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

UNLABELLED: The adenylate cyclase toxin (ACT, AC-Hly, or CyaA) plays a key role in airway infections by Bordetella pertussis and ablates the oxidative burst and opsonophagocytic capacity of sentinel phagocytes. CyaA fragments eliciting toxin-neutralizing antibodies are considered prime antigen candidates for improved acellular pertussis (aP) vaccines but their contribution to aP-mediated protection against B. pertussis infection awaits demonstration. We explored whether hybrid antigens inducing simultaneously CyaA-neutralizing and anti-Prn opsonizing antibody responses can enhance aP-elicited protection of mouse airways from infection. Fusion to the N-terminus of an RTX908 antigen derived from CyaA enabled an accelerated folding of the pertactin passenger domain (rPrn) in function of calcium loading of the RTX908 moiety and conferred on the rPrn-RTX908 fusion antigen a superior capacity to induce functional anti-Prn IgG antibodies. The rPrn-RTX908 fusion antigen also elicited CyaA neutralizing anti-RTX antibodies that relieved the toxin-imposed inhibition of oxidative burst and opsonophagocytic uptake of B. pertussis bacteria by HL-60 cells exposed to physiological concentrations of the CyaA toxin. Intranasal immunization of mice with the rPrn-RTX908 antigen admixed into a PT and FHA-based aP vaccine elicited specific sIgA responses in mucosal secretions (saliva) and conferred a significantly enhanced protection of mouse lung and nose mucosa against B. pertussis infection, yielding a significantly accelerated clearance of bacteria from the infected lungs within a single day from infection. These results demonstrate the added value of anti-CyaA antibodies elicited by intranasal application of the rPrn-RTX908 fusion antigen in the protection of the airway against B. pertussis infection. IMPORTANCE: Despite high vaccine coverage, unexpectedly massive whooping cough outbreaks are currently resurging in the most developed countries using the acellular pertussis (aP) vaccine. Accelerated development of improved aP vaccines, conferring a more complete and longer-lasting protection of the airway from Bordetella pertussis infection, is sorely needed. The highly immunosuppressive RTX adenylate cyclase toxin (CyaA) was proposed as a prime antigen candidate for inclusion into improved aP vaccines. We show here that a soluble RTX-derived antigen fused to the major opsonizing antibody target pertactin (rPrn-RTX908 hybrid) elicits opsonizing and toxin-neutralizing antibody responses that relieve the CyaA-imposed block of bactericidal opsonophagocytic uptake capacities of sentinel phagocytes. Intranasal immunization with the rPrn-RTX908 hybrid antigen then enables a significantly accelerated clearance of B. pertussis bacteria from mouse lungs and superior protection of mouse nasal mucosa from bacterial infection. These results unravel the added value of RTX antigen inclusion into the next generation of aP vaccines.

• Keywords
• Bordetella pertussis, adenylate cyclase toxin, pertactin, pertussis, protection, protein folding, whooping cough,
• MeSH
• Adenylate Cyclase Toxin * immunology   genetics   administration & dosage   MeSH
• Antigens, Bacterial * immunology   genetics   administration & dosage   MeSH
• Administration, Intranasal MeSH
• Bordetella pertussis * immunology   genetics   MeSH
• Virulence Factors, Bordetella * immunology   genetics   administration & dosage   MeSH
• Humans MeSH
• Mice, Inbred BALB C MeSH
• Mice MeSH
• Antibodies, Neutralizing blood   immunology   MeSH
• Whooping Cough * prevention & control   immunology   microbiology   MeSH
• Pertussis Vaccine * immunology   administration & dosage   genetics   MeSH
• Bacterial Outer Membrane Proteins * immunology   genetics   administration & dosage   MeSH
• Antibodies, Bacterial blood   immunology   MeSH
• Recombinant Fusion Proteins immunology   genetics   administration & dosage   MeSH
• Respiratory Mucosa * immunology   microbiology   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
• Antigens, Bacterial * MeSH
• Virulence Factors, Bordetella * MeSH
• Antibodies, Neutralizing MeSH
• pertactin MeSH Browser
• Pertussis Vaccine * MeSH
• Bacterial Outer Membrane Proteins * MeSH
• Antibodies, Bacterial MeSH
• Recombinant Fusion Proteins MeSH

Folding of the Repeats-in-toxin (RTX) domain of the bacterial adenylate cyclase toxin-hemolysin (CyaA) is critical to its toxin activities and the virulence of the whooping cough agent Bordetella pertussis. The RTX domain (RD) contains five RTX blocks (RTX-i to RTX-v) and their folding is driven by the binding of calcium. However, the detailed molecular mechanism via which the folding signal transmits within the five RTX blocks remains unknown. By combining single molecule optical tweezers, protein engineering, and toxin activity assays, here we demonstrate that the folding of the RD follows a strict hierarchy, with the folding starting from its C-terminal block RTX-v and proceeding towards the N-terminal RTX-i block sequentially. Our results reveal a strict series, templated folding mechanism, where the folding signal is transmitted along the RD in a series fashion from its C terminus continuously to the N terminus. Due to the series nature of this folding signal transmission pathway, the folding of RD can be disrupted at any given RTX block, rendering the RTX blocks located N-terminally to the disruption site and the acylation region of CyaA unfolded and abolishing CyaA's toxin activities. Our results reveal key mechanistic insights into the secretion and folding process of CyaA and may open up new potential avenues towards designing new therapeutics to abolish toxin activity of CyaA and combat B. pertussis.

• Keywords
• adenylate cyclase, bacterial toxin, optical tweezers, protein folding, single-molecule biophysics,
• Publication type
• Journal Article MeSH

The acylated Repeats in ToXins (RTX) leukotoxins, the adenylate cyclase toxin (CyaA) or α-hemolysin (HlyA), bind β2 integrins of leukocytes but also penetrate cells lacking these receptors. We show that the indoles of conserved tryptophans in the acylated segments, W876 of CyaA and W579 of HlyA, are crucial for β2 integrin-independent membrane penetration. Substitutions of W876 by aliphatic or aromatic residues did not affect acylation, folding, or the activities of CyaA W876L/F/Y variants on cells expressing high amounts of the β2 integrin CR3. However, toxin activity of CyaA W876L/F/Y on cells lacking CR3 was strongly impaired. Similarly, a W579L substitution selectively reduced HlyA W579L cytotoxicity towards cells lacking β2 integrins. Intriguingly, the W876L/F/Y substitutions increased the thermal stability (Tm) of CyaA by 4 to 8 °C but locally enhanced the accessibility to deuteration of the hydrophobic segment and of the interface of the two acylated loops. W876Q substitution (showing no increase in Tm), or combination of W876F with a cavity-filling V822M substitution (this combination decreasing the Tm closer to that of CyaA), yielded a milder defect of toxin activity on erythrocytes lacking CR3. Furthermore, the activity of CyaA on erythrocytes was also selectively impaired when the interaction of the pyrrolidine of P848 with the indole of W876 was ablated. Hence, the bulky indoles of residues W876 of CyaA, or W579 of HlyA, rule the local positioning of the acylated loops and enable a membrane-penetrating conformation in the absence of RTX toxin docking onto the cell membrane by β2 integrins.

• Keywords
• RTX toxin, acylated segment, adenylate cyclase toxin, cytotoxicity, hydrogen/deuterium exchange, thermal stability, tryptophan residue, α-hemolysin, β(2) integrins,
• MeSH
• Adenylate Cyclase Toxin * chemistry   genetics   metabolism   MeSH
• CD18 Antigens * genetics   metabolism   MeSH
• Bordetella pertussis MeSH
• Cell Membrane metabolism   MeSH
• Erythrocytes metabolism   MeSH
• Conserved Sequence MeSH
• Tryptophan * chemistry   genetics   metabolism   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Adenylate Cyclase Toxin * MeSH
• CD18 Antigens * MeSH
• Tryptophan * 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

Pore-forming repeats in toxins (RTX) are key virulence factors of many Gram-negative pathogens. We have recently shown that the aromatic side chain of the conserved tyrosine residue 940 within the acylated segment of the RTX adenylate cyclase toxin-hemolysin (CyaA, ACT or AC-Hly) plays a key role in target cell membrane interaction of the toxin. Therefore, we used a truncated CyaA-derived RTX719 construct to analyze the impact of Y940 substitutions on functional folding of the acylated segment of CyaA. Size exclusion chromatography combined with CD spectroscopy revealed that replacement of the aromatic side chain of Y940 by the side chains of alanine or proline residues disrupted the calcium-dependent folding of RTX719 and led to self-aggregation of the otherwise soluble and monomeric protein. Intriguingly, corresponding alanine substitutions of the conserved Y642, Y643 and Y639 residues in the homologous RtxA, HlyA and ApxIA hemolysins from Kingella kingae, Escherichia coli and Actinobacillus pleuropneumoniae, affected the membrane insertion, pore-forming (hemolytic) and cytotoxic capacities of these toxins only marginally. Activities of these toxins were impaired only upon replacement of the conserved tyrosines by proline residues. It appears, hence, that the critical role of the aromatic side chain of the Y940 residue is highly specific for the functional folding of the acylated domain of CyaA and determines its capacity to penetrate target cell membrane.

• MeSH
• Adenylate Cyclase Toxin genetics   MeSH
• Bordetella bronchiseptica * genetics   metabolism   MeSH
• Bordetella pertussis * genetics   metabolism   MeSH
• Cell Membrane metabolism   MeSH
• Hemolysis MeSH
• Bordetella Infections microbiology   MeSH
• Humans MeSH
• Mice, Inbred BALB C MeSH
• Mice MeSH
• THP-1 Cells MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Mice MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Adenylate Cyclase Toxin 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

The whooping cough agent Bordetella pertussis secretes an adenylate cyclase toxin (CyaA) that through its large carboxy-proximal Repeat-in-ToXin (RTX) domain binds the complement receptor 3 (CR3). The RTX domain consists of five blocks (I-V) of characteristic glycine and aspartate-rich nonapeptides that fold into five Ca2+-loaded parallel β-rolls. Previous work indicated that the CR3-binding structure comprises the interface of β-rolls II and III. To test if further portions of the RTX domain contribute to CR3 binding, we generated a construct with the RTX block II/III interface (CyaA residues 1132-1294) linked directly to the C-terminal block V fragment bearing the folding scaffold (CyaA residues 1562-1681). Despite deletion of 267 internal residues of the RTX domain, the Ca2+-driven folding of the hybrid block III/V β-roll still supported formation of the CR3-binding structure at the interface of β-rolls II and III. Moreover, upon stabilization by N- and C-terminal flanking segments, the block III/V hybrid-comprising constructs competed with CyaA for CR3 binding and induced formation of CyaA toxin-neutralizing antibodies in mice. Finally, a truncated CyaAΔ1295-1561 toxin bound and penetrated erythrocytes and CR3-expressing cells, showing that the deleted portions of RTX blocks III, IV, and V (residues 1295-1561) were dispensable for CR3 binding and for toxin translocation across the target cell membrane. This suggests that almost a half of the RTX domain of CyaA is not involved in target cell interaction and rather serves the purpose of toxin secretion.

• Keywords
• Bordetella pertussis, CD11b/CD18 integrin receptor, RTX toxin, adenylate cyclase toxin,
• MeSH
• Acylation MeSH
• Adenylate Cyclase Toxin metabolism   MeSH
• Bordetella pertussis pathogenicity   MeSH
• CHO Cells MeSH
• Cricetulus MeSH
• Epitopes metabolism   MeSH
• Humans MeSH
• Macrophage-1 Antigen chemistry   metabolism   MeSH
• Antibodies, Neutralizing metabolism   MeSH
• Protein Domains MeSH
• Protein Folding MeSH
• Amino Acid Sequence MeSH
• THP-1 Cells MeSH
• Calcium metabolism   MeSH
• Protein Binding MeSH
• Structure-Activity Relationship MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Adenylate Cyclase Toxin MeSH
• Epitopes MeSH
• Macrophage-1 Antigen MeSH
• Antibodies, Neutralizing MeSH
• Calcium MeSH