Effect of Different Forms of Adenylate Cyclase Toxin of Bordetella pertussis on Protection Afforded by an Acellular Pertussis Vaccine in a Murine Model

ABSTRACT Four recombinant forms of the cell-invasive adenylate cyclase toxin (CyaA) of Bordetella pertussis were compared for the ability to enhance protection against B. pertussis in mice when coadministered with an acellular pertussis vaccine (ACV). The four forms were as follows: fully functional CyaA, a CyaA form lacking adenylate cyclase enzymatic activity (CyaA*), and the nonacylated forms of these toxins, i.e., proCyaA and proCyaA*, respectively. None of these forms alone conferred significant (P > 0.05) protection against B. pertussis in a murine intranasal challenge model. Mice immunized with ACV alone showed significant (P < 0.05) reductions in bacterial numbers in the lungs after intranasal challenge compared with those for control mice. When administered with ACV, both CyaA and CyaA* further reduced bacterial numbers in the lungs of mice after intranasal challenge compared with those for ACV-immunized mice, but the enhanced protection was only significant (P < 0.05) with CyaA*. Coadministration of CyaA* with ACV caused a significant (P < 0.05) increase in immunoglobulin G2a antibody levels against pertactin compared with those in mice immunized with ACV alone. Spleen cells from mice immunized with ACV plus CyaA* secreted larger amounts of interleukin-5 (IL-5), IL-6, gamma interferon (IFN-γ), and granulocyte-macrophage colony-stimulating factor (GM-CSF) than did cells from mice immunized with ACV plus CyaA or ACV alone after stimulation in vitro with a mixture of B. pertussis antigens. Spleen cells from mice immunized with ACV plus CyaA* also secreted larger amounts of IFN-γ and GM-CSF than did cells from mice immunized with CyaA* alone after stimulation in vitro with CyaA*. Macrophages from mice immunized with ACV plus CyaA* produced significantly (P < 0.05) higher levels of nitric oxide than did macrophages from mice immunized with CyaA* alone, ACV alone, or ACV plus CyaA after stimulation in vitro with a mixture of B. pertussis antigens or heat-killed B. pertussis cells. These data suggest that the enhancement of protection provided by CyaA* was due to an augmentation of both Th1 and Th2 immune responses to B. pertussis antigens.

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

mBio ◽

10.1128/mbio.01743-19 ◽

2019 ◽

Vol 10 (5) ◽

Cited By ~ 3

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.

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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

Infection and Immunity ◽

10.1128/iai.72.3.1568-1579.2004 ◽

2004 ◽

Vol 72 (3) ◽

pp. 1568-1579 ◽

Cited By ~ 89

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.

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

Infection and Immunity ◽

10.1128/iai.00937-16 ◽

2017 ◽

Vol 85 (6) ◽

Cited By ~ 18

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.

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Bordetella pertussis adenylate cyclase toxin: proCyaA and CyaC proteins synthesised separately in Escherichia coli produce active toxin in vitro

Gene ◽

10.1016/s0378-1119(96)00412-x ◽

1996 ◽

Vol 180 (1-2) ◽

pp. 91-99 ◽

Cited By ~ 29

Author(s):

Gareth D. Westrop ◽

E.Kalantar Hormozi ◽

Nuno A. Da Costa ◽

Roger Parton ◽

John G. Coote

Keyword(s):

Escherichia Coli ◽

Adenylate Cyclase ◽

Bordetella Pertussis ◽

Adenylate Cyclase Toxin

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Identification by in vitro complementation of regions required for cell-invasive activity of Bordetella pertussis adenylate cyclase toxin

Molecular Microbiology ◽

10.1111/j.1365-2958.1995.mmi_17061015.x ◽

1995 ◽

Vol 17 (6) ◽

pp. 1015-1024 ◽

Cited By ~ 57

Author(s):

Masaaki Iwaki ◽

Agnes Ullmann ◽

Peter Sebo

Keyword(s):

Adenylate Cyclase ◽

Bordetella Pertussis ◽

Adenylate Cyclase Toxin

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Adenylate Cyclase Toxin From Bordetella pertussis Enhances Cisplatin-Induced Apoptosis to Lung Cancer Cells In Vitro

Oncology Research Featuring Preclinical and Clinical Cancer Therapeutics ◽

10.3727/096504005776568273 ◽

2005 ◽

Vol 15 (9) ◽

pp. 423-430 ◽

Cited By ~ 5

Author(s):

David Johansson ◽

Per Bergström ◽

Roger Henriksson ◽

Kjell Grankvist ◽

Anders Johansson ◽

...

Keyword(s):

Lung Cancer ◽

Adenylate Cyclase ◽

Cancer Cells ◽

Bordetella Pertussis ◽

Lung Cancer Cells ◽

Adenylate Cyclase Toxin ◽

Induced Apoptosis

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Faculty Opinions recommendation of Selective translocation of the Bordetella pertussis adenylate cyclase toxin across the basolateral membranes of polarized epithelial cells.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.2063966.1647064 ◽

2010 ◽

Author(s):

Drusilla L Burns

Keyword(s):

Epithelial Cells ◽

Adenylate Cyclase ◽

Bordetella Pertussis ◽

Basolateral Membranes ◽

Adenylate Cyclase Toxin ◽

Polarized Epithelial Cells

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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 ◽

10.1128/cvi.00370-16 ◽

2016 ◽

Vol 24 (1) ◽

Cited By ~ 4

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.

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In vitro effect of ochratoxin A and deoxynivalenol on the expression of interleukin 5 and interferon-gamma in broiler chicken lymphocytes

World Mycotoxin Journal ◽

10.3920/wmj2014.1852 ◽

2016 ◽

Vol 9 (2) ◽

pp. 299-304 ◽

Cited By ~ 1

Author(s):

C. Lautert ◽

L. Ferreiro ◽

M.I. Azevedo ◽

S.A. Botton ◽

J.T. Santos ◽

...

Keyword(s):

Ochratoxin A ◽

Interferon Gamma ◽

Broiler Chickens ◽

Broiler Chicken ◽

Fungal Metabolites ◽

Interleukin 5 ◽

T Helper 2 ◽

Ifn Γ ◽

Vitro Effect

Cytokines are proteins secreted by cells of innate and acquired immunity, produced in response to various antigens and responsible for mediating several function of these cells. Our study evaluated the profile of cytokines interleukin 5 (IL-5) and interferon gamma (IFN-γ), induced in lymphocytes of broiler chickens in response to secondary fungal metabolites ochratoxin A (OTA) and deoxynivalenol (DON) at concentrations of 0.001, 0.01, 0.1 and 1 μg/ml. The quantification of the cytokines was analysed at 24, 48 and 72 h after incubation with mycotoxins, using real-time PCR (qRT-PCR). The results obtained showed that OTA induced mRNA synthesis of IL-5 at concentrations 0.001, 0.1 and 1 μg/ml after 24 h of lymphocyte incubation, while at 48 h only the expression of the IL-5 cytokine at a concentration of 1 μg/ml (P<0.05) was detected. DON in a concentration of 1 μg/ml induced the expression of IL-5 in the lymphocytes only at 48 h post-incubation period (P<0.05). Regarding IFN-γ, gene expression was not observed in the lymphocytes of broiler chickens incubated with OTA and DON. The data obtained represent a profile of response mediated by T helper 2 cells to the exposure of broiler chicken immune cells to different concentrations of OTA and DON.

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