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 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 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 Pore-Forming and Enzymatic Activities of Bordetella pertussis Adenylate Cyclase Toxin Synergize in Promoting Lysis of Monocytes

2006 ◽  
Vol 74 (4) ◽  
pp. 2207-2214 ◽  
Author(s):  
Marek Basler ◽  
Jiri Masin ◽  
Radim Osicka ◽  
Peter Sebo
Keyword(s):  
Cyclic Amp ◽  
Adenylate Cyclase ◽  
Bordetella Pertussis ◽  
Hemolytic Activity ◽  
Enzymatic Activities ◽  
Membrane Pores ◽  
Threefold Increase ◽  
Adenylate Cyclase Toxin ◽  
Cytolytic Action

ABSTRACT Bordetella adenylate cyclase (AC) toxin-hemolysin (CyaA) targets myeloid phagocytes expressing the αMβ2 integrin (CD11b/CD18) and delivers into their cytosol an AC enzyme that converts ATP into cyclic AMP (cAMP). In parallel, CyaA acts as a hemolysin, forming small membrane pores. Using specific mutations, we dissected the contributions of the two activities to cytolytic potency of CyaA on J774A.1 murine monocytes. The capacity of AC to penetrate cells and deplete cytosolic ATP was essential for promoting lysis and the enzymatically inactive but fully hemolytic CyaA-AC− toxoid exhibited a 15-fold-lower cytolytic capacity on J774A.1 cells than intact CyaA. Moreover, a two- or fourfold drop of specific hemolytic activity of the CyaA-E570Q and CyaA-E581P mutants was overpowered by an intact capacity to dissipate cytosolic ATP into cAMP, allowing the less hemolytic proteins to promote lysis of J774A.1 cells as efficiently as intact CyaA. However, an increased hemolytic activity, due to lysine substitutions of glutamates 509, 516, and 581 in the pore-forming domain, conferred on AC− toxoids a correspondingly enhanced cytolytic potency. Moreover, a threefold increase in hemolytic activity could override a fourfold drop in capacity to convert cellular ATP to cAMP, conferring on the CyaA-E581K construct an overall twofold increased cytolytic potency. Hence, although appearing auxiliary in cytolytic action of the toxin on nucleated cells, the pore-forming activity can synergize with ATP-depleting activity of the cell-invasive AC enzyme and complement its action toward maximal cytotoxicity.

scholarly journals Translocation-Specific Conformation of Adenylate Cyclase Toxin from Bordetella pertussis Inhibits Toxin-Mediated Hemolysis

2001 ◽  
Vol 183 (20) ◽  
pp. 5904-5910 ◽  
Author(s):  
Mary C. Gray ◽  
Sang-Jin Lee ◽  
Lloyd S. Gray ◽  
Franca R. Zaretzky ◽  
Angela S. Otero ◽  
...  
Keyword(s):  
Amino Acids ◽  
Adenylate Cyclase ◽  
Bordetella Pertussis ◽  
Hemolytic Activity ◽  
Catalytic Domain ◽  
Target Cells ◽  
Wild Type ◽  
Free System ◽  
Rtx Toxins ◽  
A Cell

ABSTRACT Bordetella pertussis adenylate cyclase (AC) toxin belongs to the RTX family of toxins but is the only member with a known catalytic domain. The principal pathophysiologic function of AC toxin appears to be rapid production of intracellular cyclic AMP (cAMP) by insertion of its catalytic domain into target cells (referred to as intoxication). Relative to other RTX toxins, AC toxin is weakly hemolytic via a process thought to involve oligomerization of toxin molecules. Monoclonal antibody (MAb) 3D1, which binds to an epitope (amino acids 373 to 399) at the distal end of the catalytic domain of AC toxin, does not affect the enzymatic activity of the toxin (conversion of ATP into cAMP in a cell-free system) but does prevent delivery of the catalytic domain to the cytosol of target erythrocytes. Under these conditions, however, the ability of AC toxin to cause hemolysis is increased three- to fourfold. To determine the mechanism by which the hemolytic potency of AC toxin is altered, we used a series of deletion mutants. A mutant toxin, ΔAC, missing amino acids 1 to 373 of the catalytic domain, has hemolytic activity comparable to that of wild-type toxin. However, binding of MAb 3D1 to ΔAC enhances its hemolytic activity three- to fourfold similar to the enhancement of hemolysis observed with 3D1 addition to wild-type toxin. Two additional mutants, ΔN489 (missing amino acids 6 to 489) and ΔN518 (missing amino acids 6 to 518), exhibit more rapid hemolysis with quicker onset than wild-type toxin does, while ΔN549 (missing amino acids 6 to 549) has reduced hemolytic activity compared to wild-type AC toxin. These data suggest that prevention of delivery of the catalytic domain or deletion of the catalytic domain, along with additional amino acids distal to it, elicits a conformation of the toxin molecule that is more favorable for hemolysis.

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 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.

scholarly journals Use of a Toxin Neutralization Assay To Characterize the Serologic Response to Adenylate Cyclase Toxin after Infection with Bordetella pertussis

2016 ◽  
Vol 24 (1) ◽  
Author(s):  
Joshua C. Eby ◽  
Mary C. Gray ◽  
Jason M. Warfel ◽  
Tod J. Merkel ◽  
Erik L. Hewlett
Keyword(s):  
Adenylate Cyclase ◽  
Bordetella Pertussis ◽  
Neutralization Assay ◽  
Enzyme Linked Immunosorbent Assay ◽  
Immunologic Response ◽  
Serum Samples ◽  
Content Type ◽  
Adenylate Cyclase Toxin ◽  
Strong Candidate ◽  
Candidate Antigen

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.

scholarly journals Heterodimerization Between the Lutropin and Follitropin Receptors is Associated With an Attenuation of Hormone-Dependent Signaling

Endocrinology ◽  
2013 ◽  
Vol 154 (10) ◽  
pp. 3925-3930 ◽  
Author(s):  
Xiuyan Feng ◽  
Meilin Zhang ◽  
Rongbin Guan ◽  
Deborah L. Segaloff
Keyword(s):  
Protein Interactions ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
G Protein Coupled Receptors ◽  
Fsh Receptor ◽  
Bioluminescence Resonance Energy Transfer ◽  
Protein Protein Interactions ◽  
Lh Receptor ◽  
G Protein Coupled ◽  
Stimulation Of

The LH receptor (LHR) and FSH receptor (FSHR) are each G protein-coupled receptors that play critical roles in reproductive endocrinology. Each of these receptors has previously been shown to self-associate into homodimers and oligomers shortly after their biosynthesis. As shown herein using bioluminescence resonance energy transfer to detect protein-protein interactions, our data show that the LHR and FSHR, when coexpressed in the same cells, specifically heterodimerize with each other. Further experiments confirm that at least a portion of the cellular LHR/FSHR heterodimers are present on the cell surface and are functional. We then sought to ascertain what effects, if any, heterodimerization between the LHR and FSHR might have on signaling. It was observed that when the LHR was expressed under conditions promoting the heterodimerization with FSHR, LH or human chorionic gonadotropin (hCG) stimulation of Gs was attenuated. Conversely, when the FSHR was expressed under conditions promoting heterodimerization with the LHR, FSH-stimulated Gs activation was attenuated. These results demonstrate that the coexpression of the LHR and FSHR enables heterodimerizaton between the 2 gonadotropin receptors and results in an attenuation of signaling through each receptor.

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