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Peter Sebo - One of the best experts on this subject based on the ideXlab platform.
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Simultaneous Determination of Antibodies to Pertussis Toxin and Adenylate Cyclase Toxin Improves Serological Diagnosis of Pertussis.
Diagnostics (Basel Switzerland), 2021Co-Authors: Aapo Knuutila, Radim Osicka, Alex-mikael Barkoff, Jussi Mertsola, Peter SeboAbstract: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.
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Adenylate Cyclase Toxin Tinkering With Monocyte-Macrophage Differentiation
Frontiers in immunology, 2020Co-Authors: Jawid Nazir Ahmad, Peter SeboAbstract: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.
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negative charge of the ac to hly linking segment modulates calcium dependent membrane activities of bordetella Adenylate Cyclase Toxin
Biochimica et Biophysica Acta, 2020Co-Authors: Anna Sukova, Ladislav Bumba, Peter Sebo, Radovan Fiser, Jana Holubova, Pavel Srb, Vaclav Veverka, Ondrej Stanek, Josef Chmelik, Jiri MasinAbstract: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.
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Bordetella Adenylate Cyclase Toxin Inhibits Monocyte-to-Macrophage Transition and Dedifferentiates Human Alveolar Macrophages into Monocyte-like Cells.
mBio, 2019Co-Authors: Jawid Nazir Ahmad, Olga Kofronova, Oldrich Benada, Jana Holubova, Ludek Stehlik, Martina Vasakova, Peter SeboAbstract:ABSTRACT Monocytes arriving at the site of infection differentiate into functional effector macrophages to replenish the resident sentinel cells. Bordetella pertussis, the pertussis agent, secretes an Adenylate Cyclase Toxin-hemolysin (CyaA) that binds myeloid phagocytes through complement receptor 3 (CD11b/CD18) and swiftly delivers its adenylyl Cyclase enzyme domain into phagocytes. This ablates the bactericidal capacities of phagocytes through massive and unregulated conversion of cytosolic ATP into the key signaling molecule cAMP. We show that exposure of primary human monocytes to as low a concentration as 22.5 pM CyaA, or a low (2:1) multiplicity of infection by CyaA-producing B. pertussis bacteria, blocks macrophage colony-stimulating factor (M-CSF)-driven differentiation of monocytes. CyaA-induced cAMP signaling mediated through the activity of protein kinase A (PKA) efficiently blocked expression of macrophage markers, and the monocytes exposed to 22.5 pM CyaA failed to acquire the characteristic intracellular complexity of mature macrophage cells. Neither M-CSF-induced endoplasmic reticulum (ER) expansion nor accumulation of Golgi bodies, mitochondria, or lysosomes was observed in Toxin-exposed monocytes, which remained small and poorly phagocytic and lacked pseudopodia. Exposure to 22.5 pM CyaA Toxin provoked loss of macrophage marker expression on in vitro differentiated macrophages, as well as on primary human alveolar macrophages, which appeared to dedifferentiate into monocyte-like cells with upregulated CD14 levels. This is the first report that terminally differentiated tissue-resident macrophage cells can be dedifferentiated in vitro. The results suggest that blocking of monocyte-to-macrophage transition and/or dedifferentiation of the sentinel cells of innate immunity through cAMP-elevating Toxin action may represent a novel immune evasion strategy of bacterial pathogens. IMPORTANCE Macrophages are key sentinel cells of the immune system, and, as such, they are targeted by the Toxins produced by the pertussis agent Bordetella pertussis. The Adenylate Cyclase Toxin (CyaA) mediates immune evasion of B. pertussis by suspending the bactericidal activities of myeloid phagocytes. We reveal a novel mechanism of potential subversion of host immunity, where CyaA at very low (22 pM) concentrations could inhibit maturation of human monocyte precursors into the more phagocytic macrophage cells. Furthermore, exposure to low CyaA amounts has been shown to trigger dedifferentiation of mature primary human alveolar macrophages back into monocyte-like cells. This unprecedented capacity is likely to promote survival of the pathogen in the airways, both by preventing maturation of monocytes attracted to the site of infection into phagocytic macrophages and by dedifferentiation of the already airway-resident sentinel cells.
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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.
Erik L. Hewlett - One of the best experts on this subject based on the ideXlab platform.
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Albumin, in the Presence of Calcium, Elicits a Massive Increase in Extracellular Bordetella Adenylate Cyclase Toxin.
Infection and immunity, 2017Co-Authors: Laura A. Gonyar, Mary C. Gray, Gregory J. Christianson, Borna Mehrad, Erik L. HewlettAbstract: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.
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Fine Epitope Mapping of Two Antibodies Neutralizing the Bordetella Adenylate Cyclase Toxin
Biochemistry, 2017Co-Authors: Xianzhe Wang, Erik L. Hewlett, James A. Stapleton, Justin R. Klesmith, Timothy A. Whitehead, Jennifer A. MaynardAbstract: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...
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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, 2017Co-Authors: Joshua C. Eby, Mary C. Gray, Jason M. Warfel, Tod J. Merkel, Erik L. HewlettAbstract: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.
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Bordetella Adenylate Cyclase Toxin interacts with filamentous haemagglutinin to inhibit biofilm formation in vitro.
Molecular microbiology, 2016Co-Authors: Casandra L. Hoffman, Mary C. Gray, Peggy A. Cotter, Joshua C. Eby, F. Heath Damron, Jeffrey A. Melvin, Erik L. HewlettAbstract:Summary Bordetella pertussis, the causative agent of whooping cough, secretes and releases Adenylate Cyclase Toxin (ACT), which is a protein bacterial Toxin that targets host cells and disarms immune defenses. ACT binds filamentous haemagglutinin (FHA), a surface-displayed adhesin, and until now, the consequences of this interaction were unknown. A B. bronchiseptica mutant lacking ACT produced more biofilm than the parental strain; leading Irie et al. to propose the ACT-FHA interaction could be responsible for biofilm inhibition. Here we characterize the physical interaction of ACT with FHA and provide evidence linking that interaction to inhibition of biofilm in vitro. Exogenous ACT inhibits biofilm formation in a concentration-dependent manner and the N-terminal catalytic domain of ACT (AC domain) is necessary and sufficient for this inhibitory effect. AC Domain interacts with the C-terminal segment of FHA with ∼650 nM affinity. ACT does not inhibit biofilm formation by Bordetella lacking the mature C-terminal domain (MCD), suggesting the direct interaction between AC domain and the MCD is required for the inhibitory effect. Additionally, AC domain disrupts preformed biofilm on abiotic surfaces. The demonstrated inhibition of biofilm formation by a host-directed protein bacterial Toxin represents a novel regulatory mechanism and identifies an unprecedented role for ACT.
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Cyclic AMP-Mediated Suppression of Neutrophil Extracellular Trap Formation and Apoptosis by the Bordetella pertussis Adenylate Cyclase Toxin
Infection and immunity, 2014Co-Authors: Joshua C. Eby, Mary C. Gray, Erik L. HewlettAbstract:ABSTRACT The Adenylate Cyclase Toxin (ACT) of Bordetella pertussis intoxicates target cells by generating supraphysiologic levels of intracellular cyclic AMP (cAMP). Since ACT kills macrophages rapidly and potently, we asked whether ACT would also kill neutrophils. In fact, ACT prolongs the neutrophil life span by inhibiting constitutive apoptosis and preventing apoptosis induced by exposure to live B. pertussis. Imaging of B. pertussis-exposed neutrophils revealed that B. pertussis lacking ACT induces formation of neutrophil extracellular traps (NETs), whereas wild-type B. pertussis does not, suggesting that ACT suppresses NET formation. Indeed, ACT inhibits formation of NETs by generating cAMP and consequently inhibiting the oxidative burst. Convalescent-phase serum from humans following clinical pertussis blocks the ACT-mediated suppression of NET formation. These studies provide novel insight into the phagocyte impotence caused by ACT, which not only impairs neutrophil function but also inhibits death of neutrophils by apoptosis and NETosis.
Radim Osicka - One of the best experts on this subject based on the ideXlab platform.
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Simultaneous Determination of Antibodies to Pertussis Toxin and Adenylate Cyclase Toxin Improves Serological Diagnosis of Pertussis.
Diagnostics (Basel Switzerland), 2021Co-Authors: Aapo Knuutila, Radim Osicka, Alex-mikael Barkoff, Jussi Mertsola, Peter SeboAbstract: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.
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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 Disrupts Functional Integrity of Bronchial Epithelial Layers.
Infection and immunity, 2017Co-Authors: Shakir Hasan, Peter Sebo, Radim Osicka, Irena Linhartova, Nikhil Nitin Kulkarni, Arni Asbjarnarson, Gudmundur H. GudmundssonAbstract: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 Toxin-hemolysin (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.
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Structure-Function Relationships Underlying the Capacity of Bordetella Adenylate Cyclase Toxin to Disarm Host Phagocytes
Toxins, 2017Co-Authors: Jakub Novák, Ladislav Bumba, Jiri Masin, Peter Sebo, Adriana Osickova, Irena Linhartova, Ondrej Cerny, Radim OsickaAbstract:Bordetellae, pathogenic to mammals, produce an immunomodulatory Adenylate Cyclase Toxin–hemolysin (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 adenosine monophosphate (cAMP) subverts the physiology of phagocytic cells.
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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.
Jiri Masin - One of the best experts on this subject based on the ideXlab platform.
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negative charge of the ac to hly linking segment modulates calcium dependent membrane activities of bordetella Adenylate Cyclase Toxin
Biochimica et Biophysica Acta, 2020Co-Authors: Anna Sukova, Ladislav Bumba, Peter Sebo, Radovan Fiser, Jana Holubova, Pavel Srb, Vaclav Veverka, Ondrej Stanek, Josef Chmelik, Jiri MasinAbstract: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.
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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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Structure-Function Relationships Underlying the Capacity of Bordetella Adenylate Cyclase Toxin to Disarm Host Phagocytes
Toxins, 2017Co-Authors: Jakub Novák, Ladislav Bumba, Jiri Masin, Peter Sebo, Adriana Osickova, Irena Linhartova, Ondrej Cerny, Radim OsickaAbstract:Bordetellae, pathogenic to mammals, produce an immunomodulatory Adenylate Cyclase Toxin–hemolysin (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 adenosine monophosphate (cAMP) subverts the physiology of phagocytic cells.
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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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Transmembrane segments of complement receptor 3 do not participate in cytotoxic activities but determine receptor structure required for action of Bordetella Adenylate Cyclase Toxin.
Pathogens and disease, 2016Co-Authors: Tomas Wald, Jiri Masin, Peter Sebo, Adriana Osickova, Petra Liskova, Inga Petry-podgorska, Tomáš Matoušek, Radim OsickaAbstract:Adenylate Cyclase Toxin-hemolysin (CyaA, ACT or AC-Hly) of the whooping cough agent Bordetella pertussis penetrates phagocytes expressing the integrin complement receptor 3 (CR3, CD11b/CD18, αMβ2 or Mac-1). CyaA translocates its Adenylate Cyclase (AC) enzyme domain into cell cytosol and catalyzes unregulated conversion of ATP to cAMP, thereby subverting cellular signaling. In parallel, CyaA forms small cation-selective membrane pores that permeabilize cells for potassium efflux, contributing to cytotoxicity of CyaA and eventually provoking colloid-osmotic cell lysis. To investigate whether the single-pass α-helical transmembrane segments of CR3 subunits CD11b and CD18 do directly participate in AC domain translocation and/or pore formation by the Toxin, we expressed in CHO cells variants of CR3 that contained artificial transmembrane segments, or lacked the transmembrane segment(s) at all. The results demonstrate that the transmembrane segments of CR3 are not directly involved in the cytotoxic activities of CyaA but serve for maintaining CR3 in a conformation that is required for efficient Toxin binding and action.
Helena Ostolaza - One of the best experts on this subject based on the ideXlab platform.
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Irreversible versus repairable membrane poration: differences in permeabilization elicited by Bordetella Adenylate Cyclase Toxin and its hemolysin domain in macrophages
The FEBS journal, 2019Co-Authors: Asier Etxaniz, David González-bullón, Cesar Martin, Maria Teresa Alonso, Helena OstolazaAbstract:Rapid plasma membrane repair in response to pore-forming Toxins is crucial for cell survival, but the molecular mechanisms employed by eukaryotic nucleated cells to maintain membrane integrity and the specificities of such pathways remain poorly understood. Here, we have explored the permeabilization elicited by the Bordetella pertussis Adenylate Cyclase Toxin, a 200-kDa protein Toxin with α-helical pore-forming domain that forms pores of tunable size, and evaluated the response of target macrophages to such Toxin poration. We show here that the response and the fate of target macrophages depend on Toxin pore width. We find that the Toxin's hemolysin moiety induces a transient membrane permeabilization by forming wide enough pores allowing Ca2+ influx into the target cell cytosol. This activates a Ca2+ -dependent cellular response involving exocytosis and endocytosis steps eliminating Toxin pores and restoring membrane integrity. In contrast, the full-length native Toxin, at low concentrations, forms very small pores that cause insidious perturbation of cell ion homeostasis that escapes control by the macrophage membrane repair response, eventually leading to cell death. Our data reveal that permeability to Ca2+ and ATP are key elements in the membrane repair pathway for eliminating α-helical pores of bacterial origin.
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Characterization of the Intrinsic Phospholipase A1 Activity of Bordetella pertussis Adenylate Cyclase Toxin.
Toxins, 2018Co-Authors: David González-bullón, Cesar Martin, Helena OstolazaAbstract:Adenylate Cyclase Toxin (ACT, CyaA) is one of the important virulence factors secreted by the whooping cough bacterium Bordetella pertussis, and it is essential for the colonization of the human respiratory tract by this bacterium. Cytotoxicity by ACT results from the synergy between Toxin’s two main activities, production of supraphysiological cAMP levels by its N-terminal Adenylate Cyclase domain (AC domain), and cell membrane permeabilization, induced by its C-terminal pore-forming domain (hemolysin domain), which debilitate the host defenses. In a previous study we discovered that purified ACT is endowed with intrinsic phospholipase A1 (PLA) activity and that Ser in position 606 of the ACT polypeptide is a catalytic site for such hydrolytic activity, as part of G-X-S-X-G catalytic motif. Recently these findings and our conclusions have been directly questioned by other authors who claim that ACT-PLA activity does not exist. Here we provide new data on ACT phospholipase A1 characteristics. Based on our results we reaffirm our previous conclusions that ACT is endowed with PLA activity; that our purified ACT preparations are devoid of any impurity with phospholipase A activity; that ACT-S606A is a PLA-inactive mutant and thus, that Ser606 is a catalytic site for the Toxin hydrolytic activity on phospholipids, and that ACT-PLA activity is involved in AC translocation.
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Understanding the Mechanism of Translocation of Adenylate Cyclase Toxin across Biological Membranes.
Toxins, 2017Co-Authors: Helena Ostolaza, Cesar Martin, Kepa B. Uribe, David González-bullón, Asier EtxanizAbstract:Adenylate Cyclase Toxin (ACT) is one of the principal virulence factors secreted by the whooping cough causative bacterium Bordetella pertussis, and it has a critical role in colonization of the respiratory tract and establishment of the disease. ACT targets phagocytes via binding to the CD11b/CD18 integrin and delivers its N-terminal Adenylate Cyclase (AC) domain directly to the cell cytosol, where it catalyzes unregulated conversion of cytosolic ATP into cAMP upon activation by binding to cellular calmodulin. High cAMP levels disrupt bactericidal functions of the immune cells, ultimately leading to cell death. In spite of its relevance in the ACT biology, the mechanism by which its ≈400 amino acid-long AC domain is transported through the target plasma membrane, and is released into the target cytosol, remains enigmatic. This article is devoted to refresh our knowledge on the mechanism of AC translocation across biological membranes. Two models, the so-called “two-step model” and the recently-proposed “toroidal pore model”, will be considered.
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Phospholipase A activity of Adenylate Cyclase Toxin mediates translocation of its Adenylate Cyclase domain
Proceedings of the National Academy of Sciences of the United States of America, 2017Co-Authors: David González-bullón, Kepa B. Uribe, Cesar Martin, Helena OstolazaAbstract:Adenylate Cyclase Toxin (ACT or CyaA) plays a crucial role in respiratory tract colonization and virulence of the whooping cough causative bacterium Bordetella pertussis . Secreted as soluble protein, it targets myeloid cells expressing the CD11b/CD18 integrin and on delivery of its N-terminal Adenylate Cyclase catalytic domain (AC domain) into the cytosol, generates uncontrolled toxic levels of cAMP that ablates bactericidal capacities of phagocytes. Our study deciphers the fundamentals of the heretofore poorly understood molecular mechanism by which the ACT enzyme domain directly crosses the host cell membrane. By combining molecular biology, biochemistry, and biophysics techniques, we discover that ACT has intrinsic phospholipase A (PLA) activity, and that such activity determines AC translocation. Moreover, we show that elimination of the ACT–PLA activity abrogates ACT toxicity in macrophages, particularly at Toxin concentrations close to biological reality of bacterial infection. Our data support a molecular mechanism in which in situ generation of nonlamellar lysophospholipids by ACT–PLA activity into the cell membrane would form, likely in combination with membrane-interacting ACT segments, a proteolipidic toroidal pore through which AC domain transfer could directly take place. Regulation of ACT–PLA activity thus emerges as novel target for therapeutic control of the disease.
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Adenylate Cyclase Toxin promotes bacterial internalisation into non phagocytic cells.
Scientific reports, 2015Co-Authors: Cesar Martin, Felix M Goni, Kepa B. Uribe, David González-bullón, Asier Etxaniz, Aitor Etxebarria, Jon Arlucea, Juan Aréchaga, Helena OstolazaAbstract:Bordetella pertussis causes whooping cough, a respiratory infectious disease that is the fifth largest cause of vaccine-preventable death in infants. Though historically considered an extracellular pathogen, this bacterium has been detected both in vitro and in vivo inside phagocytic and non-phagocytic cells. However the precise mechanism used by B. pertussis for cell entry, or the putative bacterial factors involved, are not fully elucidated. Here we find that Adenylate Cyclase Toxin (ACT), one of the important Toxins of B. pertussis, is sufficient to promote bacterial internalisation into non-phagocytic cells. After characterization of the entry route we show that uptake of “Toxin-coated bacteria” proceeds via a clathrin-independent, caveolae-dependent entry pathway, allowing the internalised bacteria to survive within the cells. Intracellular bacteria were found inside non-acidic endosomes with high sphingomyelin and cholesterol content, or “free” in the cytosol of the invaded cells, suggesting that the ACT-induced bacterial uptake may not proceed through formation of late endolysosomes. Activation of Tyr kinases and Toxin-induced Ca2+-influx are essential for the entry process. We hypothesize that B. pertussis might use ACT to activate the endocytic machinery of non-phagocytic cells and gain entry into these cells, in this way evading the host immune system.
