|
|
Adenylate-cyclase toxin of Bordetella pertussis as a marker for the study of the complement receptor CD11b/CD18 endocytosis.
Chvojková, Věra ; Bumba, Ladislav (advisor) ; Černý, Jan (referee)
Bordetella pertussis is an important human pathogen that causes an infection disease called whooping cough. This gram-negative bacterium produces an adenylate cyclase toxin (CyaA) that recognizes an integrin receptor CD11b/CD18 present on the surface of myeloid phagocytes and delivers an adenylate cyclase (AC) domain into the cell cytosol. This thesis deals with the endocytic machinery of CyaA and its potential use as a specific marker for endocytosis of the CD11b/CD18 receptor molecule. Detoxified mutant of CyaA, CyaA-AC- , that has the capacity to promote calcium influx as well the potassium efflux, was shown to trigger activation of the integrin receptor CD11b/CD18 followed with endocytic uptake by clathrin-dependent pathway. On the other side, the inactive mutant CyaA-KP-AC- that is unable to provoke integrin activation was endocytosed by clathrin-independent pathway. These results suggest that the various endocytic pathways of the CD11b/CD18 are determined by different conformational states of the receptor molecule.
|
|
|
Assessing biochemical properties of PDE8A1: Design of experimental system in living cells"
Galica, Tomáš ; Černý, Jan (advisor) ; Mašek, Tomáš (referee)
4 Abstract Phosphodiesterases (PDEs), enzymes that hydrolyze cyclic nucleotides, are important components of signal transduction pathways in eukaryotic cells. Second messenger 3'-5'- cyclic adenosine monophosphate (cAMP) is hydrolyzed by specific PDEs. By controlling concentration levels of cAMP in cell, PDEs preserve favorable environment for successful transmission of the cAMP signal. Moreover, PDEs are activated by protein kinase A (PKA) in response to elevated cAMP concentration, which is a feature crucial for signal termination. PDE8A1 is a high-affinity cAMP-specific IBMX insensitive phosphodiesterase, an enzyme important for cAMP signaling. However, mostly due to a lack of specific inhibitor, its role has not been assessed in detail. This thesis reports cloning of PDE8A1, identification of its posttranslational modifications and subcellular localization, as well as an alternative approach to address PDE biology by the use of cyclase toxin from Bordetella pertussis. Keywords: phosphodiesterase, cAMP, posttranslational modification, myristoylation, palmitoylation, adenylate cyclase toxin
|
|
|
Immunomodulation of dendritic cells by adenylate cyclase toxin from B. pertussis
Jáňová, Hana ; Brdička, Tomáš (referee) ; Adkins, Irena (advisor)
Adenylate cyclase toxin (CyaA) produced by the causative agent of whooping cough Bordetella pertussis, is a key virulence factor important for colonization of the host. CyaA targets preferentially myeloid phagocytes expressing CD11b/CD18 integrin. By elevating cytosolic cAMP in the host cells, CyaA interferes with their phagocytic, chemotactic and oxidative burst capacities. Furthermore, CyaA modulates the secretion of cytokines and the maturation state in LPS-stimulated dendritic cells (DC) by affecting the expression of costimulatory molecules. In this study, we investigated the effects of CyaA on the capacity of murine bone-marrow DC to prime CD4+ and CD8+ T cells in response to ovalbumin epitopes delivered by the CyaA-AC- toxoid, as a model antigen. Further, we examined the possible impact of CyaA on the antigen uptake and processing for MHC class I and II-restricted presentation by DC, as we previously observed a decreased T cell stimulatory capacity of CyaA-treated DC in response to soluble ovalbumin. We found out that the high levels of cAMP generated by CyaA in LPS-stimulated DC account for the decreased presentation of ovalbumin epitopes carried by CyaA-AC- toxoid on MHC class I and II molecules, thereby impairing the CD8+ and CD4+ T cell responses. Whereas CyaA did not influence the...
|
|
|
Construction and characterization of recombinant adenylate cyclase toxoid of bacterium Bordetella pertussis carrying mycobacterial antigen TB7.7
Mikulecký, Pavel ; Vopálenský, Václav (referee) ; Staněk, Ondřej (advisor)
Bacterium Mycobacterium tuberculosis is an etiological agent of a deadly disease called tuberculosis that presents a global problem. According to The World Health Organization there are more than 2 billions people infected with latent tuberculosis all over the world. There is still need of specific, sensitive, quick and economic available method for identification of infected individuals. Currently in vitro blood tests are considered to be the best way of diagnosis. They are based on restimulation of specific T lymphocytes by mycobacterial antigens derived from virulent strains. There are several different approaches for enhancing of direct antigen delivery into antigen presenting cells and promising one is a genetically detoxified adenylate cyclase toxin (CyaA) of bacteria Bordetella pertussis. The main aim of the thesis includes construction and subsequent characterization of biological properties of CyaA protein carrying specific mycobacterial antigen TB7.7 in translocating domain. Here is shown that fusion protein CyaA-TB7.7 can form cation selective pores in target cell membranes and is able to deliver antigens into the cytosol of APC to be presented on surface with molecules MHC class II. Genetically detoxified CyaA- TB7.7 protein will be used to supplement current approaches such as also in vitro...
|
|
|
Signalization of adenylate cyclase toxin of Bordetella pertussis in macrophages.
Černý, Ondřej ; Kuthan, Martin (referee) ; Kamanová, Jana (advisor)
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...
|
guest ::
