Stable expression of foreign antigens from the chromosome of Salmonella typhimurium vaccine strains

Gene, 88 (1990) 57-63 Elsevier

57

GENE 03486

Stable expression of foreign antigens from the chromosome of Salmonella typhimurium vaccine strains (Recombinant DNA; homologous recombination; integration; tetanus toxin; Treponema pallidum; aro genes)

Richard A. Strugnell*, Duncan Maskell'*, Neil Fairweather', Derek Pickard', Alan Ceckayne u, Cinr!~ Penn b and Gordon Dougan" ° Department ofMolecular Biology, WellcomeBiotech, Beckenham, Kent BR3 3BS (U.K.) Tel. (01)658 2211 and b School of Biological Sciences, University of Birmingham, Birmingham, B I 5 2 TT (U.K.) Tel. (021)4146563 Received by J.-P. Lecocq: 21 August 1989 Revised: 20 November 1989 Accepted: 21 November 1989

SUMMARY

A simple and versatile system has been developed using a new cloning vector which can serve as a vehicle for integrating DNA fragments, which direct the expression of heterologous antigens, into the aroC gene on the Salmonella chromosome. The system is based on Escherichia coli plasmid vectors which contain the DNA fragment, cloned from the chromosome of S. typhimurium C5, which encodes the aroC gene. The aroC gene was modified using synthetic oligodeoxyribonucleotides so that it contained several unique restriction sites into which DNA, directing the expression of heterologous antigens, could be cloned. DNA was integrated into the S. typhimurium chromosome at aroC by transferring the vectors into S. typhimurium polA mutants and allowing homologous recombination to occur between the cloned and chromosomal aroC genes. The vectors were used to integrate nucleotide sequences into the S. typhimur~um chromosome which directed the expression of tetanus toxin fragment C and the Treponemapallidum lipoprotein. The expression of both antigens was detected by Western blotting.

INTRODUCTION

Attenuated Salmonella strains are finding increasing use as experimental and applied live oral vaccines (Hoiseth and Stocker, 1981; Dougan et al., 1988). They can be used to elicit immune responses in several animal species including humans (Levine et al., 1987), cattle (Smith et al., 1984), sheep (Mukkur et al., 1987) and mice (Kiilar and Eisenstein, 1985). Some attenuated Salmonella strains are highly immunogenic in the host and can stimulate humoral antiCorrespondence to: Dr. R. Strugneil, Department of Molecular Biology, Wellcome Biotech, Beckenham, Kent BR3 3BS (U.K.) Tel. (01)6582211; Fax (01)6580890. * Present address: Molecular Infectious Disease Group, Institute of Molecular Medicine, John Radcliffe Hospital, Headington, Oxford, OX3 9DU (U.K.) Tel. 0865 752345.

0378-11191901503.50 © 1990Elsevier Science Publishers B.V. (BiomedicalDivision)

body, local secretory antibody and cellular immune responses after oral feeding (Dougan et al., 1986). Attenuated Salmonella strains may be used to carry and deliver heterologous antigens to the immune system (Dougan et al., 1988). The heterologous antigens are normarly expressed from genes encoded on recombinant plasmids introduced as extrachromosomal DNA into the vaccine strains. Some plasmid constructs are lost from the live carrier once antibiotic selective pressure is relieved in vivo, probably because the bacteria undergo a number of rounds Abbreviations: aroC,gene encoding chorismate synthase; Ap, ampicillin; bp, base pair(s); fragment C, C-terminal 50-kDa fragment of tetanus toxin; kb, 1000 bp; Kin, kanamycin; LB, Luria-Bertani (medium); MCS, multiple cloning sites; nt, nucleotide(s); oligo, oligodeoxyribonucleotide; PAGE, polyacrylamide-gel electrophoresis; R resistant; s, sensitive; S., Salmonella; SDS, sodium dodecyl sulfate; T., Treponema; Tc, tetracycline; q,d, gene encoding the lipoprotein from 7'. pa///dum; [ ], denotes plasmid-carrier state.

58 of replication in the host (O'Callaghan et al., 1988). One approach to stabilising the expression of heterologous antigens in vivo would be to place the heterologous genes on the Salmonella chromosome. The aromatic biosynthetic pathway is the only route by which most bacteria can synthesise aromatic compounds. A series of genes (are genes) encode enzymes active in this pathway. Salmonella spp. which harbour stable mutations in area (Hoiseth and Stocker, 1981), aroC (Dougan et al., 1989), aroD (Miller et al., 1989) or combinations of these mutations (Dougan et al., 1988) are highly attenuated and effective oral vaccines. The aim of this study was to clone the Salmonella aroCgene and to use it for integrating foreign genes into the Salmonella chromosome, obtaining stable constitutive expression of the foreign gene and introducing an additional attenuating lesion into vaccine strains.

MATERIALS AND METHODS

(a) Bacterial strains, plasmids, media and growth conditions The bacterial strains and plasmids used in this study are listed in Table I. E. coil and S. typhimurium strains were routinely cultured on LB agar or in LB broth (Davis et al., 1980). Minimal medium consisted of l mM MgSO4/0.1mM CaC12/0.2~ (w/v) glucose/M9 salts/ 1.5 % (w/v) Noble agar (Difco) (Davis et al., 1980). Supplements for growth of are mutants were added as 'aromix' comprising (fmal concentration) 40 pg/ml each of phenylalanine, tryptophan, tyrosine; 10pg/ml p-aminobenzoic acid and dihydroxybenzoic acid. Antibiotics and aromix constituents were obtained from Sigma. Antibiotics were used at final concentrations of Ap, 100pg/ml; Km

40 g/ml. TABLE I Bacterial strains,plasmids and oligodeoxyribonucleotides Strains

Genotype

Bacteria $. typhimurium strains BRD207 SL5388flaA66 rectA22,trp-2, xyl.401, (r-m +) leu-, polA 1 SL3261 areA, his DPI BRD207 with pDELl/tetK integrated into aroC, ApR KmR DP2 Aps, Kmn derivativeof DP! DP3 SL3261 transductantfrom DP2 Aps, Km~ DP4 BRD207 with 7'.pal/idum tpd gene from pMJB30.0Sintegrated into the chromosome. ApR KmR £. coil strains BRD049 AB2849 aroC355, ~- , sup£42, tsx.357 HU835 r-m*, (Ab2,redB3, c!857, $7) TGI d(lac.proAB), sup£, thi, hsdD$, [F'v'aD36, proAB ÷, lad q ZAMI$]

Source or reference

B.A.D. Stocker Hoiseth and Stocker (1981) This study (Fig.2B) This study (Fig.2C) This study This study £. coil Genetic Stock Centre Miller et al. (1989) I.G. Charles

Genotype/phenotype Plasmids pHC79 pUCI8 pTMCI6H pTMC20H pTMC24H pTMC28H pDEL! pDEL2 pDEL/tetK pMJB30.0$ pTETtac2 pUCAK

Apa, Tca, cosmid ApR pCH79 replicon, aroC, Aps pUCI8 replicon, aroC, ApR derivativeof pTMC20, KmR, Aps derivative of pTMC24H containingMCS, ApR Kpnl deletionof pTMC28H, ApR derivative of pDELI expressingfragment C, KmR derivativeof pDELI expressingfragment C, KinR pATi53 derivativeexpressing~d, ApR plasmid expressingtetanus toxin fragmentC from Ptac promoter, ApR Kma contained betweentwo MCS

Oliges Syntheticlinker containingMCS 5'-GATCGGTACCGGATCCCCCGGGGAATTCCTGCAGC_Y 3'- C CATGGC CTAGGGGGC C CCTTAAGGACGTCGTYAA-5' Kpnl BamHl Smal EcoRl PstI

Hohn and Collins (1980) Pharmacia This study This study This study This study This study This study This study Bailey et al. (1989) Makoff et al. (1989) Pharmacia

59

(b) Isolation of DNA, DNA manipulation techniques and cosmid cloning DNA manipulations including cosmid cloning and Southern blotting were carried out as described in Maniatis et al. (1982). The cosmid gene bank of 5. typhimurium C5 was prepared as described previously (M~ller et al., 1989). Transposon Tn.S insertion mutants in recombinant plasmids were isolated as described previously (Kehoe et al., 1981) using phage Z467 rex::Tn5 c1857 029 P80 as a source of TrL5 (Coleman and Foster, 1981). Tetanus toxin fragment-C-specific DNA was detected using as a probe a 1.2-kb EcoRI fragment internal to the fragment C-encoding gene (Makoffet al., 1989). Restriction endonucleases were used according to the manufacturers' instructions.

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(c) SDS-PAGE and Western blotting SDS-PAGE was carried out using the method of Laernmli (1970). Western immunoblotting was by the method of Towbin et al. (1979)using antibody conjugates supplied by ICN Biochemicals. Anti-fragment-C antibody was raised in rabbits (Fairweather et al., 1986) and was used in immunoblots at a concentration of 1:1000.

(d) Transformation and transduetion Transformation of E. coli and 5. typhimurium and P22mediated transductions were carried out as described elsewhere (Davis et al., 1980).

RESULTS AND DISCUSSION

(a) Cloning and mapping of the aroC gone of Salmonella typhimurium CS A genomic library of 5. typhimurium C5 whole-cell DNA was prepared using the cosmid pHC79 and was repackaged into bacteriophage particles using E. coli strain HU835. The repackaged cosmid bank was used to infect E. coil BRD049 aroC and recombinants which complemented the aroC lesion were isolated by plating transfected cells on minimal medium containing Ap. DNA for one aroCcomplementing cosmid over 40 kb in size was purified, cleaved with HindIII and the fragments generated were recloned into pHC79. A plasmid harbouring a 5.8-kb HindIII fragment insert was isolated which complemented the aroC lesion of BRD049 and this plasmid was named pTMC16H. Tn~ mutagenesis was used to locate the position of the aroC gene on the 5.8-kb HindIII restriction fragment (Fig. 1). Two Tn~ inserts which were mapped close to the unique Nrul site inactivated aroC expression from pTMCI6H. The 5. typhi aroC gene contains an NruI site about 300 bp from the C-terminal end (Charles et al., 1990). DNA sequencing was used to confirm that the NruI site on pTMC16H maps at a similar location within the

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Fig. 1. Physical maps of vectors constructed in this study. Blackened bars represent cloning vectors: pHC79 (Hohn and Collins, 1980) is the vector for pTMCI6H and pUCI8 (Yanisch-Perron et al., 1985) is the vector for the other plasmids. Horizontal lines represent cloned $. typhimurJumC5 DNA, the open box represents the aroC gone and the hatched box the Km a gone. B, BamHl; E, ~.'coRl; H, Hindlll; K, Kpnl; N, Nrul; P, Pstl; S, Sinai. Upward arrows in pTMCI6H indicate the positions of the Tn.$ insertions which inactivate the aroC gone.

5. typhimurium aroC gene (I.G. Charles, unpublished results).

(h) Construction of the chromosomal vector To make pMTCI6H more suitable for use as a vector for integrating genes into the Salmonella chromosome, the repficon was changed to a higher copy plasmid, and a number of unique restriction-enzyme target sites were introduced into the nt sequence encoding the aroC gene, utilizing the Nrul site. The 5.8-kb HindIII fragment containing the aroC gene was cloned into the HindIIl site of a pUC 18 derivative from which part of the polylinker had been deleted but which retained the HindIII site. The resultant plasmid was named pTMC20H; its structure and subsequent manipulations are shown in Fig. 1. The Nrul site of pTMC20H was removed by cleavage with NruI followed by insertion of a cassette encoding KmR derived from pUC4K. The cassette was removed from pUC4K by EcoRI cleavage, followed by removal of singlestranded ends using T4 DNA polymerase. One recombi-

60

DNA which are located on either side of the aroC gene. These flanking regions can be used to integrate the foreign DNA into the Salmonella chromosome utilising homologous recombination. To select for chromosomal integration the vector is transformed into the S. ty'phimuriumpoll strain BRD207 with selection for KmR. BRD207 will not support the replication of ColEl-based repficons such as pDEL2 and thus KmR can only be maintained if the resistance gene is integrated into the Salmonella genome, most likely at the site of the aroC gene where a homologous region exists. Once KmR transformants are selected they are initially screened for the expression ofthe foreign antigen and the site of chromosomal integration is analysed by Southern blotting. When clones with the correct structure are identified, the foreign gene can be moved into the S. typhimurium area vaccine strain SL3261 using P22mediated transduction. Two examples of integration of foreign genes into the aroC gene of S. typhimurium are presented here. The gene for tetanus toxin has been cloned from Clostridium tetani (Fairweather et al., 1986) and previous studies have shown that the 50-kDa C-terminal fragment of this toxin (fragment

nant, subsequently named pTMC24H, was selected in which only one EcoRI site had been regenerated following cloning. The KmR cassette was removed from pTMC24H by cleaving the plasmid with BamHI + EcoRI and replacing the KmR cassette with a pair of oligos encoding several restriction sites. The resulting plasmid named pTMC28H has unique sites for BamHI, Sinai, EcoRI and PstI (Fig. 1). A further derivative named pI)ELI was obtained by deleting the 1.7-kb KpnI fragment encoded within the cloned S. typhimurium DNA which effectively removes a large portion of the S. typhimurium aroC gene. Finally, the KmR cassette from pUC4K was reintegrated as a PstI fragment into the unique PstI site of pDELI to create the plasmid pDEL2. (c) Insertion of foreign genes into the chromosome of Salmonella To use pDEL2 as a chromosomal integration vector, the foreign genes along with expression signals must be cloned into the unique restriction sites contained within the polylinker of pDEL2. The resultant plasmid will contain the foreign DNA, flanked by two regions of S. typhimurium

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Fig. 2. Integration of tetanus toxin fragment C-encoding gene into the chromosome of 8. ~phimu~um. (A) DNA encoding fragment C of tetanus toxin was cloned between the 8mal and BamHI sites ofpDELl and the Km R cassette inserted into the flanking PstI site producing pDELl/tetK. S. o~phimu~um BRD207 polA - was transformed with pDELl/tetK and transformants selected on Km were patched to agar containing Ap + Km. Only Ap R colonies, e.g., S. o~himur~um DPI, were detected, suggesting that single cross-overs had occurred during recombination. (B) An example of a single cross-over is represented where homologous regions a on pDELl/tetK and a' on the S. O~phimun'umchromosome recombine, resulting in ~he integration ofthe whole of pDELI/tetK into the chromosome. (C) Following growth in LB containing Km, an Ap s colony was detected, S. ~phimurium DP2. Recombination ofregions b and b' resulted in loss ofthe replicon region and Ap R. The Ap s, Km R recombinant DP2 was analysed further by Southern blotting. Distances between H/ndlll sites are shown. Stippled box, tetanus toxin fragment C-encoding gene; open box, truncated remnant ofaroC; hatched box, Km R gene; blackened box, pUC-based replicon.

61 C) is immunogenic in mice and will elicit protective antibodies against tetanus to:~tin (Helting and Zwisler, 1977; Makoff et al., 1989). A 1.6-kb EcoRV-BamHI fragment which encodes the gen¢ for fragment C under the control of the Ptac promoter (Makoff et al., 1989) was subcloned between the Sinai and B a m H I sites of p D E L I . The Km R cassette was cloned into the flanking PstI site and the recombinant plasmid p D E L l / t e t K (Fig. 2) was used to transform BRD207. Transformants were selected on LB plates containing K m and were patched onto LB plates containing K m + Ap to detect Ap s double cross-overs. None were detected in initial screens and therefore an Ap R colony (S. typhimurium DPl), presumed to be a single cross-over was selected and grown overnight in LB broth containing Km. The culture was plated onto LB plates containing Km, and single colonies were patched onto LB plates containing Ap. One Ap s colony (S. typhimurium DP2), presumed to be a double cross-over, was selected. The Km R marker from this strain was transduced into S. typhimurium SL3261 using P22-mediated transduction to construct S. typhimurium DP3 and these strains were analysed further. For Southern blotting chromosomal D N A was prepared from DPI, DP2, DP3, and the parent Salmonella, digested with HindIII, electrophoresed, transferred to nitrocellulose

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and probed with the 5.8-kb H/ndlll fragment containing the aroC gene from p T M C I 6 H (Fig. 3, lane A). The probe reacted with restriction fragments of 5.8, 4.1 and 2.6 kb in organisms which were K m R and Ap R (DPI), consistent with a single cross-over event. Such a single cross-over could result from homologous recombination between areas a and a ' , as shown in Fig. 2, or by recombination between areas b and b' (not shown). Ap s, Km R S. typh/mur/um strains (DP2 and DP3) lacked the 5.8-kb fragment suggesting that resolution of the single cross-over to a double cross-over resulted in loss of the intact aroC gene. Resolution would result from recombination between regions b and b' as shown in Fig. 2. Analysis of the S. typhimurium aroA double cross-over derivative by Southern blotting with a probe made from the fragment-C gene revealed binding of the probe to a 4.1-kb fragment (Fig. 3, lane B). These fragment lengths are consistent with those expected from homologous recombination (Fig. 2). Western immunoblots of cells from each stage of the integration process were prepared using rabbit sera raised against purified fragment C from C. tetani (Fig. 4). Bacteria containing the unintegrated expressing plasmid synthesised predominantly fragment C and degradation products ofthe protein, in addition to higher Mr forms. Following integration, the level of expressed protein was reduced and no high Mr products were observed. No differences in expression levels were detected between the constructs that

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14Fig. 3. Southern blotting of S. typhimurium chromosomal DNA after integration of the tetanus toxin fragmentC-encodinggene. Chromosomal DNA was prepared from $. typhimurium DPI, DP2 and DP3 and the parent Salmonella, cut with H/ndlll; the products were separated by electrophoresis in a 1% agarose gel followedby transfer to nitrocellulose. DNA in lanes A-C was hybridised to 32P-labelledprobe DNA from the 5.8-kb Hindlll fragment of pTMCI6H containing the aroC gene and bands were detected by autoradiography. Lanes: A, ApR, KinR $. typhimurium DPI expressing fragment C (Fig. 2B); B, Aps, KinR S. typhimurium DP2 expressing fragment C (Fig. 2C); C, Aps, KmR S. typhimurium DP3 expressing fragment C; D, $. typhimurium BRD207 p,dA; E, S. typhimurium SL3261aroA.DNA in lanes F-G was hybridised to 32P-labelledprobe DNA from a 1.6-kbfragmentcontainingthe tetanus fr:~gment-Cgene. Lanes: F, Aps, KmR S. typhimuvium DP3 expressing fragment C; G, S. typhimurium SL3261 aroA.

Fig. 4. Western blotting of $. typhbnugum after integration of tetanus toxin fragmentC-encodinggene. Whole-celllysates from 8. typhbnurium DPI, DP2, DP3 and parent Salmonella were fractionated by 0.1% SDS-10% PAGE, transferred to nitrocellulose and reacted in Western immunoblots with rabbit serum raised against purified fragmentC from C. taant. The commonbands present in all lanes are due to the presence of anti-E, coliantibodies in the serum. The arrow indicates the position of fragmentC. Protein size markers (Amersham)are indicated in kDa on the le~ margin. Lanes: A, E. coli TG-I [pTETtac2]; B, ApR, Kms S. typhimurium DPI expressing C fragment (Fig. 2B); C, Aps, KmR $. o~phbnuriwn DP2 expressing C fragment (Fig. 2C); D, Aps, KmR $. typhimurium DP3 expressing C fragment; E, S. typhimwium BRD207 polA; F, S. typhimurium SL3261 aroA; G, E. coliTG-I.

62 contained single or double cross-overs of integrated foreign DNA. (d) Integration of Treponema pallidum tpd gene into the Salmonella chromosome In a second series of experiments the T. pallidum gene tpd (Bailey et al., 1989) was subcloned into pDEL2 (Fig. 5, legend) and the gene integrated into the chromosome of S. typhimurium SL3261. Expression of the tpd lipoprotein gene product, which runs on SDS-PAGE as a diffuse band of around 36 kDa, was analysed using Western blotting (Fig. 5). The levels of antigen expression from the multicopy plasmid pMJB30.05 is shown in lane B, whereas expression from the chromosome is shown in lane C. Although the levels of expression oftpd are reduced when originating in the chromosome, the antigen is still clearly visible. (e) Conclusions and discussion (1) We have described here a genetic system which relies on homologous recombination to insert foreign genes into the Salmonella chromosome. An added feature of the sys-

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Fig. 5. Western blotting of$. typhimuriumstrains after integlc~ion oftpd gene from T.pallidum. A 1.2-kb EcoRI-BamHI fragm~,.t from pMJB30.05 containing the tpd gene was ligated into pDEL2, yielding pDEL2/tpd. 8. typhimurlum BRD207 polA was transformed with pDEL2/tpd and recombinants selected on Kin, A recombinant, S. typhimurlum DP4, expressing tpd as a result of integration into the chromosome was analysed further. Western immunoblots of whole-cell lysates were developed using sera from syphilitic rabbits. The tpd.specific band is bracketed and arrowed. Experimental condkions were as described in Fig. 4. Lanes: A, Treponemapallidum (Nichols strain); B, $. typhimurium SL3261[pMJB30.05]; C, $. typhOnuriumDP4 expressing tpd; D, S. typh~muriumBRD207 polA (see also Fig. 4 legend).

tem is that integration of the gene creates an additional attenuating lesion in the vaccine strain. If an area or aroD strain is used as the recipient the resultant recombinant strain will harbour two attenuating lesions. We have recently shown that strains of Salmonella harbouring single or double are lesions have very similar immunogenicproperties (Dougan et al., 1989). (2) The use of plasmids to carry foreign genes in such strains can result in plasmid segregation in vivo, which has been a major problem in these systems. Increased genetic stability in these strains may be achieved by integrating the foreign gene into the host chromosome. An alternative approach was taken by Nakayama etal. (1988) who recently reported a system for stabilising recombinant plasmids in vivo which relied on a requirement for the asd gene. Hone et al. (1988) reported the use of the h/s gene to integrate foreign antigens into the chromosome of S. typhimurium, however, these authors detected some instability associated with the expression of a K88 determinant in this system. To date we have found 100% stability both in vivo and in vitro of chromosomal constructs produced by replacement of aroC (data not shown). Work is in progress to determine the level of promoter activity that is sufficient to enable single-copy expression of foreign genes to induce humeral and cellular immune responses in the host. (3) Plasmid pDEL2 harbours a number of conveniently located restriction sites which will simplify the cloning of foreign protein determinants into the vector. We have cloned a number of determinants into the vector including tetanus toxin fragment C, the tpd and tpe genes from T. pallldum and malaria circumsporozoite/merozoite fusion antigen (here and our unpublished results). It should be possible to construct derivatives of pDEL2 which harbour a sucrase determinant instead of KmR which can be used to select for integration without antibiotic resistance. (4) Finally, we have shown that are lesions can be used to attenuate other bacterial pathogens including Yersinia enterocolitica (Bowe et al., 1989) and Bordetella pertussis (Roberts et al., 1990). We have isolated the area gene from these organisms and thus it is possible that similar systems could be developed to integrate foreign genes into a variety of attenuated bacterial strains. ACKNOWLEDGEMENTS

R.A.S. is a C.J. Martin Fellow of the Medical Research Council of Australia. This work was supported in part by the M.R.C. Technical assistance provided by Dr. S. Chatfield and Mr. C. Hayward is gratefully acknowledged. Thanks to Tina Silva for typing the manuscript.

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