Vol. 56, No. 11INFECTION AND IMMUNITY, Nov. 1988, p. 2942-29470019-9567/88/112942-06$02.00/0Copyright C) 1988, American Society for Microbiology

Chemical and Structural Studies of Serotype PolysaccharideAntigens of Streptococcus sobrinus 6715tKAZUKO TAKADA,* TETSUO SHIOTA, AND TADASHI IKEDA

Department of Microbiology, Nihon University School of Dentistry at Matsudo, Matsudo, Chiba 271, Japan

Received 3 May 1988/Accepted 12 August 1988

The g antigen of Streptococcus sobrinus 6715 was previously shown to consist of polysaccharides of variousmolecular weights. In this study, two such polysaccharides, LII and LIII, were purified by gel filtration andaffinity chromatography procedures. By a double immunodiffusion analysis, fraction LII was found to containa region in the serotype-specific g site not present in the serospecific g site of fraction LIII. This region was

designated x. In addition to the serotype-specific g site, the cross-reactive sites, g-a, g-d, and g-(a-d), were allpresent on a single molecule of fractions LII and LIII. Polysaccharides LII and LIII were composed ofgalactose, glucose, and rhamnose. Analysis of inhibition of the precipitin reaction suggested that the serotypeg site of fraction LII (the putative form of g antigen) may consist of two immunodominant regions, one a

galactose-containing region (region x) and the other a glucose-containing region, while the serotype g site offraction LIII appeared to have one immunodominant region containing a glucose determinant. The methylationand '3C nuclear magnetic resonance analyses of LII and LIII fractions provided information on the linkage andthe anomeric structures of the sugar components of the polysaccharides.

Strains of Streptococcus mutans are serologically groupedinto eight serotypes, a through h (2, 5, 23). Recently, newspecies names were proposed for serotypes d and g and forserotype a by Coykendall (7), namely, Streptococcus so-brinus and Streptococcus cricetus, respectively. Antibodiesspecific to serotype polysaccharide antigens have beenshown to inhibit adherence (21) and block the binding ofglucosyltransferase to cells (19). This indicated that theantigens were components of the surfaces of the cells.Antisera prepared from serotypes a, d, and g have beenfound to cross-react with antigen preparations from the threeserotypes (12, 17, 18, 23, 27). Similar cross-reactivity hasalso been found among serotypes c, e, and f (12, 17, 18).Chemical characterization studies of these polysaccharideantigens have been carried out, and they have providedinformation regarding the basis for cross-reactivity (12, 17,18, 25). Such information concerning serotype specificityand cross-reactivity may be of great importance in thedevelopment of a vaccine to prevent dental caries.A previous study (27) showed that the serotype polysac-

charide antigen from strain 6715 (serotype g) consists of theserotype-specific g site and the cross-reactive sites g-a, g-d,and g-(a-d). By gel filtration, six polysaccharide fractions,with various molecular weights, were isolated. Fractions LI,LII, and LIII were similar to the serotype antigen; that is,these antigens contain the serotype-specific g site in additionto the three cross-reactive sites. However, fractions SI, SII,and SIII were incomplete, each lacking the serotype-specificg site but containing three cross-reactive sites [g-a, g-d, andg-(a-d)], two cross-reactive sites [g-d and g-(a-d)], and onecross-reactive site [g-(a-d)], respectively.

In this study, the polysaccharide antigens LII and LIII,which were purified using specific anti-serotype g serum-Sepharose 4B affinity column and gel filtration column, were

* Corresponding author.t This publication is dedicated to the memory of Tetsuo Shiota,

who passed away on 11 July 1988.

characterized serologically, chemically, and structurally.The present report corroborates the previous proposal forthe presence of the serotype-specific g site and the cross-reactive g-a, g-d, and g-(a-d) sites on a single molecule of theantigen and the existence of multiple forms, with variousmolecular weights, and provides additional information onthe identification of the immunogenic regions within theserotype-specific site of fractions LII and LIII and theanomeric and linkage structures of sugars in fractions LIIand LIII.

MATERIALS AND METHODS

Maintenance and growth of cultures. S. cricetus HS6(serotype a) and S. sobrinus B13 (d), 6715 (g), and C307, amutant defective in polysaccharide g derived from strain6715 (11), were maintained in brain heart infusion agar (DifcoLaboratories, Detroit, Mich.) supplemented with solidCaCO3. For experimental purposes, cultures were grown inPD-glucose medium (14) and cells were dried by lyophiliza-tion.

Preparation of sera. Anti-6715 serum was prepared in NewZealand White rabbits. The immunoglobulin G fraction wasabsorbed with cells of C307 to prepare anti-g serum (27). Thespecific anti-serotype g serum was obtained by the absorp-tion of anti-g serum with cells of B13, followed by a secondabsorption with cells of HS6. Additional absorbed sera usedin this study were obtained by methods described previously(27).

Preparation of Sepharose 4B bonded with specific serotype gantibody. Specific anti-serotype g serum-Sepharose 4B wasprepared by reacting 15 g of CNBr-activated Sepharose 4B(Pharmacia Fine Chemicals, Inc., Piscataway, N.J.) withspecific anti-serotype g serum (372 mg of protein) by theprocedure provided by Pharmacia. The affinity column waswashed with water before loading the sample.

Preparation of polysaccharide fractions LII and LIII. Theprocedure adopted to obtain fractions LII and LIII includedaffinity chromatography and gel filtration. To the specific

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anti-serotype g serum-Sepharose 4B affinity column (3.0 by7.0 cm), a three-column-purified antigen preparation from aRantz Randall extract from strain 6715 (27) was added at40C. The column was washed with water and eluted with 4 MMgCI2. There were no differences in the efficacy of theaffinity column when the column was washed with water orwashing buffer in the procedure described by Pharmacia.The eluted fractions from the affinity column were monitoredfor carbohydrate by a phenol-sulfuric acid method and forantigenic activity by a double immunodiffusion method. Thefractions from the affinity column were pooled, dialyzedagainst water, and lyophilized. The fraction eluted from thespecific anti-serotype g serum-affinity column correspondedto L. The column was regenerated by washing sequentiallywith 0.1 M acetate buffer containing 0.5 M NaCl (pH 4.0),0.1 M sodium bicarbonate buffer containing 0.5 M NaCl (pH8.3), and water.Samples of the specific anti-serotype g antibody-affinity-

purified preparation L were applied to an Ultrogel A4(fractionation range of 55,000 to 9,000,000; LKB Instru-ments, Inc., Rockville, Md.) column to obtain fractions LIIand LIII. This column was also used for estimation ofmolecular weights of the polysaccharide antigens. The gelfiltration procedures and the standard materials used werepreviously described (27).

Serological analysis. For the quantitative precipitation as-say, specific antisera and affinity column-purified antigenswere used. Samples of specific anti-serotype g serum (89.0,ug of protein) were incubated with increasing concentrationsof either LII or LIII antigen in a total volume of 250 RI for 2h at room temperature and then overnight at 4°C. Precipi-tates were collected by centrifugation, washed twice withsaline, and dissolved in 0.1 N NaOH, and protein contentwas determined. For quantitative inhibition assays, a reac-tion mixture contained affinity column-purified LII antigen(1.16 ,ug of carbohydrate), 5 or 25 ,umol or no inhibitor,specific anti-serotype g serum (89.0 ,ug of protein), and salinein a total volume of 250 RI. When affinity column-purifiedfraction LIII antigen (0.75 ,ug) was used, the reaction mix-ture contained 5 or 20 ,umol or no inhibitor, specific anti-serotype g serum (89.0 ,ug), and saline in a total volume of250 RI. The mixtures were incubated for 2 h at roomtemperature and then overnight at 4°C. The antigen-antibodyprecipitates were collected and washed, and their proteincontent was determined.The procedure for the double immunodiffusion agar

method was as described previously (27).Chemical analysis. Total sugar was determined by the

phenol-sulfuric acid method with glucose as the standard (8)and protein by either the method of dye binding (Bio-RadLaboratories, Richmond, Calif.) or by the method of Lowryet al. (20) with bovine serum albumin as the standard. Totalphosphorus was measured by the method of Bartlett (1).Total hexosamine was quantitated by the procedure ofStrominger et al. (26). Quantitative determination of specificsugars was performed by gas-liquid chromatography (GLC)with appropriate standards.A sample of purified antigen (1 mg) was hydrolyzed in 1 N

H2SO4 for 8 h at 100°C. The hydrolysate was neutralizedwith BaCO3 and centrifuged. The supernatant fluid waspassed through a column of Dowex 50 x 8 (H+ form, 1 nil),and the resin was washed with water. The effluent andwashings were combined and concentrated in a vacuumrotator at 40°C. The sugars were reduced with sodiumborohydride and converted to their alditol acetate deriva-tives with a mixture of pyridine and acetic anhydride (1:1

[vol/vol]) by the method of Lindberg (16). Xylose, which wasused as an internal standard, was also converted to its alditolacetate. The resulting products were dissolved in chloroformand separated on a Shimadzu model GC-9A GLC with aglass column (0.3 by 210 cm) packed with 3% OV-225. Thechromatography was performed isothermally at 190°C withnitrogen as the carrier gas. Peaks were quantitated using aShimadzu Chromatopack model C-R3A integrator.

Methylation analyses of the purified antigens by GLC andGLC-mass spectrometry. The purified antigens were methyl-ated by the method of Hakomori (10), as modified byLindberg (16). Samples of purified LII and LIII (2.0 mg)were methylated completely by the above procedure, asjudged by an infrared Spectroscope IR-A3 (Nihon BunkouCo.). The methylated polysaccharides were hydrolyzed with2 ml of 90% formic acid for 2 h at 100°C and then with 0.25M sulfuric acid for 8 h at 100°C. The hydrolysates werereduced and acetylated as described for the analysis ofsugars in acid hydrolysates. The methylated alditol acetateswere analyzed by GLC using the same column and sameconditions as described for the sugar analyses. The capillarycolumn (0.32 mm by 25 m) was packed with OV-101; thecolumn temperature was adjusted to 170°C, held for 1 min,and then raised at 2°C per min to 230°C. Retention timesrelative to 1,5-di-O-acetyl-2,3,4,6-tetra-O-methyl-D-glucitolwere determined by interpolation of known standards. The1,5-di-O-acetyl-2,3,4,6-tetra-O-methyl-D-glucitol was pre-pared from 2,3,4,6-tetra-O-methyl-D-glucose (Sigma Chemi-cal Co., St. Louis, Mo.). 1,5-Di-O-acetyl-2,3,4,6-tetra-O-methyl-glucitol, 1,3,5-tri-O-acetyl-2,4,6-tri-O-methyl-gluci-tol, 1,4,5-tri-O-acetyl-2,3,6-tri-O-methyl-glucitol, 1,5,6-tri-O-acetyl-2, 3,4-t-Ot-methyl-glucitol, and 1,3, 5,6-tetra-O-acetyl-2,4-di-O-methyl-glucitol were prepared from apolysaccharide of Grifola frondosa (22) and were kindlyprovided by N. Ohno of the Tokyo College of Pharmacy,Tokyo, Japan.Combined GLC-mass spectrometry was performed with a

Hitachi M-80, using the 3% OV-225 column (0.3 by 210 cm)and 10% SILAR-SCP column (0.3 by 100 cm) and anionization potential of 70 eV.

"3C NMR analysis. '3C nuclear magnetic resonance (13CNMR) spectra were recorded with a JEOL-FX 200 spec-trometer operating at 50.1 MHz in the pulsed, Fourier-transform mode with complete proton decoupling. Chemicalshifts are reported in parts per million (ppm) downfield fromthe external tetramethylsilane. Polysaccharides were exam-ined as solutions in D20 (20 mg/ml) using 10-mm-diameteregg-type sampling tubes at room temperature. Spectra wererecorded with 8,000 data points and a spectral width of 12.0kHz.

RESULTS

Double immunodiffusion analyses of polysaccharides a, d, g,LII, and LIII with various antisera preparations. Since thepurpose of preparing the specific sera was to prepare affinitycolumns to purify polysaccharide antigens, it was mostimportant to establish the specificity of each antiserum. Thedouble immunodiffusion precipitin reaction pattern in Fig.1A shows that the specific anti-serotype g serum preparationreacted with only polysaccharide g and not with polysaccha-rides a and d. The fact that a band was formed with onlypolysaccharide g indicated that the antiserum preparationused did not contain any antibodies against the cross-reactive sites, g-a, g-d, and g-(a-d), but contained only theantibody against the serotype g site. Thus, the preparation of

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2944 TAKADA ET AL.

A9

i ....9RAN. It %,-.: .

B C9

d a

DUI L.

FIG. 1. Double immunodiffusion analyses of three-column-purified polysaccharides a (5 ,ug of carbohydrate) and d (5 jig) and affinitycolumn-purified polysaccharides g (2 jig), Lll (1.5 ,ug), and LIII (1.8 ,ug). Wells designated 1 and 2 contain specific anti-serotype g serum (22jig of protein) and anti-g serum (40 ,ug), respectively.

the specific anti-serotype g contained only the single type ofantibody, and the preparation was found to be most suitablein preparing an affinity column to purify appropriate anti-gens.

Figures 1B and C illustrate the analysis of the affinity-purified polysaccharide g for antigenic sites. The g antigenpreparation was placed in a well between wells containingpolysaccharides a and d (Fig. 1B) and in a well adjacent to awell containing both polysaccharides a and d (Fig. 1C). Allthese antigens were then tested against anti-g serum. Thewells which contained the affinity-purified polysaccharide gshowed partial identity with the well containing polysaccha-ride a or d and with the well containing both a and d. Theseresults suggested that the affinity-purified polysaccharide ghad not only the g site but also the cross-reactive sites, g-a,g-d, and g-(a-d), all on a single molecule.The affinity column-purified polysaccharides, LII and

LIII, were previously shown to be high-molecular-weightpolysaccharides with serotype-specific g sites and the cross-reactive sites, g-a, g-d, and g-(a-d) (27). When LII and LIIIwere tested against the specific anti-serotype g serum (Fig.1D), the partial identity seen between fractions LII and LIIIindicated that fraction LII contained an additional immuno-genic region which we had designated x (27).Sugar content of fractions LII and LIII. The results of the

sugar analysis of the waffinity-purified antigens are summa-rized in Table 1. Fractions LII and LIII were found to becomposed of galactose, glucose, and rhamnose in molarratios of approximately 5.02:1:0.18 and 4.65:1:0.39, respec-tively. Hexosamine, protein, and phosphorus were not de-tected.

Inhibition of the precipitin reaction. The reactions of LIIand LIII antigens with the specific anti-serotype g serumwere examined by the quantitative precipitin assay. Thestandard precipitin curves obtained from reacting specificanti-serotype g serum with increasing concentrations ofeither LII or LIII antigen are shown in Fig. 2A and B,respectively. At their equivalence points, 1.16 jig of LIIantigen and 0.75 jig of LIII antigen precipitated 27.8 and 21.0jig of antibody protein, respectively.

TABLE 1. Sugar composition of antigens Lll and Llll

Composition (,umol/mg)Component

Lll Llll

Galactose 4.62 2.14Glucose 0.92 0.46Rhamnose 0.17 0.18Protein ND" NDPhosphorus ND NDHexosamine ND ND

"ND, Not detected.

Experiments on the inhibition of the precipitin reactioninvolving the specific anti-serotype g serum with either theaffinity-purified LII or LIII fraction were performed. Table 2shows the results of inhibiting the reaction of the specificanti-serotype g serum with purified LII antigen. Galactoseand galactose-containing oligosaccharides were more inhib-itory than glucose-containing disaccharides. The higher con-centration of raffinose and melibiose used gave greaterinhibitions, 48.5 and 43.4%, respectively, whereas lactoseproduced 28.4% inhibition. The (x- and P-methylgalactopy-ranosides showed similar inhibitory activities. Maltose andcellobiose, both at the low and at the high concentrations,were effective glucose-containing inhibitors. Similarly, P-gentiobiose and isomaltose were also relatively effectiveinhibitors, 26.5 and 22.4%, respectively, but were effectiveonly at the higher concentration. The cx- and 3-methylglu-copyranoside showed little or no effect.

Table 3 shows the results of the test for inhibition of thereaction between the specific anti-serotype g serum and theaffinity column-purified LIII antigen. The 1,4-linked digluco-sides, maltose and cellobiose, gave the greatest inhibition,40.0 and 40.4% at the high concentration and 39.1 and 22.6%at the low concentration, respectively. The degree of inhibi-tion obtained was higher than those for LII antigen. Melibi-ose and 3-methylgalactopyranoside were moderately effec-tive inhibitors, but lactose was a very poor inhibitor.GLC and GLC-mass spectrometry analysis of fractions LII

and LIII. The partially methylated sugars as their alditolacetates prepared from fractions LII and LIII were identifiedafter analyzing their retention times by GLC and massspectrometry. The results of analyses for fractions LII and

30 A B

020

C

.10

0.

0 0.5 1.0 1.5 2.0 0 0.5 1.0 1.5 2.0

Antigen jigFIG. 2. Quantitative precipitation of specific anti-serotype g se-

rum (89.0 jig of protein) by increasing concentrations of Lll (A) orLill antigen (B).

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TABLE 2. Inhibition of the precipitin reaction between Lllfraction and specific anti-serotype-g serum

Inhibition (§)Inhibitor"

5 ,umol 25 pmol

D-Galactose 38.2 38.2Raffinose (GalX1 6G1c1i2PFruf) 20.0 48.5Melibiose (Gal'1,6Glc) 17.4 43.4Stachyose (Gaiot1,6Gal.tL,6Glcot12PFruf) 12.6 31.6Lactose (Gal1,4Glc) 24.1 28.4ox-Methylgalactopyranoside 0 29.43-Methylgalactopyranoside 8.8 26.3D-Glucose 16.2 16.2Maltose (Glct114Glc) 20.6 27.4Cellobiose (GIcP1'4Glc) 22.1 32.4Isomaltose (Glcot6Glc) 8.8 22.4

P-Gentiobiose (Glc1_6Glc) 11.8 26.5x-Methylglucopyranoside 2.9 13.2,-Methylglucopyranoside 2.6 16.2cx-Rhamnose 0 3.2

' Abbreviations: Gal. galactose; Glc, glucose; Fruf. fructofuranose.

A

tw_-s <~~~~~~~ KLI

.0 I.20..10..I140 120 100 so0 6 40

p.-...C

cocn

00u.)u

:, X s

LIII were similar and are summarized in Table 4. Five typesof galactose residues and one type of glucose residue wereobserved: a nonreducing terminal galactopyranose, a 1,3-linked galactopyranose, a 1,3-linked galactofuranose, a 1,6-linked galactopyranose, a branching 1,3,6-linked galactopy-ranose or 1,2,6-linked galactofuranose, and a 1,6-linkedglucopyranose. Also, trace amounts of 1,2-linked rhamnopy-ranose, 1,2,3-linked rhamnopyranose, and nonreducing ter-minal glucopyranose were detected in both LII and LIIIfractions. The differences between LII and LIII were in theamounts of glucose, nonreducing terminal galactose, andbranching galactose.

13C NMR study. Figures 3A and B show the13C NMRspectra of LII and LIII fractions. The close similarity of thespectra indicated that the two fractions have the same sugarresidues. The assignment of the 13C NMR data for identifi-

TABLE 3. Inhibition of the precipitin reaction between Llllfraction and specific anti-serotype-g serum

Inhibition (%)Inhibitor"

5 pmol 20',mol

D-Galactose 3.0 17.4Raffinose (Gala1'6Glc- "2 Fruf) 2.1 18.3Melibiose (Gail -6Glc) 21.3 20.9Stachyose (Galot1,6Gal°(1,6GlcOt12PFruf) 19.2 19.6Lactose (Galp"4Glc) 10.6 13.0x-Methylgalactopyranoside 0 0,B-Methylgalactopyranoside 7.2 25.2D-Glucose 0 7.2Maltose (Glc'1,4Glc) 39.1 40.0Cellobiose (GlcP1'4Glc) 22.6 40.4Isomaltose (Glca1,6G0c) 10.6 23.0,-Gentiobiose (Glc1 6Glc) 4.3 12.8o-Methylglucopyranoside 0 13.6P-Methylglucopyranoside 0 13.9x-Rhamnose 0 10.6

Abbreviations: Gal. galactose; Glc, glucose: Fruf. fructoefuranose.

120 110 105 100 95 90 85

I0 70 V 60i 60

8c 7S 70 65 ,0

FIG. 3. '3C NMR spectra of LlI fraction (A) and LllI fraction (B)and the spectrum showing anomeric carbons in Lll fraction (C).

cation of the structures of the sugars of the polysaccharideswere based on published reported values (3, 4). There are nostandard materials available to assign chemical shifts ofanomeric configurations for galactose-glucose polymers.The examination of the 13C NMR spectra of the two Lfractions for signals with ppm values greater than 95, whichare given by anomeric carbon atoms, showed signals at109.7, 106.2, 103.6, and 102.3 ppm, suggesting the presenceof 1*3-linked 3-galactofuranose, 1,3-linked 3-galactopy-ranose, nonreducing terminal 3-galactopyranose, and 1,6-linked P-glucopyranose, respectively. The large signal at98.4 ppm was assigned to 1,6-linked ox-galactopyranose.According to the methylation analysis and the large peakarea with a chemical shift of 98.4 ppm, observed in the 13CNMR spectrum, the unassigned residue was also indicativeof a branching residue with an ox anomeric configuration, thatis, a 1,2,6-linked ox-galactofuranose or a 1,3,6-linked cx-galactopyranose.

DISCUSSION

The serological study of the polysaccharide antigen of thecell wall of S. sobrinius Kl by Perch et al. (23) classified thisstrain as serotype g. This strain was made streptomycinresistant, strain K1-R, from which strain 6715 was obtainedby passage through animals (9). Subsequently, strain 6715was shown to cross-react with antisera of antigens belongingto two other serotypes, namely, a and d (12, 17). Thus, fromthese studies and our previous findings (27), we proposedthat the polysaccharide antigens of strains 6715, HS6, and

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TABLE 4. Methylation analysis of LII and LIII antigens

Relative peak area (%)Derivative Structurea

LII LIII

1,5-di-O-acetyl-2,3,4,6-tetra-O-methyl-galactitol 14.1 11.5 Gal '1,3,4-tri-O-acetyl-2,5,6-tri-O-methyl-galactitol 14.6 16.0 -3GalfaI1,3,5-tri-O-acetyl-2,4,6-tri-O-methyl-galactitol 21.8 21.8 3 Gal'1,5,6-tri-O-acetyl-2,3,4-tri-O-methyl-galactitol 15.3 16.2 6Gal1-1,3,5,6-tetra-O-acetyl-2,4-di-O-methyl-galactitol or 14.0 10.0 -Gal -> or

1,2,4,6-tetra-O-acetyl-3,5-di-O-methyl-galactitol 3

6Galf-*2

1,5,6-tri-O-acetyl-2,3,4-tri-O-methyl-glucitol 18.2 22.6 6Glc1a Galf, G41actofuranose; Gal, galactopyranose; Glc, glucopyranose.

B13 consist of the following: the serotype-specific g and thecross-reactive sites g-a, g-d, and g-(a-d); the serotype-spe-cific a and the cross-reactive sites g-a and g-(a-d); and theserotype-specific d and the cross-reactive sites g-d andg-(a-d), respectively. Furthermore, in strain 6715, the poly-saccharide g antigen was shown to occur in various molec-ular weights, the L fQrms being similar to the putative gantigen and the smaller S forms being incomplete antigens,each lacking the serotype g site and also one or more of thecross-reactive sites.The studies on the sugar composition of fractions LII and

LIII showed that these two polysaccharides contained ga-lactose, glucose, and rhamnose in approximate ratios of 5:1:trace. Although rhamnose has not been reported as a con-stituent sugar of g antigen (13, 17), the deoxysugar is a majorconstituent of the rhamnose-glucose polymer which ispresent in extracts of cells of strain 6715-T2, which lacks theserotype-specific g antigen (24). Brown and Bleiweis (6)showed that the a antigen purified by lectin affinity columnwas composed of galactose and glucose in a molar ratio of3.4:1, with a trace amount of rhamnose and phosphorus. Theserotype antigen of strain 6715, which was obtained as abuffer-boiled extract purified by gel filtration, was reportedby Iacono et al. (13) to contain galactose and glucose in aratio of 5.9:1.0, with 9.5% protein, a trace of phosphorus,and no rhamnose.Brown and Bleiweis (6) reported seven structural forms of

galactose and glucose in the serotype a antigen from strainAHT and six structural forms of the same two sugars in theserotype d antigen from strain B13. Of the seven structuralforms of the sugars in antigen a and six structural forms ofthe sugars in antigen d, six and four forms, respectively,were found in fractions LII and LIII. The presence of similarstructural forms of the two sugars in the three serotypes, a,d, and g, is consistent with the similarity of various proper-ties found among strains belonging to these serotypes.The patterns of precipitin bands of the L fractions ob-

tained by a double immunodiffusion analysis presented pre-viously (27) and also in this paper indicated that the LIIfraction contained an immunodominant region x, which isabsent in the LIII fraction. The results of the experiment onthe inhibition of the precipitin reaction suggested that thereare two regions within the serotype-specific g site of LII, onecontaining a galactose determinant and the other a glucosedeterminant. However, within the serotype-specific g site inLIII fraction, the region corresponding to the galactose-containing region in LII fraction is deficient in being anti-genic, but the glucose-containing region is sufficient and is

antigenic. Thus, this result indicated that region x, which isabsent in LIII, is most likely the galactose-containing region.The results obtained for the linkage and the anomeric

structural forms of the sugars in fraction LII (Table 4 andFig. 3) strongly suggest that the galactose-containing regionconsists of repeating units of 1,3-linked ,B-galactofuranose,1,6-linked ot-galactopyranose, and a branched a-galactofu-ranose or a-galactopyranose substituted in position 2 or 3with a nonreducing 3-galactopyranose.

In the presence of the specific anti-serotype g serum andthe LII or LIII fraction, maltose and cellobiose inhibited theprecipitin reaction (Tables 2 and 3), even though the '3CNMR analysis of LII fraction and LIII fraction (Fig. 3) didnot show the presence of ot-1,4 and ,B-1,4 linkages. The factthat maltose and cellobiose showed strong inhibitory activityand lactose showed weak inhibitory activity, in the case ofLIII fraction (Table 3), suggested that C-1 and not C-4 maybe important in the structure of the determinant of theglucose-containing region. The presence of relatively highlevels of P-1,6 glucose and 13-1,3 galactose, based on meth-ylation and 13C-NMR analyses (Table 4, Fig. 3), suggestedthat the glucose-containing region may possibly consist ofthese two sugars.The antigenic specificity is defined by the oligosaccharide

structure within the polysaccharide antigen (15). Galactoseand glucose play major roles in the specificity of the gantigen, and many factors, their anomeric structure, positionof linkage, whether one of the sugars is at a reducing ornonreducing end or is within the chain of sugars, andwhether the sugar constitutes a branch point with a sidechain, all contribute to the antigenic expression of thepolysaccharide.

LITERATURE CITED1. Bartlett, G. R. 1959. Phosphorus assay in column chromatogra-

phy. J. Biol. Chem. 234:466-468.2. Beighton, D., R. R. B. Russell, and H. Hayday. 1981. The

isolation and characterization of Streptococcus mutans sero-type h from dental plaque of monkeys (Macaca facicularis). J.Gen. Microbiol. 124:271-279.

3. Bock, K., and C. Pedersen. 1983. Carbon-13 nuclear magneticresonance spectroscopy of monosaccharides. Adv. Carbohydr.Chem. Biochem. 41:27-66.

4. Bock, K., C. Pedersen, and H. Pedersen. 1984. Carbon-13nuclear magnetic resonance data for oligosaccharides. Adv.Carbohydr. Chem. Biochem. 42:193-225.

5. Bratthall, D. 1970. Demonstration of five serological groups ofstreptococcal strains resembling Streptococcus mutans. Odon-tol. Revy 21:143-152.

6. Brown, T. A., and A. S. Bleiweis. 1979. Chemical, immunochem-

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ical, and structural studies of the cross-reactive antigens ofStreptococcus mutans AHT and B13. Infect. Immun. 24:326-336.

7. Coykendall, A. L. 1977. Proposal to elevate the subspecies ofStreptococcus mutans to species status, based on their molec-ular composition. Int. J. Syst. Bacteriol. 27:26-30.

8. Dubois, M., K. A. Gilles, J. K. Hamilton, P. A. Rebers, and F.Smith. 1956. Colorimetric method for determination of sugarsand related substances. Anal. Chem. 28:350-356.

9. Gibbons, R. J., and R. J. Fitzgerald. 1969. Dextran-inducedagglutination of Streptococcus mutans, and its potential role inthe formation of microbial plaque. J. Bacteriol. 98:341-346.

10. Hakomori, S. 1964. A rapid permethylation of glycolipid, andpolysaccharide catalyzed by methylsulfinyl carbanion in di-methylsulfoxide. J. Biochem. 55:205-207.

11. Hirasawa, M., H. Kiyono, T. Shiota, S. M. Michalek, and J. R.McGhee. 1980. Virulence of Streptococcus mutans: immuno-chemical characterization of a serotype g-defective mutant(C307). Infect. Immun. 27:697-699.

12. Iacono, V. J., M. A. Taubman, D. J. Smith, P. R. Garant, and J.Pollock. 1976. Structure and function of the type-specific poly-saccharide of Streptococcuis mutans 6715, p. 75-90. In W. H.Bowen, R. J. Genco, and T. C. O'Brien (ed.), Proceedings:immunologic aspects of dental caries (a special supplement toMicrobiology Abstracts). Information Retrieval, Inc., Washing-ton, D.C.

13. Iacono, V. J., M. A. Taubman, D. J. Smith, and M. J. Levine.1975. Isolation and immunochemical characterization of thegroup-specific antigen of Streptococcus mutans 6715. Infect.Immun. 11:117-128.

14. Ikeda, T., T. Shiota, J. R. McGhee, S. Otake, S. M. Michalek, K.Ochiai, M. Hirasawa, and K. Sugimoto. 1978. Virulence ofStreptococcus mutans: comparison of the effects of a couplingsugar and sucrose on certain metabolic activities and cariogeni-city. Infect. Immun. 19:477-480.

15. Jann, K., and B. Jann. 1977. Bacterial polysaccharide antigens,p. 247-287. In I. W. Sutherland (ed.), Surface carbohydrates ofthe prokaryotic cell. Academic Press, Inc. (London), Ltd.,London.

16. Lindberg, B. 1972. Methylation analysis of polysaccharides.Methods Enzymol. 28:178-195.

17. Linzer, R. 1976. Serotype polysaccharide antigens of Strepto-coccus mutans: composition and serological cross reactions, p.91-99. In W. H. Bowen, R. J. Genco, and T. C. O'Brien (ed.),Proceedings: immunologic aspects of dental caries (a specialsupplement to Microbiology Abstracts). Information Retrieval,

Inc., Washington, D.C.18. Linzer, R., M. S. Reddy, and M. J. Levine. 1986. Immunochem-

ical aspects of serotype carbohydrate antigens of Streptococcusmutans, p. 29-38. In S. Hamada et al. (ed.), Molecular micro-biology and immunobiology of Streptococcus mutans. Proceed-ings of an International Conference on Cellular, Molecular andClinical Aspects of Streptococcus mutans. Elsevier SciencePublishers, Amsterdam.

19. Linzer, R., and H. D. Slade. 1976. Characterization of anantiglucosyltransferase serum specific for insoluble glucan syn-thesis by Streptococcus mutans. Infect. Immun. 13:494-500.

20. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall.1951. Protein measurement with the Folin phenol reagent. J.Biol. Chem. 193:265-275.

21. Mukasa, H., and H. D. Slade. 1973. Mechanism of adherence ofStreptococcus mutans to smooth surface. I. Roles of insolubledextran-levan synthetase enzymes and cell wall polysaccharideantigen in plaque formation. Infect. Immun. 8:555-562.

22. Ohno, N., K. lino, S. Oikawa, K. Sato, M. Ohsawa, and T.Yadomae. 1986. Fractionation of acidic antitumor ,B-glucan ofGrifolafrondosa by anion exchange chromatography using ureasolutions of low and high ionic strengths. Chem. Pharm. Bull.34:3328-3332.

23. Perch, B., E. Kjems, and T. Ravn. 1974. Biochemical andserological properties of Streptococcus mutans from varioushuman and animal sources. Acta Pathol. Microbiol. Scand. 82:357-370.

24. Prakobphol, A., and R. Linzer. 1980. Purification and immuno-logical characterization of a rhamnose-glucose antigen fromStreptococcus mutans 6715-T, (serotype g). Infect. Immun. 30:140-146.

25. Pritchard, D. G., R. L. Gregory, S. M. Michalek, and J. R.McGhee. 1986. Biochemical aspects of serotype carbohydrateantigens of Streptococcus mutans, p. 39-49. In S. Hamada et al.(ed.), Molecular microbiology and immunobiology of Strepto-coccus mutans. Proceedings of an International Conference onCellular, Molecular and Clinical Aspects of Streptococcus mu-tans. Elsevier Science Publishers, Amsterdam.

26. Strominger, J. L., J. T. Park, and R. E. Thompson. 1959.Composition of the cell wall of Staphylococcus aureus: itsrelation to the mechanism of action of penicillin. J. Biol. Chem.234:3263-3268.

27. Takada, K., J. Wyszomirska, and T. Shiota. 1984. Serologicalcharacterization of Streptococcus mutans serotype polysaccha-ride g and its different molecular weight forms. Infect. Immun.45:464-469.

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