Core Oligosaccharide of Plesiomonas shigelloides PCM 2231 (Serotype O17) Lipopolysaccharide—Structural and Serological Analysis

The herein presented complete structure of the core oligosaccharide of lipopolysaccharide (LPS) P. shigelloides Polish Collection of Microorganisms (PCM) 2231 (serotype O17) was investigated by 1H, 13C NMR spectroscopy, mass spectrometry, chemical analyses and serological methods. The core oligosaccharide is composed of an undecasaccharide, which represents the second core type identified for P. shigelloides serotype O17 LPS. This structure is similar to that of the core oligosaccharide of P. shigelloides strains 302-73 (serotype O1) and 7-63 (serotype O17) and differs from these only by one sugar residue. Serological screening of 55 strains of P. shigelloides with the use of serum against identified core oligosaccharide conjugated with bovine serum albumin (BSA) indicated the presence of similar structures in the LPS core region of 28 O-serotypes. This observation suggests that the core oligosaccharide structure present in strain PCM 2231 could be the most common type among P. shigelloides lipopolysaccharides.


Isolation of Lipopolysaccharide and Core Oligosaccharide
The lipopolysaccharide of P. shigelloides strain PCM 2231 (serotype O17) was isolated by phenol/water extraction and purified as previously reported [7]. Yield of LPS was 1.8% of the dry bacterial mass. The SDS-polyacrylamide gel electrophoresis (SDS-PAGE) analysis showed the smooth character of LPS, with a characteristic ladder-like pattern indicating different degree of polymerization of the O-antigen. The O-specific polysaccharides and different oligosaccharides were released by mild acid hydrolysis of the LPS (200 mg) and separated by gel filtration on Bio-Gel P-10. Five fractions of polysaccharide region: PSI (31.2 mg), PSII (3.7 mg), PSIII (3.1 mg), PSIV (3.8 mg), PSV (11.6 mg), and three fractions of oligosaccharide region: OSI (5.4 mg), OSII (2.0 mg) and OSIII (≤0.5 mg) were obtained ( Figure 1, inset structure). Analysis of the fractions, with the use of MALDI-TOF MS, showed that PSII, PSIII, PSIV, and PSV consisted of the core OS substituted with four, three, two, and one repeating units of the O-specific PS, respectively. The PSI consisted of the core OS substituted with at least five RUs of the O-specific PS. Fraction OSIII was a mixture of low molecular weight oligosaccharides containing 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo) released during mild acid hydrolysis of the LPS.

Chemical Analysis of the Oligosaccharides PSV and OSI
Initial NMR analysis of the OSI indicated the presence of uronic acid, Kdo, and two non-acetylated hexosamine residues. Thus all subsequent sugar and methylation analyses were carried out on N-acetylated and carboxyl-reduced oligosaccharide to detect these residues. Compositional analysis of the N-acetylated core oligosaccharide (OSI) together with determination of the absolute configuration revealed the presence of L,D-Hep, D-Glc, D-Gal, D-GalA, and D-GlcN. Methylation analysis performed on the carboxyl-reduced and N-acetylated OSI indicated the presence of 2,3,7-trisubstituted L,D-Hepp, 3,7-disubstituted L,D-Hepp, 3,4-disubstituted L,D-Hepp, 7-substituted L,D-Hepp, terminal D-GlcpN, 6-substituted D-GlcpN, terminal D-Glcp, terminal D-Galp, 4-substituted D-GalpA, and 5-substituted Kdo. The PSV fraction consisted of all residues identified for OSI, and additionally 4-substituted D-GlcpN and terminal AltpNAcA were detected.

MALDI-TOF MS Analyses of the PSV, OSI and OSII
The MALDI-TOF mass spectrum of the OSI showed two main clusters of two pairs of ions at  Complete structure of the core OS substituted with one RU of the O-specific PS was presented as the inset structure with marked OSII, OSI and PSV fractions. Heterogeneity related residues are marked with asterisk. Spectra were obtained in the negative reflectron mode with 2,4,6-trihydroxyacetophenone as a matrix. m/z values represent monoisotopic masses.
The mass spectrum of the oligosaccharide OSII was obtained for fraction analysed by NMR prior to mass spectrometry ( Figure 1A and inset structure). m/z values of observed ions were ~1 Da higher than calculated masses of ions as a result of incomplete exchange of deuterium to hydrogen. Therefore major ions at m/z 1812.5 and 1650.5 corresponded to the core oligosaccharide devoid of one Ions at m/z 2255.9 and 2237.9 corresponded to the structure devoid of one hexose, resembling previously reported heterogeneity of the OSI and OSII. Finally, the presence of glycine was suggested by ions at m/z 2294.9, 2312.9, 2456.9, and 2474.9 separated by 57 Da with reference to corresponding ions at m/z 2237.9, 2255.9, 2399.9, and 2417.9.

NMR Analysis of the PSV
The fraction PSV consisted of the core oligosaccharide substituted with one repeating unit of the O-specific chain was analysed by 1D and 2D 1 H, 13 C-NMR spectroscopy. All the spin systems ( Table 1) were assigned by COSY, TOCSY with different mixing times, HSQC-DEPT (Figure 2), HSQC-TOCSY, NOESY and HMBC spectra. Experimental chemical shift values were compared with previously published NMR data for respective monosaccharides and oligosaccharides [8,14,19].    The HSQC-DEPT spectrum obtained for PSV fraction contained signals for twelve major anomeric protons and carbons, and a Kdo spin system ( Figure 2, Table 1).
Residue Residue H′ was also identified due to heterogeneity of the structure (see text below) ( Table 1). Residue K was identified as a 5-substituted Kdo based on the characteristic deoxy proton signals of H-3a (δ 1.86 ppm), H-3b (δ 2.22 ppm) and a high chemical shift of the C-5 signal (δ 75.3 ppm).
Residue L′ with the H-1/C-1 signals at δ 5.38/99.7 ppm, J C-1,H-1 ~176 Hz, was recognized as the 2,3,7-trisubstituted L-glycero-α-D-manno-Hepp based on 1 H and 13 C chemical shifts, the small vicinal couplings between H-1, H-2, and H-3, and the relatively high chemical shifts of the C-2 (δ 78.9), C-3 (δ 79.3), and C-7 (δ 73.6) signals. Residue L was also identified due to heterogeneity (see text below) ( Table 1) In the fraction PSV, glycine was identified by the presence of an additional carbonyl resonance at δ C 168.7 ppm in the HMBC spectrum and a negative CH 2 crosspeak (δ H 3.98 ppm, δ C 40.9 ppm) in the HSQC-DEPT spectrum. The presence of glycine in the PSV component was further supported by amino acid analysis and mass spectrometry; however connectivity between the glycine and the oligosaccharide was not determined.
These studies demonstrate the structure of the complete core oligosaccharide and linkage between the core region and O-specific PS of lipopolysaccharide P. shigelloides PCM 2231 (serotype O17). The structure of the complete core region of P. shigelloides PCM 2231 is similar to that of the core OSs of P. shigelloides strains 7-63 (serotype O17) [14] and 302-73 (serotype O1) [10]. The core OS of strain 7-63 (serotype O17) differs only by a single terminal residue of branched chain, that is, terminal α-D-GalpN was present instead of the terminal α-D-GlcpN (residue B) in strain PCM 2231 (serotype O17). The core OS of P. shigelloides 302-73 (serotype O1) contains Kdo as a residue linking the O-specific polysaccharide with the outer core region instead of β-D-GlcpNAc (residue N) present in strain PCM 2231 (serotype O17).

Serological Studies
The core oligosaccharide (OSI) was conjugated with BSA (OSI-BSA) with the use of high temperature conjugation described by Boratynski et al. [20]. The polyclonal antisera obtained by immunization of rabbits with the conjugate were used to scan LPSs isolated from 55 strains of P. shigelloides for the presence of epitopes similar to those found in LPS of P. shigelloides PCM 2231 (serotype O17). The obtained conjugate was a good immunogen inducing high level of specific anti-core OS antibodies as was shown in a solid-phase ELISA with homologous LPS as an antigen (data not shown). LPSs were isolated by hot phenol/water extraction. Most of them were recovered from both the water and the phenol phase. LPSs were separated by SDS-PAGE ( Figure 3A) and stained using the method of Tsai [21]. The reactivities of anti-conjugate sera with LPSs isolated from various P. shigelloides strains were examined with the use of immunoblotting ( Figure 3B). Results for LPSs which were recovered from the phenol phases were identical in comparison with the water phase derived LPSs (data not shown). To simplify presented data, results for the water phase derived LPSs were shown with the exception of LPSs CNCTC 39/89, 102/89, 5112, recovered from the phenol phase.
The antibodies against the OSI-BSA conjugate reacted mainly with fast migrating LPS fractions, representing LPS with unsubstituted core oligosaccharide. However reactions with core OS epitopes present in smooth lipopolysaccharides were also observed. Serological screening of different strains of P. shigelloides indicates that similar epitopes might also be present in the core OS of the 28 out of 55 strains. Intensities of cross-reactions observed for different LPSs were diversified.   Bacteria were grown and harvested as described previously [22].

Lipopolysaccharide and Core Oligosaccharide
The LPS was extracted from bacterial cells by the hot phenol/water method [23] and purified as previously reported [7]. The yield of LPS was ~1.8% of the dry bacterial mass. LPS (200 mg) was degraded by treatment with 1.5% acetic acid containing 2% SDS at 100 °C for 15 min. The reaction mixture was freeze-dried, the SDS removed by extraction with 96% ethanol, and the residue suspended in water and centrifuged. The poly-and oligosaccharides were separated by gel permeation chromatography, performed on Bio-Gel 10 column, equilibrated with 0.05 M pyridine/acetic acid buffer of pH 5.6. Eluates were monitored with a Knauer differential refractometer and all fractions were freeze-dried and checked by MALDI-TOF mass spectrometry.

Analytical Methods
Monosaccharides were analysed as their alditol acetates by GC-MS [24]. Absolute configurations of the monosaccharides were determined as described by Gerwig et al. [25,26] using (−)-2-butanol for the formation of 2-butyl glycosides. The trimethylsilylated butyl glycosides produced from authentic samples were used as standards. Carboxyl reduction of the core oligosaccharide fractions was carried out according to the method of Taylor et al. [27] as previously described [22]. Methylation analyses were performed on both native and carboxyl reduced oligosaccharides according to the method of Hakomori [28]. Alditol acetates and partially methylated alditol acetates were analysed with a Hewlett-Packard 5972 system using the HP-1 fused-silica capillary column (0.2 mm × 12.5 m), He as carrier gas, flow rate 1 mL· min −1 and a temperature program 150 → 270 °C at 8 °C· min −1 .

NMR Spectroscopy
All NMR spectra were obtained on Bruker 600 MHz spectrometer (Laboratory of Structural Analyses, Wroclaw University of Technology, Wroclaw, Poland). The fraction PSV containing the core oligosaccharides substituted with one repeating unit of the O-specific chain was first repeatedly exchanged with 2 H 2 O (99.9%) with intermediate lyophilisation. NMR spectra were obtained for 2 H 2 O solutions at 27 °C using acetone (δ H 2.225 ppm, δ C 31.05 ppm) as internal reference. The signals were assigned by one-and two-dimensional experiments (COSY, TOCSY, NOESY, HMBC, HSQC-DEPT and HSQC-TOCSY). The J C-1,H-1 values were obtained from non-decoupled HSQC-DEPT experiment. In the TOCSY experiments the mixing times were 60, 90 and 120 ms. The delay time in the HMBC was 60 ms and the mixing time in the NOESY experiment was 60 ms.
All spectra were acquired and processed using standard Bruker software. The processed 2D spectra were assigned using the SPARKY program [29].

Preparation of Oligosaccharide Conjugate with BSA
The core oligosaccharide (OSI) was oxidised and purified as describe previously [30]. Briefly, the core oligosaccharide OSI (10 mg) was oxidised with 0.1 M NaIO 4 (1 mL) at 21 °C in the dark for 60 min. Then ethylene glycol (10 μL) was added, and the solution was incubated at 21 °C for an additional 60 min. The reaction mixture was then applied directly on the Sephadex G-10 column, equilibrated with 0.05 M pyridine/acetic acid buffer of pH 5.6, and the first eluted fraction was lyophilised. The conjugation was carried out as described previously [20]. The oxidised core oligosaccharide OSI (3.8 mg) dissolved in H 2 O (200 μL) was mixed with an equal volume of BSA solution in H 2 O (2 mg/200 μL). Dimethylformamide was added to a final concentration of 2%, and the mixture was freeze-dried. Dry preparation was heated at 105 °C for 30 min, dissolved in H 2 O (1 mL), and dialyzed against H 2 O. The antigenic properties of the product were analysed by immunoblotting, using polyclonal rabbit serum against P. shigelloides PCM 2231.

Immunization Procedures
Rabbits were housed at the animal facility of the Institute of Immunology and Experimental Therapy (Wroclaw, Poland), and all the experiments were carried out according to the procedures approved by the Local Ethical Commission. Rabbits were immunized with the OSI-BSA conjugate, suspended in a complete Freund's adjuvant, and polyclonal antibodies against the conjugate were obtained by the procedures previously described [31].

ELISA
Enzyme-Linked immunosorbent assay (ELISA), using LPS as solid-phase antigen, was performed with a modification [30] of the method described by Voller et al. [32]. The detection system consisted of a goat anti-rabbit IgG conjugated with alkaline phosphatase (Bio-Rad, Hercules, CA, USA) as a second antibody and p-nitrophenyl phosphate as a substrate.

SDS-PAGE
The LPS was analysed by SDS-PAGE according to the method of Laemmli [33] with modifications described previously [24] and LPS bands were visualised by the silver staining method [21].

Immunoblotting
Immunoblotting was performed on the SDS-PAGE-separated LPS fractions as previously described [34]. A goat anti-rabbit IgG conjugated with alkaline phosphatase (Bio-Rad) was used as the secondary antibody, and 5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium was applied as a detection system.

Conclusions
Here we presented the structure of a complete core oligosaccharide composed of an undecasaccharide, which represents the second core type for serotype O17 of P. shigelloides. The core OS of P. shigelloides PCM 2231 is heterogeneous. The heterogeneity corresponded to the absence of terminal β-D-Glcp residue. Three minor glycoforms represent the complete core OS devoid of β-D-GlcpNAc residue, both terminal β-D-Glcp and hexosamine residues, and both terminal β-D-Glcp and β-D-GlcpNAc residues.
Serological screening of different strains of P. shigelloides indicates that identical or similar epitopes to P. shigelloides PCM 2231 might also be present in the core region of 28 out of 55 strains (51%). This observation suggests that the core structure in LPS of strain PCM 2231 (serotype O17) could be the most common type in P. shigelloides.