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.

Institute of Microbiology Academy of Sciences of the Czech Republic Prague Czech Republic

Institute of Microbiology Academy of Sciences of the Czech Republic Prague Czech Republic; University of Chemistry and Technology Prague Prague Czech Republic

Zobrazit více v PubMed

Novak J., Cerny O., Osickova A., Linhartova I., Masin J., Bumba L., Sebo P., Osicka R. Structure-function relationships underlying the capacity of Bordetella adenylate cyclase toxin to disarm host phagocytes. Toxins. 2017;9:300. PubMed PMC

Guiso N. Bordetella adenylate cyclase-hemolysin toxins. Toxins. 2017;9:277. PubMed PMC

Sakamoto H., Bellalou J., Sebo P., Ladant D. Bordetella pertussis adenylate cyclase toxin. Structural and functional independence of the catalytic and hemolytic activities. J. Biol. Chem. 1992;267:13598–13602. PubMed

Hackett M., Guo L., Shabanowitz J., Hunt D.F., Hewlett E.L. Internal lysine palmitoylation in adenylate cyclase toxin from Bordetella pertussis. Science. 1994;266:433–435. PubMed

Basar T., Havlicek V., Bezouskova S., Halada P., Hackett M., Sebo P. The conserved lysine 860 in the additional fatty-acylation site of Bordetella pertussis adenylate cyclase is crucial for toxin function independently of its acylation status. J. Biol. Chem. 1999;274:10777–10783. PubMed

Osickova A., Khaliq H., Masin J., Jurnecka D., Sukova A., Fiser R., Holubova J., Stanek O., Sebo P., Osicka R. Acyltransferase-mediated selection of the length of the fatty acyl chain and of the acylation site governs activation of bacterial RTX toxins. J. Biol. Chem. 2020;295:9268–9280. PubMed PMC

Bumba L., Masin J., Fiser R., Sebo P. Bordetella adenylate cyclase toxin mobilizes its β2 integrin receptor into lipid rafts to accomplish translocation across target cell membrane in two steps. PLoS Pathog. 2010;6 PubMed PMC

Holubova J., Kamanova J., Jelinek J., Tomala J., Masin J., Kosova M., Stanek O., Bumba L., Michalek J., Kovar M., Sebo P. Delivery of large heterologous polypeptides across the cytoplasmic membrane of antigen-presenting cells by the Bordetella RTX hemolysin moiety lacking the adenylyl cyclase domain. Infect. Immun. 2012;80:1181–1192. PubMed PMC

Voegele A., O'Brien D.P., Subrini O., Sapay N., Cannella S.E., Enguene V.Y.N., Hessel A., Karst J., Hourdel V., Perez A.C.S., Davi M., Veneziano R., Chopineau J., Vachette P., Durand D. Translocation and calmodulin-activation of the adenylate cyclase toxin (CyaA) of Bordetella pertussis. Pathog. Dis. 2018;76:fty085. PubMed

Fedele G., Schiavoni I., Adkins I., Klimova N., Sebo P. Invasion of dendritic cells, macrophages and neutrophils by the Bordetella adenylate cyclase toxin: A subversive move to fool host immunity. Toxins. 2017;9:293. PubMed PMC

Ahmad J.N., Holubova J., Benada O., Kofronova O., Stehlik L., Vasakova M., Sebo P. Bordetella adenylate cyclase toxin inhibits monocyte-to-macrophage transition and dedifferentiates human alveolar macrophages into monocyte-like cells. mBio. 2019;10 e01743-19. PubMed PMC

Knapp O., Benz R. Membrane activity and channel formation of the adenylate cyclase toxin (CyaA) of Bordetella pertussis in lipid bilayer membranes. Toxins. 2020;12:169. PubMed PMC

Fiser R., Masin J., Bumba L., Pospisilova E., Fayolle C., Basler M., Sadilkova L., Adkins I., Kamanova J., Cerny J., Konopasek I., Osicka R., Leclerc C., Sebo P. Calcium influx rescues adenylate cyclase-hemolysin from rapid cell membrane removal and enables phagocyte permeabilization by toxin pores. PLoS Pathog. 2012;8 PubMed PMC

Wald T., Petry-Podgorska I., Fiser R., Matousek T., Dedina J., Osicka R., Sebo P., Masin J. Quantification of potassium levels in cells treated with Bordetella adenylate cyclase toxin. Anal. Biochem. 2014;450:57–62. PubMed

Gray M., Szabo G., Otero A.S., Gray L., Hewlett E. Distinct mechanisms for K+ efflux, intoxication, and hemolysis by Bordetella pertussis AC toxin. J. Biol. Chem. 1998;273:18260–18267. PubMed

Ehrmann I.E., Gray M.C., Gordon V.M., Gray L.S., Hewlett E.L. Hemolytic activity of adenylate cyclase toxin from Bordetella pertussis. FEBS Lett. 1991;278:79–83. PubMed

Basler M., Masin J., Osicka R., Sebo P. Pore-forming and enzymatic activities of Bordetella pertussis adenylate cyclase toxin synergize in promoting lysis of monocytes. Infect. Immun. 2006;74:2207–2214. PubMed PMC

Sukova A., Bumba L., Srb P., Veverka V., Stanek O., Holubova J., Chmelik J., Fiser R., Sebo P., Masin J. Negative charge of the AC-to-Hly linking segment modulates calcium-dependent membrane activities of Bordetella adenylate cyclase toxin. Biochim. Biophys. Acta Biomembr. 2020;1862:183310. PubMed

Subrini O., Sotomayor-Perez A.C., Hessel A., Spiaczka-Karst J., Selwa E., Sapay N., Veneziano R., Pansieri J., Chopineau J., Ladant D., Chenal A. Characterization of a membrane-active peptide from the Bordetella pertussis CyaA toxin. J. Biol. Chem. 2013;288:32585–32598. PubMed PMC

Masin J., Roderova J., Osickova A., Novak P., Bumba L., Fiser R., Sebo P., Osicka R. The conserved tyrosine residue 940 plays a key structural role in membrane interaction of Bordetella adenylate cyclase toxin. Sci. Rep. 2017;7:9330. PubMed PMC

Masin J., Osickova A., Jurnecka D., Klimova N., Khaliq H., Sebo P., Osicka R. Retargeting from the CR3 to the LFA-1 receptor uncovers the adenylyl cyclase enzyme-translocating segment of Bordetella adenylate cyclase toxin. J. Biol. Chem. 2020;295:9349–9365. PubMed PMC

Baumann U. Structure-function relationships of the repeat domains of RTX Toxins. Toxins. 2019;11:E657. PubMed PMC

Chenal A., Karst J.C., Sotomayor Perez A.C., Wozniak A.K., Baron B., England P., Ladant D. Calcium-induced folding and stabilization of the intrinsically disordered RTX domain of the CyaA toxin. Biophys. J. 2010;99:3744–3753. PubMed PMC

Motlova L., Klimova N., Fiser R., Sebo P., Bumba L. Continuous assembly of β-roll structures is implicated in the Type I-dependent secretion of large Repeat-in-Toxins (RTX) proteins. J. Mol. Biol. 2020;432:5696–5710. PubMed

Bumba L., Masin J., Macek P., Wald T., Motlova L., Bibova I., Klimova N., Bednarova L., Veverka V., Kachala M., Svergun D.I., Barinka C., Sebo P. Calcium-driven folding of RTX domain β-rolls ratchets translocation of RTX proteins through Type I secretion ducts. Mol. Cell. 2016;62:47–62. PubMed

Guermonprez P., Khelef N., Blouin E., Rieu P., Ricciardi-Castagnoli P., Guiso N., Ladant D., Leclerc C. The adenylate cyclase toxin of Bordetella pertussis binds to target cells via the αMβ2 integrin (CD11b/CD18) J. Exp. Med. 2001;193:1035–1044. PubMed PMC

El-Azami-El-Idrissi M., Bauche C., Loucka J., Osicka R., Sebo P., Ladant D., Leclerc C. Interaction of Bordetella pertussis adenylate cyclase with CD11b/CD18: Role of toxin acylation and identification of the main integrin interaction domain. J. Biol. Chem. 2003;278:38514–38521. PubMed

Wang X., Stapleton J.A., Klesmith J.R., Hewlett E.L., Whitehead T.A., Maynard J.A. Fine epitope mapping of two antibodies neutralizing the Bordetella adenylate cyclase toxin. Biochemistry. 2017;56:1324–1336. PubMed PMC

Osicka R., Osickova A., Hasan S., Bumba L., Cerny J., Sebo P. Bordetella adenylate cyclase toxin is a unique ligand of the integrin complement receptor 3. Elife. 2015;4 PubMed PMC

Morova J., Osicka R., Masin J., Sebo P. RTX cytotoxins recognize β2 integrin receptors through N-linked oligosaccharides. Proc. Natl. Acad. Sci. U. S. A. 2008;105:5355–5360. PubMed PMC

Hasan S., Osickova A., Bumba L., Novak P., Sebo P., Osicka R. Interaction of Bordetella adenylate cyclase toxin with complement receptor 3 involves multivalent glycan binding. FEBS Lett. 2015;589:374–379. PubMed

Iwaki M., Ullmann A., Sebo P. Identification by in vitro complementation of regions required for cell-invasive activity of Bordetella pertussis adenylate cyclase toxin. Mol. Microbiol. 1995;17:1015–1024. PubMed

Bauche C., Chenal A., Knapp O., Bodenreider C., Benz R., Chaffotte A., Ladant D. Structural and functional characterization of an essential RTX subdomain of Bordetella pertussis adenylate cyclase toxin. J. Biol. Chem. 2006;281:16914–16926. PubMed

Betsou F., Sebo P., Guiso N. The C-terminal domain is essential for protective activity of the Bordetella pertussis adenylate cyclase-hemolysin. Infect. Immun. 1995;63:3309–3315. PubMed PMC

Betsou F., Sebo P., Guiso N. CyaC-mediated activation is important not only for toxic but also for protective activities of Bordetella pertussis adenylate cyclase-hemolysin. Infect. Immun. 1993;61:3583–3589. PubMed PMC

Wang X., Gray M.C., Hewlett E.L., Maynard J.A. The Bordetella adenylate cyclase repeat-in-toxin (RTX) domain is immunodominant and elicits neutralizing antibodies. J. Biol. Chem. 2015;290:3576–3591. PubMed PMC

Stanek O., Masin J., Osicka R., Jurnecka D., Osickova A., Sebo P. Rapid purification of endotoxin-free RTX toxins. Toxins. 2019;11:339. PubMed PMC

Szabo G., Gray M.C., Hewlett E.L. Adenylate cyclase toxin from Bordetella pertussis produces ion conductance across artificial lipid bilayers in a calcium- and polarity-dependent manner. J. Biol. Chem. 1994;269:22496–22499. PubMed

Blanchet C.E., Spilotros A., Schwemmer F., Graewert M.A., Kikhney A., Jeffries C.M., Franke D., Mark D., Zengerle R., Cipriani F., Fiedler S., Roessle M., Svergun D.I. Versatile sample environments and automation for biological solution X-ray scattering experiments at the P12 beamline (PETRA III, DESY) J. Appl. Crystallogr. 2015;48:431–443. PubMed PMC

Konarev P.V., Volkov V.V., Sokolova A.V., Koch M.H.J., Svergun D.I. PRIMUS : A Windows PC-based system for small-angle scattering data analysis. J. Appl. Crystallogr. 2003;36:1277–1282.

Svergun D. Determination of the regularization parameter in indirect-transform methods using perceptual criteria. J. Appl. Crystallogr. 1992;25:495–503.

Franke D., Svergun D.I. DAMMIF, a program for rapid ab-initio shape determination in small-angle scattering. J. Appl. Crystallogr. 2009;42:342–346. PubMed PMC

Volkov V.V., Svergun D.I. Uniqueness of ab initio shape determination in small-angle scattering. J. Appl. Crystallogr. 2003;36:860–864. PubMed PMC

Ladant D. Interaction of Bordetella pertussis adenylate cyclase with calmodulin. Identification of two separated calmodulin-binding domains. J. Biol. Chem. 1988;263:2612–2618. PubMed

Karimova G., Fayolle C., Gmira S., Ullmann A., Leclerc C., Ladant D. Charge-dependent translocation of Bordetella pertussis adenylate cyclase toxin into eukaryotic cells: Implication for the in vivo delivery of CD8+ T cell epitopes into antigen-presenting cells. Proc. Natl. Acad. Sci. U. S. A. 1998;95:12532–12537. PubMed PMC

Masin J., Osickova A., Sukova A., Fiser R., Halada P., Bumba L., Linhartova I., Osicka R., Sebo P. Negatively charged residues of the segment linking the enzyme and cytolysin moieties restrict the membrane-permeabilizing capacity of adenylate cyclase toxin. Sci. Rep. 2016;6:29137. PubMed PMC

The adenylate cyclase toxin RTX domain follows a series templated folding mechanism with implications for toxin activity

A conserved tryptophan in the acylated segment of RTX toxins controls their β2 integrin-independent cell penetration

Different roles of conserved tyrosine residues of the acylated domains in folding and activity of RTX toxins