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Immunological and chemical studies of Salmonella haarlem somatic antigen epitopes. II. Serological investigations
FEMS Immunology and Medical Microbiology 21 (1998) 253^259
Immunological and chemical studies of Salmonella haarlem somatic antigen epitopes. II. Serological investigations Halina Dziadziuszko a , Danuta Kunikowska a , Renata GIosènicka Zbigniew Kaczynèski b , Janusz Szafranek 2;b a
1;a
, Jerzy Gajdus b ,
Institute of Maritime and Tropical Medicine, 9 b, Powstania Styczniowego Str., 81-519 Gdynia, Poland b Department of Chemistry, University of Gdansk, Sobieskiego 18, 80-952 Gdansk, Poland Received 24 February 1998; revised 22 May 1998; accepted 27 May 1998
Abstract Lipopolysaccharide of Salmonella haarlem was hydrolyzed and the products separated. Native O-polysaccharide antigen was oxidised with sodium periodate followed by reduction with sodium borohydride. Native and chemically modified antigens were the subject of immunochemical studies. Monoclonal antibodies against S. haarlem and polyclonal rabbit antisera against S. haarlem, S. typhi and S. anatum bacteria were produced. The serological relationship between the lipopolysaccharide of Salmonella bacteria belonging to the two different groups D2 and E1 was demonstrated using haemagglutination reactions, inhibition of haemagglutination and immunoblotting. Cross-reactions were observed in haemagglutination reactions and in immunoblotting between antisera to S. haarlem, S. typhi and S. anatum. Factors 3,9,46 were found in the S. haarlem strain and the sugar composition for the epitopes of each factor was determined. z 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Salmonella ; Lipopolysaccharide; O-polysaccharide; Structure; Rabbit antiserum; Monoclonal antibody
1. Introduction The antigenic structure of Salmonella has been unveiled mostly by cross-absorption of antisera which categorised antigens into di¡erent factors. Based on
1
Corresponding author. Tel.: +48 (58) 622 30 11; Fax: +48 (58) 622 33 54; E-mail: [email protected]
2
Corresponding author. Tel.: +48 (58) 341 52 71; Fax: +48 (58) 341 03 57; E-mail: [email protected]
these antigens and speci¢c antisera, Salmonella can be classi¢ed according to the Kau¡mann-White scheme [1]. Salmonella haarlem belongs to group D2 with somatic antigen described as factors 9,46. There are several reasons to suspect the presence of factors 3,10 in this strain [2]. Structural studies on the D2 group O-speci¢c side chains from S. strasbourg were performed by Hellerqvist et al. [3,4], and the D3 group S. zurich by Nghiem et al. [5]. Luk and Lindberg [6] demonstrated that O-speci¢c monoclonal antibodies recognised antigen epitopes corresponding to O3, O4, O5,
0928-8244 / 98 / $19.00 ß 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S 0 9 2 8 - 8 2 4 4 ( 9 8 ) 0 0 0 6 5 - 0
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O6, O7, O8, O9 and O10 described in the Kau¡mann-White scheme [1]. In these studies we have performed the immunological and structural analysis of lipopolysaccharide (LPS) and O-polysaccharide (OPS) of S. haarlem to prove the existence of factors 9,46 and 3,10 in this strain. We expected that we would be able to con¢rm the sugar composition of the somatic antigen epitopes detected by chemical analysis described in part I of this paper [7].
2. Materials and methods 2.1. Bacteria and cultivation; isolation of LPS S. haarlem (9,46) Nr KOS 1251 (StS 519), S. typhi (9,12) Nr KOS 58 (StBL O 901 W) and S. anatum (3,10) Nr KOS 78 (StBL 293) strains originated from the National Salmonella Center of Poland, KOS collection, Gdynia. Bacteria were cultivated on an enriched agar medium for 24 h at 37³C as described [7]. The isolated bacteria were washed from the agar with 0.85% NaCl, killed with acetone and dried. The LPS was obtained from bacteria with the hot phenol/water extraction procedure according to Westphal and Jann [8]. The extracts were dialyzed against distilled water, concentrated, and the RNAs were precipitated with Cetavlon at pH 7. After centrifugation the supernatant was dialyzed, and the LPS was precipitated from the solution with 80% ethanol at pH 7. The LPS was dissolved, dialyzed against water, and freeze dried. 2.2. Antisera and serological methods Antisera against S. haarlem (9,46), S. typhi (9,12) and S. anatum (3,10) were obtained by intravenous injections of not pure-bred rabbits with heat-killed bacteria (2 h, 100³C) with doses increasing from 0.5U109 to 2U109 cfu. The antisera were preserved at 4³C with the addition of thiomersalate. For monoclonal antibody preparation four 6week-old BALB/c mice (PZH, Warsaw) were immunised with the LPS-coated S. haarlem bacteria (9,46) according to Luk et al. [9]. Brie£y, 1 ml of puri¢ed LPS solution (1.0 mg ml31 ) was added to 5 ml of a suspension of heated bacteria (10.0 mg ml31 ), and
the mixture dried under vacuum, in a rotary evaporator. A ¢nal suspension of 10.0 mg ml31 of LPScoated bacteria in phosphate-bu¡ered saline (PBS) was used for immunisation. Male, 6-week-old BALB/c mice were inoculated intraperitoneally four times (the second after 24 days, the third after 64 days and the fourth booster injection after 4 days) with 0.2 ml of antigen suspended in PBS. Fusion was performed 4 days after the ¢nal injection. The mouse myeloma cell line P3U63Ag8.653 (obtained from ECACC Division of Biologics, PHLS Centre for Applied Microbiology and Research, Porton Down, Salisbury, UK, No. 85011420) was used as fusion partner. These cell lines were maintained in standard culture medium, RPMI 1640 (Gibco, Paisley, UK) supplemented with 10% foetal calf serum (Gibco), 1 mM sodium pyruvate, 2 mM L-glutamine and 100 U penicillin-streptomycin (Polfa, Poland) in a 7% CO2 , 37³C incubator. The standard medium plus hypoxanthine-aminopterin-thymidine (HAT) and hypoxanthine-thymidine (HT) was used to select hybrids. Fusions to generate antibody-producing hybridomas were performed according to standard methods. Brie£y, a mixture of mouse immunised spleen cells and myeloma cells in a ratio of 4:1 was washed with RPMI 1640, and the supernatant completely removed. 0.5 ml of 50% polyethylene glycol (PEG) 1500 (BDH, UK) was added dropwise as fusion agent into the cell mixture over 1 min followed by incubation in a warm water bath with regular agitation for 1.5 min. The fusion mixture was then slowly diluted with 25 ml of RPMI 1640 solution. After centrifugation (400Ug, 10 min), the cells were resuspended in 75 ml of HAT medium and dispensed in 200-Wl aliquots in 6U96-well plates (Corning, New York). The HAT medium was changed after 7 days to HT medium. After 10^14 days of growth in this medium, culture supernatants from those wells with surviving hybrids were tested with the haemagglutination test, using puri¢ed LPS from S. haarlem. The haemagglutination test was carried out in 96well plates by adding 10 Wl of a 2% suspension of formalised sheep erythrocytes to 50 Wl of serially diluted rabbit antisera or monoclonal antibodies. For detection of serological activity LPS and OPS antigens were treated with 0.25 N NaOH for 6 min at 56³C, neutralised with 0.25 N HCl and diluted in
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0.85% NaCl. For detection of the OPS serological activity, oxidised and reduced antigen in untreated form were used. The erythrocytes were sensitised for 1 h in a water bath at 37³C, by adding 1 volume of 2% erythrocytes to 1 volume of LPS or OPS solution. For inhibition of haemagglutination, the antisera in an appropriate dilution were preincubated with an inhibitor (LPS or OPS) at 37³C for 1 h.
rabbit immunoglobulin (Dako A/S, Denmark) diluted 1:500 in TBS-3% milk. After washing two times with TBS-3% milk, and two times with TBS, the nitrocellulose was stained with 4-chloro-1-naphthol solution in the presence of H2 O2 , dried and photographed.
2.3. SDS-PAGE and immunoblotting
Chemically modi¢ed OPS was obtained by sodium periodate oxidation. O-polysaccharide was dissolved in a 40 mM solution of NaIO4 contained in 0.1 M sodium acetate bu¡er (pH 4.0) and kept for 114 h at 4³C in the dark [13]. The reaction was terminated by the addition of 1,2-ethanediol and the product was dialyzed ¢rst against tap water and then against distilled water. The resulting product was lyophilised and subjected to sugar analyses and immunological studies. A portion of rhamnose-oxidised polysaccharide was reduced with sodium borohydride and puri¢ed as before [7].
2.4. Chemical methods
For SDS-PAGE [10], LPS suspension (1.0 mg ml31 ) mixed with a sample bu¡er (0.1 M Tris-HCl20 mM EDTA, pH 6.8, containing 8% SDS, 20% glycerol and 0.001% bromophenol blue) was boiled for 20 min and appropriate portions of LPS and OPS were applied to a gel. Electrophoresis was performed in a 15% acrylamide slab gel and a 5% acrylamide stacking gel with a constant current of 30 mA per gel. LPS and OPS were detected in the gel by the silver-staining method [11]. For immunobloting [12], the LPS and OPS separated by SDS-PAGE were transblotted from the gel into a nitrocellulose sheet (Serva, pore size 0.45 Wm). Electrophoretic transfer was done in 25 mM Tris-192 mM glycine bu¡er, pH 8.3, at 20 V for 18 h. After transfer, the nitrocellulose was blocked with 3% milk in Tris-bu¡ered saline (TBS) for 1 h. The transblots were incubated at room temperature for 1 h with antisera 9,46 and 9,12 diluted 1:8 or 3,10 antiserum diluted 1:6 in TBS-3% milk. The nitrocellulose sheet was washed three times for 10 min in TBS-3% milk and incubated at room temperature for 1 h with horseradish peroxidase conjugated with goat anti-
3. Results and discussion The purpose of this study was to investigate the immunological and structural characteristics of LPS and OPS of S. haarlem in order to de¢ne and explain the serological reactivity of their native and chemically modi¢ed antigens. The serological relationship between strains belonging to the two serological groups D2 and E has been known to create di¤culties in preparation of antisera for identi¢cation of their O-factors [1,2].
Table 1 Serological activity of the S. haarlem lipopolysaccharide (LPS) and O-polysaccharide (OPS) in haemagglutination test against homologous and heterologous rabbit antisera and monoclonal antibodies Sheep red blood cells sensitised with
Haemagglutination titre (log10 ) with : rabbit antisera against
S. S. S. S. S. S.
haarlem (9,46) LPS haarlem (9,46) OPS typhi (9,12) LPS anatum (3,10) LPS haarlem (oxid.) OPS haarlem (oxidised and reduced) OPS
monoclonal antibodies
9,46
9,12
3,10
G10 B6
G10 D5
4.2 3.3 2.8 3.6 1.6 2.8
2.2 1.7 3.1 ^ ^ ^
3.6 3.6 ^ 3.3 ^ ^
2.8 1.9 ^ 1.9 3.1 n.d.
3.1 2.5 ^ 2.5 3.6 n.d.
n.d., not determined.
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Fig. 1. Selective oxidation of S. haarlem OPS with sodium metaperiodate.
The serological investigations of LPS and OPS S. haarlem, S. typhi and S. anatum antigens were performed in haemagglutination tests with polyvalent rabbit antisera and monoclonal antibodies (Table 1). Positive results in haemagglutination tests were obtained for S. haarlem LPS and OPS with antisera against 9,46 (S. haarlem) and 3,10 (S. anatum) in a high serum dilution. S. typhi (9,12) LPS adsorbed onto the formalised sheep erythrocytes showed rather weak reactions with rabbit antiserum against S. haarlem (9,46). Serological activity of chemically modi¢ed OPS antigen of S. haarlem was also determined. Native OPS antigen was oxidised (Fig. 1) with sodium periodate and the L-rhamnose pyranose ring was cleaved
providing two aldehyde groups. Hydrated aldehydes may, in turn, react intramolecularly giving rise to Table 2 Inhibition of haemagglutination Antisera
Inhibiting antigen 3.9 Wg
% of inhibition
S. S. S. S. S. S. S.
LPS 9,46 OPS 9,46 LPS 3,10 LPS 9,46 OPS 9,46 LPS 9,46 OPS 9,46
95 87.5 80 100 100 100 100
haarlem 9,46 haarlem 9,46 haarlem 9,46 typhi 9,12 typhi 9,12 london 3,10 london 3,10
Inhibitory values were calculated as follows : inhibition % is the di¡erence between 1003y, y = b/aU100, where a = titre of serum dilution before inhibition and b = serum dilution after inhibition.
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Fig. 2. SDS-PAGE analysis of cross-reactive LPSs and OPS. A: Silver-stained gel. Samples of 8 Wg of LPSs of S. typhi (lane 1), S. haarlem (lane 2), S. anatum (lane 3) and OPS S. haarlem (lane 4) were applied to a gel. B : Immunoblot treated with antiserum to S. haarlem, samples of LPSs of S. anatum (lane 1), S. typhi (lane 2) and S. haarlem (lane 3) and OPS S. haarlem (lane 4). C: Immunoblot treated with antiserum to S. typhi, samples of LPSs of S. anatum (lane 1), S. typhi (lane 2) and S. haarlem (lane 3). D: Immunoblot treated with antiserum to S. anatum, samples of LPS S. anatum (lane 1), OPS S. haarlem (lane 2) and LPS S. haarlem (lane 3).
ring structures. The aldehyde products were also reduced furnishing an open ring structure with two alcoholic groups from the L-rhamnose residue. The OPS antigens in such forms were used for sheep erythrocyte sensitisation without treatment with NaOH. Both aldehyde and reduced species have a polymeric nature and were positive in the haemagglutination reaction only with serum against S. haarlem and negative in reactions with S. typhi and S. anatum antisera. Monoclonal antibodies recognised
LPS and OPSs of S. haarlem as well as LPS of S. anatum (Table 1). Comparison of haemagglutination inhibition of LPS and OPS S. haarlem presented in Table 2 shows that these antigens possess almost similar inhibitory e¡ect in a homologous system with S. haarlem and S. typhi antisera and with S. anatum antisera. LPS (9,46) antigen in a dose of 3.9 Wg gave 95% inhibition of S. haarlem antiserum, 100% inhibition of S. typhi and S. anatum antisera. The OPS (9,46) antigen in
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Fig. 3. Proposed structure of S. haarlem O-polysaccharide. The bold outline around the oligosaccharide unit symbolises the importance of the sugars in the structure of the epitopes (O-factors).
the same dose gave 87.5% inhibition of S. haarlem antiserum, 100% inhibition of S. typhi and S. anatum antisera. The 80% inhibition of S. haarlem antiserum by the LPS (3,10) antigen indicates that antigens of S. anatum bacteria possess signi¢cant structural similarity. Silver staining of the electrophoresis-separated LPSs from S. haarlem, S. typhi and S. anatum revealed the characteristic ladder pattern (Fig. 2A). Immunoblotting of these resolved antigens with the S. haarlem antiserum gave ladder patterns similar to those obtained in the silver-stained gel. In immunoblotting (Fig. 2B^D) the antisera to S. haarlem and S. anatum reacted with the intense zones of the bands in the upper part of the gel, which were produced by both LPS and OPS of S. haarlem. The reaction of antiserum 9,46 with the LPS of S. typhi was rather weak and no reactions between 9,12 antiserum and LPS of S. anatum were observed. No
reaction with the monoclonal antibodies was observed in immunoblotting either. The cross-reactivity of the S. haarlem LPS and OPS with serum anti-3,10 suggests a close relationship between the groups D2 and E1 as described by Kau¡mann [1]. The structure of S. anatum (3,10) OPS was proposed to be [L-Man(1C4)-KRha(1C3)-K-(OAc)-Gal-(1C6)]n [2], and this antigen was used by Luk and Lindberg [6] as immunogen in LPS-coated bacteria for preparation of LPSspeci¢c monoclonal antibodies. These MAbs showed high activity in ELISA with EO trisaccharide O:3 L-Man(1C4)-K-Rha(1C3)-K-(OAc)-Gal-(1CBSA). These results suggest the very high immunological activity of this antigen fraction. A serological relationship between LPS and OPS of S. haarlem (9,46) and LPS of S. anatum (3,10) was con¢rmed in the present work by serological tests and immunoblotting. In conclusion, we propose the following structures of S. haarlem O-polysaccharide factors (presented in Fig. 3). The presence of determinants 3,9,46 in the OPS structure in this strain suggests a close relationship between group D2 and E. This con¢rms the somatic antigen structure of S. strasbourg strain described by Lindberg and LeMinor [2] as 3,10,9,46.
Acknowledgments The ¢nancial support of the Institute of Maritime and Tropical Medicine, Gdynia (Grant Dz.S./5/97) and the University of Gdanìsk (Grant BW/8000-50187-7) of this paper is gratefully acknowledged.
References [1] Kau¡mann, F. (1975) The classi¢cation of Salmonella species. In: Classi¢cation of Bacteria (Scandinavian University Book, Ed.), pp. 15^128. Munksgaard, Copenhagen. [2] Lindberg, A.A. and LeMinor, L. (1984) Serology of Salmonella. In: Methods in Microbiology (Bergen, T., Ed.), Vol. 15, pp. 1^64. Academic Press, London. [3] Hellerqvist, C.G., Lindberg, B. and Pilotti, A. (1970) Structural studies on the O-speci¢c side-chains of cell wall lipopolysaccharide from Salmonella strasbourg. Acta Chem. Scand. 24, 1168^1174. [4] Hellerqvist, C.G., Ho¡man, J., Lindberg, B., Pilotti, A. and Lindberg, A.A. (1971) Anomeric nature of the D-mannose
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[5]
[6]
[7]
[8]
residues in the Salmonella typhi and S. strasbourg lipopolysaccharides. Acta Chem. Scand. 25, 1512^1513. Nghiem, H.O., Himmelspach, K. and Mayer, H. (1992) Immunochemical and structural analysis of the O polysaccharides of Salmonella zuerich [1,9,27,(46)]. J. Bacteriol. 174, 1904^ 1910. Luk, J.M.C. and Lindberg, A.A. (1991) Anti-Salmonella lipopolysaccharide monoclonal antibodies: characterization of Salmonella BO-, CO-, and EO-speci¢c clones and their diagnostic usefulness. J. Clin. Microbiol. 29, 2424^2433. Szafranek, J., Gajdus, J., Kaczynèski, Z., Dziadziuszko, H., Kunikowska, D., GIosènicka, R., Yoshida, T., Vihanto, J. and Pihlaja, K. (1998) Immunological and chemical studies of Salmonella haarlem somatic antigen epitopes. I. Structural studies of O-antigen. FEMS Immunol. Med. Microbiol. (in press). Westphal, O. and Jann, K. (1965) Bacterial lipopolysaccharide: extraction with phenol-water and further applications of the procedure. Methods Carbohydr. 5, 83^91.
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[9] Luk, J.M.C., Nalnue, N.A. and Lindberg, A.A. (1990) E¤cient production of mouse and rat monoclonal antibodies against the O antigens of Salmonella serogroup C1 , using LPS-coated bacteria as immunogen. J. Immunol. Methods 29, 243^250. [10] Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680^685. [11] Hitchcock, P.J. and Brown, T.M. (1983) Morphological heterogeneity among Salmonella lipopolysaccharide chemotypes in silver-stained polyacrylamide gels. J. Bacteriol. 154, 269^ 277. [12] Towbin, H., Staehelin, T. and Gordon, J. (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: Procedure and some applications. Proc. Natl. Acad. Sci. USA 76, 4340^4353. [13] Pritchard, D.G., Rener, B.P., Furner, R.L., Huang, D.H. and Krishna, N.R. (1988) Structure of the group G Streptococcal polysaccharide. Carbohydr. Res. 173, 255^262.
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Immunological and chemical studies of Salmonella haarlem somatic antigen epitopes. II. Serological investigations Halina Dziadziuszko a , Danuta Kunikowska a , Renata GIosènicka Zbigniew Kaczynèski b , Janusz Szafranek 2;b a
1;a
, Jerzy Gajdus b ,
Institute of Maritime and Tropical Medicine, 9 b, Powstania Styczniowego Str., 81-519 Gdynia, Poland b Department of Chemistry, University of Gdansk, Sobieskiego 18, 80-952 Gdansk, Poland Received 24 February 1998; revised 22 May 1998; accepted 27 May 1998
Abstract Lipopolysaccharide of Salmonella haarlem was hydrolyzed and the products separated. Native O-polysaccharide antigen was oxidised with sodium periodate followed by reduction with sodium borohydride. Native and chemically modified antigens were the subject of immunochemical studies. Monoclonal antibodies against S. haarlem and polyclonal rabbit antisera against S. haarlem, S. typhi and S. anatum bacteria were produced. The serological relationship between the lipopolysaccharide of Salmonella bacteria belonging to the two different groups D2 and E1 was demonstrated using haemagglutination reactions, inhibition of haemagglutination and immunoblotting. Cross-reactions were observed in haemagglutination reactions and in immunoblotting between antisera to S. haarlem, S. typhi and S. anatum. Factors 3,9,46 were found in the S. haarlem strain and the sugar composition for the epitopes of each factor was determined. z 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Salmonella ; Lipopolysaccharide; O-polysaccharide; Structure; Rabbit antiserum; Monoclonal antibody
1. Introduction The antigenic structure of Salmonella has been unveiled mostly by cross-absorption of antisera which categorised antigens into di¡erent factors. Based on
1
Corresponding author. Tel.: +48 (58) 622 30 11; Fax: +48 (58) 622 33 54; E-mail: [email protected]
2
Corresponding author. Tel.: +48 (58) 341 52 71; Fax: +48 (58) 341 03 57; E-mail: [email protected]
these antigens and speci¢c antisera, Salmonella can be classi¢ed according to the Kau¡mann-White scheme [1]. Salmonella haarlem belongs to group D2 with somatic antigen described as factors 9,46. There are several reasons to suspect the presence of factors 3,10 in this strain [2]. Structural studies on the D2 group O-speci¢c side chains from S. strasbourg were performed by Hellerqvist et al. [3,4], and the D3 group S. zurich by Nghiem et al. [5]. Luk and Lindberg [6] demonstrated that O-speci¢c monoclonal antibodies recognised antigen epitopes corresponding to O3, O4, O5,
0928-8244 / 98 / $19.00 ß 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S 0 9 2 8 - 8 2 4 4 ( 9 8 ) 0 0 0 6 5 - 0
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O6, O7, O8, O9 and O10 described in the Kau¡mann-White scheme [1]. In these studies we have performed the immunological and structural analysis of lipopolysaccharide (LPS) and O-polysaccharide (OPS) of S. haarlem to prove the existence of factors 9,46 and 3,10 in this strain. We expected that we would be able to con¢rm the sugar composition of the somatic antigen epitopes detected by chemical analysis described in part I of this paper [7].
2. Materials and methods 2.1. Bacteria and cultivation; isolation of LPS S. haarlem (9,46) Nr KOS 1251 (StS 519), S. typhi (9,12) Nr KOS 58 (StBL O 901 W) and S. anatum (3,10) Nr KOS 78 (StBL 293) strains originated from the National Salmonella Center of Poland, KOS collection, Gdynia. Bacteria were cultivated on an enriched agar medium for 24 h at 37³C as described [7]. The isolated bacteria were washed from the agar with 0.85% NaCl, killed with acetone and dried. The LPS was obtained from bacteria with the hot phenol/water extraction procedure according to Westphal and Jann [8]. The extracts were dialyzed against distilled water, concentrated, and the RNAs were precipitated with Cetavlon at pH 7. After centrifugation the supernatant was dialyzed, and the LPS was precipitated from the solution with 80% ethanol at pH 7. The LPS was dissolved, dialyzed against water, and freeze dried. 2.2. Antisera and serological methods Antisera against S. haarlem (9,46), S. typhi (9,12) and S. anatum (3,10) were obtained by intravenous injections of not pure-bred rabbits with heat-killed bacteria (2 h, 100³C) with doses increasing from 0.5U109 to 2U109 cfu. The antisera were preserved at 4³C with the addition of thiomersalate. For monoclonal antibody preparation four 6week-old BALB/c mice (PZH, Warsaw) were immunised with the LPS-coated S. haarlem bacteria (9,46) according to Luk et al. [9]. Brie£y, 1 ml of puri¢ed LPS solution (1.0 mg ml31 ) was added to 5 ml of a suspension of heated bacteria (10.0 mg ml31 ), and
the mixture dried under vacuum, in a rotary evaporator. A ¢nal suspension of 10.0 mg ml31 of LPScoated bacteria in phosphate-bu¡ered saline (PBS) was used for immunisation. Male, 6-week-old BALB/c mice were inoculated intraperitoneally four times (the second after 24 days, the third after 64 days and the fourth booster injection after 4 days) with 0.2 ml of antigen suspended in PBS. Fusion was performed 4 days after the ¢nal injection. The mouse myeloma cell line P3U63Ag8.653 (obtained from ECACC Division of Biologics, PHLS Centre for Applied Microbiology and Research, Porton Down, Salisbury, UK, No. 85011420) was used as fusion partner. These cell lines were maintained in standard culture medium, RPMI 1640 (Gibco, Paisley, UK) supplemented with 10% foetal calf serum (Gibco), 1 mM sodium pyruvate, 2 mM L-glutamine and 100 U penicillin-streptomycin (Polfa, Poland) in a 7% CO2 , 37³C incubator. The standard medium plus hypoxanthine-aminopterin-thymidine (HAT) and hypoxanthine-thymidine (HT) was used to select hybrids. Fusions to generate antibody-producing hybridomas were performed according to standard methods. Brie£y, a mixture of mouse immunised spleen cells and myeloma cells in a ratio of 4:1 was washed with RPMI 1640, and the supernatant completely removed. 0.5 ml of 50% polyethylene glycol (PEG) 1500 (BDH, UK) was added dropwise as fusion agent into the cell mixture over 1 min followed by incubation in a warm water bath with regular agitation for 1.5 min. The fusion mixture was then slowly diluted with 25 ml of RPMI 1640 solution. After centrifugation (400Ug, 10 min), the cells were resuspended in 75 ml of HAT medium and dispensed in 200-Wl aliquots in 6U96-well plates (Corning, New York). The HAT medium was changed after 7 days to HT medium. After 10^14 days of growth in this medium, culture supernatants from those wells with surviving hybrids were tested with the haemagglutination test, using puri¢ed LPS from S. haarlem. The haemagglutination test was carried out in 96well plates by adding 10 Wl of a 2% suspension of formalised sheep erythrocytes to 50 Wl of serially diluted rabbit antisera or monoclonal antibodies. For detection of serological activity LPS and OPS antigens were treated with 0.25 N NaOH for 6 min at 56³C, neutralised with 0.25 N HCl and diluted in
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0.85% NaCl. For detection of the OPS serological activity, oxidised and reduced antigen in untreated form were used. The erythrocytes were sensitised for 1 h in a water bath at 37³C, by adding 1 volume of 2% erythrocytes to 1 volume of LPS or OPS solution. For inhibition of haemagglutination, the antisera in an appropriate dilution were preincubated with an inhibitor (LPS or OPS) at 37³C for 1 h.
rabbit immunoglobulin (Dako A/S, Denmark) diluted 1:500 in TBS-3% milk. After washing two times with TBS-3% milk, and two times with TBS, the nitrocellulose was stained with 4-chloro-1-naphthol solution in the presence of H2 O2 , dried and photographed.
2.3. SDS-PAGE and immunoblotting
Chemically modi¢ed OPS was obtained by sodium periodate oxidation. O-polysaccharide was dissolved in a 40 mM solution of NaIO4 contained in 0.1 M sodium acetate bu¡er (pH 4.0) and kept for 114 h at 4³C in the dark [13]. The reaction was terminated by the addition of 1,2-ethanediol and the product was dialyzed ¢rst against tap water and then against distilled water. The resulting product was lyophilised and subjected to sugar analyses and immunological studies. A portion of rhamnose-oxidised polysaccharide was reduced with sodium borohydride and puri¢ed as before [7].
2.4. Chemical methods
For SDS-PAGE [10], LPS suspension (1.0 mg ml31 ) mixed with a sample bu¡er (0.1 M Tris-HCl20 mM EDTA, pH 6.8, containing 8% SDS, 20% glycerol and 0.001% bromophenol blue) was boiled for 20 min and appropriate portions of LPS and OPS were applied to a gel. Electrophoresis was performed in a 15% acrylamide slab gel and a 5% acrylamide stacking gel with a constant current of 30 mA per gel. LPS and OPS were detected in the gel by the silver-staining method [11]. For immunobloting [12], the LPS and OPS separated by SDS-PAGE were transblotted from the gel into a nitrocellulose sheet (Serva, pore size 0.45 Wm). Electrophoretic transfer was done in 25 mM Tris-192 mM glycine bu¡er, pH 8.3, at 20 V for 18 h. After transfer, the nitrocellulose was blocked with 3% milk in Tris-bu¡ered saline (TBS) for 1 h. The transblots were incubated at room temperature for 1 h with antisera 9,46 and 9,12 diluted 1:8 or 3,10 antiserum diluted 1:6 in TBS-3% milk. The nitrocellulose sheet was washed three times for 10 min in TBS-3% milk and incubated at room temperature for 1 h with horseradish peroxidase conjugated with goat anti-
3. Results and discussion The purpose of this study was to investigate the immunological and structural characteristics of LPS and OPS of S. haarlem in order to de¢ne and explain the serological reactivity of their native and chemically modi¢ed antigens. The serological relationship between strains belonging to the two serological groups D2 and E has been known to create di¤culties in preparation of antisera for identi¢cation of their O-factors [1,2].
Table 1 Serological activity of the S. haarlem lipopolysaccharide (LPS) and O-polysaccharide (OPS) in haemagglutination test against homologous and heterologous rabbit antisera and monoclonal antibodies Sheep red blood cells sensitised with
Haemagglutination titre (log10 ) with : rabbit antisera against
S. S. S. S. S. S.
haarlem (9,46) LPS haarlem (9,46) OPS typhi (9,12) LPS anatum (3,10) LPS haarlem (oxid.) OPS haarlem (oxidised and reduced) OPS
monoclonal antibodies
9,46
9,12
3,10
G10 B6
G10 D5
4.2 3.3 2.8 3.6 1.6 2.8
2.2 1.7 3.1 ^ ^ ^
3.6 3.6 ^ 3.3 ^ ^
2.8 1.9 ^ 1.9 3.1 n.d.
3.1 2.5 ^ 2.5 3.6 n.d.
n.d., not determined.
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Fig. 1. Selective oxidation of S. haarlem OPS with sodium metaperiodate.
The serological investigations of LPS and OPS S. haarlem, S. typhi and S. anatum antigens were performed in haemagglutination tests with polyvalent rabbit antisera and monoclonal antibodies (Table 1). Positive results in haemagglutination tests were obtained for S. haarlem LPS and OPS with antisera against 9,46 (S. haarlem) and 3,10 (S. anatum) in a high serum dilution. S. typhi (9,12) LPS adsorbed onto the formalised sheep erythrocytes showed rather weak reactions with rabbit antiserum against S. haarlem (9,46). Serological activity of chemically modi¢ed OPS antigen of S. haarlem was also determined. Native OPS antigen was oxidised (Fig. 1) with sodium periodate and the L-rhamnose pyranose ring was cleaved
providing two aldehyde groups. Hydrated aldehydes may, in turn, react intramolecularly giving rise to Table 2 Inhibition of haemagglutination Antisera
Inhibiting antigen 3.9 Wg
% of inhibition
S. S. S. S. S. S. S.
LPS 9,46 OPS 9,46 LPS 3,10 LPS 9,46 OPS 9,46 LPS 9,46 OPS 9,46
95 87.5 80 100 100 100 100
haarlem 9,46 haarlem 9,46 haarlem 9,46 typhi 9,12 typhi 9,12 london 3,10 london 3,10
Inhibitory values were calculated as follows : inhibition % is the di¡erence between 1003y, y = b/aU100, where a = titre of serum dilution before inhibition and b = serum dilution after inhibition.
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Fig. 2. SDS-PAGE analysis of cross-reactive LPSs and OPS. A: Silver-stained gel. Samples of 8 Wg of LPSs of S. typhi (lane 1), S. haarlem (lane 2), S. anatum (lane 3) and OPS S. haarlem (lane 4) were applied to a gel. B : Immunoblot treated with antiserum to S. haarlem, samples of LPSs of S. anatum (lane 1), S. typhi (lane 2) and S. haarlem (lane 3) and OPS S. haarlem (lane 4). C: Immunoblot treated with antiserum to S. typhi, samples of LPSs of S. anatum (lane 1), S. typhi (lane 2) and S. haarlem (lane 3). D: Immunoblot treated with antiserum to S. anatum, samples of LPS S. anatum (lane 1), OPS S. haarlem (lane 2) and LPS S. haarlem (lane 3).
ring structures. The aldehyde products were also reduced furnishing an open ring structure with two alcoholic groups from the L-rhamnose residue. The OPS antigens in such forms were used for sheep erythrocyte sensitisation without treatment with NaOH. Both aldehyde and reduced species have a polymeric nature and were positive in the haemagglutination reaction only with serum against S. haarlem and negative in reactions with S. typhi and S. anatum antisera. Monoclonal antibodies recognised
LPS and OPSs of S. haarlem as well as LPS of S. anatum (Table 1). Comparison of haemagglutination inhibition of LPS and OPS S. haarlem presented in Table 2 shows that these antigens possess almost similar inhibitory e¡ect in a homologous system with S. haarlem and S. typhi antisera and with S. anatum antisera. LPS (9,46) antigen in a dose of 3.9 Wg gave 95% inhibition of S. haarlem antiserum, 100% inhibition of S. typhi and S. anatum antisera. The OPS (9,46) antigen in
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Fig. 3. Proposed structure of S. haarlem O-polysaccharide. The bold outline around the oligosaccharide unit symbolises the importance of the sugars in the structure of the epitopes (O-factors).
the same dose gave 87.5% inhibition of S. haarlem antiserum, 100% inhibition of S. typhi and S. anatum antisera. The 80% inhibition of S. haarlem antiserum by the LPS (3,10) antigen indicates that antigens of S. anatum bacteria possess signi¢cant structural similarity. Silver staining of the electrophoresis-separated LPSs from S. haarlem, S. typhi and S. anatum revealed the characteristic ladder pattern (Fig. 2A). Immunoblotting of these resolved antigens with the S. haarlem antiserum gave ladder patterns similar to those obtained in the silver-stained gel. In immunoblotting (Fig. 2B^D) the antisera to S. haarlem and S. anatum reacted with the intense zones of the bands in the upper part of the gel, which were produced by both LPS and OPS of S. haarlem. The reaction of antiserum 9,46 with the LPS of S. typhi was rather weak and no reactions between 9,12 antiserum and LPS of S. anatum were observed. No
reaction with the monoclonal antibodies was observed in immunoblotting either. The cross-reactivity of the S. haarlem LPS and OPS with serum anti-3,10 suggests a close relationship between the groups D2 and E1 as described by Kau¡mann [1]. The structure of S. anatum (3,10) OPS was proposed to be [L-Man(1C4)-KRha(1C3)-K-(OAc)-Gal-(1C6)]n [2], and this antigen was used by Luk and Lindberg [6] as immunogen in LPS-coated bacteria for preparation of LPSspeci¢c monoclonal antibodies. These MAbs showed high activity in ELISA with EO trisaccharide O:3 L-Man(1C4)-K-Rha(1C3)-K-(OAc)-Gal-(1CBSA). These results suggest the very high immunological activity of this antigen fraction. A serological relationship between LPS and OPS of S. haarlem (9,46) and LPS of S. anatum (3,10) was con¢rmed in the present work by serological tests and immunoblotting. In conclusion, we propose the following structures of S. haarlem O-polysaccharide factors (presented in Fig. 3). The presence of determinants 3,9,46 in the OPS structure in this strain suggests a close relationship between group D2 and E. This con¢rms the somatic antigen structure of S. strasbourg strain described by Lindberg and LeMinor [2] as 3,10,9,46.
Acknowledgments The ¢nancial support of the Institute of Maritime and Tropical Medicine, Gdynia (Grant Dz.S./5/97) and the University of Gdanìsk (Grant BW/8000-50187-7) of this paper is gratefully acknowledged.
References [1] Kau¡mann, F. (1975) The classi¢cation of Salmonella species. In: Classi¢cation of Bacteria (Scandinavian University Book, Ed.), pp. 15^128. Munksgaard, Copenhagen. [2] Lindberg, A.A. and LeMinor, L. (1984) Serology of Salmonella. In: Methods in Microbiology (Bergen, T., Ed.), Vol. 15, pp. 1^64. Academic Press, London. [3] Hellerqvist, C.G., Lindberg, B. and Pilotti, A. (1970) Structural studies on the O-speci¢c side-chains of cell wall lipopolysaccharide from Salmonella strasbourg. Acta Chem. Scand. 24, 1168^1174. [4] Hellerqvist, C.G., Ho¡man, J., Lindberg, B., Pilotti, A. and Lindberg, A.A. (1971) Anomeric nature of the D-mannose
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[5]
[6]
[7]
[8]
residues in the Salmonella typhi and S. strasbourg lipopolysaccharides. Acta Chem. Scand. 25, 1512^1513. Nghiem, H.O., Himmelspach, K. and Mayer, H. (1992) Immunochemical and structural analysis of the O polysaccharides of Salmonella zuerich [1,9,27,(46)]. J. Bacteriol. 174, 1904^ 1910. Luk, J.M.C. and Lindberg, A.A. (1991) Anti-Salmonella lipopolysaccharide monoclonal antibodies: characterization of Salmonella BO-, CO-, and EO-speci¢c clones and their diagnostic usefulness. J. Clin. Microbiol. 29, 2424^2433. Szafranek, J., Gajdus, J., Kaczynèski, Z., Dziadziuszko, H., Kunikowska, D., GIosènicka, R., Yoshida, T., Vihanto, J. and Pihlaja, K. (1998) Immunological and chemical studies of Salmonella haarlem somatic antigen epitopes. I. Structural studies of O-antigen. FEMS Immunol. Med. Microbiol. (in press). Westphal, O. and Jann, K. (1965) Bacterial lipopolysaccharide: extraction with phenol-water and further applications of the procedure. Methods Carbohydr. 5, 83^91.
259
[9] Luk, J.M.C., Nalnue, N.A. and Lindberg, A.A. (1990) E¤cient production of mouse and rat monoclonal antibodies against the O antigens of Salmonella serogroup C1 , using LPS-coated bacteria as immunogen. J. Immunol. Methods 29, 243^250. [10] Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680^685. [11] Hitchcock, P.J. and Brown, T.M. (1983) Morphological heterogeneity among Salmonella lipopolysaccharide chemotypes in silver-stained polyacrylamide gels. J. Bacteriol. 154, 269^ 277. [12] Towbin, H., Staehelin, T. and Gordon, J. (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: Procedure and some applications. Proc. Natl. Acad. Sci. USA 76, 4340^4353. [13] Pritchard, D.G., Rener, B.P., Furner, R.L., Huang, D.H. and Krishna, N.R. (1988) Structure of the group G Streptococcal polysaccharide. Carbohydr. Res. 173, 255^262.
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