FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2023521873> ?p ?o ?g. }

• W2023521873 endingPage "12429" @default.
• W2023521873 startingPage "12419" @default.
• W2023521873 abstract "Bordetella adenylate cyclase toxin-hemolysin (CyaA, AC-Hly, or ACT) permeabilizes cell membranes by forming small cation-selective (hemolytic) pores and subverts cellular signaling by delivering into host cells an adenylate cyclase (AC) enzyme that converts ATP to cAMP. Both AC delivery and pore formation were previously shown to involve a predicted amphipathic α-helix502–522 containing a pair of negatively charged Glu509 and Glu516 residues. Another predicted transmembrane α-helix565–591 comprises a Glu570 and Glu581 pair. We examined the roles of these glutamates in the activity of CyaA. Substitutions of Glu516 increased specific hemolytic activity of CyaA by two different molecular mechanisms. Replacement of Glu516 by positively charged lysine residue (E516K) increased the propensity of CyaA to form pores, whereas proline (E516P) or glutamine (E516Q) substitutions extended the lifetime of open single pore units. All three substitutions also caused a drop of pore selectivity for cations. Substitutions of Glu570 and Glu581 by helix-breaking proline or positively charged lysine residue reduced (E570K, E581P) or ablated (E570P, E581K) AC membrane translocation. Moreover, E570P, E570K, and E581P substitutions down-modulated also the specific hemolytic activity of CyaA. In contrast, the E581K substitution enhanced the hemolytic activity of CyaA 4 times, increasing both the frequency of formation and lifetime of toxin pores. Negative charge at position 570, but not at position 581, was found to be essential for cation selectivity of the pore, suggesting a role of Glu570 in ion filtering inside or close to pore mouth. The pairs of glutamate residues in the predicted transmembrane segments of CyaA thus appear to play a key functional role in membrane translocation and pore-forming activities of CyaA. Bordetella adenylate cyclase toxin-hemolysin (CyaA, AC-Hly, or ACT) permeabilizes cell membranes by forming small cation-selective (hemolytic) pores and subverts cellular signaling by delivering into host cells an adenylate cyclase (AC) enzyme that converts ATP to cAMP. Both AC delivery and pore formation were previously shown to involve a predicted amphipathic α-helix502–522 containing a pair of negatively charged Glu509 and Glu516 residues. Another predicted transmembrane α-helix565–591 comprises a Glu570 and Glu581 pair. We examined the roles of these glutamates in the activity of CyaA. Substitutions of Glu516 increased specific hemolytic activity of CyaA by two different molecular mechanisms. Replacement of Glu516 by positively charged lysine residue (E516K) increased the propensity of CyaA to form pores, whereas proline (E516P) or glutamine (E516Q) substitutions extended the lifetime of open single pore units. All three substitutions also caused a drop of pore selectivity for cations. Substitutions of Glu570 and Glu581 by helix-breaking proline or positively charged lysine residue reduced (E570K, E581P) or ablated (E570P, E581K) AC membrane translocation. Moreover, E570P, E570K, and E581P substitutions down-modulated also the specific hemolytic activity of CyaA. In contrast, the E581K substitution enhanced the hemolytic activity of CyaA 4 times, increasing both the frequency of formation and lifetime of toxin pores. Negative charge at position 570, but not at position 581, was found to be essential for cation selectivity of the pore, suggesting a role of Glu570 in ion filtering inside or close to pore mouth. The pairs of glutamate residues in the predicted transmembrane segments of CyaA thus appear to play a key functional role in membrane translocation and pore-forming activities of CyaA. Bordetella pertussis, the etiological agent of whooping cough, secretes an adenylate cyclase toxin (CyaA, 3The abbreviations used are: CyaA, adenylate cyclase toxin; AC, adenylate cyclase; Hly, hemolysin; BSA, bovine serum albumin; pS, picosiemens. adenylate cyclase-hemolysin (AC-Hly), or ACT) that is a key virulence factor of the bacteria during early phases of respiratory tract colonization (1Weiss J. Hewlett E.L. Cronin M.J. Biochem. Biophys. Res. Commun. 1986; 136: 463-469Crossref PubMed Scopus (5) Google Scholar, 2Harvill E.T. Cotter P.A. Yuk M.H. Miller J.F. Infect. Immun. 1999; 67: 1493-1500Crossref PubMed Google Scholar, 3Goodwin M.S. Weiss A.A. Infect. Immun. 1990; 58: 3445-3447Crossref PubMed Google Scholar, 4Khelef N. Sakamoto H. Guiso N. Microb. Pathog. 1992; 12: 227-235Crossref PubMed Scopus (95) Google Scholar). The toxin paralyzes bactericidal activities of host phagocytes and induces macrophage apoptosis, possibly enabling the pathogen to escape host immune surveillance (5Hanski E. Trends Biochem. Sci. 1989; 14: 459-463Abstract Full Text PDF PubMed Scopus (67) Google Scholar, 6Confer D.L. Eaton J.W. Science. 1982; 217: 948-950Crossref PubMed Scopus (291) Google Scholar, 7Khelef N. Zychlinsky A. Guiso N. Infect. Immun. 1993; 61: 4064-4071Crossref PubMed Google Scholar, 8Khelef N. Guiso N. FEMS Microbiol. Lett. 1995; 134: 27-32Crossref PubMed Google Scholar). CyaA is synthesized as a single polypeptide of 1706 residues and consists of an amino-terminal AC domain of about 400 residues and a pore-forming RTX (repeat in toxin) Hly moiety of about 1306 residues (9Glaser P. Sakamoto H. Bellalou J. Ullmann A. Danchin A. EMBO J. 1988; 7: 3997-4004Crossref PubMed Scopus (282) Google Scholar). The Hly bears a C-terminal secretion signal (10Masure H.R. Au D.C. Gross M.K. Donovan M.G. Storm D.R. Biochemistry. 1990; 29: 140-145Crossref PubMed Scopus (38) Google Scholar, 11Sebo P. Ladant D. Mol. Microbiol. 1993; 9: 999-1009Crossref PubMed Scopus (58) Google Scholar) and consists of a hydrophobic pore-forming domain (residues 500–700) (12Benz R. Maier E. Ladant D. Ullmann A. Sebo P. J. Biol. Chem. 1994; 269: 27231-27239Abstract Full Text PDF PubMed Google Scholar), of a calcium-binding glycine- and aspartate-rich nonapeptide repeat domain (last 700 residues), and of a fatty acyl activation domain (residues 800–1000). In the last, the essential covalent post-translational modification is taking place (13Hackett M. Guo L. Shabanowitz J. Hunt D.F. Hewlett E.L. Science. 1994; 266: 433-435Crossref PubMed Scopus (196) Google Scholar) in the presence of the accessory acyltransferase, CyaC (14Barry E.M. Weiss A.A. Ehrmann I.E. Gray M.C. Hewlett E.L. Goodwin M.S. J. Bacteriol. 1991; 173: 720-726Crossref PubMed Google Scholar). The AC domain is enzymatically active by itself (15Glaser P. Elmaoglou-Lazaridou A. Krin E. Ladant D. Barzu O. Danchin A. EMBO J. 1989; 8: 967-972Crossref PubMed Scopus (71) Google Scholar). The entire toxin is, however, needed for AC delivery into target cells (16Bellalou J. Sakamoto H. Ladant D. Geoffroy C. Ullmann A. Infect. Immun. 1990; 58: 3242-3247Crossref PubMed Google Scholar, 17Iwaki M. Ullmann A. Sebo P. Mol. Microbiol. 1995; 17: 1015-1024Crossref PubMed Scopus (61) Google Scholar), where AC is activated by cytosolic calmodulin and catalyzes unregulated conversion of ATP to the key signal molecule, cAMP (18Hanski E. Farfel Z. J. Biol. Chem. 1985; 260: 5526-5532Abstract Full Text PDF PubMed Google Scholar, 19Wolff J. Cook G.H. Goldhammer A.R. Berkowitz S.A. Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 3841-3844Crossref PubMed Scopus (193) Google Scholar). In turn, membrane insertion and pore-forming (hemolytic) activities of CyaA do not require the AC domain and are located to the Hly portion (20Sakamoto H. Bellalou J. Sebo P. Ladant D. J. Biol. Chem. 1992; 267: 13598-13602Abstract Full Text PDF PubMed Google Scholar). This exhibits hemolytic activity on erythrocytes (16Bellalou J. Sakamoto H. Ladant D. Geoffroy C. Ullmann A. Infect. Immun. 1990; 58: 3242-3247Crossref PubMed Google Scholar, 21Glaser P. Danchin A. Ladant D. Barzu O. Ullmann A. Tokai J. Exp. Clin. Med. 1988; 13: 239-252PubMed Google Scholar) and can form small cation-selective membrane pores (12Benz R. Maier E. Ladant D. Ullmann A. Sebo P. J. Biol. Chem. 1994; 269: 27231-27239Abstract Full Text PDF PubMed Google Scholar, 22Szabo G. Gray M.C. Hewlett E.L. J. Biol. Chem. 1994; 269: 22496-22499Abstract Full Text PDF PubMed Google Scholar). CyaA binds target cells primarily via the αMβ2 integrin receptor known as CD11b/CD18, complement receptor 3 (CR3), or Mac-1 (23Guermonprez P. Khelef N. Blouin E. Rieu P. Ricciardi-Castagnoli P. Guiso N. Ladant D. Leclerc C. J. Exp. Med. 2001; 193: 1035-1044Crossref PubMed Scopus (274) Google Scholar). This is expressed by granulocytes/neutrophils, macrophages, myeloid dendritic cells, NK cells, and certain subsets of CD8+ T and of B cells. It is noteworthy, however, that the toxin can penetrate and intoxicate to detectable levels also a variety of other cell types lacking the CD11b/CD18 receptor and/or endocytic mechanisms, such as mammalian erythrocytes (5Hanski E. Trends Biochem. Sci. 1989; 14: 459-463Abstract Full Text PDF PubMed Scopus (67) Google Scholar, 6Confer D.L. Eaton J.W. Science. 1982; 217: 948-950Crossref PubMed Scopus (291) Google Scholar, 16Bellalou J. Sakamoto H. Ladant D. Geoffroy C. Ullmann A. Infect. Immun. 1990; 58: 3242-3247Crossref PubMed Google Scholar, 24Gordon V.M. Young Jr., W.W. Lechler S.M. Gray M.C. Leppla S.H. Hewlett E.L. J. Biol. Chem. 1989; 264: 14792-14796Abstract Full Text PDF PubMed Google Scholar). Because of a lack of structural data, the mechanisms of membrane insertion of CyaA, its capacity to cross directly the cytoplasmic membrane, and its ability to form cation-selective membrane pores are poorly understood. Indirect evidence suggests that translocation of the AC domain across membrane and formation of CyaA pores are two independent and separable membrane activities, resulting from membrane insertion of CyaA (22Szabo G. Gray M.C. Hewlett E.L. J. Biol. Chem. 1994; 269: 22496-22499Abstract Full Text PDF PubMed Google Scholar, 25Rogel A. Schultz J.E. Brownlie R.M. Coote J.G. Parton R. Hanski E. EMBO J. 1989; 8: 2755-2760Crossref PubMed Scopus (59) Google Scholar, 26Rogel A. Meller R. Hanski E. J. Biol. Chem. 1991; 266: 3154-3161Abstract Full Text PDF PubMed Google Scholar, 27Sebo P. Glaser P. Sakamoto H. Ullmann A. Gene (Amst.). 1991; 104: 19-24Crossref PubMed Scopus (69) Google Scholar, 28Rogel A. Hanski E. J. Biol. Chem. 1992; 267: 22599-22605Abstract Full Text PDF PubMed Google Scholar, 29Betsou F. Sebo P. Guiso N. Infect. Immun. 1993; 61: 3583-3589Crossref PubMed Google Scholar, 30Hackett M. Walker C.B. Guo L. Gray M.C. Van Cuyk S. Ullmann A. Shabanowitz J. Hunt D.F. Hewlett E.L. Sebo P. J. Biol. Chem. 1995; 270: 20250-20253Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar, 31Rose T. Sebo P. Bellalou J. Ladant D. J. Biol. Chem. 1995; 270: 26370-26376Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar, 32Gray M. Szabo G. Otero A.S. Gray L. Hewlett E. J. Biol. Chem. 1998; 273: 18260-18267Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar, 33Basar T. Havlicek V. Bezouskova S. Halada P. Hackett M. Sebo P. J. Biol. Chem. 1999; 274: 10777-10783Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). Recently, we demonstrated that both of these activities involve a transmembrane α-helix predicted between residues 502 and 522 of the poreforming domain of CyaA. This harbors a pair of negatively charged glutamate residues, Glu509 and Glu516. A helix-breaking proline substitution E509P within this segment abolished the capacity of CyaA to deliver the AC domain into erythrocytes. Moreover, the charge-reversing lysine substitutions E509K and E516K strongly enhanced the specific hemolytic activity of CyaA, by up-modulating its capacity to form pores while ablating AC domain translocation into cells. In addition, combination of the substitutions in CyaA-E509K/E516K strongly decreased the cation selectivity of formed pores, indicating that Glu509 and Glu516 are located within or close to the membrane pore (34Osickova A. Osicka R. Maier E. Benz R. Sebo P. J. Biol. Chem. 1999; 274: 37644-37650Abstract Full Text Full Text PDF PubMed Google Scholar). On this basis, we suggested that prior to interaction with target membrane, two conformational isomers of CyaA might form, one being a translocation precursor allowing AC domain delivery into cell cytosol and the other being a pore precursor, whose insertion into membranes yields formation of oligomeric membrane pores (34Osickova A. Osicka R. Maier E. Benz R. Sebo P. J. Biol. Chem. 1999; 274: 37644-37650Abstract Full Text Full Text PDF PubMed Google Scholar). Here, we show that AC membrane translocation and poreforming (hemolytic) activity of CyaA further depends on an additional predicted transmembrane amphipathic α-helix, localized between residues 565 and 591 of the pore-forming domain and comprising yet another pair of glutamate residues, Glu570 and Glu581. Moreover, we show for the first time that different amino acid substitutions of the glutamate residue at position 516 can increase the hemolytic activity of CyaA by two different molecular mechanisms. Bacterial Strains, Growth Conditions, and Plasmids—Escherichia coli XL1-Blue (Stratagene) was used throughout this work for DNA manipulation and CyaA expression. Bacteria were grown in Luria-Bertani medium supplemented with 150 μg/ml ampicillin. Wild-type CyaA and its mutant derivatives were expressed from the pCACT3 plasmid (29Betsou F. Sebo P. Guiso N. Infect. Immun. 1993; 61: 3583-3589Crossref PubMed Google Scholar). Site-directed Mutagenesis—Single amino acid substitutions were introduced into the cyaA gene by site-directed PCR mutagenesis using the TaqDNA polymerase and suitable pairs of mutagenic PCR primers (supplemental Table 1). PCR products with introduced substitutions were subcloned into the pCACT3, and the absence of other undesired mutations was verified by DNA sequencing. Complete sequences and detailed schemes of the plasmid constructs will be provided on request. Production and Purification of the CyaA-derived Proteins—Intact CyaA and its mutant variants were produced in E. coli transformed with appropriate pCACT3-derived constructs. Exponential 500-ml cultures were grown at 37 °C and induced by isopropyl 1-thio-β-d-galactopyranoside (1 mm) for 4 h before the cells were washed in 50 mm Tris-HCl (pH 8), 150 mm NaCl, resuspended in 50 mm Tris-HCl (pH 8), 0.2 mm CaCl2, and disrupted by sonication. The insoluble cell debris was resuspended in 8 m urea, 50 mm Tris-HCl (pH 8.0), 50 mm NaCl, 0.2 mm CaCl2. Upon centrifugation at 25,000 × g for 20 min, clarified urea extracts were loaded onto a DEAE-Sepharose column equilibrated with 8 m urea, 50 mm Tris-HCl (pH 8.0), 120 mm NaCl. After washing, the CyaA-derived proteins were eluted with 8 m urea, 50 mm Tris-HCl (pH 8.0), 2 m NaCl, diluted four times with the 50 mm Tris-HCl (pH 8.0), 1 m NaCl buffer, and further purified on a phenyl-Sepharose column equilibrated with the same buffer. Unbound proteins were washed out with 50 mm Tris-HCl (pH 8.0), and the CyaA-derived proteins were eluted with 8 m urea, 50 mm Tris-HCl (pH 8.0), 2 mm EDTA and stored at –20 °C. Assay of AC, Cell Binding, and Cytotoxic and Hemolytic Activities—Adenylate cyclase activities were measured in the presence of 1 μm calmodulin as previously described (35Ladant D. J. Biol. Chem. 1988; 263: 2612-2618Abstract Full Text PDF PubMed Google Scholar). One unit of AC activity corresponds to 1 μmol of cAMP formed per min at 30 °C, pH 8.0. Because toxin alterations outside the AC domain do not affect the specific AC activity, concentrations of mutant CyaAs in the extracts and in the purified preparations were equalized on the basis of their AC content prior to activity testing. Cell-invasive AC and hemolytic activities were measured as previously described (16Bellalou J. Sakamoto H. Ladant D. Geoffroy C. Ullmann A. Infect. Immun. 1990; 58: 3242-3247Crossref PubMed Google Scholar) by determining the amount of AC enzyme activity protected against externally added trypsin upon internalization into erythrocytes and by hemoglobin release in time upon toxin incubations with washed sheep erythrocytes (5 × 108/ml), respectively. Erythrocyte binding of the toxins was determined as described in detail previously (17Iwaki M. Ullmann A. Sebo P. Mol. Microbiol. 1995; 17: 1015-1024Crossref PubMed Scopus (61) Google Scholar). Determination of the Intracellular cAMP Level—Cytotoxic activity on sheep erythrocytes was determined as the capacity of various CyaAs to raise intracellular cAMP levels in 5 × 108/ml of sheep red blood cells upon incubation with 5 μg/ml of the CyaA proteins for 30 min at 37 °C in HBSS (10 mm Na-HEPES, pH 7.4, 10 mm KCl, 140 mm NaCl, 3 mm MgCl2, 2 mm CaCl2, and 5 mm d-glucose) containing 100 μm 3-isobutyl-1-methylxanthin. The reaction was stopped by the addition of 100 mm HCl in 0.1% Tween 20, and the samples were boiled for 15 min at 100 °C to denature cellular proteins (cAMP is heat- and acid-resistant). The samples were neutralized by the addition of 150 mm unbuffered imidazol, passed through an Al2O3 column (aluminum oxide 90 active neutral, activity stage I; Merck), and cAMP concentration was determined by a competition immunoassay. Microtiter enzyme-linked immunosorbent assay plates (Nunc-Immuno; Maxisorp) were coated with a cAMP-BSA conjugate (gift of D. Ladant) diluted to 5 μg/ml in 0.1 m Na2CO3, pH 9.5. The plate wells were washed twice in 50 mm Tris-HCl, 0.15 m NaCl, 0.1% Tween 20, pH 8.0 (TBS-Tween), saturated for 3 h with 2% BSA in TBS (TBS-BSA), and washed three times with TBS-Tween. 100 μl of sample or of the cAMP standard (Sigma) were directly added to the plate wells coated with cAMP-BSA and containing 50 μl of anti-cAMP rabbit antibodies (gift of A. Ullmann) diluted at 1:3000 in 2% TBS-BSA. Upon incubation at 4 °C overnight, the plates were washed four times with TBS-Tween, and anti-rabbit peroxidase conjugate (1:1000) was added in TBS-BSA. After incubation at 37 °C for 2 h, the wells were washed four times with TBS-Tween, and the peroxidase activity was determined using o-phenylenediamine (Sigma) as a substrate. Lipid Bilayer Experiments—The methods used for black lipid bilayer experiments have been described previously (36Benz R. Janko K. Lauger P. Biochim. Biophys. Acta. 1979; 551: 238-247Crossref PubMed Scopus (243) Google Scholar). The experimental setup consisted of a Teflon cell with two water-filled compartments connected by a small circular hole. The hole had an area of about 0.4 mm2. Membranes were formed across the hole from a 1% solution of asolectin (lecithin type IIIs from soy beans from Sigma) in n-decane. The temperature was maintained at 20 °C during all experiments. All salts were obtained from Merck (analytical grade) and were buffered with 10 mm HEPES-KOH to a pH of ∼7. The electrical measurements were performed using Ag/AgCl electrodes (with salt bridges) connected in series to a voltage source and a homemade current-to-voltage converter. The amplified signal was recorded on a strip chart or tape recorder. For the selectivity measurements, the membranes were formed in a 100 mm KCl solution. Toxin was added to both sides of the membrane, and the increase of the membrane conductance due to insertion of pores was observed with the electrometer (Keithley 617). After incorporation of 10–100 pores into a membrane, the instrumentation was switched to the measurement of the zero current potential, and a KCl gradient was established by adding 3 m KCl solution to one side of the membrane. Analysis of the zero current membrane potential was performed using the Goldman-Hodgkin-Katz equation (36Benz R. Janko K. Lauger P. Biochim. Biophys. Acta. 1979; 551: 238-247Crossref PubMed Scopus (243) Google Scholar). For activity measurements, CyaA or CyaA mutants were added to both sides of the membrane, and the membrane conductance was taken 30 min after the addition, when further conductance increase in time was negligible (12Benz R. Maier E. Ladant D. Ullmann A. Sebo P. J. Biol. Chem. 1994; 269: 27231-27239Abstract Full Text PDF PubMed Google Scholar). The aqueous phase contained 1 m KCl and was also buffered to pH 7. The applied membrane potential was 50 mV. Statistical Analysis—Significance of differences in values was assessed by Student's t test. Substitutions of Glutamate 516 Increase Hemolytic Activity of CyaA by Extending Lifetime or by Enhancing Formation Frequency of CyaA Pores—In previous work, we showed that AC membrane translocation, as well as formation and cation selectivity of CyaA pores, depend on the structure and net charge of a potential amphipathic α-helical transmembrane segment (34Osickova A. Osicka R. Maier E. Benz R. Sebo P. J. Biol. Chem. 1999; 274: 37644-37650Abstract Full Text Full Text PDF PubMed Google Scholar). This is predicted to form between amino acid residues 502 and 522 (α-helix502–522) and comprises a pair of negatively charged glutamate residues, Glu509 and Glu516 (Fig. 1A). To analyze the function of this α-helix in membrane activities of CyaA in more detail, the glutamate 516 was replaced here by a helix-breaking proline residue and by a neutral glutamine residue. The mutant CyaA proteins were expressed in E. coli and purified by a combination of ion exchange and hydrophobic chromatography (Fig. 2), and their specific cell-binding, cell-invasive, and hemolytic activities were compared with those of intact CyaA, using sheep erythrocytes as target cells. The cell-invasive AC activity of CyaA constructs was first determined as the amount of the AC enzyme activity internalized into erythrocytes and protected against digestion by externally added trypsin (17Iwaki M. Ullmann A. Sebo P. Mol. Microbiol. 1995; 17: 1015-1024Crossref PubMed Scopus (61) Google Scholar).FIGURE 2SDS-PAGE analysis of purified wild-type CyaA and of its mutant variants. The proteins were expressed in recombinant E. coli K12 strains transformed with the pCACT3-derived plasmids and purified from urea extracts of cell debris by a combination of ion exchange and hydrophobic chromatography as previously described (41Karimova G. Fayolle C. Gmira S. Ullmann A. Leclerc C. Ladant D. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 12532-12537Crossref PubMed Scopus (76) Google Scholar). Proteins were separated on a 7.5% acrylamide gel and visualized by Coomassie staining.View Large Image Figure ViewerDownload Hi-res image Download (PPT) As shown in Table 1, substitution of Glu516 by the proline residue modestly reduced the capacity of CyaA-E516P to bind erythrocytes while causing ∼4-fold reduction in the specific cell-invasive activity of the CyaA-E516P mutant, as compared with intact CyaA. In contrast, substitution of Glu516 by the neutral glutamine residue, not expected to disrupt the α-helical structure of the mutagenized segment, had little effect on the cell association capacity of the CyaA-E516Q mutant and on the membrane translocation capacity of its AC domain (Table 1). These results are consistent with those obtained previously for proline and glutamine substitutions of the glutamate residue in position 509 in the same segment (34Osickova A. Osicka R. Maier E. Benz R. Sebo P. J. Biol. Chem. 1999; 274: 37644-37650Abstract Full Text Full Text PDF PubMed Google Scholar). The data, hence, further show that residues Glu509 and Glu516 are part of an α-helical structure that is crucial for AC domain translocation across target cell membrane.TABLE 1Activities of different CyaA mutants on erythrocytes and black lipid bilayer membranesToxinPercentage of activity of CyaA on erythrocytesaAll activities are expressed as percentages of intact CyaA activity and represent average values ± S.D. from at least three independent determinations performed in duplicate with three different toxin preparations. An asterisk indicates values significantly different from wild-type CyaA (*, p < 0.05; **, p < 0.001).Pore properties of CyaA in lipid bilayersBindingbSheep erythrocytes (5 × 108/ml) were incubated at 37 °C with 2 units/ml purified CyaA proteins, and after 30 min, aliquots were taken for determinations of the cell-associated AC activity and of the AC activity internalized into erythrocytes and protected against digestion by externally added trypsin (17).Cell invasivenessbSheep erythrocytes (5 × 108/ml) were incubated at 37 °C with 2 units/ml purified CyaA proteins, and after 30 min, aliquots were taken for determinations of the cell-associated AC activity and of the AC activity internalized into erythrocytes and protected against digestion by externally added trypsin (17).HemolysiscThe hemolysis was measured photometrically at 541 nm as hemoglobin released in time upon incubation of sheep erythrocytes with the indicated toxins (17).Most frequent single pore conductancedThe membranes were formed from asolectin dissolved in n-decane. The 1 m KCl solution was buffered by 10 mm HEPES and had a pH of 7.0. The applied voltage was 50 mV, and the temperature was 20 °C. The most frequent single-pore conductance, G, was determined from at least 100 single pore opening events. The error corresponds to the half of the counting interval of the single pore conductance histogram.LifetimeeThe number of pores with a given lifetime was plotted as a function of the mean lifetime. The graph was fitted with an exponential decay function, n = Noexp(-t/τ), and the lifetime was calculated as lifetime = τln2.Cation selectivityfZero-current membrane potentials were determined for a 10-fold gradient of KCl. The aqueous salt solutions were buffered by 10 mm HEPES and had a pH of 7.0; T = 20 °C. The permeability ratio Pcation/Panion was calculated with the Goldman-Hodgkin-Katz equation (36) from the mean of at least three individual experiments. An asterisk indicates values significantly different from wild-type CyaA (*, p < 0.05; **, p < 0.001). (Pcation/Panion)Overall membrane activitygThe membrane activity of the proteins was compared after 30 min of incubation with the membranes at a protein concentration of 13 ng/ml. The number of plus signs refers to the overall conductance of the membrane/cm2 induced by the various CyaA proteins under these conditions in asolectin membranes and reflects the size (conductance), the lifetime, and the specific frequency of formation of pores by the various CyaA constructs.pSsCyaA100 ± 7100 ± 5100 ± 942 ± 52.810.8 ± 0.9+++(+)CyaA-E516P76 ± 14*26 ± 8**129 ± 11**8 ± 218.32.5 ± 0.4*+++(+)CyaA-E516Q97 ± 686 ± 9*200 ± 20**30 ± 216.83.0 ± 0.3*++++CyaA-E516KhThe CyaA-E516K mutant toxin was originally constructed and characterized in detail by Osickova et al. (34), showing that despite reduced cell association capacity, the protein forms CyaA pores with importantly enhanced frequency as compared with intact CyaA. In this work, activities of CyaA-E516K were measured again under the same experimental conditions as those used for other mutants (pH 7).54 ± 6**29 ± 4**206 ± 18**40 ± 53.53.5 ± 0.4*++++CyaA-E570P68 ± 8**1 ± 1**15 ± 1**40 ± 52.83.3 ± 0.2*+CyaA-E570Q103 ± 1197 ± 649 ± 11**24 ± 22.44.4 ± 0.2*++CyaA-E570K90 ± 965 ± 3**58 ± 3**24 ± 23.13.6 ± 0.2*++(+)CyaA-E581P79 ± 13*50 ± 11**25 ± 11**32 ± 22.9NDiNot determined due to the extremely low membrane activity of the CyaA-E581P mutant.+CyaA-E581Q102 ± 1085 ± 24125 ± 11*32 ± 23.28.2 ± 0.4+++(+)CyaA-E581K74 ± 15*2 ± 2**400 ± 20**20 ± 210.511.4 ± 2.6+++++a All activities are expressed as percentages of intact CyaA activity and represent average values ± S.D. from at least three independent determinations performed in duplicate with three different toxin preparations. An asterisk indicates values significantly different from wild-type CyaA (*, p < 0.05; **, p < 0.001).b Sheep erythrocytes (5 × 108/ml) were incubated at 37 °C with 2 units/ml purified CyaA proteins, and after 30 min, aliquots were taken for determinations of the cell-associated AC activity and of the AC activity internalized into erythrocytes and protected against digestion by externally added trypsin (17Iwaki M. Ullmann A. Sebo P. Mol. Microbiol. 1995; 17: 1015-1024Crossref PubMed Scopus (61) Google Scholar).c The hemolysis was measured photometrically at 541 nm as hemoglobin released in time upon incubation of sheep erythrocytes with the indicated toxins (17Iwaki M. Ullmann A. Sebo P. Mol. Microbiol. 1995; 17: 1015-1024Crossref PubMed Scopus (61) Google Scholar).d The membranes were formed from asolectin dissolved in n-decane. The 1 m KCl solution was buffered by 10 mm HEPES and had a pH of 7.0. The applied voltage was 50 mV, and the temperature was 20 °C. The most frequent single-pore conductance, G, was determined from at least 100 single pore opening events. The error corresponds to the half of the counting interval of the single pore conductance histogram.e The number of pores with a given lifetime was plotted as a function of the mean lifetime. The graph was fitted with an exponential decay function, n = Noexp(-t/τ), and the lifetime was calculated as lifetime = τln2.f Zero-current membrane potentials were determined for a 10-fold gradient of KCl. The aqueous salt solutions were buffered by 10 mm HEPES and had a pH of 7.0; T = 20 °C. The permeability ratio Pcation/Panion was calculated with the Goldman-Hodgkin-Katz equation (36Benz R. Janko K. Lauger P. Biochim. Biophys. Acta. 1979; 551: 238-247Crossref PubMed Scopus (243) Google Scholar) from the mean of at least three individual experiments. An asterisk indicates values significantly different from wild-type CyaA (*, p < 0.05; **, p < 0.001).g The membrane activity of the proteins was compared after 30 min of incubation with the membranes at a protein concentration of 13 ng/ml. The number of plus signs refers to the overall conductance of the membrane/cm2 induced by the various CyaA proteins under these conditions in asolectin membranes and reflects the size (conductance), th" @default.
• W2023521873 created "2016-06-24" @default.
• W2023521873 creator A5000090788 @default.
• W2023521873 creator A5017167729 @default.
• W2023521873 creator A5035190614 @default.
• W2023521873 creator A5043208950 @default.
• W2023521873 creator A5043639307 @default.
• W2023521873 creator A5043880451 @default.
• W2023521873 creator A5069862319 @default.
• W2023521873 creator A5073524785 @default.
• W2023521873 date "2007-04-01" @default.
• W2023521873 modified "2023-10-15" @default.
• W2023521873 title "Segments Crucial for Membrane Translocation and Pore-forming Activity of Bordetella Adenylate Cyclase Toxin" @default.
• W2023521873 cites W1492682055 @default.
• W2023521873 cites W1502276768 @default.
• W2023521873 cites W1517788279 @default.
• W2023521873 cites W1526838717 @default.
• W2023521873 cites W1528686474 @default.
• W2023521873 cites W1572028032 @default.
• W2023521873 cites W1583032072 @default.
• W2023521873 cites W1586520658 @default.
• W2023521873 cites W1678098555 @default.
• W2023521873 cites W1745436806 @default.
• W2023521873 cites W186907087 @default.
• W2023521873 cites W1897188075 @default.
• W2023521873 cites W1899077083 @default.
• W2023521873 cites W1903755278 @default.
• W2023521873 cites W1946864347 @default.
• W2023521873 cites W1973454831 @default.
• W2023521873 cites W1978952396 @default.
• W2023521873 cites W1990669291 @default.
• W2023521873 cites W1994806280 @default.
• W2023521873 cites W2001600700 @default.
• W2023521873 cites W2021793865 @default.
• W2023521873 cites W2026582002 @default.
• W2023521873 cites W2035859531 @default.
• W2023521873 cites W2036846779 @default.
• W2023521873 cites W2045203765 @default.
• W2023521873 cites W2046499903 @default.
• W2023521873 cites W2049338606 @default.
• W2023521873 cites W2056530947 @default.
• W2023521873 cites W2076288433 @default.
• W2023521873 cites W2076477984 @default.
• W2023521873 cites W2078849651 @default.
• W2023521873 cites W2080870740 @default.
• W2023521873 cites W2081924677 @default.
• W2023521873 cites W2109351754 @default.
• W2023521873 cites W2120943171 @default.
• W2023521873 cites W2123941208 @default.
• W2023521873 cites W2154909011 @default.
• W2023521873 cites W2167805501 @default.
• W2023521873 cites W2172260392 @default.
• W2023521873 doi "https://doi.org/10.1074/jbc.m611226200" @default.
• W2023521873 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/17347146" @default.
• W2023521873 hasPublicationYear "2007" @default.
• W2023521873 type Work @default.
• W2023521873 sameAs 2023521873 @default.
• W2023521873 citedByCount "64" @default.
• W2023521873 countsByYear W20235218732012 @default.
• W2023521873 countsByYear W20235218732013 @default.
• W2023521873 countsByYear W20235218732014 @default.
• W2023521873 countsByYear W20235218732015 @default.
• W2023521873 countsByYear W20235218732016 @default.
• W2023521873 countsByYear W20235218732017 @default.
• W2023521873 countsByYear W20235218732018 @default.
• W2023521873 countsByYear W20235218732019 @default.
• W2023521873 countsByYear W20235218732020 @default.
• W2023521873 countsByYear W20235218732021 @default.
• W2023521873 countsByYear W20235218732022 @default.
• W2023521873 countsByYear W20235218732023 @default.
• W2023521873 crossrefType "journal-article" @default.
• W2023521873 hasAuthorship W2023521873A5000090788 @default.
• W2023521873 hasAuthorship W2023521873A5017167729 @default.
• W2023521873 hasAuthorship W2023521873A5035190614 @default.
• W2023521873 hasAuthorship W2023521873A5043208950 @default.
• W2023521873 hasAuthorship W2023521873A5043639307 @default.
• W2023521873 hasAuthorship W2023521873A5043880451 @default.
• W2023521873 hasAuthorship W2023521873A5069862319 @default.
• W2023521873 hasAuthorship W2023521873A5073524785 @default.
• W2023521873 hasBestOaLocation W20235218731 @default.
• W2023521873 hasConcept C104317684 @default.
• W2023521873 hasConcept C12554922 @default.
• W2023521873 hasConcept C138626823 @default.
• W2023521873 hasConcept C180526460 @default.
• W2023521873 hasConcept C181199279 @default.
• W2023521873 hasConcept C185592680 @default.
• W2023521873 hasConcept C2777367657 @default.
• W2023521873 hasConcept C2777582894 @default.
• W2023521873 hasConcept C2780069284 @default.
• W2023521873 hasConcept C41625074 @default.
• W2023521873 hasConcept C523546767 @default.
• W2023521873 hasConcept C54355233 @default.
• W2023521873 hasConcept C55493867 @default.
• W2023521873 hasConcept C86803240 @default.
• W2023521873 hasConcept C89423630 @default.
• W2023521873 hasConcept C95444343 @default.
• W2023521873 hasConceptScore W2023521873C104317684 @default.
• W2023521873 hasConceptScore W2023521873C12554922 @default.

• next