scholarly journals Bioengineering of Bordetella pertussis Adenylate Cyclase Toxin for Vaccine Development and Other Biotechnological Purposes

Toxins ◽  
2021 ◽  
Vol 13 (2) ◽  
pp. 83
Author(s):  
Daniel Ladant
Keyword(s):  
Adenylate Cyclase ◽  
Protein Interactions ◽  
Bordetella Pertussis ◽  
Catalytic Domain ◽  
Vaccine Development ◽  
Whooping Cough ◽  
Bacterial Colonization ◽  
Protein Protein Interactions ◽  
Adenylate Cyclase Toxin ◽  
Antigen Presenting

The adenylate cyclase toxin, CyaA, is one of the key virulent factors produced by Bordetella pertussis, the causative agent of whooping cough. This toxin primarily targets innate immunity to facilitate bacterial colonization of the respiratory tract. CyaA exhibits several remarkable characteristics that have been exploited for various applications in vaccinology and other biotechnological purposes. CyaA has been engineered as a potent vaccine vehicle to deliver antigens into antigen-presenting cells, while the adenylate cyclase catalytic domain has been used to design a robust genetic assay for monitoring protein–protein interactions in bacteria. These two biotechnological applications are briefly summarized in this chapter.

scholarly journals Stimulation of Bordetella pertussisAdenylate Cyclase Toxin Intoxication by Its Hemolysin Domain

2000 ◽  
Vol 68 (6) ◽  
pp. 3727-3730 ◽  
Author(s):  
Masaaki Iwaki ◽  
Kazunari Kamachi ◽  
Toshifumi Konda
Keyword(s):  
Adenylate Cyclase ◽  
Protein Interactions ◽  
Bordetella Pertussis ◽  
Hemolytic Activity ◽  
Cytoplasmic Membrane ◽  
Catalytic Domain ◽  
Protein Protein Interactions ◽  
Adenylate Cyclase Toxin ◽  
Stimulation Of

ABSTRACT The internalization of the N-terminal catalytic domain ofBordetella pertussis adenylate cyclase toxin (ACT) across the cytoplasmic membrane has been considered to occur independently from protein-protein interactions which can lead to oligomerization required for hemolytic activity by its C-terminal hemolysin domain. Here we report that when added in excess, this hemolysin domain stimulates the internalization, suggesting the involvement of protein-protein interactions in cell-invasive activity of ACT, as well as its hemolytic activity.

scholarly journals Bioengineering of Bordetella pertussis Adenylate Cyclase Toxin for Antigen-Delivery and Immunotherapy

Toxins ◽  
2018 ◽  
Vol 10 (7) ◽  
pp. 302 ◽  
Author(s):  
Alexandre Chenal ◽  
Daniel Ladant
Keyword(s):  
Adenylate Cyclase ◽  
Bordetella Pertussis ◽  
Catalytic Domain ◽  
Whooping Cough ◽  
Basic Knowledge ◽  
Target Cells ◽  
Specific Cell ◽  
Antigen Delivery ◽  
Adenylate Cyclase Toxin

The adenylate cyclase toxin (CyaA) is one of the major virulence factors of Bordetella pertussis, the causative agent of whooping cough. CyaA is able to invade eukaryotic cells where, upon activation by endogenous calmodulin, it synthesizes massive amounts of cAMP that alters cellular physiology. The CyaA toxin is a 1706 residues-long bifunctional protein: the catalytic domain is located in the 400 amino-proximal residues, whereas the carboxy-terminal 1306 residues are implicated in toxin binding to the cellular receptor, the αMβ2 (CD11b/CD18) integrin, and subsequently in the translocation of the catalytic domain across the cytoplasmic membrane of the target cells. Indeed, this protein is endowed with the unique capability of delivering its N-terminal catalytic domain directly across the plasma membrane of eukaryotic target cells. These properties have been exploited to engineer the CyaA toxin as a potent non-replicating vector able to deliver antigens into antigen presenting cells and elicit specific cell-mediated immune responses. Antigens of interest can be inserted into the CyaA protein to yield recombinant molecules that are targeted in vivo to dendritic cells, where the antigens are processed and presented by the major class I and class II histocompatibility complexes (MHC-I and II). CyaA turned out to be a remarkably effective and versatile vaccine vector capable of inducing all the components of the immune response (T-CD4, T-CD8, and antibody). In this chapter, we summarize the basic knowledge on the adenylate cyclase toxin and then describe the application of CyaA in vaccinology, including some recent results of clinical trials of immunotherapy using a recombinant CyaA vaccine.

scholarly journals Phospholipase A activity of adenylate cyclase toxin mediates translocation of its adenylate cyclase domain

2017 ◽  
Vol 114 (33) ◽  
pp. E6784-E6793 ◽  
Author(s):  
David González-Bullón ◽  
Kepa B. Uribe ◽  
César Martín ◽  
Helena Ostolaza
Keyword(s):  
Cell Membrane ◽  
Adenylate Cyclase ◽  
Molecular Mechanism ◽  
Catalytic Domain ◽  
Whooping Cough ◽  
Phospholipase A ◽  
Host Cell Membrane ◽  
Adenylate Cyclase Toxin ◽  
Causative Bacterium ◽  
Domain Transfer

Adenylate cyclase toxin (ACT or CyaA) plays a crucial role in respiratory tract colonization and virulence of the whooping cough causative bacteriumBordetella 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.

scholarly journals Adenylate Cyclase Toxin from Bordetella pertussis Synergizes with Lipopolysaccharide To Promote Innate Interleukin-10 Production and Enhances the Induction of Th2 and Regulatory T Cells

2004 ◽  
Vol 72 (3) ◽  
pp. 1568-1579 ◽  
Author(s):  
Pádraig J. Ross ◽  
Ed C. Lavelle ◽  
Kingston H. G. Mills ◽  
Aoife P. Boyd
Keyword(s):  
T Cell ◽  
Adenylate Cyclase ◽  
Bordetella Pertussis ◽  
Bacterial Colonization ◽  
Class Ii ◽  
Cell Surface Expression ◽  
Major Histocompatibility ◽  
Necrosis Factor Alpha ◽  
Adenylate Cyclase Toxin

ABSTRACT Adenylate cyclase toxin (CyaA) from Bordetella pertussis can subvert host immune responses allowing bacterial colonization. Here we have examined its adjuvant and immunomodulatory properties and the possible contribution of lipopolysaccharide (LPS), known to be present in purified CyaA preparations. CyaA enhanced antigen-specific interleukin-5 (IL-5) and IL-10 production and immunoglobulin G1 antibodies to coadministered antigen in vivo. Antigen-specific CD4+-T-cell clones generated from mice immunized with antigen and CyaA had cytokine profiles characteristic of Th2 or type 1 regulatory T (Tr1) cells. Since innate immune cells direct the induction of T-cell subtypes, we examined the influence of CyaA on activation of dendritic cells (DC) and macrophages. CyaA significantly augmented LPS-induced IL-6 and IL-10 and inhibited LPS-driven tumor necrosis factor alpha and IL-12p70 production from bone marrow-derived DC and macrophages. CyaA also enhanced cell surface expression of CD80, CD86, and major histocompatibility class II on immature DC. The stimulatory activity of our CyaA preparation for IL-10 production and CD80, CD86, and major histocompatibility complex class II expression was attenuated following the addition of polymyxin B or with the use of DC from Toll-like receptor (TLR) 4-defective mice. However, treatment of DC with LPS alone at the concentration present in the CyaA preparation (0.2 ng/ml) failed to activate DC in vitro. Our findings demonstrate that activation of innate cells in vitro by CyaA is dependent on a second signal through a TLR and that CyaA can promote Th2/Tr1-cell responses by inhibiting IL-12 and promoting IL-10 production by DC and macrophages.

scholarly journals Cyclic AMP-Elevating Capacity of Adenylate Cyclase Toxin-Hemolysin Is Sufficient for Lung Infection but Not for Full Virulence of Bordetella pertussis

2017 ◽  
Vol 85 (6) ◽  
Author(s):  
Karolina Skopova ◽  
Barbora Tomalova ◽  
Ivan Kanchev ◽  
Pavel Rossmann ◽  
Martina Svedova ◽  
...  
Keyword(s):  
Cyclic Amp ◽  
Adenylate Cyclase ◽  
Bordetella Pertussis ◽  
Whooping Cough ◽  
Lung Parenchyma ◽  
Lung Pathology ◽  
Phagocytic Cells ◽  
Content Type ◽  
Adenylate Cyclase Toxin

ABSTRACT The adenylate cyclase toxin-hemolysin (CyaA, ACT, or AC-Hly) of Bordetella pertussis targets phagocytic cells expressing the complement receptor 3 (CR3, Mac-1, αMβ2 integrin, or CD11b/CD18). CyaA delivers into cells an N-terminal adenylyl cyclase (AC) enzyme domain that is activated by cytosolic calmodulin and catalyzes unregulated conversion of cellular ATP into cyclic AMP (cAMP), a key second messenger subverting bactericidal activities of phagocytes. In parallel, the hemolysin (Hly) moiety of CyaA forms cation-selective hemolytic pores that permeabilize target cell membranes. We constructed the first B. pertussis mutant secreting a CyaA toxin having an intact capacity to deliver the AC enzyme into CD11b-expressing (CD11b+) host phagocytes but impaired in formation of cell-permeabilizing pores and defective in cAMP elevation in CD11b− cells. The nonhemolytic AC+ Hly− bacteria inhibited the antigen-presenting capacities of coincubated mouse dendritic cells in vitro and skewed their Toll-like receptor (TLR)-triggered maturation toward a tolerogenic phenotype. The AC+ Hly− mutant also infected mouse lungs as efficiently as the parental AC+ Hly+ strain. Hence, elevation of cAMP in CD11b− cells and/or the pore-forming capacity of CyaA were not required for infection of mouse airways. The latter activities were, however, involved in bacterial penetration across the epithelial layer, enhanced neutrophil influx into lung parenchyma during sublethal infections, and the exacerbated lung pathology and lethality of B. pertussis infections at higher inoculation doses (>107 CFU/mouse). The pore-forming activity of CyaA further synergized with the cAMP-elevating activity in downregulation of major histocompatibility complex class II (MHC-II) molecules on infiltrating myeloid cells, likely contributing to immune subversion of host defenses by the whooping cough agent.

scholarly journals Role of CD11b/CD18 in the Process of Intoxication by the Adenylate Cyclase Toxin of Bordetella pertussis

2011 ◽  
Vol 80 (2) ◽  
pp. 850-859 ◽  
Author(s):  
Joshua C. Eby ◽  
Mary C. Gray ◽  
Annabelle R. Mangan ◽  
Gina M. Donato ◽  
Erik L. Hewlett
Keyword(s):  
Adenylate Cyclase ◽  
Bordetella Pertussis ◽  
Catalytic Domain ◽  
Chinese Hamster ◽  
K562 Cells ◽  
Cell Types ◽  
Target Cells ◽  
Content Type ◽  
Intracellular Atp ◽  
Adenylate Cyclase Toxin

ABSTRACTThe adenylate cyclase toxin (ACT) ofBordetella pertussisdoes not require a receptor to generate intracellular cyclic AMP (cAMP) in a broad range of cell types. To intoxicate cells, ACT binds to the cell surface, translocates its catalytic domain across the cell membrane, and converts intracellular ATP to cAMP. In cells that express the integrin CD11b/CD18 (CR3), ACT is more potent than in CR3-negative cells. We find, however, that the maximum levels of cAMP accumulation inside CR3-positive and -negative cells are comparable. To better understand how CR3 affects the generation of cAMP, we used Chinese hamster ovary and K562 cells transfected to express CR3 and examined the steps in intoxication in the presence and absence of the integrin. The binding of ACT to cells is greater in CR3-expressing cells at all concentrations of ACT, and translocation of the catalytic domain is enhanced by CR3 expression, with ∼80% of ACT molecules translocating their catalytic domain in CR3-positive cells but only 25% in CR3-negative cells. Once in the cytosol, the unregulated catalytic domain converts ATP to cAMP, and at ACT concentrations >1,000 ng/ml, the intracellular ATP concentration is <5% of that in untreated cells, regardless of CR3 expression. This depletion of ATP prevents further production of cAMP, despite the CR3-mediated enhancement of binding and translocation. In addition to characterizing the effects of CR3 on the actions of ACT, these data show that ATP consumption is yet another concentration-dependent activity of ACT that must be considered when studying how ACT affects target cells.

scholarly journals Characterization of the Intrinsic Phospholipase A1 Activity of Bordetella pertussis Adenylate Cyclase Toxin

Toxins ◽  
2018 ◽  
Vol 10 (12) ◽  
pp. 514 ◽  
Author(s):  
David González-Bullón ◽  
César Martín ◽  
Helena Ostolaza
Keyword(s):  
Adenylate Cyclase ◽  
Bordetella Pertussis ◽  
Whooping Cough ◽  
Hydrolytic Activity ◽  
Catalytic Site ◽  
Phospholipase A1 ◽  
Human Respiratory Tract ◽  
Cell Membrane Permeabilization ◽  
Adenylate Cyclase Toxin ◽  
Catalytic Motif

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.

scholarly journals Membrane Restructuring by Bordetella pertussis Adenylate Cyclase Toxin, a Member of the RTX Toxin Family

2004 ◽  
Vol 186 (12) ◽  
pp. 3760-3765 ◽  
Author(s):  
César Martín ◽  
M.-Asunción Requero ◽  
Jiri Masin ◽  
Ivo Konopasek ◽  
Félix M. Goñi ◽  
...  
Keyword(s):  
Adenylate Cyclase ◽  
Bordetella Pertussis ◽  
Whooping Cough ◽  
Cell Types ◽  
Permeability Barrier ◽  
Dependent Manner ◽  
Flip Flop ◽  
Protein Receptor ◽  
Adenylate Cyclase Toxin ◽  
Lipid Structures

ABSTRACT Adenylate cyclase toxin (ACT) is secreted by Bordetella pertussis, the bacterium causing whooping cough. ACT is a member of the RTX (repeats in toxin) family of toxins, and like other members in the family, it may bind cell membranes and cause disruption of the permeability barrier, leading to efflux of cell contents. The present paper summarizes studies performed on cell and model membranes with the aim of understanding the mechanism of toxin insertion and membrane restructuring leading to release of contents. ACT does not necessarily require a protein receptor to bind the membrane bilayer, and this may explain its broad range of host cell types. In fact, red blood cells and liposomes (large unilamellar vesicles) display similar sensitivities to ACT. A varying liposomal bilayer composition leads to significant changes in ACT-induced membrane lysis, measured as efflux of fluorescent vesicle contents. Phosphatidylethanolamine (PE), a lipid that favors formation of nonlamellar (inverted hexagonal) phases, stimulated ACT-promoted efflux. Conversely, lysophosphatidylcholine, a micelle-forming lipid that opposes the formation of inverted nonlamellar phases, inhibited ACT-induced efflux in a dose-dependent manner and neutralized the stimulatory effect of PE. These results strongly suggest that ACT-induced efflux is mediated by transient inverted nonlamellar lipid structures. Cholesterol, a lipid that favors inverted nonlamellar phase formation and also increases the static order of phospholipid hydrocarbon chains, among other effects, also enhanced ACT-induced liposomal efflux. Moreover, the use of a recently developed fluorescence assay technique allowed the detection of trans-bilayer (flip-flop) lipid motion simultaneous with efflux. Lipid flip-flop further confirms the formation of transient nonlamellar lipid structures as a result of ACT insertion in bilayers.

scholarly journals Functional and structural consequences of epithelial cell invasion by Bordetella pertussis adenylate cyclase toxin

2020 ◽  
Author(s):  
Christelle Angely ◽  
Daniel Ladant ◽  
Emmanuelle Planus ◽  
Bruno Louis ◽  
Marcel Filoche ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Adenylate Cyclase ◽  
Bordetella Pertussis ◽  
Immune Cells ◽  
Catalytic Domain ◽  
Innate Immune ◽  
Alveolar Epithelial Cells ◽  
Innate Immune Cells ◽  
Alveolar Epithelial ◽  
Adenylate Cyclase Toxin

AbstractBordetella 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.

Sign in / Sign up

Export Citation Format

Share Document