Bordetella adenylate cyclase toxin-hemolysin (CyaA) penetrates the cytoplasmic membrane of phagocytes and employs two distinct conformers to exert its multiple activities. One conformer forms cation-selective pores that permeabilize phagocyte membrane for efflux of cytosolic potassium. The other conformer conducts extracellular calcium ions across cytoplasmic membrane of cells, relocates into lipid rafts, translocates the adenylate cyclase enzyme (AC) domain into cells and converts cytosolic ATP to cAMP. We show that the calcium-conducting activity of CyaA controls the path and kinetics of endocytic removal of toxin pores from phagocyte membrane. The enzymatically inactive but calcium-conducting CyaA-AC⁻ toxoid was endocytosed via a clathrin-dependent pathway. In contrast, a doubly mutated (E570K+E581P) toxoid, unable to conduct Ca²⁺ into cells, was rapidly internalized by membrane macropinocytosis, unless rescued by Ca²⁺ influx promoted in trans by ionomycin or intact toxoid. Moreover, a fully pore-forming CyaA-ΔAC hemolysin failed to permeabilize phagocytes, unless endocytic removal of its pores from cell membrane was decelerated through Ca²⁺ influx promoted by molecules locked in a Ca²⁺-conducting conformation by the 3D1 antibody. Inhibition of endocytosis also enabled the native B. pertussis-produced CyaA to induce lysis of J774A.1 macrophages at concentrations starting from 100 ng/ml. Hence, by mediating calcium influx into cells, the translocating conformer of CyaA controls the removal of bystander toxin pores from phagocyte membrane. This triggers a positive feedback loop of exacerbated cell permeabilization, where the efflux of cellular potassium yields further decreased toxin pore removal from cell membrane and this further enhances cell permeabilization and potassium efflux.

Faculty of Science Charles University Prague Czech Republic

Zobrazit více v 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

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

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

Gray M, Szabo G, Otero AS, 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

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

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

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

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

Sebo P, Glaser P, Sakamoto H, Ullmann A. High-level synthesis of active adenylate cyclase toxin of Bordetella pertussis in a reconstructed Escherichia coli system. Gene. 1991;104:19–24. 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

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

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

Bumba L, Masin J, Fiser R, Sebo P. Bordetella adenylate cyclase toxin mobilizes its beta2 integrin receptor into lipid rafts to accomplish translocation across target cell membrane in two steps. PLoS Pathog. 2010;6:e1000901. PubMed PMC

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

Guermonprez P, Ladant D, Karimova G, Ullmann A, Leclerc C. Direct delivery of the Bordetella pertussis adenylate cyclase toxin to the MHC class I antigen presentation pathway. J Immunol. 1999;162:1910–1916. PubMed

Schlecht G, Loucka J, Najar H, Sebo P, Leclerc C. Antigen targeting to CD11b allows efficient presentation of CD4+ and CD8+ T cell epitopes and in vivo Th1-polarized T cell priming. J Immunol. 2004;173:6089–6097. PubMed

Khelef N, Gounon P, Guiso N. Internalization of Bordetella pertussis adenylate cyclase-haemolysin into endocytic vesicles contributes to macrophage cytotoxicity. Cell Microbiol. 2001;3:721–730. PubMed

Martin C, Uribe KB, Gomez-Bilbao G, Ostolaza H. Adenylate cyclase toxin promotes internalisation of integrins and raft components and decreases macrophage adhesion capacity. PLoS One. 2011;6:e17383. 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

Mousavi SA, Malerod L, Berg T, Kjeken R. Clathrin-dependent endocytosis. Biochem J. 2004;377:1–16. PubMed PMC

Benmerah A, Bayrou M, Cerf-Bensussan N, Dautry-Varsat A. Inhibition of clathrin-coated pit assembly by an Eps15 mutant. J Cell Sci. 1999;112(Pt 9):1303–1311. PubMed

Bain J, Plater L, Elliott M, Shpiro N, Hastie CJ, et al. The selectivity of protein kinase inhibitors: a further update. Biochem J. 2007;408:297–315. PubMed PMC

Macia E, Ehrlich M, Massol R, Boucrot E, Brunner C, et al. Dynasore, a cell-permeable inhibitor of dynamin. Dev Cell. 2006;10:839–850. PubMed

Wang LH, Rothberg KG, Anderson RG. Mis-assembly of clathrin lattices on endosomes reveals a regulatory switch for coated pit formation. J Cell Biol. 1993;123:1107–1117. PubMed PMC

Lo-Man R, Langeveld JP, Deriaud E, Jehanno M, Rojas M, et al. Extending the CD4(+) T-cell epitope specificity of the Th1 immune response to an antigen using a Salmonella enterica serovar typhimurium delivery vehicle. Infect Immun. 2000;68:3079–3089. PubMed PMC

Havlicek V, Higgins L, Chen W, Halada P, Sebo P, et al. Mass spectrometric analysis of recombinant adenylate cyclase toxin from Bordetella pertussis strain 18323/pHSP9. J Mass Spectrom. 2001;36:384–391. PubMed

Dunne A, Ross PJ, Pospisilova E, Masin J, Meaney A, et al. Inflammasome activation by adenylate cyclase toxin directs Th17 responses and protection against Bordetella pertussis. J Immunol. 2010;185:1711–1719. PubMed

Madshus IH, Sandvig K, Olsnes S, van Deurs B. Effect of reduced endocytosis induced by hypotonic shock and potassium depletion on the infection of Hep 2 cells by picornaviruses. J Cell Physiol. 1987;131:14–22. PubMed

Larkin JM, Brown MS, Goldstein JL, Anderson RG. Depletion of intracellular potassium arrests coated pit formation and receptor-mediated endocytosis in fibroblasts. Cell. 1983;33:273–285. PubMed

Prior S, Corbel MJ, Xing DK. Development of an approach for the laboratory toxicological evaluation of Bordetella pertussis adenylate cyclase genetic toxoid constructs as multipurpose vaccines. Hum Vaccin. 2005;1:151–159. PubMed

Saron MF, Fayolle C, Sebo P, Ladant D, Ullmann A, et al. Anti-viral protection conferred by recombinant adenylate cyclase toxins from Bordetella pertussis carrying a CD8+ T cell epitope from lymphocytic choriomeningitis virus. Proc Natl Acad Sci U S A. 1997;94:3314–3319. PubMed PMC

Fayolle C, Ladant D, Karimova G, Ullmann A, Leclerc C. Therapy of murine tumors with recombinant Bordetella pertussis adenylate cyclase carrying a cytotoxic T cell epitope. J Immunol. 1999;162:4157–4162. PubMed

Fayolle C, Osickova A, Osicka R, Henry T, Rojas MJ, et al. Delivery of multiple epitopes by recombinant detoxified adenylate cyclase of Bordetella pertussis induces protective antiviral immunity. J Virol. 2001;75:7330–7338. PubMed PMC

Dadaglio G, Moukrim Z, Lo-Man R, Sheshko V, Sebo P, et al. Induction of a polarized Th1 response by insertion of multiple copies of a viral T-cell epitope into adenylate cyclase of Bordetella pertussis. Infect Immun. 2000;68:3867–3872. PubMed PMC

Cheung GY, Dickinson P, Sing G, Craigon M, Ghazal P, et al. Transcriptional responses of murine macrophages to the adenylate cyclase toxin of Bordetella pertussis. Microb Pathog. 2008;44:61–70. 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

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

Karimova G, Pidoux J, Ullmann A, Ladant D. A bacterial two-hybrid system based on a reconstituted signal transduction pathway. Proc Natl Acad Sci U S A. 1998;95:5752–5756. PubMed PMC

Boes M, Cerny J, Massol R, Op den Brouw M, Kirchhausen T, et al. T-cell engagement of dendritic cells rapidly rearranges MHC class II transport. Nature. 2002;418:983–988. PubMed

Lutz MB, Kukutsch N, Ogilvie AL, Rossner S, Koch F, et al. An advanced culture method for generating large quantities of highly pure dendritic cells from mouse bone marrow. J Immunol Methods. 1999;223:77–92. PubMed

Kingella kingae RtxA Cytotoxin in the Context of Other RTX Toxins

Almost half of the RTX domain is dispensable for complement receptor 3 binding and cell-invasive activity of the Bordetella adenylate cyclase toxin

Distinct Spatiotemporal Distribution of Bacterial Toxin-Produced Cellular cAMP Differentially Inhibits Opsonophagocytic Signaling

Residues 529 to 549 participate in membrane penetration and pore-forming activity of the Bordetella adenylate cyclase toxin

Bordetella Pertussis Adenylate Cyclase Toxin Does Not Possess a Phospholipase A Activity; Serine 606 and Aspartate 1079 Residues Are Not Involved in Target Cell Delivery of the Adenylyl Cyclase Enzyme Domain

Bordetella pertussis Adenylate Cyclase Toxin Disrupts Functional Integrity of Bronchial Epithelial Layers

Phosphoproteomics of cAMP signaling of Bordetella adenylate cyclase toxin in mouse dendritic cells

Structure-Function Relationships Underlying the Capacity of Bordetella Adenylate Cyclase Toxin to Disarm Host Phagocytes

Invasion of Dendritic Cells, Macrophages and Neutrophils by the Bordetella Adenylate Cyclase Toxin: A Subversive Move to Fool Host Immunity

The conserved tyrosine residue 940 plays a key structural role in membrane interaction of Bordetella adenylate cyclase toxin

Design and Synthesis of Fluorescent Acyclic Nucleoside Phosphonates as Potent Inhibitors of Bacterial Adenylate Cyclases

Pore-formation by adenylate cyclase toxoid activates dendritic cells to prime CD8+ and CD4+ T cells

Bordetella adenylate cyclase toxin: a unique combination of a pore-forming moiety with a cell-invading adenylate cyclase enzyme

Amidate prodrugs of 9-[2-(phosphonomethoxy)ethyl]adenine as inhibitors of adenylate cyclase toxin from Bordetella pertussis

Differences in purinergic amplification of osmotic cell lysis by the pore-forming RTX toxins Bordetella pertussis CyaA and Actinobacillus pleuropneumoniae ApxIA: the role of pore size