Gene, 104 (1991) 19-24 0

1991 Elsevier

GENE

Science

Publishers

B.V. All rights reserved

19

0378-l 119/91/$03.50

05075

High-level synthesis of active adenylate Escherichia coli system (Recombinant

DNA;

holotoxin

activation;

cyclase

invasiveness;

toxin of Bordetellu

hemolytic

pertussis

in a reconstructed

activity)

Peter Sebo a, Philippe Glaser b, Hiroshi Sakamoto a and Agnes Ullmann a ” Unite de Biochimie des Rt!gulationsCellulaires,and h UmitPde R&ulation de I’Expression GPnPtique,Dipartement de Biochimie et GMtique Mokulaire. Institut Pasteur, 75724 Paris Cedex 15 (France) Received by J.-P. Lecocq: 18 February 1991 Revised/Accepted: 19 April/l 8 May 199 1 Received at publishers: 27 May 1991

SUMMARY

The Bordetellu pertussis adenylate cyclase(Cya) toxin-encoding locus (cya) is composed of five genes. The cyaA gene encodes a virulence factor (CyaA), exhibiting adenylate cyclase, hemolytic and invasive activities. The cyuB, D and E gene products are necessary for CyaA transport, and the cyaC gene product is required to activate CyaA. We reconstructed, in Escherichia coli, the cya locus of B. pertussis by cloning the different genes on appropriate vectors under the control of strong promoters and E. coli-specific translation initiation signals. We show that in the absence of additional gene products, CyaA is synthesized at high levels, is endowed with adenylate cyclase activity, but is devoid of invasive and hemolytic activities. CyaC is sufftcient to confer upon the adenylate cyclase holotoxin full invasive and partial hemolytic activities. Coexpression of the cy&I, D and E genes neither stimulates nor potentiates the activation brought about by CyaC. This reconstructed system should help to elucidate both the mechanism and the structural requirements of holotoxin activation.

INTRODUCTION

The extracellular adenylate cyclase toxin (CyaA) produced by B. pertussis, the causative agent of whooping cough, is now considered as one of the major virulence Correspondence to: Dr. A. Ullmann, Unite de Biochimie des Regulations Institut Pasteur, 28 rue du Docteur Roux, 75724 Paris Cedex

Cellulaires,

15 (France) Tel.: (33-1)45688385;

Fax: (33-1)43069835.

Abbreviations:

A,,,,, absorbance at 600 mm; aa, amino acid(s); Ap, B., Border&z; bp, base pair(s); CAMP, cyclic AMP; Cya,

ampicillin; adenylate

cyclase; cya, gene encoding

isopropyl-p-D-thiogalactopyranoside; nucleotide(s);

oligo,

Cya; Cm, chloramphenicol; kb,

kilobase

oligodeoxyribonucleotide;

or ORF,

IPTG,

1000 bp; open

nt,

reading

frame; ori, origin of DNA replication; PAGE, polyacrylamide-gel electrophoresis; P,,,< , lac promoter; PC,,, hybrid trp-lacpromoter; R, resistant; RBS, ribosome-binding site; rrnB, ribosomal RNA transcriptional unit; SDS, sodium

dodecyl

sulfate;

u, unit(s);

wt, wild type.

factors involved in the pathogenesis (St. Mary Goodwin and Weiss, 1990). The toxin penetrates various mammalian cells and on activation by the intracellular calmodulin, elicits a dramatic increase in the intracellular CAMP concentration resulting in impairment of cellular functions (see Weiss and Hewlett, 1986, for review). The nt sequence of the cyaA gene, encoding the toxin, and the characterization of the gene product revealed that CyaA is a bifunctional protein of 1706 aa exhibiting both invasive adenylate cyclase and hemolysin activities (Glaser et al., 1988a,b; Rogel et al., 1989; Hewlett et al., 1989; Bellalou et al., 1990). The CyaA protein is composed of two regions. The N-terminal 400-aa domain corresponds to the calmodulin-activated adenylate cyclase activity (Ladant et al., 1989); the C-terminal 1306 aa, which share 25% sequence homology with E. coli x-hemolysin, are essential for invasiveness and hemolytic activity of the CyaA toxin (Bellalou et al., 1990). The secretion of the CyaA protein

20

(A)

Plac

pDIA5240

(4.0

oril

kb)

AP R pACT7

(7.9

DPSG I5

(9.8

kb)

pPSGlE

(9.8

kb)

pPSG2E

(I 0.7 kb)

pPSG3CE

(1 I .6b)

(E/N)

pPS4C

(5.4

ori 1

kb)

WN)

i A

kb)

(B) pDlA5240

pPSGlE Fig. 1. Construction in pTZl8

of plasmids

(Pharmacia)

for adenylate

was used as starting

ofcyaA on pDIA5227 ATCAGG

,..

pDIA5240,

form of CyaA consisting

of the two N-terminal

by insertion

and A&II sites around

of pHSG575

Aat _--. II ,!f&&AACAGACCaLgACGTCG-3’.

cyclase toxin expression.

contained

(Takeshita

(CYaS, D, El

of plasmids strand.

The construction

of E. coli fi-galactosidase fragment

in plasmid

codon

in pPSGl5.

(Gilles et al., 1990) bearing the first 399 codons ofc.VuA cloned

for cyaA expression.

In the first step, the 93 nt preceding

ofpDIA5211

pACT7.

fused to the N-terminal

the ATG

the entire fragment

the C-terminal

1437 aa of CyaA, between

of the cyaB, D and E genes we used the unique

BspHI-&I

fragment

of pDlA5211

RBS and start codon (underlined)

of the 6.2-kb AatII-Hind111

fragment

of cyuA.

of the truncated

399 aa of the toxin, In the second step the CJVJA

(Glaser et al., 1988a), encoding

For subcloning

Then, a synthetic

of the cyaB gene, by insertion

was verified by resequencing

of lac.2 fused to the ORF of cyaA; it allowed the overproduction

the start codon of cyaB. First, a 6.2-kb blunt-ended

et al., 1987) resulting

was fused to the second

of the complementary

the RBS and two codons

resulting

*)

(Taylor et al., 1985) using a 34-mer S’-dGAAACAGCTATGACCATGCAGCAATCGC-

mutagenesis

residues

(cyaA

Plasmid pDIA5227

for the construction

of a 4389-bp Bell-BspHI

the BclI and EcoRI sites of pDIA5240, @HI

5’-

were deleted by site-directed

plasmid,

gene was completed

BAACAGCT&f&ACCi&gCAGAA-3’

material

as a primer for the in vitro synthesis

The resulting

5’-

was inserted

sequence

of pPSGl5

and overlapping

into the BarnHI

site

5’-AGGAAACAGACCG

into the blunt-ended

Nrol site of the

pKK233-2 vector (Pharmacia) by the use of a synthetic adaptor S’-ACGT, to preserve the AarII site. This resulted in pPSGIE. Finally, the 7.1-kb EcoRI-ScaI fragment ofpPSGlE was inserted between the EcoRI and Hind111 sites of pHSG575, yielding pPSG2E. The restored EcoRI site of pPSG2E was used for insertion were removed standard

of the 0.9-kb NaeI fragment

from pPSG3CE

protocols

(Sambrook

by an AatII-Hind111

of pDIA5211, deletion,

et al., 1989). (A) Schematic

containing

resulting

the cyaC gene, resulting

in pPS4C.

representation

The recombinant

of the constructed

in pPSG3CE.

In the end, the cyaB, D and E genes

DNA manipulations

plasmids.

(B) Sequences

were performed

according

of the newly introduced

to

RBSes

of cyuA and cyaB genes. The start codons of cyaA and cyaB genes are indicated by lower-case underlined characters. Symbols: cyaA *, first 399 codons of evaA; P,,,, I’,,,, promoters; oril, pMBl-derived origin of replication; ori2, pSClOl-derived origin of replication; r, T2, transcription terminators of the rrnB region. A,AotII; by ligation

Bc, BclI; E,EcoRI;

of blunt ends. (A/H).

H, HindIII;

N, NueI; SC, ScaI; Sm, SmnI; Xh,XhoI.

(E/N), (SC/H) are sites erased

by ligation

of blunt ends.

Restriction

sites in parentheses

are those that are destroyed

21 requires the products

of three genes, cyaB, D and E, located

downstream from the cyaA gene (Glaser et al., 1988b). Rogel et al. (1989) showed that the expression of the cyaA

12

kDa

345

C yaA*

- 205

gene in E. coli leads to the production of a catalytically active, 200-kDa CyaA devoid of invasive and hemolytic activities. They proposed that a post-translational moditication, which occurs in B. pertussis but not in E. coli, confers upon the CyaA protein the toxic properties. Recently, a cyaC gene homologous to the hlyC gene, required for the activation of the hlyA gene product in E. cob, has been discovered in B. pertussis and shown to be necessary for hemolytic and toxin activities of the cyaA gene product (Barry et al., 1991). In B. pertussis, however, the secretion genes cyaB, D and E, are coexpressed with cyaC; therefore their involvement in the activation of CyaA protein could not be excluded. The aim of the present work was to produce active CyaA toxin in a reconstructed E. coli system. We show that, whereas the cvaC gene product is sufficient to confer full invasive activity on the CyaA protein, additional and yet unidentified factors are probably required to restore full hemolytic activity.

analysis

DISCUSSION

Ap/ml

toxins. Bacteria

-

66

-

45

of recombinant

Table I were grown at 37°C to an absorbance with

were harvested,

disrupted

by sonication.

proteins

were pelleted

containing

resuspended

cyclase activity were resuspended

were stored at -20°C.

1 ml of 8 M urea extracts washed

at -20°C.

(7.5%) according

1 ml calmodulin-agarose

The proteins

to Laemmli

urea extract B.

columns.

were

separated

(1970). Cell-associated

2 mM EDTA

CyaA protein from

to Bellalou et al. (1990). Lanes: 1,8 M

PS30 and PS40, respectively;

pertussis;M, standards

The col-

by SDS-PAGE

of cell debris of PS40; 2, 3. 4, afftnity-purified

of PSlO,

of the

four times with

with 2 M urea in Buffer A and the CyaA

B. pertussis was purified according extracts

in 1 ml of

For the purification was diluted

were eluted with 8 M urea in Buffer A containing

and stored

CyaA

at 16000 x g. The pellets

HCl pH S.OjO.2 mM CaCI, (Buffer A). At this

Buffer A and passed through proteins

(AhO,, = 2.0 f 0.2) the

and membrane-associated

with the cell debris

60”/, of adenylate

listed in

= 0.2 and induced

in M63 medium (Miller, 1972) and

The aggregated

8 M urea in 50 mM Tris

with 150 ng

strains

of&,,

1mM IPTG. After an additional 4 h ofgrowth

bacteria

were grown in

et al., 1989) supplemented

and 12 pg Cm/ml. The cultures

umns were extensively

(a) Design of an Escherichia coli system for expression of the Bordetella pertussis cya genes The cya locus of B. pertussis is composed of five genes: cyaA encodes the bifunctional CyaA protein, whose secretion requires the expression of the downstream cyaB, D, and E genes (Glaser et al., 1988b). Activation of CyaA requires the expression of a fifth gene, cyaC, located upstream from cyaA (Barry et al., 1991). B. pertussis is a slow-growing organism and the tools for its genetic analysis are still limited. As a first step toward the understanding of the mechanism of activation of the CyaA toxin we developed a system in E. coli that would enable the expression of CyaA endowed with invasive and hemolytic activities. Therefore, we cloned the five known genes of the cya locus in E. coli to assess the contribution of each one to the toxin activity. Preliminary experiments showed that expression of the cyaA gene cloned in E. coli on multicopy plasmids under control of strong inducible promoters was surprisingly low, presumably due to inefficient recognition of the cyaA translation initiation signals. We therefore constructed a plasmid (pACT7, Fig. 1) which contained the cyaA gene expressed under the control of the transcription and translation initiation signals of IucZ. The cyaB, D, E and C genes were cloned in a low copy number vector (five to six copies per cell), pHSG575, compatible with the pMB 1 replicon derived plasmids, such as pACT7. A strong inducible promoter, P,,,. and a synthetic

of purified

liquid 2 x YT medium (Sambrook

stage the urea extracts AND

97.4

- 29 Fig. 2. SDS-PAGE

CyaA proteins, RESULTS

-

are indicated

toxins from

5, toxin purified

from

on the right margin.

RBS were placed upstream from the ATG start codon of cyaB, and two consecutive transcriptional terminators of the rrnB gene were placed downstream from the cyaE gene (pPSG2E, Fig. 1). The cyaC gene was cloned under the control of the lac promoter, either upstream from the cyaB, D, E genes (pPSG3CE, Fig. l), or separately in a modified pHSG75 vector (pPS4C, Fig. 1). (b) Purification of CyaA proteins expressed in Escherichia coli The recombinant strains harboring different combinations of plasmids are shown in Table I. The expression from the pACT7 plasmid led to high level production of CyaA, which represented about 2% of the E. coli total proteins. Furthermore, over 60% of the adenylate cyclase activity was recovered within the cell debris after sonication. The presence or absence of the cyaC, B, D, and E genes encoding activation and secretion functions did not affect the apparent aggregation or membrane association of the CyaA proteins, produced in E. coli. It is surprising that,

22 TABLE

I

Invasive

and hemolytic

Recombinant

activities

Plasmid

of CyaA proteins

content h

in Escherichia co11

produced

Gene

strains I’

Adenylate activity’

context

cyclase

Hemolytic

(Cya)

Hemolytic

activity d

Internalized

activity Gya activity

( ‘, ) Internalized

Input u/ml

u/ml

PSlO

pACT7

f pHSG575

cyuA

PS20

pACT7

+ pPSG2E

cyuA,B.D,E

PS30

pACT7

+ pPSG3CE

cyaA,C,B,D,E

940

7.3

14

1.91

PS40

pACT7

+ pPS4C

cyaA,C

861

11.2

31

2.76

” The recombinant I~clqZAM15, h Plasmids

strains

are described

‘ Adenylate

(Stratagene)

of erythrocytes 5 The ratios

activities

by transformation

Xl-l

0.05

ND

-

0.07

ND

-

{en&l,

hsdRl7, supE44, thi-I, I

with the indicated

pairs of plasmids,

, recAl, gyrA96, relA1, d(lac-proB)[F’,

described

proAB

’.

in Fig. 1.

in Fig. 1 legend.

cyclase activities

were measured

at 30°C and pH 8. lnternalized d Hemolytic

from E. co/i strain

were derived

TnZO(tetR)]}

885 1400

adenylate

were determined

as previously cyclase

described

activities

by incubation

(Ladant

were determined

of sheep erythrocytes

et al., 1989). One unit (u) corresponds after 30 min incubation,

to 1 nmol of CAMP formed per min

as described

previously

(Bellalou

et al., 1990).

(5 x 10s cells/ml) with the toxins at 37°C for 270 min and expressed

in “,,

lysed. ND, not detectable of 14/7.3 and 31/l 1.2.

even though we have preliminary evidence, in a minicell system, that the cyaB, D and E genes were expressed in E. coli, the CyaA protein was not secreted. In addition, when a replicative derivative of pPSG 1E (Fig. 1) was introduced into a secretion-deficient B. pertussis mutant, CyaA secretion was restored to wt level. The aggregation of the CyaA proteins enabled a simple purification procedure by extracting the cell debris with 8 M urea (Glaser et al., 1989), followed by single-step affinity chromatography on calmodulin-agarose. As shown in Fig. 2, the purified preparations of the 200-kDa CyaA protein contained also some lower-M, forms; they correspond to degradation products still bearing the N-terminal calmodulin-binding domain, as assessed by immunoblots (not shown). The yields of CyaA purification ranged between 40 and 50% and about 1 mg of the CyaA proteins, expressed in different genetic backgrounds, could be obtained from lOO-ml cultures with specific activities ranging from 175 to 200 pmol cAMP/min/mg. TABLE

II

Invasive

and hemolytic

Strains”

activities

of the purified

toxins Adenylate

Gene

(c) Invasive activity of CyaA toxin produced in Eschevichia coli We expressed various combinations of c:,la genes in E. coli and in a first set of experiments we used crude extracts of CyaA proteins to assess the invasive adenylate cyclase and hemolytic activities. The results, summarized in Table I, show that CyaA produced alone in E. coli has no invasive or hemolytic activity and that the cyaC gene product is sufftcient to render the CyaA holotoxin invasive and hemolytic in the reconstructed system in E. coli. Coexpression of cyaB, D and E genes in tram to cyaA does not confer invasiveness and hemolytic activity upon the holotoxin, nor does it potentiate the activities brought about by the c_vaC gene product. The nature of the CyaC-mediated activation of the toxin is still unknown. However, the high degree of homology between CyaC and HlyC (Barry et al., 1991) known to activate E. coli x-hemolysin by post-translational modilication (Nicaud et al., 1985; Wagner et al., 1988) strongly sug-

cyclase (Cya) activityb

Hemolytic

Hemolytic activity’

context Input

Internalized

Ui’d

u/ml

Internalized

activity Cya activity

(Y0) ______~

B. pertussis

wild type

PSlO PS30

cyaA

PS40 .’ B. pertussis is strain h See Table I, footnote ‘ See Table I, footnote

cyaA,C,B,D,E cyaA,C 18323 (Pittman,

900

5.9

51

900

0.075

ND

8.64 -

1040 906

5.04 9.48

6 15

1.19 1.58

1984). E. coli strains

c. d; ND, not detectable.

PSlO, PS30, PS40 are described

in Table 1, footnote

a

23 tion by CyaC or to some unknown the cyaB, D, and E products. 60

2

u. 40

0

20

determinants

0

100

TIME

Fig. 3. Time-dependent cytes (5x lo8 cells/ml)

200

OF

hemolytic

toxins (900 u/ml adenylate

strains

activity

are described

(min)

of CyaA toxins. The purified

were incubated

and hemolytic

as described

300

INCUBATION

cyclase)

used for toxin production

B. pertusk)

within the CyaA toxin are involved in invasive

and hemolytic

0

determined

for the

invasive activity, although their hemolytic activity was considerably reduced. This suggests that distinct structural

L

was

by one of

decreased hemolytic activity. Our data confirm the results of Bellalou et al. (1990) that invasive and hemolytic activities of CyaA toxin can be separated. They showed that a truncated toxin, expressed in B. pertussis, was devoid of invasive activity, while still preserving 50% of wt hemolytic activity. Here we show that the CyaC-activated toxins produced in E. coli retained full

u,

is 2 5 p

modification

This could account

activity

previously

with sheep erythro-

(6% of erythrocyte

(Bellalou

and purification

lysis)

et al., 1990). The

activities.

The decreased

hemolytic

activity

of the toxin produced in E. coli could also be accounted for by a difference in the nature of the post-translational modifications taking place in the two organisms. It can, however, not be excluded that an additional factor, present in B. pertussis, is required to confer full hemolytic activity upon the toxin.

(PS 10, PS30, PS40,

in Tables I and II.

gests that the CyaC-mediated activation of CyaA is also due to post-translational modification. The fact that this modification is not lost during toxin purification from B. pertussis by a procedure including 8 M urea extraction or SDSPAGE separation (Hewlett et al., 1989) indicates that the modification is covalent. (d) Hemolytic activity of CyaA toxin produced in Escherichia coli To compare the invasive and hemolytic activities of CyaA toxins produced in E. coli and in B. pertussis we used purified proteins from both organisms (Fig. 2). The results are summarized in Table II. It is striking, that for similar invasive adenylate cyclase activities the CyaC-activated proteins produced in E. coli (strains PS30 and PS40) show five times lower hemolytic activity than the toxin produced in B. pertussis. The ratio of hemolytic vs. invasive activity did not change significantly with the purification of the proteins, indicating that the decreased hemolytic activity is not due to some inhibitory factor present in E. cob extracts, but rather reflects an intrinsic property of the toxins produced in E. coli. Two additional arguments support this interpretation: first, the time course of hemolysis is almost linear, regardless of the source of the toxins (Fig. 3) indicating that the stabilities of the proteins purified from E. coli and B. pertussis are similar. Second, some possible differences in the initial conformation of the toxins produced in the two organisms, could be ruled out because the purification procedures involved complete denaturation in 8 M urea. It is, however, possible that the aggregation observed in E. coli might render the protein refractive to full modifica-

(e) Conclusions We have reconstructed in E. coli an expression system, consisting of all known genes of the cya locus of B. pertussis, which enabled production of active CyaA toxin endowed with catalytic, invasive and hemolytic activities. This system should allow us to uncover the mechanism of activation of CyaA by the cyaCgene product and to identify the structural domains of the toxin involved in the invasive and hemolytic activities, without requiring time-consuming genetic manipulation in B. pertussis. We are currently carrying out studies to identify additional factors required to reconstruct a functional system for secretion of CyaA toxin from E. cob.

ACKNOWLEDGEMENTS

We thank D. Ladant for the gift of purified toxin and for helpful advice, E. Krin for performing the site-directed mutagenesis, J. Pidoux for technical assistance, S. Goyard, J. Bellalou and A. Danchin for stimulating discussions, S. Cole for critical reading of the manuscript, J. Lortholary for graphic work and M. Ferrand for secretarial help. This work was supported by the Institut Pasteur and the Centre National de la Recherche Scientifique (URA 1129). P.S. was financed by a fellowship from the Minis&e de la Recherche et de la Technologie.

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