Microbial Pathogenesis 1991 ; 11 : 179-188
Epitope differences in toxin-coregulated pili produced by classical and El Tor Vibrio cholerae 01 Gunhild Jonson, Jan Holmgren and Ann-Mari Svennerholm* Department of Medical Microbiology and Immunology, University of Goteborg, S-413 46 Goteborg, Sweden (Received February 28, 1991 ; accepted in revised form May 2, 1991)
Jonson, G . (Dept of Medical Microbiology and Immunology, University of Goteborg, S-413 46 Goteborg, Sweden), J . Holmgren and A.-M . Svennerholm . Epitope differences in toxincoregulated pili produced by classical and El Tor Vibrio cholerae 01 . Microbial Pathogenesis 1991 ; 11 : 179-188 . A toxin -coregulated pilus (TCP), that is important for intestinal colonization of Vibrio cholerae 01, may be produced by vibrios of both classical and El Tor biotypes . By comparing TCP produced by various strains of the two biotypes in immunoblotting and enzyme-linked immunosorbent assays (ELISA) using monoclonal antibodies (mAbs) and polyclonal antisera against TCP from classical vibrios, we have found biotype-related epitope differences in TCP . Our results indicate that TCP of classical strains has an epitope in the TcpA-subunit (20 .5 kDa) that is missing in El Tor TcpA, and an additional epitope that is more strongly expressed in classical TcpA . A polyclonal antiserum reacted strongly with TcpA from strains of both biotypes in immunoblotting suggesting both the presence of major shared TcpA epitopes and that the low or absent reactivity of El Tor TcpA with the mAbs was not due to lower production of TcpA by El Tor strains . Whereas all the TcpA-positive classical strains inhibited the binding of polyclonal antiserum and mAbs to solid phase-bound TCP-positive bacteria in an inhibition ELISA, practically no inhibition was observed with TcpA-positive El Tor strains . This together with findings in immunoelectron microscopy studies that TCP 'bundles' were only detected on classical strains, suggest that TCP is poorly expressed on El Tor vibrios . Key words: Vibrio cholerae ; pilus ; biotype ; monoclonal antibody; epitope .
Introduction Vibrio cholerae 01 exists as two biotypes, classical and El Tor, and as two serotypes, Ogawa and Inaba . Vibrios of either biotype and serotype may cause severe cholera diarrhoeal disease by producing cholera toxin . An important early step in the pathogenesis of V. cho/erae is penetration of the intestinal mucus layer and colonization of the mucosal surface . A pilus named toxin-coregulated pilus (TCP), which is positively regulated together with cholera toxin and several outer membrane proteins by a transmembrane protein, ToxR,' has been shown to be important for intestinal colonization of V. cholerae 01 bacteria in humans . 2 Toxin-coregulated pilus increases the virulence of V. cholerae in mice, both when expressed by classical vibrios and when cloned from a classical strain into El Tor bacteria ." TCP was first described in classical strains and although El Tor strains Author to whom correspondence should be addressed . 0882-4010/91/090179+10 $03 .00/0
© 1991 Academic Press Limited
1 80
G . Jonson et al[
harbour DNA that hybridizes both with TCP- and ToxR-probes, 3-5 several studies have failed to show expression of TCP by V. cholerae El Tor . s,7 This might be explained by the fact that more stringent growth conditions than those used for classical strains are needed to activate ToxR in El Tor strains . Thus, when growing El Tor bacteria at 30 ° C in AKI medium, which has been used by Iwanaga et al.' to increase cholera toxin production by this biotype, TCP was found also in El Tor strains ."' The only El Tor strain reported to express TCP during 'classical culture conditions', e .g . growth on colonization factor antigen (CFA) agar at 25°C for 36 h, carried a tcp gene cloned from a classical strain .' Antibodies to TCP from classical strains have failed to protect against El Tor infections in infant mice, whereas these antibodies were highly effective in protecting against classical strains .' , ' In a recent report, however, Sun et al.' claimed that mice infected with El Tor vibrios were also protected when antibodies to classical TCP were used at a high concentration . In the present study, we have tested whether there are biotype-related epitope differences in TCP produced by classical and El Tor V. cholerae 01 . This was done by studying the reactivity of different monoclonal antibodies (mAbs) and polyclonal antisera against TCP with TCP and its TcpA subunit as expressed by a number of V . cholerae 01 strains of classical and El Tor biotypes .
Results The expression of TCP and TcpA epitopes in V. cholerae 01 of different biotypes was compared . Seventeen V. cholerae 01 strains of either biotype were studied for reactivity with mAbs or rabbit polyclonal antiserum against TCP using immunoblotting and inhibition ELISA techniques . Antiserum was produced by first immunizing with classical V. cholerae 01 bacteria expressing TCP and then extensively absorbing the immune serum with TCP-negative bacteria ; the titer of the absorbed serum was 30000 when tested in enzyme-linked immunosorbent assay (ELISA) against vibrios expressing TCP and 600 and 200, respectively, when TCP-negative vibrios or Iipopolysaccharide (LPS) were used as antigens . In immunoblot analyses this antiserum gave a strong reaction with the TcpAsubunit (20 .5 kDa) of the TCP-positive reference strain JS1569(pJS752-3) (a rifampicin resistant derivative of the classical strain CVD103, harbouring a plasmid encoding for CTB) . In addition, the serum developed a number of weaker bands, one of which (50 kDa) seemed to be coregulated with TcpA and not detected in TCPnegative bacteria . The two anti-TCP mAbs used, Tc 20 :2 and Tc 21 :1, were both of IgG1 isotype . The concentrations of immunoglobulin in culture medium were 57 and 41 pg ml -1 , respectively . Although the ELISA titers against homologous TCP-positive bacteria were very similar for the two mAbs, 13000 and 10000, respectively, they differed markedly in their ability to detect TcpA in different vibrio strains . Immunoblot analyses of TcpA produced by classical and El Tor V . cholerae When using the rabbit anti-TCP serum in immunoblot analyses there seemed to be no marked differences in the amounts of TcpA produced by classical and El Tor strains of either serotype, although the classical strain 569B produced considerably higher quantities of TcpA than any of the other strains studied . The TcpA-subunit was detected in all strains except one (which also produced a low level of toxin) [Figs . 1 and 2(A) ; see also Table 1 ] . In addition, another band at 50 kDa was found in all Tcp A-positive strains (Fig . 1 ) ; this protein may be the same as the 50 kDa protein reported to be coregulated with TcpA and Omp U . 1 Although there seemed to be no
Biotype-related epitope differences in V. cholerae 01 TCP
1 81
Fig . 1 . Immunoblot analyses showing TCP production in classical and El Tor V. cholerae 01 bacteria grown at 30°C by the AKI method . 8 Washed bacteria (1010 cells ml -1 ) were boiled in presence of SDS and 2-mercaptoethanol and separated by SDS-PAGE : R, Biorad low molecular mass references : 97 .4, 66 .2, 42 .7, 31, 21 .5 and 14 .4 kDa, respectively . Samples : classical strains : 1, 395; 2, 569B ; 3, Cairo 48; 4, Cairo 50 ; 5, X28214 ; El Tor strains : 6,T19479; 7, N16961 ; 8, E7946 ; 9, Phil 6973 ; 10, X25049 . The nitrocellulose sheet was developed with an absorbed rabbit antiserum (diluted 1 :800) against TCP-positive classical V . cholerae 01 bacteria (a derivative of strain 569B) . The positions for the TcpA subunit (20 .5 kDa) of TCP and the 50 kDa pilus-coregulated protein are indicated by arrows .
Fig . 2 . Immunoblot analyses showing differences in expression of TcpA epitopes in classical and El Tor V. cholerae . Bacteria were prepared as in Fig . 1 ; the samples in (D) were boiled without 2-mercaptoethanol .
Ten different SDS-PAGE separated strains were transblotted and developed with : (A) rabbit anti-TCP (as in Fig . 1) ; (B) mAb Tc 20 :2 (12 Mg IgG1 ml - ') ; (C) and (D) mAbTc 21 :1 (250 jug IgG1 ml - '), respectively . Samples : classical strains : 1, 395 ; 2, C21 ; 3, T19766 ; 4, X23332; 5, VM47760 ; El Tor strains : 6, N16961 ; 7, E7946; 8, C5 ; 9, X24761 ; 10, VM1 2260 . It can be seen from (D) that TcpA with retained disulphide bonds reacts much more strongly than reduced TcpA (C) with mAb Tc 21 :1 .
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G . Jonson et a/.
Table 1 TCP production by V. cho/erae 01 strains as detected by immunoblotting or inhibition ELISA using polyclonal antiserum and different monoclonal antibodies Immunoblot reactivity' Rabbit anti-TCP d
Strain` Classical 395 569B Cairo 48 Cairo 50 X28214 C21 T19766 X23332 VM47760
(0)e (I) (I) (0) (1) (0) (0) (I) (0)
El Tor T19479 N16961 E7946 Phil 6973 X25049 C5 X24761 VM12260
(I) (I) (0) (I) (0) (0) (I) (0)
Reference JS1569 (pJS752-3)'
mAb Tc20 :2
Inhibition ELISA titer'
mAb Tc21 :1
Rabbit anti-TCP'
mAb Tc20 :2
mAb Tc21 :1
+(+)
1 15 2
Epitope differences in toxin-coregulated pili produced by classical and El Tor Vibrio cholerae 01 Gunhild Jonson, Jan Holmgren and Ann-Mari Svennerholm* Department of Medical Microbiology and Immunology, University of Goteborg, S-413 46 Goteborg, Sweden (Received February 28, 1991 ; accepted in revised form May 2, 1991)
Jonson, G . (Dept of Medical Microbiology and Immunology, University of Goteborg, S-413 46 Goteborg, Sweden), J . Holmgren and A.-M . Svennerholm . Epitope differences in toxincoregulated pili produced by classical and El Tor Vibrio cholerae 01 . Microbial Pathogenesis 1991 ; 11 : 179-188 . A toxin -coregulated pilus (TCP), that is important for intestinal colonization of Vibrio cholerae 01, may be produced by vibrios of both classical and El Tor biotypes . By comparing TCP produced by various strains of the two biotypes in immunoblotting and enzyme-linked immunosorbent assays (ELISA) using monoclonal antibodies (mAbs) and polyclonal antisera against TCP from classical vibrios, we have found biotype-related epitope differences in TCP . Our results indicate that TCP of classical strains has an epitope in the TcpA-subunit (20 .5 kDa) that is missing in El Tor TcpA, and an additional epitope that is more strongly expressed in classical TcpA . A polyclonal antiserum reacted strongly with TcpA from strains of both biotypes in immunoblotting suggesting both the presence of major shared TcpA epitopes and that the low or absent reactivity of El Tor TcpA with the mAbs was not due to lower production of TcpA by El Tor strains . Whereas all the TcpA-positive classical strains inhibited the binding of polyclonal antiserum and mAbs to solid phase-bound TCP-positive bacteria in an inhibition ELISA, practically no inhibition was observed with TcpA-positive El Tor strains . This together with findings in immunoelectron microscopy studies that TCP 'bundles' were only detected on classical strains, suggest that TCP is poorly expressed on El Tor vibrios . Key words: Vibrio cholerae ; pilus ; biotype ; monoclonal antibody; epitope .
Introduction Vibrio cholerae 01 exists as two biotypes, classical and El Tor, and as two serotypes, Ogawa and Inaba . Vibrios of either biotype and serotype may cause severe cholera diarrhoeal disease by producing cholera toxin . An important early step in the pathogenesis of V. cho/erae is penetration of the intestinal mucus layer and colonization of the mucosal surface . A pilus named toxin-coregulated pilus (TCP), which is positively regulated together with cholera toxin and several outer membrane proteins by a transmembrane protein, ToxR,' has been shown to be important for intestinal colonization of V. cholerae 01 bacteria in humans . 2 Toxin-coregulated pilus increases the virulence of V. cholerae in mice, both when expressed by classical vibrios and when cloned from a classical strain into El Tor bacteria ." TCP was first described in classical strains and although El Tor strains Author to whom correspondence should be addressed . 0882-4010/91/090179+10 $03 .00/0
© 1991 Academic Press Limited
1 80
G . Jonson et al[
harbour DNA that hybridizes both with TCP- and ToxR-probes, 3-5 several studies have failed to show expression of TCP by V. cholerae El Tor . s,7 This might be explained by the fact that more stringent growth conditions than those used for classical strains are needed to activate ToxR in El Tor strains . Thus, when growing El Tor bacteria at 30 ° C in AKI medium, which has been used by Iwanaga et al.' to increase cholera toxin production by this biotype, TCP was found also in El Tor strains ."' The only El Tor strain reported to express TCP during 'classical culture conditions', e .g . growth on colonization factor antigen (CFA) agar at 25°C for 36 h, carried a tcp gene cloned from a classical strain .' Antibodies to TCP from classical strains have failed to protect against El Tor infections in infant mice, whereas these antibodies were highly effective in protecting against classical strains .' , ' In a recent report, however, Sun et al.' claimed that mice infected with El Tor vibrios were also protected when antibodies to classical TCP were used at a high concentration . In the present study, we have tested whether there are biotype-related epitope differences in TCP produced by classical and El Tor V. cholerae 01 . This was done by studying the reactivity of different monoclonal antibodies (mAbs) and polyclonal antisera against TCP with TCP and its TcpA subunit as expressed by a number of V . cholerae 01 strains of classical and El Tor biotypes .
Results The expression of TCP and TcpA epitopes in V. cholerae 01 of different biotypes was compared . Seventeen V. cholerae 01 strains of either biotype were studied for reactivity with mAbs or rabbit polyclonal antiserum against TCP using immunoblotting and inhibition ELISA techniques . Antiserum was produced by first immunizing with classical V. cholerae 01 bacteria expressing TCP and then extensively absorbing the immune serum with TCP-negative bacteria ; the titer of the absorbed serum was 30000 when tested in enzyme-linked immunosorbent assay (ELISA) against vibrios expressing TCP and 600 and 200, respectively, when TCP-negative vibrios or Iipopolysaccharide (LPS) were used as antigens . In immunoblot analyses this antiserum gave a strong reaction with the TcpAsubunit (20 .5 kDa) of the TCP-positive reference strain JS1569(pJS752-3) (a rifampicin resistant derivative of the classical strain CVD103, harbouring a plasmid encoding for CTB) . In addition, the serum developed a number of weaker bands, one of which (50 kDa) seemed to be coregulated with TcpA and not detected in TCPnegative bacteria . The two anti-TCP mAbs used, Tc 20 :2 and Tc 21 :1, were both of IgG1 isotype . The concentrations of immunoglobulin in culture medium were 57 and 41 pg ml -1 , respectively . Although the ELISA titers against homologous TCP-positive bacteria were very similar for the two mAbs, 13000 and 10000, respectively, they differed markedly in their ability to detect TcpA in different vibrio strains . Immunoblot analyses of TcpA produced by classical and El Tor V . cholerae When using the rabbit anti-TCP serum in immunoblot analyses there seemed to be no marked differences in the amounts of TcpA produced by classical and El Tor strains of either serotype, although the classical strain 569B produced considerably higher quantities of TcpA than any of the other strains studied . The TcpA-subunit was detected in all strains except one (which also produced a low level of toxin) [Figs . 1 and 2(A) ; see also Table 1 ] . In addition, another band at 50 kDa was found in all Tcp A-positive strains (Fig . 1 ) ; this protein may be the same as the 50 kDa protein reported to be coregulated with TcpA and Omp U . 1 Although there seemed to be no
Biotype-related epitope differences in V. cholerae 01 TCP
1 81
Fig . 1 . Immunoblot analyses showing TCP production in classical and El Tor V. cholerae 01 bacteria grown at 30°C by the AKI method . 8 Washed bacteria (1010 cells ml -1 ) were boiled in presence of SDS and 2-mercaptoethanol and separated by SDS-PAGE : R, Biorad low molecular mass references : 97 .4, 66 .2, 42 .7, 31, 21 .5 and 14 .4 kDa, respectively . Samples : classical strains : 1, 395; 2, 569B ; 3, Cairo 48; 4, Cairo 50 ; 5, X28214 ; El Tor strains : 6,T19479; 7, N16961 ; 8, E7946 ; 9, Phil 6973 ; 10, X25049 . The nitrocellulose sheet was developed with an absorbed rabbit antiserum (diluted 1 :800) against TCP-positive classical V . cholerae 01 bacteria (a derivative of strain 569B) . The positions for the TcpA subunit (20 .5 kDa) of TCP and the 50 kDa pilus-coregulated protein are indicated by arrows .
Fig . 2 . Immunoblot analyses showing differences in expression of TcpA epitopes in classical and El Tor V. cholerae . Bacteria were prepared as in Fig . 1 ; the samples in (D) were boiled without 2-mercaptoethanol .
Ten different SDS-PAGE separated strains were transblotted and developed with : (A) rabbit anti-TCP (as in Fig . 1) ; (B) mAb Tc 20 :2 (12 Mg IgG1 ml - ') ; (C) and (D) mAbTc 21 :1 (250 jug IgG1 ml - '), respectively . Samples : classical strains : 1, 395 ; 2, C21 ; 3, T19766 ; 4, X23332; 5, VM47760 ; El Tor strains : 6, N16961 ; 7, E7946; 8, C5 ; 9, X24761 ; 10, VM1 2260 . It can be seen from (D) that TcpA with retained disulphide bonds reacts much more strongly than reduced TcpA (C) with mAb Tc 21 :1 .
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G . Jonson et a/.
Table 1 TCP production by V. cho/erae 01 strains as detected by immunoblotting or inhibition ELISA using polyclonal antiserum and different monoclonal antibodies Immunoblot reactivity' Rabbit anti-TCP d
Strain` Classical 395 569B Cairo 48 Cairo 50 X28214 C21 T19766 X23332 VM47760
(0)e (I) (I) (0) (1) (0) (0) (I) (0)
El Tor T19479 N16961 E7946 Phil 6973 X25049 C5 X24761 VM12260
(I) (I) (0) (I) (0) (0) (I) (0)
Reference JS1569 (pJS752-3)'
mAb Tc20 :2
Inhibition ELISA titer'
mAb Tc21 :1
Rabbit anti-TCP'
mAb Tc20 :2
mAb Tc21 :1
+(+)
1 15 2
