Bordetella pertussis Adenylate Cyclase Toxin - The Journal of ...

THEJOURNAL OF BIOLOGICAL CHEMISTRY (0 1992 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 267, No. 19,Issue of July 5 , pp. 13598-13602, 1992 Printed in U.S.A.

Bordetella pertussisAdenylate CyclaseToxin STRUCTURAL AND FUNCTIONALINDEPENDENCE OF T H E CATALYTICANDHEMOLYTIC ACTIVITIES* (Received for publication, February 3, 1992)

Hiroshi SakamotoS, Jacques Bellalou, Peter Sebo, and Daniel Ladant From the Unite de Biochimie des Regulations Cellulaires, Znstitut Pasteur, 75724 Paris, Cedex 15, France

The Bordetella pertussis calmodulin-dependent adenylate cyclase (CyaA) is a 1706-residue-long toxin, endowed with hemolytic activity. We have constructed B. pertussis mutant strains producing modified CyaAs devoid of adenylate cyclase activity. Our results show that such modified CyaAs display hemolytic activity identical to the wild-type toxin, thus demonstrating that the hemolytic activity is independent of the adenylate cyclase activity. Furthermore, B. pertussis and Escherichia coli strains producing CyaA lacking the catalytic domain (residues 1-373) were constructed. The truncated protein exhibits hemolytic activity comparable to the wild-type toxin, thus establishing that the carboxyl-terminal 1332 residues alone are endowed with hemolytic activity. Together, these findings show that adenylate cyclase and hemolytic activities are located in two distinct regions of the molecule (respectively, approximately amino acids 1-400 and 401-1706) and that the two regionsof CyaA are functionally independent.

(Ladant et al., 1986). The remaining carboxyl-terminal 1306 residues, that show 25% homology with the Escherichia coli a-hemolysin (HlyA), areinvolved in hemolytic and cytotoxic activities of CyaA (Glaser et al., 1988b; Bellalou et al., 1990; Rogel et al., 1991). We recently demonstrated that the hemolytic activity of CyaA is weak as compared to the E. coli HlyA, and that hemolysis only starts long after the intracellular cAMP accumulation in the targetcells has reached its maximum (Bellalou et al., 1990). On the basis of these observations, it was suggested that thehemolytic activity of CyaA was the consequence of cAMP accumulation within erythrocytes, leading to cell lysis. To test this hypothesis, we constructed B. pertussis and E. coli mutant strains producing full length modified CyaA devoid of adenylate cyclase activity, or truncated CyaA molecules lackingthecatalyticdomain(residues 1373). Both typeof modified CyaA display hemolytic activities identical to thewild-type toxin, thus demonstrating that the hemolytic and adenylate cyclase activities are located in two distinct and functionally independentdomains. Implications of these findings, with respect to the physiological relevance of the hemolytic activity of CyaA, are discussed.

Bordetellapertussis, the etiological agent of whooping EXPERIMENTALPROCEDURES cough, produces a variety of virulence factors including pertussis toxin, filamentous hemagglutinin, and adenylate cyConstruction of Plasmids and Strains-Plasmid pRTPl (Stibitz et clase-hemolysin (CyaA)’ (reviewedbyWeiss and Hewlett, al., 1986) derivatives carrying fragments of the cya locus of B. pertussis 1986). CyaA is a toxin which can invade eukaryoticcells and, (see Fig. 1) were constructed according to conventionalmethods DNA uponactivation by the calmodulin (CaM) of target cells, (Sambrook et al., 1989). Plasmid pHSP70 harborsa4-kb fragment with a marker encoding kanamycin resistance, cloned into produces supraphysiologicallevels of CAMP, leading to drastic the cyaA gene between codons 188 and 189 (EcoRV site). Plasmid damage of cell functions (cytotoxic activity) (Confer and pHSP87 harbors a 3.6-kb DNA fragment in which the cyaA gene was Eaton, 1982; Hanski and Farfel, 1985). This toxin is a major mutated by changing the codon at position 58 from AAA to CAA virulence factor, and mutant strains devoid of CyaA are less (Glaser et al., 1989). Plasmid pRAC3 contains a 1.6-kb DNA fragment in which the cyaA gene was mutated by cloning an oligonucleotide virulent thanwild-type strains, when tested in animal systems (Weiss et al., 1983; Khelef et al., 1992). CyaA is a secreted (CTGCAG) between codons 188 and 189 (Ladant et al., 1992). The 1706-amino acids (aa)-long multifunctional protein,endowed suicide plasmids derived from pRTPl were introduced into B. pertussis strain 18323 (Pittman, 1984) by conjugation, as already dewithadenylate cyclase, hemolytic, and cytotoxic activities scribed (Bellalou et al., 1990). The E. coli strain S17-1 (Simon et al., (Wolff et al., 1980; Ladant et al., 1986; Glaser et al., 1988a; 1983) was used as the donor, and the conjugation was performed on Bellalou et al., 1990; Rogel et al., 1991; Hewlett et al., 1991). Bordet-Gengou agar plates. Selection of the insertion of the suicide The amino-terminal 400 residues constitute the catalyticdo- plasmid into theB. pertussis chromosome, and counterselection of E. main, and canbe purified as a proteolytic fragmentof 45 kDa coli were performed by plating on ampicillin (30 pg/ml) and nalidixic acid (50 pg/ml), whereas selection for the allelic exchange was done

* This work was supported by grants from the Institut Pasteur and by plating ampicillin-resistant clones on streptomycin (200 wg/ml). Centre National de la RechercheScientifique URA-1129, Grant 91.0615 from the Institut National de la SantC. et de la Recherche Scientifique, and Grant 91.048 (to A. U.) from the Direction des Recherches-Etudes et Techniques. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. $ To whom correspondence should be addressed. The abbreviations used are: CyaA, adenylate cyclase; HlyA, crhemolysin; LktA, leukotoxin; aa, amino acid(s); kb, kilobase pair(s); SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis.

This procedure was used to construct strains 18HS10 (cyaA::Km), 18HS22 (cyaA KmQ), and 18HS24 (cyaA D I ~ [ L Q J I ~ ~ . The nonhemolytic B. pertussis strain (348HS3) which did not express the cyaA gene, but still expressed the cyaCBDE genes, was constructed as follows. A pUC18 derivative harboring the entire cya locus (genes cyaABCDE) was first constructed. The cyaA gene was then completely deleted by Bal31 nuclease digestion, yielding plasmid pHSP49. Plasmid pHSP49 was introduced into BP348 (Weiss et al., 1983) by electroporation, selecting for ampicillin resistance (Zealey et al., 1988). Ampicillin-resistant clones resulted from single recombination events in the chromosome, as pHSP49 does not replicate in BP348. The resulting strain 348HS3 was tested for the production of

13598

-

B. pertussis Adenylate Cyclase-Hemolysin Toxin

18323

I

CYlA

1

13599

A

t at

348HS3 A

pHSP70-d

D

Tn5

V

IC1

A

I BIDIE

c

c

PcyaC

PcyaC Km

T7

18HsIo

-

B

pSSP87-d

E

MAAAKLTF,

I

pJB1 IBHS22

w

I*

CytA

I

A

'A

1

-

PTC

C FIG. 1. Schematic representation of the construction of B. pertussis strains. Left, cyaA mutations of the catalytic domain. Horizontal bars represent E. pertussis DNA, either on the chromosome (thick bars), or on the suicide plasmids (thin bars). Open boxes are entire or truncatedcyaA genes. The DNA fragment encoding resistance to kanamycin is represented by the triangle Km, and the pointshows the position of the mutation to be introduced on the chromosome (i.e. Ks8Q). Vertical arrows represent recombination events. A, first allelic exchange resulting from the action of plasmid pHSP70 on E. pertussis strain 18323, and yielding strain 18HS10; E , second allelic exchange, between homologous regions of strain 18HS10 and plasmid pHSP87, yielding, C, the final strain 18HS22, which harbors on its chromosome the mutation (ie. K58Q). Right, 5"truncated cyaA gene. Horizontal bars represent E. pertussis chromosomal DNA, open boxes cya genes. The T n 5 transposon (open triangle) of strain BP348abolishes the expression of the cy&, E , D , E genes. In strain 348HS3 ( D ) ,the integration in the cya locus of a pUC18 derivative plasmid, carrying the cyaC, E , D, E genes under the control of cyaC or cyaA promoters (PcyaC, PcyaA), provides the secretion apparatus. Theclosed triangle represents pUC18 vector DNA. The truncatedcyaA gene encoding the last 1332 residues of CyaA is harbored on plasmid p J B l ( E ) and expressed under the control of the trc promoter (Ptrc). Lys374indicates the 374th codon of cyaA. Additional amino acids, introduced by the linker used during the construction. are indicated. a n active secretion apparatus by transcomplementation with the cyaA gene which restored the hemolytic phenotype on blood agar plates. Plasmid pCTER95 andp J B l were constructed as follows. Starting from plasmid pACT7 (Sebo et al., 1991) which harbors the entire cyaA gene, the unique EstBI site (codons 373 and 374 in cyaA) was converted to a Hind111 site by the insertion of an oligonucleotide linker 5"CGTAAGCTTA. The HindIII-ScaI fragment encoding the last 1332 residues of CyaAwas then subcloned in the expression vector pKK233-2 (PharmaciaLKB Biotechnology Inc.), yielding plasmid pCTER95. This resulted in an in-frame fusion of the truncated cyaA gene to the translation initiation sequence of pKK233-2. Thus plasmid pCTER95 encodes a protein of 1339 residues expressed under the control of the inducible trc promoter. The truncated CyaA molecule starting at residue Lys374is synthesized as a fusion protein with the linker-encoded amino-terminal peptide Met-Ala-Ala-AlaLys-Leu-Thr. For the transfer of the construction into B. pertussis, the BamHI-ScaIfragment from pCTER95, encoding the 1332-aa carboxyl-terminal part of CyaA, was subcloned into the EamHI-SmaI sites of the conjugative vector pMB208 (Moraleset al., 1991), yielding plasmid pJB1. Plasmid p J B l was introduced into strain 348HS3 by conjugation using E. coli SMlO (Simon et al., 1983), as previously described (Glaser et al., 1988b). Plasmid pPS4C, harboring the cyaC gene, and E. coli strain XLI were previously described (Sebo et al., 1991). Bacterial Growth Conditions and CyaA Purification-E. pertussis strains were grown for 24 h at 36 "C in modified Stainer and Scholte liquid medium, as already described (Bellalou et al., 1990). The pellet (1.3 g of bacteria) was resuspended in 10 ml of buffer A (50 mM Tris hydrochloride, pH 8,0.2 mM CaCl?, 5 M urea), and incubated at 20 "C for 60 min. After centrifugation, the supernatant was used for purification of CyaA on calmodulin-agarose, as already described (Bellalou et al., 1990). E. coli strains XLI/pPS4C/pACT7 or XLI/pPS4C/ pCTER95 were grown at 37 "C in 2YT medium (Sambrook et al., 1989) containing100 pg/ml ampicillin and 20 pg/ml chloramphenicol, to an optical density (Aso0)of0.5-0.7. Then isopropyl-1-thio-j3-Dgalactopyranoside was added (1 mM final concentration), to induce the synthesis of wild-type or truncated CyaA molecules. After 4 h of induction, the cells were collected, resuspended in 2% of the initial culture volume of buffer B (50 mM Tris-HC1, 0.2 mM CaCl,, pH 8), and disrupted by sonication. The sonicated extract was centrifuged for 10 min at 13,000 rpm at 4 "C. The pellet, which contained about

70% of total adenylate cyclase activity, was resuspended in 1%of the initial culture volume of 8 M urea in buffer B and shaken for 1 h at 4 "C. After 10 min of centrifugation at 13,000 rpm, the supernatant ("urea extract") was collected. Wild-type CyaA waspurified by CaMagarose chromatography as previously described (Sebo et al., 1991). The truncated protein was purified by chromatography on DEAESepharose in denaturing conditions as follows: NaCl was added to 2 ml of urea extract (0.1 M final concentration), and the preparation was loaded at a flow rate of 2ml/h on a1-mlDEAE-Sepharose column equilibrated in buffer B containing 8 M urea and 0.1 M NaCl. After extensive washing with the same buffer, the truncated protein was eluted with 0.2 M NaCl in buffer B containing 8 M urea. Adenylate cyclase activity was measured according to Ladant etal. (1988). One unit of enzymatic activity correspondsto 1pmol of CAMP formed per min at 30 "C and pH8. Proteins were measured according to Bradford (1976). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed according to Laemmli (1970). Immunoblots were performed as described by Monneron et al. (1988), using monoclonal antibodies directed againstthe carboxylterminal part of CyaA and detected with anti-mouse immunoglobulin labeled with alkaline phosphatase. Hemolysis and Cytotoxicity Assays-These assays were performed as already described (Bellalou et al., 1990). Briefly, various amounts of purified CyaA protein were incubated at 37 "C for 18 h with 3 X 10' sheeperythrocytes, in afinal volume of 1 ml. The extent of hemolysis was determined by measuring the of the sample and comparing with a sample incubated with water or excess listeriolysin, taken as a100% value. The cytotoxicity assay was performed by incubating 2 pg of CyaA protein with 3 X 10' sheep erythrocytes at 37 "C for 30 min, in a final volume of 1 ml, and the intracellular CAMP concentration was determined by radioimmunoassay as already described (Bellalou et al., 1990). Materials-Bovine brain CaM, isopropyl-l-thio-j3-D-galactopyranoside, and antibiotics were from Sigma. Enzymes for routine DNA manipulations were obtained from Bethesda Research Labs, Pharmacia, United States Biochemicals, or New England Biolabs. Radiolabeled ATP was from the Radiochemical Center, Amersham (UK). Oligonucleotides were synthesized by the Organic Chemistry Unit, Institut Pasteur (Paris).Urea was a product of Schwarz/Mann. Antimouse immunoglobulin labeled with alkaline phosphatase was ob-

B. Adenylate pertussis Cyclase-Hemolysin Toxin

13600 tained from Biosys(France). product.

DEAE-Sepharose was a Pharmacia (

RESULTS

P

I

”

CyaA Lacking Adenylate Cyclase Activity Exhibits Wild-type Hemolytic Activity-The catalytic domain of CyaA (aa 1-400) has been the subject of intense studies from various laboratories. Several mutations have been constructed in E. coli on a truncated c y d gene encoding modified, stable 45-kDa proteins affected intheir adenylate cyclase activity (Glaser et al., 1989; Au et al., 1989; Glaser et al., 1991; Ladant et al., 1992). Two such mutations were further used. The first one (KSRQ) is areplacement of Lys at position 58 by Gln, the corresponding modified protein exhibiting a drastically reduced adenylate cyclase activity (0.1% of wild type) (Glaser et al., 1989). The second one (Dlm[LQ]IIs9)corresponds to the insertion of two amino acids (Leu and Gln) between residues 188 (Asp) and 189 (Ile), resulting in a complete loss of adenylate cyclase activity (Ladant et al., 1992). These two mutations were introduced into the B. pertussis chromosome by allelic exchange, as shown in Fig. IA. In a first step, a selectable marker (i.e. kanamycin resistance gene, K m ) was introduced into thec y d gene, closeto themutations t o be transferred. The resulting strain (18HS10) was used as the recipient for further mutations to be introduced. During the second allelic exchange the K m marker can be replaced by a mutated c y d sequence, yielding a strain that could be easily screened by its sensitivity to kanamycin. This strain should produce a modified CyaA devoid of adenylate cyclase activity. Strains 18HS22 (harboring mutation KSRQ)and 18HS24 (harboring mutation DIB[LQ]1189) were obtained this way. The two recombinant strains 18HS22 and 18HS24 displayed no detectable adenylate cyclase activity (Table I) and, as shown by immunoblots (Fig. 2 A ) , they synthetized a 200kDa CyaA protein, which wassecreted in the culturemedium as efficiently as thewild-type toxin. As the CaM binding sites of the modified CyaA were intact, we were able to purify the two modifiedproteins using the standard CaM-agarose affinity chromatography procedure devised previously forthe purification of the wild-type toxin (Bellalou et al. 1990).Fig. 2B shows a SDS-PAGE analysis of the purified CyaA preparations, from wild-typeand mutant strains,which werefurther used for in vitro hemolysis and cytotoxicity assays. Fig. 3, A and B, shows dose-response hemolysis curves and kinetics of lysis, respectively. From these experiments, it can be concluded that the modified CyaAs exhibited similar hemolytic activities to the wild-type toxin. As expected, the modified CyaA did not increase the intracellular cAMP level of erythTABLE I Characterization of CyaA proteins from wild-typeor mutant B. pertussis strains Strains

Adenylate cyclase activity”

CyaA secretionb

Hemolytic activity’

unitslml

Pglml

%

Cytotoxic activity

-Wild type 400 1.5 100 3300 18HS22