Tài liệu Báo cáo khoa học: A unique variant of streptococcal group O-antigen (C-polysaccharide) that lacks phosphocholine ppt

A unique variant of streptococcal group O-antigen (C-polysaccharide) that lacks phosphocholine Niklas Bergstro¨m 1 , Per-Erik Jansson 1 , Mogens Kilian 2 and Uffe B. Skov Sørensen 2 1 Clinical Research Centre, Analytical Unit, Karolinska Institute, Huddinge Hospital, Sweden; 2 Department of Medical Microbiology and Immunology, University of Aarhus, Denmark Streptococcus mitis strain SK598, which represents a sub- group of biovar 1, possesses a unique variant of the C-polysaccharide found in the cell wall of all strains of Streptococcus pneumoniae and in some strains of S. mitis. This new variant lacks the choline methyl groups in contrast to the previously characterized forms of C-polysaccharide, which all contain one or two choline residues per repeat. The following structure of the repeating unit of the SK598 polysaccharide was established: where AAT is 2-acetamido-4-amino-2,4,6-trideoxy- D - galactose. This structure is identical to the double choline-substi- tuted form of C-polysaccharide, except that it is substituted with ethanolamine instead of choline. This extends the number of recognized C-polysaccharide variants to four. Keywords: cell wall polysaccharide; C-polysaccharide; Strepto- coccus pneumoniae; phosphocholine; Streptococcus mitis. Previous serological analysis of the mitis group streptococci suggested that C-polysaccharide is a common antigen of Streptococcus pneumoniae and of most Streptococcus mitis biovar 1 strains. Different reaction patterns, however, emerged among the mitis group streptococci when exam- ined by using a combination of two monoclonal antibodies in an enzyme linked immunoassay that recognize phospho- choline moieties and the backbone of C-polysaccharide, respectively. Positive reactions with both monoclonals were interpreted as the presence of the classical C-polysaccharide with one or more phosphocholine residues attached, as confirmed by structural analysis of polysaccharide prepared from S. mitis strain SK137 [1]. Reaction with both of the monoclonals was observed for all strains of S. pneumoniae andforamajorityofS. mitis biovar 1 strains. However, other strains reacted with one of the two monoclonals only, and some S. mitis biovar 2 did not react with any of them. The structure of the polysaccharide prepared from S. mitis strain SK598, which represents strains that reacted with the monoclonal antibody directed to the backbone of C-polysaccharide but not with monoclonal antibody to phosphocholine, is examined in the present study. It is concluded that this S. mitis biovar 1 strain possesses a unique variant of double choline-substituted C-polysaccha- ride that lacks only the methyl groups in choline, i.e. is substituted with ethanolamine residues. This new structural variant extends the number of recognized C-polysaccharide forms to four. Materials and methods Bacterial strain The S. mitis biovar 1 strain SK598 used for preparation of polysaccharide was from our own strain collection. This strain was selected as it was negative for the presence of phosphocholine, although it seemed to possess a C-poly- saccharide like molecule when examined by ELISA and by immunoelectrophoresis [1]. Strain SK598 was characterized and identified as previously described [1,2]. It belongs to Lancefield serogroup O as an extract from SK598 reacts with streptococcal group O-antiserum purchased from Statens Serum Institut, Copenhagen, Denmark. Preparation of polysaccharide The S. mitis biovar 1 strain SK598 was cultured overnight at 37 °C in 5 L laboratory flasks each containing 2.5 L Todd-Hewitt broth (CM189, Oxoid, Basingstoke, UK). The bacterial cells were harvested by centrifugation Correspondence to P E. Jansson, Karolinska Institute, Clinical Research Centre, Novum, Huddinge University Hospital, S-141 86 Huddinge, Sweden. Fax: + 46 8585 83820, Tel.: + 46 8585 83821, E-mail: pererik.jansson@kfc.hs.sll.se (Received 5 September 2002, revised 6 March 2003, accepted 13 March 2003) Eur. J. Biochem. 270, 2157–2162 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03569.x (10 000 g, 30 min) and pooled from a total of 30 L broth culture. The cells were washed twice in saline and suspended in 50 mL of lysis buffer [0.1 M NaCl, 1 m M MgCl 2 ,0.05 M Hepes pH 7.0, mutanolysin 100 UÆmL )1 and lysozyme 1mgÆmL )1 (M-9901 and L-6876, respectively, Sigma, St Louis, MI, USA)]. Sodium azide (1 mgÆmL )1 ) was added to the suspension as a preservative, and the bacterial cells were digested at 37 °C for 18 h. Cell debris was removed from the digest by centrifugation and the supernatant was heated to 50 °C for 30 min to kill viable cells. Crude polysaccharide was prepared by removal of most protein and lipids from the lysate by chloroform/butanol treatment followed by precipitation with ethanol [3]. The precipitate was re-dissolved in MilliQ water, clarified by centrifugation and lyophilized. The crude polysaccharide was treated with DNAse, RNAse and proteinase K according to the manu- facturer’s instructions and was then fractionated by size exclusion chromatography on a Sephacryl S-300 column. NMR spectroscopy 1 Hand 13 C NMR spectra were recorded with a JEOL JNM ECP500 spectrometer, using standard pulse sequences. Spectra of samples in 20 m M phosphate buffers of pD 7.4 were recorded at 35 °C. Chemical shifts are reported in p.p.m., using sodium 3-trimethylsilylpropanoate-d 4 (d H 0.00) or acetone (d C 31.00) or aqueous 2% phosphoric acid (d P 0.00) as internal references. For 13 Cand 31 P the reference measurement was made with a separate tube before the actual measurement. Chemical shifts were taken from 1D spectra when possible, or else from 1 H, 1 H-correlated 2D NMR spectra, i.e. 1 H, 1 H-COSY and 1 H, 1 H-TOCSY (40 ms spin lock time). The mixing time in the NOESY experiment was 300 ms. The J H1,H2 values were obtained from the 1D spectra, other couplings from the COSY spectrum. The proton-carbon correlated spectrum (HMQC), and the long-range proton-carbon correlated spectrum (HMBC) were obtained with decoupling [4] using delay times of 42 and 97 ms using JEOL standard pulse sequences. The delay time in the HMQC-TOCSY experiment was 20 ms. The decoupled proton-phosphorus correlated spectra com- prised a delay time of 71 ms, corresponding to 7 Hz couplings. Sugar and methylation analyses For sugar analysis, alditol acetates were prepared by hydrolysis of the polysaccharide using 2 M trifluoroacetic acid at 120 °C for 2 h or 4 M HCl at 120 °Cfor1h, followed by reduction with NaBH 4 or NaBD 4 ,and acetylation. For methylation analysis, methylation was performed with methyl iodide in the presence of sodium methyl sulfinyl methanide, and the methylated products were purified using Sep-Pak C 18 -cartridges. For GLC, a Hewlett-Packard 5890 instrument fitted with a flame- ionization detector was used. Separation of alditol acetates was performed on a DB-5 capillary column (30 m · 0.25 mm) using a temperature program 160 °C(1min)fi 250 °Cat3°CÆmin )1 . GLC-MS (EI) was performed on a Hewlett-Packard 5890/Nermag R10–10H quadrupole instrument. Partially methylated alditol acetates were separated on a DB-5 capillary column (25 m · 0.20 mm), using the same temperature program as described for alditol acetates. The absolute configurations of the sugar residues were determined by GLC-MS of the trimethylsilylated (+)-2-butyl glycosides [5], using the same temperature program as described for alditol acetates. HF degradation A solution of the crude cell wall polysaccharide (69 mg) in aqueous 48% HF (1 mL) was kept for 48 h at 18 °C, blown to dryness with dry air and residual traces of acid were neutralized with 1 M ammonia, and the resulting material fractionated on a column of Bio-Gel P-4 eluted with 0.1 M pyridinium acetate buffer at pH 5.3. Polymeric material (minor) was recovered at the void volume and oligomeric material at 1.4 void volumes (major). Mass spectrometry ESI-MS was performed in the negative mode using an LCQ iontrap (Thermo Finnigan) with aqueous 50% acetonitrile as the mobile phase at a flow rate of 10 lLÆmin )1 .Samples were dissolved in aqueous 50% acetonitrile at a concentra- tion about 1 mgÆmL )1 ,and10lL was injected via a syringe pump into the electrospray source. Results Size exclusion chromatography of the crude polysaccharide from S. mitis SK598, pretreated to remove proteins, lipids and nucleic acids, gave two partially overlapping peaks that appeared at 1.3 (PSI) and 1.7 (PSII) void volumes in the eluate from a Sephacryl S-300 column. The unseparated material showed on hydrolysis with trifluoroacetic acid ribitol, glucose, galactose, glucosamine and galactosamine in the proportions, 1 : 1.8 : 1.4 : 1 : 0.2. PSI was a minor fraction only (< 10%) and it was not investigated in detail as it was a complex mixture of probably peptides and polysaccharides. On trifluoroacetic acid hydrolysis it gave ribitol, glucose, galactose in the ratio 1 : 3.5 : 3.5 and some minor amounts of other monosaccharides. The latter major fraction, PSII was hydrolyzed with 4 M hydrochloric acid and showed glucose and galactosamine in the proportions 1 : 4.5. This hydrolysis enhances amino sugars but ribitol is not detected. The absolute configuration of the sugars was D , as demonstrated by GLC of the trimethylsilylated (+)-2-butyl glycosides. In order to main- tain a constant pD to get reproducible spectra in the NMR studies, the solution of PSII was buffered at pD 7.4 (pH 7.0). The 1 H-NMR spectrum of PSII showed five major peaks in the anomeric region corresponding to approximately one proton each, and some smaller signals (Fig. 1). The five large signals in the anomeric region appeared at d 5.17, 4.94, 4.77, 4.64 and 4.62 (Table 1). This could be recognized as closely similar but not identical to signals in the anomeric region from the C-polysaccharide purified from S. pneumoniae [1,3,6–8]. Four of the signals could be shown to be anomeric and appeared at d 5.17 (J 1,2 3.5 Hz, 1H), 4.94 (J 1,2 3.5 Hz, 1H), 4.64 (J 1,2 7.3 Hz, 1H), and 4.62 (J 1,2 7.3 Hz, 1H) and the corresponding sugar residues were designated A–D, respectively. A signal at d 4.77, which was an obscured quartet, could be assigned to 2158 N. Bergstro ¨ m et al. (Eur. J. Biochem. 270) Ó FEBS 2003 H-5 of a 2-acetamido-4-amino-2,4,6-trideoxy- D -galactose residue (AAT) (see below). Asignalatd 3.29–3.30 (4 H) was assigned to two N-linked methylene groups in two phosphoethanolamine residues (see below). Four signals for anomeric carbons, virtually coinciding with those reported previously for the C-polysaccharide [1,3,6–8], were observed in the 13 C-NMR spectrum at d 104.6, 102.1, 98.9, and 94.2. For residues A and D it was possible to follow the spin- systems from H-1 up to H-4 in the COSY spectrum. For residues B and C it was possible to follow the whole spin- system in the COSY spectrum, these assignments were then verified in the TOCSY spectrum. Residue A (H-1 d 5.17) could be assigned to a 4,6-disubstituted GalNAc residue with the a configuration, as evident from its J 1,2 -value of 3.5 Hz. The galacto configuration was apparent as the H-3– H-4 coupling was small. That C-2 was linked to nitrogen was indicated by a correlation in the HMQC spectrum to a signal at d 50.1. The C-5 signal was identified from a correlation from H-1 in the HMBC spectrum. H-5 and H-6 were both identified by a correlation to C-4 in the HMBC spectrum; correlations between H-5/C-6 and H-6/C-5 verified the assignments. Substituted positions in the residue were indicated from the large glycosylation shifts, 7.8 and 1.9 p.p.m., for the C-4 and C-6 signals, respectively, when compared to unsubstituted a- D -GalNAc. Residue B (H-1 Fig. 1. 1 H NMR spectrum (35 °C, 500 MHz) of the cell wall polysaccharide from S. mitis SK598. A–D refer to anomeric protons as described in the text. Table 1. 1 H- and 13 C-NMR data for the C-polysaccharide (PSII) of S. mitis. SK598 obtained at pD 7.4. Sugar residue Chemical shifts (p.p.m.) 1 2345 6a6b fi6)-a-GalpNAc(1fi A 5.17 [3,5] a 4.32 3.93 4.11 4.01 4.02 4.02 4 94.2 50.1 67.5 77.4 71.3 64.0 › fi3)-a-AATp(1fi B 4.98 [3,5] 4.23 4.39 3.94 4.77 1.24 98.9 49.0 75.6 55.3 63.7 16.0 fi6)-b-Glcp-(1fi C 4.64 [3,7] 3.35 3.51 3.52 3.57 4.10 4.14 104.6 73.5 76.0 69.4 75.1 65.0 fi6)-b-GalpNAc(1fi D 4.62 [3,7] 4.11 3.86 4.18 3.84 4.07 4.07 3 102.1 51.1 75.0 63.9 74.0 65.0 › fi1)-Ribitol(5fi E 3.89, 3.99 3.77 3.91 3.77 3.98, 4.07 71.3 72.2 71.4 72.2 67.0 Ethanolamine F 4.09 3.29 62.5 40.7 Ethanolamine G 4.13 3.30 62.5 40.7 a J 1,2 -values are given in brackets. Ó FEBS 2003 Cell wall polysaccharides of S. mitis biovar 1 (Eur. J. Biochem. 270) 2159 d 4.98) was assigned to a 3-substituted 2-acetamido-4-amino- 2,4,6-trideoxy-galacto-pyranose (AAT) residue also with the a configuration, as indicated from its J 1,2- value. The C-2 and C-4 in AAT were linked to nitrogen, due to correlations in the HMQC spectrum to signals at d 49.0 and 55.3, respectively. The substitution of B was indicated by the high numerical value of the chemical shift of the C-3 signal, d 75.6. The AAT residue had the D configuration and a free 4-amino group as was strongly indicated by the similarities between the chemical shifts of this AAT and that in the S. mitis SK137 C-polysaccharide [1]. Residue C (H-1 d 4.64) was assigned to a 6-substituted b-Glc residue as all ring proton couplings in the ring system were large, thereby demonstrating an all-axial proton relation and the anomeric configuration was b,astheJ 1,2 - value was 7.3 Hz. In the NOESY spectrum H-3 and H-5 signals could be assigned from correlations to H-1. Further assignments were obtained from the HMQC-TOCSY spec- trum, where correlations H-2/C-3 and C-4/H-5 were evident. The residue was determined to be 6-substituted because of a large glycosylation shift, 3.2 p.p.m., for the C-6 signal. Residue D (H-1 d 4.62) was assigned to a 3,6-disubsti- tuted GalNAc residue with the b configuration (J 1,2 -value of 7.3 Hz) and the galacto-configuration being evident with a small coupling between H-3 and H-4. The C-2 was linked to nitrogen indicated by a correlation in the HMQC spectrum to signal at d 51.1. In the NOESY spectrum H-3 and H-5 signals were assigned from correlations to H-1. C-6 was determined from a correlation to H-5 and H-6 was confirmed by a correlation to C-5, both in the HMQC- TOCSY spectrum. The 3,6-disubstitution was indicated by the chemical shifts of the C-3 and C-6 which were shifted 3.0 and 3.1 p.p.m., respectively. Residue E was determined to be a 1,5-disubstituted ribitol residue as all proton and carbon signals could be assigned with the aid of the COSY, NOESY and HMQC spectra by which a pentitol residue was evident. A good correspon- dence with previous data from C-polysaccharide was also observed. Substantial downfield shifts of signals for C-1 and C-5 indicated substitution at those positions (see below). Residues F and G were assigned to two-carbon units as only one correlation was observed for each unit in of the COSY and HMBC spectra. The proton and carbon chemical shifts are in accord with methylene groups next to oxygen and to nitrogen, thus fitting with ethanolamine. The signal for carbon next to the amino group is observed at d 40.7 compared to d  67 in choline. The strong methyl- signal found in choline at d 55 is absent as well, thereby showing that residues F and G are ethanolamine substitu- ents. Strictly, mono- and dimethylated ethanolamine deri- vatives are not excluded but no signals corresponding to such moieties were observed in the 13 C-NMR spectrum. In the HMBC spectrum the following interresidue correlations were observed (Table 2): d 5.17/75.0 (A-1/ D-3), d 4.98/77.0 and d 98.9/4.11 (B-1/A-4), d 4.64/75.4 and d104.6/4.38 (C-1/B-3), d 4.62/71.3 and d 102.1/3.88,3.98 (D-1/E-1). Thus, from these data the following structural element could be established: CBADE. A NOESY experiment revealed inter alia H-1/H-3 and H-1/H-5 intraresidue correlations in residues C and D further demonstrating their anomeric configurations as b. The following five interresidue correlations between H-1 and linkage protons were observed: d 5.17/3.86 (A H-1/D H-3), d 4.98/4.11 (B H-1/A H-4), d 4.64/4.38 (C H-1/B H-3), d 4.62/3.88 (D H-1/E H-1a), and d 4.62/3.98 (D H-1/E H-1b). The NOESY data could thereby confirm the structural element CBADE. The 31 P-NMR spectrum showed three signals of equal intensity at d 1.33, 0.33, and )0.04 (Fig. 2). All three signals could be assigned to a polysaccharide similar to C-polysaccharide. The signal at d 1.33 was assigned to a phosphate group bridging the ribitol and Glc residues. The value is close to that observed for C-polysaccharide. Thus, correlations in the H,P-HMQC spectrum from phosphorus to protons with signals at d 4.15, 4.09 (H-6a and H-6b of residue C), 4.06, and 3.98 (H-5a and H-5b in the ribitol, Fig. 2. 31 PNMRspectrum(35°C, 200 MHz) of the cell wall poly- saccharide from S. mitis SK598. Table 2. Inter-residue connectivities observed in HMBC and NOESY spectra for C-polysaccharide of S. mitis SK598. Residue Chemical shifts (H/C) Anomeric nucleus Inter-residue correlations d ( 1 H) d ( 13 C) d ( 1 H) d ( 13 C) Residue, atom HMBC A 5.17 75.0 D, C-3 94.2 – B 4.98 77.4 A, C-4 98.9 4.11 A, H-4 C 4.64 75.6 B, C-3 104.6 4.38 B, H-3 D 4.62 71.3 E, C-1 102.1 3.88, 3.98 E, H-1a, H-1b NOESY A 5.17 3.86 D, H-3 B 4.98 4.11 A, H-4 C 4.64 4.38 B, H-3 D 4.62 3.88, 3.98 E, H-1a, H-1b 2160 N. Bergstro ¨ m et al. (Eur. J. Biochem. 270) Ó FEBS 2003 residue E) were observed. The structural element CBADE can thus be shown to be the repeating unit in a teichoic acid. The remaining two signals, at d P 0.33 and )0.04, were assigned to two phosphate groups linked to GalNAc and ethanolamine moieties as the signal at d P 0.33 correlates to d H 4.09 (H-1a and H-1b, of F)andtod H 4.02 (H-6a and H-6b, of A)andasthesignalatd P – 0.04 correlated to d H 4.13 (H-1a and H-1b, of G)andtod H 4.07 (H-6a and H-6b, of D). The two phosphoethanolamine groups are therefore linked to the 6-positions of residues A and D. Two peaks were obtained in the chromatogram when the crude material was treated with aqueous 48% HF for 48 h at )18 °C and fractionated on a column of Bio-Gel P-4. The first peak contained PSI and was a polymeric fraction eluted at 1.2 void volumes. The second peak was an oligosaccha- ride fraction eluted at 1.4 void volumes (PSII-OLS). From the 1 H-NMR spectrum it was clear that the oligosaccharide fraction was a mixture and that it was the same mixture as that obtained from pneumococcal C-polysaccharide when treated with aqueous 48% HF under same conditions. The phosphoethanolamine and phosphate groups were absent as a result of that all phosphate ester linkages were broken. From the 1 H-NMR spectrum it was clear that the fraction contained a major and a minor compound, where the major compound showed anomeric signals at d 5.23 (D, 0.45H), 5.15 (A, 0.45H), 5.13 (A, 0.55H), 4.93 (B,1H),and4.59 (C and D, 1.35H), thus exposing a reducing end, indicating that the ribitol residue was hydrolyzed off. This gives twinning of the signal at d  5.14, as observed also in previous degradations of pneumococcal C-polysaccharide. A larger yield of ribitol-containing oligosaccharide may be obtained if the temperature is kept lower when evaporating the HF to dryness. Analysis of PSII-OLS by ESI-MS in positive mode, showed singly charged species [M + H] + ,withtwo major peaks at m/z 773 and 795 and two minor peaks at m/z 907 and 929. The peak at m/z 773 corresponds to an oligosaccharide comprised of one hexose, two acetamido- hexoses, and one AAT residue, the peak at m/z 795 corresponds to its sodium adduct. The peaks at m/z 907 and 929 corresponded to the same oligosaccharide plus one ribitol residue, the latter corresponding to the sodium adduct. Thus the ESI-MS clearly showed that the majority of the material constituted of a tetrasaccha- ride and a smaller amount of a tetrasaccharide-ribitol. The data shows that the AAT residue is indeed an acetamido-amino derivative and supports the postulated repeat as CBADE. From the combined data obtained from NMR and mass spectrometry the following structure was concluded for the cell wall polysaccharide from S. mitis strain SK598: Discussion We previously interpreted reactivity of a streptococcal cell wall polysaccharide preparation with both of two monoclonal antibodies that detect phosphocholine and the backbone of pneumococcal C-polysaccharide, respect- ively, as an indication of the presence of an antigen identical or closely similar to C-polysaccharide. This interpretation was validated for S. mitis strain SK137 [1]. Structural analysis demonstrated that this S. mitis biovar 1 strain possesses a true C-polysaccharide in addition to a unique glycan. The C-polysaccharide found in all S. pneumoniae strains and in most S. mitis biovar 1 strains was shown to represent the streptococcal sero- group O antigen [1]. We have now investigated the structure of a polysaccha- ride prepared from another S. mitis biovar 1 strain that differs from the previously examined strain by failing to react with the monoclonal antibody against phospho- choline. As expected, the predominant polysaccharide demonstrated in strain SK598 was found to be a cell wall polysaccharide similar but not identical to pneumococcal C-polysaccharide. The structures are identical except that the characteristic phosphocholine residues of pneumo- coccal C-polysaccharide are absent from the new S. mitis C-polysaccharide, which is substituted with ethanolamine (structure 1). Choline is a strict nutritional requirement for pneumo- cocci although mutant strains that have acquired the ability to grow in the absence of choline have been described [9,10]. When grown in a chemically defined medium containing ethanolamine but no choline, such strains generate phos- phocholine-free teichoic acid like the one we describe for strain SK598. However, the normal physiology of pneumo- cocci is clearly affected under these growth conditions because ethanolamine cannot functionally replace choline [10]. Interestingly, the S. mitis biovar 1 strain SK598 generates phosphocholine-free C-polysaccharide under nor- mal conditions even when grown in a choline rich medium as Todd-Hewitt broth, and cells of this strain display a normal morphology when examined in Gram-stained smears. The fact that four out of 43 natural isolates of S. mitis biovar 1 were found to lack phosphocholine in the C-polysaccharide structure suggests that this is not a rare phenomenon [1]. Separation of the polysaccharide from strain SK598 by size exclusion chromatography initially revealed an addi- tional but minor high molecular weight fraction (PSI). This fraction was believed to contain a teichoic acid, as ribitol, glucose, galactose and some other monosaccha- rides were detected after hydrolysis. This conclusion is Ó FEBS 2003 Cell wall polysaccharides of S. mitis biovar 1 (Eur. J. Biochem. 270) 2161 further supported by the finding that this fraction was decomposed upon treatment with 48% HF, which indicates the presence of phosphate diester linkage. It is interesting that S. mitis strain SK598, like S. mitis strain SK137 [1], possibly possesses two different kinds of polysaccharides. Unfortunately, the limited amount of material precluded structural analysis of this additional polysaccharide. In the present paper we have used the designation ÔC-polysaccharideÕ for any polysaccharide, irrespective of the number and nature of the substituted residues, that have the following main structure or ÔbackboneÕ: 6Þ-b-d-Glcp-ð1!3Þ-a-AATp-ð1!4Þ-a-d-GalpNAc- ð1!3Þ-b-d-GalpNAc-ð1!1Þ-ribitol-5-P-ðOÞ in which one or both Gal are amino sugars that may or may not be N-acetylated. The finding of a new C-polysaccharide structure extends the number recognized of C-polysaccharide variants. The first found contained only one phosphocholine group and one GalNH 2 residue, which is normally N-acetylated [6]. Subsequently, a polysaccharide with only N-acetylated GalN residues and with two phosphocholine residues was reported [7]. More recently a polysaccharide with the same backbone but with one phosphocholine group was identified [3]. The polysaccharide with two phosphoetha- nolamine groups described in this communication extends the list to four. We suggest that streptococcal strains, including pneumococci, which possess one of these C-polysaccharide variants are referred to as Lancefield serogroup O [1]. Acknowledgements This work was supported by grants from the Karolinska institutets fonder (to P.E.J.) and by the Danish Medical Research Council grant # FOR 9702265 (to M.K.). References 1. Bergstro ¨ m, N., Jansson, P.E., Kilian, M. & Skov Sørensen, U.B. (2000) Structures of two cell wall-associated polysaccharides of a Streptococcus mitis biovar 1 strain. A unique teichoic acid-like polysaccharide and the group O antigen which is a C-poly- saccharide in common with pneumococci. Eur J. Biochem. 267, 7147–7157. 2. Kilian, M., Mikkelsen, L. & Henrichsen, J. (1989) Taxonomic study of viridans streptococci: Description of Streptococcus gor- donii sp. nov. & emended descriptions of Streptococcus sanguis (White and Niven 1946), Streptococcus oralis (Bridge and Sneath 1982), and Streptococcus mitis (Andrewes and Horder 1906). Int. J. Syst. Bacteriol. 39, 471–484. 3. Karlsson, C., Jansson, P.E. & Sørensen, U.B. (1999) The pneu- mococcal common antigen C-polysaccharide occurs in different forms. Mono-substituted or di-substituted with phosphocholine. Eur. J. Biochem. 265, 1–8. 4. Bax, A. & Summers, M.F. (1986) Proton and carbon-13 assign- ments from sensitivity-enhanced detection of heteronuclear mul- tiple-bond connectivity by 2D multiple quantum NMR. J. Am. Chem. Soc. 108, 2093–2094. 5. Gerwig, G.J., Kamerling, J.P. & Vliegenthart, J.F.G. (1978) Determination of the D and L configuration of neutral mono- saccharides by high resolution capillary GLC. Carbohydrate Res. 62, 349–357. 6. Jennings, H.J., Lugowski, C. & Young, N.M. (1980) Structure of the complex polysaccharide C-substance from Streptococcus pneumoniae type 1. Biochemistry 19, 4712–4719. 7. Kulakowska, M., Brisson, J.R., Griffith, D.W., Young, N.M. & Jennings, H.J. (1993) High–resolution NMR spectroscopic ana- lysis of the C-polysaccharide of Streptococcus pneumoniae. Can. J. Chem. 71, 644–648. 8. Fischer,W.,Behr,T.,Hartmann,R.,Peter-Katalinic,J.&Egge,H. (1993) Teichoic acid and lipoteichoic acid of Streptococcus pneu- moniae possess identical chain structures. A reinvestigation of teichoic acid (C-polysaccharide). Eur. J. Biochem. 215, 851–857. 9. Yother,J.,Leopold,K.,White,J.&Fisher,W.(1998)Generation and properties of a Streptococcus pneumoniae mutant which does not require choline or analogs for growth. J. Bacteriol. 180, 2093–2101. 10. Fisher, W. (2000) Phosphocholine of pneumococcal teichoic acids: role in bacterial physiology and pneumococcal infection. Res. Microbiol. 151, 421–427. 2162 N. Bergstro ¨ m et al. (Eur. J. Biochem. 270) Ó FEBS 2003 . of a tetrasaccha- ride and a smaller amount of a tetrasaccharide-ribitol. The data shows that the AAT residue is indeed an acetamido-amino derivative and. A unique variant of streptococcal group O-antigen (C-polysaccharide) that lacks phosphocholine Niklas Bergstro¨m 1 , Per-Erik Jansson 1 , Mogens Kilian 2 and