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:
(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.
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