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Saccharide-protein covalent conjugates: immunochemical characterization of Citrobacter 036 core oligosaccharide-tetanus toxoid conjugates
FEMS Microbiology Immunology 76 (1991) 1-6 © 1991 Federation of European Microbiological Societies 0920-8534/91/$03.50 Published by Elsevier ADONIS 0920853491000510 FEMSIM 00125
Saccharide-protein covalent conjugates: immunochemical characterization of Citrobacter 036 core oligosaccharide-tetanus toxoid conjugates Czeslaw Lugowski, Malgorzata Kulakowska and El~bieta R o m a n o w s k a Laboratory of Microbial Immunochemistry, Ludwik Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Wroclaw, Poland Received 17 July 1990 Accepted 27 July 1990
Key words: Lipopolysaccharide; Citrobacter; Saccharide-protein conjugate
1. SUMMARY
2. INTRODUCTION
Core oligosaccharides (complete and incomplete) isolated from Citrobacter 036 lipopolysaccharide were covalently conjugated with tetanus toxoid. Serological examination of the Citrobacter 036 core oligosaccharide-tetanus toxoid conjugates showed that they are strong immunogens. The monospecific anti-conjugate sera prepared by immunization of rabbits, were used to study the antigenic relations between lipopolysaccharide core regions of 8 strains of Citrobacter. Immunoelectrophoresis, immunoblotting and quantitative microprecipitation were performed in the experiments.
In our previous papers concerning core oligosaccharide-protein conjugates the cores isolated from Shigella sonnei phase II [1] and Citrobacter PCM 1487 strain [2] lipopolysaccharides were used. In Citrobacter 036 lipopolysaccharide (LPS) the complete and incomplete core regions occur simultaneously. The complete core (0360SII) is decasaccharide, and the incomplete one (0360SIII) heptasaccharide, as their recently established structures proved [3]. The lipopolysaccharide of rough mutant (R36LPS) also contained complete and incomplete core oligosaccharides. During immunization of rabbits with R36 cells a mixture of antibodies directed to both oligosaccharide structures was raised. Moreover, the anti-R36 serum contained mainly IgM antibodies active in passive hemagglutination [3], but inapplicable in modern serological tests. In order to obtain monospecific, immune antiLPS core sera of a high level of IgG antibodies the covalent conjugates of Citrobacter 036 core oligosaccharides with tetanus toxoid (TT) were prepared. For conjugation the preparations of both
Correspondence to: Czeslaw Lugowski, Laboratory of Microbial Immunochemistry, Ludwik Hirszfeld Institute of Immunology and Experimental Therapy, ul. Czerska 12, 53-114 Wroc/aw, Poland. A preliminary account of a part of this work is in press in Excerpta Medica International Congress Series (ICS), No. 923, published by Elsevier Science Publishers, Amsterdam.
core fractions were scrupulously purified by rechromatography on a Bio-Gel P-4 column. The application of this approach to other Citrobacter strains is of considerable interest to better understanding the structural and serological interrelationships between bacteria.
3. MATERIALS A N D M E T H O D S
Citrobacterfreundii strains: negSTc s (036), R36, PCM 1487, 52/57 (04), 87/57 (023), 1556 (R23), 1560 (027), 114/66 (036) and PCM 1488 (036) used in this study, the tetanus toxoid preparation, the preparation of lipopolysaccharides and core oligosaccharides, and of anti-R36 serum have been previously described [2,3]. Conjugation of 036 core oligosaccharides with tetanus toxoid, analytical determinations, immunization procedure, quantitative microprecipitation (QMP), double immunodiffusion, SDS-gel electrophoresis and immunoblotting were performed as described previously [1,2]. Rocket immunoelectrophoresis was carried out according to Weeke [4] with 1% agarose gel in 0.02 M barbital buffer, pH 8.6, containing 2% polyethylene glycol 6000. The thickness of the gel layer was 2 mm. The antibody gel contained 10% of the appropriate antiserum. After electrophoresis gels were washed to free of excess reagent, dried and photographed after staining with 1% amino black 10B.
4. RESULTS A N D DISCUSSION
4.1. Preparation of 0360SII-TT and 0360SLI1-TT conjugates and their characteristics Complete and incomplete core fractions obtained after mild acid hydrolysis of Citrobacter 036 LPS isolated from negSTc s strain and separation of a carbohydrate material on a Bio-Gel P-4 column were rechromatographed to enhance their purity. Pure core fractions 0360SII and 0360SIII were oxidized with periodate under controlled conditions to obtain the product containing one reactive aldehyde group in heptose region. The oxidized
Fig. 1. Double immunodiffusionof Citrobacter 036 core oligosaccharide-tetanus toxoid conjugates and R36 lipopolysaccharide with anti-R36 serum (1); 1:R36 LPS (500 ~tg/ml); 2: 0360SII-TT (2.5/~g/ml); 3: 0360SIII-TT (2.5 t~g/ml).
oligosaccharides were conjugated covalently with tetanus toxoid using the reductive amination procedure. Sugar and protein analyses of the conjugates showed that in 0360SII-T1~ as well as in 0360SIII-TT conjugate the molar ratio of core oligosaccharide to tetanus toxoid amounted to 6:1. The positive reaction of the conjugates with anti-bacterial R36 serum in the double immunodiffusion test (Fig. 1) indicated the antigenic activity of their core oligosaccharide components. However, 0 3 6 0 S I I - T T conjugate reacted much stronger than 0360SIII-TT giving evidence that the prevailing amount of antibodies of anti-R36 serum raised against the 036 complete core.
4.2. Immunogenicity of the conjugates The immunogenicity of 0 3 6 0 S I I - T T and 0360SIII-TT conjugates was examined in rabbits. The total amount of antibodies in the anti-conjugate sera was measured by the quantitative microprecipitin test (Fig. 2). In the equivalence point, R36 LPS precipitated from anti-0360SII-TT conjugate 1.1 mg of antibody per ml of serum, and from anti-0360SIII-TT conjugate 0.9 mg of antibody per ml of serum.
24 ? .7..
20
I'1
16
I--
12
U.J ,v* Q..
8 O )--
tt
5
10
20
Fig. 3. Rocket immunoelectrophoresis comparison of lipopolysaccharides of Citrobacter strains: 1:1560 (R27); 2: Tc R (R36); 3: PCM 1487; 4: negSTcs (036); 5:1556 (R23); 6: 52/57 (04); 7 : 8 7 / 5 7 (023); 8 : 1 1 4 / 6 6 (036). (A) Anti0360SII-TT; B: Anti-0360SIII-TT conjugate sera (10% in gel). Sample contained 10 #g of the alkalized LPS.
ANTIGEN (pq)
Fig. 2. Precipitation of anti-0360SlI-TT (o) and anti0360SIII-TT (O) conjugate sera by R36 lipopolysaccharide.
In order to examine the specificity of the anticonjugate sera rocket immunoelectrophoresis was carried out using lipopolysaccharides of 8 Citrobacter strains as antigens (Fig. 3). As seen, the homologous lipopolysaccharides 036 and R36 of strain negSTc s, but also LPSs of 114/66 (036) and 52/57 (04) strains reacted with anti-conjugate sera positively. The cross-reactivity of LPS 114/66 and LPS 52/57 with anti-0360SIII-TT conjugate was A
I
stronger than with anti-0360SII-TT conjugate serum. To characterize the anti-conjugate sera and the Citrobacter LPS epitopes, their immunoblotting analysis was carried out. SDS-gel electrophoresis (SDS-PAGE) of 8 Citrobacter lipopolysaccharides showed their great heterogeneity (Fig. 4A). The silver-stained electro-
B
a
b
tl-. 1
2
3
4
5
6
7
8
3
6
2
5
4
6
2
7
5
4
Fig. 4. (A) Silver-stained SDS-polyacrylamide gel electrophoresis of Citrobacter lipopolysaccharides isolated from strains: 1:1560 (R27); 2 : 5 2 / 5 7 (04); 3 : 8 7 / 5 7 (023); 4: Tc R (R36); 5: negSTc s (036); 6: PCM 1487; 7 : 1 1 4 / 6 6 (036); 8:1556 (R23). (B) Immunoblots of the lipopolysaccharides with anti-0360SII-TT (a) and anti-036IlI-TT (b) conjugate sera.
phoresis pattern of LPSs of smooth strains showed the presence of high molecular bands of 0-specific polysaccharide LPS as well as fast-migrating bands related to core LPS. 036 LPS and R36 LPS in S D S - P A G E showed 4 fast-migrating bands resulting from the presence of 2 types of core regions (complete and incomplete), and moreover from the partial splitting of diphosphorylethanolamine residues of the cores. On the other hand, only 2 fast-migrating bands related to core region were found in the remaining iipopolysaccharides during SDS-PAGE. Immunoblotting analysis (Fig. 4B) confirmed the results of rocket immunoelectrophoresis: the anti-conjugate sera reacted with 036 and 04 lipopolysaccharides. However, both anti-conjugate sera gave no reaction with 0-specific polysaccharide LPS fraction, but it should be emphasized C [TROBACTER
036
CORE
COMPLETE
OGala DClcfl 14
DClcal
PPktN" LDHeDII 17
Oq cOMPLETE
DCalo 16
PPEtN 17
DGIc~
Ll)Hepa
14
PPt LN 14
17
2DGIcal-JLDtIepol
DCIc~
PP[tN
DGIcfl
Ll)Hepa
PPEtN
14
}tDHepal-bdOelA
PP[tN 14 17 I4 DCa 1NAc~I -M)Ga ] NAca 1- JOGI cNAcp 1-41)Gi ca I - 2DG i c~ 1- JL Dltepa 1 - Jl OHepa 1- Sd[Ic I A
014 INCONPLETE
DCIea
I~
I~qlc~
PPEtN
17
tl~tep~
PPEtN
DC I e e l - 2DC I cG1- Jt DHepa 1- ~1 I)Hepa 1- 5dOc l A
027 PCM 1 ~ 7
PPELN 17 D(;BINAcQ LDPlepa PPEtN I~ 17 j4 DGl c u 1 - 2 1 ~ I cu1-21)Ca l u l - SDCIcQ1 - ~LDI4ep(ll - 5LDt~eI:~l -b@13e[ ~
023
OGalAfl
i7 17
LDHepa DGlc~l
80
g
~0
p-
z
20 m
1
2
ma
5
10
II1
20
mq
50 100 200 500
INHIBITOR
(pg)
Fig. 6. Inhibition by the core oligosaccharides of precipitation of anti-0360SII-TT conjugate serum by R36 lipopolysaccharide: (©) 0360S incomplete, (@) 0360S complete, (1) 040S incomplete, (A) 040S complete, (D) PCM 14870S, (I) 0230S.
REGIONS
DCalNAc@I-M~aINAcal-SDC]cNAcpl-4DClcal-2DClcal-JLDitepal-JLOitepal-SdOclA++ O ~ INCOMPLETE
100
PPELN
14
2DGalal-2DClcal-41)Clcal-JLDHep(ll-$LDftepal-bdOclA
Fig. 5. Chemical structures of Citrobacter core oligosaccharides isolated from serotypes 036 and 04 [3], PCM 1487 [2] and serotype 023 (unpublished results). * PPEtN, diphosphorylethanolamine; * * dOclA, 3-deoxy-D-OCtUlosonicacid.
that in agreement with their specificity, the anti0 3 6 0 S I I I - T T conjugate reacted predominantly with fast-migrating fractions of LPS preparation related to incomplete core LPS, and the anti0 3 6 0 S I I - T T conjugate with fast-migrating fractions belonging to complete core LPS. LPS 52/57 (04) which did not show the presence of complete core LPS fraction in SDS-gel electrophoresis, gave no reaction in the place of this fraction on the blot. Besides strong immunoblots obtained in the homologous systems: complete core LPS 036-anti0 3 6 0 S I I - T T conjugate and incomplete core LPS 0 3 6 - a n t i - 0 3 6 0 S I I I - T T conjugate, also weaker cross-reactions were observed in the systems: 036 complete core LPS-anti-0360SIII-TT conjugate and 036 incomplete core LPS-anti-0360SII-TT conjugate. These interactions result from the marked structural similarity of both 036 core oligosaccharides (Fig. 5). Citrobacter core oligosaccharides, the structures of which are established (Fig. 5), were tested in Q M P as inhibitors of the systems: R36 LPS-anti0 3 6 0 S I I - T T conjugate (Fig. 6) and R36 LPS-anti0 3 6 0 S I I I - T T conjugate (Fig. 7). The best inhibitor of the system of 036 complete core was obviously 0360SII (50% inhibition - 1.6 /~g); the complete
100
80 v z 0
60
F--
z N
40
20
1
Nil
roll
2
5
Ill
10
20
IIn
50
tained antibodies directed against incomplete or complete LPS core region only, and not against other c o m p o n e n t s of the bacterial cell wall. The subtle structural differences between various C i t r o b a c t e r core oligosaccharides (Fig. 5) were reflected in their inhibitory activities against the anti-conjugate sera. The cross-reactivity between 036 and 04 cores was a consequence of their close similarity. T h e y differ in one structural element only: side-chain glucose is present in 04 oligosaccharide in the place of galactose in the 0360S. The lack of inhibitory activity of the core oligosaccharides from P C M 1487 strain and 023 serotype was due to their structures being very m u c h different from that of the 036 core.
100 200
INHIBITOR (vq)
Fig. 7. Inhibition by the core oligosaccharides of precipitation of anti-0360SIII-TT conjugate serum by R36 hpopolysaccharide (symbols as in Fig. 6). core oligosaccharide of 5 2 / 5 7 (04) showed slightly lower inhibitory power (50% inhibition - 7 #g). Incomplete core oligosaccharides of 036 and 04 serotypes were rather weak inhibitors of this system. In the case of the incomplete core system (Fig. 7), the best inhibitor was 0360SIII (50% inhibition - 2.3 /~g) whereas 0360SII and incomplete core oligosaccharide of 5 2 / 5 7 (04) were somewhat weaker inhibitors (50% inhibition - 5/~g and 9 ttg, respectively). Core oligosaccharides of P C M 1487 and 8 7 / 5 7 (023) were inactive as inhibitors of b o t h systems. The serological results indicated that the antic o r e - T T conjugate sera are monospecific and con-
REFERENCES [1] Lugowski, C., Kulakowska, M. and Romanowska, E. (1986) Characterization and diagnostic application of a lipopolysaccharide core oligosaccharide protein conjugate. J. Immunol. Methods 95, 187-194. [2] Lugowski, C., Kulakowska, M. and Romanowska, E. (1990) Immunochemical characterization of Citrobacter strain PCM 1487 0-specific polysaccharide- and core oligosaccharide-protein conjugates. FEMS Immunol. Microbiol. (in press). [3] Romanowska, E., Gamian, A., Lugowski, C., Romanowska, A., Dabrowski, J., Hauck, M., Opferkuch, H.J. and von der Lieth, C.W. (1988) Structure elucidation of the core region from Citrobacter 04 and 036 lipopolysaccharides by chemical and enzymatic methods, gas chromatography/mass spectrometry, and NMR spectroscopy at 500 MHz. Biochemistry 27, 4153-4161. [4] Weeke, B. (1973) Rocket immunoelectrophoresis. Scand. J. Immunol. 2 (Suppl.), 37-46.
Saccharide-protein covalent conjugates: immunochemical characterization of Citrobacter 036 core oligosaccharide-tetanus toxoid conjugates Czeslaw Lugowski, Malgorzata Kulakowska and El~bieta R o m a n o w s k a Laboratory of Microbial Immunochemistry, Ludwik Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Wroclaw, Poland Received 17 July 1990 Accepted 27 July 1990
Key words: Lipopolysaccharide; Citrobacter; Saccharide-protein conjugate
1. SUMMARY
2. INTRODUCTION
Core oligosaccharides (complete and incomplete) isolated from Citrobacter 036 lipopolysaccharide were covalently conjugated with tetanus toxoid. Serological examination of the Citrobacter 036 core oligosaccharide-tetanus toxoid conjugates showed that they are strong immunogens. The monospecific anti-conjugate sera prepared by immunization of rabbits, were used to study the antigenic relations between lipopolysaccharide core regions of 8 strains of Citrobacter. Immunoelectrophoresis, immunoblotting and quantitative microprecipitation were performed in the experiments.
In our previous papers concerning core oligosaccharide-protein conjugates the cores isolated from Shigella sonnei phase II [1] and Citrobacter PCM 1487 strain [2] lipopolysaccharides were used. In Citrobacter 036 lipopolysaccharide (LPS) the complete and incomplete core regions occur simultaneously. The complete core (0360SII) is decasaccharide, and the incomplete one (0360SIII) heptasaccharide, as their recently established structures proved [3]. The lipopolysaccharide of rough mutant (R36LPS) also contained complete and incomplete core oligosaccharides. During immunization of rabbits with R36 cells a mixture of antibodies directed to both oligosaccharide structures was raised. Moreover, the anti-R36 serum contained mainly IgM antibodies active in passive hemagglutination [3], but inapplicable in modern serological tests. In order to obtain monospecific, immune antiLPS core sera of a high level of IgG antibodies the covalent conjugates of Citrobacter 036 core oligosaccharides with tetanus toxoid (TT) were prepared. For conjugation the preparations of both
Correspondence to: Czeslaw Lugowski, Laboratory of Microbial Immunochemistry, Ludwik Hirszfeld Institute of Immunology and Experimental Therapy, ul. Czerska 12, 53-114 Wroc/aw, Poland. A preliminary account of a part of this work is in press in Excerpta Medica International Congress Series (ICS), No. 923, published by Elsevier Science Publishers, Amsterdam.
core fractions were scrupulously purified by rechromatography on a Bio-Gel P-4 column. The application of this approach to other Citrobacter strains is of considerable interest to better understanding the structural and serological interrelationships between bacteria.
3. MATERIALS A N D M E T H O D S
Citrobacterfreundii strains: negSTc s (036), R36, PCM 1487, 52/57 (04), 87/57 (023), 1556 (R23), 1560 (027), 114/66 (036) and PCM 1488 (036) used in this study, the tetanus toxoid preparation, the preparation of lipopolysaccharides and core oligosaccharides, and of anti-R36 serum have been previously described [2,3]. Conjugation of 036 core oligosaccharides with tetanus toxoid, analytical determinations, immunization procedure, quantitative microprecipitation (QMP), double immunodiffusion, SDS-gel electrophoresis and immunoblotting were performed as described previously [1,2]. Rocket immunoelectrophoresis was carried out according to Weeke [4] with 1% agarose gel in 0.02 M barbital buffer, pH 8.6, containing 2% polyethylene glycol 6000. The thickness of the gel layer was 2 mm. The antibody gel contained 10% of the appropriate antiserum. After electrophoresis gels were washed to free of excess reagent, dried and photographed after staining with 1% amino black 10B.
4. RESULTS A N D DISCUSSION
4.1. Preparation of 0360SII-TT and 0360SLI1-TT conjugates and their characteristics Complete and incomplete core fractions obtained after mild acid hydrolysis of Citrobacter 036 LPS isolated from negSTc s strain and separation of a carbohydrate material on a Bio-Gel P-4 column were rechromatographed to enhance their purity. Pure core fractions 0360SII and 0360SIII were oxidized with periodate under controlled conditions to obtain the product containing one reactive aldehyde group in heptose region. The oxidized
Fig. 1. Double immunodiffusionof Citrobacter 036 core oligosaccharide-tetanus toxoid conjugates and R36 lipopolysaccharide with anti-R36 serum (1); 1:R36 LPS (500 ~tg/ml); 2: 0360SII-TT (2.5/~g/ml); 3: 0360SIII-TT (2.5 t~g/ml).
oligosaccharides were conjugated covalently with tetanus toxoid using the reductive amination procedure. Sugar and protein analyses of the conjugates showed that in 0360SII-T1~ as well as in 0360SIII-TT conjugate the molar ratio of core oligosaccharide to tetanus toxoid amounted to 6:1. The positive reaction of the conjugates with anti-bacterial R36 serum in the double immunodiffusion test (Fig. 1) indicated the antigenic activity of their core oligosaccharide components. However, 0 3 6 0 S I I - T T conjugate reacted much stronger than 0360SIII-TT giving evidence that the prevailing amount of antibodies of anti-R36 serum raised against the 036 complete core.
4.2. Immunogenicity of the conjugates The immunogenicity of 0 3 6 0 S I I - T T and 0360SIII-TT conjugates was examined in rabbits. The total amount of antibodies in the anti-conjugate sera was measured by the quantitative microprecipitin test (Fig. 2). In the equivalence point, R36 LPS precipitated from anti-0360SII-TT conjugate 1.1 mg of antibody per ml of serum, and from anti-0360SIII-TT conjugate 0.9 mg of antibody per ml of serum.
24 ? .7..
20
I'1
16
I--
12
U.J ,v* Q..
8 O )--
tt
5
10
20
Fig. 3. Rocket immunoelectrophoresis comparison of lipopolysaccharides of Citrobacter strains: 1:1560 (R27); 2: Tc R (R36); 3: PCM 1487; 4: negSTcs (036); 5:1556 (R23); 6: 52/57 (04); 7 : 8 7 / 5 7 (023); 8 : 1 1 4 / 6 6 (036). (A) Anti0360SII-TT; B: Anti-0360SIII-TT conjugate sera (10% in gel). Sample contained 10 #g of the alkalized LPS.
ANTIGEN (pq)
Fig. 2. Precipitation of anti-0360SlI-TT (o) and anti0360SIII-TT (O) conjugate sera by R36 lipopolysaccharide.
In order to examine the specificity of the anticonjugate sera rocket immunoelectrophoresis was carried out using lipopolysaccharides of 8 Citrobacter strains as antigens (Fig. 3). As seen, the homologous lipopolysaccharides 036 and R36 of strain negSTc s, but also LPSs of 114/66 (036) and 52/57 (04) strains reacted with anti-conjugate sera positively. The cross-reactivity of LPS 114/66 and LPS 52/57 with anti-0360SIII-TT conjugate was A
I
stronger than with anti-0360SII-TT conjugate serum. To characterize the anti-conjugate sera and the Citrobacter LPS epitopes, their immunoblotting analysis was carried out. SDS-gel electrophoresis (SDS-PAGE) of 8 Citrobacter lipopolysaccharides showed their great heterogeneity (Fig. 4A). The silver-stained electro-
B
a
b
tl-. 1
2
3
4
5
6
7
8
3
6
2
5
4
6
2
7
5
4
Fig. 4. (A) Silver-stained SDS-polyacrylamide gel electrophoresis of Citrobacter lipopolysaccharides isolated from strains: 1:1560 (R27); 2 : 5 2 / 5 7 (04); 3 : 8 7 / 5 7 (023); 4: Tc R (R36); 5: negSTc s (036); 6: PCM 1487; 7 : 1 1 4 / 6 6 (036); 8:1556 (R23). (B) Immunoblots of the lipopolysaccharides with anti-0360SII-TT (a) and anti-036IlI-TT (b) conjugate sera.
phoresis pattern of LPSs of smooth strains showed the presence of high molecular bands of 0-specific polysaccharide LPS as well as fast-migrating bands related to core LPS. 036 LPS and R36 LPS in S D S - P A G E showed 4 fast-migrating bands resulting from the presence of 2 types of core regions (complete and incomplete), and moreover from the partial splitting of diphosphorylethanolamine residues of the cores. On the other hand, only 2 fast-migrating bands related to core region were found in the remaining iipopolysaccharides during SDS-PAGE. Immunoblotting analysis (Fig. 4B) confirmed the results of rocket immunoelectrophoresis: the anti-conjugate sera reacted with 036 and 04 lipopolysaccharides. However, both anti-conjugate sera gave no reaction with 0-specific polysaccharide LPS fraction, but it should be emphasized C [TROBACTER
036
CORE
COMPLETE
OGala DClcfl 14
DClcal
PPktN" LDHeDII 17
Oq cOMPLETE
DCalo 16
PPEtN 17
DGIc~
Ll)Hepa
14
PPt LN 14
17
2DGIcal-JLDtIepol
DCIc~
PP[tN
DGIcfl
Ll)Hepa
PPEtN
14
}tDHepal-bdOelA
PP[tN 14 17 I4 DCa 1NAc~I -M)Ga ] NAca 1- JOGI cNAcp 1-41)Gi ca I - 2DG i c~ 1- JL Dltepa 1 - Jl OHepa 1- Sd[Ic I A
014 INCONPLETE
DCIea
I~
I~qlc~
PPEtN
17
tl~tep~
PPEtN
DC I e e l - 2DC I cG1- Jt DHepa 1- ~1 I)Hepa 1- 5dOc l A
027 PCM 1 ~ 7
PPELN 17 D(;BINAcQ LDPlepa PPEtN I~ 17 j4 DGl c u 1 - 2 1 ~ I cu1-21)Ca l u l - SDCIcQ1 - ~LDI4ep(ll - 5LDt~eI:~l -b@13e[ ~
023
OGalAfl
i7 17
LDHepa DGlc~l
80
g
~0
p-
z
20 m
1
2
ma
5
10
II1
20
mq
50 100 200 500
INHIBITOR
(pg)
Fig. 6. Inhibition by the core oligosaccharides of precipitation of anti-0360SII-TT conjugate serum by R36 lipopolysaccharide: (©) 0360S incomplete, (@) 0360S complete, (1) 040S incomplete, (A) 040S complete, (D) PCM 14870S, (I) 0230S.
REGIONS
DCalNAc@I-M~aINAcal-SDC]cNAcpl-4DClcal-2DClcal-JLDitepal-JLOitepal-SdOclA++ O ~ INCOMPLETE
100
PPELN
14
2DGalal-2DClcal-41)Clcal-JLDHep(ll-$LDftepal-bdOclA
Fig. 5. Chemical structures of Citrobacter core oligosaccharides isolated from serotypes 036 and 04 [3], PCM 1487 [2] and serotype 023 (unpublished results). * PPEtN, diphosphorylethanolamine; * * dOclA, 3-deoxy-D-OCtUlosonicacid.
that in agreement with their specificity, the anti0 3 6 0 S I I I - T T conjugate reacted predominantly with fast-migrating fractions of LPS preparation related to incomplete core LPS, and the anti0 3 6 0 S I I - T T conjugate with fast-migrating fractions belonging to complete core LPS. LPS 52/57 (04) which did not show the presence of complete core LPS fraction in SDS-gel electrophoresis, gave no reaction in the place of this fraction on the blot. Besides strong immunoblots obtained in the homologous systems: complete core LPS 036-anti0 3 6 0 S I I - T T conjugate and incomplete core LPS 0 3 6 - a n t i - 0 3 6 0 S I I I - T T conjugate, also weaker cross-reactions were observed in the systems: 036 complete core LPS-anti-0360SIII-TT conjugate and 036 incomplete core LPS-anti-0360SII-TT conjugate. These interactions result from the marked structural similarity of both 036 core oligosaccharides (Fig. 5). Citrobacter core oligosaccharides, the structures of which are established (Fig. 5), were tested in Q M P as inhibitors of the systems: R36 LPS-anti0 3 6 0 S I I - T T conjugate (Fig. 6) and R36 LPS-anti0 3 6 0 S I I I - T T conjugate (Fig. 7). The best inhibitor of the system of 036 complete core was obviously 0360SII (50% inhibition - 1.6 /~g); the complete
100
80 v z 0
60
F--
z N
40
20
1
Nil
roll
2
5
Ill
10
20
IIn
50
tained antibodies directed against incomplete or complete LPS core region only, and not against other c o m p o n e n t s of the bacterial cell wall. The subtle structural differences between various C i t r o b a c t e r core oligosaccharides (Fig. 5) were reflected in their inhibitory activities against the anti-conjugate sera. The cross-reactivity between 036 and 04 cores was a consequence of their close similarity. T h e y differ in one structural element only: side-chain glucose is present in 04 oligosaccharide in the place of galactose in the 0360S. The lack of inhibitory activity of the core oligosaccharides from P C M 1487 strain and 023 serotype was due to their structures being very m u c h different from that of the 036 core.
100 200
INHIBITOR (vq)
Fig. 7. Inhibition by the core oligosaccharides of precipitation of anti-0360SIII-TT conjugate serum by R36 hpopolysaccharide (symbols as in Fig. 6). core oligosaccharide of 5 2 / 5 7 (04) showed slightly lower inhibitory power (50% inhibition - 7 #g). Incomplete core oligosaccharides of 036 and 04 serotypes were rather weak inhibitors of this system. In the case of the incomplete core system (Fig. 7), the best inhibitor was 0360SIII (50% inhibition - 2.3 /~g) whereas 0360SII and incomplete core oligosaccharide of 5 2 / 5 7 (04) were somewhat weaker inhibitors (50% inhibition - 5/~g and 9 ttg, respectively). Core oligosaccharides of P C M 1487 and 8 7 / 5 7 (023) were inactive as inhibitors of b o t h systems. The serological results indicated that the antic o r e - T T conjugate sera are monospecific and con-
REFERENCES [1] Lugowski, C., Kulakowska, M. and Romanowska, E. (1986) Characterization and diagnostic application of a lipopolysaccharide core oligosaccharide protein conjugate. J. Immunol. Methods 95, 187-194. [2] Lugowski, C., Kulakowska, M. and Romanowska, E. (1990) Immunochemical characterization of Citrobacter strain PCM 1487 0-specific polysaccharide- and core oligosaccharide-protein conjugates. FEMS Immunol. Microbiol. (in press). [3] Romanowska, E., Gamian, A., Lugowski, C., Romanowska, A., Dabrowski, J., Hauck, M., Opferkuch, H.J. and von der Lieth, C.W. (1988) Structure elucidation of the core region from Citrobacter 04 and 036 lipopolysaccharides by chemical and enzymatic methods, gas chromatography/mass spectrometry, and NMR spectroscopy at 500 MHz. Biochemistry 27, 4153-4161. [4] Weeke, B. (1973) Rocket immunoelectrophoresis. Scand. J. Immunol. 2 (Suppl.), 37-46.