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 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

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 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

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

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

The airway epithelium restricts the penetration of inhaled pathogens into the underlying tissue and plays a crucial role in the innate immune defense against respiratory infections. The whooping cough agent, Bordetella pertussis, adheres to ciliated cells of the human airway epithelium and subverts its defense functions through the action of secreted toxins and other virulence factors. We examined the impact of B. pertussis infection and of adenylate cyclase toxin-hemolysin (CyaA) action on the functional integrity of human bronchial epithelial cells cultured at the air-liquid interface (ALI). B. pertussis adhesion to the apical surface of polarized pseudostratified VA10 cell layers provoked a disruption of tight junctions and caused a drop in transepithelial electrical resistance (TEER). The reduction of TEER depended on the capacity of the secreted CyaA toxin to elicit cAMP signaling in epithelial cells through its adenylyl cyclase enzyme activity. Both purified CyaA and cAMP-signaling drugs triggered a decrease in the TEER of VA10 cell layers. Toxin-produced cAMP signaling caused actin cytoskeleton rearrangement and induced mucin 5AC production and interleukin-6 (IL-6) secretion, while it inhibited the IL-17A-induced secretion of the IL-8 chemokine and of the antimicrobial peptide beta-defensin 2. These results indicate that CyaA toxin activity compromises the barrier and innate immune functions of Bordetella-infected airway epithelia.

• Keywords
• Bordetella pertussis, CyaA, adenylate cyclase toxin, airway epithelia, antimicrobial peptides, cyclic AMP, epithelial cells, immunomodulatory cytokines, tight junctions,
• MeSH
• Adenylate Cyclase Toxin genetics   metabolism   toxicity   MeSH
• Cyclic AMP metabolism   MeSH
• Bordetella pertussis genetics   metabolism   MeSH
• Bronchi cytology   metabolism   microbiology   MeSH
• Cytoskeleton metabolism   MeSH
• Epithelial Cells metabolism   microbiology   MeSH
• Interleukin-6 metabolism   MeSH
• Humans MeSH
• Mucin 5AC metabolism   MeSH
• Whooping Cough genetics   metabolism   microbiology   MeSH
• Signal Transduction drug effects   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
• Interleukin-6 MeSH
• Mucin 5AC MeSH

Bordetellae, pathogenic to mammals, produce an immunomodulatory adenylate cyclase toxin-hemolysin (CyaA, ACT or AC-Hly) that enables them to overcome the innate immune defense of the host. CyaA subverts host phagocytic cells by an orchestrated action of its functional domains, where an extremely catalytically active adenylyl cyclase enzyme is delivered into phagocyte cytosol by a pore-forming repeat-in-toxin (RTX) cytolysin moiety. By targeting sentinel cells expressing the complement receptor 3, known as the CD11b/CD18 (αMβ₂) integrin, CyaA compromises the bactericidal functions of host phagocytes and supports infection of host airways by Bordetellae. Here, we review the state of knowledge on structural and functional aspects of CyaA toxin action, placing particular emphasis on signaling mechanisms by which the toxin-produced 3',5'-cyclic adenosine monophosphate (cAMP) subverts the physiology of phagocytic cells.

• Keywords
• Bordetella, CD11b/CD18, adenylate cyclase toxin, cAMP, cell signaling, complement receptor 3, innate immunity, membrane pores, repeats-in-toxin, β2 integrins,
• MeSH
• Adenylate Cyclase Toxin chemistry   MeSH
• Macrophages, Alveolar cytology   MeSH
• Cyclic AMP chemistry   MeSH
• Bordetella pertussis MeSH
• Dendritic Cells cytology   MeSH
• Phagocytes chemistry   MeSH
• Syk Kinase MeSH
• Humans MeSH
• Macrophage-1 Antigen MeSH
• Neutrophils cytology   MeSH
• Protein Domains MeSH
• Signal Transduction * MeSH
• Protein Structure, Tertiary MeSH
• Structure-Activity Relationship MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Review MeSH
• Names of Substances
• Adenylate Cyclase Toxin MeSH
• Cyclic AMP MeSH
• Syk Kinase MeSH
• Macrophage-1 Antigen MeSH
• SYK protein, human MeSH Browser

The adenylate cyclase toxin-hemolysin (CyaA, ACT or AC-Hly) translocates its adenylate cyclase (AC) enzyme domain into target cells in a step that depends on membrane cholesterol content. We thus examined what role in toxin activities is played by the five putative cholesterol recognition amino acid consensus (CRAC) motifs predicted in CyaA hemolysin moiety. CRAC-disrupting phenylalanine substitutions had no impact on toxin activities and these were not inhibited by free cholesterol, showing that the putative CRAC motifs are not involved in cholesterol binding. However, helix-breaking proline substitutions in these segments uncovered a structural role of the Y632, Y658, Y725 and Y738 residues in AC domain delivery and pore formation by CyaA. Substitutions of Y940 of the fifth motif, conserved in the acylated domains of related RTX toxins, did not impact on fatty-acylation of CyaA by CyaC and the CyaA-Y940F mutant was intact for toxin activities on erythrocytes and myeloid cells. However, the Y940A or Y940P substitutions disrupted the capacity of CyaA to insert into artificial lipid bilayers or target cell membranes. The aromatic ring of tyrosine 940 side chain thus appears to play a key structural role in molecular interactions that initiate CyaA penetration into target membranes.

• MeSH
• Adenylate Cyclase Toxin genetics   metabolism   MeSH
• Amino Acid Motifs MeSH
• Cell Membrane metabolism   MeSH
• Cell Line MeSH
• Cholesterol metabolism   MeSH
• Erythrocytes metabolism   MeSH
• Macrophages metabolism   MeSH
• DNA Mutational Analysis MeSH
• Mice MeSH
• Amino Acid Substitution MeSH
• Protein Transport MeSH
• Tyrosine genetics   metabolism   MeSH
• Protein Binding 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
• Cholesterol MeSH
• Tyrosine MeSH

The whooping cough agent, Bordetella pertussis, secretes an adenylate cyclase toxin-hemolysin (CyaA) that plays a crucial role in host respiratory tract colonization. CyaA targets CR3-expressing cells and disrupts their bactericidal functions by delivering into their cytosol an adenylate cyclase enzyme that converts intracellular ATP to cAMP. In parallel, the hydrophobic domain of CyaA forms cation-selective pores that permeabilize cell membrane. The invasive AC and pore-forming domains of CyaA are linked by a segment that is unique in the RTX cytolysin family. We used mass spectrometry and circular dichroism to show that the linker segment forms α-helical structures that penetrate into lipid bilayer. Replacement of the positively charged arginine residues, proposed to be involved in target membrane destabilization by the linker segment, reduced the capacity of the toxin to translocate the AC domain across cell membrane. Substitutions of negatively charged residues then revealed that two clusters of negative charges within the linker segment control the size and the propensity of CyaA pore formation, thereby restricting the cell-permeabilizing capacity of CyaA. The 'AC to Hly-linking segment' thus appears to account for the smaller size and modest cell-permeabilizing capacity of CyaA pores, as compared to typical RTX hemolysins.

• MeSH
• Adenylate Cyclase Toxin chemistry   genetics   metabolism   MeSH
• Adenylyl Cyclases chemistry   genetics   MeSH
• Cyclic AMP metabolism   MeSH
• Bordetella pertussis chemistry   pathogenicity   MeSH
• Hemolysin Proteins genetics   MeSH
• Humans MeSH
• Lipid Bilayers chemistry   metabolism   MeSH
• Perforin chemistry   MeSH
• Cell Membrane Permeability drug effects   MeSH
• Whooping Cough genetics   microbiology   pathology   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
• Adenylyl Cyclases MeSH
• Cyclic AMP MeSH
• Hemolysin Proteins MeSH
• Lipid Bilayers MeSH
• Perforin MeSH