Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:4.6.1.1 (adenylate cyclase)
19,190 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The Bordetella adenylate cyclase toxoid (CyaA) targets cells expressing the alphaMbeta2 integrin receptor CD11b/CD18 (CR3 or Mac-1) and can penetrate into cytosol of professional antigen-presenting cells, such as dendritic cells. This allows us to use CyaA for delivery of passenger antigens into the cytosolic pathway of processing and MHC class I-restricted presentation, which can promote induction of antigen-specific CD8+ cytotoxic T-lymphocyte immune responses. We show here that vaccination with a genetically detoxified CyaA336/E7 protein, carrying the full-length oncoprotein E7 of the human papilloma virus 16 inserted at position 336 of the cell-invasive AC domain of CyaA, induces an E7-specific CD8+ T-cell immune response and confers on mice protective, as well as therapeutic immunity against challenge with TC-1 tumor cells expressing the E7 oncoprotein. The therapeutic efficacy of priming with the CyaA336/E7 vaccine could further be enhanced by a heterologous booster immunization with a highly attenuated modified vaccinia virus Ankara (MVA) expressing the E7 protein fused to the lysosome-associated membrane protein (LAMP1). These results establish the potential of CyaA as a new antigen delivery tool for prime/boost immunotherapy of tumors.
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PMID:Prime/boost immunotherapy of HPV16-induced tumors with E7 protein delivered by Bordetella adenylate cyclase and modified vaccinia virus Ankara. 1592 77

The Bordetella adenylate cyclase toxin-hemolysin (CyaA, ACT, or AC-Hly) forms cation-selective membrane channels and delivers into the cytosol of target cells an adenylate cyclase domain (AC) that catalyzes uncontrolled conversion of cellular ATP to cAMP. Both toxin activities were previously shown to depend on post-translational activation of proCyaA to CyaA by covalent palmitoylation of the internal Lys983 residue (K983). CyaA, however, harbors a second RTX acylation site at residue Lys860 (K860), and the role of K860 acylation in toxin activity is unclear. We produced in E. coli the CyaA-K860R and CyaA-K983R toxin variants having the Lys860 and Lys983 acylation sites individually ablated by arginine substitutions. When examined for capacity to form membrane channels and to penetrate sheep erythrocytes, the CyaA-K860R acylated on Lys983 was about 1 order of magnitude more active than CyaA-K983R acylated on Lys860, although, in comparison to intact CyaA, both monoacylated constructs exhibited markedly reduced activities in erythrocytes. Channels formed in lipid bilayers by CyaA-K983R were importantly less selective for cations than channels formed by CyaA-K860R, intact CyaA, or proCyaA, showing that, independent of its acylation status, the Lys983 residue may play a role in toxin structures that determine the distribution of charged residues at the entry or inside of the CyaA channel. While necessary for activity on erythrocytes, acylation of Lys983 was also sufficient for the full activity of CyaA on CD11b+ J774A.1 monocytes. In turn, acylation of Lys860 alone did not permit toxin activity on erythrocytes, while it fully supported the high-affinity binding of CyaA-K983R to the toxin receptor CD11b/CD18 and conferred on CyaA-K983R a reduced but substantial capacity to penetrate and kill the CD11b+ cells. This is the first evidence that acylation of Lys860 may play a role in the biological activity of CyaA, even if redundant to the acylation of Lys983.
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PMID:Acylation of lysine 860 allows tight binding and cytotoxicity of Bordetella adenylate cyclase on CD11b-expressing cells. 1617 90

CR3 (CD11b/CD18) is expressed on neutrophils, and the engagement of CR3 can promote phagocytosis. CR3 serves as the receptor for the Bordetella pertussis adhesin filamentous hemagglutinin (FHA) and for the adenylate cyclase toxin (ACT), which blocks neutrophil function. The influence of CR3, FHA, and ACT on the phagocytosis of B. pertussis by human neutrophils was examined. The surface expression and function of CR3 are regulated. Tumor necrosis factor alpha (TNF-alpha) and gamma interferon (IFN-gamma) increased CR3 surface expression, but only TNF-alpha increased the ability of neutrophils to phagocytose B. pertussis, suggesting that elevated CR3 expression alone is not sufficient to promote phagocytosis. Purified FHA and pertussis toxin also increased the surface expression of CR3 on neutrophils, while ACT and the B subunit of pertussis toxin did not affect CR3 expression. FHA-mediated attachment to CR3 can lead to phagocytosis, especially in the absence of ACT. FHA mutants failed to attach and were not phagocytosed by neutrophils. Similarly, an antibody to CR3 blocked both attachment and phagocytosis. The addition of exogenous FHA enhanced the attachment and phagocytosis of wild-type B. pertussis and FHA mutants. Mutants lacking the SphB1 protease, which cleaves FHA and allows the release of FHA from the bacterial surface, were phagocytosed more efficiently than wild-type bacteria. ACT mutants were efficiently phagocytosed, but wild-type B. pertussis or ACT mutants plus exogenous ACT resisted phagocytosis. These studies suggest that the activation and surface expression of CR3, FHA expression, and the efficiency of ACT internalization all influence whether B. pertussis will be phagocytosed and ultimately killed by neutrophils.
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PMID:Influence of CR3 (CD11b/CD18) expression on phagocytosis of Bordetella pertussis by human neutrophils. 1623 29

Bordetella that infect mammals produce a multifunctional repeat in toxin (RTX) adenylate cyclase toxin known as CyaA, an excellent example of bacterial sophistication in subverting host defense. Recent reports show that interaction of CyaA with tracheal epithelial cells aids adhesion of Bordetella to ciliated mucosa and induces production of the pro-inflammatory cytokine interleukin, IL-6. Myeloid phagocytes, attracted to the site of infection are the target of freshly secreted CyaA that binds to the alpha(M)beta2 integrin (CD11b/CD18), penetrates cells and promptly suppresses their bactericidal functions by converting cellular ATP to cAMP. Such uncontrolled cAMP signaling can also drive CD11b-expressing immature dendritic cells into a semi-mature state, possibly hijacking them to shape the local adaptive immune response towards tolerance of the pathogen.
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PMID:Bordetella adenylate cyclase toxin: a swift saboteur of host defense. 1640 75

Bordetella adenylate cyclase (AC) toxin-hemolysin (CyaA) targets myeloid phagocytes expressing the alphaMbeta2 integrin (CD11b/CD18) and delivers into their cytosol an AC enzyme that converts ATP into cyclic AMP (cAMP). In parallel, CyaA acts as a hemolysin, forming small membrane pores. Using specific mutations, we dissected the contributions of the two activities to cytolytic potency of CyaA on J774A.1 murine monocytes. The capacity of AC to penetrate cells and deplete cytosolic ATP was essential for promoting lysis and the enzymatically inactive but fully hemolytic CyaA-AC- toxoid exhibited a 15-fold-lower cytolytic capacity on J774A.1 cells than intact CyaA. Moreover, a two- or fourfold drop of specific hemolytic activity of the CyaA-E570Q and CyaA-E581P mutants was overpowered by an intact capacity to dissipate cytosolic ATP into cAMP, allowing the less hemolytic proteins to promote lysis of J774A.1 cells as efficiently as intact CyaA. However, an increased hemolytic activity, due to lysine substitutions of glutamates 509, 516, and 581 in the pore-forming domain, conferred on AC- toxoids a correspondingly enhanced cytolytic potency. Moreover, a threefold increase in hemolytic activity could override a fourfold drop in capacity to convert cellular ATP to cAMP, conferring on the CyaA-E581K construct an overall twofold increased cytolytic potency. Hence, although appearing auxiliary in cytolytic action of the toxin on nucleated cells, the pore-forming activity can synergize with ATP-depleting activity of the cell-invasive AC enzyme and complement its action toward maximal cytotoxicity.
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PMID:Pore-forming and enzymatic activities of Bordetella pertussis adenylate cyclase toxin synergize in promoting lysis of monocytes. 1655 51

Recombinant, genetically-detoxified adenylate cyclase toxin (CyaA) constructs from Bordetella pertussis have been developed as potential antigen delivery systems and as promising antigen candidates for inclusion in acellular pertussis vaccines. The major toxic effects of native CyaA are attributed to its enzymatic activity following delivery to cells of the innate immune system via the CD11b/CD18 (CR3) cell receptor. In view of the potential use of detoxified CyaA in vaccinology, a complement dependent in vitro model was used to investigate the potential effects of the interaction of detoxified CyaA with CD11b/CD18 (CR3) on phagocytic function. Interaction of CyaA with CD11b/CD18 (CR3) on human pro-myelocytic NB-4 cells differentiated to a neutrophil-like phenotype was measured as inhibition of binding of a monoclonal antibody to the receptor. This interaction was dose-dependent and required acylation of CyaA. Treatment of the cells with either acylated or non-acylated detoxified CyaA constructs inhibited their phagocytic function. Washing the cells allowed recovery of phagocytic function after treatment with non-acylated toxin but not for cells treated with acylated CyaA constructs. However, availability of CD11b/CD18 receptors on acylated CyaA-treated cells was restored after washing and further incubation. The results suggest that the interaction of detoxified CyaA constructs to the CD11b/CD18 (CR3) receptor may temporarily influence the complement-dependent phagocytic function in neutrophil leukocytes.
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PMID:Evaluation of adenyl cyclase toxin constructs from Bordetella pertussis as candidate vaccine components in an in vitro model of complement-dependent intraphagocytic killing. 1667 78

The Bordetella adenylate cyclase toxin-hemolysin (CyaA) targets phagocytes expressing the alpha(M)beta2 integrin (CD11b/CD18), permeabilizes their membranes by forming small cation-selective pores, and delivers into cells a calmodulin-activated adenylate cyclase (AC) enzyme that dissipates cytosolic ATP into cAMP. We describe here a third activity of CyaA that yields elevation of cytosolic calcium concentration ([Ca2+]i) in target cells. The CyaA-mediated [Ca2+]i increase in CD11b+ J774A.1 monocytes was inhibited by extracellular La3+ ions but not by nifedipine, SK&F 96365, flunarizine, 2-aminoethyl diphenylborinate, or thapsigargin, suggesting that influx of Ca2+ into cells was not because of receptor signaling or opening of conventional calcium channels by cAMP. Compared with intact CyaA, a CyaA-AC- toxoid unable to generate cAMP promoted a faster, albeit transient, elevation of [Ca2+]i. This was not because of cell permeabilization by the CyaA hemolysin pores, because a mutant exhibiting a strongly enhanced pore-forming activity (CyaA-E509K/E516K), but unable to deliver the AC domain into cells, was also unable to elicit a [Ca2+]i increase. Further mutations interfering with AC translocation into cells, such as proline substitutions of glutamate residues 509 or 570 or deletion of the AC domain as such, reduced or ablated the [Ca2+]i-elevating capacity of CyaA. Moreover, structural alterations within the AC domain, because of insertion of various oligopeptides, differently modulated the kinetics and extent of Ca2+ influx elicited by the respective AC- toxoids. Hence, the translocating AC polypeptide itself appears to participate in formation of a novel type of membrane path for calcium ions, contributing to action of CyaA in an unexpected manner.
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PMID:Third activity of Bordetella adenylate cyclase (AC) toxin-hemolysin. Membrane translocation of AC domain polypeptide promotes calcium influx into CD11b+ monocytes independently of the catalytic and hemolytic activities. 1714 36

Bordetella pertussis causes a profound inflammatory response in lungs of infected individuals. The adenylate cyclase toxin (ACT) of B. pertussis is a potent enzyme that converts cytosolic ATP into cAMP, and is required for virulence in vivo. During infection, secreted ACT binds to macrophages utilizing the beta2 integrin, Mac-1 (CR3, CD11b/CD18), and subsequent intoxication by ACT inhibits essential antibacterial activities of macrophages. Additionally, Mac-1 has been reported to be a co-receptor for TLR4 required for the full induction of some LPS-responsive genes, including pro-inflammatory cyclooxygenase 2 (COX-2). We have examined the effect of ACT on COX-2 expression in HEK293T cells expressing Mac-1 and in murine macrophages. We report that ACT induces COX-2 in a manner that absolutely requires the catalytic activity of this enzyme and Mac-1 expression dramatically enhanced the sensitivity of cells to ACT-dependent COX-2 induction. The mechanism of COX-2 induction by ACT utilizes the cAMP-PKA-CREB-dependent pathway. Finally, ACT and TLR2 or TLR4 act synergistically to increase COX-2 expression. These data suggest that ACT contributes significantly to the inflammatory response induced by B. pertussis infection by augmenting COX-2 expression and provides evidence against the concept that ACT functions exclusively via its inhibitory effects on phagocytic leucocytes.
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PMID:Bordetella pertussis adenylate cyclase toxin (ACT) induces cyclooxygenase-2 (COX-2) in murine macrophages and is facilitated by ACT interaction with CD11b/CD18 (Mac-1). 1792 97

Bordetella pertussis adenylate cyclase (AC) toxin-hemolysin (Hly) (CyaA, ACT, or AC-Hly) is a cytotoxin of the RTX (repeat in toxin) family. It delivers into target cells an AC domain that catalyzes uncontrolled conversion of ATP to cAMP, a key signaling molecule subverting phagocyte functions. CyaA utilizes a heavily N-glycosylated beta(2) integrin receptor CD11b/CD18 (alpha(M)beta(2), Mac-1, or CR3). We show that deglycosylation of cell surface proteins by glycosidase treatment, or inhibition of protein N-glycosylation by tunicamycin, ablates CyaA binding and penetration of CD11b-expressing cells. Furthermore, binding of CyaA to cells was strongly inhibited in the presence of free saccharides occurring as building units of integrin oligosaccharide complex, whereas saccharides absent from integrin oligosaccharide chains failed to inhibit CyaA binding to CD11b/CD18-expressing cells. CyaA, hence, selectively recognized sugar residues of N-linked oligosaccharides of integrins. Moreover, glycosylation of CD11a/CD18, another receptor of the beta(2) integrin family, was also essential for cytotoxic action of other RTX cytotoxins, the leukotoxin of Aggregatibacter actinomycetemcomitans (LtxA) and the Escherichia coli alpha-Hly (HlyA). These results show that binding and killing of target cells by CyaA, LtxA, and HlyA depends on recognition of N-linked oligosaccharide chains of beta(2) integrin receptors. This sets a new paradigm for action of RTX cytotoxins.
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PMID:RTX cytotoxins recognize beta2 integrin receptors through N-linked oligosaccharides. 1837 64

Adenylate cyclase toxin (CyaA) of Bordetella pertussis binds to CD11b/CD18 on macrophages and dendritic cells (DC) and confers virulence to the bacteria by subverting innate immune responses of the host. We have previously demonstrated that CyaA promotes the induction of IL-10-secreting regulatory T cells in vivo by modulating DC activation. Here, we examine the mechanism of immune subversion, specifically, the modulation of TLR signaling pathways in DC. We found that CyaA synergized with LPS to induce IL-10 mRNA and protein expression in DC but significantly inhibited IL-12p70 production. CyaA enhanced LPS-induced phosphorylation of p38 MAPK and ERK in DC, and inhibitors of p38 MAPK, MEK, or NF-kappaB suppressed IL-10 production in response to LPS and CyaA. However, inhibition of p38 MAPK, MEK, and NF-kappaB did not reverse the inhibitory effect of CyaA on TLR agonist-induced IL-12 production. Furthermore, CyaA suppression of IL-12 was independent of IL-10. In contrast, CyaA suppressed LPS- and IFN-gamma-induced IFN-regulatory factor-1 (IRF-1) and IRF-8 expression in DC. The modulatory effects of CyaA were dependent on adenylate cyclase activity and induction of intracellular cAMP, as an enzyme-inactive mutant of CyaA failed to modulate TLR-induced signaling in DC, whereas the effects of the wild-type toxin were mimicked by stimulation of the DC with PGE2. Our findings demonstrate that CyaA modulates TLR agonist-induced IL-10 and IL-12p70 production in DC by, respectively, enhancing MAPK phosphorylation and inhibiting IRF-1 and IRF-8 expression and that this is mediated by elevation of intercellular cAMP concentrations.
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PMID:Adenylate cycalse toxin of Bordetella pertussis inhibits TLR-induced IRF-1 and IRF-8 activation and IL-12 production and enhances IL-10 through MAPK activation in dendritic cells. 1840 Oct 6


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