scholarly journals Cyclic AMP-Mediated Suppression of Neutrophil Extracellular Trap Formation and Apoptosis by the Bordetella pertussis Adenylate Cyclase Toxin

2014 ◽  
Vol 82 (12) ◽  
pp. 5256-5269 ◽  
Author(s):  
Joshua C. Eby ◽  
Mary C. Gray ◽  
Erik L. Hewlett
Keyword(s):  
Cyclic Amp ◽  
Adenylate Cyclase ◽  
Bordetella Pertussis ◽  
Neutrophil Function ◽  
Neutrophil Extracellular Trap ◽  
Target Cells ◽  
Content Type ◽  
Extracellular Traps ◽  
Adenylate Cyclase Toxin ◽  
Insight Into

ABSTRACTThe adenylate cyclase toxin (ACT) ofBordetella pertussisintoxicates 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 liveB. pertussis. Imaging ofB. pertussis-exposed neutrophils revealed thatB. pertussislacking ACT induces formation of neutrophil extracellular traps (NETs), whereas wild-typeB. pertussisdoes 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.

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 The Eukaryotic Host Factor 14-3-3 Inactivates Adenylate Cyclase Toxins ofBordetella bronchisepticaandB. parapertussis, but NotB. pertussis

mBio ◽  
2018 ◽  
Vol 9 (4) ◽  
Author(s):  
Aya Fukui-Miyazaki ◽  
Hirono Toshima ◽  
Yukihiro Hiramatsu ◽  
Keisuke Okada ◽  
Keiji Nakamura ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Adenylate Cyclase ◽  
Virulence Factors ◽  
Virulence Factor ◽  
Bordetella Pertussis ◽  
Host Factor ◽  
Target Cells ◽  
Content Type ◽  
Functional Differences ◽  
Adenylate Cyclase Toxin

ABSTRACTBordetella pertussis,Bordetella bronchiseptica, andBordetella parapertussisshare highly homologous virulence factors and commonly cause respiratory infections in mammals; however, their host specificities and disease severities differ, and the reasons for this remain largely unknown. Adenylate cyclase toxin (CyaA) is a homologous virulence factor that is thought to play crucial roles inBordetellainfections. We herein demonstrate that CyaAs function as virulence factors differently betweenB. bronchiseptica/B. parapertussisandB. pertussis.B.bronchisepticaCyaA bound to target cells, and its enzyme domain was translocated into the cytosol similarly toB.pertussisCyaA. The hemolytic activity ofB.bronchisepticaCyaA on sheep erythrocytes was also preserved. However, in nucleated target cells,B.bronchisepticaCyaA was phosphorylated at Ser375, which constitutes a motif (RSXpSXP [pS is phosphoserine]) recognized by the host factor 14-3-3, resulting in the abrogation of adenylate cyclase activity. Consequently, the cytotoxic effects ofB.bronchisepticaCyaA based on its enzyme activity were markedly attenuated.B.parapertussisCyaA carries the 14-3-3 motif, indicating that its intracellular enzyme activity is abrogated similarly toB.bronchisepticaCyaA; however,B.pertussisCyaA has Phe375instead of Ser, and thus, was not affected by 14-3-3. In addition,B.pertussisCyaA impaired the barrier function of epithelial cells, whereasB.bronchisepticaCyaA did not. Rat infection experiments suggested that functional differences in CyaA are related to differences in pathogenicity betweenB. bronchiseptica/B.parapertussisandB. pertussis.IMPORTANCEBordetella pertussis,B. bronchiseptica, andB. parapertussisare bacterial respiratory pathogens that are genetically close to each other and produce many homologous virulence factors; however, their host specificities and disease severities differ, and the reasons for this remain unknown. Previous studies attempted to explain these differences by the distinct virulence factors produced by eachBordetellaspecies. In contrast, we indicated functional differences in adenylate cyclase toxin, a homologous virulence factor ofBordetella. The toxins ofB. bronchisepticaand presumablyB. parapertussiswere inactivated by the host factor 14-3-3 after phosphorylation in target cells, whereas theB. pertussistoxin was not inactivated because of the lack of the phosphorylation site. This is the first study to show that 14-3-3 inactivates the virulence factors of pathogens. The present results suggest that pathogenic differences inBordetellaare attributed to the different activities of adenylate cyclase toxins.

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 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 BordetellaAdenylate Cyclase Toxin Inhibits Monocyte-to-Macrophage Transition and Dedifferentiates Human Alveolar Macrophages into Monocyte-like Cells

mBio ◽  
2019 ◽  
Vol 10 (5) ◽  
Author(s):  
Jawid Nazir Ahmad ◽  
Jana Holubova ◽  
Oldrich Benada ◽  
Olga Kofronova ◽  
Ludek Stehlik ◽  
...  
Keyword(s):  
Adenylate Cyclase ◽  
Immune Evasion ◽  
Bordetella Pertussis ◽  
Alveolar Macrophages ◽  
Human Monocyte ◽  
Content Type ◽  
Macrophage Cells ◽  
Human Alveolar Macrophages ◽  
Adenylate Cyclase Toxin

ABSTRACTMonocytes 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-producingB. pertussisbacteria, 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 onin vitrodifferentiated 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 dedifferentiatedin 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.IMPORTANCEMacrophages are key sentinel cells of the immune system, and, as such, they are targeted by the toxins produced by the pertussis agentBordetella pertussis. The adenylate cyclase toxin (CyaA) mediates immune evasion ofB. pertussisby 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.

scholarly journals Quantification of the Adenylate Cyclase Toxin of Bordetella pertussisIn Vitroand during Respiratory Infection

2013 ◽  
Vol 81 (5) ◽  
pp. 1390-1398 ◽  
Author(s):  
Joshua C. Eby ◽  
Mary C. Gray ◽  
Jason M. Warfel ◽  
Christopher D. Paddock ◽  
Tara F. Jones ◽  
...  
Keyword(s):  
Adenylate Cyclase ◽  
Respiratory Tract ◽  
Whooping Cough ◽  
Extensive Study ◽  
Target Cells ◽  
Human Infants ◽  
Content Type ◽  
Adenylate Cyclase Toxin

ABSTRACTWhooping cough results from infection of the respiratory tract withBordetella pertussis, and the secreted adenylate cyclase toxin (ACT) is essential for the bacterium to establish infection. Despite extensive study of the mechanism of ACT cytotoxicity and its effects over a range of concentrationsin vitro, ACT has not been observed or quantifiedin vivo, and thus the concentration of ACT at the site of infection is unknown. The recently developed baboon model of infection mimics the prolonged cough and transmissibility of pertussis, and we hypothesized that measurement of ACT in nasopharyngeal washes (NPW) from baboons, combined with human andin vitrodata, would provide an estimate of the ACT concentration in the airway during infection. NPW contained up to ∼108CFU/mlB. pertussisand 1 to 5 ng/ml ACT at the peak of infection. Nasal aspirate specimens from two human infants with pertussis contained bacterial concentrations similar to those in the baboons, with 12 to 20 ng/ml ACT. When ∼108CFU/ml of a laboratory strain ofB. pertussiswas culturedin vitro, ACT production was detected in 60 min and reached a plateau of ∼60 ng/ml in 6 h. Furthermore, when bacteria were brought into close proximity to target cells by centrifugation, intoxication was increased 4-fold. Collectively, these data suggest that at the bacterium-target cell interface during infection of the respiratory tract, the concentration of ACT can exceed 100 ng/ml, providing a reference point for future studies of ACT and pertussis pathogenesis.

scholarly journals Bordetella pertussis Inhibition of Interleukin-12 (IL-12) p70 in Human Monocyte-Derived Dendritic Cells Blocks IL-12 p35 through Adenylate Cyclase Toxin-Dependent Cyclic AMP Induction

2006 ◽  
Vol 74 (5) ◽  
pp. 2831-2838 ◽  
Author(s):  
Fabiana Spensieri ◽  
Giorgio Fedele ◽  
Cecilia Fazio ◽  
Maria Nasso ◽  
Paola Stefanelli ◽  
...  
Keyword(s):  
Immune Response ◽  
Cyclic Amp ◽  
Adenylate Cyclase ◽  
Bordetella Pertussis ◽  
Interleukin 12 ◽  
Protein Chain ◽  
Cell Functions ◽  
A Subunit ◽  
Adenylate Cyclase Toxin ◽  
The Impact

ABSTRACT Bordetella pertussis, the causative agent of whooping cough, possesses an array of virulence factors, including adenylate cyclase toxin (ACT), relevant in the establishment of infection. Here we better define the impact of cyclic AMP (cAMP) intoxication due to the action of ACT on dendritic cell (DC)-driven immune response, by infecting monocyte-derived DC (MDDC) with an ACT-deficient B. pertussis mutant (ACT−18HS19) or its parental strain (WT18323). Both strains induced MDDC maturation and antigen-presenting cell functions; however, only ACT−18HS19 infected MDDC-induced production of interleukin-12 (IL-12) p70. Gene expression analysis of the IL-12 cytokine family subunits revealed that both strains induced high levels of p40 (protein chain communal to IL-12 p70 and IL-23) as well as p19, a subunit of IL-23. Conversely only ACT−18HS19 infection induced consistent transcription of IL-12 p35, a subunit of IL-12 p70. Addition of the cAMP analogous d-butyril-cAMP (d-cAMP) abolished IL-12 p70 production and IL-12 p35 expression in ACT−18HS19-infected MDDC. ACT−18HS19 infection induced the expression of the transcription factors interferon regulatory factor 1 (IRF-1) and IRF-8 and of beta interferon, involved in IL-12 p35 regulation, and the expression of these genes was inhibited by d-cAMP addition and in WT18323-infected MDDC. The concomitant expression of IL-12 p70 and IL-23 allowed ACT−18HS19 to trigger a more pronounced T helper 1 polarization compared to WT18323. The present study suggests that ACT-dependent cAMP induction leads to the inhibition of pathways ultimately leading to IL-12 p35 production, thus representing a mechanism for B. pertussis to escape the host immune response.

scholarly journals The Adenylate Cyclase Toxin of Bordetella pertussis Binds to Target Cells via the αMβ2 Integrin (Cd11b/Cd18)

2001 ◽  
Vol 193 (9) ◽  
pp. 1035-1044 ◽  
Author(s):  
Pierre Guermonprez ◽  
Nadia Khelef ◽  
Eric Blouin ◽  
Philippe Rieu ◽  
Paola Ricciardi-Castagnoli ◽  
...  
Keyword(s):  
Cell Death ◽  
Monoclonal Antibodies ◽  
Adenylate Cyclase ◽  
Cell Lines ◽  
Bordetella Pertussis ◽  
Chinese Hamster Ovary Cells ◽  
Human Neutrophils ◽  
Target Cells ◽  
Intracellular Camp ◽  
Adenylate Cyclase Toxin

The adenylate cyclase toxin (CyaA) of Bordetella pertussis is a major virulence factor required for the early phases of lung colonization. It can invade eukaryotic cells where, upon activation by endogenous calmodulin, it catalyzes the formation of unregulated cAMP levels. CyaA intoxication leads to evident toxic effects on macrophages and neutrophils. Here, we demonstrate that CyaA uses the αMβ2 integrin (CD11b/CD18) as a cell receptor. Indeed, the saturable binding of CyaA to the surface of various hematopoietic cell lines correlated with the presence of the αMβ2 integrin on these cells. Moreover, binding of CyaA to various murine cell lines and human neutrophils was specifically blocked by anti-CD11b monoclonal antibodies. The increase of intracellular cAMP level and cell death triggered by CyaA intoxication was also specifically blocked by anti-CD11b monoclonal antibodies. In addition, CyaA bound efficiently and triggered intracellular cAMP increase and cell death in Chinese hamster ovary cells transfected with αMβ2 (CD11b/CD18) but not in cells transfected with the vector alone or with the αXβ2 (CD11c/CD18) integrin. Thus, the cellular distribution of CD11b, mostly on neutrophils, macrophages, and dendritic and natural killer cells, supports a role for CyaA in disrupting the early, innate antibacterial immune response.

scholarly journals Newly secreted adenylate cyclase toxin is responsible for intoxication of target cells by Bordetella pertussis

2004 ◽  
Vol 53 (6) ◽  
pp. 1709-1719 ◽  
Author(s):  
M. C. Gray ◽  
G. M. Donato ◽  
F. R. Jones ◽  
T. Kim ◽  
E. L. Hewlett
Keyword(s):  
Adenylate Cyclase ◽  
Bordetella Pertussis ◽  
Target Cells ◽  
Adenylate Cyclase Toxin

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.

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