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Cloning and sequence of the Bordetella bronchiseptica adenylate cyclase-hemolysin-encoding gene: comparison with the Bordetella pertussis gene
Gene, 162 (1995) 165-166 © 1995 Elsevier Science B.V. All rights reserved. 0378-1119/95/$09.50
165
GENE 09057
Cloning and sequence of the Bordetella bronchiseptica adenylate cyclasehemolysin-encoding gene: comparison with the Bordetella pertussis gene * (Virulence; pathogen; RTX toxin; protective antigen)
Fotini Betsou a, Odile Sismeiro b, Antoine Danchin b and Nicole Guiso
a
a Unitk de Bactkriologie Molkculaire et M~dicale, Institut Pasteur, 75724 Paris Cedex 15, France; and b Unitk de R~gulation de l'Expression Gkn~tique, lnstitut Pasteur, 75724 Paris Cedex 15, France
Received by A.J. Podhajska: 16 March 1995; Revised/Accepted: 28 April/10 May 1995; Received at publishers: 15 May 1995
SUMMARY
The cyaA gene from Bordetella bronchiseptica (Bb), encoding the adenylate cyclase-hemolysin (AC-Hly), has been cloned and its complete nucleotide sequence has been determined. The deduced amino-acid sequence was compared to the AC-Hly from B. pertussis (Bp) and the main differences were found in the C-terminal repeat region of the molecule.
The human pathogen Bordetella pertussis (Bp) (agent of whooping cough) and the animal pathogen B. bronchiseptica (Bb) (agent of piglet atrophic rhinitis and canine kennel cough) are closely related species that both synthesize and secrete a number of well characterized virulence factors, including filamentous hemagglutinin (FHA), agglutinogens (AGG), pertactin (PRN) and adenylate cyclase-hemolysin (AC-Hly). The Bp AC-Hly has recently been shown to be important for bacterial pathogenesis (Khelef et al., 1992; 1993b) and immunization with Bp or Bb AC-Hly induces the synthesis of protective Ab in mice (Khelef et al., 1993a; Gueirard and Guiso, 1993). The Bp AC-Hly, encoded by cyaA, is a member of the RTX toxin family with extensive Gly-rich repeat structures in the C-terminal region (Glaser, 1988). This enzyme possesses Correspondence to: Dr. N. Guiso, Unit6 de Bact6riologie Mol6culaire et M6dicale, Institut Pasteur, 28 rue du Dr. Roux, 75724 Paris Cedex 15, France. Tel. (33-1) 4568-8334; Fax (33-1) 4061-3001; e-mail: [email protected] * On request, the authors will supply detailed experimental evidence for the conclusions reached in this Brief Note.
Abbrevations: aa, amino acid(s); Ab, antibody(ies); AC, adenylate cyclase; AC-HIy, AC-hemolysin(s); B., Bordetella; Bb, B. bronchiseptica; bp, base pair(s); Bp, B. pertussis; CaM, calmodulin; kb, kilobase(s) or 1000 bp; cyaA, gene encoding AC-Hly; nt, nucleotide(s); re-, recombinant; RTX, repeat in toxins. SSDI 0 3 7 8 - 1 1 1 9 ( 9 5 ) 0 0 3 3 9 - 8
calmodulin (CaM)-dependent AC activity, hemolytic activity and an invasive activity. The last two activities require the presence of Ca 2÷ and a post-translational acylation (Sebo et al., 1991; Hackett et al., 1994). We have recently shown that this post-translational modification is not only important for AC-Hly hemolytic and invasive activities, but also for its protective activity (Betsou et al., 1993). It has been demonstrated that both the Bp and Bb AC-Hly possess AC, hemolytic and invasive activities (Glaser et al., 1988; Gueirard and Guiso, 1993; and data not shown). Yet, they exhibit immunological differences, as well as different protective activities (Gueirard and Guiso, 1993). This suggests that at least one or more epitopes important for inducing synthesis of protective Ab are different between Bp and Bb. Here, we report the cloning and sequencing of the Bb cyaA. Bb cyaA was cloned in a pBR322 vector employing the AC-Hly-CaM interaction strategy previously used to clone the Bp cyaA (Glaser et al., 1988) and, in a reverse experiment, mouse CaM (Danchin et al., 1989). Subcloned fragments were sequenced using M13mpl8 and M13mpl9 vectors. The ORF and the ATG start codons were defined according to the Bp cyaA sequence. The nt sequence has GenBank accession No. U22953. This nt sequence is
166 about 98% identical to that of Bp cyaA. However, Bb cyaA encodes a protein of 1705 aa, while Bp cyaA encodes a protein of 1706 aa. The Ala 546 is present in Bp AC-Hly, but is missing in the Bb AC-Hly. The AC domain contains four aa differences, which are not located in either the ATP or CaM-binding regions (aa 291, 369, 370, 374). Five conservative aa differences are observed in the hydrophobic domain (aa 546, 799, 807, 909, 1000). More differences (36) are found in the third domain of the protein, containing the highly repetitive structure of Gly + Asp-rich motifs and the C-terminal secretion signal. This part of the molecule supposedly interacts with the eucaryotic cell membrane, allowing the membrane-spanning hydrophobic segment to form a pore through which the catalytically active N-terminal portion of the toxin is translocated (Rogel and Hanski, 1992). One difference is that Thr 977 is replaced by a Lys in the Bb AC-HIy. It was recently shown that Bp AC-Hly is post-translationally modified by addition of a palmitate on its Lys 983 (Hackett et al., 1994). This residue is identical in both enzymes and is followed by one conservative change at aa 1000. Eight differences are assembled in the first two groups of Gly- and Asp-rich repeats (aa 1034, 1054, 1058, 1139, 1143, 1149, 1156, 1163). Ten aa changes are assembled in the long stretch between the second and third group of Gly-Asp repeats (aa 1217, 1219, 1236, 1237, 1243, 1244, 1245, 1247, 1252, 1255). Five aa changes (aa 1335, 1346, 1356, 1362, 1364), including a Pro which replaces a Gln ~335 and the replacement of an Asp 1346 by an Ala, are located in the area between the third and the fourth group of repeats. This area is followed by three aa changes (aa 1480, 1522, 1530), including the replacement of Arg ~48° by an Asn. In spite of these aa changes, the repeats of Gly-Asp motifs are unaffected, However, Bb AC-Hly has one repeat less than Bp AC-Hly, since in the fifth group of repeats, a whole Gly~-~55-Gly-Ala-Gly ~558 motif is replaced by Ala-Ala-Pro-Ala. This modification is situated in the aa 1552-1592 region. This suggests that this repeat may not be essential for hemolytic and/or invasive activities exhibited by both enzymes, but may instead be involved in protective activity. This region is followed by three conservative aa changes (aa 1593. 1654, 1662). Finally, the Gln 1664, located in the C-terminal secretion signal of Bp AC-Hly is replaced by a Pro residue in Bb AC-Hly. The important aa differences between Bp and Bb AC-HIy are nearly all clustered between the second and third group of repeats and in the second group of the Gly-rich motif. It is tempting to speculate that these domains are surface accessible, in agreement with the previous model of Rogel and Hanski (1992), and thus more
susceptible to antigenic variation. One may also hypothesize that, since these regions supposedly interact with a receptor on target cells, Bp AC-Hly receptor may be different than that of Bp AC-HIy. In conclusion, the AC-Hly of Bp and Bb contain identical domains for AC activity, CaM-binding, poreforming activity, Ca2~--binding, and translocation inside the cell. Furthermore, one finds no alteration of the presence and spacing of acidic aa of the C-terminal part of the molecule, suspected of being an important feature of RTX-secretion signals (Sebo and Ladant, 1993). These aa might have an essential role of in secretion. Our results suggest that surface accessible portions of AC-Hly molecule containing repeats that vary between Bp and Bb species are immunodominant and important for protective activity. We are currently testing this hypothesis by examining the ability of fragments of the protein to induce both immune and protective responses. We thank Benjamin Gold for correcting the English language of the manuscript.
REFERENCES Betsou, V.. Sebo, P. and Guiso, N.: CyaC-mediatedactivation is importam not only for toxic but also for protective activities of Bordetella pertussis adenylate cyclase-hemolysin. Infect. Immun. 61 (19931 3583-3589. Danchin, A., Sezer, O., Glaser, P., Chalon, P. and Caput, D.: Cloning and expression of mouse-brain calmodulin as an activator of Bortedella pertussis adenylate cyclase in Escheriehia coli. Gene 80 (1989) 145 149. Glaser, P., Ladant, D., Sezer, O., Pichot, F.. Ullmann, A. and Danchin, A.: The calmodulin-sensitive adenylate cyclaseof Bordetella pertussis: cloning and expression in Escherichia coli. Mol. Microbiol. 2 11988) 19 30. Gueirard, P. and Guiso, N.: Virulence of Bordetella bronchiseptica: role of adenylate cyclase-hemolysin.Infect. Immun.61 ( 1993) 4072-4078. Hacken, M., Guo, L., Shabanowitz, J., Hunt, D.F. and Hewlett, E.L.: Internal lysine palmitoylation in adenylate cyclase toxin from Bordetella pertussis. Science 266 (1994}433 435. Khelef, N., Sakamoto, H. and Guiso, N.: Both adenylate cyclase and hemolytic activities are required by Bordetella pertussis to initiate infection. Microb. Pathogen. 12 (1992) 227 235. Khelef, N.. Danve, B., Quentin-Millet, M.J. and Guiso, N.: Bordetella pertussis and Bordetellaparapertus~sis: two immunologicallydistinct species. Infect. Immun. 61 (1993a) 486-490. Khelef, N., Zychlinsky, A. and Guiso, N.: Bordetella pertussis induces apoptosis in macrophages: role of adenylate cyclase-hemolysin. Infect. lmmun. 61 (1993b) 4064-4(171. Rogel, A. and Hanski, E.: Distinct steps in the penetration of adenylate cyclase toxin of Bordetella pertussis into sheep erythrocytes.J. Biol. Chem. 267 (1992) 22599 22605. ,%bo, P. and Ladant. D.: Repeat sequences in the Bordetella pertussis adenylate cyclase toxin can be recognized as alternative carboxyproximal secretion signals by the Escherichiaeoli ~-hemolysintranslocator. Mol. Microbiol. 9 (1993) 999-1009.
165
GENE 09057
Cloning and sequence of the Bordetella bronchiseptica adenylate cyclasehemolysin-encoding gene: comparison with the Bordetella pertussis gene * (Virulence; pathogen; RTX toxin; protective antigen)
Fotini Betsou a, Odile Sismeiro b, Antoine Danchin b and Nicole Guiso
a
a Unitk de Bactkriologie Molkculaire et M~dicale, Institut Pasteur, 75724 Paris Cedex 15, France; and b Unitk de R~gulation de l'Expression Gkn~tique, lnstitut Pasteur, 75724 Paris Cedex 15, France
Received by A.J. Podhajska: 16 March 1995; Revised/Accepted: 28 April/10 May 1995; Received at publishers: 15 May 1995
SUMMARY
The cyaA gene from Bordetella bronchiseptica (Bb), encoding the adenylate cyclase-hemolysin (AC-Hly), has been cloned and its complete nucleotide sequence has been determined. The deduced amino-acid sequence was compared to the AC-Hly from B. pertussis (Bp) and the main differences were found in the C-terminal repeat region of the molecule.
The human pathogen Bordetella pertussis (Bp) (agent of whooping cough) and the animal pathogen B. bronchiseptica (Bb) (agent of piglet atrophic rhinitis and canine kennel cough) are closely related species that both synthesize and secrete a number of well characterized virulence factors, including filamentous hemagglutinin (FHA), agglutinogens (AGG), pertactin (PRN) and adenylate cyclase-hemolysin (AC-Hly). The Bp AC-Hly has recently been shown to be important for bacterial pathogenesis (Khelef et al., 1992; 1993b) and immunization with Bp or Bb AC-Hly induces the synthesis of protective Ab in mice (Khelef et al., 1993a; Gueirard and Guiso, 1993). The Bp AC-Hly, encoded by cyaA, is a member of the RTX toxin family with extensive Gly-rich repeat structures in the C-terminal region (Glaser, 1988). This enzyme possesses Correspondence to: Dr. N. Guiso, Unit6 de Bact6riologie Mol6culaire et M6dicale, Institut Pasteur, 28 rue du Dr. Roux, 75724 Paris Cedex 15, France. Tel. (33-1) 4568-8334; Fax (33-1) 4061-3001; e-mail: [email protected] * On request, the authors will supply detailed experimental evidence for the conclusions reached in this Brief Note.
Abbrevations: aa, amino acid(s); Ab, antibody(ies); AC, adenylate cyclase; AC-HIy, AC-hemolysin(s); B., Bordetella; Bb, B. bronchiseptica; bp, base pair(s); Bp, B. pertussis; CaM, calmodulin; kb, kilobase(s) or 1000 bp; cyaA, gene encoding AC-Hly; nt, nucleotide(s); re-, recombinant; RTX, repeat in toxins. SSDI 0 3 7 8 - 1 1 1 9 ( 9 5 ) 0 0 3 3 9 - 8
calmodulin (CaM)-dependent AC activity, hemolytic activity and an invasive activity. The last two activities require the presence of Ca 2÷ and a post-translational acylation (Sebo et al., 1991; Hackett et al., 1994). We have recently shown that this post-translational modification is not only important for AC-Hly hemolytic and invasive activities, but also for its protective activity (Betsou et al., 1993). It has been demonstrated that both the Bp and Bb AC-Hly possess AC, hemolytic and invasive activities (Glaser et al., 1988; Gueirard and Guiso, 1993; and data not shown). Yet, they exhibit immunological differences, as well as different protective activities (Gueirard and Guiso, 1993). This suggests that at least one or more epitopes important for inducing synthesis of protective Ab are different between Bp and Bb. Here, we report the cloning and sequencing of the Bb cyaA. Bb cyaA was cloned in a pBR322 vector employing the AC-Hly-CaM interaction strategy previously used to clone the Bp cyaA (Glaser et al., 1988) and, in a reverse experiment, mouse CaM (Danchin et al., 1989). Subcloned fragments were sequenced using M13mpl8 and M13mpl9 vectors. The ORF and the ATG start codons were defined according to the Bp cyaA sequence. The nt sequence has GenBank accession No. U22953. This nt sequence is
166 about 98% identical to that of Bp cyaA. However, Bb cyaA encodes a protein of 1705 aa, while Bp cyaA encodes a protein of 1706 aa. The Ala 546 is present in Bp AC-Hly, but is missing in the Bb AC-Hly. The AC domain contains four aa differences, which are not located in either the ATP or CaM-binding regions (aa 291, 369, 370, 374). Five conservative aa differences are observed in the hydrophobic domain (aa 546, 799, 807, 909, 1000). More differences (36) are found in the third domain of the protein, containing the highly repetitive structure of Gly + Asp-rich motifs and the C-terminal secretion signal. This part of the molecule supposedly interacts with the eucaryotic cell membrane, allowing the membrane-spanning hydrophobic segment to form a pore through which the catalytically active N-terminal portion of the toxin is translocated (Rogel and Hanski, 1992). One difference is that Thr 977 is replaced by a Lys in the Bb AC-HIy. It was recently shown that Bp AC-Hly is post-translationally modified by addition of a palmitate on its Lys 983 (Hackett et al., 1994). This residue is identical in both enzymes and is followed by one conservative change at aa 1000. Eight differences are assembled in the first two groups of Gly- and Asp-rich repeats (aa 1034, 1054, 1058, 1139, 1143, 1149, 1156, 1163). Ten aa changes are assembled in the long stretch between the second and third group of Gly-Asp repeats (aa 1217, 1219, 1236, 1237, 1243, 1244, 1245, 1247, 1252, 1255). Five aa changes (aa 1335, 1346, 1356, 1362, 1364), including a Pro which replaces a Gln ~335 and the replacement of an Asp 1346 by an Ala, are located in the area between the third and the fourth group of repeats. This area is followed by three aa changes (aa 1480, 1522, 1530), including the replacement of Arg ~48° by an Asn. In spite of these aa changes, the repeats of Gly-Asp motifs are unaffected, However, Bb AC-Hly has one repeat less than Bp AC-Hly, since in the fifth group of repeats, a whole Gly~-~55-Gly-Ala-Gly ~558 motif is replaced by Ala-Ala-Pro-Ala. This modification is situated in the aa 1552-1592 region. This suggests that this repeat may not be essential for hemolytic and/or invasive activities exhibited by both enzymes, but may instead be involved in protective activity. This region is followed by three conservative aa changes (aa 1593. 1654, 1662). Finally, the Gln 1664, located in the C-terminal secretion signal of Bp AC-Hly is replaced by a Pro residue in Bb AC-Hly. The important aa differences between Bp and Bb AC-HIy are nearly all clustered between the second and third group of repeats and in the second group of the Gly-rich motif. It is tempting to speculate that these domains are surface accessible, in agreement with the previous model of Rogel and Hanski (1992), and thus more
susceptible to antigenic variation. One may also hypothesize that, since these regions supposedly interact with a receptor on target cells, Bp AC-Hly receptor may be different than that of Bp AC-HIy. In conclusion, the AC-Hly of Bp and Bb contain identical domains for AC activity, CaM-binding, poreforming activity, Ca2~--binding, and translocation inside the cell. Furthermore, one finds no alteration of the presence and spacing of acidic aa of the C-terminal part of the molecule, suspected of being an important feature of RTX-secretion signals (Sebo and Ladant, 1993). These aa might have an essential role of in secretion. Our results suggest that surface accessible portions of AC-Hly molecule containing repeats that vary between Bp and Bb species are immunodominant and important for protective activity. We are currently testing this hypothesis by examining the ability of fragments of the protein to induce both immune and protective responses. We thank Benjamin Gold for correcting the English language of the manuscript.
REFERENCES Betsou, V.. Sebo, P. and Guiso, N.: CyaC-mediatedactivation is importam not only for toxic but also for protective activities of Bordetella pertussis adenylate cyclase-hemolysin. Infect. Immun. 61 (19931 3583-3589. Danchin, A., Sezer, O., Glaser, P., Chalon, P. and Caput, D.: Cloning and expression of mouse-brain calmodulin as an activator of Bortedella pertussis adenylate cyclase in Escheriehia coli. Gene 80 (1989) 145 149. Glaser, P., Ladant, D., Sezer, O., Pichot, F.. Ullmann, A. and Danchin, A.: The calmodulin-sensitive adenylate cyclaseof Bordetella pertussis: cloning and expression in Escherichia coli. Mol. Microbiol. 2 11988) 19 30. Gueirard, P. and Guiso, N.: Virulence of Bordetella bronchiseptica: role of adenylate cyclase-hemolysin.Infect. Immun.61 ( 1993) 4072-4078. Hacken, M., Guo, L., Shabanowitz, J., Hunt, D.F. and Hewlett, E.L.: Internal lysine palmitoylation in adenylate cyclase toxin from Bordetella pertussis. Science 266 (1994}433 435. Khelef, N., Sakamoto, H. and Guiso, N.: Both adenylate cyclase and hemolytic activities are required by Bordetella pertussis to initiate infection. Microb. Pathogen. 12 (1992) 227 235. Khelef, N.. Danve, B., Quentin-Millet, M.J. and Guiso, N.: Bordetella pertussis and Bordetellaparapertus~sis: two immunologicallydistinct species. Infect. Immun. 61 (1993a) 486-490. Khelef, N., Zychlinsky, A. and Guiso, N.: Bordetella pertussis induces apoptosis in macrophages: role of adenylate cyclase-hemolysin. Infect. lmmun. 61 (1993b) 4064-4(171. Rogel, A. and Hanski, E.: Distinct steps in the penetration of adenylate cyclase toxin of Bordetella pertussis into sheep erythrocytes.J. Biol. Chem. 267 (1992) 22599 22605. ,%bo, P. and Ladant. D.: Repeat sequences in the Bordetella pertussis adenylate cyclase toxin can be recognized as alternative carboxyproximal secretion signals by the Escherichiaeoli ~-hemolysintranslocator. Mol. Microbiol. 9 (1993) 999-1009.