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.

• Klíčová slova
• Bordetella pertussis, adenylate cyclase toxin, pertactin, pertussis, protection, protein folding, whooping cough,
• MeSH
• adenylátcyklasový toxin * imunologie   genetika   aplikace a dávkování   MeSH
• antigeny bakteriální * imunologie   genetika   aplikace a dávkování   MeSH
• aplikace intranazální MeSH
• Bordetella pertussis * imunologie   genetika   MeSH
• faktory virulence rodu Bordetella * imunologie   genetika   aplikace a dávkování   MeSH
• lidé MeSH
• myši inbrední BALB C MeSH
• myši MeSH
• neutralizující protilátky krev   imunologie   MeSH
• pertuse * prevence a kontrola   imunologie   mikrobiologie   MeSH
• pertusová vakcína * imunologie   aplikace a dávkování   genetika   MeSH
• proteiny vnější bakteriální membrány * imunologie   genetika   aplikace a dávkování   MeSH
• protilátky bakteriální krev   imunologie   MeSH
• rekombinantní fúzní proteiny imunologie   genetika   aplikace a dávkování   MeSH
• respirační sliznice * imunologie   mikrobiologie   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• adenylátcyklasový toxin * MeSH
• antigeny bakteriální * MeSH
• faktory virulence rodu Bordetella * MeSH
• neutralizující protilátky MeSH
• pertactin MeSH Prohlížeč
• pertusová vakcína * MeSH
• proteiny vnější bakteriální membrány * MeSH
• protilátky bakteriální MeSH
• rekombinantní fúzní proteiny 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.

• Klíčová slova
• adenylate cyclase, bacterial toxin, optical tweezers, protein folding, single-molecule biophysics,
• Publikační typ
• časopisecké články 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.

• Klíčová slova
• RTX toxin, acylated segment, adenylate cyclase toxin, cytotoxicity, hydrogen/deuterium exchange, thermal stability, tryptophan residue, α-hemolysin, β(2) integrins,
• MeSH
• adenylátcyklasový toxin * chemie   genetika   metabolismus   MeSH
• antigeny CD18 * genetika   metabolismus   MeSH
• Bordetella pertussis MeSH
• buněčná membrána metabolismus   MeSH
• erytrocyty metabolismus   MeSH
• konzervovaná sekvence MeSH
• tryptofan * chemie   genetika   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• adenylátcyklasový toxin * MeSH
• antigeny CD18 * MeSH
• tryptofan * 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
• adenylátcyklasový toxin genetika   MeSH
• Bordetella bronchiseptica * genetika   metabolismus   MeSH
• Bordetella pertussis * genetika   metabolismus   MeSH
• buněčná membrána metabolismus   MeSH
• hemolýza MeSH
• infekce bakteriemi rodu Bordetella mikrobiologie   MeSH
• lidé MeSH
• myši inbrední BALB C MeSH
• myši MeSH
• THP-1 buňky MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• adenylátcyklasový toxin MeSH