Most cited article - PubMed ID 26391732
Bordetella adenylate cyclase toxin: a unique combination of a pore-forming moiety with a cell-invading adenylate cyclase enzyme
Acylated domains (ADs), like that of the Bordetella pertussis adenylate cyclase toxin (CyaA), are structures found in all pore-forming toxins from the family of Repeat-in-ToXin (RTX) proteins. These AD segments are fatty-acylated on ε-amino groups of conserved lysine residues, such as the K860 and K983 residues of CyaA. The ε-amide-linked acyl chains are essential for toxin activity and promote irreversible membrane insertion of the CyaA molecule, thus enabling the toxin to translocate its N-terminal adenyl cyclase enzyme domain into the host cell cytoplasm. In parallel, the membrane-inserted CyaA molecules can oligomerize into cation-selective pores in the plasma membrane. Here, we show that the attached acyl chains are not only crucial for membrane insertion of the toxin but also play an important role in CyaA folding. We demonstrate that assembly of the noncanonical β-roll structure in the C-terminal segment of the AD of CyaA is cooperatively directed by the Ca2+-driven folding of the adjacent RTX domain. In contrast, the N-terminal AD segment consists of an α-helical structure that folds independently of Ca2+ ion binding and may form one or two acyl binding site(s) accommodating the acyl chains protruding from the C-terminal AD segment. This acyl-mediated interaction between the N- and C-terminal segments promotes local structural rearrangements within the AD that significantly enhances the stability of the toxin molecule. These findings highlight the critical role of the acyl modification in membrane interaction capacity and structural stability of the CyaA toxin.
- Keywords
- Bordetella pertussis, RTX toxin, acylation, adenylate cyclase toxin, protein folding,
- MeSH
- Acylation MeSH
- Adenylate Cyclase Toxin * metabolism chemistry genetics MeSH
- Bordetella pertussis * metabolism enzymology genetics MeSH
- Cell Membrane * metabolism MeSH
- Humans MeSH
- Protein Domains MeSH
- Protein Folding MeSH
- Calcium metabolism MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Names of Substances
- Adenylate Cyclase Toxin * MeSH
- Calcium MeSH
The pertussis agent Bordetella pertussis produces a number of virulence factors, of which the filamentous hemagglutinin (FhaB) plays a role in B. pertussis adhesion to epithelial and phagocytic cells. Moreover, FhaB was recently found to play a crucial role in nasal cavity infection and B. pertussis transmission to new hosts. The 367 kDa FhaB protein translocates through an FhaC pore to the outer bacterial surface and is eventually processed to a ~220 kDa N-terminal FHA fragment by the SphB1 protease. A fraction of the mature FHA then remains associated with bacterial cell surface, while most of FHA is shed into the bacterial environment. Previously reported indirect evidence suggested that FHA, or its precursor FhaB, may bind the β2 integrin CD11b/CD18 of human macrophages. Therefore, we assessed FHA binding to various cells producing or lacking the integrin and show that purified mature FHA does not bind CD11b/CD18. Further results then revealed that the adhesion of B. pertussis to cells does not involve an interaction between the bacterial surface-associated FhaB and/or mature FHA and the β2 integrin CD11b/CD18. In contrast, FHA binding was strongly inhibited at micromolar concentrations of heparin, corroborating that the cell binding of FHA is ruled by the interaction of its heparin-binding domain with sulfated glycosaminoglycans on the cell surface.
- Keywords
- Bordetella pertussis, CD11b/CD18, adenylate cyclase toxin, filamentous hemagglutinin, heparin, integrin,
- MeSH
- CD18 Antigens MeSH
- Bacterial Adhesion MeSH
- Adhesins, Bacterial metabolism MeSH
- Bordetella pertussis * metabolism MeSH
- Virulence Factors, Bordetella MeSH
- Glycosaminoglycans MeSH
- Hemagglutinins metabolism MeSH
- Heparin MeSH
- Integrins MeSH
- Humans MeSH
- Macrophage-1 Antigen MeSH
- Whooping Cough * MeSH
- Peptide Hydrolases MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Names of Substances
- CD18 Antigens MeSH
- Adhesins, Bacterial MeSH
- Virulence Factors, Bordetella MeSH
- Glycosaminoglycans MeSH
- Hemagglutinins MeSH
- Heparin MeSH
- Integrins MeSH
- Macrophage-1 Antigen MeSH
- Peptide Hydrolases MeSH
The adenylate cyclase (ACT) and the pertussis (PT) toxins of Bordetella pertussis exert potent immunomodulatory activities that synergize to suppress host defense in the course of whooping cough pathogenesis. We compared the mouse lung infection capacities of B. pertussis (Bp) mutants (Bp AC- or Bp PT-) producing enzymatically inactive toxoids and confirm that ACT action is required for maximal bacterial proliferation in the first days of infection, whereas PT action is crucial for persistence of B. pertussis in mouse lungs. Despite accelerated and near complete clearance from the lungs by day 14 of infection, the PT- bacteria accumulated within the lymphoid tissue of lung-draining mediastinal lymph nodes (mLNs). In contrast, the wild type or AC- bacteria colonized the lungs but did not enter into mLNs. Lung infection by the PT- mutant triggered an early arrival of migratory conventional dendritic cells with associated bacteria into mLNs, where the PT- bacteria entered the T cell-rich paracortex of mLNs by day 5 and proliferated in clusters within the B-cell zone (cortex) of mLNs by day 14, being eventually phagocytosed by infiltrating neutrophils. Finally, only infection by the PT- bacteria triggered an early production of anti-B. pertussis serum IgG antibodies already within 14 days of infection. These results reveal that action of the pertussis toxin blocks DC-mediated delivery of B. pertussis bacteria into mLNs and prevents bacterial colonization of mLNs, thus hampering early adaptive immune response to B. pertussis infection.
- MeSH
- Bordetella pertussis * MeSH
- Dendritic Cells pathology MeSH
- Lymph Nodes pathology MeSH
- Mice, Inbred BALB C MeSH
- Mice MeSH
- Whooping Cough * MeSH
- Pertussis Toxin MeSH
- Lung MeSH
- Animals MeSH
- Check Tag
- Mice MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Names of Substances
- Pertussis Toxin MeSH
The Gram-negative bacterium Kingella kingae is part of the commensal oropharyngeal flora of young children. As detection methods have improved, K. kingae has been increasingly recognized as an emerging invasive pathogen that frequently causes skeletal system infections, bacteremia, and severe forms of infective endocarditis. K. kingae secretes an RtxA cytotoxin, which is involved in the development of clinical infection and belongs to an ever-growing family of cytolytic RTX (Repeats in ToXin) toxins secreted by Gram-negative pathogens. All RTX cytolysins share several characteristic structural features: (i) a hydrophobic pore-forming domain in the N-terminal part of the molecule; (ii) an acylated segment where the activation of the inactive protoxin to the toxin occurs by a co-expressed toxin-activating acyltransferase; (iii) a typical calcium-binding RTX domain in the C-terminal portion of the molecule with the characteristic glycine- and aspartate-rich nonapeptide repeats; and (iv) a C-proximal secretion signal recognized by the type I secretion system. RTX toxins, including RtxA from K. kingae, have been shown to act as highly efficient 'contact weapons' that penetrate and permeabilize host cell membranes and thus contribute to the pathogenesis of bacterial infections. RtxA was discovered relatively recently and the knowledge of its biological role remains limited. This review describes the structure and function of RtxA in the context of the most studied RTX toxins, the knowledge of which may contribute to a better understanding of the action of RtxA in the pathogenesis of K. kingae infections.
- Keywords
- Kingella kingae, RTX toxin, RtxA, membrane, pore-forming, β2 integrins,
- Publication type
- Journal Article MeSH
- Review MeSH
The whooping cough agent, Bordetella pertussis, secretes an adenylate cyclase toxin-hemolysin (CyaA, ACT, or AC-Hly) that catalyzes the conversion of intracellular ATP to cAMP and through its signaling annihilates the bactericidal activities of host sentinel phagocytes. In parallel, CyaA permeabilizes host cells by the formation of cation-selective membrane pores that account for the hemolytic activity of CyaA. The pore-forming activity contributes to the overall cytotoxic effect of CyaA in vitro, and it has previously been proposed to synergize with the cAMP-elevating activity in conferring full virulence on B. pertussis in the mouse model of pneumonic infection. CyaA primarily targets myeloid phagocytes through binding of their complement receptor 3 (CR3, integrin αMβ2, or CD11b/CD18). However, with a reduced efficacy, the toxin can promiscuously penetrate and permeabilize the cell membrane of a variety of non-myeloid cells that lack CR3 on the cell surface, including airway epithelial cells or erythrocytes, and detectably intoxicates them by cAMP. Here, we used CyaA variants with strongly and selectively enhanced or reduced pore-forming activity that, at the same time, exhibited a full capacity to elevate cAMP concentrations in both CR3-expressing and CR3-non-expressing target cells. Using B. pertussis mutants secreting such CyaA variants, we show that a selective enhancement of the cell-permeabilizing activity of CyaA does not increase the overall virulence and lethality of pneumonic B. pertussis infection of mice any further. In turn, a reduction of the cell-permeabilizing activity of CyaA did not reduce B. pertussis virulence any importantly. These results suggest that the phagocyte-paralyzing cAMP-elevating capacity of CyaA prevails over the cell-permeabilizing activity of CyaA that appears to play an auxiliary role in the biological activity of the CyaA toxin in the course of B. pertussis infections in vivo.
- Keywords
- Bordetella pertussis, RTX toxin, adenylate cyclase toxin, cAMP intoxication, lung colonization, lung inflammation, pore-forming activity, virulence,
- MeSH
- Adenylate Cyclase Toxin metabolism MeSH
- Cyclic AMP metabolism MeSH
- Bordetella pertussis pathogenicity physiology MeSH
- Phagocytes metabolism microbiology MeSH
- Host-Pathogen Interactions MeSH
- Humans MeSH
- Mice, Inbred BALB C MeSH
- Mice MeSH
- Sheep MeSH
- Cell Membrane Permeability MeSH
- Whooping Cough metabolism microbiology pathology MeSH
- Virulence MeSH
- Animals MeSH
- Check Tag
- Humans MeSH
- Mice MeSH
- Female MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Names of Substances
- Adenylate Cyclase Toxin MeSH
- Cyclic AMP MeSH
The mucus layer protects airway epithelia from damage by noxious agents. Intriguingly, Bordetella pertussis bacteria provoke massive mucus production by nasopharyngeal epithelia during the initial coryza-like catarrhal stage of human pertussis and the pathogen transmits in mucus-containing aerosol droplets expelled by sneezing and post-nasal drip-triggered cough. We investigated the role of the cAMP-elevating adenylate cyclase (CyaA) and pertussis (PT) toxins in the upregulation of mucin production in B. pertussis-infected airway epithelia. Using human pseudostratified airway epithelial cell layers cultured at air-liquid interface (ALI), we show that purified CyaA and PT toxins (100 ng/mL) can trigger production of the major airway mucins Muc5AC and Muc5B. Upregulation of mucin secretion involved activation of the cAMP response element binding protein (CREB) and was blocked by the 666-15-Calbiochem inhibitor of CREB-mediated gene transcription. Intriguingly, a B. pertussis mutant strain secreting only active PT and producing the enzymatically inactive CyaA-AC- toxoid failed to trigger any important mucus production in infected epithelial cell layers in vitro or in vivo in the tracheal epithelia of intranasally infected mice. In contrast, the PT- toxoid-producing B. pertussis mutant secreting the active CyaA toxin elicited a comparable mucin production as infection of epithelial cell layers or tracheal epithelia of infected mice by the wild-type B. pertussis secreting both PT and CyaA toxins. Hence, the cAMP-elevating activity of B. pertussis-secreted CyaA was alone sufficient for activation of mucin production through a CREB-dependent mechanism in B. pertussis-infected airway epithelia in vivo.
- Keywords
- Bordetella, CREB, adenylate cyclase toxin, cAMP, epithelium, mucin, pertussis toxin,
- MeSH
- Adenylate Cyclase Toxin toxicity MeSH
- Bordetella pertussis metabolism pathogenicity MeSH
- Cell Line MeSH
- Respiratory System metabolism microbiology MeSH
- Epithelial Cells metabolism microbiology MeSH
- Humans MeSH
- Mucin 5AC metabolism MeSH
- Mice, Inbred BALB C MeSH
- Mice MeSH
- Whooping Cough metabolism microbiology MeSH
- Cyclic AMP Response Element-Binding Protein metabolism MeSH
- Animals MeSH
- Check Tag
- Humans MeSH
- Mice MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Names of Substances
- Adenylate Cyclase Toxin MeSH
- Mucin 5AC MeSH
- Cyclic AMP Response Element-Binding Protein MeSH
Myeloid phagocytes have evolved to rapidly recognize invading pathogens and clear them through opsonophagocytic killing. The adenylate cyclase toxin (CyaA) of Bordetella pertussis and the edema toxin (ET) of Bacillus anthracis are both calmodulin-activated toxins with adenylyl cyclase activity that invade host cells and massively increase the cellular concentrations of a key second messenger molecule, 3',5'-cyclic adenosine monophosphate (cAMP). However, the two toxins differ in the kinetics and mode of cell entry and generate different cAMP concentration gradients within the cell. While CyaA rapidly penetrates cells directly across their plasma membrane, the cellular entry of ET depends on receptor-mediated endocytosis and translocation of the enzymatic subunit across the endosomal membrane. We show that CyaA-generated membrane-proximal cAMP gradient strongly inhibits the activation and phosphorylation of Syk, Vav, and Pyk2, thus inhibiting opsonophagocytosis. By contrast, at similar overall cellular cAMP levels, the ET-generated perinuclear cAMP gradient poorly inhibits the activation and phosphorylation of these signaling proteins. Hence, differences in spatiotemporal distribution of cAMP produced by the two adenylyl cyclase toxins differentially affect the opsonophagocytic signaling in myeloid phagocytes.
- Keywords
- 3′,5′-cyclic adenosine monophosphate (cAMP), Pyk2, Syk, Vav, adenylate cyclase toxin, edema toxin, opsonophagocytosis, phagocytes, signaling pathway,
- MeSH
- Adenylate Cyclase Toxin toxicity MeSH
- Cyclic AMP metabolism MeSH
- Antigens, Bacterial toxicity MeSH
- Bacterial Toxins toxicity MeSH
- Spatio-Temporal Analysis MeSH
- Phagocytosis drug effects MeSH
- Phagocytes drug effects metabolism MeSH
- Phosphorylation drug effects MeSH
- Humans MeSH
- Actin Cytoskeleton drug effects MeSH
- Opsonin Proteins pharmacology MeSH
- Receptors, Immunologic metabolism MeSH
- Signal Transduction drug effects MeSH
- THP-1 Cells MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Names of Substances
- Adenylate Cyclase Toxin MeSH
- Cyclic AMP MeSH
- anthrax toxin MeSH Browser
- Antigens, Bacterial MeSH
- Bacterial Toxins MeSH
- opsonin receptor MeSH Browser
- Opsonin Proteins MeSH
- Receptors, Immunologic MeSH
The adenylate cyclase toxin-hemolysin (CyaA, ACT or AC-Hly) of pathogenic Bordetellae delivers its adenylyl cyclase (AC) enzyme domain into the cytosol of host cells and catalyzes uncontrolled conversion of cellular ATP to cAMP. In parallel, the toxin forms small cation-selective pores that permeabilize target cell membrane and account for the hemolytic activity of CyaA on erythrocytes. The pore-forming domain of CyaA is predicted to consist of five transmembrane α-helices, of which the helices I, III, IV and V have previously been characterized. We examined here the α-helix II that is predicted to form between residues 529 to 549. Substitution of the glycine 531 residue by a proline selectively reduced the hemolytic capacity but did not affect the AC translocating activity of the CyaA-G531P toxin. In contrast, CyaA toxins with alanine 538 or 546 replaced by diverse residues were selectively impaired in the capacity to translocate the AC domain across cell membrane but remained fully hemolytic. Such toxins, however, formed pores in planar asolectin bilayer membranes with a very low frequency and with at least two different conducting states. The helix-breaking substitution of alanine 538 by a proline residue abolished the voltage-activated increase of membrane activity of CyaA in asolectin bilayers. These results reveal that the predicted α-helix comprising the residues 529 to 549 plays a key role in CyaA penetration into the target plasma membrane and pore-forming activity of the toxin.
- MeSH
- Adenylate Cyclase Toxin chemistry genetics toxicity MeSH
- Bordetella enzymology MeSH
- Cell Membrane drug effects MeSH
- Erythrocytes drug effects MeSH
- Hemolysis MeSH
- Protein Conformation, alpha-Helical MeSH
- Cells, Cultured MeSH
- Mice MeSH
- Sheep MeSH
- Amino Acid Substitution MeSH
- Animals MeSH
- Check Tag
- Mice MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Names of Substances
- Adenylate Cyclase Toxin MeSH
Kingella kingae is a member of the commensal oropharyngeal flora of young children. Improvements in detection methods have led to the recognition of K. kingae as an emerging pathogen that frequently causes osteoarticular infections in children and a severe form of infective endocarditis in children and adults. Kingella kingae secretes a membrane-damaging RTX (Repeat in ToXin) toxin, RtxA, which is implicated in the development of clinical infections. However, the mechanism by which RtxA recognizes and kills host cells is largely unexplored. To facilitate structure-function studies of RtxA, we have developed a procedure for the overproduction and purification of milligram amounts of biologically active recombinant RtxA. Mass spectrometry analysis revealed the activation of RtxA by post-translational fatty acyl modification on the lysine residues 558 and/or 689 by the fatty-acyltransferase RtxC. Acylated RtxA was toxic to various human cells in a calcium-dependent manner and possessed pore-forming activity in planar lipid bilayers. Using various biochemical and biophysical approaches, we demonstrated that cholesterol facilitates the interaction of RtxA with artificial and cell membranes. The results of analyses using RtxA mutant variants suggested that the interaction between the toxin and cholesterol occurs via two cholesterol recognition/interaction amino acid consensus motifs located in the C-terminal portion of the pore-forming domain of the toxin. Based on our observations, we conclude that the cytotoxic activity of RtxA depends on post-translational acylation of the K558 and/or K689 residues and on the toxin binding to cholesterol in the membrane.
- MeSH
- Acylation MeSH
- Bacterial Toxins genetics metabolism MeSH
- Cell Membrane metabolism MeSH
- Cell Line MeSH
- Cholesterol metabolism MeSH
- Kingella kingae enzymology genetics MeSH
- Humans MeSH
- Lysine chemistry MeSH
- Protein Processing, Post-Translational * MeSH
- Recombinant Proteins metabolism MeSH
- Transaminases genetics metabolism MeSH
- Protein Binding MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Names of Substances
- Bacterial Toxins MeSH
- Cholesterol MeSH
- Lysine MeSH
- Recombinant Proteins MeSH
- Transaminases MeSH
The adenylate cyclase toxin-hemolysin (CyaA, ACT, or AC-Hly) plays a crucial role in virulence and airway colonization capacity of the whooping cough agent Bordetella pertussis. The toxin penetrates target cell membranes and exhibits three distinct biological activities. A population of CyaA conformers forms small cation-selective pores that permeabilize the cell membrane for potassium efflux, which can provoke colloid-osmotic (oncotic) cell lysis. The other two activities are due to CyaA conformers that transiently form calcium influx conduits in the target cell membrane and translocate the adenylate cyclase (AC) enzyme into cytosol of cells. A fourth putative biological activity has recently been reported; an intrinsic phospholipase A (PLA) activity was claimed to be associated with the CyaA polypeptide and be involved in the mechanism of translocation of the AC enzyme polypeptide across cell membrane lipid bilayer. However, the conclusions drawn by the authors contradicted their own results and we show them to be erroneous. We demonstrate that highly purified CyaA is devoid of any detectable phospholipase A1 activity and that contrary to the published claims, the two putative conserved phospholipase A catalytic residues, namely the Ser606 and Asp1079 residues, are not involved in the process of membrane translocation of the AC domain of CyaA across target membranes.
- Keywords
- AC domain translocation, adenylate cyclase toxin, phospholipase A activity,
- MeSH
- Adenylate Cyclase Toxin metabolism toxicity MeSH
- Bordetella pertussis MeSH
- Cell Line MeSH
- Erythrocytes MeSH
- Phospholipases A metabolism MeSH
- Hemolysis MeSH
- Aspartic Acid MeSH
- Mice MeSH
- Sheep MeSH
- Serine MeSH
- Animals MeSH
- Check Tag
- Mice MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Names of Substances
- Adenylate Cyclase Toxin MeSH
- Phospholipases A MeSH
- Aspartic Acid MeSH
- Serine MeSH