© INSTITUTPASTEUR/ELsEVIER Paris 1990
Res. Microbiol. 1990, 141, 1013-1017
F I M B R I A E O F E S C H E R I C H I A C O L I AS C A R R I E R S O F HETEROLOGOUS ANTIGENIC SEQUENCES
P. Klemm and L. Hedegaard Department of Microbiology, The Technical University of Denmark, 2800 Lyngby (Denmark)
Most commensal and parasitic bacteria express specific e,dnesins wlaich enable them to colonize various surfaces. In many cases such adhesins are part of thread-like organelles, fimbriae, found in great numbers on the bacterial surface. Such fimbriae mediate the binding of E. coli strains to specific epithelial receptors (for reviews, see Klemm, 1985; Mooi and de Graaf, 1985), thereby giving the fimbriated bacteria a specific target-finding ability, enabling it to localize and to colonize a specific host surface. Fimbriae are often associated with various pathogenic strains. However, in this respect, they are only serving as colonization factors and are in themselves completely non-toxic proteins. Vaccines based on fimbriae have been highly successful, for example, in protecting against various diarrhoea-causing enterotoxigenic E. coil strains. Such results have indicated that fimbriae normally are very good immunogens both in the context of live vaccines and as purified proteins. We have been interested in type 1 fimbriae. A single such fimbria consists of approximately 1000 identical subunits and a small amount of minor components, which are responsible for the adhesive properties of the fimbria. However, these latter proteins are per se not necessary for the actual formation of the fimbriae (Klemm and Christiansen, 1987). Several gene products are required for the expression, bioassembly and expressional control of the fimbriae. In figure 1 is given a short overview of the fire genes responsible for the synthesis of type 1 fimbriae. Comparisons of subunit protein sequences from related fimbriae have identified peptide segments which are highly variable. One of the foundations of the present vaccine concept was, therefore, that since these systems could naturally tolerate a good deal of sequence variability, one might be able to go a step further and introduce completely foreign sequence segments representing antigenic determinants from various selected pathogens into the structural proteins.
Results. In order to evaluate which regions of the maior structural protein of the type 1 fimbriae that could tolerate insertions, mutagenesis of the corresponding genefimA was carried out. This was done by in-frame insertion of small synthetic oligonucleotide linkers (6-12 bp) in virtually all available restriction sites in the gene (Hedegaard and Klemm, 1989). The mutants were subsequently assayed for their ability to produce
1014
P. K L E M M
A N D L. H E D E G A A R D
Bcl I Pvull
Regulation
Major subunit
~ Transport & as~;embly
/ Adhesin Minor components
FIG. I. - - Overview of the tim gene cluster with a brief description of the gene products. ThefimA gene encodes the major subunit protein (Klemm, 1984) used in this study for the insertion of foreign epitopes. The products of the fimB and timE genes are involved in the regulation of fimbriae expression by controlling the configuration of the phase switch in front of thefimA gene (Klemm, 1986; Pallesen et al., 1989). The FimC and FimD proteins are required for transport and surface location of the fimbriae (Kiemm et al., 1985 ; Klemm and Christiansen, 1990). The products of thefimF, fling andfimH genes are required for biological activity of the fimbriae (Klemm and Christiansen, 1987; Krogfelt et al., 1990).
fimbriae. This was tested by reactions with specific antiserum against type 1 fimbriae, by ability of the recombinant strains to agglutinate erythrocytes and, in some cases, by electron microscopy. The results showed that some regions of the FimA protein seemed to be essentially unaffected by the insertions. In particular, the mutants with insertions between residues 28/29, 57/58 and 103/104 exhibited a normal fimbriation phenotype. Based on these data, a few sites, i.e. position 28/29 and 57/58 in FimA, were selected for insertions of oligo nucleotides encoding foreign antigenic determinants in f i m A . Six sets of oligonucleotides were made, encoding known antigenic determinants of various viral surface proteins (fig. 2). These represented aa 122-137, aa 125-136 and aa 140-146 of the hepatitis B virus surface antigen (HBsAg) and aa 133-145 of the pre S2-region of HBsAg (Charnay et al., 1980; Tiollais et al., 1985). A 39-bp oligo-set, encoding aa 93-103 of the VP1 capsid protein from type 1 polio virus representing the C3 epitope (Emini et al., 1983 ; Van der Weft et al., 1983) and a 60-bp oligo-set coding for an epitope of the VP 1 capsid protein of foot-and-mounth disease virus (FMDV) (Bittle et al., 1982). The oiigos were inserted into the above mentioned positions in relevant plasmids and transformed into the E. coli t i m strain HB101. Firstly, the resulting clones were screened for their ability to produce fimbriae. In all cases, except for the clone carrying the aa 122-137 HBsAg insert i n f i m A , timbriated phenotypes resulted. Secondly, the hybrid fimbriae were investigated for their ability to be recognized by the appropriate antisera directed against the foreign inserts. The recombinant fimbriae containing the aa 125-136 segment of HBsAg in FimA were not recognized by serum directed against the HBsAg. However, the type 1 hybrid fimbriae containing aa 140-146 of HBsAg, aa 133-145 of the preS2 HBsAg, the C3 polio epitope and the segment of FMDV capsid protein fused into FimA were all recognized by respective antisera directed against the respective parent vira (table I).
E. COLI FIMBRIAE
AS Ag SEQUENCE
CARRIERS
1015
GTT GAT CCT ACC AAA CCT TCG GAC GGA AAT GAT Val Asp Pro Thr Lys Pro Set Asp G1y Asn Asp 28 29 HBsAg (aa 140-146)
GTTGATCCCCGGACCTGCATGATCATCGCTCAAGGAACCTCTATGTATCCCTCCTGTTGGGGGGAT ValAspProArgThrCysMetThrThrAlaGlnGlyThrSerMetTyrProSerCysTr~GlyAsp
28
29 HBsAg (aa 122-137)
CAG MG ATC ATC GCT CAA GGA ACC TCT ATG TAT CCC TCC CTG Gln Met Thr Thr Ala Gln Gly Thr Set Met Tyr Pro Ser Leu 57 58 HBsAg (aa 125-136)
GTT GAT CCT CGA GTT AGA GGT CTC TAC TTC CCG GCA GGT GGC GAT Val Asp Pro APg Val Arg Gly Leu Tyr Phe Pro Ala Ely Gly Asp
28
preS2-HBsAg (aa 133-145)
~9
GTT 6AT CCA 6AT)~AC COG BeG TC6 ACC ACG AAT AAG GAT AAG GAT Val Asp Pro Asp Asn Pro Ala Set Thr Thr Asn Lys Asp Lys Asp 28 29 C3-polio
GTTGATCCAAACCTGCGTGGAGATCTCCAGGTTCTGGCTCAGAAAGTTGCTCGTACCTTACCGGAT ValAspProAsnLeuArgGlyAspLeuGlnValLeuAlaGlnLysValAlaAr9ThrLeuProAsp 28 29
FMDV, VPI (aa 142-160)
F]o. 2. - - The nucleotide and aa sequences o f viral DNA insertions into the sites corresponding to aa 28/29 and 57/58 in the mature FimA protein. The inserted nucleotide sequences and corresponding aa are indicated in bold letters. The enlarged section of limA shows insertion sites for foreign epitope encoding o!igonucleotides.
1016
P. K L E M M ']'ABLE I.
Foreign epitope insert HBsAg 122-137 HBsAg 125-137 HBsAg 140-146 preS2 133-145 C3 polio FMDV,VPI 142-160
-
A N D L. H E D E G A A R D
Summary of the immunological reactions of FimA epitope hybrid fimbriae. -
Insert Plasmid position designation 28/29 57/58 28/29 28/29 28/29 28/29
pLiH182 pLiH115 pLiH208 pLiH240 pLiH220 pLiH240
Reaction with sera: anti-tim anti-HBsAg anti-polio anti-FMDV (+) + + + +
(+) + ---
----+ --
-----+
Conclusions. The aim o f the present study has been to insert small, viral, peptide sequences known to represent antigenic determinants into specific regions o f the FimA protein. The major advantages of this presentation concept are: (1) the antigen molecules wi!! be transported to the cell surface in high numbers; (2) the hybrid protein may possess both antigenic and adhesive properties (for live vaccines); and (3) fimbriae are usually strong immunogens, which may be important for the stimulation o f an immune response directed against the inserted epitope. The present results indicate that it is possible to construct E. coli strains expressing such recombinant fimbriae carrying immunologicaUy active forcign sequences without seriously affecting the properties o f fimbriae, provided a sequence is used which has been identified as a functional epitope. However, there may be limitations due to actual aa compositions. KEY-WORDS: Escherichia coli, Fimbria; Bacterial surface, Fusion.
References. BITTLE, J.L., KOUGHTEN,R.A., ALEXANDER,H., SHINNICK,J.M., LERNER,R., ROWLANS,D.J. & BROWN,F. (i~32), Protection against foot-and-mouth disease by immunization with chemically synthesized peptide predicted from the viral nucleotide sequence. Nature (Lond.), 298, 30-33. CHARNAY,P.~ GERVAIS,M., LouisE, A., (~ALIBERT,F. & TIOLLAIS,P. (1980), Biosynthesis of hepatitis B virus surface antigen in Escherichia coil Nature (Lond.), 286, 893-895. EMINI,E.A., JAMESON,B.A. & WIMMER,E. (1983), Priming for and induction of antipoliovirus neutralizing antibodies by synthetic peptides. Nature (Lond.), 304, 699-?03. HEDEGAARD,L. & KLEMM,P. (1989), Type 1 fimbriae of Escherichia coli as carriers of heterologous antigenic sequences. Gene, 85, 115-124. KLEMM,P. (1984), ThefimA gene encoding the type 1 fimbrial subunit of Escherichia coli: nucleotide sequence and primary structure of the protein. Europ. J. Biochem., 143, 395-399. KLEMM,P. (1985), Fimbrial at~hesions of Escherichia coli. Rev. infect. Dis., 7, 321-339. KLEMM,P., JI~RGENSEN,B.J., V~N Die, I. DE REE, H. & BERGMANS,H. (1985), Thefim genes responsible for synthesis of type 1 fimbriae in Escherichia coll. Mol. gen. Genetics, 199, 410-414.
E. COLI F I M B R I A E A S A g S E Q U E N C E C A R R I E R S
1017
KLEMM,P. (1986), Two regulatory tim genes, fimB and-timE, control the phase variation of type 1 fimbriae in Escherichia coli. EMBO J., 5, 1389-1393. KLEMM,P. & CHRISTIANSEN,G. (1987), Threetim genes required for the regulation of length and mediation of adhesion of Escherichia coil type I fimbriae. Mol. gen. Genetics, 208, 439-445. KLEMM, P. & CHRISTIANSE~,G. (1990), The fireD gene required for surface localization of Escherichia coli type 1 fimbriae. MoL gen. Genetics, 220, 334-338. KROOFELT,K.A., BEROMANS,H. & KLEMM,P. (1990), Direct evidence that the FimH protein is the mannose-specific adhesin of Escherichia coli type I fimhriae. Infect. Immun., 5g, 1995-1998. Mool, F.R. & DE fJRAAF, F.K. (1953), Molecular biology of fimhriae of enterotoxigenic Escherichia coll. Current Top. Microbiol. ImmunoL, l l g , 119-138. PALLESEN,L., MADSEN,O. & KLEMM,P. (1989), Regulation of the phase switch controlling expression of type 1 fimbriae in Escherichia coil Mol. MicrobioL, 3, 925-931. TIOLtAIS, P., POURC~L,C. & DEJEAN, A. (1985), The hepatitis B virus. Nature (Lond.), 31"/, 489-495. VANDERWERF, S., WYCHOWSKI,C., BRr0NEAt3,P., BLONDEL,B., CRAINIC,R., HORODNICEAtSrd,F. & GIRARO,M. (1983), Localization of a poliovirus type 1 neutralization epitope in viral capsid polypeptide VPI. Proc. nat. Acad. Sci. (Wash.), 80, 5080-5084.
Res. Microbiol. 1990, 141, 1013-1017
F I M B R I A E O F E S C H E R I C H I A C O L I AS C A R R I E R S O F HETEROLOGOUS ANTIGENIC SEQUENCES
P. Klemm and L. Hedegaard Department of Microbiology, The Technical University of Denmark, 2800 Lyngby (Denmark)
Most commensal and parasitic bacteria express specific e,dnesins wlaich enable them to colonize various surfaces. In many cases such adhesins are part of thread-like organelles, fimbriae, found in great numbers on the bacterial surface. Such fimbriae mediate the binding of E. coli strains to specific epithelial receptors (for reviews, see Klemm, 1985; Mooi and de Graaf, 1985), thereby giving the fimbriated bacteria a specific target-finding ability, enabling it to localize and to colonize a specific host surface. Fimbriae are often associated with various pathogenic strains. However, in this respect, they are only serving as colonization factors and are in themselves completely non-toxic proteins. Vaccines based on fimbriae have been highly successful, for example, in protecting against various diarrhoea-causing enterotoxigenic E. coil strains. Such results have indicated that fimbriae normally are very good immunogens both in the context of live vaccines and as purified proteins. We have been interested in type 1 fimbriae. A single such fimbria consists of approximately 1000 identical subunits and a small amount of minor components, which are responsible for the adhesive properties of the fimbria. However, these latter proteins are per se not necessary for the actual formation of the fimbriae (Klemm and Christiansen, 1987). Several gene products are required for the expression, bioassembly and expressional control of the fimbriae. In figure 1 is given a short overview of the fire genes responsible for the synthesis of type 1 fimbriae. Comparisons of subunit protein sequences from related fimbriae have identified peptide segments which are highly variable. One of the foundations of the present vaccine concept was, therefore, that since these systems could naturally tolerate a good deal of sequence variability, one might be able to go a step further and introduce completely foreign sequence segments representing antigenic determinants from various selected pathogens into the structural proteins.
Results. In order to evaluate which regions of the maior structural protein of the type 1 fimbriae that could tolerate insertions, mutagenesis of the corresponding genefimA was carried out. This was done by in-frame insertion of small synthetic oligonucleotide linkers (6-12 bp) in virtually all available restriction sites in the gene (Hedegaard and Klemm, 1989). The mutants were subsequently assayed for their ability to produce
1014
P. K L E M M
A N D L. H E D E G A A R D
Bcl I Pvull
Regulation
Major subunit
~ Transport & as~;embly
/ Adhesin Minor components
FIG. I. - - Overview of the tim gene cluster with a brief description of the gene products. ThefimA gene encodes the major subunit protein (Klemm, 1984) used in this study for the insertion of foreign epitopes. The products of the fimB and timE genes are involved in the regulation of fimbriae expression by controlling the configuration of the phase switch in front of thefimA gene (Klemm, 1986; Pallesen et al., 1989). The FimC and FimD proteins are required for transport and surface location of the fimbriae (Kiemm et al., 1985 ; Klemm and Christiansen, 1990). The products of thefimF, fling andfimH genes are required for biological activity of the fimbriae (Klemm and Christiansen, 1987; Krogfelt et al., 1990).
fimbriae. This was tested by reactions with specific antiserum against type 1 fimbriae, by ability of the recombinant strains to agglutinate erythrocytes and, in some cases, by electron microscopy. The results showed that some regions of the FimA protein seemed to be essentially unaffected by the insertions. In particular, the mutants with insertions between residues 28/29, 57/58 and 103/104 exhibited a normal fimbriation phenotype. Based on these data, a few sites, i.e. position 28/29 and 57/58 in FimA, were selected for insertions of oligo nucleotides encoding foreign antigenic determinants in f i m A . Six sets of oligonucleotides were made, encoding known antigenic determinants of various viral surface proteins (fig. 2). These represented aa 122-137, aa 125-136 and aa 140-146 of the hepatitis B virus surface antigen (HBsAg) and aa 133-145 of the pre S2-region of HBsAg (Charnay et al., 1980; Tiollais et al., 1985). A 39-bp oligo-set, encoding aa 93-103 of the VP1 capsid protein from type 1 polio virus representing the C3 epitope (Emini et al., 1983 ; Van der Weft et al., 1983) and a 60-bp oligo-set coding for an epitope of the VP 1 capsid protein of foot-and-mounth disease virus (FMDV) (Bittle et al., 1982). The oiigos were inserted into the above mentioned positions in relevant plasmids and transformed into the E. coli t i m strain HB101. Firstly, the resulting clones were screened for their ability to produce fimbriae. In all cases, except for the clone carrying the aa 122-137 HBsAg insert i n f i m A , timbriated phenotypes resulted. Secondly, the hybrid fimbriae were investigated for their ability to be recognized by the appropriate antisera directed against the foreign inserts. The recombinant fimbriae containing the aa 125-136 segment of HBsAg in FimA were not recognized by serum directed against the HBsAg. However, the type 1 hybrid fimbriae containing aa 140-146 of HBsAg, aa 133-145 of the preS2 HBsAg, the C3 polio epitope and the segment of FMDV capsid protein fused into FimA were all recognized by respective antisera directed against the respective parent vira (table I).
E. COLI FIMBRIAE
AS Ag SEQUENCE
CARRIERS
1015
GTT GAT CCT ACC AAA CCT TCG GAC GGA AAT GAT Val Asp Pro Thr Lys Pro Set Asp G1y Asn Asp 28 29 HBsAg (aa 140-146)
GTTGATCCCCGGACCTGCATGATCATCGCTCAAGGAACCTCTATGTATCCCTCCTGTTGGGGGGAT ValAspProArgThrCysMetThrThrAlaGlnGlyThrSerMetTyrProSerCysTr~GlyAsp
28
29 HBsAg (aa 122-137)
CAG MG ATC ATC GCT CAA GGA ACC TCT ATG TAT CCC TCC CTG Gln Met Thr Thr Ala Gln Gly Thr Set Met Tyr Pro Ser Leu 57 58 HBsAg (aa 125-136)
GTT GAT CCT CGA GTT AGA GGT CTC TAC TTC CCG GCA GGT GGC GAT Val Asp Pro APg Val Arg Gly Leu Tyr Phe Pro Ala Ely Gly Asp
28
preS2-HBsAg (aa 133-145)
~9
GTT 6AT CCA 6AT)~AC COG BeG TC6 ACC ACG AAT AAG GAT AAG GAT Val Asp Pro Asp Asn Pro Ala Set Thr Thr Asn Lys Asp Lys Asp 28 29 C3-polio
GTTGATCCAAACCTGCGTGGAGATCTCCAGGTTCTGGCTCAGAAAGTTGCTCGTACCTTACCGGAT ValAspProAsnLeuArgGlyAspLeuGlnValLeuAlaGlnLysValAlaAr9ThrLeuProAsp 28 29
FMDV, VPI (aa 142-160)
F]o. 2. - - The nucleotide and aa sequences o f viral DNA insertions into the sites corresponding to aa 28/29 and 57/58 in the mature FimA protein. The inserted nucleotide sequences and corresponding aa are indicated in bold letters. The enlarged section of limA shows insertion sites for foreign epitope encoding o!igonucleotides.
1016
P. K L E M M ']'ABLE I.
Foreign epitope insert HBsAg 122-137 HBsAg 125-137 HBsAg 140-146 preS2 133-145 C3 polio FMDV,VPI 142-160
-
A N D L. H E D E G A A R D
Summary of the immunological reactions of FimA epitope hybrid fimbriae. -
Insert Plasmid position designation 28/29 57/58 28/29 28/29 28/29 28/29
pLiH182 pLiH115 pLiH208 pLiH240 pLiH220 pLiH240
Reaction with sera: anti-tim anti-HBsAg anti-polio anti-FMDV (+) + + + +
(+) + ---
----+ --
-----+
Conclusions. The aim o f the present study has been to insert small, viral, peptide sequences known to represent antigenic determinants into specific regions o f the FimA protein. The major advantages of this presentation concept are: (1) the antigen molecules wi!! be transported to the cell surface in high numbers; (2) the hybrid protein may possess both antigenic and adhesive properties (for live vaccines); and (3) fimbriae are usually strong immunogens, which may be important for the stimulation o f an immune response directed against the inserted epitope. The present results indicate that it is possible to construct E. coli strains expressing such recombinant fimbriae carrying immunologicaUy active forcign sequences without seriously affecting the properties o f fimbriae, provided a sequence is used which has been identified as a functional epitope. However, there may be limitations due to actual aa compositions. KEY-WORDS: Escherichia coli, Fimbria; Bacterial surface, Fusion.
References. BITTLE, J.L., KOUGHTEN,R.A., ALEXANDER,H., SHINNICK,J.M., LERNER,R., ROWLANS,D.J. & BROWN,F. (i~32), Protection against foot-and-mouth disease by immunization with chemically synthesized peptide predicted from the viral nucleotide sequence. Nature (Lond.), 298, 30-33. CHARNAY,P.~ GERVAIS,M., LouisE, A., (~ALIBERT,F. & TIOLLAIS,P. (1980), Biosynthesis of hepatitis B virus surface antigen in Escherichia coil Nature (Lond.), 286, 893-895. EMINI,E.A., JAMESON,B.A. & WIMMER,E. (1983), Priming for and induction of antipoliovirus neutralizing antibodies by synthetic peptides. Nature (Lond.), 304, 699-?03. HEDEGAARD,L. & KLEMM,P. (1989), Type 1 fimbriae of Escherichia coli as carriers of heterologous antigenic sequences. Gene, 85, 115-124. KLEMM,P. (1984), ThefimA gene encoding the type 1 fimbrial subunit of Escherichia coli: nucleotide sequence and primary structure of the protein. Europ. J. Biochem., 143, 395-399. KLEMM,P. (1985), Fimbrial at~hesions of Escherichia coli. Rev. infect. Dis., 7, 321-339. KLEMM,P., JI~RGENSEN,B.J., V~N Die, I. DE REE, H. & BERGMANS,H. (1985), Thefim genes responsible for synthesis of type 1 fimbriae in Escherichia coll. Mol. gen. Genetics, 199, 410-414.
E. COLI F I M B R I A E A S A g S E Q U E N C E C A R R I E R S
1017
KLEMM,P. (1986), Two regulatory tim genes, fimB and-timE, control the phase variation of type 1 fimbriae in Escherichia coli. EMBO J., 5, 1389-1393. KLEMM,P. & CHRISTIANSEN,G. (1987), Threetim genes required for the regulation of length and mediation of adhesion of Escherichia coil type I fimbriae. Mol. gen. Genetics, 208, 439-445. KLEMM, P. & CHRISTIANSE~,G. (1990), The fireD gene required for surface localization of Escherichia coli type 1 fimbriae. MoL gen. Genetics, 220, 334-338. KROOFELT,K.A., BEROMANS,H. & KLEMM,P. (1990), Direct evidence that the FimH protein is the mannose-specific adhesin of Escherichia coli type I fimhriae. Infect. Immun., 5g, 1995-1998. Mool, F.R. & DE fJRAAF, F.K. (1953), Molecular biology of fimhriae of enterotoxigenic Escherichia coll. Current Top. Microbiol. ImmunoL, l l g , 119-138. PALLESEN,L., MADSEN,O. & KLEMM,P. (1989), Regulation of the phase switch controlling expression of type 1 fimbriae in Escherichia coil Mol. MicrobioL, 3, 925-931. TIOLtAIS, P., POURC~L,C. & DEJEAN, A. (1985), The hepatitis B virus. Nature (Lond.), 31"/, 489-495. VANDERWERF, S., WYCHOWSKI,C., BRr0NEAt3,P., BLONDEL,B., CRAINIC,R., HORODNICEAtSrd,F. & GIRARO,M. (1983), Localization of a poliovirus type 1 neutralization epitope in viral capsid polypeptide VPI. Proc. nat. Acad. Sci. (Wash.), 80, 5080-5084.
