The Experts below are selected from a list of 1671 Experts worldwide ranked by ideXlab platform
Daniel Ladant - One of the best experts on this subject based on the ideXlab platform.
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Functional and structural consequences of epithelial cell invasion by Bordetella pertussis adenylate cyclase toxin
PLoS ONE, 2020Co-Authors: Christelle Angely, And Alexandre Chenal, Daniel Ladant, Emmanuelle Planus, Bruno Louis, Marcel Filoche, Daniel IsabeyAbstract:Bordetella pertussis, the causative agent of whopping cough, produces an adenylate cyclase toxin (CyaA) that plays a key role in the host colonization by targeting innate immune cells which express CD11b/CD18, the cellular receptor of CyaA. CyaA is also able to invade non-phagocytic cells, via a unique entry pathway consisting in a direct translocation of its catalytic domain across the cytoplasmic membrane of the cells. Within the cells, CyaA is activated by calmodulin to produce high levels of cyclic adenosine monophosphate (cAMP) and alter cellular physiology. In this study, we explored the effects of CyaA toxin on the cellular and molecular structure remodeling of A549 alveolar epithelial cells. Using classical imaging techniques, biochemical and functional tests, as well as advanced cell mechanics method, we quantify the structural and functional consequences of the massive increase of intracellular cyclic AMP induced by the toxin: cell shape rounding associated to adhesion weakening process, actin structure remodeling for the cortical and dense components, increase in cytoskeleton stiffness, and inhibition of migration and repair. We also show that, at low concentrations (0.5 nM), CyaA could significantly impair the migration and wound healing capacities of the intoxicated alveolar epithelial cells. As such concentrations might be reached locally during B. pertussis infection, our results suggest that the CyaA, beyond its major role in disabling innate immune cells, might also contribute to the local alteration of the epithelial barrier of the respiratory tract, a hallmark of pertussis.
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functional and structural consequences of epithelial cell invasion by bordetella pertussis adenylate cyclase toxin
bioRxiv, 2020Co-Authors: Christelle Angely, And Alexandre Chenal, Daniel Ladant, Emmanuelle Planus, Bruno Louis, Marcel Filoche, Daniel IsabeyAbstract:Bordetella pertussis, the causative agent of whopping cough, produces an adenylate cyclase toxin (CyaA) that plays a key role in the host colonization by targeting innate immune cells which express CD11b/CD18, the cellular receptor of CyaA. CyaA is also able to invade non-phagocytic cells, via a unique entry pathway consisting in a direct translocation of its catalytic domain across the cytoplasmic membrane of the cells. Within the cells, CyaA is activated by calmodulin to produce high levels of cyclic adenosine monophosphate (cAMP) and alter cellular physiology. In this study, we explored the effects of CyaA toxin on the cellular and molecular structure remodeling of A549 alveolar epithelial cells. Using classical imaging techniques, biochemical and functional tests, as well as advanced cell mechanics method, we quantify the structural and functional consequences of the massive increase of intracellular cyclic AMP induced by the toxin: cell shape rounding associated to adhesion weakening process, actin structure remodeling for the cortical and dense components, increase in cytoskeleton stiffness, and inhibition of migration and repair. We also show that, at the low concentrations that may be found in vivo during B. pertussis infection, CyaA impairs the migration and wound healing capacities of the intoxicated alveolar epithelial cells. Our results suggest that the CyaA, beyond its major role in disabling innate immune cells, might also contribute to the local alteration of the epithelial barrier of the respiratory tract, that is an hallmark of pertussis.
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Post-translational acylation controls the folding and functions of the CyaA RTX toxin
FASEB Journal, 2019Co-Authors: Darragh O'brien, Alexis Voegele, Marilyne Davi, Thibaut Douche, Mariette Matondo, Sebastien Brier, Daniel Ladant, Sara Cannella, Dorothée Raoux-barbot, And Alexandre ChenalAbstract:The adenylate cyclase (CyaA) toxin is a major virulence factor of Bordetella pertussis, the causative agent of whooping cough. CyaA is synthetized as a pro-toxin, pro-CyaA, and converted into its cytotoxic form upon acylation of two lysines. After secretion, CyaA invades eukaryotic cells and produces cAMP, leading to host defense subversion. To gain further insights into the effect of acylation, we compared the functional and structural properties of pro-CyaA and CyaA proteins. HDX-MS results show that the refolding process of both proteins upon progressive urea removal is initiated by calcium binding to the C-terminal RTX domain. We further identified a critical hydrophobic segment, distal from the acylation region, that folds at higher urea concentration in CyaA than in pro-CyaA. Once refolded into monomers, CyaA is more compact and stable than pro-CyaA, due to a complex set of interactions between domains. Our HDX-MS data provide direct evidence that the presence of acyl chains in CyaA induces a significant stabilization of the apolar segments of the hydrophobic domain and of most of the acylation region. We propose a refolding model dependent on calcium and driven by local and distal acylation-dependent interactions within CyaA. Therefore, CyaA acylation is not only critical for cell intoxication, but also for protein refolding into its active conformation. Our data shed light on the complex relationship between post-translational modifications, structural disorder and protein folding. Coupling calcium-binding and acylation-driven folding is likely pertinent for other repeat-in-toxin cytolysins produced by many Gram-negative bacterial pathogens.-O'Brien, D. P., Cannella, S. E., Voegele, A., Raoux-Barbot, D., Davi, M., Douché, T., Matondo, M., Brier, S., Ladant, D., Chenal, A. Post-translational acylation controls the folding and functions of the CyaA RTX toxin.
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post translational acylation controls the folding and functions of the CyaA rtx toxin
The FASEB Journal, 2019Co-Authors: Darragh P Obrien, Sara E Cannella, Alexis Voegele, Dorothee Raouxbarbot, Marilyne Davi, Thibaut Douche, Mariette Matondo, Sebastien Brier, Daniel Ladant, And Alexandre ChenalAbstract:The adenylate cyclase (CyaA) toxin is a major virulence factor of Bordetella pertussis, the causative agent of whooping cough. CyaA is synthetized as a pro-toxin, pro-CyaA, and converted into its c...
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The Adenylate Cyclase (CyaA) Toxin from Bordetella pertussis Has No Detectable Phospholipase A (PLA) Activity In Vitro
MDPI AG, 2019Co-Authors: Alexis Voegele, Thibaut Douche, Mariette Matondo, Daniel Ladant, Dorothée Raoux-barbot, Mirko Sadi, And Alexandre ChenalAbstract:The adenylate cyclase (CyaA) toxin produced in Bordetella pertussis is the causative agent of whooping cough. CyaA exhibits the remarkable capacity to translocate its N-terminal adenyl cyclase domain (ACD) directly across the plasma membrane into the cytosol of eukaryotic cells. Once translocated, calmodulin binds and activates ACD, leading to a burst of cAMP that intoxicates the target cell. Previously, Gonzalez-Bullon et al. reported that CyaA exhibits a phospholipase A activity that could destabilize the membrane to facilitate ACD membrane translocation. However, Bumba and collaborators lately reported that they could not replicate these results. To clarify this controversy, we assayed the putative PLA activity of two CyaA samples purified in two different laboratories by using two distinct fluorescent probes reporting either PLA2 or both PLA1 and PLA2 activities, as well as in various experimental conditions (i.e., neutral or negatively charged membranes in different buffers.) However, we could not detect any PLA activity in these CyaA batches. Thus, our data independently confirm that CyaA does not possess any PLA activity
Peter Sebo - One of the best experts on this subject based on the ideXlab platform.
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Residues 529 to 549 participate in membrane penetration and pore-forming activity of the Bordetella adenylate cyclase toxin.
Scientific reports, 2019Co-Authors: Jana Roderova, Peter Sebo, Adriana Osickova, Radim Osicka, Radovan Fiser, Anna Sukova, Gabriela Mikušová, Jiri MasinAbstract:The adenylate cyclase toxin-hemolysin (CyaA, ACT or AC-Hly) of pathogenic Bordetellae delivers its adenylyl cyclase (AC) enzyme domain into the cytosol of host cells and catalyzes uncontrolled conversion of cellular ATP to cAMP. In parallel, the toxin forms small cation-selective pores that permeabilize target cell membrane and account for the hemolytic activity of CyaA on erythrocytes. The pore-forming domain of CyaA is predicted to consist of five transmembrane α-helices, of which the helices I, III, IV and V have previously been characterized. We examined here the α-helix II that is predicted to form between residues 529 to 549. Substitution of the glycine 531 residue by a proline selectively reduced the hemolytic capacity but did not affect the AC translocating activity of the CyaA-G531P toxin. In contrast, CyaA toxins with alanine 538 or 546 replaced by diverse residues were selectively impaired in the capacity to translocate the AC domain across cell membrane but remained fully hemolytic. Such toxins, however, formed pores in planar asolectin bilayer membranes with a very low frequency and with at least two different conducting states. The helix-breaking substitution of alanine 538 by a proline residue abolished the voltage-activated increase of membrane activity of CyaA in asolectin bilayers. These results reveal that the predicted α-helix comprising the residues 529 to 549 plays a key role in CyaA penetration into the target plasma membrane and pore-forming activity of the toxin.
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Bordetella Pertussis Adenylate Cyclase Toxin Does Not Possess a Phospholipase A Activity; Serine 606 and Aspartate 1079 Residues Are Not Involved in Target Cell Delivery of the Adenylyl Cyclase Enzyme Domain.
Toxins, 2018Co-Authors: Ladislav Bumba, Adriana Osickova, Radim Osicka, Jiri Masin, Peter SeboAbstract:The adenylate cyclase toxin-hemolysin (CyaA, ACT, or AC-Hly) plays a crucial role in virulence and airway colonization capacity of the whooping cough agent Bordetella pertussis. The toxin penetrates target cell membranes and exhibits three distinct biological activities. A population of CyaA conformers forms small cation-selective pores that permeabilize the cell membrane for potassium efflux, which can provoke colloid-osmotic (oncotic) cell lysis. The other two activities are due to CyaA conformers that transiently form calcium influx conduits in the target cell membrane and translocate the adenylate cyclase (AC) enzyme into cytosol of cells. A fourth putative biological activity has recently been reported; an intrinsic phospholipase A (PLA) activity was claimed to be associated with the CyaA polypeptide and be involved in the mechanism of translocation of the AC enzyme polypeptide across cell membrane lipid bilayer. However, the conclusions drawn by the authors contradicted their own results and we show them to be erroneous. We demonstrate that highly purified CyaA is devoid of any detectable phospholipase A1 activity and that contrary to the published claims, the two putative conserved phospholipase A catalytic residues, namely the Ser606 and Asp1079 residues, are not involved in the process of membrane translocation of the AC domain of CyaA across target membranes.
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Phosphoproteomics of cAMP signaling of Bordetella adenylate cyclase toxin in mouse dendritic cells
Scientific reports, 2017Co-Authors: Jakub Novák, Ivo Fabrik, Irena Linhartova, Marek Link, Ondřej Černý, Jiří Stulík, Peter SeboAbstract:The adenylate cyclase toxin (CyaA) of the whooping cough agent Bordetella pertussis subverts immune functions of host myeloid cells expressing the αMβ2 integrin (CD11b/CD18, CR3 or Mac-1). CyaA delivers into cytosol of cells an extremely catalytically active adenylyl cyclase enzyme, which disrupts the innate and adaptive immune functions of phagocytes through unregulated production of the key signaling molecule cAMP. We have used phosphoproteomics to analyze cAMP signaling of CyaA in murine bone marrow-derived dendritic cells. CyaA action resulted in alterations of phosphorylation state of a number of proteins that regulate actin cytoskeleton homeostasis, including Mena, Talin-1 and VASP. CyaA action repressed mTOR signaling through activation of mTORC1 inhibitors TSC2 and PRAS40 and altered phosphorylation of multiple chromatin remodelers, including the class II histone deacetylase HDAC5. CyaA toxin action further elicited inhibitory phosphorylation of SIK family kinases involved in modulation of immune response and provoked dephosphorylation of the transcriptional coactivator CRTC3, indicating that CyaA-promoted nuclear translocation of CRTC3 may account for CyaA-induced IL-10 production. These findings document the complexity of subversive physiological manipulation of myeloid phagocytes by the CyaA toxin, serving in immune evasion of the pertussis agent.
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the conserved tyrosine residue 940 plays a key structural role in membrane interaction of bordetella adenylate cyclase toxin
Scientific Reports, 2017Co-Authors: Jiri Masin, Ladislav Bumba, Peter Sebo, Adriana Osickova, Jana Roderova, Petr Novak, Radovan Fiser, Radim OsickaAbstract:The adenylate cyclase toxin-hemolysin (CyaA, ACT or AC-Hly) translocates its adenylate cyclase (AC) enzyme domain into target cells in a step that depends on membrane cholesterol content. We thus examined what role in toxin activities is played by the five putative cholesterol recognition amino acid consensus (CRAC) motifs predicted in CyaA hemolysin moiety. CRAC-disrupting phenylalanine substitutions had no impact on toxin activities and these were not inhibited by free cholesterol, showing that the putative CRAC motifs are not involved in cholesterol binding. However, helix-breaking proline substitutions in these segments uncovered a structural role of the Y632, Y658, Y725 and Y738 residues in AC domain delivery and pore formation by CyaA. Substitutions of Y940 of the fifth motif, conserved in the acylated domains of related RTX toxins, did not impact on fatty-acylation of CyaA by CyaC and the CyaA-Y940F mutant was intact for toxin activities on erythrocytes and myeloid cells. However, the Y940A or Y940P substitutions disrupted the capacity of CyaA to insert into artificial lipid bilayers or target cell membranes. The aromatic ring of tyrosine 940 side chain thus appears to play a key structural role in molecular interactions that initiate CyaA penetration into target membranes.
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negatively charged residues of the segment linking the enzyme and cytolysin moieties restrict the membrane permeabilizing capacity of adenylate cyclase toxin
Scientific Reports, 2016Co-Authors: Jiri Masin, Ladislav Bumba, Adriana Osickova, Radim Osicka, Radovan Fiser, Irena Linhartova, Anna Sukova, Petr Halada, Peter SeboAbstract:The whooping cough agent, Bordetella pertussis, secretes an adenylate cyclase toxin-hemolysin (CyaA) that plays a crucial role in host respiratory tract colonization. CyaA targets CR3-expressing cells and disrupts their bactericidal functions by delivering into their cytosol an adenylate cyclase enzyme that converts intracellular ATP to cAMP. In parallel, the hydrophobic domain of CyaA forms cation-selective pores that permeabilize cell membrane. The invasive AC and pore-forming domains of CyaA are linked by a segment that is unique in the RTX cytolysin family. We used mass spectrometry and circular dichroism to show that the linker segment forms α-helical structures that penetrate into lipid bilayer. Replacement of the positively charged arginine residues, proposed to be involved in target membrane destabilization by the linker segment, reduced the capacity of the toxin to translocate the AC domain across cell membrane. Substitutions of negatively charged residues then revealed that two clusters of negative charges within the linker segment control the size and the propensity of CyaA pore formation, thereby restricting the cell-permeabilizing capacity of CyaA. The ‘AC to Hly-linking segment’ thus appears to account for the smaller size and modest cell-permeabilizing capacity of CyaA pores, as compared to typical RTX hemolysins.
Claude Leclerc - One of the best experts on this subject based on the ideXlab platform.
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Induction of anti-Tat neutralizing antibodies by the CyaA vector targeting dendritic cells: Influence of the insertion site and of the delivery of multicopies of the dominant Tat B-cell epitope
Vaccine, 2010Co-Authors: Catherine Fayolle, Marilyne Davi, Daniel Ladant, Hui Dong, Dorothea Ritzel, Aurélie Le Page, Friederike Knipping, Laleh Majlessi, Claude LeclercAbstract:Abstract HIV-Tat based vaccines have been proposed as an attractive option to prevent or treat AIDS. A vaccine to induce optimal anti-Tat neutralizing antibody responses was designed by inserting this protein, or its dominant B-cell epitope, into the CyaA vector, which targets dendritic cells (DC). Tat was inserted into various sites of CyaA, including regions that do not translocate into the cytosol of the targeted DC. The presentation of the Tat CD4 + T-cell epitope delivered by the CyaA-Tat proteins was observed with a recombinant CyaA in which the entire AC domain was replaced by the entire Tat protein (Tat-Δ373 CyaA) but was abolished with large deletions of the N-terminal region. Moreover, CyaA carrying multiple copies of the dominant Tat: 1–21 B-cell epitope were shown to induce high titers of anti-Tat antibodies, even after a single immunization, that persisted up to 10 weeks post-immunization.
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Delivery of the HIV-1 Tat protein to dendritic cells by the CyaA vector induces specific Th1 responses and high affinity neutralizing antibodies in non human primates.
Vaccine, 2006Co-Authors: Laurent Mascarell, Daniel Ladant, Cécile Bauche, Catherine Fayolle, Ousmane M Diop, Monique Dupuy, Nolwenn Nougarede, Ronald Perraut, Claude LeclercAbstract:The human immunodeficiency virus type 1 (HIV-1) Tat is a key protein playing a major role in the infectivity of the virus. Thus, HIV-Tat based vaccines have been proposed as an attractive option to treat AIDS. Recently, we have shown that the recombinant detoxified adenylate cyclase (CyaA) from Bordetella pertussis carrying HIV-Tat (CyaA-E5-Tat), targets dendritic cells (DCs) and induces specific Th1 polarized and neutralizing antibody responses in mice. To further explore the potentialities of this prototype vaccine for human use, we analyzed the CyaA-E5-Tat induced antibody responses in non-human primates and established the biological characteristics of these antibodies. African Green Monkeys (AGM) were immunized with CyaA-E5-Tat in the presence or in the absence of alum adjuvant. First, we showed that the anti-CyaA antibodies induced by such immunization does not interfere with the binding of CyaA-E5-Tat to its receptor at the DC surface, the alphaMbeta2 integrin. Monkeys immunized with CyaA-E5-Tat, with or without alum, produced anti-Tat antibodies that mainly recognized the N-terminal domain of the Tat protein. Importantly, all sera obtained after three immunizations displayed the capacity to bind to Tat and neutralize its transactivating function in vitro. Finally, in the absence of alum, CyaA-E5-Tat, induced Th1 Tat specific T cell responses. These findings reveal that CyaA-E5-Tat is efficiently delivered in non-human primates and had a significant impact on the generation of neutralizing anti-Tat antibodies. These observations are, thus, encouraging for the use of the CyaA vector in human and also suggest that CyaA-E5-Tat might be a useful tool to decipher the biological characteristic of such antibodies.
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Acylation of lysine 860 allows tight binding and cytotoxicity of Bordetella adenylate cyclase on CD11b-expressing cells.
Biochemistry, 2005Co-Authors: Jiri Masin, Roland Benz, Marek Basler, Oliver Knapp, Mohammed El-azami-el-idrissi, Elke Maier, Ivo Konopásek, Claude Leclerc, Peter SeboAbstract: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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Induction of neutralizing antibodies and Th1-polarized and CD4-independent CD8+ T-cell responses following delivery of human immunodeficiency virus type 1 Tat protein by recombinant adenylate cyclase of Bordetella pertussis.
Journal of virology, 2005Co-Authors: Laurent Mascarell, Daniel Ladant, Cécile Bauche, Catherine Fayolle, Claude LeclercAbstract:HIV-Tat, a conserved protein playing a key role in the early life cycle of the human immunodeficiency virus (HIV) has been proposed as a potential AIDS vaccine. An HIV-Tat-based vaccine should elicit a broad, long-lasting, and neutralizing immune response. We have previously demonstrated that the adenylate cyclase (CyaA) from Bordetella pertussis targets dendritic cells and delivers CD8+ and CD4+ T-cell epitopes into the major histocompatibility complex class I and class II presentation pathways. We have also showed that CyaA induced specific and protective cytotoxic T cell responses in vivo. Here, we designed a prototype vaccine based on the HIV type 1 Tat delivered by CyaA (CyaA-E5-Tat) and tested its capacity to induce HIV-Tat-specific cellular as well as antibody responses. We showed that immunization of mice by CyaA-E5-Tat in the absence of adjuvant elicited strong and long-lasting neutralizing anti-Tat antibody responses more efficient than those obtained after immunization with Tat toxoid in aluminum hydroxide adjuvant. Analyses of the anti-Tat immunoglobulin G isotypes and the cytokine pattern showed that CyaA-E5-Tat induced a Th1-polarized immune response in contrast to the Th2-polarized immune responses obtained with the Tat toxoid. In addition, our data demonstrated that HIV-Tat-specific gamma interferon-producing CD8+ T cells were generated after vaccination with CyaA-E5-Tat in a CD4+ T-cell-independent manner. Based on these findings, CyaA-E5-Tat represents an attractive vaccine candidate for both preventive and therapeutic vaccination involving CyaA as an efficient nonreplicative vector for protein delivery.
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Efficient Ex Vivo Stimulation of Mycobacterium tuberculosis-Specific T Cells by Genetically Detoxified Bordetella pertussis Adenylate Cyclase Antigen Toxoids
Infection and immunity, 2005Co-Authors: Katalin A. Wilkinson, Peter Sebo, Claude Leclerc, Marcela Simsova, Elisabeth H. Schölvinck, H. Martin Vordermeier, Stuart J. Dickson, Jillian R. Brown, Robert N. Davidson, Geoffrey PasvolAbstract:Mycobacterium tuberculosis is a significant threat to global health. Mycobacterium bovis BCG vaccine provides only partial protection, and the skin test reagent used to aid diagnosis of both active and latent tuberculosis, purified protein derivative (PPD), lacks specificity and sensitivity. The use of genetically detoxified Bordetella pertussis adenylate cyclase toxin (CyaA) as a delivery system for two immunodominant proteins of M. tuberculosis that are of greater specificity than PPD, early-secreted antigenic target 6-kDa protein (ESAT-6) and culture filtrate protein 10 (CFP-10), was therefore investigated. CyaA toxoids incorporating these antigens were able to restimulate T cells from more than 91% tuberculosis patients and healthy sensitized donors. Delivery of antigen by CyaA decreased by 10-fold the amount of ESAT-6 and CFP-10 required to restimulate T cells, and in low responders, the overall frequency of gamma interferon-producing cells detected by enzyme-linked immunospot assay was increased (P < 0.01 for both antigens). Delivery of ESAT-6 and CFP-10 by CyaA enabled the detection of both CD4+ and CD8+ T cells: these responses could be blocked by inhibition of major histocompatibility complex class II or class I, respectively. Covalent linkage of antigen to the CyaA vector was required for enhancement to occur, as a mixture of mock CyaA toxoid plus recombinant ESAT-6 did not lead to enhancement. In a simplified whole-blood model to detect tuberculosis infection, the frequency of positive responses to CFP-10 was increased by CyaA delivery, a potentially important attribute that could facilitate the identification of latent infection.
And Alexandre Chenal - One of the best experts on this subject based on the ideXlab platform.
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Functional and structural consequences of epithelial cell invasion by Bordetella pertussis adenylate cyclase toxin
PLoS ONE, 2020Co-Authors: Christelle Angely, And Alexandre Chenal, Daniel Ladant, Emmanuelle Planus, Bruno Louis, Marcel Filoche, Daniel IsabeyAbstract:Bordetella pertussis, the causative agent of whopping cough, produces an adenylate cyclase toxin (CyaA) that plays a key role in the host colonization by targeting innate immune cells which express CD11b/CD18, the cellular receptor of CyaA. CyaA is also able to invade non-phagocytic cells, via a unique entry pathway consisting in a direct translocation of its catalytic domain across the cytoplasmic membrane of the cells. Within the cells, CyaA is activated by calmodulin to produce high levels of cyclic adenosine monophosphate (cAMP) and alter cellular physiology. In this study, we explored the effects of CyaA toxin on the cellular and molecular structure remodeling of A549 alveolar epithelial cells. Using classical imaging techniques, biochemical and functional tests, as well as advanced cell mechanics method, we quantify the structural and functional consequences of the massive increase of intracellular cyclic AMP induced by the toxin: cell shape rounding associated to adhesion weakening process, actin structure remodeling for the cortical and dense components, increase in cytoskeleton stiffness, and inhibition of migration and repair. We also show that, at low concentrations (0.5 nM), CyaA could significantly impair the migration and wound healing capacities of the intoxicated alveolar epithelial cells. As such concentrations might be reached locally during B. pertussis infection, our results suggest that the CyaA, beyond its major role in disabling innate immune cells, might also contribute to the local alteration of the epithelial barrier of the respiratory tract, a hallmark of pertussis.
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functional and structural consequences of epithelial cell invasion by bordetella pertussis adenylate cyclase toxin
bioRxiv, 2020Co-Authors: Christelle Angely, And Alexandre Chenal, Daniel Ladant, Emmanuelle Planus, Bruno Louis, Marcel Filoche, Daniel IsabeyAbstract:Bordetella pertussis, the causative agent of whopping cough, produces an adenylate cyclase toxin (CyaA) that plays a key role in the host colonization by targeting innate immune cells which express CD11b/CD18, the cellular receptor of CyaA. CyaA is also able to invade non-phagocytic cells, via a unique entry pathway consisting in a direct translocation of its catalytic domain across the cytoplasmic membrane of the cells. Within the cells, CyaA is activated by calmodulin to produce high levels of cyclic adenosine monophosphate (cAMP) and alter cellular physiology. In this study, we explored the effects of CyaA toxin on the cellular and molecular structure remodeling of A549 alveolar epithelial cells. Using classical imaging techniques, biochemical and functional tests, as well as advanced cell mechanics method, we quantify the structural and functional consequences of the massive increase of intracellular cyclic AMP induced by the toxin: cell shape rounding associated to adhesion weakening process, actin structure remodeling for the cortical and dense components, increase in cytoskeleton stiffness, and inhibition of migration and repair. We also show that, at the low concentrations that may be found in vivo during B. pertussis infection, CyaA impairs the migration and wound healing capacities of the intoxicated alveolar epithelial cells. Our results suggest that the CyaA, beyond its major role in disabling innate immune cells, might also contribute to the local alteration of the epithelial barrier of the respiratory tract, that is an hallmark of pertussis.
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Post-translational acylation controls the folding and functions of the CyaA RTX toxin
FASEB Journal, 2019Co-Authors: Darragh O'brien, Alexis Voegele, Marilyne Davi, Thibaut Douche, Mariette Matondo, Sebastien Brier, Daniel Ladant, Sara Cannella, Dorothée Raoux-barbot, And Alexandre ChenalAbstract:The adenylate cyclase (CyaA) toxin is a major virulence factor of Bordetella pertussis, the causative agent of whooping cough. CyaA is synthetized as a pro-toxin, pro-CyaA, and converted into its cytotoxic form upon acylation of two lysines. After secretion, CyaA invades eukaryotic cells and produces cAMP, leading to host defense subversion. To gain further insights into the effect of acylation, we compared the functional and structural properties of pro-CyaA and CyaA proteins. HDX-MS results show that the refolding process of both proteins upon progressive urea removal is initiated by calcium binding to the C-terminal RTX domain. We further identified a critical hydrophobic segment, distal from the acylation region, that folds at higher urea concentration in CyaA than in pro-CyaA. Once refolded into monomers, CyaA is more compact and stable than pro-CyaA, due to a complex set of interactions between domains. Our HDX-MS data provide direct evidence that the presence of acyl chains in CyaA induces a significant stabilization of the apolar segments of the hydrophobic domain and of most of the acylation region. We propose a refolding model dependent on calcium and driven by local and distal acylation-dependent interactions within CyaA. Therefore, CyaA acylation is not only critical for cell intoxication, but also for protein refolding into its active conformation. Our data shed light on the complex relationship between post-translational modifications, structural disorder and protein folding. Coupling calcium-binding and acylation-driven folding is likely pertinent for other repeat-in-toxin cytolysins produced by many Gram-negative bacterial pathogens.-O'Brien, D. P., Cannella, S. E., Voegele, A., Raoux-Barbot, D., Davi, M., Douché, T., Matondo, M., Brier, S., Ladant, D., Chenal, A. Post-translational acylation controls the folding and functions of the CyaA RTX toxin.
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post translational acylation controls the folding and functions of the CyaA rtx toxin
The FASEB Journal, 2019Co-Authors: Darragh P Obrien, Sara E Cannella, Alexis Voegele, Dorothee Raouxbarbot, Marilyne Davi, Thibaut Douche, Mariette Matondo, Sebastien Brier, Daniel Ladant, And Alexandre ChenalAbstract:The adenylate cyclase (CyaA) toxin is a major virulence factor of Bordetella pertussis, the causative agent of whooping cough. CyaA is synthetized as a pro-toxin, pro-CyaA, and converted into its c...
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The Adenylate Cyclase (CyaA) Toxin from Bordetella pertussis Has No Detectable Phospholipase A (PLA) Activity In Vitro
MDPI AG, 2019Co-Authors: Alexis Voegele, Thibaut Douche, Mariette Matondo, Daniel Ladant, Dorothée Raoux-barbot, Mirko Sadi, And Alexandre ChenalAbstract:The adenylate cyclase (CyaA) toxin produced in Bordetella pertussis is the causative agent of whooping cough. CyaA exhibits the remarkable capacity to translocate its N-terminal adenyl cyclase domain (ACD) directly across the plasma membrane into the cytosol of eukaryotic cells. Once translocated, calmodulin binds and activates ACD, leading to a burst of cAMP that intoxicates the target cell. Previously, Gonzalez-Bullon et al. reported that CyaA exhibits a phospholipase A activity that could destabilize the membrane to facilitate ACD membrane translocation. However, Bumba and collaborators lately reported that they could not replicate these results. To clarify this controversy, we assayed the putative PLA activity of two CyaA samples purified in two different laboratories by using two distinct fluorescent probes reporting either PLA2 or both PLA1 and PLA2 activities, as well as in various experimental conditions (i.e., neutral or negatively charged membranes in different buffers.) However, we could not detect any PLA activity in these CyaA batches. Thus, our data independently confirm that CyaA does not possess any PLA activity
Chanan Angsuthanasombat - One of the best experts on this subject based on the ideXlab platform.
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preferential modification of CyaA hemolysin by cyac acyltransferase through the catalytic ser30 his33 dyad in esterolysis of palmitoyl donor substrate devoid of acyl carrier proteins
Archives of Biochemistry and Biophysics, 2020Co-Authors: Mattayaus Yentongchai, Chompounoot Imtong, Niramon Thamwiriyasati, Chanan AngsuthanasombatAbstract:Abstract We previously demonstrated that the ∼130-kDa CyaA-hemolysin domain (CyaA-Hly) from Bordetella pertussis co-expressed with CyaC-acyltransferase in Escherichia coli was acylated at Lys983 and thus activated its hemolytic activity. Here, attempts were made to provide greater insights into such toxin activation via fatty-acyl modification by CyaC-acyltransferase. Non-acylated CyaA-Hly (NA/CyaA-Hly) and CyaC were separately expressed in E. coli and subsequently purified by FPLC to near homogeneity. When effects of acyl-chain length were comparatively evaluated through CyaC-esterolysis using various p-nitrophenyl (pNP) derivatives, Michaelis-Menten steady-state kinetic parameters (KM and kcat) of CyaC-acyltransferase revealed a marked preference for myristoyl (C14:0) and palmitoyl (C16:0) substrates of which catalytic efficiencies (kcat/KM) were roughly the same (∼1.5 × 103 s−1mM−1). However, pNP-palmitate (pNPP) gave the highest hemolytic activity of NA/CyaA-Hly after being acylated in vitro with a range of acyl-donor substrates. LC-MS/MS analysis confirmed such CyaC-mediated palmitoylation of CyaA-Hly occurring at Lys983, denoting no requirement of an acyl carrier protein (ACP). A homology-based CyaC structure inferred a role of a potential catalytic dyad of conserved Ser30 and His33 residues in substrate esterolysis. CyaC-ligand binding analysis via molecular docking corroborated high-affinity binding of palmitate with its carboxyl group oriented toward such a dyad. Ala-substitutions of each residue (S30A or H33A) caused a drastic decrease in kcat/KM of CyaC toward pNPP, and hence its catalytic malfunction through palmitoylation-dependent activation of NA/CyaA-Hly. Altogether, our present data evidently provide such preferential palmitoylation of CyaA-Hly by CyaC-acyltransferase through the enzyme Ser30-His33 nucleophile-activation dyad in esterolysis of palmitoyl-donor substrate, particularly devoid of a natural acyl-ACP donor.
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structural requirement of the hydrophobic region of the bordetella pertussis CyaA hemolysin for functional association with cyac acyltransferase in toxin acylation
Biochemical and Biophysical Research Communications, 2018Co-Authors: Veerada Raksanoh, Panchika Prangkio, Chompounoot Imtong, Niramon Thamwiriyasati, Kittipong Suvarnapunya, Lalida Shank, Chanan AngsuthanasombatAbstract:Abstract Previously, we demonstrated that the ∼130-kDa CyaA-hemolysin (CyaA-Hly, Met482-Arg1706) from Bordetella pertussis was palmitoylated at Lys983 when co-expressed with CyaC-acyltransferase in Escherichia coli, and thus activated its hemolytic activity. Here, further investigation on a possible requirement of the N-terminal hydrophobic region (HP, Met482-Leu750) for toxin acylation was performed. The ∼100-kDa RTX (Repeat-in-ToXin) fragment (CyaA-RTX, Ala751-Arg1706) containing the Lys983-acylation region (AR, Ala751-Gln1000), but lacking HP, was co-produced with CyaC in E. coli. Hemolysis assay indicated that CyaA-RTX showed no hemolytic activity. Additionally, MALDI-TOF/MS and LC-MS/MS analyses confirmed that CyaA-RTX was non-acylated, although the co-expressed CyaC-acyltransferase was able to hydrolyze its chromogenic substrate−p-nitrophenyl palmitate and acylate CyaA-Hly to become hemolytically active. Unlike CyaA-RTX, the ∼70-kDa His-tagged CyaA-HP/BI fragment which is hemolytically inactive and contains both HP and AR was constantly co-eluted with CyaC during IMAC-purification as the presence of CyaC was verified by Western blotting. Such potential interactions between the two proteins were also revealed by semi-native PAGE. Moreover, structural analysis via electrostatic potential calculations and molecular docking suggested that CyaA-HP comprising α1-α5 (Leu500-Val698) can interact with CyaC through several hydrogen and ionic bonds formed between their opposite electrostatic surfaces. Overall, our results demonstrated that the HP region of CyaA-Hly is conceivably required for not only membrane-pore formation but also functional association with CyaC-acyltransferase, and hence effective palmitoylation at Lys983.
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Contributions of the Hydrophobic Helix 2 of the Bordetella pertussis CyaA-hemolysin to Membrane Permeabilization.
Protein and peptide letters, 2018Co-Authors: Panchika Prangkio, Sirikran Juntapremjit, Melanie Koehler, Peter Hinterdorfer, Chanan AngsuthanasombatAbstract:BACKGROUND Adenylate cyclase (CyaA) is one of the major virulence factors of Bordetella pertussis that plays a key role in whooping cough pathogenesis. A putative transmembrane helical hairpin (α2-loop-α3), encompassing residues 529-594 of CyaA hemolysin (CyaA-Hly) domain, was previously proposed to be crucially involved in hemolytic activity against target erythrocytes. OBJECTIVE The main objective of this study was to gain more insight into membrane permeabilization of this toxin. Membrane-permeabilizing abilities of the purified 130-kDa CyaA-Hly and synthetic peptides corresponding to the helical component of interest, were evaluated. METHODS Synthetic peptides corresponding to the critical helical component, i.e. α2 (W528-G550), α3 (G568-R594) and α2-loop-α3 (W528-R594), were examined on various membrane models in comparison with the purified 130-kDa CyaA-Hly. The peptides were commercially synthesized and the purified toxin was obtained from recombinant plasmid construction and expression in Escherichia coli, followed by purification via immobilized-metal affinity chromatography. Membrane permeabilization or hemolysis of the peptides or the purified toxin were determined by liposomal leakage, hemolysis assays and atomic force microscopy (AFM) imaging. RESULTS Our results showed that the truncated 130-kDa CyaA-Hly, the synthetic peptides α2, α3 and the α2-loop-α3 hairpin exhibited distinct membrane-permeabilizing capacities in different membrane models. We demonstrated that the CyaA-Hly toxin and the peptides, especially the α2 peptide, caused nonspecific liposomal leakage as monitored by fluorescence dequenching of sulforhodamine B-loaded lipid vesicles. Notably, α2 peptide showed a predominant effect of membrane permeabilization when compared to α2-loop-α3 hairpin and α3 peptides. In addition, AFM imaging demonstrates lipid membrane disruption induced by the CyaA-Hly toxin or the peptidic α2-loop-α3 hairpin. CONCLUSION Overall, the study provides the supporting evidence that the putative helical α2-loop-α3 hairpin could interact with the lipid membranes while the helical α2 peptide strongly induced liposomal leakage and hemolysis, as compared with the helical α3 or the α2-loop-α3 peptides, suggesting that the helix 2 from the hydrophobic region of CyaA-Hly is a crucial component that contributes to membrane permeabilization.
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Zn 2+ -dependent autocatalytic activity of the Bordetella pertussis CyaA-hemolysin
Biochemical and biophysical research communications, 2017Co-Authors: Veerada Raksanoh, Panchika Prangkio, Chompounoot Imtong, Lalida Shank, Mattayaus Yentongchai, Somsri Sakdee, Chanan AngsuthanasombatAbstract:Proteolytic degradation of the ∼100-kDa isolated RTX (Repeat-in-ToXin) subdomain (CyaA-RTX) of the Bordetella pertussis CyaA-hemolysin (CyaA-Hly) was evidently detected upon solely-prolonged incubation. Here, a truncated CyaA-Hly fragment (CyaA-HP/BI) containing hydrophobic and acylation regions connected with the first RTX block (BI1015–1088) was constructed as a putative precursor for investigating its potential autocatalysis. The 70-kDa His-tagged CyaA-HP/BI fragment which was over-expressed in Escherichia coli as insoluble aggregate was entirely solubilized with 4 M urea. After re-naturation in a Ni2+-NTA affinity column, the purified-refolded CyaA-HP/BI fragment in HEPES buffer (pH 7.4) supplemented with 2 mM CaCl2 was completely degraded upon incubation at 37 °C for 3 h. Addition of 1,10-phenanthroline‒an inhibitor of Zn2+-dependent metalloproteases markedly reduced the extent of degradation for CyaA-HP/BI and CyaA-RTX, but the degradative effect was clearly enhanced by addition of 100 mM ZnCl2. Structural analysis of a plausible CyaA-HP/BI model revealed a potential Zn2+-binding His-Asp cluster located between the acylation region and RTX-BI1015–1088. Moreover, Arg997‒one of the identified cleavage sites of the CyaA-RTX fragment was located in close proximity to the Zn2+-binding catalytic site. Overall results demonstrated for the first time that the observed proteolysis of CyaA-HP/BI and CyaA-RTX fragments is conceivably due to their Zn2+-dependent autocatalytic activity.
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Acylation of the Bordetella pertussis CyaA-hemolysin: Functional implications for efficient membrane insertion and pore formation.
Biochimica et biophysica acta. Biomembranes, 2016Co-Authors: Kanungsuk Meetum, Chompounoot Imtong, Gerd Katzenmeier, Chanan AngsuthanasombatAbstract:Abstract Previously, the ~ 130-kDa CyaA-hemolysin domain (CyaA-Hly) from Bordetella pertussis co-expressed with CyaC-acyltransferase in Escherichia coli was demonstrated to be palmitoylated at Lys983 and thus activated its hemolytic activity against target erythrocytes. Here, we report the functional importance of Lys983-palmitoylation for membrane insertion and pore formation of CyaA-Hly. Intrinsic fluorescence emissions of both non-acylated CyaA-Hly (NA/CyaA-Hly) and CyaA-Hly were indistinguishable, suggesting no severe conformational change upon acylation at Lys983. Following pre-incubation of sheep erythrocytes with NA/CyaA-Hly, there was a drastic decrease in CyaA-Hly-induced hemolysis. Direct interactions between NA/CyaA-Hly and target erythrocyte membranes were validated via membrane-binding assays along with Western blotting, suggestive of acylation-independent capability of NA/CyaA-Hly to interact with erythrocyte membranes. As compared with CyaA-Hly, NA/CyaA-Hly displayed a slower rate of incorporation into DOPC:DOPE:Ch or DiPhyPC bilayers under symmetrical conditions (1 M KCl, 10 mM HEPES, pH 7.4) and formed channels exhibiting different conductance. Further analysis revealed that channel-open lifetime in DOPC:DOPE:Ch bilayers of NA/CyaA-Hly was much shorter than that of the acylated form, albeit slightly shorter lifetime found in DiPhyPC bilayers. Sequence alignments of the Lys983-containing CyaA-segment with those of related RTX-cytolysins revealed a number of highly conserved hydrophobic residues and a Lys/Arg cluster that is predicted be important for toxin-membrane interactions. Altogether, our data disclosed that the Lys983-linked palmitoyl group is not directly involved in either binding to target erythrocyte membranes or toxin-induced channel conductivity, but rather required for efficient membrane insertion and pore formation of the acylated CyaA-Hly domain.
