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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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Interaction of the adenylate cyclase toxin with complement receptor 3 - Relation of structure and function
Morová, Jana ; Šebo, Peter (advisor) ; Konvalinka, Jan (referee) ; Bezouška, Karel (referee)
4 CoNcl-usroxs PnonucrroN oF tNrEcRtN CDI lb/CDlg F Four fragments of the subunit cDilb were produced and purified and then they were used for immunization of mice and for preparation of specific antibodíes. detected that 52 cells are not able to transport effectively this subunit to cell surface or to secrete its extracellular domain into medium and not even it has a signal peptide specific for 52 cells. Further it has been shorvn, that the level of production of CDl8 subunit was not affected by usage of constitutive or inducible plasmid. ANALYSIS oF THE |NTERÁCTIoN oF RTx ToxINs w|TH p2 INTEGRINS - THE RoLE oF THT, GLYCOSYLATION OF RECEPTORS IN BINDINC ON RTX TOXIN of cell surface glycoproteins, suggesting that cyaA binding to the cell surface- expressed cDllb/cDl8 integrin fully depends on its glycolsylation. It has been a|so demonstrated. that the deglycosylation did not affected ťormation of CD I I b/CD l8 heterodimer or its expression to cell surface. inhibited in the presence ofonly saccharide units that occur in the oligosaccharide chain of integrin molecule. This demonstrates, that cyaA directry recognizes the N-linked oligosaccharide chain ofits p2 integrin recepror. CD l I b/CD I 8-expressing cells. cytotoxic activity of others RTX toxins. l5...
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Regulatory roles of PAG and CSK in FcɛRI signaling of mast cells
Potůčková, Lucie ; Dráber, Petr (advisor) ; Šebo, Peter (referee) ; Holáň, Vladimír (referee)
8 1 ABSTRACT (EN) This thesis is focused mainly on understanding mechanisms of regulatory roles of C-terminal Src kinase (CSK) and phosphoprotein associated with glycosphingolipid- enriched microdomains (PAG) in the high-affinity IgE receptor (FcɛRI)-mediated signaling of murine mast cells. FcɛRI activation is initiated by aggregation of the receptor by complexes of multivalent antigen with IgE, followed by activation and enhanced activities of protein tyrosine kinases, phosphatases, adaptor proteins and number of other signal transduction molecules. The signaling events result in mast cell degranulation and release of variety of proinflammatory mediators, responsible for initiation of allergy and other inflammatory diseases. Understanding the function of key regulatory molecules controlling FcεRI-mediated mast cell activation, degranulation, and cytokines production could have therapeutic impact. CSK is a major negative regulator of Src family tyrosine kinases (SFKs) that play a critical role in various immunoreceptor signaling events. However, its function in mast cell activation has not been completely understood. Because of its cytoplasmic localization, CSK was assumed to be brought to the vicinity of the plasma membrane- bound SFKs via binding to membrane-bound adaptors and PAG was a major candidate....
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Mechanism of action of adenylate cyclase toxin on immune function of dendritic cells
Švédová, Martina ; Šebo, Peter (advisor) ; Černý, Jan (referee) ; Stulík, Jiří (referee)
The adenylate cyclase toxin (CyaA) is a key virulence factor of the whooping cough agent Bordetella pertussis. CyaA primarily penetrates CR3-expressing myeloid phagocytes and subverts cellular signaling by a rapid conversion of ATP to cAMP. In parallel, CyaA can form cation-selective pores within cellular membrane, provoking massive potassium efflux from cell cytosol. An enzymatically inactive adenylate cyclase toxoid (CyaA-AC- ) has then been abundantly used as an efficient antigen delivery tool over the past 20 years. This work focused mainly on the mechanism of action of CyaA toxin and of its toxoid on dendritic cells. We studied the potency of the CyaA toxoid to act as adjuvant, its penetration capacity and its potential use in delivery of influenza epitopes. We show that the pore-forming activity and the activation of MAP kinases JNK and p38 were crucial for the adjuvant effects of the CyaA-AC- , which provokes maturation of dendritic cells (DC) independently of Toll-like receptor (TLR) or inflammasome signaling. Furthermore, such CyaA-AC- -stimulated DC acquired the ability to induce CD8+ and CD4+ T cells responses, as was determined both in vitro and in vivo. We further showed that the first 371 amino acids are dispensable for the capacity of CyaA to deliver its AC domain with inserted...
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The study of the pathogenesis of infection caused by bacteria from Burkholderia cepacia complex in patients with cystic fibrosis
Kalferstová, Lucie ; Dřevínek, Pavel (advisor) ; Kolář, Milan (referee) ; Šebo, Peter (referee)
Summary: Bacteria from Burkholderia cepacia complex (Bcc) and bacteria Pseudomonas aeruginosa belong among the most serious pathogens causing lung infections in patients with cystic fibrosis (CF). These bacteria are highly resistant to almost all of the available antibiotics. Another serious problem is the ability of certain strains to spread among the patients, which can cause an epidemic infection. Some of the Bcc strains are capable of entering the bloodstream and causing serious septic condition called cepacia syndrome. One of these strains is the Czech epidemic strain B. cenocepacia ST32, which spread among Czech patients with CF in the 90s of the 20th century. The aim of this study was to compare transcriptome profiles of isolates gained from blood of patients with cepacia syndrome with transcriptome profiles of isolates gained from sputum of patients in the stable phase eventually exacerbation, and to choose the most appropriate genes with the different expression, which could be used as a possible marker for detection of arising cepacia syndrome. Another aim of this study was to do further study of function and influence on virulence of chosen marker (which is coding generally known virulence factor) in the time of cepacia syndrome. The last aim was to assess the epidemiologic situation of bacteria...
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Signaling effects of adenylate cyclase toxin action on phagocytes
Černý, Ondřej ; Šebo, Peter (advisor) ; Černý, Jan (referee) ; Dráber, Petr (referee)
The adenylate cyclase toxin (CyaA) plays a key role in the virulence of Bordetella pertussis. CyaA penetrates CR3-expressing phagocytes and catalyzes the uncontrolled conversion of cytosolic ATP to the key second messenger molecule cAMP. This paralyzes the capacity of neutrophils and macrophages to kill bacteria by oxidative burst and opsonophagocytic mechanisms. Here we show that CyaA suppresses the production of bactericidal reactive oxygen and nitrogen species in neutrophils and macrophages, respectively. The inhibition of reactive oxygen species (ROS) production is most-likely achieved by the combined PKA-dependent inhibition of PLC and Epac-dependent dysregulation of NADPH oxidase assembly. Activation of PKA or Epac interfered with fMLP-induced ROS production and the inhibition of PKA partially reversed the CyaA-mediated inhibition of ROS production. CyaA/cAMP signaling then inhibited DAG formation, while the PIP3 formation was not influenced. These results suggest that cAMP produced by CyaA influences the composition of target membranes. We further show here that cAMP signaling through the PKA pathway activates the tyrosine phosphatase SHP-1 and suppresses the production of reactive nitrogen species (RNS) in macrophages. Selective activation of PKA interfered with LPS- induced iNOS expression...
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Structure-function relationships and use of RTX proteins of Gram-negative bacteria
Sadílková, Lenka ; Šebo, Peter (advisor) ; Stulík, Jiří (referee) ; Weiser, Jaroslav (referee)
RTX (Repeat in ToXin) superfamily consists of many proteins divided into several groups according to their different functions and characteristics: toxins, metalloproteases, lipases, proteins of the S-layer, bacteriocins and proteins with unknown function. However, all of them can be characterized by the following features: i) they contain tandemly repeated (6-50) nonapeptide glycine-rich calcium-binding consensus sequences GGXGXDX[L/I/V/W/Y/F]X (where X is any amino acid residue) in the C-terminal part of the protein. The presence of these repeats is a sine qua non condition for RTX protein family membership; ii) secretion from the cell occurs without a periplasmic intermediate by a mechanism which involves recognition of a signal sequence at the C-terminus of the protein by membrane-associated proteins that export the toxin across a channel spanning the entire bacterial envelope directly to the outside of the cell (Type I Secretion System); iii) the genes for protein synthesis, activation and secretion are mostly grouped together on the chromosome and form rtx operons. RTX toxins are the largest protein group of the RTX family. To this group belong mostly the proteins with molecular weight ranging from 100 to 200 kDa, with posttranslational fatty acid acylation mediated by a specific activating...
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Topography of signaling molecules on the plasma membrane in the course of mast cell activation
Lebduška, Pavel ; Dráber, Petr (advisor) ; Šebo, Peter (referee) ; Benada, Oldřich (referee) ; Tučková, Ludmila (referee)
presented technique of plasma membrane sheet preparation from nonadherent cells may facilitate research in this field. It must be, however, mentioned that a plastic view ofsignal transduction across the plasma membrane can be achieved only by combination of various mutually complementary approaches. Conclusions Three techniques of lsoladon of plasmr m€mbrane sh$ts from nonadherent BMMC mast cells have bmn developed. one of them, based on edsorption ofl€ukocýes to glass suďace, turned out to be very promlsing md provided many scientifrc datr(article E). Actlvation of RBL m9st c€l|s by FGRI raeptor dimerintion led to increme of Grb2 adaptor content in the Plasma membrane. Ilowever' by contřast to the case of receptor mu|t|merintion, this Grb2 did not sign|ficantly colocallz€ w|th FERI' and' by |mmuno|rbeling of membrane she€ts, distribution of FC5RI wrs not d|stinguishabl€ from the disfibution on nonact|vrted ce|ls (article A). BMMC, In contrast to RBL cells, after multimerization of FaRI did not form larger aggregat€s ofthis r€c€ptor thrn nonact|vat€d cells did. FGRI muldmer|ation led to lts int€rna|iation of comparable intensity rnd overa|l dynemics ln BMMC end RBL cel|s' but loce| redistribut|on of FaRI fundamentďly differed betwcn these two c€|| wes (article E). Established mode| oflrrg€ (8pproxim8t€|y...
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