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.

• Klíčová slova
• Bordetella pertussis, RTX toxin, acylation, adenylate cyclase toxin, protein folding,
• MeSH
• acylace MeSH
• adenylátcyklasový toxin * metabolismus   chemie   genetika   MeSH
• Bordetella pertussis * metabolismus   enzymologie   genetika   MeSH
• buněčná membrána * metabolismus   MeSH
• lidé MeSH
• proteinové domény MeSH
• sbalování proteinů MeSH
• vápník metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• adenylátcyklasový toxin * MeSH
• vápník 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.

• 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