Bordetella adenylate cyclase toxin (CyaA) binds the alpha(M)beta(2) integrin (CD11b/CD18, Mac-1, or CR3) of myeloid phagocytes and delivers into their cytosol an adenylate cyclase (AC) enzyme that converts ATP into the key signaling molecule cAMP. We show that penetration of the AC domain across cell membrane proceeds in two steps. It starts by membrane insertion of a toxin 'translocation intermediate', which can be 'locked' in the membrane by the 3D1 antibody blocking AC domain translocation. Insertion of the 'intermediate' permeabilizes cells for influx of extracellular calcium ions and thus activates calpain-mediated cleavage of the talin tether. Recruitment of the integrin-CyaA complex into lipid rafts follows and the cholesterol-rich lipid environment promotes translocation of the AC domain across cell membrane. AC translocation into cells was inhibited upon raft disruption by cholesterol depletion, or when CyaA mobilization into rafts was blocked by inhibition of talin processing. Furthermore, CyaA mutants unable to mobilize calcium into cells failed to relocate into lipid rafts, and failed to translocate the AC domain across cell membrane, unless rescued by Ca(2+) influx promoted in trans by ionomycin or another CyaA protein. Hence, by mobilizing calcium ions into phagocytes, the 'translocation intermediate' promotes toxin piggybacking on integrin into lipid rafts and enables AC enzyme delivery into host cytosol.

Institute of Microbiology AS CR v v i Prague Czech Republic

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Friedman RL, Fiederlein RL, Glasser L, Galgiani JN. Bordetella pertussis adenylate cyclase: effects of affinity-purified adenylate cyclase on human polymorphonuclear leukocyte functions. Infect Immun. 1987;55:135–140. PubMed PMC

Kamanova J, Kofronova O, Masin J, Genth H, Vojtova J, et al. Adenylate cyclase toxin subverts phagocyte function by RhoA inhibition and unproductive ruffling. J Immunol. 2008;181:5587–5597. PubMed

Confer DL, Eaton JW. Phagocyte impotence caused by an invasive bacterial adenylate cyclase. Science. 1982;217:948–950. PubMed

Weingart CL, Weiss AA. Bordetella pertussis virulence factors affect phagocytosis by human neutrophils. Infect Immun. 2000;68:1735–1739. PubMed PMC

Goodwin MS, Weiss AA. Adenylate cyclase toxin is critical for colonization and pertussis toxin is critical for lethal infection by Bordetella pertussis in infant mice. Infect Immun. 1990;58:3445–3447. PubMed PMC

Bellalou J, Sakamoto H, Ladant D, Geoffroy C, Ullmann A. Deletions affecting hemolytic and toxin activities of Bordetella pertussis adenylate cyclase. Infect Immun. 1990;58:3242–3247. PubMed PMC

Benz R, Maier E, Ladant D, Ullmann A, Sebo P. Adenylate cyclase toxin (CyaA) of Bordetella pertussis. Evidence for the formation of small ion-permeable channels and comparison with HlyA of Escherichia coli. J Biol Chem. 1994;269:27231–27239. 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

Hewlett EL, Donato GM, Gray MC. Macrophage cytotoxicity produced by adenylate cyclase toxin from Bordetella pertussis: more than just making cyclic AMP! Mol Microbiol. 2006;59:447–459. PubMed

Vojtova-Vodolanova J, Basler M, Osicka R, Knapp O, Maier E, et al. Oligomerization is involved in pore formation by Bordetella adenylate cyclase toxin. Faseb J. 2009;23:2831–2843. PubMed

Vojtova J, Kamanova J, Sebo P. Bordetella adenylate cyclase toxin: a swift saboteur of host defense. Curr Opin Microbiol. 2006;9:69–75. PubMed

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

Hackett M, Walker CB, Guo L, Gray MC, Van Cuyk S, et al. Hemolytic, but not cell-invasive activity, of adenylate cyclase toxin is selectively affected by differential fatty-acylation in Escherichia coli. J Biol Chem. 1995;270:20250–20253. PubMed

Rose T, Sebo P, Bellalou J, Ladant D. Interaction of calcium with Bordetella pertussis adenylate cyclase toxin. Characterization of multiple calcium-binding sites and calcium-induced conformational changes. J Biol Chem. 1995;270:26370–26376. PubMed

Gordon VM, Leppla SH, Hewlett EL. Inhibitors of receptor-mediated endocytosis block the entry of Bacillus anthracis adenylate cyclase toxin but not that of Bordetella pertussis adenylate cyclase toxin. Infect Immun. 1988;56:1066–1069. PubMed PMC

Gmira S, Karimova G, Ladant D. Characterization of recombinant Bordetella pertussis adenylate cyclase toxins carrying passenger proteins. Res Microbiol. 2001;152:889–900. PubMed

Otero AS, Yi XB, Gray MC, Szabo G, Hewlett EL. Membrane depolarization prevents cell invasion by Bordetella pertussis adenylate cyclase toxin. J Biol Chem. 1995;270:9695–9697. PubMed

Fiser R, Masin J, Basler M, Krusek J, Spulakova V, et al. Third activity of Bordetella adenylate cyclase (AC) toxin-hemolysin. Membrane translocation of AC domain polypeptide promotes calcium influx into CD11b+ monocytes independently of the catalytic and hemolytic activities. J Biol Chem. 2007;282:2808–2820. PubMed

Basler M, Knapp O, Masin J, Fiser R, Maier E, et al. Segments crucial for membrane translocation and pore-forming activity of Bordetella adenylate cyclase toxin. J Biol Chem. 2007;282:12419–12429. PubMed

Osickova A, Osicka R, Maier E, Benz R, Sebo P. An amphipathic alpha-helix including glutamates 509 and 516 is crucial for membrane translocation of adenylate cyclase toxin and modulates formation and cation selectivity of its membrane channels. J Biol Chem. 1999;274:37644–37650. PubMed

Ladant D, Ullmann A. Bordatella pertussis adenylate cyclase: a toxin with multiple talents. Trends Microbiol. 1999;7:172–176. PubMed

Paccani SR, Dal Molin F, Benagiano M, Ladant D, D'Elios MM, et al. Suppression of T-lymphocyte activation and chemotaxis by the adenylate cyclase toxin of Bordetella pertussis. Infect Immun. 2008;76:2822–2832. PubMed PMC

Gordon VM, Young WW, Jr, Lechler SM, Gray MC, Leppla SH, et al. Adenylate cyclase toxins from Bacillus anthracis and Bordetella pertussis. Different processes for interaction with and entry into target cells. J Biol Chem. 1989;264:14792–14796. PubMed

Morova J, Osicka R, Masin J, Sebo P. RTX cytotoxins recognize beta2 integrin receptors through N-linked oligosaccharides. Proc Natl Acad Sci U S A. 2008;105:5355–5360. PubMed PMC

Harris ES, McIntyre TM, Prescott SM, Zimmerman GA. The leukocyte integrins. J Biol Chem. 2000;275:23409–23412. PubMed

Hynes RO. Integrins: bidirectional, allosteric signaling machines. Cell. 2002;110:673–687. PubMed

Leitinger B, Hogg N. The involvement of lipid rafts in the regulation of integrin function. J Cell Sci. 2002;115:963–972. PubMed

Krauss K, Altevogt P. Integrin leukocyte function-associated antigen-1-mediated cell binding can be activated by clustering of membrane rafts. J Biol Chem. 1999;274:36921–36927. PubMed

Stefanova I, Horejsi V, Ansotegui IJ, Knapp W, Stockinger H. GPI-anchored cell-surface molecules complexed to protein tyrosine kinases. Science. 1991;254:1016–1019. PubMed

Simons K, Ikonen E. Functional rafts in cell membranes. Nature. 1997;387:569–572. PubMed

Brown DA. Lipid rafts, detergent-resistant membranes, and raft targeting signals. Physiology (Bethesda) 2006;21:430–439. PubMed

Jacobson K, Mouritsen OG, Anderson RG. Lipid rafts: at a crossroad between cell biology and physics. Nat Cell Biol. 2007;9:7–14. PubMed

Helms JB, Zurzolo C. Lipids as targeting signals: lipid rafts and intracellular trafficking. Traffic. 2004;5:247–254. PubMed

Lafont F, van der Goot FG. Bacterial invasion via lipid rafts. Cell Microbiol. 2005;7:613–620. PubMed

Manes S, del Real G, Martinez AC. Pathogens: raft hijackers. Nat Rev Immunol. 2003;3:557–568. PubMed

Fivaz M, Abrami L, van der Goot FG. Landing on lipid rafts. Trends Cell Biol. 1999;9:212–213. PubMed

Lee SJ, Gray MC, Guo L, Sebo P, Hewlett EL. Epitope mapping of monoclonal antibodies against Bordetella pertussis adenylate cyclase toxin. Infect Immun. 1999;67:2090–2095. PubMed PMC

Rogel A, Hanski E. Distinct steps in the penetration of adenylate cyclase toxin of Bordetella pertussis into sheep erythrocytes. Translocation of the toxin across the membrane. J Biol Chem. 1992;267:22599–22605. PubMed

Gray MC, Lee SJ, Gray LS, Zaretzky FR, Otero AS, et al. Translocation-specific conformation of adenylate cyclase toxin from Bordetella pertussis inhibits toxin-mediated hemolysis. J Bacteriol. 2001;183:5904–5910. PubMed PMC

Sampath R, Gallagher PJ, Pavalko FM. Cytoskeletal interactions with the leukocyte integrin beta2 cytoplasmic tail. Activation-dependent regulation of associations with talin and alpha-actinin. J Biol Chem. 1998;273:33588–33594. PubMed PMC

Stewart MP, McDowall A, Hogg N. LFA-1-mediated adhesion is regulated by cytoskeletal restraint and by a Ca2+-dependent protease, calpain. J Cell Biol. 1998;140:699–707. PubMed PMC

Guermonprez P, Khelef N, Blouin E, Rieu P, Ricciardi-Castagnoli P, et al. The adenylate cyclase toxin of Bordetella pertussis binds to target cells via the alpha(M)beta(2) integrin (CD11b/CD18). J Exp Med. 2001;193:1035–1044. PubMed PMC

Vang T, Torgersen KM, Sundvold V, Saxena M, Levy FO, et al. Activation of the COOH-terminal Src kinase (Csk) by cAMP-dependent protein kinase inhibits signaling through the T cell receptor. J Exp Med. 2001;193:497–507. PubMed PMC

Willoughby D, Wong W, Schaack J, Scott JD, Cooper DM. An anchored PKA and PDE4 complex regulates subplasmalemmal cAMP dynamics. Embo J. 2006;25:2051–2061. PubMed PMC

Tasken K, Stokka AJ. The molecular machinery for cAMP-dependent immunomodulation in T-cells. Biochem Soc Trans. 2006;34:476–479. PubMed

Steed LL, Akporiaye ET, Friedman RL. Bordetella pertussis induces respiratory burst activity in human polymorphonuclear leukocytes. Infect Immun. 1992;60:2101–2105. PubMed PMC

Lafont F, Abrami L, van der Goot FG. Bacterial subversion of lipid rafts. Curr Opin Microbiol. 2004;7:4–10. PubMed

Parton RG. Ultrastructural localization of gangliosides; GM1 is concentrated in caveolae. J Histochem Cytochem. 1994;42:155–166. PubMed

Fivaz M, Vilbois F, Thurnheer S, Pasquali C, Abrami L, et al. Differential sorting and fate of endocytosed GPI-anchored proteins. Embo J. 2002;21:3989–4000. PubMed PMC

Waheed AA, Shimada Y, Heijnen HF, Nakamura M, Inomata M, et al. Selective binding of perfringolysin O derivative to cholesterol-rich membrane microdomains (rafts). Proc Natl Acad Sci U S A. 2001;98:4926–4931. PubMed PMC

Osickova A, Masin J, Fayolle C, Krusek J, Basler M, et al. Adenylate cyclase toxin translocates across target cell membrane without forming a pore. Mol Microbiol. 2010;75:1550–1562. PubMed

Vojtova J, Kofronova O, Sebo P, Benada O. Bordetella adenylate cyclase toxin induces a cascade of morphological changes of sheep erythrocytes and localizes into clusters in erythrocyte membranes. Microsc Res Tech. 2006;69:119–129. PubMed

Martin C, Requero MA, Masin J, Konopasek I, Goni FM, et al. Membrane restructuring by Bordetella pertussis adenylate cyclase toxin, a member of the RTX toxin family. J Bacteriol. 2004;186:3760–3765. PubMed PMC

Ahmed SN, Brown DA, London E. On the origin of sphingolipid/cholesterol-rich detergent-insoluble cell membranes: physiological concentrations of cholesterol and sphingolipid induce formation of a detergent-insoluble, liquid-ordered lipid phase in model membranes. Biochemistry. 1997;36:10944–10953. PubMed

Silvius JR, del Giudice D, Lafleur M. Cholesterol at different bilayer concentrations can promote or antagonize lateral segregation of phospholipids of differing acyl chain length. Biochemistry. 1996;35:15198–15208. PubMed

Scotto AW, Zakim D. Reconstitution of membrane proteins: catalysis by cholesterol of insertion of integral membrane proteins into preformed lipid bilayers. Biochemistry. 1986;25:1555–1561. PubMed

Goll DE, Thompson VF, Li H, Wei W, Cong J. The calpain system. Physiol Rev. 2003;83:731–801. PubMed

Fong KP, Pacheco CM, Otis LL, Baranwal S, Kieba IR, et al. Actinobacillus actinomycetemcomitans leukotoxin requires lipid microdomains for target cell cytotoxicity. Cell Microbiol. 2006;8:1753–1767. PubMed PMC

Davies EV, Hallett MB. High micromolar Ca2+ beneath the plasma membrane in stimulated neutrophils. Biochem Biophys Res Commun. 1998;248:679–683. PubMed

Franken KL, Hiemstra HS, van Meijgaarden KE, Subronto Y, den Hartigh J, et al. Purification of his-tagged proteins by immobilized chelate affinity chromatography: the benefits from the use of organic solvent. Protein Expr Purif. 2000;18:95–99. PubMed

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, et al. 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

Osicka R, Osickova A, Basar T, Guermonprez P, Rojas M, et al. Delivery of CD8(+) T-cell epitopes into major histocompatibility complex class I antigen presentation pathway by Bordetella pertussis adenylate cyclase: delineation of cell invasive structures and permissive insertion sites. Infect Immun. 2000;68:247–256. PubMed PMC

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