scholarly journals Calcium, Acylation, and Molecular Confinement Favor Folding of Bordetella pertussis Adenylate Cyclase CyaA Toxin into a Monomeric and Cytotoxic Form

2014 ◽  
Vol 289 (44) ◽  
pp. 30702-30716 ◽  
Author(s):  
Johanna C. Karst ◽  
V. Yvette Ntsogo Enguéné ◽  
Sara E. Cannella ◽  
Orso Subrini ◽  
Audrey Hessel ◽  
...  
Keyword(s):  
Adenylate Cyclase ◽  
Bordetella Pertussis ◽  
Whooping Cough ◽  
Active Form ◽  
Size Exclusion ◽  
Target Cells ◽  
Cytotoxic Activities ◽  
Experimental Conditions ◽  
Molecular Approaches ◽  
Molecular Confinement

The adenylate cyclase (CyaA) toxin, a multidomain protein of 1706 amino acids, is one of the major virulence factors produced by Bordetella pertussis, the causative agent of whooping cough. CyaA is able to invade eukaryotic target cells in which it produces high levels of cAMP, thus altering the cellular physiology. Although CyaA has been extensively studied by various cellular and molecular approaches, the structural and functional states of the toxin remain poorly characterized. Indeed, CyaA is a large protein and exhibits a pronounced hydrophobic character, making it prone to aggregation into multimeric forms. As a result, CyaA has usually been extracted and stored in denaturing conditions. Here, we define the experimental conditions allowing CyaA folding into a monomeric and functional species. We found that CyaA forms mainly multimers when refolded by dialysis, dilution, or buffer exchange. However, a significant fraction of monomeric, folded protein could be obtained by exploiting molecular confinement on size exclusion chromatography. Folding of CyaA into a monomeric form was found to be critically dependent upon the presence of calcium and post-translational acylation of the protein. We further show that the monomeric preparation displayed hemolytic and cytotoxic activities suggesting that the monomer is the genuine, physiologically active form of the toxin. We hypothesize that the structural role of the post-translational acylation in CyaA folding may apply to other RTX toxins.

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 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 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 The role of four cholesterol-recognition motifs localized between amino acid residues 400-550 in regulating translocation and lytic activity of Adenylate Cyclase Toxin

2021 ◽  
Author(s):  
Jone Amuategi ◽  
Rocio Alonso ◽  
Helena Ostolaza
Keyword(s):  
Adenylate Cyclase ◽  
Whooping Cough ◽  
Cell Signalling ◽  
Lipid Vesicles ◽  
Cell Types ◽  
Target Cells ◽  
Amino Acid Residues ◽  
Cyclase Domain ◽  
Adenylate Cyclase Toxin ◽  
Recognition Motifs

Adenylate Cyclase Toxin (ACT or CyaA) is an important virulence factor secreted by Bordetella pertussis, the bacterium causative of whooping cough, playing an essential role in the establishment of infection in the respiratory tract. ACT is a pore-forming cytolysin belonging to the RTX (Repeats in ToXin) family of leukotoxins, capable of permeabilizing several cell types and pure lipid vesicles. Besides, the toxin delivers its N-terminal adenylate cyclase domain into the target cytosol, where catalyzes the conversion of ATP into cAMP, which affects cell signalling. In this study we have made two major observations. First, we show that ACT binds free cholesterol, and identify in its sequence 38 potential cholesterol-recognition motifs. Second, we reveal that four of those motifs are real, functional cholesterol-binding sites. Mutations of the central phenylalanine residues in said motifs have an important impact on the ACT lytic and translocation activities, suggesting their direct intervention in cholesterol recognition and toxin functionality. From our data a likely transmembrane topology can be inferred for the ACT helices constituting the translocation and the hydrophobic regions. From this topology a simple and plausible mechanism emerges by which ACT could translocate its AC domain into target cells, challenging previous views in the field. Blocking the ACT-cholesterol interactions might thus be an effective approach for inhibiting ACT toxicity on cells, and this could help in mitigating the severity of pertussis disease in humans.

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 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 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 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 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.

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