1. Printed in U.S.A.. Purification and Immunochemical Characterization of ... glucose-glucose dimer is the predominant antigenic determinant of the e ... against distilled water, and the insoluble materials .... They are most likely nucleoprotein polymers. The eI and eII column fractions ... Only the first peak, which coincided.
Vol. 14, No. 1 Printed in U.S.A.
INFECTION AND IMMUNITY, JUlY 1976, p. 68-76 Copyright ©D 1976 American Society for Microbiology
Purification and Immunochemical Characterization of Type e Polysaccharide Antigen of Streptococcus mutans SHIGEYUKI HAMADA AND HUITON D. SLADE* Department of Microbiology-Immunology, Northwestern University Medical and Dental Schools, Chicago, Illinois 60611 Received for publication 1 March 1976
The type-specific antigen of Streptococcus mutans strain MT703, serotype e, has been chromatographically purified and characterized. Two chromatographic fractions were obtained from saline extracts which reacted with both anti-MT703 whole-cell serum and Lancefield group E serum. The major fraction (eI) was identified as a polysaccharide composed of 37% glucose, 56% rhamnose, 5% protein, and 0.3% phosphorus, whereas the minor fraction (eII) contained 66% protein in addition to 10% glucose and 17% rhamnose. The immunological specificity of these antigens was found to be the same by immunodiffusion in agar gel. Another fraction with a negative charge (eIII) reacted with polyglycerophosphate antisera from Streptococcus mutans and Streptococcus pyogenes. For comparison, the MT703 antigen in a hot trichloroacetic acid extract (eA) and the group E antigen from a saline extract of cells of strain K129 (EI) were similarly purified by anionic ion-exchange chromatography. Although the ratio of glucose and rhamnose in eA was 1:0.9 and in eI and eII approximately 1:1.5, reactions of identity were obtained in gel diffusion against specific anti-e serum. This difference in ratio is probably a result of the extraction procedures. Both the type e and group E antisera were reactive with both eI and EI antigens. The adsorption of group E antiserum with MT703 cells removed all E antibody, whereas type e-specific antibody remained after adsorption with K129 cells. These results suggest that eI antigen possesses both e and E specificities, whereas EI possesses E only. These findings were supported by the quantitative precipitin test and immunodiffusion and/or immunoelectrophoretic patterns in agar gel. Methyl-,-i->glucopyranoside markedly inhibited the precipitin reaction in both type e and group E sera. However, a significantly stronger inhibition by cellobiose of type e serum than of group E serum indicates that a ,-linked glucose-glucose dimer is the predominant antigenic determinant of the e specificity. The presence of both e and E specificities on a single polysaccharide molecule was demonstrated by the use of purified e antigen released from a specific e-anti-e complex. This antigen reacted with a group E-specific serum as well as a type e-specific serum. An examination of five S. mutans type e strains showed the presence of group E specificity also, whereas the I, II, and IV serotypes of group E streptococci only possessed the group E specificity. Strains of Streptococcus mutans, an agent that develops dental caries in animals and probably humans, have been subdivided into seven immunological types (2, 30). The antigens of types a (27), b (26), c (16, 42), d (13, 18) and f (8a) are located in the cell wall and are polysaccharides composed of rhamnose and glucose (type c and f), rhamnose and galactose (type b), and glucose and galactose (types a and d). S. mutans strains do not show any immunological cross-reaction with the streptococcal groups except group E (3, 30). Bratthall (3) considered that type e S. mutans possessed the same antigenic determinant as that of the group E streptococcal antigen
(38). This conclusion was derived from the fact that the antigen extract of type e S. mutans gave a precipitin line in agar gel against antiserum specific for group E streptococci which fused with that formed between group E antigen and group E antiserum. However, we have found (10) that antiserum obtained by immunizing rabbits with type e S. mutans cells showed type e specificity in addition to group E specificity. Type e-specific antibody was obtained by adsorption of antiserum against type e S. mutans cells with group E streptococcal cells. The adherence of type e S. mutans cells to a glass surface in the presence of sucrose and 68
VOL. 14, 1976
POLYSACCHARIDE TYPE e ANTIGEN OF S. MUTANS
crude glucosyltransferases was strongly inhibited by pretreatment of the cells with type especific antibody, whereas no significant inhibition of adherence by group E-specific antibody was obtained (10). The present paper describes the immunological and chemical characterization of the type e antigen and a comparison of its properties with those of the group E polysaccharide antigen (38). MATERIALS AND METHODS Streptococcal strains and culture conditions. S. mutans strain MT703 and streptococcus group E strain K129 were used. The former strain was isolated from the dental plaque of a carious tooth of a Japanese child and was identified as serotype e (9, 10). The latter strain was isolated from milk and was kindly supplied by R. C. Lancefield, Rockefeller University, New York. Both strains were grown in Todd-Hewitt broth (Difco) supplemented with glucose and salts (12). The cells were collected by centrifugation at 10,000 x g for 20 min, washed once with saline and then twice with water, and lyophilized. Extraction of the antigens. The type e streptococcal antigen was extracted from the whole cells of strain MT703 according to a modified method of Rantz and Randall (32). The cells were suspended in saline (10 g [dry weight]/200 ml) and autoclaved at 120 C for 30 min. These cells were extracted two additional times under the same conditions. All centrifuged supernatants were combined and dialyzed against distilled water, and the insoluble materials that appeared during dialysis were removed by one additional centrifugation at 6,000 x g for 30 min. The superrtatant was passed through a membrane filter (Millipore Corp., 0.45 am) and lyophilized. The crude antigen weighed 1.58 g. Group E streptococcal cells were similarly treated as described above. MT703 whole cells were also extracted with 5% trichloroacetic acid (37). Chromatographic purification of the crude antigen. The crude saline-extracted MT703 antigen (1.58 g) was dissolved in 12 ml of 0.05 M ammonium carbonate solution (ph 8.5) and dialyzed against 2 liters of the same solution to remove traces of NaCl which could affect the chromatographic profile. The antigen was then applied to a column (30 by 1.5 cm) of diethylaminoethyl (DEAE)-Sephadex A-25 (Pharmacia Fine Chemicals, Inc., Piscataway, N.J.) which had been equilibrated with 0.05 M ammonium carbonate. The elution was performed using the following buffers: 100 ml of 0.05 M ammonium carbonate, linear gradient of 0.05 and 1.0 M ammonium carbonate (each 200 ml), followed by 200 ml of ammonium bicarbonate containing 1 M NaCl. The fractions that reacted with anti-type e and antigroup E sera were combined, dialyzed against distilled water, and lyophilized. Further purification was attempted using Sephadex G-200 (Pharmacia) and carboxymethylcellulose (CM)-Sephadex A-25 (Pharmacia). In the gel filtration experiment, a Sephadex G-200 column (60 by 2.5 cm) was used and eluted with saline. In the second approach a column of CM-Sephadex A-25 (30 cm by 1.5 cm) was equili-
69
brated with 0.01 M ammonium bicarbonate. Elution was started with 100 ml of this solution, followed by a linear gradient of 0.01 M ammonium bicarbonate and 0.01 M ammonium bicarbonate containing 1 M sodium chloride (200 ml each). The trichloroacetic acid-extracted antigen of type e S. mutans and saline-treated group E antigen were also purified by passing through DEAE-Sephadex A-25 column as described above. The fractions reactive with specific antisera were combined, dialyzed against water, and lyophilized. No further purification was attempted. Antisera. Antiserum specific for type e S. mutans was obtained by immunizing rabbits with merthiolate-killed whole cells of strain MT703. Intravenous injections were performed as described previously (10). It was found that the antiserum was also reactive with polyglycerophosphate (PGP) in addition to type e and group E antigens. Therefore, the antiserum was adsorbed with the whole cells of group A S. pyogenes strain Richard before use to eliminate the antibody against PGP (10). To obtain type e-specific antibody, antiserum against MT703 whole cells was adsorbed with whole cells of group E streptococcus strain K129. For this purpose, 2 ml of the antiserum was added to 100 mg (dry weight) of K129 cells and incubated at 37 C for 1 h and 4 C overnight with occasional shaking. The cells were removed by centrifugation, and the supernatant was used as type e antibody. Group E antiserum was provided by the Communicable Disease Center, Atlanta, Ga. Immunochemical procedures. Capillary precipitin tests and immunodiffusion in agar gel were done as described previously (10). Immunoelectrophoresis was carried out in Noble agar (Difco) (1% Lwt/vol]) containing 0.05 M barbital-hydrochloride, pH 8.2, on a microscopic slide under the constant current of 6 mA for 1.5 h and then stored in the cold for 18 h. To establish the nonidentity of the antigenic determinant between type e S. mutans and group E streptococci, a modified immunoelectrophoresis (41) procedure was performed as described (2). The quantitative precipitin reaction (37) was carried out as follows: antigen in saline was added to 2.5 A1 of type e antiserum or 10 M1 of group E antiserum, and the final volume was adjusted to 50 MA1. The mixture was incubated at 37 C for 1 h and then 4 C for 24 h. The precipitate was washed three times with cold saline, and the protein content was determined (20). Inhibition of the quantitative precipitin test was measured by the addition of 2 mol of inhibitor dissolved in 10 ML1 of saline to 10 M1 of fourfold diluted anti-MT703 serum and incubated at 37 C for 1 h. Two micrograms of purified type e antigen was then added to the mixture and further incubated at 37 C for 1 h and 4 C for 24 h. The protein content of the precipitate was determined as described above. Demonstration of type-specific antigen by immunoadsorption. The procedure was modified from that previously described (17, 22). To 150 Mul of the type e antibody, 150 Ml of saline and 60 M1 of chromatographically purified e antigen solution (1 Mg/Ml) were mixed and incubated at 37 C for 1 h and 4 C for 18 h. After incubation the mixture was centrifuged in the cold and washed three times with 40 MAl of
70
INFECT. IMMUN.
HAMADA AND SLADE
saline. One hundred and fifty microliters of 0.02 N HCI was then added to the precipitate to dissociate the antigen-antibody complex. After frequent mixing at room temperature for 15 min, 150 1AI of 20% trichloroacetic acid was added to precipitate the antibody. The supernatant was neutralized with 2 N NaOH and dialyzed against saline, and the immunological specificity was examined for type e and group E antigens. Lability of type e antigen with acid and glucosidases. Chromatographically purified type e antigen was dissolved in 400 ,1. of cold 0.2 N HCI in a final concentration of 5 ,g/,ul. Aliquots (60 ,1 each) were taken in small test tubes, and acid hydrolysis of the antigen was carried out at 100 C for 15, 30, 45, 60, 90, and 120 min in sealed ampoules. After hydrolysis, they were dried in vacuo, and 30 ,ul of distilled water was added to each tube. Aliquots (5 ,u) of the hydrolysates were spotted on a thin-layer plate (Cellulose MN300, Brinkmann Instruments Inc., Westbury, N.Y.) and developed in butanol-pyridinewater (6:4:3, [vol/vol]) (38) to determine the sugars released by hydrolysis of the antigen. Ten microliters of each hydrolysate was diluted with 90 ,ul of saline, and twofold dilutions were tested by the capillary precipitin procedure against type e antiserum. One milligram of type e antigen was dissolved in 400 ,ul of 0.01 M acetate buffer (pH 5.6) containing 20 ,ug of 18-glucosidase (Worthington Biochemicals, Freehold, N.J., 2.5 U/mg) and incubated for 2 h at 37 C. The same quantity of antigen was treated with a-glucosidase (type I, Sigma Chemical Co., St. Louis, Mo., 3 U/mg) in 400 ,l. of phosphate buffer (0.01 M, pH 6.8). The reactions were stopped by
holding in boiling water for 2 min. Twofold dilutions were made in saline. Antigenic activity was checked by the capillary precipitin test (39) against type especific serum. Chemical analyses. Total hexose was estimated by the anthrone method (35), and total phosphorus and protein were estimated according to the procedures of Lowry et al. (19, 20). Ribonucleic acid (RNA) was estimated by method of Mejbaum (23), using ribose as standard. Quantitative determination of sugars were done by using gas-liquid chromatography (25).
RESULTS Chemical purification of type e antigen. Autoclaved saline extract of S. mutans MT703 cells was applied to a column of DEAE-Sephadex A-25 and eluted as described above. The elution profile is shown in Fig. 1. The fractions which contained type e antigen were monitored by the capillary precipitin test against type e and group E antiserum. The main antigenic fraction (eI) passed through the column without adsorption to the resin and was essentially composed of sugars with a small amount of protein. The second antigenic peak (eII) appeared with gradient elution. Both peaks reacted with both type e and group E antisera. The third peak (eWIM) reacted with anti-PGP (glycerol teichoic acid) serum. This fraction contained phosphorus, but no detectable amounts of protein, hexose, and ribose were found. The remaining one peak was composed
FRACTION NUMBER (10 mI/TUBE) FIG. 1. Chromatographic separation on a DEAE-Sephadex A-25 column of hot saline-extracted antigen from type e S. mutans MT703. Crude extract in 0.05 M ammonium carbonate was loaded on a column (1.5 by 35 cm) and the column was washed with the starting buffer (110 ml), followed by a linear gradient elution of 0.05 to 1.0 M ammonium carbonate (200 ml each). Then, the column was eluted with 1.0 M ammonium bicarbonate containing 1 M NaCl (200 ml). Total hexose, total phosphorus, protein, and ribose were determined in each fraction. Serologically active fractions were monitored by capillary precipitin test, and the active fractions are marked eI, eII, and eIII.
POLYSACCHARIDE TYPE
VOL. 14, 1976
essentially of phosphorus and ribose and did not immunological activity. They are most likely nucleoprotein polymers. The eI and eII column fractions were dialyzed against distilled water and lyophilized. Passage through Sephadex G-200 did not achieve a further separation. No further purification of eII was attempted. eI was applied to a CM-Sephadex C-25 column in bicarbonate buffer and found to be eluted without adsorption. This suggested that eI was chromatographically homogenous, although it reacted with both type e and group E antisera. The elution pattern on a trichloroacetic acid extract of MT703 cells from a column of DEAE-Sephadex A-25 was essentially similar to that of the saline-autoclave extract. Only the first peak, which coincided with eI, was used for comparison and was designated as eA. A saline-autoclave extract of Lancefield group E streptococcus K129 cells was similarly chromatographed. Again the polysaccharide peak (EI) showed reactivity against anti-group E and anti-MT703 sera. The EI preparation was used as group E reference antigen. All these antigenic materials were dialyzed against distilled water and lyophilized. Chemical composition of the antigens. Chemical and gas-liquid chromatographic determinations revealed that el, eA, and EI were composed principally of rhamnose and glucose with 2 to 5% protein and 0.1 to 0.4% phosphorus (Table 1). eII contained 65% protein. The antigen from the autoclave extract showed a rhamnose-glucose ratio of 1:150, whereas the tricholoroacetic acid antigen showed a 1:0.91 ratio. The composition of EI antigen was very similar to that of the trichloroacetic acid-extracted E antigen reported previously (38). Immunochemical characterization of the antigens. Immunodiffusion tests with MT703 serum revealed an identical specificity for el, eII, and eA. Although EI cross-reacted with anti-MT703 serum, it lacked the major precipitin line which appeared between homologous type e antigen and anti-MT703 serum (Fig. 2). The difference in antigenicity between type e and group E polysaccharide was shown by adsorption of the antisera with homologous cells. This procedure resulted in the complete loss of
possess any
e
ANTIGEN OF S. MUTANS
antibodies specific for type e and group E polysaccharides. When the antiserum against MT703 cells (type e) was adsorbed with K129 (group E) cells, the adsorbed serum was reactive with type e antigen only. On the other hand, adsorption of the K129 antiserum with whole cells removed group E antibody from the antiserum. These results suggest that type e S. mutans has two immunological specificities, factors e and E, whereas group E streptococcus has only one immunological factor. The immunological relationship between e and E specificities was investigated in more detail by immunoelectrophoresis. Figure 3a shows that el migrated toward the cathode and formed precipitin arcs with the same mobility against anti-MT703 serum that had been adsorbed with either group A S. pyogenes (upper trough) or group E (lower trough) whole cells, although the intensity of the precipitin arc of the former combination was stronger than that of the latter. The nonidentity between e and E specificity is demonstrated in Fig. 3b and 3c. eI was first subjected to electrophoresis in the usual manner and EI was then placed in both upper troughs. Anti-MT703 serum adsorbed with group A S. pyogenes cells (Fig. 3b) or group E (Fig. 3c) was added to the lower troughs and immunodiffusion was allowed to develop in the cold room. EI formed a precipitin line in parallel with the troughs against anti-e (MT703) serum adsorbed with group A streptococcal cells (Fig. 3b). However, the precipitin arc which formed between eI and the antiserum showed the nonidentity of the two antigens. On the
FIG. 2. Immunodiffusion in agar gel showing the different specificity of type e (0.6 pg) (upper left well) and group E (0.6 pAg) (upper right well). The lower well contains 10 p1 of anti-MT703 serum adsorbed with group A S. pyogenes whole cells.
TABLE 1. Chemical composition of various antigens purified by chromatography Weight (%)
Antigen
Extraction method Rhamnose
eI eII eA EI
Autoclave-saline Autoclave-saline Trichloroacetic acid Autoclave-saline
55.7 17.1 45.5 64.3
71
Glucose
37.1 10.2 49.8 30.8
Protein
Phosphorus
Total
5.4 65.9 2.0 4.5
0.28 1.5 0.08 0.39
98.4 94.7 97.4 99.7
72
HAMADA AND SLADE
INFECT. IMMUN.
ANTI-e ADSORBED W~ITH A CELL a
10
el o
*-)
ANTI-E ADSORBED WNITH E CELL
EI b
eIo ADSORBED WITH A CE elTI-F
%-0
Q)
0
5
ANTI-El ADSORBED VflTH A CELL
EI 0
c
eI o ANT= eA ANTI-fl ADSORBED WI1TH E CELL
Antigen (pg)
FIG. 4. Quantitative precipitin curves for eI and EI and anti-MT703 serum adsorbed with group A S. pyogenes cells and anti-group E serum in different FIG. 3. Immunoelectrophoresis demonstration of combinations. For each test, 2.5 and 10 p1 of antisera the type e and group E specificities. Two micrograms specific for type e and group E antigens were added of eI in each center well was subjected to 6 mAlslide with different amounts of antigens. Final volume for 90 min. To the upper trough of slide 1 was then was adjusted to 50 ,l. Antigen-antibody combinaadded 50 pi of anti-MT703 serum which had been tions: *-*, anti-type e versus eI; 0----0, antiadsorbed with group A streptococcal cells, and to the group E versus EI; 0 O, anti-type e versus EI; lower trough was added 50 pi of anti-MT703 serum and @----@, anti-group E versus eL. which had been adsorbed with group E streptococcal cells. To the upper trough of slide 2 was added 50 pg of EI in saline, and to the lower trough was added 50 i1 anti-MT703 serum which had been adsorbed with group A streptococcal cells. To the upper trough of TABLE 2. Hapten inhibition of the quantitative slide 3 was added EI (50 pgli00 pi), and to the lower precipitin reaction between eI and anti-type e seruma trough was added anti-MT703 serum (100 pI) which had been adsorbed with group E streptococcal cells. Hapten Inhibition (%) Diffusion then proceeded for 18 h in each case. Rhamnose ....................... 7 Glucose ......................... 77 15 other hand, Fig. 3c shows that EI formed no a-Methyl-D-glucopyranoside 76 precipitin line with the e antiserum adsorbed 13-Methyl-D-glucopyranoside 15 with group E cells although a precipitin arc Sucrose (fructose-glucose) Melibiose (galactose-a-1,6-glucose) 7 appeared between eI and the antiserum. Maltose 49 Quantitative precipitin reactions were per- Isomaltose(glucose-a-1,4-glucose) (glucose-a-1,6-glucose). 51 formed (Fig. 4) using anti-MT703 serum ad- Raffinose (galactose-a-1,6-glucosesorbed with group A cells and anti-group E 24 ,8-1,2-fructose) ................. serum and antigens eI and EI. Four different Cellobiose (glucose-f3-1,4-glucose). 85 combinations were used. Homologous reactions " e serum Anti-type (2.5 ,ul) was incubated with 2 between anti-MT703 serum and eI and between ,umol of the hapten for 1 h at 37 C, followed by anti-group E serum and EI reacted more in- addition of the purified eI (2 ug). Final volume of the the tensely than the heterologous reactions be- reaction mixture was adjusted to 50 ul. After 18 h of tween anti-MT703 serum and EI, and between incubation at 4 C the precipitate was assayed for anti-group E serum and eL. protein.
POLYSACCHARIDE TYPE e ANTIGEN OF S. MUTANS
VOL. 14, 1976
Quantitative precipitin inhibition tests showed that cellobiose, glucose, and f3-methylD-glucopyranoside were the best inhibitors of the reaction between eI and anti-MT703 wholecell serum (Table 2). It is of interest that maltose and isomaltose also significantly inhibited the precipitin reaction, although they are both a-linked glucose-glucose dimers. The results indicate that a 8-linked glucose-glucose contributes to the type e specificity. Identification of E and e specificities on the type e polysaccharide molecule. To determine whether the E and e specificities resided on the same antigen molecule, eI was mixed with type e-specific serum that contained no group E antibody. The complex was dissociated with acid, and type e-specific antigen was recovered from the antigen-antibody complex. The reactivity of this antigen with anti-group E and anti-type e sera was then tested using the capillary precipitin test. A strongly positive reaction was observed with both antisera. This indicates that the eI polysaccharide possessed the E and e specificities. Lability of the antigen and release of immunodeterminant sugars. eI was submitted to acid hydrolysis for varying times, and both the release of monosaccharide and the loss of anti-
73
genicity were monitored by the capillary precipitin test. Hydrolysis in 0.2 N HCl at 100 C for 15 min resulted in a release of glucose (Fig. 5) with concomitant loss of antigenicity (Table 3). Hydrolysis for 1 h abolished the antigenicity, although the release of rhamnose and glucose continued. However, enzymatic hydrolysis with a- and 8-glucosidase failed to release any detectable glucose or to reduce the antigenicity of eL. DISCUSSION The presence of type e-specific antigen was demonstrated in the hot saline or trichloroacetic acid extract of the whole cells of S. mutans strain MT703 and in the enzymatic lysate of cell walls (10). Hardie and Bowden (11) reported that rhamnose and glucose were present in the wall of type e strains. The rhamnose-glucose concentration (93%) in eI polysaccharide (Table 1) indicates that this antigen may be a major polysaccharide of the cell wall. Our finding that the E preparation (38) and the e antigen (Table 2) have a similar sugar composition is in agreement with our conclusion that the E and e immunological specificities reside in the same molecule. Therefore, it
rhoamnose Vr v Ig(u cose
l
WV
w *
V
I
S 15 3045 S
6090120
TIME (MIN) FIG. 5. Thin-layer chromatogram showing the release of sugars from ej with partial acid hydrolysis (cf. Table 3).
74
HAMADA AND SLADE
INFECT. IMMUN.
TABLE 3. Loss of immunological activity of eI during partial acid hydrolysisa Capillary precipitin reaction Time (min) of hydrolysis
(dilution x 2n) 0
1
2
3
4
5
6
7
8
9
1+
2+ -
3+ 2+ 1+
3+ 3+ 2+ 1+
3+ 2+ 1+ 1+
2+ 2+ -
2+ 1+ -
1+
-
-
-
-
-
60
-
-
4+ 3+ 1+ 1+ 1+
-
-
-
-
-
-
90
-
-
_
_
_
_
_
120
-
-
-
-
-
-
-
0 15 30 45
-
-
-
-
-
eI was hydrolyzed in 0.2 N HCI at 100 C, and an aliquot was removed at the time indicated. The final concentration of the antigen was adjusted to 1 mg/ml with saline and twofold dilutions were made.
was of interest to compare the nature of the determinant in each case, as assessed by the method of ligand inhibition. f3-Methyl-D-glucoside was an excellent inhibitor in both cases. Cellobiose (,8-1,4), however, was 85% effective on the e site (Table 2) and only 38% effective on the E site (38). Maltose (a-1,4) was 49% effective on the e site and 16% effective on the E site. These results indicate that 83-linked glucose dimers are more likely than the a-linked type to represent the dominant group of the e specificity, whereas /3-linked single glucose units may be the dominant group in the E specificity (38). It is likely, however, that type e and group E specificities depend in part on the stereochemical configuration of the terminal and nonterminal glucosidic linkages (5). This explanation is also supported by the quantitative precipitin reaction as shown in Fig. 4. This type of multiple specificity on the single antigen molecule has been demonstrated in the polysaccharide antigens from type a and d S. mutants (17, 27) and type f S. mutans (8a). The presence of glucose as a determinant in the antibody combining site is further illustrated by the rapid release of the hexose during acid hydrolysis (Fig. 5, Table 3). The slow release of rhamnose and its inability to inhibit the precipitin reaction (Table 2) indicates that it possesses a minor role in the antigenic specificity. Cross-reactions between extracts of c, e, and f S. mutans cells and whole-cell antibody have been reported (10, 16, 30). In each case, however, the purified antigen does not show such reactions. Rhamnose and glucose are the principal components of the antigen in each of these antigens. The c and f antigens possess an alinked glucose specificity, whereas the e is probably /3-linked. A further difference in the nature of the c and e determinants as compared with the f is illustrated by failure of the c and e antigens to react with concanavalin A-Sepha-
rose. These results suggest that the presence of a-glucosidic linkages does not assure a reaction with concanavalin A. Genetic homology as shown by deoxyribonucleic acid (DNA)DNA hybridization for e and c strains has been shown (7), although e possesses a /-linked determinant and c possesses an a-linked determinant. The difference of the rhamnose-to-glucose ratio between eI and eA may be due to the different extraction methods used. The hot saline extraction (32) of MT703 whole cells was used because of its simplicity and reproducibility. The usefulness of this method has been confirmed with cells from different Lancefield groups of streptococci by several workers (14, 28, 33) in addition to S. mutans (9). It should be noted, however, that the saline extract was separated into three antigenic fractions on an anionic column. The first two antigenic fractions (eI and eII) were shown to be immunologically identical, although elI contained more than 60% protein. Therefore, eII could be considered to represent a fragment of eI which contained cellular protein. Similar findings were reported on the type c and f antigens (8a, 16, 42) which had been extracted with hot trichloroacetic acid, formamide, or saline. eII and another peak containing phosphorus and ribose appeared during linear gradient elution (Fig. 1). eIII contained phosphorus but no detectable ribose and showed specific reactivity against anti-PGP (glycerol teichoic acid without substituted sugars) serum. The second phosphorus peak showed no serological activity, and the conclusion was drawn that it was nucleic acid in nature. PGP was found to be the cross-reacting antigen among many gram-positive bacteria (6, 21, 36), including S. mutans (4, 16, 18, 27). Recently, we have found that the addition of DEAE-Sephadex resin to a crude antigen extract selectively adsorbed the cross-reacting PGP without de-
VOL. 14, 1976
POLYSACCHARIDE TYPE e ANTIGEN OF S. MUTANS
tectable reduction of its antigenicity (Hamada, Tai, and Slade, unpublished data). Although it was found that cross-reacting antibody directed against PGP in the whole-cell antiserum couild be adsorbed with group A S. pyogenes cells (10), this adsorption was also accomplished with S. mutans cells from all immunological types. The presence of PGP in numerous streptococci (21) supports the use of these two species for the adsorption of PGP antibodies. The identity of the I, II, III, and IV antigens (24, 29, 43) of the group E streptococcus remains to be elucidated. Our experiments show that hot saline extracts of type I (strain K129), II (K131), and IV (Newson) cells did not give cross-reactions against anti-MT703 e-specific serum. These results indicate that the type antigens of group E cells are different in antigenic specificity from the type e antigen of S. mutans.
It is of interest to note that type e S. mutans occurs on tooth surfaces and occasionally is recovered from cases of subacute endocarditis. Group E streptococcus, however, is mostly of animal origin and is a frequent cause of cervical lymphadenitis in swine (1, 8, 31, 34, 40). Although no information is available on a DNA homology between group E and type e streptococci, their many common biochemical characteristics indicate a phylogenetic relationship. ACKNOWLEDGMENTS This investigation was supported by Public Health Service research grants HE-03709-18 from the National Heart and Lung Institute and DE-03615-04 from the National Institute of Dental Research, and by grants from the Grainger Fund, the Pioneer Fund, and the Hemac Fund. H.D.S. is the recipient of Public Health Service research career award K6-GM-16284 from the National Institute of General Medical Sciences. LITERATURE CITED 1. Armstrong, C. H., and J. B. Pyne. 1969. Bacteria recovered from swine affected with cervical lymphadenitis (jowl abscess). Am. J. Vet. Res. 30:1607-1612. 2. Bratthall, D. 1969. Immunodiffusion studies on the serological specificity of streptococci resembling Streptococcus mutans. Odontol. Revy 20:231-244. 3. Bratthall, D. 1970. Demonstration of five serological groups of streptococcal strains resembling Streptococcus mutans. Odontol. Revy 21:143-152. 4. Chorpenning, F. W., H. R. Cooper, and S. Rosen. 1975. Cross-reactions of Streptococcus mutans due to cell wall teichoic acid. Infect. Immun. 12:586-591. 5. Cisar, J., E. A. Kabat, M. M. Dorner, and J. Liao. 1975. Binding properties of immunoglobulin combining sites specific for terminal or nonterminal antigenic determinants in dextran. J. Exp. Med. 142:435-459. 6. Coley, J., M. Duckworth, and J. Baddiley. 1972. The occurrence of li?oteichoic acid in the membranes of gram-positive bacteria. J. Gen. Microbiol. 73:587591. 7. Coykendall, A. L. 1974. Four types of Streptococcus mutans based on their genetic, antigenic and bio-
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