Structural studies of the putative O-specific polysaccharide of Serratia marcescens O9

Abstract

A polymeric fraction containing the putative 0-antigen has been isolated from the lipopolysaccharide of the reference strain (CDC 4534-60) for serogroup 0 9 of Serratia marcescens. The major component of the fraction was a polymer with a disaccharide repeating-unit of L-rhamnose (Rha) and 2-acetamido-2-deoxy-~-galactose (GalNAc) with the following structure: -+3)~-GalpNAc(~1+3)~-Rhap(crl+. Evidence for the presence in the fraction of a similar, minor polymer containing 4-substituted rhamnose residues was provided by the NMR spectra, methylation analysis, and Smith degradation.Although the serotyping of heat-stable (0) antigens is widely used for the characterisation of clinical isolates of Serratia marcescens, the organism has a reputation [l] for producing a complex array of surface carbohydrates, and extensive cross-reactions have been reported by some workers [2, 31. As part of a systematic study of the 0-antigens of S. marcescens [4 -lo], we have isolated and studied the lipopolysaccharide of the 0 9 reference strain (CDC 4534-60). Serological cross-reactions do not seem to be a major problem with 0 9 strains, although the reference strain apparently contains antigenic factors absent from another 0 9 strain, CDC 2870-67 [3, 111. Here we report the structure of the major polymer from the reference strain, and the presence of a minor polymer in the lipopolysaccharide preparation. MATERIALS AND METHODS Growth of bacteria, and extraction and fractionation of lipopolysaccharideSerratia marcescens 0 9 (CDC 4534-60) was grown for 16 hat 30°C in nutrient broth (20 1, Oxoid no. 2) with aeration at 20 1 min-l. The cells (170 g wet weight) were disintegrated mechanically, and the lipopolysaccharide (1.08 g) was isolated from the purified cell walls (4.66 g) as in previous studies [lo].The water-soluble products of mild hydrolysis (1 YO acetic acid, 100°C, 2.25 h) were fractionated on Sephadex G-50. The polymeric fraction obtained was further eluted from a column of DEAE-Sepharose with water or 0.1 M NaC1, and the products were recovered by dialysis and freeze-drying. pyridinelwater (1 31514, by vol.), or B, ethyl acetateipyridinei acetic acidiwater (51511 /3, by vol.). High-voltage paper electrophoresis was carried out at pH 5.3, and the detection reagents used on chromatograms and electrophoretograms were those listed previously [lo]. Gas-liquid chromatography of alditol acetates was carried out using a packed column with OV-275 as the stationary phase. A fused silica capillary column (25 m) of BPlO (S.G.E.) was used for chromatography of methylated alditol acetates, and mass spectra were obtained using a Finnigan model 1020B instrument fitted with this column. NMR spectra (lH and 13C) were recorded for samples in D20 (containing 10% dimethyl sulphoxide in the case of fraction SD2) with a Bruker WH-400 spectrometer. 'H spectra were recorded at about 80°C with 4,4-dimethyl-4-silapentane-1-sulphonate as an external standard. 3C spectra were recorded at about 50°C with tetramethylsilane as an external standard. Polyacry...

Structural and serological studies of lipopolysaccharides from proposed new serotypes (O25 and O26) of Serratia marcescens

“…This result closely parallels that for the 015 antigen [19] and, with components in the inverse ratio, for the 09 antigen [18]. This result closely parallels that for the 015 antigen [19] and, with components in the inverse ratio, for the 09 antigen [18].…”

“…Methylation analysis of the polymeric material (batch B) showed the presence of both 3-substituted Rhap residues (minor) and 4-substituted Rhap residues (major), as well as 3-substituted GalpNAc residues. This result closely parallels that for the 015 antigen [19] and, with components in the inverse ratio, for the 09 antigen [18]. To confirm this resemblance, and to obtain evidence for two separate polymeric fractions in the 026 product, a Smith degradation was carried out.…”

“…Antiserum 026 cross-reacted in ELISA only with 09 and 015, known to possess the same two neutral polysaccharides, albeit in different proportions [18,19]. Like 025, the 026 strain was negative in the Quellung test, but with this strain specific results were seen in ELISA and immunoblots.…”

“…Similar profiles have been obtained for comparable hydrolysates of LPS from 5. marcescens 09 [18] and 015 [191. The polymeric fraction obtained on mild acid hydrolysis of the LPS (yield: A, 22%; B, 17%) gave an elongated elution profile on Sephadex G-50, extending almost to the oligomeric region, indicating a varied but relatively low degree of polymerisation.…”

“…The polymeric fraction obtained on mild acid hydrolysis of the LPS (yield: A, 22%; B, 17%) gave an elongated elution profile on Sephadex G-50, extending almost to the oligomeric region, indicating a varied but relatively low degree of polymerisation. Similar profiles have been obtained for comparable hydrolysates of LPS from 5. marcescens 09 [18] and 015 [191.…”