Skov Sørensen2 1 Clinical Research Centre, Analytical Unit, Karolinska Institute, Huddinge Hospital, Sweden;2Department of Medical Microbiology and Immunology, University of Aarhus, Denm
Trang 1A unique variant of streptococcal group O-antigen (C-polysaccharide) that lacks phosphocholine
Niklas Bergstro¨m1, Per-Erik Jansson1, Mogens Kilian2and Uffe B Skov Sørensen2
1
Clinical Research Centre, Analytical Unit, Karolinska Institute, Huddinge Hospital, Sweden;2Department of Medical Microbiology and Immunology, University of Aarhus, Denmark
Streptococcus mitisstrain 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 pneumoniaeand 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 formof C-polysaccharide, except that it is substicholine-substi-tuted 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; StreptoStrepto-coccus mitis
Previous serological analysis of the mitis group streptococci
suggested that C-polysaccharide is a common antigen of
Streptococcus pneumoniaeand 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
and for a m ajority of S 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 fromour 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 fromSK598 reacts with streptococcal group O-antiserumpurchased from Statens SerumInstitut, Copenhagen, Denmark
Preparation of polysaccharide The S mitis biovar 1 strain SK598 was cultured overnight
at 37C 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)
Trang 2(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 [0.1MNaCl, 1 mMMgCl2, 0.05M
Hepes pH 7.0, mutanolysin 100 UÆmL)1 and lysozyme
1 m gÆmL)1 (M-9901 and L-6876, respectively, Sigma,
St Louis, MI, USA)] Sodiumazide (1 mgÆmL)1) was added
to the suspension as a preservative, and the bacterial cells
were digested at 37C for 18 h Cell debris was removed
fromthe digest by centrifugation and the supernatant was
heated to 50C 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
1H and13C NMR spectra were recorded with a JEOL JNM
ECP500 spectrometer, using standard pulse sequences
Spectra of samples in 20 mMphosphate buffers of pD 7.4
were recorded at 35C Chemical shifts are reported
in p.p.m., using sodium 3-trimethylsilylpropanoate-d4 (dH
0.00) or acetone (dC31.00) or aqueous 2% phosphoric acid
(dP0.00) as internal references For13C and31P the reference
measurement was made with a separate tube before the
actual measurement Chemical shifts were taken from 1D
spectra when possible, or else from1H,1H-correlated 2D
NMR spectra, i.e.1H,1H-COSY and1H,1H-TOCSY (40 ms
spin lock time) The mixing time in the NOESY experiment
was 300 ms The JH1,H2values were obtained fromthe 1D
spectra, other couplings fromthe 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 2M trifluoroacetic
acid at 120C for 2 h or 4M HCl at 120C for 1 h,
followed by reduction with NaBH4 or NaBD4, 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 C18-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 160C (1 m in) fi
250C at 3 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 programas 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 18C, blown
to dryness with dry air and residual traces of acid were neutralized with 1Mammonia, and the resulting material fractionated on a column of Bio-Gel P-4 eluted with 0.1M pyridiniumacetate 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 Sam ples were dissolved in aqueous 50% acetonitrile at a concentra-tion about 1 mgÆmL)1, and 10 lL 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 froma 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 4M 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 1H-NMR spectrumof 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 (J1,2 3.5 Hz, 1H), 4.94 (J1,23.5 Hz, 1H), 4.64 (J1,27.3 Hz, 1H), and 4.62 (J1,2 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
Trang 3H-5 of a 2-acetamido-4-amino-2,4,6-trideoxy-D-galactose
residue (AAT) (see below)
A signal at d 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 the13C-NMR
spectrumat 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-systemin 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 fromits J1,2-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 spectrumto a signal at d 50.1 The C-5 signal was identified froma correlation fromH-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 fromthe 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- -GalNAc Residue B (H-1
Fig 1.1H NMR spectrum (35 °C, 500 MHz) ofthe cell wall polysaccharide from S mitis SK598 A–D refer to anomeric protons as described in the text.
Table 1.1H- and13C-NMR data for the C-polysaccharide (PSII) of S mitis SK598 obtained at pD 7.4.
Sugar residue
Chemical shifts (p.p.m.)
fi6)-a-GalpNAc(1fi A 5.17 [3,5]a 4.32 3.93 4.11 4.01 4.02 4.02
›
fi3)-a-AATp(1fi B 4.98 [3,5] 4.23 4.39 3.94 4.77 1.24
fi6)-b-Glcp-(1fi C 4.64 [3,7] 3.35 3.51 3.52 3.57 4.10 4.14
fi6)-b-GalpNAc(1fi D 4.62 [3,7] 4.11 3.86 4.18 3.84 4.07 4.07
›
fi1)-Ribitol(5fi E 3.89, 3.99 3.77 3.91 3.77 3.98, 4.07
a
J 1,2 -values are given in brackets.
Trang 4d 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 fromits J1,2-value The C-2 and
C-4 in AAT were linked to nitrogen, due to correlations
in the HMQC spectrumto 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 mitisSK137 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, as the J1,2
-value was 7.3 Hz In the NOESY spectrumH-3 and H-5
signals could be assigned fromcorrelations 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 (J1,2-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 spectrumH-3 and H-5
signals were assigned fromcorrelations 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 fromC-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 the13C-NMR spectrum
In the HMBC spectrumthe 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, fromthese 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 confirmthe structural element CBADE
The31P-NMR spectrumshowed 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 spectrumfromphosphorus
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 2.31P NMR spectrum (35 °C, 200 MHz) ofthe 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 (1H) d (13C) d (1H) d (13C) Residue, atom HMBC
94.2 –
98.9 4.11 A, H-4
104.6 4.38 B, H-3
102.1 3.88, 3.98 E, H-1a, H-1b NOESY
D 4.62 3.88, 3.98 E, H-1a, H-1b
Trang 5residue 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 dP 0.33 and )0.04, were
assigned to two phosphate groups linked to GalNAc and
ethanolamine moieties as the signal at dP0.33 correlates to
dH4.09 (H-1a and H-1b, of F) and to dH4.02 (H-6a and
H-6b, of A) and as the signal at dP – 0.04 correlated to
dH4.13 (H-1a and H-1b, of G) and to dH4.07 (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
the1H-NMR spectrumit was clear that the oligosaccharide
fraction was a mixture and that it was the same mixture as
that obtained frompneumococcal 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
Fromthe1H-NMR spectrumit 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), and 4.59
(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]+, with two
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 sodiumadduct The peaks at m/z 907
and 929 corresponded to the same oligosaccharide plus
one ribitol residue, the latter corresponding to the
sodiumadduct Thus the ESI-MS clearly showed that
the majority of the material constituted of a
tetrasaccha-ride and a smaller amount of a tetrasacchatetrasaccha-ride-ribitol
The data shows that the AAT residue is indeed an
acetamido-amino derivative and supports the postulated
repeat as CBADE
Fromthe combined data obtained fromNMR 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 fromanother S mitis biovar 1 strain that differs fromthe 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 fromthe 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 mitisbiovar 1 were found to lack phosphocholine in the C-polysaccharide structure suggests that this is not a rare phenomenon [1]
Separation of the polysaccharide fromstrain 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
Trang 6further 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
C-polysaccharide for any polysaccharide, irrespective of
the number and nature of the substituted residues, that have
the following main structure or backbone:
6Þ-b-d-Glcp-ð1 ! 3Þ-a-AATp-ð1 !
4Þ-a-d-GalpNAc-ð1 ! 3Þ-b-d-GalpNAc-4Þ-a-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 GalNH2 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 fromthe Karolinska institutets
fonder (to P.E.J.) and by the Danish Medical Research Council grant
# FOR 9702265 (to M.K.).
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