title: 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.
abstract: The Bordetella adenylate cyclase toxin-hemolysin (CyaA) targets phagocytes expressing the alpha(M)beta2 integrin (CD11b/CD18), permeabilizes their membranes by forming small cation-selective pores, and delivers into cells a calmodulin-activated adenylate cyclase (AC) enzyme that dissipates cytosolic ATP into cAMP. We describe here a third activity of CyaA that yields elevation of cytosolic calcium concentration ([Ca2+]i) in target cells. The CyaA-mediated [Ca2+]i increase in CD11b+ J774A.1 monocytes was inhibited by extracellular La3+ ions but not by nifedipine, SK&F 96365, flunarizine, 2-aminoethyl diphenylborinate, or thapsigargin, suggesting that influx of Ca2+ into cells was not because of receptor signaling or opening of conventional calcium channels by cAMP. Compared with intact CyaA, a CyaA-AC- toxoid unable to generate cAMP promoted a faster, albeit transient, elevation of [Ca2+]i. This was not because of cell permeabilization by the CyaA hemolysin pores, because a mutant exhibiting a strongly enhanced pore-forming activity (CyaA-E509K/E516K), but unable to deliver the AC domain into cells, was also unable to elicit a [Ca2+]i increase. Further mutations interfering with AC translocation into cells, such as proline substitutions of glutamate residues 509 or 570 or deletion of the AC domain as such, reduced or ablated the [Ca2+]i-elevating capacity of CyaA. Moreover, structural alterations within the AC domain, because of insertion of various oligopeptides, differently modulated the kinetics and extent of Ca2+ influx elicited by the respective AC- toxoids. Hence, the translocating AC polypeptide itself appears to participate in formation of a novel type of membrane path for calcium ions, contributing to action of CyaA in an unexpected manner.
authors.affiliation: Department of Genetics and Microbiology, Faculty of Science, Charles University, CZ-128 44, Prague 2.
authors.forename: Radovan
authors.initials: R
authors.lastname: Fiser
authors.forename: Jirí
authors.initials: J
authors.lastname: Masín
authors.forename: Marek
authors.initials: M
authors.lastname: Basler
authors.forename: Jan
authors.initials: J
authors.lastname: Krusek
authors.forename: Veronika
authors.initials: V
authors.lastname: Spuláková
authors.forename: Ivo
authors.initials: I
authors.lastname: Konopásek
authors.forename: Peter
authors.initials: P
authors.lastname: Sebo
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D037361:Adenylate Cyclase Toxin / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D000262:Adenylyl Cyclases / Q000378:metabolism*; D019943:Amino Acid Substitution; D000818:Animals; D001692:Biological Transport; D039481:CD11b Antigen / Q000502:physiology*; D002118:Calcium / Q000378:metabolism*; D002384:Catalysis; D002460:Cell Line; D002462:Cell Membrane / Q000502:physiology; D000242:Cyclic AMP / Q000378:metabolism; D006461:Hemolysis; D008264:Macrophages / Q000502:physiology; D051379:Mice; D009000:Monocytes / Q000502:physiology*; D016297:Mutagenesis, Site-Directed; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012756:Sheep
chemical_list: D037361:Adenylate Cyclase Toxin; D039481:CD11b Antigen; D011994:Recombinant Proteins; D000255:Adenosine Triphosphate; D000242:Cyclic AMP; D000262:Adenylyl Cyclases; D002118:Calcium