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Immunochemical characterization of Citrobacter strain PCM 1487 O-specific polysaccharide- and core oligossaccharide-protein conjugates
FEMS Microbiology Immunology 89 (1992) 201-208 © 1992 Federation of European Microbiological Societies 0920-8534/92/$05.00 Published by Elsevier
201
FEMSIM 00205
Immunochemical characterization of Citrobacter strain PCM 1487 O-specific polysaccharideand core oligosaccharide-protein conjugates Czestaw Lugowski, Matgorzata Kutakowska 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, Wroctaw, Poland Received 17 July 1990 Revision received 28 November 1991 Accepted 14 January 1992
Key words: Lipopolysaccharide; Citrobacter; Saccharide-protein conjugate
1. SUMMARY O-specific-polysaccharide and core oligosaccharide were isolated from Citrobacter strain PCM 1487 lipopolysaccharide and purified. The polysaccharide was selectively devoid of 4-deoxyD-arabinohexose residues. Covalent conjugates of the modified O-specific polysaccharide and of the core oligosaccharide
with tetanus toxoid were prepared. Immunochemical characterization of these conjugates proved that they are strong immunogens. Using monospecific rabbit antisera raised against the conjugates, the antigenic relationships between lipopolysaccharides of various strains of Citrobacter, Shigella sonnei, and Shigella flexneri were studied by quantitative microprecipitin and quantitative microprecipitin inhibition and immunoblotting tests.
Correspondence to: C. Lugowski, Laboratory of Microbial Immunochemistry, Ludwik Hirszfeld Institute of Immunology and Experimental Therapy, ul. Czerska 12, 53-114 Wrodaw, Poland. A preliminary account of a part of this work is published in Excerpta Medica International Congress Series (ICS No. 923) 1990, Endotoxin Research Series Vol. 1, Edited by A. Nowotny, J. J. Spitzer and E.J. Ziegler, Elsevier, Amsterdam.
2. INTRODUCTION In the studies on carbohydrate moiety of Citrobacter PCM 1487 strain lipopolysaccharide (LPS) the structures of O-specific polysaccharide (PS) and of core oligosaccharide (OS) were estab-
202 lished [1-3]. The O-specific polysaccharide consists of the trisaccharide repeating units:
may yield the preliminary information helpful in further structural studies.
-4-D-GalNAcp a 1-6-D-glcNAcp a 1-
13
3. MATERIALS AND METHODS
4-deoxy-D-araHexp/3 where 4-deoxy-D-araHex stands for 4-deoxy-Darabinohexose. The formula of the core oligosaccharide is the following: PPEtN 7 oGalNAcpa 3
LoHeppa 7
PPEtN 4
oGlcpctl-2oGlcpal-2oGalpctl-3oGlcpctl-3LDHeppcd-3LoHeppcd-5dOclA dOclA, 3-deoxyoctulosonic acid; PPEtN, diphosphorylethanolamine.
Rabbit antiserum obtained by immunization with Citrobacter microorganisms is directed against the O-specific polysaccharide chains. The antiserum shows the presence of IgM antibodies and cross-reactivity with homopolymers of/3(1-2) linked 4-deoxy-o-arabinohexose residues originated from O-antigens of Citrobacter serotypes 04, 027, and 036 which is a disadvantage in its application to modern serological tests. In our earlier studies on the monospecific antiserum raised to Shigella sonnei phase II core oligosaccharide-protein conjugate the distinct relationship between serological reactions and the structure of core regions of the lipopolysaccharides examined was observed [4]. The aim of the present work has been to obtain covalent conjugates of the O-specific polysaccharide selectively devoid of 4-deoxy-Darabinohexose and of the core oligosaccharide with tetanus toxoid (TT). 4-deoxy-D-arabinohexose was removed from the PCM 1487-specific polysaccharide to avoid undesired cross-reactions of the monospecific anti-PCM 1487PS-TT conjugate serum. The application of the monospecific antiCitrobacter saccharide-protein conjugate sera in examination of the relationship between O-antigens of Citrobacter and other Enterobacteriaceae
Citrobacter freundii strains PCM 1487 and 1488 (036) derived from the stock collection of the Institute of Immunology and Experimental Therapy; strain negSTc s (036) and its rough mutant (R36) were obtained from the Institute of Microbiology, Wrodaw University; strains 52/57 (04), 87/57 (023), 1556 (R mutant of 023 serotype), 1560 (027) and 114/66 (036) from the Czechoslovakian National Collection of Type Cultures, Prague; Shigella sonnei phase I strain 9773 and Shigella flexneri 4b from the Dysentary Reference Laboratory, London. Crude tetanus toxoid preparation was obtained from Wytw6rnia Surowic i Szczepionek, Warsaw and purified on Sepharose 6B column (2.6 × 100 cm) equilibrated with PBS. The preparation of lipopolysaccharides, Ospecific polysaccharides and core oligosaccharides were performed as previously described [1,2,5]. Chemical degradation of the PCM 1487specific polysaccharide was carried out according to Gamian et al. [1]. After degradation the PCM 1487PS is devoid of 4-deoxy-D-arabinohexose and shows no cross-reactivity with Citrobacter 036 LPS. De-O-acetylation of lipopolysaccharide: LPS preparation dissolved in 0.25 M NaOH was heated at 56°C for 1 h. Analytical methods: protein was determined by the Lowry et al. method [6] and sugar by the phenol method [7]. Conjugation of PCM 1487OS and degraded PCM 1487PS with tetanus toxoid and purification of the conjugates: the conjugation was carried out according to Jennings and Lugowski [8]. The oligoor polysaccharide was selectively oxidized with NalO 4 using the procedure of Lehmann et al. [9]. Conjugation of PCM 14870S-TT. The aqueous solution of PCM 1487OS (40 mg/ml) was mixed with 1 ml 0.75% NaIO 4 and incubated in the dark at 4°C with magnetic stirring for 45 min. The excess periodate was destroyed by adding ethy-
203 lene glycol. The oxidized PCM 1487OS was desalted on a column of Sephadex G-25 (2.6 × 90 cm) equilibrated with 0.02 M p y r i d i n e / C H 3 C O O H buffer pH 5.4. Fractions containing oxidized PCM 1487OS (35 mg) were lyophilized, and then dissolved in 1 ml 0.5 M K 2 H P O 4 pH 9.0. Tetanus toxoid (3.3 mg), 35 mg N a C N B H 3 and 1 drop of chloroform were added to the solution. The reaction mixture was kept in a sealed vial for 12 days at 37°C and then, for purification, it was applied to Sephadex G-100 (1.6 × 100 cm) column equilibrated with PBS. Fractions containing PCM 1487OS-TT conjugate were concentrated by ultrafiltration to give a yield of 3.5 mg.
Conjugation of degraded PCM 1487PS (30 mg) with TT (3.3 mg). This was performed by the same procedure. The conjugate PCM 1487PS-TT was purified on Sepharose 6B column and concentrated by ultrafiltration. The yield of the conjugate amounted to 4.2 mg. Immunization procedure: Rabbits were immunized in the footpads with the antigens suspended in Freund's complete adjuvant, as described earlier by Lugowski et al. [10]. Serological tests. A quantitative microprecipitin test was carried out as in [10]. Inhibition of precipitation was performed by incubation of serum with increasing concentrations of inhibitors for 1 h at 37°C prior to adding antigen. The optimal concentration of antigen required for maximal antibody precipitation was determined from the precipitin curve. D o t - i m m u n o b i o t t i n g : nitrocellulose filter (Schleicher-Schuell, 0.45 /zm) was washed for 10 min by gentle agitation in TBS, then air-dried at room temperature for 10 rain. LPS solution, 100 n g / 1 /zl TBS, was placed into the dry filter. The filter again was dried very thoroughly and then incubated in 3% gelatin in TBS for 0.5 h at 30°C to block nonspecific binding sites on the nitrocellulose. The filter was incubated with the anti-conjugate serum diluted 1:1000 (primary antibody) for 2 h at 30°C, then it was washed once with water and twice with TBS, for 10 min each washing. The filter was incubated with a second antibody: peroxidase-linked goat antirabbit IgG (diluted 1:3000). The second antibody was re-
moved. After washing, as above, the nitrocellulose filter was developed in 4-chloro-l-naphtol (Bio-Rad) solution in the presence of H 2 0 2. SDS-gel electrophoresis and immunoblotting were performed as described previously [4].
4. R E S U L T S AND DISCUSSION
4.1. Preparation of the PCM 14870S-TT and PCM 1487PS- TT conjugates and their characteristics The core oligosaccharide of Citrobacter PCM 1487 was submitted to the controlled periodate oxidation. During this process one of the three heptosyl residues can be oxidized between C6 and C7 and 3-deoxyoctulosonic acid (dOclA) residue between C7 and C8, but the rest of the sugar components remained intact. The oxidized oligosaccharide containing on average one reactive aldehyde group in the heptose-dOciA region was linked covalently with tetanus toxoid in a reductive amination p r o c e d u r e and P C M 1487OS-TT conjugate was formed. The same procedure was also used in the case of degraded PCM 1487-specific polysaccharide (devoid of 4deoxy-D-arabinohexose) to obtain PCM 1487PST T conjugate. Due to the fact that O-specific polysaccharide preparation is always linked to core oligosaccharide, its conjugation with tetanus toxoid could be realized through the core region as described above. Based on the results of sugar and protein determinations the molar ratio of PCM 1487OS to T T in the conjugate was calculated to be 8 : 1. In the conjugate PCM 1487PS-TT this ratio amounted to 2: 1.
4.2. Immunogenicity of the conjugates The immunogenicity of the conjugates was tested in rabbits. Antibodies raised against PCM 1487OS-TT as well as against PCM 1487PS-TT conjugate were determined by quantitative microprecipitation (QMP) (Fig. 1). In the equivalence point, PCM 1487 LPS precipitated from anti-PCM 1487OS-TT 1.8 mg of antibody per ml of serum, and from anti-PCM 1487PS-q-T 2.05 mg of antibody per ml of serum.
204
v
[10
I,--
i
20
i! ¸
I.
5 10 20
50
100
150
ANTIGEN (pq)
Fig. 1. Precipitation of anti-PCM 1487PS-TT ( o ©) and anti-PCM 1487OS-TT (e e) conjugate sera by the homologous lipopolysaccharide.
4.3. Characteristics of anti-PCM 14870S-TT and anti-PCM 1487PS- TT conjugate sera In order to examine the specificity of the anticonjugate sera obtained, dot-immunoblotting was carried out using lipopolysaccharides of nine strains of Citrobacter, S. sonnei phase I and S. flexneri 4b as antigens. Only the homologous PCM 1487 LPS reacted with both antisera. Citrobacter 027 LPS and S. flexneri 4b LPS cross-reacted with anti-conjugate serum directed against PCM 1487 core oligosaccharide. The remaining lipopolysaccharides showed no cross-reactivity. The SDS-gel electrophoresis of lipopolysaccharides of some Citrobacter strains and S. flexneri 4b indicated their great heterogeneity (Fig. 2). In the lipopolysaccharides of smooth strains high-molecular bands with O-specific chains and fast-migrating bands related to core LPS could be differentiated; in R mutant lipopolysaccharides fast-migrating bands of core LPS were observed only. The lipopolysaccharides reacting positively in dot-immunoblotting (Fig. 2) were separated in SDS-gel electrophoresis, then they were transblotted from the gel into nitrocellulose and incubated with the anti-conjugate sera. As shown in Fig. 3 anti-PCM 1487PS-TT conjugate serum gave a strong positive immunoblot with high-molecular O-specific chain LPS fraction from Citrobacter PCM 1487 only. On the other hand, the anti-PCM 1487OS-TT conjugate serum reacted with the
ll.
A
.
B
+
.
.
-
.
-I-
+
-
+
Fig. 2. I: Silver stained SDS-gel electrophoresis of lipopolysaccharides (1 /~g sample) from Citrobacter strains and Shigella flexneri 4b. II: Dot-immunoblotting of the lipopolysaccharides with anti-PCM 1487PS-TT (A) and anti-PCM 1487OS-TT (B) conjugate sera.
fast-migrating core LPS fraction from the homologous PCM 1487 strain as well as with those isolated from Citrobacter 027 and S. flexneri 4b. S
A
i!i
B
S
A
B
S
A
B
i
i n 1
2
o 3
Fig. 3. Silver stained SDS-gel electrophoresis of lipopolysaccharides (S) from Citrobacter strains PCM 1487 (1) and 1560 (027) (3) and S. flexneri 4b (2), and their immunoblots with anti-PCM 1487PS-TT (A) and anti-PCM 1487OS-TT (B) sera. LPS sample amounted to 200 ng.
205
qO
100
v
20
m 50 ==
5 10 20 5
10 20
50 100 200
INHIBITOR (~g)
Fig. 4. Precipitation inhibition of the system Citrobacter PCM 1487LPS--anti-PCM 1487PS-TT conjugate by PCM 1487specific polysaccharide ( • •), the degraded PCM 1487specific polysaccharide (© 0) and PCM 1487-core oligosaccharide ( • - • ).
T h e reactivity of the latter antiserum with the homologous LPS of Citrobacter 027 LPS was of the same intensity, whilst it was weaker with S. flexneri 4b LPS. To characterize more precisely the anti-conjugate sera quantitative microprecipitin and QMP inhibition tests were carried out. The PCM 1487 LPS-anti-PCM 1487PS-TF conjugate system was examined in Q M P inhibition using native and degraded PCM 1487-specific polysaccharides as inhibitors (Fig. 4). As could be predicted, the degraded PS was a better inhibitor than the native one: 3.8 ~ g of the degraded PS but 23/xg of the native PS gave 50% inhibition. This anti-conjugate serum was not inhibited by PCM 1487 core oligosaccharide; the cross-reactivity with Citrobacter 036 LPS was also not observed. A n t i - P C M 1487OS-TT conjugate serum showed cross-reactions in QMP: Citrobacter 027 LP precipitated 1.85 mg of antibody per ml of serum, but S. flexneri 4b LPS gave only 1.4 mg of antibody per ml of the same serum (Fig. 5). Core oligosaccharides derived from Citrobacter strains: PCM 1487, 027, 036, S. flexneri 4b and S. sonnei phase I, the structures of which are established, were used as inhibitors in QMP assay employing PCM 1487LPS--anti-PCM 1487OST T conjugate system (Fig. 6). The best inhibitor
50
100
ANTIGEN (~9) Fig. 5. Cross-precipitation of anti-PCM 1487OS-TT conjugate serum by lipopolysaccharides from Citrobacter strain 1560 (027) (© ©) and S. flexneri 4b (e e).
of the system was the homologous core oligosaccharide: 10.6/xg of PCM 1487OS gave 50% inhibition, whereas 027OS inhibited somewhat more weakly (50% inhibition--22 /xg of 027OS). The inhibitory power of S. flexneri 4b OS was significantly lower (50% inhibition-188/xg of S. flexneri 4b OS). Citrobacter 036OS (incomplete) and S. sonnei phase I OS were inactive as inhibitors of this system. The serological results obtained with the anti-conjugate sera were in full agreement with the structures (Fig. 7) of the core oligosaccharides and the O-specific polysaccharide used
100
g i,-
m
50
~
5
rill
10 20
~
~
50 100
200
INHIBITOR C~)
Fig. 6. Precipitation inhibition of the system Citrobacter PCM 1487LPS--anti-PCM 1487OS-TT conjugate by core oligosaccharides of Citrobacter PCM 1487 (© ©), Citrobacter 1560 (027) (o e), Citrobacter 036 incomplete ( [] [] ), S. flexneri 4b ( • • ) and S. sonnei phase
I(B
B).
206
ity with anti-PCM148OS-TT conjugate serum was similar. Some differences in the activity are probably caused by intrastrain microheterogeneity. Core oligosaccharides of S. flexneri 4b were nearly
as inhibitors. As seen, core oligosaccharides of Citrobacter 027 and PCM 1487 strains have structures which cannot be distinguished by using chemical and spectroscopic methods. Their activCITROBACTER
O-SPECIFIC
P O L Y S A C C H A R IDE
PEH 1487
-4DGalNAca1-6DGlcNAcal
-
13
4deoxyDaraHexl3
CITROBACTER
DEGRADED
O-SPECIFIC
POLYSACCHARIDE
PCH 1487 -4DGalNAcal-6DGlcNAcal-
CORE OL IGOSACCHAR IDES
CITROBACTER PCM 1487
PPEEN
CITROBACTER 027
17
DGalNAca 3
LDHep~ 17
PPEtN 4
**
DGlcal-2DGlcal-2DG~lal-3DGIcal-3LDHepal-3LDH~pal-SdOclA SHIGELLA FLEXNERI 4b DGlcNAca
13
DGlcal-2DGlc~l-2DGalal-3D(;lcal-Hep...
CITROBACTER 036 INCOMPLETE
DGalu
16 DGlcp 14
PPELN
17 17
LDHepa
PPELN
14
DGlcul-2DGlcal-3LDHep~I-3LDHep~I-SdOclA
SHIGELLA SONNEI PHASE I
D(;lcl3
13
DGlcNa1-7LDHepa
17
PPEt.N
14
DGalul'ZDGalQ1-2DGlc~1-3DG]cal-3LDH@p-1-3LDHep¢I -SdOclA Fig. 7. Chemical structures of O-specific polysaccharide, degraded polysaccharide and core oligosaccharide of Citrobacter PCM 1487 strain [1,2], and of core oligosaccharides of Citrobacter 027 [11], S. flexneri 4b [12], Citrobacter 036 [13] and S. sonnei phase I [14]. * PPEtN, diphosphorylethanolamine; * * dOclA, 3-deoxyoctulosonic acid.
207
20 times weaker inhibitor than PCM 1487OS but they differ in one terminal sugar: S. flexneri 4b OS has N-acetylglucosamine, and PCM 1487OS N-acetylgalactosamine (Fig. 7). In conclusion the results obtained proved that: 1. The anti-PCM 1487OS-TT and anti-PCM 1487PS-TT conjugate sera can be used in quantitative microprecipitin and immunoblotting tests; 2. The anti-conjugate sera are monospecific and contain antibodies directed against the hexose region of the core (anti-OS-TY serum) or against the O-specific polysaccharide devoid of 4-deoxy-o-arabinohexose residues (anti-PS-T-F serum). The latter serum does not give unwanted cross-reactions with lipopolysaccharides containing homopolymers of 4-deoxy-Darabinohexose; 3. The anti-conjugate sera can be used for examination of the antigenic relationships between lipopolysaccharides of various origin and for detection of rough mutants containing LPS with complete core region; 4. The anti-conjugate sera can be useful in the detection of degradation products of bacteria and in rapid diagnostic tests in infectious diseases. REFERENCES [1] Gamian, A., Romanowska, E., Romanowska, A., Lugow° ski, C., Dabrowski, J. and Trauner, K. (1985) Citrobacter lipopolysaccharides; structure elucidation of the Ospecific polysaccharide from strain PCM 1487 by mass spectrometry, one-dimensional and two-dimensional 1HNMR spectroscopy and methylation analysis. Eur. J. Biochem. 146, 641-647. [2] Romanowska, E., Gamian, A. and Dabrowski, J. (1986) Core region of Citrobacter lipopolysaccharide from strain PCM 1487. Structure elucidation by two-dimensional IHNMR spectroscopy at 500 MHz and methylation analysis/mass spectrometry. Eur. J. Biochem. 161, 557-564.
[3] Gamian, A. and Romanowska, E. (1990) Structure of the heptose-3-deoxyoctulosonic acid region of Citrobacter lipopolysaccharide core. Carbohydr. Res. 198, 381-383. [4] Lugowski, C., Kulakowska, M. and Romanowska, E. (1986) Characterization and diagnostic application of a lipopolysaccharide core oligosaccharide protein conjugate. J. Immunol. Meth. 95, 187-194. [5] Romanowska, E., Romanowska, A., Lugowski, C. and Katenzenellenbogen, E. (1981) Structural and serological analysis of Citrobacter 036-specific polysaccharide, the homopolymer of (1-2) fl-linked 4-deoxy-o-arabinohexopyranosyl unit. Eur. J. Biochem. 121, 119-123. [6] Lowry, O.H., Rosenbrough, N.J., Farr, A.L. and Randall, R.J. (1951) Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265-275. [7] Dubois, H., Gilles, K.A., Hamilton, J.K., Rebers, P. and Smith, F. (1956) Colorimetric method for determination of sugars and related substances. Anal. Chem. 28, 350356. [8] Jennings, H.J. and Lugowski, C. (1981) Immunochemistry of groups A, B, and C meningococcal polysaccharidetetanus toxoid conjugates. J. Immunol. 127, 1011-1018. [9] Lehmann, V., Liideritz, O. and Westphal, O. (1971) The linkage of pyrophosphorylethanolamine to heptose with the core of Salmonella minnesota lipopolysaccharide. Eur. J. Biochem. 21,339-347. [10] Lugowski, C., Kutakowska, M. and Romanowska, E. (1983) Enterobacterial common antigen/tetanus toxoid conjugate as immunogen. Infect. Immun. 42, 1086-1091. [11] Romanowska, E., Romanowska, A., Dabrowski, J. and Trauner, K. (1989) The structure of the lipopolysaccharide core region of Citrobacter 027. FEMS Microbiol. Lett. 58, 107-110. [12] Jansson, P.E., Lindberg, A.A., Lindberg, B. and Wollin, R. (1981) Structural studies on the hexose region of the core in lipopolysaccharides from Enterobacteriaceae. Eur. J. Biochem. 115, 571-577. [13] 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. [14] Gamian, A. and Romanowska, E. (1982) The core structure of Shigella sonnei lipopolysaccharide and the linkage between O-specific polysaccharide and the core region. Eur. J. Biochem. 129, 105-109.
201
FEMSIM 00205
Immunochemical characterization of Citrobacter strain PCM 1487 O-specific polysaccharideand core oligosaccharide-protein conjugates Czestaw Lugowski, Matgorzata Kutakowska 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, Wroctaw, Poland Received 17 July 1990 Revision received 28 November 1991 Accepted 14 January 1992
Key words: Lipopolysaccharide; Citrobacter; Saccharide-protein conjugate
1. SUMMARY O-specific-polysaccharide and core oligosaccharide were isolated from Citrobacter strain PCM 1487 lipopolysaccharide and purified. The polysaccharide was selectively devoid of 4-deoxyD-arabinohexose residues. Covalent conjugates of the modified O-specific polysaccharide and of the core oligosaccharide
with tetanus toxoid were prepared. Immunochemical characterization of these conjugates proved that they are strong immunogens. Using monospecific rabbit antisera raised against the conjugates, the antigenic relationships between lipopolysaccharides of various strains of Citrobacter, Shigella sonnei, and Shigella flexneri were studied by quantitative microprecipitin and quantitative microprecipitin inhibition and immunoblotting tests.
Correspondence to: C. Lugowski, Laboratory of Microbial Immunochemistry, Ludwik Hirszfeld Institute of Immunology and Experimental Therapy, ul. Czerska 12, 53-114 Wrodaw, Poland. A preliminary account of a part of this work is published in Excerpta Medica International Congress Series (ICS No. 923) 1990, Endotoxin Research Series Vol. 1, Edited by A. Nowotny, J. J. Spitzer and E.J. Ziegler, Elsevier, Amsterdam.
2. INTRODUCTION In the studies on carbohydrate moiety of Citrobacter PCM 1487 strain lipopolysaccharide (LPS) the structures of O-specific polysaccharide (PS) and of core oligosaccharide (OS) were estab-
202 lished [1-3]. The O-specific polysaccharide consists of the trisaccharide repeating units:
may yield the preliminary information helpful in further structural studies.
-4-D-GalNAcp a 1-6-D-glcNAcp a 1-
13
3. MATERIALS AND METHODS
4-deoxy-D-araHexp/3 where 4-deoxy-D-araHex stands for 4-deoxy-Darabinohexose. The formula of the core oligosaccharide is the following: PPEtN 7 oGalNAcpa 3
LoHeppa 7
PPEtN 4
oGlcpctl-2oGlcpal-2oGalpctl-3oGlcpctl-3LDHeppcd-3LoHeppcd-5dOclA dOclA, 3-deoxyoctulosonic acid; PPEtN, diphosphorylethanolamine.
Rabbit antiserum obtained by immunization with Citrobacter microorganisms is directed against the O-specific polysaccharide chains. The antiserum shows the presence of IgM antibodies and cross-reactivity with homopolymers of/3(1-2) linked 4-deoxy-o-arabinohexose residues originated from O-antigens of Citrobacter serotypes 04, 027, and 036 which is a disadvantage in its application to modern serological tests. In our earlier studies on the monospecific antiserum raised to Shigella sonnei phase II core oligosaccharide-protein conjugate the distinct relationship between serological reactions and the structure of core regions of the lipopolysaccharides examined was observed [4]. The aim of the present work has been to obtain covalent conjugates of the O-specific polysaccharide selectively devoid of 4-deoxy-Darabinohexose and of the core oligosaccharide with tetanus toxoid (TT). 4-deoxy-D-arabinohexose was removed from the PCM 1487-specific polysaccharide to avoid undesired cross-reactions of the monospecific anti-PCM 1487PS-TT conjugate serum. The application of the monospecific antiCitrobacter saccharide-protein conjugate sera in examination of the relationship between O-antigens of Citrobacter and other Enterobacteriaceae
Citrobacter freundii strains PCM 1487 and 1488 (036) derived from the stock collection of the Institute of Immunology and Experimental Therapy; strain negSTc s (036) and its rough mutant (R36) were obtained from the Institute of Microbiology, Wrodaw University; strains 52/57 (04), 87/57 (023), 1556 (R mutant of 023 serotype), 1560 (027) and 114/66 (036) from the Czechoslovakian National Collection of Type Cultures, Prague; Shigella sonnei phase I strain 9773 and Shigella flexneri 4b from the Dysentary Reference Laboratory, London. Crude tetanus toxoid preparation was obtained from Wytw6rnia Surowic i Szczepionek, Warsaw and purified on Sepharose 6B column (2.6 × 100 cm) equilibrated with PBS. The preparation of lipopolysaccharides, Ospecific polysaccharides and core oligosaccharides were performed as previously described [1,2,5]. Chemical degradation of the PCM 1487specific polysaccharide was carried out according to Gamian et al. [1]. After degradation the PCM 1487PS is devoid of 4-deoxy-D-arabinohexose and shows no cross-reactivity with Citrobacter 036 LPS. De-O-acetylation of lipopolysaccharide: LPS preparation dissolved in 0.25 M NaOH was heated at 56°C for 1 h. Analytical methods: protein was determined by the Lowry et al. method [6] and sugar by the phenol method [7]. Conjugation of PCM 1487OS and degraded PCM 1487PS with tetanus toxoid and purification of the conjugates: the conjugation was carried out according to Jennings and Lugowski [8]. The oligoor polysaccharide was selectively oxidized with NalO 4 using the procedure of Lehmann et al. [9]. Conjugation of PCM 14870S-TT. The aqueous solution of PCM 1487OS (40 mg/ml) was mixed with 1 ml 0.75% NaIO 4 and incubated in the dark at 4°C with magnetic stirring for 45 min. The excess periodate was destroyed by adding ethy-
203 lene glycol. The oxidized PCM 1487OS was desalted on a column of Sephadex G-25 (2.6 × 90 cm) equilibrated with 0.02 M p y r i d i n e / C H 3 C O O H buffer pH 5.4. Fractions containing oxidized PCM 1487OS (35 mg) were lyophilized, and then dissolved in 1 ml 0.5 M K 2 H P O 4 pH 9.0. Tetanus toxoid (3.3 mg), 35 mg N a C N B H 3 and 1 drop of chloroform were added to the solution. The reaction mixture was kept in a sealed vial for 12 days at 37°C and then, for purification, it was applied to Sephadex G-100 (1.6 × 100 cm) column equilibrated with PBS. Fractions containing PCM 1487OS-TT conjugate were concentrated by ultrafiltration to give a yield of 3.5 mg.
Conjugation of degraded PCM 1487PS (30 mg) with TT (3.3 mg). This was performed by the same procedure. The conjugate PCM 1487PS-TT was purified on Sepharose 6B column and concentrated by ultrafiltration. The yield of the conjugate amounted to 4.2 mg. Immunization procedure: Rabbits were immunized in the footpads with the antigens suspended in Freund's complete adjuvant, as described earlier by Lugowski et al. [10]. Serological tests. A quantitative microprecipitin test was carried out as in [10]. Inhibition of precipitation was performed by incubation of serum with increasing concentrations of inhibitors for 1 h at 37°C prior to adding antigen. The optimal concentration of antigen required for maximal antibody precipitation was determined from the precipitin curve. D o t - i m m u n o b i o t t i n g : nitrocellulose filter (Schleicher-Schuell, 0.45 /zm) was washed for 10 min by gentle agitation in TBS, then air-dried at room temperature for 10 rain. LPS solution, 100 n g / 1 /zl TBS, was placed into the dry filter. The filter again was dried very thoroughly and then incubated in 3% gelatin in TBS for 0.5 h at 30°C to block nonspecific binding sites on the nitrocellulose. The filter was incubated with the anti-conjugate serum diluted 1:1000 (primary antibody) for 2 h at 30°C, then it was washed once with water and twice with TBS, for 10 min each washing. The filter was incubated with a second antibody: peroxidase-linked goat antirabbit IgG (diluted 1:3000). The second antibody was re-
moved. After washing, as above, the nitrocellulose filter was developed in 4-chloro-l-naphtol (Bio-Rad) solution in the presence of H 2 0 2. SDS-gel electrophoresis and immunoblotting were performed as described previously [4].
4. R E S U L T S AND DISCUSSION
4.1. Preparation of the PCM 14870S-TT and PCM 1487PS- TT conjugates and their characteristics The core oligosaccharide of Citrobacter PCM 1487 was submitted to the controlled periodate oxidation. During this process one of the three heptosyl residues can be oxidized between C6 and C7 and 3-deoxyoctulosonic acid (dOclA) residue between C7 and C8, but the rest of the sugar components remained intact. The oxidized oligosaccharide containing on average one reactive aldehyde group in the heptose-dOciA region was linked covalently with tetanus toxoid in a reductive amination p r o c e d u r e and P C M 1487OS-TT conjugate was formed. The same procedure was also used in the case of degraded PCM 1487-specific polysaccharide (devoid of 4deoxy-D-arabinohexose) to obtain PCM 1487PST T conjugate. Due to the fact that O-specific polysaccharide preparation is always linked to core oligosaccharide, its conjugation with tetanus toxoid could be realized through the core region as described above. Based on the results of sugar and protein determinations the molar ratio of PCM 1487OS to T T in the conjugate was calculated to be 8 : 1. In the conjugate PCM 1487PS-TT this ratio amounted to 2: 1.
4.2. Immunogenicity of the conjugates The immunogenicity of the conjugates was tested in rabbits. Antibodies raised against PCM 1487OS-TT as well as against PCM 1487PS-TT conjugate were determined by quantitative microprecipitation (QMP) (Fig. 1). In the equivalence point, PCM 1487 LPS precipitated from anti-PCM 1487OS-TT 1.8 mg of antibody per ml of serum, and from anti-PCM 1487PS-q-T 2.05 mg of antibody per ml of serum.
204
v
[10
I,--
i
20
i! ¸
I.
5 10 20
50
100
150
ANTIGEN (pq)
Fig. 1. Precipitation of anti-PCM 1487PS-TT ( o ©) and anti-PCM 1487OS-TT (e e) conjugate sera by the homologous lipopolysaccharide.
4.3. Characteristics of anti-PCM 14870S-TT and anti-PCM 1487PS- TT conjugate sera In order to examine the specificity of the anticonjugate sera obtained, dot-immunoblotting was carried out using lipopolysaccharides of nine strains of Citrobacter, S. sonnei phase I and S. flexneri 4b as antigens. Only the homologous PCM 1487 LPS reacted with both antisera. Citrobacter 027 LPS and S. flexneri 4b LPS cross-reacted with anti-conjugate serum directed against PCM 1487 core oligosaccharide. The remaining lipopolysaccharides showed no cross-reactivity. The SDS-gel electrophoresis of lipopolysaccharides of some Citrobacter strains and S. flexneri 4b indicated their great heterogeneity (Fig. 2). In the lipopolysaccharides of smooth strains high-molecular bands with O-specific chains and fast-migrating bands related to core LPS could be differentiated; in R mutant lipopolysaccharides fast-migrating bands of core LPS were observed only. The lipopolysaccharides reacting positively in dot-immunoblotting (Fig. 2) were separated in SDS-gel electrophoresis, then they were transblotted from the gel into nitrocellulose and incubated with the anti-conjugate sera. As shown in Fig. 3 anti-PCM 1487PS-TT conjugate serum gave a strong positive immunoblot with high-molecular O-specific chain LPS fraction from Citrobacter PCM 1487 only. On the other hand, the anti-PCM 1487OS-TT conjugate serum reacted with the
ll.
A
.
B
+
.
.
-
.
-I-
+
-
+
Fig. 2. I: Silver stained SDS-gel electrophoresis of lipopolysaccharides (1 /~g sample) from Citrobacter strains and Shigella flexneri 4b. II: Dot-immunoblotting of the lipopolysaccharides with anti-PCM 1487PS-TT (A) and anti-PCM 1487OS-TT (B) conjugate sera.
fast-migrating core LPS fraction from the homologous PCM 1487 strain as well as with those isolated from Citrobacter 027 and S. flexneri 4b. S
A
i!i
B
S
A
B
S
A
B
i
i n 1
2
o 3
Fig. 3. Silver stained SDS-gel electrophoresis of lipopolysaccharides (S) from Citrobacter strains PCM 1487 (1) and 1560 (027) (3) and S. flexneri 4b (2), and their immunoblots with anti-PCM 1487PS-TT (A) and anti-PCM 1487OS-TT (B) sera. LPS sample amounted to 200 ng.
205
qO
100
v
20
m 50 ==
5 10 20 5
10 20
50 100 200
INHIBITOR (~g)
Fig. 4. Precipitation inhibition of the system Citrobacter PCM 1487LPS--anti-PCM 1487PS-TT conjugate by PCM 1487specific polysaccharide ( • •), the degraded PCM 1487specific polysaccharide (© 0) and PCM 1487-core oligosaccharide ( • - • ).
T h e reactivity of the latter antiserum with the homologous LPS of Citrobacter 027 LPS was of the same intensity, whilst it was weaker with S. flexneri 4b LPS. To characterize more precisely the anti-conjugate sera quantitative microprecipitin and QMP inhibition tests were carried out. The PCM 1487 LPS-anti-PCM 1487PS-TF conjugate system was examined in Q M P inhibition using native and degraded PCM 1487-specific polysaccharides as inhibitors (Fig. 4). As could be predicted, the degraded PS was a better inhibitor than the native one: 3.8 ~ g of the degraded PS but 23/xg of the native PS gave 50% inhibition. This anti-conjugate serum was not inhibited by PCM 1487 core oligosaccharide; the cross-reactivity with Citrobacter 036 LPS was also not observed. A n t i - P C M 1487OS-TT conjugate serum showed cross-reactions in QMP: Citrobacter 027 LP precipitated 1.85 mg of antibody per ml of serum, but S. flexneri 4b LPS gave only 1.4 mg of antibody per ml of the same serum (Fig. 5). Core oligosaccharides derived from Citrobacter strains: PCM 1487, 027, 036, S. flexneri 4b and S. sonnei phase I, the structures of which are established, were used as inhibitors in QMP assay employing PCM 1487LPS--anti-PCM 1487OST T conjugate system (Fig. 6). The best inhibitor
50
100
ANTIGEN (~9) Fig. 5. Cross-precipitation of anti-PCM 1487OS-TT conjugate serum by lipopolysaccharides from Citrobacter strain 1560 (027) (© ©) and S. flexneri 4b (e e).
of the system was the homologous core oligosaccharide: 10.6/xg of PCM 1487OS gave 50% inhibition, whereas 027OS inhibited somewhat more weakly (50% inhibition--22 /xg of 027OS). The inhibitory power of S. flexneri 4b OS was significantly lower (50% inhibition-188/xg of S. flexneri 4b OS). Citrobacter 036OS (incomplete) and S. sonnei phase I OS were inactive as inhibitors of this system. The serological results obtained with the anti-conjugate sera were in full agreement with the structures (Fig. 7) of the core oligosaccharides and the O-specific polysaccharide used
100
g i,-
m
50
~
5
rill
10 20
~
~
50 100
200
INHIBITOR C~)
Fig. 6. Precipitation inhibition of the system Citrobacter PCM 1487LPS--anti-PCM 1487OS-TT conjugate by core oligosaccharides of Citrobacter PCM 1487 (© ©), Citrobacter 1560 (027) (o e), Citrobacter 036 incomplete ( [] [] ), S. flexneri 4b ( • • ) and S. sonnei phase
I(B
B).
206
ity with anti-PCM148OS-TT conjugate serum was similar. Some differences in the activity are probably caused by intrastrain microheterogeneity. Core oligosaccharides of S. flexneri 4b were nearly
as inhibitors. As seen, core oligosaccharides of Citrobacter 027 and PCM 1487 strains have structures which cannot be distinguished by using chemical and spectroscopic methods. Their activCITROBACTER
O-SPECIFIC
P O L Y S A C C H A R IDE
PEH 1487
-4DGalNAca1-6DGlcNAcal
-
13
4deoxyDaraHexl3
CITROBACTER
DEGRADED
O-SPECIFIC
POLYSACCHARIDE
PCH 1487 -4DGalNAcal-6DGlcNAcal-
CORE OL IGOSACCHAR IDES
CITROBACTER PCM 1487
PPEEN
CITROBACTER 027
17
DGalNAca 3
LDHep~ 17
PPEtN 4
**
DGlcal-2DGlcal-2DG~lal-3DGIcal-3LDHepal-3LDH~pal-SdOclA SHIGELLA FLEXNERI 4b DGlcNAca
13
DGlcal-2DGlc~l-2DGalal-3D(;lcal-Hep...
CITROBACTER 036 INCOMPLETE
DGalu
16 DGlcp 14
PPELN
17 17
LDHepa
PPELN
14
DGlcul-2DGlcal-3LDHep~I-3LDHep~I-SdOclA
SHIGELLA SONNEI PHASE I
D(;lcl3
13
DGlcNa1-7LDHepa
17
PPEt.N
14
DGalul'ZDGalQ1-2DGlc~1-3DG]cal-3LDH@p-1-3LDHep¢I -SdOclA Fig. 7. Chemical structures of O-specific polysaccharide, degraded polysaccharide and core oligosaccharide of Citrobacter PCM 1487 strain [1,2], and of core oligosaccharides of Citrobacter 027 [11], S. flexneri 4b [12], Citrobacter 036 [13] and S. sonnei phase I [14]. * PPEtN, diphosphorylethanolamine; * * dOclA, 3-deoxyoctulosonic acid.
207
20 times weaker inhibitor than PCM 1487OS but they differ in one terminal sugar: S. flexneri 4b OS has N-acetylglucosamine, and PCM 1487OS N-acetylgalactosamine (Fig. 7). In conclusion the results obtained proved that: 1. The anti-PCM 1487OS-TT and anti-PCM 1487PS-TT conjugate sera can be used in quantitative microprecipitin and immunoblotting tests; 2. The anti-conjugate sera are monospecific and contain antibodies directed against the hexose region of the core (anti-OS-TY serum) or against the O-specific polysaccharide devoid of 4-deoxy-o-arabinohexose residues (anti-PS-T-F serum). The latter serum does not give unwanted cross-reactions with lipopolysaccharides containing homopolymers of 4-deoxy-Darabinohexose; 3. The anti-conjugate sera can be used for examination of the antigenic relationships between lipopolysaccharides of various origin and for detection of rough mutants containing LPS with complete core region; 4. The anti-conjugate sera can be useful in the detection of degradation products of bacteria and in rapid diagnostic tests in infectious diseases. REFERENCES [1] Gamian, A., Romanowska, E., Romanowska, A., Lugow° ski, C., Dabrowski, J. and Trauner, K. (1985) Citrobacter lipopolysaccharides; structure elucidation of the Ospecific polysaccharide from strain PCM 1487 by mass spectrometry, one-dimensional and two-dimensional 1HNMR spectroscopy and methylation analysis. Eur. J. Biochem. 146, 641-647. [2] Romanowska, E., Gamian, A. and Dabrowski, J. (1986) Core region of Citrobacter lipopolysaccharide from strain PCM 1487. Structure elucidation by two-dimensional IHNMR spectroscopy at 500 MHz and methylation analysis/mass spectrometry. Eur. J. Biochem. 161, 557-564.
[3] Gamian, A. and Romanowska, E. (1990) Structure of the heptose-3-deoxyoctulosonic acid region of Citrobacter lipopolysaccharide core. Carbohydr. Res. 198, 381-383. [4] Lugowski, C., Kulakowska, M. and Romanowska, E. (1986) Characterization and diagnostic application of a lipopolysaccharide core oligosaccharide protein conjugate. J. Immunol. Meth. 95, 187-194. [5] Romanowska, E., Romanowska, A., Lugowski, C. and Katenzenellenbogen, E. (1981) Structural and serological analysis of Citrobacter 036-specific polysaccharide, the homopolymer of (1-2) fl-linked 4-deoxy-o-arabinohexopyranosyl unit. Eur. J. Biochem. 121, 119-123. [6] Lowry, O.H., Rosenbrough, N.J., Farr, A.L. and Randall, R.J. (1951) Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265-275. [7] Dubois, H., Gilles, K.A., Hamilton, J.K., Rebers, P. and Smith, F. (1956) Colorimetric method for determination of sugars and related substances. Anal. Chem. 28, 350356. [8] Jennings, H.J. and Lugowski, C. (1981) Immunochemistry of groups A, B, and C meningococcal polysaccharidetetanus toxoid conjugates. J. Immunol. 127, 1011-1018. [9] Lehmann, V., Liideritz, O. and Westphal, O. (1971) The linkage of pyrophosphorylethanolamine to heptose with the core of Salmonella minnesota lipopolysaccharide. Eur. J. Biochem. 21,339-347. [10] Lugowski, C., Kutakowska, M. and Romanowska, E. (1983) Enterobacterial common antigen/tetanus toxoid conjugate as immunogen. Infect. Immun. 42, 1086-1091. [11] Romanowska, E., Romanowska, A., Dabrowski, J. and Trauner, K. (1989) The structure of the lipopolysaccharide core region of Citrobacter 027. FEMS Microbiol. Lett. 58, 107-110. [12] Jansson, P.E., Lindberg, A.A., Lindberg, B. and Wollin, R. (1981) Structural studies on the hexose region of the core in lipopolysaccharides from Enterobacteriaceae. Eur. J. Biochem. 115, 571-577. [13] 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. [14] Gamian, A. and Romanowska, E. (1982) The core structure of Shigella sonnei lipopolysaccharide and the linkage between O-specific polysaccharide and the core region. Eur. J. Biochem. 129, 105-109.