Eur. J. Biochem. 232, 391-396 (1995)
0 FEBS 1995
Structure of the capsular polysaccharide
of Vibrio cholerae 0139 synonym Bengal
containing D-galactose 4,6-cyclophosphate
Yuriy A. KNIREL' ', Liliana PAREDES', Per-Erik JANSSON', Andrej WEINTRAUB', Goran WIDMALM4 and M. John ALBERTT
'
Karolinska Institute, Clinical Research Centre, Huddinge University Hospital, Sweden
' N. D. Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, Moscow, Russia
' Karolinska Institute, Department of Immunology, Microbiology, Pathology
and Infectious Diseases, Division of Clinical Bacteriology,
Huddinge University Hospital, Sweden
Department of Organic Chemistry, Arrhenius Laboratory, Stockholm University, Sweden
International Centre for Diarrhoea1 Diseases Research, Dhaka, Bangladesh
(Received 5 May/22 June 1995) - EJB 95 0719/5
The capsular polysaccharide (CPS) of Vibrio cholerae 0139 synonym Bengal, which is thought to
carry determinants of 0-specificity, was isolated by phenol/water extraction followed by delipidation of
the contaminating lipopolysaccharide at pH 4.2 and gel-permeation chromatography. The CPS contained
(colitose, Col), 2-acetamido-2-deoxy-D-glucose,2-acetamido-2,6D-galactose, 3,6-dideoxy-~-xylo-hexose
dideoxy-D-glucose (N-acetyl-D-quinovosamine,D-QuiNAc), D-galacturonic acid (D-GalA), and phosphate. The CPS was studied by NMR spectroscopy, methylation analysis, and selective degradations,
including partial acid hydrolysis at pH 3.1 and dephosphorylation with aqueous 48 % hydrofluoric acid,
which both resulted in complete cleavage of Col. It was concluded that the CPS is built up of hexasaccharide repeating units containing inter alia D-galaCtOSe 4,6-cyclophosphate and having the following structure
HO
0
\/
P
/\
4 6
a-Colp-( 1-2)-,!?-D-Galp
1
1
-
3
-6)-,!?-~-GlcpNAc-( 1-4)-a-~-GalpA-( 1-3)-P-D-QuipNAc-( 1
4
T
1
a-Colp
These data basically confirm the structure of the K cholerae CPS proposed on the basis of an NMR
study [L. M. Preston et al. (1995) J. Bacteriol. 177, 835-8381 and specify exactly the absolute configurations of the constituent monosaccharides and the position of the cyclic phosphate.
Keywords: Kbrio cholerae ; capsular polysaccharide ; structure ; cyclic phosphate; NMR spectroscopy.
Cholera is a secretory diarrhoea1 disease caused by Wbrio
cholerae. Until recently, the disease was known to be caused
only by % cholerae serogroup 01. There have been seven pandemics of cholera in recorded history, the last three being due
Correspondence to P.-E. Jansson, Clinical Research Centre, Novum,
Huddinge University Hospital, S-141 86 Huddinge, Sweden
Fax: +46 8 746 5230.
Abbreviations. Col, 3,6-dideoxy-~-xylo-hexose
(colitose) ; GalA, galacturonic acid; QuiNAc, 2-acetamido-2,6-dideoxyglucose(N-acetylquinovosamine) ; COSY, correlation spectroscopy ; CPS, capsular polysaccharide; FAB, fast atom bombardment; HMQC, heteronuclear multiquantum coherence ; ROESY, rotating-frame NOE spectroscopy ;
TOCSY, total correlation spectroscopy.
to % cholerae 0 1 . The seventh pandemic started in 1961 and 30
years later reached the South American continent [l]. K cholerae non-01 were not considered as epidemic agents of cholera
until late 1992. By this time, new epidemics of diarrhoea started
in India and South Bangladesh [2, 31. The causative agent was
a strain of K cholera non-01, which did not belong to any of
by that time known 138 serogroups of b! cholerae and, hence,
was named K cholerae 0139 with the synonym Bengal [4]. It
shares several properties with and in many aspects is indistinguishable from K cholerae 01 El Tor 15, 61.
The main difference between the 01 and 0139 serogroups
is the architecture of the cell envelope. Unlike K cholerae 01,
K cholerue 0139 has a lipopolysaccharide with a short O-anti-
392
Knirel et al. ( E m 1.Bioclzem. 232)
gen chain 17-91 and produces a polysaccharide capsule 15, 81.
Serological and genetic studies suggested that the capsular polysaccharide (CPS) has the same repeating unit as the 0-antigen
[10-12]. Sugar analysis revealed that the CPS contains galactose, 3,6-dideoxy-xylo-hexose, 2-amino-2-deoxyglucose, 2amino-2,6-dideoxyglucose (quinovosamine, QuiN), and galacturonic acid (GalA) [8, 131. The 3,6-dideoxyhexose has been
identified as colitose (Col) 171. The CPS has been found to include also a cyclic phosphate, and a preliminary structure has
been proposed on the basis of an NMR study [13], but absolute
configurations of the monosaccharides and the exact position of
the phosphate group on a galactose residue remained unknown.
We now report the complete structure of the CPS of R cholerue 0139 Bengal based on chemical, NMR spectroscopic, and
fast atom bombardment (FAB) mass spectrometric data.
EXPERIMENTAL PROCEDURES
Bacterium, cultivation, and isolation of CPS. R cholerue
0139 Bengal, strain Al-1838 derived from the Microbiology
Laboratory of the International Centre for Diarrhoea1 Disease
Research (Dhaka, Bangladesh), was grown in a 30-1 fermentor
(Belach AB, Sweden) in a rich tryptone/yeast extract as described earlier [8, 141. Pelleted bacterial cells were suspended in
water and extracted with hot aqueous phenol [15]. The crude
extract was dissolved in 0.1 M sodium acetate pH 4.2, heated
for 4 h at IOO"C, a precipitate removed by centrifugation, and
the CPS isolated by gel-permeation chromatography on Sephadex (3-50.
Chromatography and mass spectrometry. Gel-permeation
chromatography was performed on a column (30 cmX3 cm) of
Sephadex G-50 or on a column (60 cmX2.5 cm) of Bio-Gel
P-4 using 0.05 M pyridinium acetate pH 4.5 or water as eluent,
respectively ; monitoring was performed using a Waters differential refractometer. GLC was performed using a HewlettPackard 5890 instrument on a DB-5 fused-silica capillary column using a temperature program 160°C or 130°C (1 min) to
250°C at 3"C/min for separation of sugar alditol acetates and
partially methylated alditol acetates, respectively. GLC/MS was
performed with the same chromatograph equipped with an Hewlett-Packard 5970 mass spectrometer, using the above conditions. FAB mass spectra were recorded on a NERMAG R1010L quadrupole instrument. Ions were produced from a matrix
of thioglycerol; in negative-mode MS, a crown-ether was added
to bind cations.
NMR spectroscopy. NMR spectra of D,O solutions were
run with a JEOL EX-270 or a JEOL alpha 400 instrument at
20 "C for oligosaccharides and 70 "C for polysaccharides. Chemical shifts are reported in ppm, using internal sodium 3-trimethyl~ilyI(2,2,3,3-~H,)propionate(6, 0.00), internal dioxane (6,
67.4), and external 85 % phosphoric acid (& 0.00 pprn) as references. Two-dimensional correlation spectroscopy (COSY), relayed COSY, total correlation spectroscopy (TOCSY), rotatingframe NOE spectroscopy (ROESY) (all run in the phase-sensitive mode), and 'H-detected ' H , T heteronuclear multi-quantum
coherence spectroscopy (HMQC) experiments were performed
with standard JEOL pulse sequences. The TOCSY and ROESY
experiments were performed using mixing times of 160 ms and
250 ms, respectively. A 'H-detected 'H,"P HMQC experiment
was performed for tetrasaccharide 1 at 25°C and pD 7.6 using
a Varian Unity 500 spectrometer.
Sugar and phosphate analysis. Hydrolysis was performed
with 0.5 M trifluoroacetic acid (lOO"C, 16 h). Neutral and amino
sugars were identified by GLC as their alditol acetates [16] using
authentic samples. Phosphate-containing compounds were de-
phosphorylated before hydrolysis by treatment with aqueous
48% hydrofluoric acid (20 h at ambient temperature). Uronic
acid was identified by GLC after methanolysis with 1 M hydrogen chloride in methanol (8O"C, 16 h) and acetylation. Absolute configurations were determined by GLC of acetylated glycosides with optically active 2-butanol (for GalA) and 2-octanol
(for Col, Gal, and QuiNAc) as described 117, 181. Phosphate
was determined by the method of Chen et al. [19j.
Methylation analysis. Methylation was performed with
methyl iodide in dimethyl sulfoxide in the presence of lithium
methylsulfinylmethanide [20] ; the methylated compounds were
recovered using a SepPak C,, cartridge. Hydrolysis and dephosphorylation were performed as in sugar analysis. Partially methylated monosaccharides were reduced with sodium borodeuteride, acetylated, and analysed by GLC/MS. Identification was
performed by comparison of the mass spectra with published
data [21]. The mass spectral data, m/7: (relative intensities in
parentheses), for 1,2,5-tri-O-acetyl-3,6-dideoxy-2-O-rnethyl-[
1'HI-hexitol were: 55 (X), 57 (8), 59 (12), 70 (20), 75 (11), X3
(loo), 84 (7), 87 (11), 97 (8), 102 (30), 115 (30), 118 (12), 129
(3,157 (26), 171 ( 3 ) , 173 ( 3 ) ,217 (6).
Chemical degradations. An aqueous solution of the CPS
was acidified with acetic acid to pH 3.1, heated for 2 h at 1OO"C,
a precipitate removed by centrifugation, and the product fractionated by gel-permeation chromatography on Sephadex G-50
to give tetrasaccharide 1 and octasaccharide 2; the former was
further purified by gel-permeation chromatography on Bio-Gel
P-4.
Dephosphorylation of the CPS was performed with aqueous
48% hydrofluoric acid (4"C, 20 h). The solution was cooled,
neutralised with cold aqueous 25 % ammonia, centrifuged, and
the supernatant applied to a column of Sephadex G-50 to give
polysaccharide 3 and a number of homologous oligosaccharides,
including octasaccharide 4.
RESULTS AND DISCUSSION
The CPS of V cholerue 0239 synonym Bengal, strain AI1838, was isolated by phenol/water extraction followed by delipidation of the contaminating lipopolysaccharide at pH 4.2 and
gel-permeation chromatography on Sephadex G-50. As the polysaccharide portion of the lipopolysaccharide has a low molecular
mass, it was easily separated from the CPS.
The CPS contained phosphorus (2.5%). Sugar analysis, and
determination of the absolute configurations by GLC of acetylated glycosides with optically active secondary alcohols [17,
181, revealed the presence of ~-galactose,3,6-dideoxy-~-xylohexose (colitose), 2-amino-2-deoxy-~-gh1cose,2-amino-2,6-dideoxy-D-glucose (D-quinovosamine), and D-galacturonic acid.
Galactose was detected in the hydrolysate only when the CPS
was dephosphorylated before hydrolysis.
The 31P-NMRspectrum of the CPS contained one broad signal at -1.3 ppm. The 'H- and 'T-NMR spectra of the CPS were
practically identical to those reported for the CPS of V cholerue
0139, strain AI-1837 [13]. The presence in the "C-NMR
spectrum of six signals for anomeric carbons in the region 98105 ppm indicated a hexasaccharide repeating unit. In the
spectrum there were two signals for C6 of Col at 16.3 ppm and
16.4 ppm; the signals for C2 and C3 of Col at 64.4 ppm and
33.7 ppm, respectively (cf. the published data 1221) had double
intensity. Hence, the repeating unit of the CPS contains two Col
residues. It was evident from the spectra that the amino sugars
were N-acetylated.
Mild acid hydrolysis of the CPS at pH 3.1 resulted in cleavage of Col and depolymerisation to give tetrasaccharide 1 and
393
Knirel et al. (Eul: J. Biochem. 232)
Table 1. Data of a 'H-NMR spectrum of tetrasaccharide 1.
Residue
Chemical shift (coupling constant) for
HI
H2
4,6-P-j$~-Galp-(
1+
4.53
(JIZ
= 7.6)
+~)-~-D-GIc]~NAc-(I+ 4.68
(JI.2
+4)-~-D-GalpA-(I --f
(Ji.2
+3)-a-~-QuipNAc
=
8.5)
=
3.7)
5.41
5.10
( J , , ~= 4.0)
+3)-fi-~-QuipNAc
4.71
(Jl = 8.5)
H3
3.61
(J2.3
3.84
H4
H6a
H-6b
CH,CON
3.77
=
9.9)
(J3.4
=
3.3)
3.79
= 10)
(J3.4 =
3.75
3.96
(J2,, = 10.5) (J3,4
=
4.01
3.79
(J2.0 = 10.2) (J3,4 =
3.75
3.65
(J2.3 = 10)
(J3.4 =
(J2.3
H5
9)
2.6)
3.96
(J5.6
9)
3.52
( J S , = 6.3)
=
1.27
1.99
1.30
1.98
6.6)
9)
.IHhh,
= 22 Hz.
In the predominant tetrasaccharide with P-QuipNAc; 5.38 ppm in the tetrasaccharide with a-QuipNAc
octasaccharide 2 isolated by gel-permeation chromatography.
Chemical analysis revealed that 1 and 2 contained Gal, GalA,
GlcNAc, QuiNAc, and phosphate.
The FAB mass spectrum (run in positive mode) of 1 showed
two large peaks at m/z 831 and 853. The molecular mass of 1
with the phosphate group as a monoester is 826, and the observed ions were therefore suggested to derive from 1 with the
phosphate group as a diester with the concomitant loss of water.
The observed ions should therefore correspond to adduct ions
with one and two Na+, respectively. Accordingly, the FAB mass
spectrum of 2 contained peaks at d z 1621 and 1643. The molecular masses of 1 and 2 (808 and 3598 Da, respectively) were confirmed by FAB mass spectra run in negative mode, which
showed peaks at m/z 807 for 1 and 1597 and 1619 for 2, the last
peak corresponding to (M-2H+Na)-. The difference of 18 Da
for 1 and 36 Da for 2 may be accounted for by the presence of a
cyclic phosphate, also suggested in the CPS of strain AI-1837 [13].
The 'H-NMR spectrum of 1 was completely assigned using
two-dimensional COSY and TOCSY experiments (Table 1). In
the spectrum QuiNAc gave two series of signals for the a- and
p-anomers and, hence, it occupies the reducing end. As judged
by the coupling constants J,,* = 7.6-8.5 Hz, Gal and GlcNAc
are &linked, while GalA is a-linked (.T,.2 = 3.7 Hz).
The two-dimensional ROESY spectrum of 1 showed correlation peaks for H1 Gal/H3 GlcNAc at 6 4.53i3.79, HI GlcNAc/
H4 GalA at 6 4.W4.34, H1 GalA/H3 a-QuiNAc at 6 5.38/3.79,
and H1 GalA/H3 B-QuiNAc at 6 5.4U3.65. These data indicated
that the tetrasaccharide 1 is linear, Gal occupies the nonreducing
end, GlcNAc and QuiNAc are substituted at position 3 and GalA
is substituted at position 4, and clarified the sequence of these
sugar residues.
Low-field positions of the signals for H4, H6a, and H6b of
the terminal Gal at 4.61, 4.43, and 4.27 ppm, as compared with
their positions at 3.89, 3.72 and 3.64 ppm, respectively, in the
spectrum of P-galactopyranose [23], suggested that the phosphate group is attached at positions 4 and 6. It is worth noting
= 22 Hz,
the unusually high value of the coupling constant
which is evidently due to the anti-periplanar orientation of the
coupled atoms (Fig. 1). In contrast, values of the coupling constants JH4, and .TH6*, are low, so that splitting of the corresponding signals was not visible. The cyclic phosphate group does not
distort the pyranose ring of the galactose residue, as follows
from the values of the coupling constants of the ring protons
(Table 1) which are typical of /I-galactopyranose. This rules out
the possibility of attachment of the phosphate group at positions
0
I1
'9I
I
I
H3
H1
Fig. 1. Molecular structure of ~-D-galactOpyranOSyl4,6-cyclophosphate.
3 and 6, since such an arrangement would require inversion of
the pyranose ring and, as a result, dramatically change the coupling constant values.
With the 'H-NMR spectrum assigned, the "C-NMR
spectrum of 1 was interpreted using an HMQC experiment
(Table 2). Downfield shifts of the signals for C3 of GlcNAc, C4
of GalA, and C3 of a-and /?-QuiNAc to 83.2, 78.8, 80.4, and
81.9 ppm, as compared with their positions in the corresponding
nonsubstituted monosaccharides [22], were consistent with the
positions of the glycosidic linkages determined by the ROESY
experiment. In Gal, downfield shifts of the signals for C4 and
C6 by =7 ppm and upfield shifts of the signals for C3 and C5
by 2 ppm and 8 ppm, respectively, (cf. the published data for pgalactopyranose [22] and pyruvic acid 4,6-acetal of methyl /lgalactopyranoside [25]) confirmed the attachment of the phosphate groups at positions 4 and 6 of this residue.
The "P-NMR spectrum of 1 contained one signal at
- 1.38 ppm, which was split to a doublet, J , Hhb = 22.6 Hz, in
the coupled spectrum (cf. the data of the 'H-NMR spectrum).
As stated above, the antiperiplanar orientation of the atoms gives
rise to this unusually large coupling constant value. The 'H,"P
HMQC spectrum showed that the phosphorus signal correlated
with proton signals at 3.77, 4.27, 4.43, and 4.61 ppm, which
correspond to H3, H4, H6a, and H6b of Gal (Table 1). The coupling to the last three atoms is usual ' J coupling, in complete
agreement with the structure containing a cyclic phosphate at
394
Knirel et al. (Eui: J. Biochem. 232)
Table 2. Data of '-'C-NMRspectra. The spectrum of tetrasaccharide 1 was assigned using an HMQC experiment and the spectrum of polysaccharide
3 tentatively assigned using published data [23, 241. The assignments of the signals with a difference in the chemical shifts <0.3 ppm for 1 and
< 0.5 ppm for 3 could be interchanged.
Sugar
Chemical shift for
Residue
___________
Tetrasaccharide 1 4.6-P-p-n-GaIp-( 1 4
+3)-j?-D-GlcpNAc-(l t
t'I)-U-D-GdIpA-( 1 --f
+3)-cx-D-QuipNAc
-+3)-P-~-QuipNAc
Polysaccharide 3 [h-Galp-(1+
+)-/~-D-GIC~NAC-(
1--f
6
c1
c2
c3
c4
C5
C6
103.9
102.9
101.0"
91.7
95.4
104.3
102.7
70.6
55.4
69.1
53.5
56.4
71.7
55.5
71.8
83.2
69 .9
80.4
81.9
73.5
83.7
76.8
69.2
78.8
76.9
76.6
69.5
68.9
68.1
75.9
71.7
68.2
72.4
76.2
74.7
69.1
61.8
174.2
17.4
11.4
61.9
69.4
101.2
102.6
69.4
55.7
70.3
82.5
79.2
76.8
71.8
72.3
173.6
17.5
CH,CON
CH,CON
23.2
175.6
22.7
22.9
175.1
175.4
23.3
175.5
23.2
175.1
7
+4)-a-v-GalpA-(l t
+3)-/h-QuipNAc-( 1+
In the predominant tetrasaccharide with j?-QuipNAc; 101.3 ppm in the tetrasaccharide with a-QuipNAc
Table 3. Data of methylation analysis. 2,4-Col, 2,4-di-O-Me-Col; 4QuiN, 2-N-Me-4-di-0-methylquinovosamine
; 4-QuiN-ol, 2-N-Me-44O-methylquinovosaminitol,etc. t,, relative retention time of the corresponding alditol acetate in GLC referenced to 2,3,4,6-Gal = 1.00. 2*,
reduced, methylated and dephosphorylated 2. 4**,borohydride-reduced 4.
positions 4 and 6, but the coupling to H3 must be a four-bond
coupling which occurs due to the planar zig-zag arrangement of
the five atoms, H3, C3, C4, 04, and P (Fig. 1).
Therefore, the tetrasaccharide 1 is a modified repeating unit
of the CPS, which contains the cyclic phosphate group but is
devoid of Col, and has the following structure
HO
Sugar
TK
0
Detector response of compound
2*
\/
P
3
4**
CPS
YO
/\
~ _ _ _
4
6
P-D-GaIp
1
2,4-C0l
2x01
2,3,4,6-GaI
4-QuiN-01
2,3-Gal
4-QuiN
3-Gal
4,6-GlcN
4-GlcN
GlcN
1
3
1+4)-a-~-GalpA-(1+3)-~-QuiNAc
,h-~-GlcpNAc-(
1
The octasaccharide 2 was reduced with sodium borohydride
and methylated, and the resulting partially methylated sugars
were analysed by GLC/MS as alditol acetates (Table 3). The
methylation data confirmed the monosaccharide substitution
pattern within the tetrasaccharide repeating unit established by
the NMR study of 1. The presence of 2,3-di-O-Me-Gal, which,
like Gal in the sugar analysis, appeared only after dephosphorylation, unambiguously proved the location of the phosphate
group at positions 4 and 6 of Gal. The presence of the alditol
acetates derived from 4-0-Me- and 4,6-di-O-Me-GlcNAcMe,
showed that the site of attachment of the tetrasaccharide repeat-
HO
0.51
0.71
1.oo
1.07
1.46
1.58
1.66
1.90
2.13
2.21
26
8
58
9
26
28
36
10
19
19
21
17
23
17
18
27
19
20
ing units to each other is position 6 of GlcNAc. The glycosidic
linkage of QuiNAc within the oligosaccharide chain is /3, as followed from the "C chemical shifts 103.2 ppm and 55.5 ppm of
the signals for C1 and C2 of this residue, which are in accord
with the data for P-QuipNAc and /3-GlcpNAc [22].
Therefore, the octasaccharide 2 consists of two tetrasaccharide repeating units and has the following structure
HO
0
0
\/
\/
P
P
/\
/\
4 6
p-D-Galp
1
4 6
P-D-Galp
I
1
1
3
3
/3-~-GlcpNAc-(
1-+4)-n-~-GalpA-(
1+3)-P-~-QuipNAc-(1+6)-P-~-GlcpNAc-(1-4)-u-~-GalpA-( 1+3)-~-QuiNAc
2
Knirel et al. (Eur J. Biockem. 232)
An attempt to obtain a dephosphorylated CPS with the carbohydrate backbone unaffected by treatment with aqueous 48 %
hydrofluoric acid at 4°C failed. Dephosphorylation of the CPS
under these conditions was, however, accompanied by complete
removal of Col and by depolymerisation. Fractionation of the
products by gel-permeation chromatography on Sephadex G-50
afforded polysaccharide 3 and a number of homologous oligosaccharides, octasaccharide 4 being the least of them. Most
likely, the corresponding tetrasaccharide was also obtained but
not separated in gel-petmeation chromatography from salts. The
isolated products had the same sugar composition (they contained almost equal amounts of Gal, GlcNAc, QuiNAc, and
GalA) and were free from phosphate. As expected, the molecular mass of 4 was determined as 1474 Da by FAB MS (run in
positive mode): the mass spectrum showed major peaks at d z
1475, 1497, and 1513, which belonged to (M+H)+, (M+Na)+,
and (M+K)+, respectively. A peak at m/z 729 was also present,
which was assigned to the tetrasaccharide fragment (oligosyl
cation, A,) from the nonreducing end.
Methylation analysis of 3 and borohydride-reduced 4 (Table
3) confirmed their structures and, in particular, the lateral position of the galactose residue in the repeating unit.
The proposed structure of the polysaccharide 3 also fitted
in well with the "C-NMR data (Table 2 ) which, in particular,
confirmed that the repeating units are connected by the B(1-6)
linkage between QuiNAc and GlcNAc. Depolymerisation of the
CPS by both hot dilute acetic acid at pH 3.1 and cold aqueous
48 % hydrofluoric acid is, thus, not unexpected since P-(146)linked 2-acetamido-2-deoxyhexoses(and, certainly, also 2-acetamido-2,6-dideoxyhexoses)are known to be easily cleaved under mild acidic conditions (e.g. complete depolymerisation of
the 0-specific polysaccharide of Citrobacter 0 16, containing
a fragment /?-~-GalpNAc-(
l--t6)-~-Galp, was observed on
dephosphorylation with 48% hydrofluoric acid at 4°C [26]). Another example of the lability of glycosidic linkages towards 48%
hydrofluoric acid is the cleavage of L-Rha in the 0-specific
polysaccharide from Salmonella typhirnurium [27]. This is the
case also for 3,6-dideoxyhexoses.
Therefore, the polysaccharide 3 and the octasaccharide 4
have the following structures, 3 being a dephosphorylated CPS
devoid of Col and 4 consisting of two tetrasaccharide repeating
units of 3.
395
Col, namely, 2-0-Me-Co1, was detected. The former derivative
gave a mass spectrum identical to that published [21] (apart from
the difference by 1 Da due to borodeuteride reduction). The latter derivative showed in the mass spectrum peaks at mdz 118
and 217, which corresponded to the primary fragments C1 -C2
and C2-C6, respectively, and peaks of the secondary fragments
at d z 157, 115, and 83 formed by sequential elimination from
the ion with m/z 217 of acetic acid, ketene, and methanol.
A repeated methylation of the methylated CPS under the
same conditions resulted in an increase of the content of 2,4-di0-Me-Col and a decrease of that of 2-0-Me-Col (their ratio became 1 :0.3 instead of 1:0.75 after a single methylation). It is,
therefore, suggested that 2-0-Me-Col appeared as a result of
undermethylation of one of the terminal Col residues. A simple
molecular model shows that spatial proximity can occur between
OH4 of Col-(1-+4) and the phosphate group. We suspect that
this may prevent full methylation. The positions of the signals
for C2 and C3 at 64.4 ppm and 33.7 ppm, respectively, showed
that both Col residues are a-linked (cf. the chemical shifts
63.9 ppm and 33. 9 ppm for the corresponding signals in methyl
3,6-dideoxy-a-xylo-hexopyranoside,
but 66.4 ppm and 38.1 ppm
in methyl 3,6-dideoxy-P-xylo-hexopyranoside[22]).
Therefore, on the basis of the obtained data, it was concluded
that the CPS of K cholerae 0139 synonym Bengal, strain AI1838 has the following structure
HO
0
\/
P
/\
4 6
a-colp-( 1-2)-P-~-Galp
1
1
3
-6)-P-~-GlcpNAc-( 1-4)-u-~-GalpA-( 1 4 3 ) 4
/?-D-QuipNAc-(1-
t
1
a-Colp
While this work was in progress, a preliminary structure of
the CPS of K cholerae 0139, strain AI-1837 was reported 1131
which was basically the same but the absolute configurations of
the constituent monosaccharides and the exact position of the
cyclic phosphate was not established. We do not know of any
/?-~-Galp
other example of a bacterial polysaccharide containing a cyclic
1
phosphate.
1
It is worth noting that polyclonal antibodies against the
3
studied K cholerae 0139 strain agglutinate Salmonella
-6)-~-~-GlcpNAc-(1-4)-a-~-GalpA-(l-.3)-~-~-QuipNAc-(lgreenside and Escherichia coli 055 (authors' unpublished data).
3
P-D-Galp
1
P-D-Galp
1
1
1
3
3
p-~-GlcpNAc-(1+4)-u-~-GalpA-(143)-P-~-QuipNAc-(
1-6)-P-~-GlcpNAc-( 1+4)-a-~-GalpA-(1 - + 3 ) - ~ - Q u i N A c
4
Methylation analysis of the CPS (Table 3 ) gave the alditol
acetates from 3-0-Me-Gal and GlcNAcMe, thus indicating that
position 2 of galactose and position 4 of GlcNAc are the sites
of attachment of the Col residues which, thus, are terminal
sugars of two side chains in the CPS. Surprisingly, together with
expected 2,4-di-O-Me-Col, another methylated derivative of
The 0-antigens of these enteric bacteria are known to possess
the same structure [28, 291 which is shown below, and their
cross reactivity with K cholerae 0139 is apparently due to the
presence of a common epitope containing a side-chain disaccharide a-Colp-(1-2)-P-~-Galp linked at position 3 of p-DGlcpNAc in the backbone.
396
Knirel et al. (Eur: J. Biochem. 232)
n-Colp-( 1-2)-p-~-Galp
1
1
-
3
-6)-p-~-GlcpNAc-( 1-3)-n-~-Galp-( 1+3)-P-~-GalpNAc-(1
The authors thank Dr E. Schweda and Mrs G. Alvelius (Clinical
Research Centre) for help in NMR spectroscopy and MS, respectively,
and Mrs M. Jansson (Division of Clinical Bacteriology) for technical
assistance. This work was supported by grants from the Swedish National Board for Technical and lndustrial Development, the Swedish Natural Science Research Council, and the Swedish Medical Research
Council (B95-16X-11227-01A). Y. A. Knirel thanks Karolinska Institutet for a fellowship.
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