for the CyaA capacity to deliver a passenger OVA...
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Bordetella Adenylate Cyclase: Molecular Mechanism of Action and Its Use for Antigen Delivery
Kamanová, Jana
Dissertation title: Bordetella Adenylate Cyclase: Molecular Mechanism of Action and Its Use for Antigen Delivery Author: Jana Kamanová The first part of this PhD. thesis deals with molecular mechanism of action of the adenylate cyclase toxin (CyaA), a key virulence factor of the whooping cough agent Bordetella pertussis. CyaA belongs to the family of RTX (Repeat-in-ToXin) proteins secreted by Gram-negative bacteria and primarily targets myeloid phagocytes, expressing the CD11b/CD18 integrin receptor (also known as αMβ2, CR3 or Mac-1). Upon binding, CyaA permeabilizes cell membranes by forming small cation-selective pores, and subverts cellular signaling by delivering into host cells an adenylate cyclase (AC) enzyme that converts ATP to cAMP. Elevation of the cytosolic cAMP levels by CyaA then knocks down bactericidal functions of host innate immunity. CyaA is unique among other enzymatically active toxins in its capacity to penetrate cells directly from cell surface across the cytoplasmic membrane, without the need for endocytosis. Penetrating activity of CyaA depends on plasma membrane potential and on an intact, acylated and calcium-loaded RTX cytolysin moiety. By examining a set of 18 CyaA constructs that bear overlapping deletions within AC domain and a CD8+ OVA T-cell epitope tag, we showed that the...
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