(Received for publication, July 5, 1995; and in revised form, February 7, 1996)
From the
The type VIII capsular polysaccharide has been isolated and
purified from a newly described strain of group B Streptococcus which is a leading cause of sepsis and neonatal meningitis in
Japan. The polysaccharide contains D-glucose, D-galactose, L-rhamnose, and sialic acid in the molar
ratio 1:1:1:1. By means of high resolution H nuclear
magnetic resonance (
H NMR),
C NMR, and homo-
and heterocorrelated NMR, the repeating unit structure of the type VIII
polysaccharide was delineated as the following,
Enzymatic studies established
this polysaccharide as the first from which sialic acid, linked to a
branched -D-galactopyranosyl residue, is known to be
removed by bacterial neuraminidase.
The group B Streptococcus (GBS) ()has long
been recognized as a major cause of neonatal sepsis and
meningitis(1, 2) . GBS strains are classified into
serotypes on the basis of their type-specific capsular polysaccharides.
The strains isolated from clinical cases usually belong to one of the
major capsular types (Ia, Ib, II, and III)(2) , but five new
serotypes have recently been described: IV, V(3) ,
VI(4) , VII, and VIII. Type VIII (
)(originally
designated type M9(5) ), while not isolated in North America,
has been identified with increasing frequency over the last six years
among disease-causing isolates in Japan, where it is now a prevalent
strain (5) .
The structures of the capsular polysaccharides of GBS types Ia, Ib, II, III(6) , IV(7) , V(8) , VI(9) , and VII (10) have been elucidated. Despite their structural relatedness they are largely distinct immunologically. We report the isolation, structural analysis, and immunochemical characterization of the type VIII GBS capsular polysaccharide which like all the other type-specific GBS polysaccharides contains terminal sialic acid.
The absolute configurations of the monosaccharides were determined by a modification of the method described by Gerwig et al.(15) . The polysaccharide sample (1 mg) was hydrolyzed with 0.25 M sulfuric acid at 100 °C for 20 h, and the hydrolyzate was neutralized with barium carbonate. The sediment was removed by centrifugation and the supernatant was freeze-dried. The lyophilized residue was dissolved in 0.5 ml of(-)-2-butanol, 1 drop of trifluoroacetic acid was added, and the mixture was heated at 80 °C for 16 h. The solvents were removed by evaporation in vacuum and the dried sample was trimethylsilylated using 0.5 ml of N,O-bis-(trimethylsilyl)acetamide (Pierce) and 0.5 ml of pyridine. The trimethylsilylated(-)-2-butylglycosides of the constituent monosaccharides were analyzed by capillary gas-liquid chromatography using a Varian Saturn II GC-MS instrument equipped with a DB-17 capillary column (0.25 mm x 30 m, film thickness 0.25 µm) in the temperature program 150 to 210 °C at 2 °C/min.
Figure 1:
H NMR
spectrum of the type VIII GBS capsular polysaccharide. The signal at
2.225 ppm is that of the methyl group of internal acetone. The signal
at about 3.81 ppm belongs to the methylene groups of Tris used in the
buffer solution. The HOD signal is at 4.44
ppm.
The assignment of the H and
C NMR
signals was performed using homocorrelated two-dimensional COSY, TOCSY,
and NOESY techniques, as well as the heterocorrelated HMQC and
HMQC-TOCSY methods. The hexose components of the native repeating unit
shown in Fig. 2were designated a, b, and c according to the sequence of their anomeric signals in the
H NMR spectrum. N-Acetylneuraminic acid was
designated as unit d. The location of the H-2 signal of unit b at 3.31 ppm (doublet of doublets with large diaxial couplings J
= 7.3 Hz, J
= 9.6 Hz) unambiguously characterized unit b as
being
-D-glucose. A small coupling constant (J
3 Hz) and very low-field resonance for
H-4, together with a large value of J
coupling
for H-1c, characterized unit c as
-D-galactose. By elimination, the remaining unit a was assigned to be
-L-rhamnose. Because of the
mannopyranosyl configuration of the rhamnose ring, it was impossible to
determine its anomeric configuration on the basis of J
value. However, NOESY experiments (Table 1) showed that the
anomeric protons of all the sugar units had cross-peaks with their
respective H-3 and H-5 resonances. This finding indicated that the
rhamnopyranosyl residue had the
-anomeric configuration as well.
The complete assignment of the
H and
C NMR
signals of the native and desialylated type VIII GBS polysaccharide are
presented in Table 2and Table 3, respectively.
Figure 2: Structure of the native (top) and desialylated (bottom) type VIII polysaccharide antigens of GBS.
The
sequence of monosaccharides in the repeating unit of both the native
and desialylated type VIII GBS polysaccharides were established from
separate analyses of their two-dimensional-NOESY spectra(18) ,
and the NOE data (Table 1) are consistent with their structures
shown in Fig. 2. Independent confirmation of the sequence of
monosaccharides in the repeating unit of the desialylated
polysaccharide was also obtained using an H-detected
multiple-bond correlation experiment which showed cross-peaks between
C-1a and H-4c, C-1b and H-4a, as well as
between C-1c and H-4b. These long-range correlations are
in agreement with the substitution pattern established by the NOESY
experiment. Additional evidence for the position of sialylation (O-3)
to galactose was also obtained from the downfield displacements
exhibited by the H-3 (Table 2) and C-3 (Table 3) signals of
galactose on desialylation of the native type VIII polysaccharide.
Competition ELISA revealed that rabbit antiserum to whole cells of type VIII strain 130013 had high affinity for the native type VIII polysaccharide. The concentration of native type VIII polysaccharide that resulted in a 50% inhibition of the binding of type VIII-specific antiserum was 2.8 µM (Fig. 3). Desialylated, periodate oxidized/borohydride reduced, or carbodiimide-reduced type VIII polysaccharide failed to inhibit binding of type VIII-specific antiserum even when used at a concentration of 0.5 mM (Fig. 3).
Figure 3: ELISA inhibition of GBS type VIII rabbit antiserum with native (closed circles), carbodiimide-reduced (open circles), periodate-oxidized/borohydride-reduced (closed squares), and desialylated (open squares) type VIII polysaccharides. Values are the mean of duplicate determinations.
The structure of the type VIII polysaccharide represents a
different motif from previously studied capsular polysaccharides of
GBS. Capsular polysaccharides of types Ia, Ib, II, III, IV, V, and VII
are all composed of D-galactose, D-glucose, N-acetyl-D-glucosamine, and N-acetylneuraminic acid and contain complex repeating units
built from five to seven monosaccharides. The type VIII polysaccharide
is not only the first GBS capsular polysaccharide composed of
tetrasaccharide repeating units, but is also unique in that
-L- rhamnopyranosyl residues replace the
2-acetamido-2-deoxy-
-D-glucopyranosyl residues found in
all other GBS type-specific polysaccharides except type VI(9) .
The overall structure of the type VIII polysaccharide most closely
resembles that of the type II polysaccharide (26) in that the D-galactopyranosyl residues are situated in the backbone
rather than in the side chains, the latter being the case in all other
types(6, 7, 8, 9, 10) .
Like all the other GBS capsular polysaccharides, the type VIII
polysaccharide has terminal sialic acid residues linked to O-3
of D-galactopyranosyl residues.
Although it shares structural features with other GBS polysaccharides, the type VIII polysaccharide is antigenically distinct. No significant cross-reactions were detected by type VIII-specific ELISA after adsorption of type VIII-specific rabbit antiserum with GBS organisms of heterologous serotypes. The lack of cross-reactions among the GBS polysaccharides (7, 8, 28) indicates that sialic acid is not, in and by itself, an immunodominant epitope. The failure of sialic acid to be immunodominant might be expected in light of its ubiquity in human and animal tissues(29) . However, it is only ``non-immunodominant'' in the classical sense, that is, not being a direct epitope for antibody binding, but its presence is still crucial to the immunospecificity of antibody raised to the native type VIII polysaccharide. This was demonstrated by the inability of desialylated or otherwise chemically modified type VIII polysaccharide to bind to a significant extent to type VIII-specific antibodies. The involvement of sialic acid in the formation of an immunospecific epitope has also been reported for GBS serotypes Ia, II, III, and VI(4, 28) , and hypothesized that the explanation for this specificity lies in the ability of sialic acid to exert conformational control over the epitopic expression of these polysaccharides.
The epitopic expression based on extended helical
domains of polymers of -(2
8)-polysialic acid has been well
documented(28, 30, 31) . A similar
explanation could apply to the epitopic expression of some of the GBS
polysaccharides; in the case of the type III polysaccharide, the
chain-length dependence (number of repeating units) of the epitope,
which is a requirement for extended helical
epitopes(28, 32) , has been established(33) .
Evidence for a sialic acid-controlled conformational epitope in the
type III polysaccharide was obtained with NMR spectroscopy which
revealed significant displacements in chemical shifts of carbon signals
remote from sialic acid when the latter was removed(16) .
Similar displacements in signals in the spectrum of the type VIII
polysaccharide have been detected and may indicate the presence of
sialic acid-controlled conformational epitope. Both NMR signals of the
anomeric carbon (Table 3) and anomeric proton (Table 2) of
the polysaccharide's backbone
-D-glucopyranosyl
residue were significantly displaced (1.0 and 0.17 ppm, respectively)
when terminal sialic acid was removed. However, attachment of sialic
acid directly to the backbone of the type VIII polysaccharide puts it
in closer proximity to the backbone than it is in the type III
polysaccharide(6, 11) . Therefore, for the type VIII
polysaccharide, these displacements may also be attributable to
deshielding of the anomeric carbon and proton of the
-D-glucopyranosyl residue by the carboxylate group of the
terminal sialic acid.
GBS type II polysaccharide and G ganglioside (II
NeuAcGgOse
Cer) are known
to have in their structures terminal N-acetylneuraminic acid
residues that are linked directly to O-3 of a branched
-D-galactopyranosyl residues that are resistant to
treatment with neuraminidase(26, 34, 35) .
This resistance has been attributed to steric hindrance of the approach
of the enzyme because sialic acid is attached directly to O-3 of the
backbone
-D-galactopyranosyl residues that have vicinal
substituents in position O-2 or O-4(26) . Although the type
VIII polysaccharide also exhibits the above structural features, its
terminal sialic acid is readily removed with neuraminidase. Obviously
this effect is dependent on specific structural features surrounding
-D-galactopyranosyl residues of the type VIII
polysaccharide. In this region type VIII polysaccharide differs in
structure from both type II polysaccharide and G
ganglioside; the most pronounced difference is that its backbone
-D-galactopyranosyl residue is linked glycosidically to a
unique
-L-rhamnopyranosyl residue. It is interesting to
speculate that the enzymatic source for the biosynthesis of the
rhamnose substitution in the type VIII polysaccharide may have been
from the same enzymes used by all GBS to synthesize the common group
B-specific cell wall associated polysaccharide which consists of L-rhamnose, D-galactose, D-glucitol, and N-acetyl-D-glucosamine arranged in a complex
multiantennary structure of four structurally distinct oligosaccharides (27) .
All GBS polysaccharides whose structures have been elucidated possess side chains that are composed of or terminate with sialic acid. Sialic acid has been shown to be a critical virulence component (36) of these organisms by limiting the deposition on cells of C3b for opsonization in the absence of specific antibodies(37) . That this distinguishing characteristic has been maintained on all capsular polysaccharides of GBS isolated from human sources, including type VIII, emphasizes the importance of this sugar in GBS pathogenesis.