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Daniel Ladant - One of the best experts on this subject based on the ideXlab platform.
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a gateway compatible bacterial Adenylate Cyclase based two hybrid system
Environmental Microbiology Reports, 2014Co-Authors: Daniel Ladant, Scot P Ouellette, Emilie Gauliard, Zuzana AntosovaAbstract:Summary The bacterial Adenylate Cyclase two-hybrid (BACTH) system has been widely used to characterize protein–protein interactions in the prokaryotic world. This system relies on the interaction-mediated reconstitution of Adenylate Cyclase activity in Escherichia coli by bringing together two complementary fragments of the catalytic domain of the Adenylate Cyclase toxin of Bordetella pertussis. A limiting factor in performing large-scale two-hybrid interaction screens with full-length open reading frames (ORFs) is the need to clone each ORF individually into the plasmids used to express the hybrid proteins. The Gateway® (GW) cloning system (Life Technologies, Grand Island, NY, USA) partially circumvents this limitation, and we describe here modifications to the BACTH system for compatibility with this recombineering technology. We validated and tested the functionality of the BACTH Gateway (BACTHGW) system using several models of protein–protein interactions, focusing particularly on those involved in bacterial cell division. We further modified the BACTH plasmids to incorporate a transmembrane (TM) segment downstream of the Cyclase fragments to permit analysis of extracytoplasmic protein interactions. This approach was also useful to identify putative TM segments and to experimentally validate bioinformatically identified TM domains. The BACTHGW system will prove a useful addition to the study of protein–protein interactions.
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bordetella pertussis Adenylate Cyclase toxin structural and functional independence of the catalytic and hemolytic activities
Journal of Biological Chemistry, 1992Co-Authors: Hiroshi Sakamoto, Jacques Bellalou, Peter Sebo, Daniel LadantAbstract:The Bordetella pertussis calmodulin-dependent Adenylate Cyclase (CyaA) is a 1706-residue-long toxin, endowed with hemolytic activity. We have constructed B. pertussis mutant strains producing modified CyaAs devoid of Adenylate Cyclase activity. Our results show that such modified CyaAs display hemolytic activity identical to the wild-type toxin, thus demonstrating that the hemolytic activity is independent of the Adenylate Cyclase activity. Furthermore, B. pertussis and Escherichia coli strains producing CyaA lacking the catalytic domain (residues 1-373) were constructed. The truncated protein exhibits hemolytic activity comparable to the wild-type toxin, thus establishing that the carboxyl-terminal 1332 residues alone are endowed with hemolytic activity. Together, these findings show that Adenylate Cyclase and hemolytic activities are located in two distinct regions of the molecule (respectively, approximately amino acids 1-400 and 401-1706) and that the two regions of CyaA are functionally independent.
Nicole Guiso - One of the best experts on this subject based on the ideXlab platform.
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Bordetella Adenylate Cyclase-Hemolysin Toxins
Toxins, 2017Co-Authors: Nicole GuisoAbstract:Adenylate Cyclase-hemolysin toxin is secreted and produced by three classical species of the genus Bordetella: Bordetella pertussis, B. parapertussis and B. bronchiseptica. This toxin has several properties such as: (i) Adenylate Cyclase activity, enhanced after interaction with the eukaryotic protein, calmodulin; (ii) a pore-forming activity; (iii) an invasive activity. It plays an important role in the pathogenesis of these Bordetella species responsible for whooping cough in humans or persistent respiratory infections in mammals, by modulating host immune responses. In contrast with other Bordetella toxins or adhesins, lack of (or very low polymorphism) is observed in the structural gene encoding this toxin, supporting its importance as well as a potential role as a vaccine antigen against whooping cough. In this article, an overview of the investigations undertaken on this toxin is presented.
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bordetella pertussis induces apoptosis in macrophages role of Adenylate Cyclase hemolysin
Infection and Immunity, 1993Co-Authors: N Khelef, A Zychlinsky, Nicole GuisoAbstract:Bordetella pertussis, the causative agent of whooping cough, has been shown recently to enter and survive in epithelial cells and macrophages in vitro. In the present study, we show that B. pertussis is cytotoxic for J774A.1 cells, a monocyte-macrophage cell line, and for murine alveolar macrophages. We demonstrate that cell cytotoxicity mediated by B. pertussis occurred through apoptosis, as shown by changes in nuclear morphology and by host cell DNA fragmentation. Parental strains and a mutant deficient in pertussis toxin expression are able to induce apoptosis, whereas avirulent mutant or Adenylate Cyclase-hemolysin-deficient mutants are not cytotoxic. Both Adenylate Cyclase and hemolytic activities are required for programmed cell death. These results show that induction of apoptosis is dependent on the expression of Adenylate Cyclase-hemolysin. The infection of murine alveolar macrophages in primary culture with B. pertussis leads to apoptosis, suggesting that this process might be relevant in vivo. The ability of B. pertussis to promote cell death may be important for the initiation of infection, bacterial survival, and escape of the host immune response.
P B Molinoff - One of the best experts on this subject based on the ideXlab platform.
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beta adrenergic receptor g protein Adenylate Cyclase complex in experimental canine congestive heart failure produced by rapid ventricular pacing
Circulation Research, 1991Co-Authors: K P Marzo, Martin Frey, John R Wilson, B T Liang, D R Manning, Vita Lanoce, P B MolinoffAbstract:Changes in the beta-adrenergic receptor-G protein-Adenylate Cyclase complex were investigated in an experimental canine model of low-output heart failure produced by chronic rapid ventricular pacing. The contractile response occurring after exposure to the beta-adrenergic agonist dobutamine, measured as peak left ventricular + dP/dt, was decreased after 3 weeks of pacing. To further characterize the diminished functional responsiveness to beta-adrenergic receptor stimulation, beta-adrenergic receptor-Adenylate Cyclase coupling was investigated using membranes prepared from both control and paced animals. The density of beta-adrenergic receptors was decreased by 40% with a selective downregulation of the beta 1-subtype. The affinity of the receptor for the antagonist radioligand [125I]iodocyanopindolol remained unchanged. A defect in coupling was suggested by a decreased ability of isoproterenol, fluoride, and forskolin to stimulate Adenylate Cyclase in membranes prepared from failing hearts. Determination of the levels of Gi alpha (the alpha-subunit of Gi) by immunoblotting and pertussis toxin labeling revealed modest increases of approximately 30%. Furthermore, Mn2+ and purified Gs failed to stimulate Adenylate Cyclase in membranes prepared from failing hearts, indicating an impairment in the catalytic moiety of Adenylate Cyclase itself or in the ability of Adenylate Cyclase to couple to Gs. In contrast, complementation assay did not reveal differences in the functional activity of Gs alpha (the alpha-subunit of Gs). Taken together, these data demonstrate a selective decrease in the beta 1-subtype of adrenergic receptors and an increase in a 40-kd G1-like protein in the failing heart. Similar changes have been described in human idiopathic dilated cardiomyopathy. In addition to these changes, we identified a possible defect at the level of the catalytic subunit of Adenylate Cyclase.
Emilie Gauliard - One of the best experts on this subject based on the ideXlab platform.
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a gateway compatible bacterial Adenylate Cyclase based two hybrid system
Environmental Microbiology Reports, 2014Co-Authors: Daniel Ladant, Scot P Ouellette, Emilie Gauliard, Zuzana AntosovaAbstract:Summary The bacterial Adenylate Cyclase two-hybrid (BACTH) system has been widely used to characterize protein–protein interactions in the prokaryotic world. This system relies on the interaction-mediated reconstitution of Adenylate Cyclase activity in Escherichia coli by bringing together two complementary fragments of the catalytic domain of the Adenylate Cyclase toxin of Bordetella pertussis. A limiting factor in performing large-scale two-hybrid interaction screens with full-length open reading frames (ORFs) is the need to clone each ORF individually into the plasmids used to express the hybrid proteins. The Gateway® (GW) cloning system (Life Technologies, Grand Island, NY, USA) partially circumvents this limitation, and we describe here modifications to the BACTH system for compatibility with this recombineering technology. We validated and tested the functionality of the BACTH Gateway (BACTHGW) system using several models of protein–protein interactions, focusing particularly on those involved in bacterial cell division. We further modified the BACTH plasmids to incorporate a transmembrane (TM) segment downstream of the Cyclase fragments to permit analysis of extracytoplasmic protein interactions. This approach was also useful to identify putative TM segments and to experimentally validate bioinformatically identified TM domains. The BACTHGW system will prove a useful addition to the study of protein–protein interactions.
Scot P Ouellette - One of the best experts on this subject based on the ideXlab platform.
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a gateway compatible bacterial Adenylate Cyclase based two hybrid system
Environmental Microbiology Reports, 2014Co-Authors: Daniel Ladant, Scot P Ouellette, Emilie Gauliard, Zuzana AntosovaAbstract:Summary The bacterial Adenylate Cyclase two-hybrid (BACTH) system has been widely used to characterize protein–protein interactions in the prokaryotic world. This system relies on the interaction-mediated reconstitution of Adenylate Cyclase activity in Escherichia coli by bringing together two complementary fragments of the catalytic domain of the Adenylate Cyclase toxin of Bordetella pertussis. A limiting factor in performing large-scale two-hybrid interaction screens with full-length open reading frames (ORFs) is the need to clone each ORF individually into the plasmids used to express the hybrid proteins. The Gateway® (GW) cloning system (Life Technologies, Grand Island, NY, USA) partially circumvents this limitation, and we describe here modifications to the BACTH system for compatibility with this recombineering technology. We validated and tested the functionality of the BACTH Gateway (BACTHGW) system using several models of protein–protein interactions, focusing particularly on those involved in bacterial cell division. We further modified the BACTH plasmids to incorporate a transmembrane (TM) segment downstream of the Cyclase fragments to permit analysis of extracytoplasmic protein interactions. This approach was also useful to identify putative TM segments and to experimentally validate bioinformatically identified TM domains. The BACTHGW system will prove a useful addition to the study of protein–protein interactions.