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.
Highlights
Adenylate cyclase toxin-hemolysin (AC-hemolysin moiety (Hly) or CyaA) is a key virulence factor involved in the early stages of the respiratory tract colonization by Bordetella pertussis, a Gram-negative pathogen causing whooping cough [1]
Because CyaA possesses the capacity to form small cationselective membrane pores that account for hemolytic activity of CyaA and might potentially permeabilize cells for entry of Ca2ϩ, we first examined whether exposure to CyaA causes alterations of [Ca2ϩ]i in CD11bϩ J774A.1 monocytic cells
The presence of a control isotype antibody had no effect on [Ca2ϩ]i increase, the capacity of the toxin to elevate [Ca2ϩ]i in J774A.1 cells was completely abrogated in the presence of an excess of the CD11b-specific antibody M1/70 that competes with CyaA
Summary
Chemicals—2-APB, 2-deoxy-D-glucose (2DG), Bt2cAMP, EGTA, FCCP, flunarizine, IBMX, LaCl3, nifedipine, SK&F 96365, calmodulin, cytochalasin D, and lipopolysaccharide from Escherichia coli serotype 0111:B4 were purchased from Sigma. CyaA and derived proteins were added to J774A.1 cells by direct dilution from the concentrated stock solutions containing 8 M urea. The samples were neutralized by addition of 150 mM unbuffered imidazole, and cAMP concentration was determined by an antibody competition immunoassay as described elsewhere [53]. Fluorescence Measurement of Cytosolic Ca2ϩ—Cells grown on glass coverslips were washed in modified HBSS (140 mM NaCl, 5 mM KCl, 2 mM CaCl2, 3 mM MgCl2, 10 mM Hepes-Na, 50 mM glucose (pH 7.4)). In vivo calibrations for determination of the calcium concentration were performed as described elsewhere [54], using 10Ϫ5 M ionomycin and a Kd ϭ 224 nM value for Fura-2. The minimal cytosolic concentration of Ca2ϩ was determined by measurements in calcium-free HBSS, pH 7.4, containing 4 mM EGTA instead of 2 mM CaCl2. The maximum of [Ca2ϩ]i was determined in HBSS supplemented with 10 mM CaCl2
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