scholarly journals Antibody-mediated inhibition of Bordetella pertussis adenylate cyclase–haemolysin-induced macrophage cytotoxicity is influenced by variations in the bacterial population

Microbiology ◽  
2014 ◽  
Vol 160 (5) ◽  
pp. 962-969 ◽  
Author(s):  
N. Hegerle ◽  
N. Guiso
Keyword(s):  
Adenylate Cyclase ◽  
Bordetella Pertussis ◽  
Bacterial Population ◽  
Whooping Cough ◽  
Cell Lysis ◽  
Macrophage Cytotoxicity ◽  
Different Types ◽  
Induced Immunity

Whooping cough is a vaccine-preventable disease presenting with epidemic cycles linked to natural and/or vaccine-driven evolution of the aetiological agent of the disease, Bordetella pertussis. Adenylate cyclase–haemolysin (AC-Hly) is a major toxin produced by this pathogen, which mediates macrophage apoptosis in vitro and in vivo. While current acellular pertussis vaccine (APV) formulations do not include AC-Hly, they all contain pertussis toxin and can comprise filamentous haemagglutinin (FHA), which interacts with AC-Hly, and pertactin (PRN), which has been hypothesized also to interact with AC-Hly. We aimed to study the capacity of specific antibodies to inhibit the in vitro B. pertussis AC-Hly-mediated cytotoxicity of J774A.1 murine macrophages in a background of a changing bacterial population. We demonstrate that: (i) clinical isolates of different types or PRN phenotype are all cytotoxic and lethal in the mouse model of respiratory infection; (ii) lack of PRN production does not impact AC-Hly-related phenotypes; (iii) anti-AC-Hly antibodies inhibit cell lysis whatever the phenotype of the isolate, while anti-PRN antibodies significantly inhibit cell lysis provided the isolate produces this antigen, which might be relevant in vivo for APV-induced immunity; and (iv) anti-FHA antibodies only inhibit lysis induced by isolates collected in 2012, maybe indicating specific characteristics of epidemic lineages of B. pertussis.

scholarly journals The Adenylate Cyclase (CyaA) Toxin from Bordetella pertussis Has No Detectable Phospholipase A (PLA) Activity In Vitro

Toxins ◽  
2019 ◽  
Vol 11 (2) ◽  
pp. 111 ◽  
Author(s):  
Alexis Voegele ◽  
Mirko Sadi ◽  
Dorothée Raoux-Barbot ◽  
Thibaut Douché ◽  
Mariette Matondo ◽  
...  
Keyword(s):  
Adenylate Cyclase ◽  
Bordetella Pertussis ◽  
Whooping Cough ◽  
Adenyl Cyclase ◽  
Phospholipase A ◽  
Eukaryotic Cells ◽  
Experimental Conditions ◽  
Cyclase Domain ◽  
Charged Membranes

The adenylate cyclase (CyaA) toxin produced in Bordetella pertussis is the causative agent of whooping cough. CyaA exhibits the remarkable capacity to translocate its N-terminal adenyl cyclase domain (ACD) directly across the plasma membrane into the cytosol of eukaryotic cells. Once translocated, calmodulin binds and activates ACD, leading to a burst of cAMP that intoxicates the target cell. Previously, Gonzalez-Bullon et al. reported that CyaA exhibits a phospholipase A activity that could destabilize the membrane to facilitate ACD membrane translocation. However, Bumba and collaborators lately reported that they could not replicate these results. To clarify this controversy, we assayed the putative PLA activity of two CyaA samples purified in two different laboratories by using two distinct fluorescent probes reporting either PLA2 or both PLA1 and PLA2 activities, as well as in various experimental conditions (i.e., neutral or negatively charged membranes in different buffers.) However, we could not detect any PLA activity in these CyaA batches. Thus, our data independently confirm that CyaA does not possess any PLA activity.

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 Analysis of bvgR Expression in Bordetella pertussis

2003 ◽  
Vol 185 (23) ◽  
pp. 6902-6912 ◽  
Author(s):  
Tod J. Merkel ◽  
Philip E. Boucher ◽  
Scott Stibitz ◽  
Vanessa K. Grippe
Keyword(s):  
Bordetella Pertussis ◽  
Transcriptional Activation ◽  
Northern Blot Analysis ◽  
Whooping Cough ◽  
Transmembrane Protein ◽  
In Vitro Transcription ◽  
Environmental Signals ◽  
Transcription System

ABSTRACT Bordetella pertussis, the causative agent of whooping cough, produces a wide array of factors that are associated with its ability to cause disease. The expression and regulation of these virulence factors are dependent upon the bvg locus, which encodes three proteins: BvgA, a 23-kDa cytoplasmic protein; BvgS, a 135-kDa transmembrane protein; and BvgR, a 32-kDa protein. It is hypothesized that BvgS responds to environmental signals and interacts with BvgA, a transcriptional regulator, which upon modification by BvgS binds to specific promoters and activates transcription. An additional class of genes is repressed by the products of the bvg locus. The repression of these genes is dependent upon the third gene, bvgR. Expression of bvgR is dependent upon the function of BvgA and BvgS. This led to the hypothesis that the binding of phosphorylated BvgA to the bvgR promoter activates the expression of bvgR. We undertook an analysis of the transcriptional activation of bvgR expression. We identified the bvgR transcript by Northern blot analysis and identified the start site of transcription by primer extension. We determined that transcriptional activation of the bvgR promoter in an in vitro transcription system requires the addition of phosphorylated BvgA. Additionally, we have identified cis-acting regions that are required for BvgA activation of the bvgR promoter by in vitro footprinting and in vivo deletion and linker scanning analyses. A model of BvgA binding to the bvgR promoter is presented.

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 Development of RNAseq methodologies to profile the in vivo transcriptome of Bordetella pertussis during murine lung infection

2017 ◽  
Author(s):  
Ting Wong ◽  
Jesse Hall ◽  
Dylan Boehm ◽  
Mariette Barbier ◽  
F. Heath Damron
Keyword(s):  
Bordetella Pertussis ◽  
Whooping Cough ◽  
Expression Profiles ◽  
Gene Expression Profiles ◽  
Respiratory Pathogen ◽  
Cell Vaccine ◽  
Bacterial Cells ◽  
Murine Lung

AbstractBordetella pertussis is an obligate human respiratory pathogen that causes the disease whooping cough. A whole cell vaccine (DTP) was developed in the 1940s and was subsequently replaced in the 1990s with a protein-based subunit acellular vaccine (DTaP; tdap). Today, we are observing a resurgence of whooping cough due to evolution of the pathogen and waning vaccine immunity. The use of vaccines decreased the need for basic research on this pathogen. As a result, numerous questions on the basic pathogenesis of B. pertussis remain to be answered. Microarrays and more recently, RNA sequencing (RNAseq), have allowed the field to describe the in vitro gene expression profiles of the pathogen growing in both virulent and avirulent phases; however, no published studies have described an in vivo transcriptome of the pathogen. To address this need, we have designed and evaluated workflows to characterize the in vivo transcriptome of B. pertussis during infection of the murine lung. During our initial studies, we observed that only 0.014% of the ~100 million 2x50bp illumina reads corresponded to the pathogen, which is insufficient for analysis. Therefore, we developed a simple protocol to filter the bacteria out of the tissue homogenates and separate bacterial cells from the host tissue. RNA is then prepared, quantified, and the B. pertussis to host RNA ratio is determined. Here, we present the protocol and discuss the uses and next directions for which this RNAseq workflow can be applied. With this strategy we plan to fully characterize the B. pertussis transcriptome when the pathogen is infecting the murine lung in order to identify expressed genes that encode potential new vaccine antigens that will facilitate the development of the next generation of pertussis vaccines.

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 Integrin-mediated localization of Bordetella pertussis within macrophages: role in pulmonary colonization.

1991 ◽  
Vol 173 (5) ◽  
pp. 1143-1149 ◽  
Author(s):  
K Saukkonen ◽  
C Cabellos ◽  
M Burroughs ◽  
S Prasad ◽  
E Tuomanen
Keyword(s):  
Bordetella Pertussis ◽  
Whooping Cough ◽  
Rabbit Model ◽  
Bacterial Attachment ◽  
Lung Pathology ◽  
Relative Contribution ◽  
Pulmonary Macrophages ◽  
Complement Receptor 3

The adherence of Bordetella pertussis to human respiratory cilia is critical to the pathogenesis of whooping cough but the significance of bacterial attachment to macrophages has not been determined. Adherence to cilia and macrophages is mediated by two large, nonfimbrial bacterial proteins, filamentous hemagglutinin (FHA), and pertussis toxin (PT). PT and FHA both recognize carbohydrates on cilia and macrophages; FHA also contains an Arg-Gly-Asp (RGD) sequence which promotes bacterial association with the macrophage integrin complement receptor 3 (CR3). We determined that virulent B. pertussis enter and survive in mammalian macrophages in vitro and that CR3 is important for this uptake process. We then determined the relative contribution of CR3 versus carbohydrate-dependent interactions to in vivo pulmonary colonization using a rabbit model. B. pertussis colonized the lung as two approximately equal populations, one extracellular population attached to ciliary and macrophage surface glycoconjugates and another population within pulmonary macrophages. Loss of the CR3 interaction, either by mutation of FHA or treatment with antibody to CR3, disrupted accumulation of viable intracellular bacteria but did not prevent lung pathology. In contrast, elimination of carbohydrate-bound bacteria, either by a competitive receptor analogue or an anti-receptor antibody, was sufficient to prevent pulmonary edema. We propose that CR3-dependent localization of B. pertussis within macrophages promotes persistence of bacteria in the lung without pulmonary injury. On the other hand, the presence of extracellular bacteria adherent to cilia and macrophages in carbohydrate-dependent interactions is associated with pulmonary pathology.

scholarly journals Strain-Dependent Role of BrkA during Bordetella pertussis Infection of the Murine Respiratory Tract

2004 ◽  
Vol 72 (10) ◽  
pp. 5919-5924 ◽  
Author(s):  
Kelly D. Elder ◽  
Eric T. Harvill
Keyword(s):  
Respiratory Tract ◽  
Bordetella Pertussis ◽  
Whooping Cough ◽  
Single Gene ◽  
Wild Type ◽  
Pertussis Infection ◽  
Redundant Functions

ABSTRACT Bordetella pertussis, the causative agent of whooping cough, expresses many virulence factors believed to be involved in infection and disease progression. While these factors as a group are required for infection, deletion of individual virulence factor genes generally has limited effects on the ability of B. pertussis to efficiently infect the respiratory tract of mice, suggesting they may perform noncritical or redundant functions. We have recently observed that a B. pertussis strain, putatively with a mutation of a single gene, brkA, results in a severe defect in vivo. Although BrkA has been shown to be required for B. pertussis to resist complement-mediated killing in vitro, the relevance of these findings to the in vivo role of BrkA during infection has not been examined. Transducing this mutation into multiple wild-type B. pertussis strains allowed us to confirm the in vitro phenotype of reduced resistance to serum complement. All ΔbrkA mutants were increased in their sensitivity to complement in vitro, both in the presence and absence of antibodies. However, these strains differed substantially in their phenotypes in vivo. ΔbrkA mutants of recent clinical isolates were indistinguishable from wild-type strains in their efficient infection of respiratory organs, suggesting that the function of BrkA in these strains is noncritical or redundant. In contrast, multiple ΔbrkA strains derived from Tohama I were severely defective during the first week postinoculation compared to their wild-type parent. This defect was present even in complement-deficient mice, revealing a complement-independent phenotype for the ΔbrkA mutant in respiratory tract infection.

Epinephrine Induces von Willebrand Factor Release from Cultured Endothelial Cells: Involvement of Cyclic AMP-dependent Signalling in Exocytosis

1997 ◽  
Vol 77 (06) ◽  
pp. 1182-1188 ◽  
Author(s):  
Ulrich M Vischer ◽  
Claes B Wollheinn
Keyword(s):  
Endothelial Cells ◽  
Adenylate Cyclase ◽  
Von Willebrand Factor ◽  
Umbilical Vein ◽  
Free Calcium ◽  
Camp Content ◽  
Von Willebrand ◽  
Willebrand Factor

Summaryvon Willebrand factor (vWf) is released from endothelial cell storage granules after stimulation with thrombin, histamine and several other agents that induce an increase in cytosolic free calcium ([Ca2+]i). In vivo, epinephrine and the vasopressin analog DDAVP increase vWf plasma levels, although they are thought not to induce vWf release from endothelial cells in vitro. Since these agents act via a cAMP-dependent pathway in responsive cells, we examined the role of cAMP in vWf secretion from cultured human umbilical vein endothelial cells. vWf release increased by 50% in response to forskolin, which activates adenylate cyclase. The response to forskolin was much stronger when cAMP degradation was blocked with IBMX, an inhibitor of phosphodiesterases (+200%), whereas IBMX alone had no effect. vWf release could also be induced by the cAMP analogs dibutyryl-cAMP (+40%) and 8-bromo-cAMP (+25%); although their effect was weak, they clearly potentiated the response to thrombin. Epinephrine (together with IBMX) caused a small, dose-dependent increase in vWf release, maximal at 10-6 M (+50%), and also potentiated the response to thrombin. This effect is mediated by adenylate cyclase-coupled β-adrenergic receptors, since it is inhibited by propranolol and mimicked by isoproterenol. In contrast to thrombin, neither forskolin nor epinephrine caused an increase in [Ca2+]j as measured by fura-2 fluorescence. In addition, the effects of forskolin and thrombin were additive, suggesting that they act through distinct signaling pathways. We found a close correlation between cellular cAMP content and vWf release after stimulation with epinephrine and forskolin. These results demonstrate that cAMP-dependent signaling events are involved in the control of exocytosis from endothelial cells (an effect not mediated by an increase in [Ca2+]i) and provide an explanation for epinephrine-induced vWf release.

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