The adenylate cyclase toxin-hemolysin (CyaA, ACT, or AC-Hly) of Bordetella pertussis targets phagocytic cells expressing the complement receptor 3 (CR3, Mac-1, αMβ2 integrin, or CD11b/CD18). CyaA delivers into cells an N-terminal adenylyl cyclase (AC) enzyme domain that is activated by cytosolic calmodulin and catalyzes unregulated conversion of cellular ATP into cyclic AMP (cAMP), a key second messenger subverting bactericidal activities of phagocytes. In parallel, the hemolysin (Hly) moiety of CyaA forms cation-selective hemolytic pores that permeabilize target cell membranes. We constructed the first B. pertussis mutant secreting a CyaA toxin having an intact capacity to deliver the AC enzyme into CD11b-expressing (CD11b+) host phagocytes but impaired in formation of cell-permeabilizing pores and defective in cAMP elevation in CD11b- cells. The nonhemolytic AC+ Hly- bacteria inhibited the antigen-presenting capacities of coincubated mouse dendritic cells in vitro and skewed their Toll-like receptor (TLR)-triggered maturation toward a tolerogenic phenotype. The AC+ Hly- mutant also infected mouse lungs as efficiently as the parental AC+ Hly+ strain. Hence, elevation of cAMP in CD11b- cells and/or the pore-forming capacity of CyaA were not required for infection of mouse airways. The latter activities were, however, involved in bacterial penetration across the epithelial layer, enhanced neutrophil influx into lung parenchyma during sublethal infections, and the exacerbated lung pathology and lethality of B. pertussis infections at higher inoculation doses (>107 CFU/mouse). The pore-forming activity of CyaA further synergized with the cAMP-elevating activity in downregulation of major histocompatibility complex class II (MHC-II) molecules on infiltrating myeloid cells, likely contributing to immune subversion of host defenses by the whooping cough agent.

Centre National de la Recherche Scientifique URA3012 Paris France

Czech Centre for Phenogenomics Division BIOCEV Institute of Molecular Genetics of the CAS v v i Czech Academy of Sciences Prague Czech Republic

Institut Pasteur Molecular Prevention and Therapy of Human Diseases Unit Paris France

Institute of Microbiology of the CAS v v i Prague Czech Republic

Zobrazit více v PubMed

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

Khelef N, Bachelet CM, Vargaftig BB, Guiso N. 1994. Characterization of murine lung inflammation after infection with parental Bordetella pertussis and mutants deficient in adhesins or toxins. Infect Immun 62:2893–2900. PubMed PMC

Khelef N, Sakamoto H, Guiso N. 1992. Both adenylate cyclase and hemolytic activities are required by Bordetella pertussis to initiate infection. Microb Pathog 12:227–235. doi:10.1016/0882-4010(92)90057-U. PubMed DOI

Guermonprez P, Khelef N, Blouin E, Rieu P, Ricciardi-Castagnoli P, Guiso N, Ladant D, Leclerc C. 2001. The adenylate cyclase toxin of Bordetella pertussis binds to target cells via the alpha(M)beta(2) integrin (CD11b/CD18). J Exp Med 193:1035–1044. doi:10.1084/jem.193.9.1035. PubMed DOI PMC

Osicka R, Osickova A, Hasan S, Bumba L, Cerny J, Sebo P. 2015. Bordetella adenylate cyclase toxin is a unique ligand of the integrin complement receptor 3. eLife 4:e10766. doi:10.7554/eLife.10766. PubMed DOI PMC

Confer DL, Eaton JW. 1982. Phagocyte impotence caused by an invasive bacterial adenylate cyclase. Science 217:948–950. doi:10.1126/science.6287574. PubMed DOI

Kamanova J, Kofronova O, Masin J, Genth H, Vojtova J, Linhartova I, Benada O, Just I, Sebo P. 2008. Adenylate cyclase toxin subverts phagocyte function by RhoA inhibition and unproductive ruffling. J Immunol 181:5587–5597. doi:10.4049/jimmunol.181.8.5587. PubMed DOI

Pearson RD, Symes P, Conboy M, Weiss AA, Hewlett EL. 1987. Inhibition of monocyte oxidative responses by Bordetella pertussis adenylate cyclase toxin. J Immunol 139:2749–2754. PubMed

Prior S, Corbel MJ, Xing DK. 2005. Development of an approach for the laboratory toxicological evaluation of Bordetella pertussis adenylate cyclase genetic toxoid constructs as multipurpose vaccines. Hum Vaccin 1:151–159. doi:10.4161/hv.1.4.1972. PubMed DOI

Vojtova J, Kamanova J, Sebo P. 2006. Bordetella adenylate cyclase toxin: a swift saboteur of host defense. Curr Opin Microbiol 9:69–75. doi:10.1016/j.mib.2005.12.011. PubMed DOI

Hanski E. 1989. Invasive adenylate cyclase toxin of Bordetella pertussis. Trends Biochem Sci 14:459–463. doi:10.1016/0968-0004(89)90106-0. PubMed DOI

Eby JC, Gray MC, Warfel JM, Paddock CD, Jones TF, Day SR, Bowden J, Poulter MD, Donato GM, Merkel TJ, Hewlett EL. 2013. Quantification of the adenylate cyclase toxin of Bordetella pertussis in vitro and during respiratory infection. Infect Immun 81:1390–1398. doi:10.1128/IAI.00110-13. PubMed DOI PMC

Donato GM, Goldsmith CS, Paddock CD, Eby JC, Gray MC, Hewlett EL. 2012. Delivery of Bordetella pertussis adenylate cyclase toxin to target cells via outer membrane vesicles. FEBS Lett 586:459–465. doi:10.1016/j.febslet.2012.01.032. PubMed DOI PMC

Eby JC, Ciesla WP, Hamman W, Donato GM, Pickles RJ, Hewlett EL, Lencer WI. 2010. Selective translocation of the Bordetella pertussis adenylate cyclase toxin across the basolateral membranes of polarized epithelial cells. J Biol Chem 285:10662–10670. doi:10.1074/jbc.M109.089219. PubMed DOI PMC

Masin J, Osicka R, Bumba L, Sebo P. 2015. Bordetella adenylate cyclase toxin: a unique combination of a pore-forming moiety with a cell-invading adenylate cyclase enzyme. Pathog Dis 73:ftv075. doi:10.1093/femspd/ftv075. PubMed DOI PMC

Sebo P, Osicka R, Masin J. 2014. Adenylate cyclase toxin-hemolysin relevance for pertussis vaccines. Expert Rev Vaccines 13:1215–1227. doi:10.1586/14760584.2014.944900. PubMed DOI

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

Bumba L, Masin J, Macek P, Wald T, Motlova L, Bibova I, Klimova N, Bednarova L, Veverka V, Kachala M, Svergun DI, Barinka C, Sebo P. 2016. Calcium-driven folding of RTX domain β-rolls ratchets translocation of RTX proteins through type I secretion ducts. Mol Cell 62:47–62. doi:10.1016/j.molcel.2016.03.018. PubMed DOI

Hackett M, Guo L, Shabanowitz J, Hunt DF, Hewlett EL. 1994. Internal lysine palmitoylation in adenylate cyclase toxin from Bordetella pertussis. Science 266:433–435. doi:10.1126/science.7939682. PubMed DOI

Hackett M, Walker CB, Guo L, Gray MC, Van Cuyk S, Ullmann A, Shabanowitz J, Hunt DF, Hewlett EL, Sebo P. 1995. Hemolytic, but not cell-invasive activity, of adenylate cyclase toxin is selectively affected by differential fatty-acylation in Escherichia coli. J Biol Chem 270:20250–20253. doi:10.1074/jbc.270.35.20250. PubMed DOI

Hewlett EL, Gray L, Allietta M, Ehrmann I, Gordon VM, Gray MC. 1991. Adenylate cyclase toxin from Bordetella pertussis. Conformational change associated with toxin activity. J Biol Chem 266:17503–17508. PubMed

El-Azami-El-Idrissi M, Bauche C, Loucka J, Osicka R, Sebo P, Ladant D, Leclerc C. 2003. Interaction of Bordetella pertussis adenylate cyclase with CD11b/CD18: role of toxin acylation and identification of the main integrin interaction domain. J Biol Chem 278:38514–38521. doi:10.1074/jbc.M304387200. PubMed DOI

Hanski E, Farfel Z. 1985. Bordetella pertussis invasive adenylate cyclase. Partial resolution and properties of its cellular penetration. J Biol Chem 260:5526–5532. PubMed

Wolff J, Cook GH, Goldhammer AR, Berkowitz SA. 1980. Calmodulin activates prokaryotic adenylate cyclase. Proc Natl Acad Sci U S A 77:3841–3844. doi:10.1073/pnas.77.7.3841. PubMed DOI PMC

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

Benz R, Maier E, Ladant D, Ullmann A, Sebo P. 1994. 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 269:27231–27239. PubMed

Svedova M, Masin J, Fiser R, Cerny O, Tomala J, Freudenberg M, Tuckova L, Kovar M, Dadaglio G, Adkins I, Sebo P. 2016. Pore-formation by adenylate cyclase toxoid activates dendritic cells to prime CD8(+) and CD4(+) T cells. Immunol Cell Biol 94:322–333. doi:10.1038/icb.2015.87. PubMed DOI

Dunne A, Ross PJ, Pospisilova E, Masin J, Meaney A, Sutton CE, Iwakura Y, Tschopp J, Sebo P, Mills KH. 2010. Inflammasome activation by adenylate cyclase toxin directs Th17 responses and protection against Bordetella pertussis. J Immunol 185:1711–1719. doi:10.4049/jimmunol.1000105. PubMed DOI

Basler M, Masin J, Osicka R, Sebo P. 2006. Pore-forming and enzymatic activities of Bordetella pertussis adenylate cyclase toxin synergize in promoting lysis of monocytes. Infect Immun 74:2207–2214. doi:10.1128/IAI.74.4.2207-2214.2006. PubMed DOI PMC

Hewlett EL, Donato GM, Gray MC. 2006. Macrophage cytotoxicity produced by adenylate cyclase toxin from Bordetella pertussis: more than just making cyclic AMP! Mol Microbiol 59:447–459. doi:10.1111/j.1365-2958.2005.04958.x. PubMed DOI

Hasan S, Osickova A, Bumba L, Novak P, Sebo P, Osicka R. 2015. Interaction of Bordetella adenylate cyclase toxin with complement receptor 3 involves multivalent glycan binding. FEBS Lett 589:374–379. doi:10.1016/j.febslet.2014.12.023. PubMed DOI

Morova J, Osicka R, Masin J, Sebo P. 2008. RTX cytotoxins recognize beta2 integrin receptors through N-linked oligosaccharides. Proc Natl Acad Sci U S A 105:5355–5360. doi:10.1073/pnas.0711400105. PubMed DOI PMC

Bumba L, Masin J, Fiser R, Sebo P. 2010. Bordetella adenylate cyclase toxin mobilizes its beta2 integrin receptor into lipid rafts to accomplish translocation across target cell membrane in two steps. PLoS Pathog 6:e1000901. doi:10.1371/journal.ppat.1000901. PubMed DOI PMC

Vojtova-Vodolanova J, Basler M, Osicka R, Knapp O, Maier E, Cerny J, Benada O, Benz R, Sebo P. 2009. Oligomerization is involved in pore formation by Bordetella adenylate cyclase toxin. FASEB J 23:2831–2843. doi:10.1096/fj.09-131250. PubMed DOI

Basler M, Knapp O, Masin J, Fiser R, Maier E, Benz R, Sebo P, Osicka R. 2007. Segments crucial for membrane translocation and pore-forming activity of Bordetella adenylate cyclase toxin. J Biol Chem 282:12419–12429. doi:10.1074/jbc.M611226200. PubMed DOI

Fiser R, Masin J, Basler M, Krusek J, Spulakova V, Konopasek I, Sebo P. 2007. 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 282:2808–2820. doi:10.1074/jbc.M609979200. PubMed DOI

Osickova A, Masin J, Fayolle C, Krusek J, Basler M, Pospisilova E, Leclerc C, Osicka R, Sebo P. 2010. Adenylate cyclase toxin translocates across target cell membrane without forming a pore. Mol Microbiol 75:1550–1562. doi:10.1111/j.1365-2958.2010.07077.x. PubMed DOI

Osickova A, Osicka R, Maier E, Benz R, Sebo P. 1999. 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 274:37644–37650. PubMed

Basar T, Havlicek V, Bezouskova S, Halada P, Hackett M, Sebo P. 1999. 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 274:10777–10783. doi:10.1074/jbc.274.16.10777. PubMed DOI

Gueirard P, Druilhe A, Pretolani M, Guiso N. 1998. Role of adenylate cyclase-hemolysin in alveolar macrophage apoptosis during Bordetella pertussis infection in vivo. Infect Immun 66:1718–1725. PubMed PMC

Ehrmann IE, Weiss AA, Goodwin MS, Gray MC, Barry E, Hewlett EL. 1992. Enzymatic activity of adenylate cyclase toxin from Bordetella pertussis is not required for hemolysis. FEBS Lett 304:51–56. doi:10.1016/0014-5793(92)80587-7. PubMed DOI

Gross MK, Au DC, Smith AL, Storm DR. 1992. Targeted mutations that ablate either the adenylate cyclase or hemolysin function of the bifunctional cyaA toxin of Bordetella pertussis abolish virulence. Proc Natl Acad Sci U S A 89:4898–4902. doi:10.1073/pnas.89.11.4898. PubMed DOI PMC

Bagley KC, Abdelwahab SF, Tuskan RG, Fouts TR, Lewis GK. 2002. Pertussis toxin and the adenylate cyclase toxin from Bordetella pertussis activate human monocyte-derived dendritic cells and dominantly inhibit cytokine production through a cAMP-dependent pathway. J Leukoc Biol 72:962–969. PubMed

Boschwitz JS, Batanghari JW, Kedem H, Relman DA. 1997. Bordetella pertussis infection of human monocytes inhibits antigen-dependent CD4 T cell proliferation. J Infect Dis 176:678–686. doi:10.1086/514090. PubMed DOI

Fedele G, Spensieri F, Palazzo R, Nasso M, Cheung GY, Coote JG, Ausiello CM. 2010. Bordetella pertussis commits human dendritic cells to promote a Th1/Th17 response through the activity of adenylate cyclase toxin and MAPK-pathways. PLoS One 5:e8734. doi:10.1371/journal.pone.0008734. PubMed DOI PMC

Hickey FB, Brereton CF, Mills KH. 2008. Adenylate cyclase toxin of Bordetella pertussis inhibits TLR-induced IRF-1 and IRF-8 activation and IL-12 production and enhances IL-10 through MAPK activation in dendritic cells. J Leukoc Biol 84:234–243. doi:10.1189/jlb.0208113. PubMed DOI

Njamkepo E, Pinot F, Francois D, Guiso N, Polla BS, Bachelet M. 2000. Adaptive responses of human monocytes infected by Bordetella pertussis: the role of adenylate cyclase hemolysin. J Cell Physiol 183:91–99. doi:10.1002/(SICI)1097-4652(200004)183:1<91::AID-JCP11>3.0.CO;2-S. PubMed DOI

Ross PJ, Lavelle EC, Mills KH, Boyd AP. 2004. Adenylate cyclase toxin from Bordetella pertussis synergizes with lipopolysaccharide to promote innate interleukin-10 production and enhances the induction of Th2 and regulatory T cells. Infect Immun 72:1568–1579. doi:10.1128/IAI.72.3.1568-1579.2004. PubMed DOI PMC

Spensieri F, Fedele G, Fazio C, Nasso M, Stefanelli P, Mastrantonio P, Ausiello CM. 2006. Bordetella pertussis inhibition of interleukin-12 (IL-12) p70 in human monocyte-derived dendritic cells blocks IL-12 p35 through adenylate cyclase toxin-dependent cyclic AMP induction. Infect Immun 74:2831–2838. doi:10.1128/IAI.74.5.2831-2838.2006. PubMed DOI PMC

Ladant D, Glaser P, Ullmann A. 1992. Insertional mutagenesis of Bordetella pertussis adenylate cyclase. J Biol Chem 267:2244–2250. PubMed

Miller LS, O'Connell RM, Gutierrez MA, Pietras EM, Shahangian A, Gross CE, Thirumala A, Cheung AL, Cheng G, Modlin RL. 2006. MyD88 mediates neutrophil recruitment initiated by IL-1R but not TLR2 activation in immunity against Staphylococcus aureus. Immunity 24:79–91. doi:10.1016/j.immuni.2005.11.011. PubMed DOI

Bassinet L, Gueirard P, Maitre B, Housset B, Gounon P, Guiso N. 2000. Role of adhesins and toxins in invasion of human tracheal epithelial cells by Bordetella pertussis. Infect Immun 68:1934–1941. doi:10.1128/IAI.68.4.1934-1941.2000. PubMed DOI PMC

Perkins DJ, Gray MC, Hewlett EL, Vogel SN. 2007. Bordetella pertussis adenylate cyclase toxin (ACT) induces cyclooxygenase-2 (COX-2) in murine macrophages and is facilitated by ACT interaction with CD11b/CD18 (Mac-1). Mol Microbiol 66:1003–1015. doi:10.1111/j.1365-2958.2007.05972.x. PubMed DOI

Adkins I, Kamanova J, Kocourkova A, Svedova M, Tomala J, Janova H, Masin J, Chladkova B, Bumba L, Kovar M, Ross PJ, Tuckova L, Spisek R, Mills KH, Sebo P. 2014. Bordetella adenylate cyclase toxin differentially modulates Toll-like receptor-stimulated activation, migration and T cell stimulatory capacity of dendritic cells. PLoS One 9:e104064. doi:10.1371/journal.pone.0104064. PubMed DOI PMC

Higgs R, Higgins SC, Ross PJ, Mills KH. 2012. Immunity to the respiratory pathogen Bordetella pertussis. Mucosal Immunol 5:485–500. doi:10.1038/mi.2012.54. PubMed DOI

Miller VL, Mekalanos JJ. 1988. A novel suicide vector and its use in construction of insertion mutations: osmoregulation of outer membrane proteins and virulence determinants in Vibrio cholerae requires toxR. J Bacteriol 170:2575–2583. doi:10.1128/jb.170.6.2575-2583.1988. PubMed DOI PMC

Caro V, Hot D, Guigon G, Hubans C, Arrive M, Soubigou G, Renauld-Mongenie G, Antoine R, Locht C, Lemoine Y, Guiso N. 2006. Temporal analysis of French Bordetella pertussis isolates by comparative whole-genome hybridization. Microbes Infect 8:2228–2235. doi:10.1016/j.micinf.2006.04.014. PubMed DOI

Stainer DW, Scholte MJ. 1970. A simple chemically defined medium for the production of phase I Bordetella pertussis. J Gen Microbiol 63:211–220. doi:10.1099/00221287-63-2-211. PubMed DOI

Inatsuka CS, Xu Q, Vujkovic-Cvijin I, Wong S, Stibitz S, Miller JF, Cotter PA. 2010. Pertactin is required for Bordetella species to resist neutrophil-mediated clearance. Infect Immun 78:2901–2909. doi:10.1128/IAI.00188-10. PubMed DOI PMC

Weber C, Boursaux-Eude C, Coralie G, Caro V, Guiso N. 2001. Polymorphism of Bordetella pertussis isolates circulating for the last 10 years in France, where a single effective whole-cell vaccine has been used for more than 30 years. J Clin Microbiol 39:4396–4403. doi:10.1128/JCM.39.12.4396-4403.2001. PubMed DOI PMC

Ladant D, Brezin C, Alonso JM, Crenon I, Guiso N. 1986. Bordetella pertussis adenylate cyclase. Purification, characterization, and radioimmunoassay. J Biol Chem 261:16264–16269. PubMed

Lutz MB, Kukutsch N, Ogilvie AL, Rossner S, Koch F, Romani N, Schuler G. 1999. An advanced culture method for generating large quantities of highly pure dendritic cells from mouse bone marrow. J Immunol Methods 223:77–92. doi:10.1016/S0022-1759(98)00204-X. PubMed DOI

Karimova G, Pidoux J, Ullmann A, Ladant D. 1998. A bacterial two-hybrid system based on a reconstituted signal transduction pathway. Proc Natl Acad Sci U S A 95:5752–5756. doi:10.1073/pnas.95.10.5752. PubMed DOI PMC

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

Pertussis toxin suppresses dendritic cell-mediated delivery of B. pertussis into lung-draining lymph nodes

The Fim and FhaB adhesins play a crucial role in nasal cavity infection and Bordetella pertussis transmission in a novel mouse catarrhal infection model

Kingella kingae RtxA Cytotoxin in the Context of Other RTX Toxins

Selective Enhancement of the Cell-Permeabilizing Activity of Adenylate Cyclase Toxin Does Not Increase Virulence of Bordetella pertussis

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

Bordetella Adenylate Cyclase Toxin Elicits Airway Mucin Secretion through Activation of the cAMP Response Element Binding Protein

Lipid binding by the N-terminal motif mediates plasma membrane localization of Bordetella effector protein BteA

A Mutation Upstream of the rplN-rpsD Ribosomal Operon Downregulates Bordetella pertussis Virulence Factor Production without Compromising Bacterial Survival within Human Macrophages

Adenylate Cyclase Toxin Tinkering With Monocyte-Macrophage Differentiation

Cytotoxicity of the effector protein BteA was attenuated in Bordetella pertussis by insertion of an alanine residue

Bordetella Adenylate Cyclase Toxin Inhibits Monocyte-to-Macrophage Transition and Dedifferentiates Human Alveolar Macrophages into Monocyte-like 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