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Adenylate Cyclase Toxin

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Peter Sebo – One of the best experts on this subject based on the ideXlab platform.

  • Simultaneous Determination of Antibodies to Pertussis Toxin and Adenylate Cyclase Toxin Improves Serological Diagnosis of Pertussis.
    Diagnostics (Basel Switzerland), 2021
    Co-Authors: Aapo Knuutila, Radim Osicka, Alex-mikael Barkoff, Jussi Mertsola, Peter Sebo
    Abstract:

    Serological diagnosis of pertussis is mainly based on anti-pertussis Toxin (PT) IgG antibodies. Since PT is included in all acellular vaccines (ACV), serological assays do not differentiate antibodies induced by ACVs and infection. Adenylate Cyclase Toxin (ACT) is not included in the ACVs, which makes it a promising candidate for pertussis serology with the specific aim of separating infection- and ACV-induced antibodies. A multiplex lateral flow test with PT and ACT antigens was developed to measure serum antibodies from pertussis-seropositive patients (n = 46), healthy controls (n = 102), and subjects who received a booster dose of ACV containing PT, filamentous hemagglutinin, and pertactin (n = 67) with paired sera collected before and one month after the vaccination. If the diagnosis was solely based on anti-PT antibodies, 98.5–44.8% specificity (before and after vaccination, respectively) and 78.2% sensitivity were achieved, whereas if ACT was used in combination with PT, the sensitivity of the assay increased to 91.3% without compromising specificity. No increase in the level of anti-ACT antibodies was found after vaccination. This exploratory study indicates that the use of ACT for serology would be beneficial in combination with a lower quantitative cutoff for anti-PT antibodies, and particularly in children and adolescents who frequently receive booster vaccinations.

  • Adenylate Cyclase Toxin Tinkering With Monocyte-Macrophage Differentiation
    Frontiers in immunology, 2020
    Co-Authors: Jawid Nazir Ahmad, Peter Sebo
    Abstract:

    Circulating inflammatory monocytes are attracted to infected mucosa and differentiate into macrophage or dendritic cells endowed with enhanced bactericidal and antigen presenting capacities. In this brief Perspective we discuss the newly emerging insight into how the cAMP signaling capacity of Bordetella pertussis Adenylate Cyclase Toxin manipulates the differentiation of monocytes and trigger dedifferentiation of the alveolar macrophages to facilitate bacterial colonization of human airways.

  • negative charge of the ac to hly linking segment modulates calcium dependent membrane activities of bordetella Adenylate Cyclase Toxin
    Biochimica et Biophysica Acta, 2020
    Co-Authors: Anna Sukova, Peter Sebo, Ladislav Bumba, Radovan Fiser, Jana Holubova, Pavel Srb, Vaclav Veverka, Ondrej Stanek, Josef Chmelik, Jiri Masin
    Abstract:

    Abstract Two distinct conformers of the Adenylate Cyclase Toxin (CyaA) appear to accomplish its two parallel activities within target cell membrane. The translocating conformer would deliver the N-terminal adenylyl Cyclase (AC) enzyme domain across plasma membrane into cytosol of cells, while the pore precursor conformer would assemble into oligomeric cation-selective pores and permeabilize cellular membrane. Both Toxin activities then involve a membrane-interacting ‘AC-to-Hly-linking segment’ (residues 400 to 500). Here, we report the NMR structure of the corresponding CyaA411–490 polypeptide in dodecylphosphocholine micelles and show that it consists of two α-helices linked by an unrestrained loop. The N-terminal α-helix (Gly418 to His439) remained solvent accessible, while the C-terminal α-helix (His457 to Phe485) was fully enclosed within detergent micelles. CyaA411–490 weakly bound Ca2+ ions (apparent KD 2.6 mM) and permeabilized negatively charged lipid vesicles. At high concentrations (10 μM) the CyaA411–490 polypeptide formed stable conductance units in artificial lipid bilayers with applied voltage, suggesting its possible transmembrane orientation in the membrane-inserted Toxin. Mutagenesis revealed that two clusters of negatively charged residues within the ‘AC-to-Hly-linking segment’ (Glu419 to Glu432 and Asp445 to Glu448) regulate the balance between the AC domain translocating and pore-forming capacities of CyaA in function of calcium concentration.

Erik L. Hewlett – One of the best experts on this subject based on the ideXlab platform.

  • Albumin, in the Presence of Calcium, Elicits a Massive Increase in Extracellular Bordetella Adenylate Cyclase Toxin.
    Infection and immunity, 2017
    Co-Authors: Laura A. Gonyar, Mary C. Gray, Gregory J. Christianson, Borna Mehrad, Erik L. Hewlett
    Abstract:

    ABSTRACT Pertussis (whooping cough), caused by Bordetella pertussis, is resurging in the United States and worldwide. Adenylate Cyclase Toxin (ACT) is a critical factor in establishing infection with B. pertussis and acts by specifically inhibiting the response of myeloid leukocytes to the pathogen. We report here that serum components, as discovered during growth in fetal bovine serum (FBS), elicit a robust increase in the amount of ACT, and ≥90% of this ACT is localized to the supernatant, unlike growth without FBS, in which ≥90% is associated with the bacterium. We have found that albumin, in the presence of physiological concentrations of calcium, acts specifically to enhance the amount of ACT and its localization to the supernatant. Respiratory secretions, which contain albumin, promote an increase in amount and localization of active ACT that is comparable to that elicited by serum and albumin. The response to albumin is not mediated through regulation of ACT at the transcriptional level or activation of the Bvg two-component system. As further illustration of the specificity of this phenomenon, serum collected from mice that lack albumin does not stimulate an increase in ACT. These data, demonstrating that albumin and calcium act synergistically in the host environment to increase production and release of ACT, strongly suggest that this phenomenon reflects a novel host-pathogen interaction that is central to infection with B. pertussis and other Bordetella species.

  • Fine Epitope Mapping of Two Antibodies Neutralizing the Bordetella Adenylate Cyclase Toxin
    Biochemistry, 2017
    Co-Authors: Xianzhe Wang, Erik L. Hewlett, James A. Stapleton, Justin R. Klesmith, Timothy A. Whitehead, Jennifer A. Maynard
    Abstract:

    Adenylate Cyclase Toxin (ACT) is an important Bordetella pertussis virulence factor that is not included in current acellular pertussis vaccines. We previously demonstrated that immunization with the repeat-in-Toxin (RTX) domain of ACT elicits neutralizing antibodies in mice and discovered the first two antibodies to neutralize ACT activities by occluding the receptor-binding site. Here, we fully characterize these antibodies and their epitopes. Both antibodies bind ACT with low nanomolar affinity and cross-react with ACT homologues produced by B. parapertussis and B. bronchiseptica. Antibody M1H5 binds B. pertussis RTX751 ∼100-fold tighter than RTX751 from the other two species, while antibody M2B10 has similar affinity for all three variants. To initially map the antibody epitopes, we generated a series of ACT chimeras and truncation variants, which implicated the repeat blocks II–III. To identify individual epitope residues, we displayed randomly mutated RTX751 libraries on yeast and isolated clones wi…

  • Use of a Toxin neutralization assay to characterize the serologic response to Adenylate Cyclase Toxin after infection with Bordetella pertussis
    Clinical and vaccine immunology : CVI, 2017
    Co-Authors: Joshua C. Eby, Mary C. Gray, Tod J. Merkel, Jason M. Warfel, Erik L. Hewlett
    Abstract:

    Adenylate Cyclase Toxin (ACT) is an essential virulence factor of Bordetella pertussis, and antibodies to ACT protect against B. pertussis infection in mice. The Toxin is therefore a strong candidate antigen for addition to future acellular pertussis vaccines. In order to characterize the functionality of the immunologic response to ACT after infection, we developed an assay for testing the ability of serum samples from subjects infected with B. pertussis to neutralize ACT-induced cytotoxicity in J774 macrophage cells. Baboons develop neutralizing anti-ACT antibodies following infection with B. pertussis, and all sera from baboons with positive anti-ACT IgG enzyme-linked immunosorbent assay (ELISA) results neutralized ACT cytotoxicity. The Toxin neutralization assay (TNA) was positive in some baboon sera in which ELISA remained negative. Of serum samples obtained from humans diagnosed with pertussis by PCR, anti-ACT IgG ELISA was positive in 72%, and TNA was positive in 83%. All samples positive for anti-ACT IgG ELISA were positive by TNA, and none of the samples from humans without pertussis neutralized Toxin activity. These findings indicate that antibodies to ACT generated following infection with B. pertussis consistently neutralize Toxin-induced cytotoxicity and that TNA can be used to improve understanding of the immunologic response to ACT after infection or vaccination.

Radim Osicka – One of the best experts on this subject based on the ideXlab platform.

  • Simultaneous Determination of Antibodies to Pertussis Toxin and Adenylate Cyclase Toxin Improves Serological Diagnosis of Pertussis.
    Diagnostics (Basel Switzerland), 2021
    Co-Authors: Aapo Knuutila, Radim Osicka, Alex-mikael Barkoff, Jussi Mertsola, Peter Sebo
    Abstract:

    Serological diagnosis of pertussis is mainly based on anti-pertussis Toxin (PT) IgG antibodies. Since PT is included in all acellular vaccines (ACV), serological assays do not differentiate antibodies induced by ACVs and infection. Adenylate Cyclase Toxin (ACT) is not included in the ACVs, which makes it a promising candidate for pertussis serology with the specific aim of separating infection- and ACV-induced antibodies. A multiplex lateral flow test with PT and ACT antigens was developed to measure serum antibodies from pertussis-seropositive patients (n = 46), healthy controls (n = 102), and subjects who received a booster dose of ACV containing PT, filamentous hemagglutinin, and pertactin (n = 67) with paired sera collected before and one month after the vaccination. If the diagnosis was solely based on anti-PT antibodies, 98.5–44.8% specificity (before and after vaccination, respectively) and 78.2% sensitivity were achieved, whereas if ACT was used in combination with PT, the sensitivity of the assay increased to 91.3% without compromising specificity. No increase in the level of anti-ACT antibodies was found after vaccination. This exploratory study indicates that the use of ACT for serology would be beneficial in combination with a lower quantitative cutoff for anti-PT antibodies, and particularly in children and adolescents who frequently receive booster vaccinations.

  • Residues 529 to 549 participate in membrane penetration and pore-forming activity of the Bordetella Adenylate Cyclase Toxin.
    Scientific reports, 2019
    Co-Authors: Jana Roderova, Radim Osicka, Peter Sebo, Adriana Osickova, Radovan Fiser, Anna Sukova, Gabriela Mikušová, Jiri Masin
    Abstract:

    The Adenylate Cyclase Toxinhemolysin (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 resiresidue 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.

  • Bordetella pertussis Adenylate Cyclase Toxin Disrupts Functional Integrity of Bronchial Epithelial Layers.
    Infection and immunity, 2017
    Co-Authors: Shakir Hasan, Radim Osicka, Peter Sebo, Irena Linhartova, Nikhil Nitin Kulkarni, Arni Asbjarnarson, Gudmundur H. Gudmundsson
    Abstract:

    The airway epithelium restricts the penetration of inhaled pathogens into the underlying tissue and plays a crucial role in the innate immune defense against respiratory infections. The whooping cough agent, Bordetella pertussis, adheres to ciliated cells of the human airway epithelium and subverts its defense functions through the action of secreted Toxins and other virulence factors. We examined the impact of B. pertussis infection and of Adenylate Cyclase Toxinhemolysin (CyaA) action on the functional integrity of human bronchial epithelial cells cultured at the air-liquid interface (ALI). B. pertussis adhesion to the apical surface of polarized pseudostratified VA10 cell layers provoked a disruption of tight junctions and caused a drop in transepithelial electrical resistance (TEER). The reduction of TEER depended on the capacity of the secreted CyaA Toxin to elicit cAMP signaling in epithelial cells through its adenylyl Cyclase enzyme activity. Both purified CyaA and cAMP-signaling drugs triggered a decrease in the TEER of VA10 cell layers. Toxin-produced cAMP signaling caused actin cytoskeleton rearrangement and induced mucin 5AC production and interleukin-6 (IL-6) secretion, while it inhibited the IL-17A-induced secretion of the IL-8 chemokine and of the antimicrobial peptide beta-defensin 2. These results indicate that CyaA Toxin activity compromises the barrier and innate immune functions of Bordetella-infected airway epithelia.

Jiri Masin – One of the best experts on this subject based on the ideXlab platform.

  • negative charge of the ac to hly linking segment modulates calcium dependent membrane activities of bordetella Adenylate Cyclase Toxin
    Biochimica et Biophysica Acta, 2020
    Co-Authors: Anna Sukova, Peter Sebo, Ladislav Bumba, Radovan Fiser, Jana Holubova, Pavel Srb, Vaclav Veverka, Ondrej Stanek, Josef Chmelik, Jiri Masin
    Abstract:

    Abstract Two distinct conformers of the Adenylate Cyclase Toxin (CyaA) appear to accomplish its two parallel activities within target cell membrane. The translocating conformer would deliver the N-terminal adenylyl Cyclase (AC) enzyme domain across plasma membrane into cytosol of cells, while the pore precursor conformer would assemble into oligomeric cation-selective pores and permeabilize cellular membrane. Both Toxin activities then involve a membrane-interacting ‘AC-to-Hly-linking segment’ (residues 400 to 500). Here, we report the NMR structure of the corresponding CyaA411–490 polypeptide in dodecylphosphocholine micelles and show that it consists of two α-helices linked by an unrestrained loop. The N-terminal α-helix (Gly418 to His439) remained solvent accessible, while the C-terminal α-helix (His457 to Phe485) was fully enclosed within detergent micelles. CyaA411–490 weakly bound Ca2+ ions (apparent KD 2.6 mM) and permeabilized negatively charged lipid vesicles. At high concentrations (10 μM) the CyaA411–490 polypeptide formed stable conductance units in artificial lipid bilayers with applied voltage, suggesting its possible transmembrane orientation in the membrane-inserted Toxin. Mutagenesis revealed that two clusters of negatively charged residues within the ‘AC-to-Hly-linking segment’ (Glu419 to Glu432 and Asp445 to Glu448) regulate the balance between the AC domain translocating and pore-forming capacities of CyaA in function of calcium concentration.

  • Residues 529 to 549 participate in membrane penetration and pore-forming activity of the Bordetella Adenylate Cyclase Toxin.
    Scientific reports, 2019
    Co-Authors: Jana Roderova, Radim Osicka, Peter Sebo, Adriana Osickova, Radovan Fiser, Anna Sukova, Gabriela Mikušová, Jiri Masin
    Abstract:

    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.

  • Structure-Function Relationships Underlying the Capacity of Bordetella Adenylate Cyclase Toxin to Disarm Host Phagocytes
    Toxins, 2017
    Co-Authors: Jakub Novák, Peter Sebo, Adriana Osickova, Ladislav Bumba, Jiri Masin, Irena Linhartova, Ondrej Cerny, Radim Osicka
    Abstract:

    Bordetellae, pathogenic to mammals, produce an immunomodulatory Adenylate Cyclase Toxinhemolysin (CyaA, ACT or AC-Hly) that enables them to overcome the innate immune defense of the host. CyaA subverts host phagocytic cells by an orchestrated action of its functional domains, where an extremely catalytically active adenylyl Cyclase enzyme is delivered into phagocyte cytosol by a pore-forming repeat-in-Toxin (RTX) cytolysin moiety. By targeting sentinel cells expressing the complement receptor 3, known as the CD11b/CD18 (αMβ2) integrin, CyaA compromises the bactericidal functions of host phagocytes and supports infection of host airways by Bordetellae. Here, we review the state of knowledge on structural and functional aspects of CyaA Toxin action, placing particular emphasis on signaling mechanisms by which the Toxin-produced 3′,5′-cyclic adenadenosineophosphate (cAMP) subverts the physiology of phagocytic cells.

Helena Ostolaza – One of the best experts on this subject based on the ideXlab platform.