INFFCTION AND IMMUNITY, Jan. 1977, p. 91-103 Copyright © 1977 American Society for Microbiology

Vol. 15, No. 1 Printed in U.S.A.

Antigenic Relatedness of Glucosyltransferase Enzymes from Streptococcus mutans DANIEL J. SMITH* AND MARTIN A. TAUBMAN Department of Immunology, Forsyth Dental Center, Boston, Massachusetts 02115 Received for publication 7 July 1976

The antigenic relationship of glucosyltransferases (GTF) produced by different serotypes of Streptococcus mutans was studied by using a functional inhibition assay. Rat, rabbit, or hamster immune fluids, directed to cell-associated or supernatant-derived GTF, were tested against ammonium sulfate-precipitated culture supernatants containing GTF from seven'strains ofS. mutans representing six different serotypes. An antigenic relationship was shown to exist among GTF from serotypes a, d, and g, since both rat and rabbit antisera directed to serotype a or g GTF inhibited GTF of serotypes d and g similarly and both antisera also inhibited serotype a GTF. Furthermore, serum inhibition patterns indicated that GTF of serotypes c and e, and possibly b, are antigenically related to each other, but are antigenically distinct from GTF of serotype a, d, or g. Serum antibody directed to antigens other than enzyme (e.g., serotype-specific antigen or teichoic acid) had little effect on the inhibition assay. Salivas from rats immunized with cell-associated or supernatant-derived GTF exhibited low but consistent inhibition of GTF activity, which generally corresponded to the serum patterns. The sera of two groups of hamsters immunized with GTF (serotype g), enriched either in water-insoluble or water-soluble glucan synthetic activity, gave patterns of inhibition quite similar to those seen with sera from more heterogeneous cell-associated or crude supernatant-derived GTF preparations. Both groups of hamster sera also gave virtually identical patterns, suggesting that the two enzyme forms used as antigen share common antigenic determinants. The results from the three animal models suggest that among the cariogenic organisms tested, two (serotypes a, d, g and b, c, e), or perhaps three (serotypes a, d, g; b; and c, e), different subsets of GTF exist that have distinct antigenic determinants within a subset.

Cariogenic Streptococcus mutans produce glucosyltransferase (GTF) enzymes, which are present both extracellularly (45) and associated with the cell surface (20, 32). These enzymes synthesize the glucose polymers (glucans) which seem to be required for the adherence of S. mutans to the tooth surface, leading to the formation of dental plaque (for a review, see reference 18). Both water-soluble and waterinsoluble glucans are products of GTF synthetic activity (6). The critical role of GTF in the pathogenesis of dental caries caused by S. mutans has given rise to their use as antigens in in vivo immunization experiments designed to determine the effectiveness of these enzymes in eliciting a caries-protective immune response (2, 19, 24, 41). Experiments using GTF derived from S. mutans serotype g have demonstrated a measure of protection with these antigens in rodent models (41). At least seven distinct S. mutans serotypes have been described (3, 36). Representative strains from each serotype have been isolated

from the human oral cavity. Serotypes c and d have been recovered from humans in the United States with a frequency of approximately 80 and 10%, respectively (42). The extracellular GTF elaborated by S. mutans of each serotype do not appear to be identical based on electrophoretic mobilities of the enzymes themselves (7) or on analysis of their glucan products (35). Assays of functional inhibition of GTF activity using rabbit antisera directed to whole cells of five serotypes (12, 16) or directed to a purified GTF preparation (serotype a) (15) indicated that certain similarities existed between GTF of serotypes a and d. However, functional (12) or structural (15) measurements of GTF relatedness using these antisera suggested that serotypes a and d were immunochemically distinct from other S. mutans serotypes tested. On the other hand, an antiserum to another GTF preparation from serotype a could be shown to react with GTF from five different serotypes (31). Since GTF have potential as antigens that 91

92

SMITH AND TAUBMAN

can elicit caries-protective antibody, their apparent functional and biochemical heterogeneity necessitates a clarification of the antigenic relationships among GTF of the various S. mutans serotypes. Therefore, the experiments described herein were designed to investigate the ability of anti-GTF sera to interfere with glucan formation by GTF derived from six serotypes of S. mutans. Antisera were directed to (i) cell-associated GTF, (ii) crude preparations of extracellular GTF, or (iii) GTF preparations enriched in water-insoluble or water-soluble product synthesis. Furthermore, since potential anti-GTF immune protective effects may be mediated by saliva, these secretions were also

studied for enzyme-inhibitory properties. MATERIALS AND METHODS Bacteria. S. mutans strains E49 (serotype a), FA1 (serotype b), and Ingbritt (serotype c) were obtained from B. Krasse. Strains HS6 (serotype a), B13 (serotype d), and LM7 (serotype e) were obtained from H. Jordan. S. mutans strain 6715 (serotype g) (23, 25, 36) was originally obtained from J. van Houte. Strains E49 and 6715 were both resistant to streptomycin at a concentration 2,000 ug/ml. Enzymes from all strains formed water-insoluble polysaccharide from sucrose and incorporated ['4C]glucose (from [14C]glucosyl-labeled sucrose) into ethanol-insoluble polysaccharide. Cells to be used as a vaccine were grown for 16 h at 370C in Trypticase soy broth (Bioquest) supplemented with 0.8% glucose and then centrifuged, washed, and formalinized as described previously (40). A completely dialyzable culture medium for growth of bacterial cells was obtained by using the dialysate of brain heart infusion (Bioquest), which had been supplemented with 0.8% glucose and 3 g of K2HPO4 per liter (5). The medium was used to prepare enzyme for the ['4C]glucose incorporation assay. After anaerobic growth of bacteria for 16 to 24 h at 37°C, cultures were centrifuged (18,000 x g) to obtain a cell-free supernatant. Supernatants were precipitated at 40C with ammonium sulfate to 55% saturation. The precipitate was washed with 60% ammonium sulfate, dissolved in distilled water, and extensively dialyzed against distilled water. The dialyzed precipitates were lyophilized and stored at -20°C until use. Antigens. Washed S. mutans cells containing cell-associated GTF were prepared and formalinized as previously described (40). Crude enzyme antigens (CEA) from serotype g (strain 6715) and serotype a (strain E49) were prepared for rat and rabbit injection. CEA from S. mutans strain 6715 was obtained as previously described (41). Briefly, 6 to 10 liters of S. mutans culture supernatant was concentrated, dialyzed, and chromatographed on diethylaminoethyl-cellulose in 0.01 M sodium phosphate buffer, pH 6.8, and eluted in a NaCl gradient to 0.6 M in phosphate buffer, pH 6.8. Fractions forming water-insoluble polysaccharide from 0.125 M sucrose (4 h at 37°C)

INFECT. IMMUN. were pooled. Some of these fractions also formed water-soluble (EtOH-insoluble) polysaccharide. After concentration by negative pressure, the pool was gel filtered on a column of Sepharose 4B (Pharmacia Fine Chemicals, Inc.). Volid volume (V0) fractions formed water-insoluble polysaccharide, whereas fractions having a relative elution volume (Ve/Vo) of 2 formed EtOH-insoluble polysaccharide. Both fractions contained GTF activity as evaluated by chemical assays (41) and by the ['4C]glucose incorporation assay. To include all antigenic forms of GTF, the two fractions were pooled for injection. No serotype carbohydrate antigen could be detected in the combined pool (5 mg/ml) when tested in gel diffusion and immunoelectrophoresis using purified rabbit immunoglobulin G (IgG) antibody (1.5 mg/ ml) to the serotype g antigen. This fraction did react in gel diffusion with rabbit antisera to the glycerolphosphate backbone of streptococcal teichoic acid (obtained from T. Myoda, Dupont Research Institute). CEA from S. mutans strain E49 was prepared by J. Bulkacz using the method of Guggenheim and Newbrun (20). Culture supernatants of strain E49 were eluted stepwise (0.2 and 0.5 M sodium phosphate) on hydroxylapatite equilibrated with phosphate buffer, pH 6.1. The enzyme-containing fractions, which eluted in 0.5 M phosphate, were pooled and isoelectrically focused. One major peak of enzymatic activity (determined by chemical assay) was obtained at pH 4.5 and used for rabbit injection. The solubility of the glucan synthesized by this fraction and the presence of serotype carbohydrate antigen or teichoic acid were not evaluated before injection. Two defined enzyme antigens (DEA) to be used for hamster injection were also obtained as previously described (41). Diethylaminoethyl-cellulose chromatography and gel filtration on Sepharose 4B were used in the preparation of these antigens. DEA-1 was a high-molecular-weight (eluting at the V0 of the Sepharose 4B column) GTF preparation that formed a flocculent water-insoluble polysaccharide product and only trace amounts of water-soluble polysaccharide. One band of protein corresponding to this enzymatic activity entered a 7% gel after polyacrylamide disc gel electrophoresis. DEA-1 also contained enzyme that did not penetrate the gel. DEA-2 eluted from the column of Sepharose 4B with a relative elution volume = 1.9. This fraction formed some finely dispersed water-insoluble polysaccharide and considerable water-soluble polysaccharide. Several protein-staining bands were observed after disc gel electrophoresis (7% gel) of DEA-2. Immunization. Groups of weanling SpragueDawley rats (CD strain, Charles River) were injected subcutaneously in the salivary gland vicinity with formalinized S. mutans. These rats did not harbor indigenous S. mutans. For injection, 5 x 108 cells in phosphate-buffered saline were incorporated (vol/vol) into complete Freund adjuvant and injected in a total volume of 0.2 ml. Four additional injections at 7- to 10-day intervals followed. Approximately 7 days after this immunization regimen, rats were bled from the retroorbital sinus and salivated under pilocarpine stimulation (0.1 mg/100 kg of body weight). In all cases sera from rats immunized with

VOL. 15, 1977

ANTIGENIC RELATEDNESS OF GLUCOSYLTRANSFERASE

formalinized S. mutans agglutinated cells of the homologous bacterial strains to reciprocal titers (geometric means) of 63 (strain HS6), 5,309 (strain Ingbritt), and 501 (strain 6715). These agglutinations were probably indicative of reaction with the type-specific antigen (25, 27) and teichoic acid. Other groups of rats were injected in a similar fashion with CEA of serotype g (strain 6715). These rats received 0.5 mg of CEA (containing 2 dextransucrase units [321 of activity) in 0.1 ml of phosphatebuffered saline, combined with an equal volume of adjuvant. All immune sera from rats injected with CEA showed at least one precipitin band with homologous culture supernatant in gel diffusion. However, when immune rat sera were tested in gel diffusion against purified serotype g carbohydrate (26), dextran T-10 (Pharmacia Fine Chemicals, Inc.), lipoteichoic acid, or partially hydrolyzed mutan (20), no reaction was observed. Groups of weanling NIH white hamsters were injected subcutaneously in the salivary gland vicinity with DEA-1 and DEA-2. A volume of 0.1 ml of each antigen (containing 1.6 dextransucrase units) was incorporated into adjuvant (vol/vol) prior to injection. Three additional injections at 7- to 10-day intervals were administered, followed 7 days later by bleeding as described for the rats. New Zealand white rabbits were also used to obtain antisera to CEA from serotypes g and a. For this purpose injections were made in the four footpads 'and the scruff of the neck with 1 mg of serotype g CEA (containing 18 dextransucrase units) in 0.5 ml of phosphate-buffered saline incorporated into 0.5 ml of adjuvant. Two subsequent injections were given; the first was given 1 month later and another was given 3 months later. Two weeks after the last injection, rabbits were exsanguinated and sera were prepared for use. These sera formed at least three precipitin bands with serotype g CEA in gel diffusion and immunoelectrophoresis. At least one band could be attributed to enzyme, but none were attributed to serotype g carbohydrate, dextran T-10, lipoteichoic acid, or partially hydrolyzed mutan. Another rabbit was injected in a similar fashion with 0.24 mg of serotype a CEA incorporated (vol/vol) with adjuvant. One additional injection was given 2 weeks later. Two weeks after the second injection, the rabbit was exsanguinated to obtain sera for assay. In gel diffusion analysis this antiserum formed one precipitin band with serotype a CEA but formed no bands with purified serotype g carbohydrate, boiled extracts of serotype a cells, dextran T-10, lipoteichoic acid, or partially hydrolyzed mutan. After the immunization protocols described above, the sera of rats, hamsters, and rabbits injected with enzymes derived from culture supernatants (CEA and DEA) contained similar levels of mean GTF inhibitory activity directed to the respective homologous enzyme antigens. Group means ranged from 52 to 59% inhibition of [14C]glucose incorporation into EtOH-insoluble glucan, with the exception of rabbit anti-serotype g CEA (mean inhibition, 89%). Assay for inhibition of GTF activity. The procedure for measuring inhibition of GTF activity was a

93

modification of the technique described by Evans and Genco (12). GTF activity was determined by [14C]glucose incorporation from glucosyl-labeled sucrose into ethanol-insoluble polysaccharide. In the assay for the measurement of inhibiting activity in the serum, 50 ,ul of serum (1:10 dilution) was preincubated with 50 ,ld of ammonium sulfate-precipitated enzyme preparations (0.1 to 0.4 mg/50 ,l) for 1 h at 37°C in a shaking water bath. Enzyme was dissolved in 0.05 M sodium phosphate buffer, pH 6.8. A 5C-ttg portion of primer dextran (average molecular weight, 20,000; Pharmacia Fine Chemicals, Inc.) and 0.018 ,ug of [14C]glucosyl-labeled sucrose (specific activity, 275 mCi/mmol; New England Nuclear Corp.) contained in 0.2 ml ofbuffer were then added. This mixture was then incubated at 37°C for 2 h. The reaction was stopped by precipitation with the addition of EtOH to a final concentration of 75% (vol/ vol). After centrifugation of the precipitate at 13,000 x g, the pellet was dissolved in distilled water and reprecipitated with EtOH. The centrifuged pellet was redissolved in 0.2 ml of distilled water, mixed with 4 ml of Ready-Solv solution VI (Beckman Instruments, Inc.), and counted in a liquid scintillation spectrometer (model LS-100C, Beckman Instruments, Inc.). This technique reflects total glucan synthesized by GTF. GTF in the ammonium sulfate precipitates from the seven serotypes of S. mutans based in the assay incorporated, per milligram, 2,000 to 16,000 cpm into '4C-labeled glucan. All serum assays were performed in duplicate. Salivary inhibiting activity was assayed by incubating 10 ,ul of dialyzed, unconcentrated saliva with 10 ,ul of enzyme for 30 min at 37°C in a shaking water bath. A 50-,ug amount of primer dextran and 0.018 .tg of ['4C]glucosyl-labeled sucrose were then added in a buffer volume of 50 jl. Mixtures were incubated for 5 h, at which time polysaccharide was precipitated, washed, and counted as described for the serum assay. All saliva assays were performed in triplicate. The effect of immune fluids on enzyme function was expressed as the percentage of reduction in the individual counts incorporated by enzyme incubated with immune sera or salivas compared to the mean counts of enzyme incubated with from 4 to 13 sera or salivas from nonimmunized animals. Evidence for the specificity of the assay was obtained in the following manner. A 1:10 dilution (representing 5 ,ul of antiserum in the assay) of a rabbit antiserum, which was directed to the serotypeg-specific carbohydrate, did not inhibit the incorporation of ['4C]glucose into EtOH-insoluble glucan by CEA of serotype g when compared with normal rabbit sera controls. A similar amount of a rabbit antiserum directed against the glycerol-phosphate backbone of streptococcal teichoic acid (obtained from T. Myoda, Dupont Research Institute) also did not inhibit GTF activity of this serotype. Evidence for the fact that inhibition in this assay reflects specific antibody activity was determined by the use in the inhibition assay of rabbit IgG antibody directed against cell-associated antigens of S. mutans serotype g. Antibody was prepared with the use of cellular immunoadsorbants and gel filtration on Sephadex G-200 as described

94

SMITH AND TAUBMAN

previously (26). Five microliters of this antibody (150 ,ug/100 1d) showed greater than 80% inhibition of enzyme from serotype g in the functional assay. This observation is in agreement with other reports, which have shown both the inhibitory effect of purified IgG anti-GTF antibody in a similar assay (38) and the resultant decrease in inhibition when antibody and immunoglobulin were specifically precipitated and removed from immune fluids (13). Adsorption of antisera. Antisera directed to CEA derived from either serotype a (strain E49) or g (strain 6715) were adsorbed with water-insoluble polysaccharide of serotype g. Water-insoluble polysaccharide was synthesized by extracellular GTF of S. mutans strain 6715, essentially as described by Newbrun (35). After synthesis, polysaccharide was extensively washed with both buffer and 6 M guanidine HCl to remove bound protein before use in adsorption experiments. After a final two washings in phosphate buffer, pH 6.8, the centrifuged polysaccharide pellet was suspended in three times its volume in buffer. A sample of this suspension, containing approximately 2 mg of carbohydrate as measured by the phenol-sulfuric acid procedure (9), was combined (vol/vol) with undiluted antiserum directed to the CEA preparations of either serotype a or g, followed by incubation for 4 h at 4°C with stirring. Unadsorbed antiserum and normal serum controls were similarly diluted with phosphate buffer (all to a final dilution of 1:10 as used in the assay) and incubated. After removal of the polysaccharide by centrifugation, the adsorbed and unadsorbed antisera were compared for their ability to inhibit the incorporation of ["4C]glucose from sucrose into ethanol-insoluble polysaccharide by enzyme from both serotypes. Materials. Purified serotypeg (strain 6715) carbohydrate was prepared as previously described (27). Lipoteichoic acid (obtained from E. Leiberman) had been prepared by cold phenol extraction of cell membranes of S. sanguis (strain Blackburn) (44). This preparation was identified as teichoic acid by its phosphate content and by the use of specific antiglycerol-phosphate backbone antisera. Solubilization of the water-insoluble polysaccharide of strain 6715 (prepared as described above) for gel diffusion analysis was accomplished by limited acid hydrolysis in 1 N HCl at 10000 for 20 min. The pH of the solubilized polysaccharide was adjusted to neutrality with 1 N NaOH.

RESULTS

Cell-associated GTF. The pattern of reactivity of antibody to cell-associated GTF was determined by assaying rat antisera, produced by immunizing with formalinized cells of S. mutans strain HS6 (serotype a), strain Ingbritt (serotype c), or strain 6715 (serotypeg), against ammonium sulfate-precipitated culture supernatants from S. mutans strains E49, HS6, FA-1, Ingbritt, B13, LM7, and 6715. The results of these experiments are shown in Fig. 1. Antisera (1:10 dilution representing 5 ,ul of antise-

INFECT. IMMUN.

rum used per test) from rats immunized with

HS6 (serotype a) cells inhibited [14C]glucose incorporation into ethanol-insoluble polysaccharide by enzyme from organisms of serotypes d (36%) and g (43%). Virtually no inhibition occurred when these sera were incubated with enzyme from serotype b, c, or e. Antisera to Ingbritt (c) cells showed inhibition of enzyme from serotypes c (48%), b (33%), and e (27%). Antisera to serotype g strain 6715 showed significant inhibition of the same enzymes that were inhibited by the anti-serotype a sera. These antisera were strongly inhibitory both in the homologous system (71%) and with serotype d enzyme (62%). A low level of inhibition was also seen with the two serotype a strains (15%, 22%). This level of inhibition was considered to be significant, since radioactivity incorporated from [14Cjglucose by enzyme in the presence of control sera varied by a mean of less than 6%. These experiments indicate that antisera to cell-associated GTF of serotype a org react with enzyme of serotypes a, d, and g, whereas antisera to cell-associated GTF of serotype c seem to react chiefly with enzyme of serotypes e and b. These patterns of reactivity were also seen when salivas of rats from the above groups were examined for inhibitory activity (Fig. 2). Much lower levels of inhibition occurred, reflecting the lower concentrations of immunoglobulin and antibody in rat saliva compared with rat serum (33, 40). However, a consistent pattern of inhibition was obtained through the use of multiple saliva samples assayed in triplicate. Salivas from the HS6 (serotype a) cellinjected rats showed inhibition of the homologous serotype a enzyme (12%) and slight inhibition with enzyme of serotypes d (6%) and possiblyg (4%). Salivas of rats injected with Ingbritt (serotype c) cells showed little inhibitory activity. Enzyme only from serotypes c (6%) and e (7%) showed mean values of inhibition that exceeded the error of control values. Salivas of rats injected with strain 6715 (g) cells demonstrated greater inhibitory activity and reflected a pattern similar to that of the corresponding sera of these animals (Fig. 1). These salivas clearly inhibited enzyme fromg (15%), d (23%), and a (11%) serotypes, but not from b, c, or e serotypes. Therefore, the data obtained with antisera and immune salivas directed to cellassociated GTF (and other cell-associated antigens) seem to suggest that GTF enzymes of serotypes a, d, and g share some antigenic determinants, as do enzymes of serotypes c and e (and possibly b). Supernatant-derived CEA. The inhibition patterns seen among antisera of rats immunized with whole cells reflected the cross-reac-

VOL. 15, 1977

ANTIGENIC RELATEDNESS OF GLUCOSYLTRANSFERASE 6 5

95

Anti.Q-r r ct IedI Directed To

4

HS6 Cells

4

6

0

.M

E49

-66

FA-1

kg

B13

LM7

6715

0

~~~~~~~~~~~~~~~1

12

z

5

Ingbritt Cells

3 w

~~8

O

12

I-5 E49 H66 w 0-

FA-1

kVg

613

LM7

6715

0

6715 CellIs

6491H6

SEROTYPE

FA-1

kg

B13

LM7

6715

]

FIG. 1. Percentage of inhibition of['4C]glucose incorporation into ethanol-insoluble polysaccharide by rat antisera directed to cell-associated GTF. Bars indicate percentage of inhibition, which is reported as: mean [individual counts per minute incorporated in the presence of immune serum (50 p± of a 1:10 dilution representing 5 pl of serum)/mean counts per minute incorporated in the presence of an equal amount of appropriate control sera] x 100. Brackets enclose two standard errors of the group mean. Sera were assayed in duplicate. Two to five control rat sera were used in the assay of each enzyme. Mean counts per minute standard error incorporated by 50 p1 of ammonium sulfate-precipitated enzymes (01 to 0.4 mg/50 PI) from the strains of S. mutans assayed in the presence of 1:10 dilutions of control rat sera were: 5,314 260 (strain E49); 273 16 (strain HS6); 2,465 126 (strain FA-1); 2,188 126 (strain Ingbritt); 1,473 3 (strain B13); 849 69 (strain LM7); and 3,309 140 (strain 6715). Strains of S. mutans from which the enzymes used in the respective assays were obtained are indicated under the respective bars. Serotypes to which strains belong are indicated below the bottom set of bars. Rat antisera against which the ammonium sulfate-precipitated enzymes were assayed are indicated to the right of the respective bar graphs. The number of immune rat sera used in each experiment is shown above each bracket. The open portion of each bar indicates one standard error of the mean of control sera values. ±

±

±

±

±

±

tions observed among the serotype carbohy- culture supernatant were studied for inhibitory drate antigens of S. mutans (25, 30). The sera activity. The results of inhibition experiments using used in the experiments reported above probably also contained antibody directed to the re- the antisera of up to 23 rats (Fig. 3A) or individspective type-specific antigens. Therefore, anti- ual rabbits (Fig. 3B and C) that had been insera from rats and rabbits injected with extra- jected with CEA are shown in Fig. 3. The sera cellular crude GTF antigen derived from the of the rats and the rabbit injected with CEA

96

INFECT. IMMUN.

SMITH AND TAUBMAN

Immune Saliva Directed To

20

4

10J z 0 co

0O

E49

FAl

Ing

4

B13

LM7

HS6 Cells

6715

z

w I-

Ingbritt Cells z

w cc w

6: E49

2

Fkrl

3

kV

B13

L

6715

LM7

6715

a.

6715 Cells

Pb El 1 a I FIG. 2. Percentage of inhibition of ['4C]glucose incorporation into ethanol-insoluble polysaccharide by undiluted dialyzed salivas of rats immunized with cell-associated GTF. Bars indicate percentage of inhibition, which is reported as: mean (individual counts per minute incorporated in the presence of 10 pi of immune salivalmean counts per minute incorporated in the presence of an equal amount of appropriate control salivas)

SEROTYPE T

x 100. Brackets enclose two standard errors of the mean. Salivas were assayed in triplicate. Two to five control rat salivas were used for each assay. Mean counts per minute + standard error incorporated by 50 p of ammonium sulfate-precipitated enzymes (01 to 0.4 mgI50 pi) from the strains of S. mutans assayed in the presence of 10 1i1 of undiluted control rat salivas were: 6,380 ± 212 (strain E49); 1,175 + 110 (strain FA-1); 3,911 ± 13 (strain Ingbritt); 4,077 ± 125 (strain B13); 2,501 ± 46 (strain LM7); and 7,653 ± 143 (strain 6715). Strains of S. mutans from which the enzymes used in the respective assays were obtained are indicated under the respective bars. Serotypes to which strains belong are indicated below the bottom set of bars. Immune rat salivas against which enzymes were assayed are indicated to the right of the respective bar graphs. The number of immune rat salivas used in each experiment is shown above each bracket. The open portion of each bar indicates one standard error of the mean of control saliva values.

from serotype g strain 6715 strongly inhibited the homologous enzyme (57 and 89%, respectively) and that of serotype d strain B13 (52 and 72%, respectively). Inhibition of serotype a enzymes also occurred (13 and 26%). Minimal inhibition of the enzyme from other serotypes tested with the rat antisera may indicate that a slight degree of antigenic relatedness exists among GTF of these serotypes and that of serotype g. However, the rabbit antisera showed no inhibition whatsoever of serotype b, c, or e GTF. In support of the previous observations with antisera to cell-associated GTF, the serum of a rabbit injected with a CEA preparation from strain E49 (serotype a) showed significant

cross-reaction only with d (30%) and g (25%) serotype enzyme. Therefore, these observations with the rat and rabbit anti-CEA antisera reinforced the conclusion that GTF of serotypes a, d, and g are immunochemically similar. Furthermore, the pattern of inhibition seen apparently is not the result of antibody directed to the serotype carbohydrate since (i) these antisera, which are directed to extracellular enzyme preparations and are free of serotype antigens, inhibited GTF in a fashion similar to the antisera directed to cell-associated GTF, (ii) these antisera did not react with purified serotype g carbohydrate (2 mg/ml) in gel diffusion analyses, and (iii) antisera directed to the sero-

ANTIGENIC RELATEDNESS OF GLUCOSYLTRANSFERASE

VOL. 15, 1977

type g carbohydrate did not inhibit GTF of this serotype (see Materials and Methods). To eliminate the possibility that antibody directed to the water-insoluble polysaccharide product (which might contaminate some of the CEA preparations) was interfering in the inhibition, an adsorption experiment was per-

97 formed. Washed water-insoluble polysaccharide, synthesized by CEA from S. mutans strain 6715, was used to adsorb antisera directed to CEA of serotype a org. The inhibiting abilities of both adsorbed and unadsorbed antisera (Table 1) were generally similar when these sera were tested against either homolo-

623

60A

Rat Antisera

Directed To

40-

CEA-6715 20-

23

In6 Fk1 HS6 E49 E49 HS6

FM-

Ing

B13

LIM7

6715

z 0 Im-

Rabbit Antisera Directed To

F-

CEA-6715

0 z

LLJ

C) w: 0-

60 C 40CEA- E49

20

n FAM

E49

SEROTYPE or

Ing

B13

n LM7

6715

iii

FIG. 3. Percentage of inhibition of ['4C]glucose incorporation into ethanol-insoluble polysaccharide by rat rabbit antisera directed to CEA. Symbols are as described for Fig. 1. (A) Inhibition with rat antisera

directed to CEA

of extracellular GTF from S.

mutans strain 6715

(serotypeg) is reported

as

expressed for Fig.

1. Two to thirteen control rat sera were used for each assay. Mean counts per minute standard error S. incorporated by 50 pi of ammonium sulfate-precipitated enzymes (0.1 to 0.4 mg/50 pa) from the strains of + mutans assayed in the presence of 1:10 dilutions of control rat sera were: 2,946 + 130 (strain E49); 138 14 (strain HS6); 191 2 (strain FA-1); 1,339 -+- 27 (strain Ingbritt); 1,468 + 144 (strain B13); 1,201 + 99 (strain LM7); and 1,311 44 (strain 6715). (B) Inhibition with a rabbit antiserum directed to CEA of extracellular GTF from S. mutans strain 6715 (serotype g). A pool of normal rabbit sera was used as the control serum to determine 100% incorporation. Immune and normal sera were assayed in duplicate. Counts per minute incorporated by 50 p. of ammonium sulfate-precipitated enzymes (0.1 to 0.4 mg/50 p1) in the presence of 1:10 dilutions of the normal rabbit serum pool were: 3,691 (strain E49); 269 (strain HS6); 3,755 (strain FA-1);a 1,433 (strain Ingbritt); 1,595 (strain B13); 1,458 (strain LM7); and 3,958 (strain 6715). (C) Inhibition with of rabbit antiserum directed to CEA of extracellular GTF from S. mutans strain E49 (serotype a). Calculation percentage of inhibition and indication of counts per minute incorporated in the presence of the normal rabbit serum control were identical to those described for (B).

98

INFECT. IMMUN.

SMITH AND TAUBMAN

TABLE 1. Effect of preadsorption with water-insoluble polysaccharide (mutan) from S. mutans 6715 (serotype g) on enzymatic inhibitory activity of antisera directed to CEA % Inhibition of ['4C]glucose incorporation into EtOH-insoluble polysaccharide Antisera Adsorbent 6715

Rat anti-CEA (6715) Rabbit anti-CEA (E49)

None

35.3 ± 5.2a

Serotype g mutan (2 mg)

37.3 ± 1.7

None

29.2 ± 3.1 29.1 ± 2.2

Serotypeg mutan (2 mg) a

E49 51.2 + 2.1

37.7 + 6.2 37.1 + 2.6 43.8 ± 0.3

Mean ± standard error.

gous or heterologous enzyme sources. This indicated that if antibody against glucan was present, it did not significantly influence the assay, thereby reinforcing the conclusion that functional inhibition reflects the presence of antibody directed chiefly to the enzyme antigens. In addition, the salivas of rats injected in the vicinity of the four major salivary glands with CEA from S. mutans 6715 (serotype g) were assayed against enzyme of five serotypes of S. mutans (Fig. 4). Salivas from the serotype g CEA-injected rats interfered with the function of enzyme from serotypes a (14%), d (10%), and g (13%) but not of serotypes b and c. Therefore, the pattern of inhibition with salivas (Fig. 4) and sera (Fig. 3) of CEA-injected rats was virtually identical to that observed for the salivas and sera of rats injected with cell-associated GTF (Fig. 1 and 2). Both antigenic forms of these GTF enzymes, derived either from serotype a or g, elicited antibody that inhibited enzyme from serotype a, d, or g but showed little reaction with enzyme from serotype b, c, or e.

DEA. A recent report (31) described a pattern of inhibition of a rabbit antiserum directed against an extracellular enzyme complex that specifically synthesized water-insoluble glucans from sucrose. This complex was derived from S. mutans strain HS6 (serotype a). The antiserum inhibited insoluble glucan synthesis by enzyme from serotypes a, b, c, d, e, and g and also inhibited the total glucan synthesis (soluble plus insoluble) of all serotypes except d (strain B13). To determine the reactivity of antisera directed to a serotype g GTF preparation that also synthesized primarily a water-insoluble product, 13 hamsters were injected with DEA-1 from S. mutans strain 6715. The inhibition pattern of sera from these animals is shown in Fig. 5. Sera directed against DEA-1 exhibited a pattern of inhibition that paralleled the spectrum of cross-reactions seen for antisera directed to GTF contained in the more heterogeneous CEA or cell-associated antigen

Z 29

O

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SEROTYPER n n g E FIG. 4. Percentage of inhibition of['4C]glucose incorporation into ethanol-insoluble polysaccharide by undiluted, dialyzed salivas of rats injected with extracellular CEA from S. mutans strain 6715 (serotype g). Symbols are as described in Fig. 2. Salivas were assayed in triplicate. Two to eleven control rat salivas were used for each assay. Mean counts per minute + standard error incorporated by 50 pl of ammonium sulfate-precipitated enzymes (0.1 to 0.4 mg/50 pi) from the strains of S. mutans assayed in the presence of 10 p1 of undiluted rat saliva were: 7,735 ± 649 (strain E49); 6,896 + 101 (strain FA-1); 3,636 + 174 (strain Ingbritt); 4,199 + 100 (strain B13); and 3,359 + 88 (strain 6715).

preparations. DEA-1 elicited antibody that extensively inhibited the total glucan synthesis of enzyme from serotype d (71%) and gave less inhibition with serotype a enzyme (20 and 25%) but did not inhibit enzyme from serotype b, c, or e. The lack of response with the latter serotypes occurred despite the use of hyperimmune antisera, a fact that further supports the specificity of the inhibition assay. In the past, isolation of enzyme with GTF activity from several serotypes of S. mutans has often yielded a high-molecular-weight fraction that formed insoluble glucan and a lowermolecular-weight fraction that formed soluble product (14, 28, 34). The comparability in the GTF assay of antisera elicited by two such GTF fractions purified from S. mutans strain 6715, one forming water-insoluble (DEA-1) and one forming primarily water-soluble product, was studied after injecting 13 additional hamsters with a DEA-2 preparation that primarily synthesized water-soluble glucan. The inhibition pattern of these anti-DEA-2 sera (Fig. 5) is quite similar to that seen in Fig. 5 for anti-

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ANTIGENIC RELATEDNESS OF GLUCOSYLTRANSFERASE

99

reactivity among enzyme-containing antigens used for injection and to avoid the idiosyncracies of individual animal responses. These studies revealed several interesting relationships between GTF that was cell-associated and extracellular GTF that was obtained from culture supernatants. Both forms of GTF gave rise to serum antibody that inhibited enzyme antigen from representative S. mutans serotypes in a similar pattern and to a similar extent. This would indicate that both forms can give rise to an equivalent response and that the GTF associated with the cell is antigenically quite similar (if not identical) to that GTF found extracellularly, as has been suggested by Guggenheim and Newbrun (20). Antisera to cell-associated or supernatantderived GTF displayed rather specific patterns of reactivity in the functional assay. GTF of serotypes d and g was inhibited to a similar extent with antisera to GTF from serotypes a andg. GTF of serotype a appeared to be related, but not identical, to GTF of serotypes d and g, based (i) on the consistent inhibition of enzyme from serotype a (HS6 or E49) by antiserum to serotype g GTF and (ii) on the considerable cross-reaction of antisera directed to either cellassociated enzyme or CEA from serotype a

DEA-1 sera, both with respect to the absence of reaction with serotype b, c, or e and in the extent of inhibition of enzyme from serotypes a and d. Thus, serotype g GTF populations synthesizing either water-soluble or water-insoluble glucans appear to contain common antigenic determinants which give rise to antibody that inhibits in a similar pattern. DISCUSSION The antigenic relationship of GTF produced by strains of S. mutans representative of the different serotypes was studied using a functional inhibition assay. Since inhibition of glucan formation by anti-GTF sera in the functional assay has been closely correlated with the ability of such antisera to interfere with GTF-mediated adherence of S. mutans to glass surfaces (16, 31), it was felt that the use of this assay might yield information with potential biological significance. In addition, this technique has been reported to be more discriminatory among antisera directed to GTF of different specificities when compared with several other serological techniques (4). Multiple samples of hyperimmune sera (40) from three animal models were tested to optimize potential

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FRI 9 SEROTYPE 1 FIG. 5. Percentage of inhibition of [14C]glucose incorporation into ethanol-insoluble polysaccharide by hamster antisera directed to extracellular DEA obtained from S. mutans strain 6715 (serotype g). Symbols are as described in Fig. 1. Sera were assayed in duplicate. One to six appropriate control hamster sera were used for assay of each ammonium sulfate-precipitated enzyme. Mean counts per minute + standard error incorporated by 50 pi of enzyme (01 to 0.4 mg/50 pi) from the strains of S. mutans assayed in the presence of 1:10 dilutions of control hamster sera were: 636 38 (strain E49); 238 14 (strain HS6); 1,993 5 (strain FA-1); 1,214 (strain Ingbritt); 766 64 (strain B13); 1,133 (strain LM7); and 2,405 200 (strain 6715).

100

SMITH AND TAUBMAN

(Fig. 1 and 3C) with enzyme of serotypes d and g. Both Genco and his co-workers, who used the functional assay to study anti-whole-cell sera to serotype d andg organisms, and Fukui and his colleagues, who used the functional assay and gel diffusion analyses to determine the activity of an antiserum to purified dextransucrase from strain HS6 (serotype a), disclosed a similar relationship among GTF of these three serotypes (15, 16). On the other hand, our studies seem to suggest that GTF of serotypes b, c, and e is antigenically dissimilar to GTF of serotypes a, d, and g. The serum (Fig. 1) and salivary (Fig. 2) inhibition patterns indicate that GTF of serotypes c and e can cross-react with one another, and possibly with GTF from serotype b. To summarize the patterns of inhibition displayed by antisera to GTF of S. mutans, we suggest that among the cariogenic organisms tested two (serotypes a, d, g and b, c, e), or perhaps three (serotypes a, d, g; b; and c, e), different subsets of GTF exist which have distinct antigenic determinants within a subset. Within serotype g, the GTF that formed water-insoluble glucan (DEA-1) and the GTF that formed predominantly water-soluble glucan (DEA-2) elicited antibody that showed quite similar patterns of inhibition when incubated with enzyme of homologous and heterologous serotypes (Fig. 5). The immunological similarity of these two GTF fractions has also been suggested from preliminary immunoelectrophoretic analyses followed by incubation with sucrose (data not shown). In these analyses both antisera (anti-DEA-1 and -2) reacted with a common enzyme antigen in the DEA-2 fraction, although the DEA-1 also contained an additional enzyme antigen not present in DEA2. It could be that these two GTF fractions simply represent the same enzyme in two different states of aggregation. On the other hand, if the two GTF fractions were indeed structurally as well as functionally different, the observed inhibition phenomena also could result from the sharing of common antigenic determinants on two otherwise dissimilar enzymes. Alternatively, perhaps only one of the two GTF enzymes elicits the important inhibitory antibody response. The similarity in the pattern of antibody-mediated inhibition might then be explained by the presence of this enzyme in both GTF pools used to prepare the antisera, perhaps being prominent in one but "contaminating" the other pool. Regarding the similarity of inhibition of GTF from S. mutans of strains B13 and 6715 by antisera directed to cell-associated or extracellular GTF of serotypes a and g (Fig. 1, 3, and 5), it might be concluded that an identical enzyme(s) or enzymes with very simi-

INFECT. IMMUN.

lar antigenic determinants were being inhibited in either strain. This would be in agreement with the reported similarity of these two strains for other biochemical and serological criteria (3, 8, 36). Enzyme of serotype a strains HS6 and E49 was also inhibited by antisera to GTF of serotypeg. These antisera inhibited enzyme of serotype a to about one-third of the extent of inhibition with the homologous serotype g enzyme. If one assumes that different enzymes are responsible for the synthesis of water-soluble glucan and water-insoluble glucan, and that serotype a and g strains share only one of these enzymes in common, then antibody against serotype g GTF would inhibit only the shared serotype a enzyme. Therefore, in our ['4Clglucose assay, which measures total (both water-soluble and water-insoluble) glucan synthesis, such a reaction would result in partial inhibition of total glucan synthesis. Alternatively, the decreased inhibitory power of the anti-serotype g GTF antibody for the serotype a GTF enzymes might be accounted for by differences in the structure, the number, or the location of antigenic determinants on a similarly functioning enzyme(s) of the two serotypes. Apparently, serotype a, d, or g GTF does not share antigenic determinants with serotype b, c, or e GTF, which are important in antibodymediated enzyme inhibition. The similarities within and differences between these subsets of GTF have also been noted when other characteristics of GTF have been studied. Ciardi and co-workers (7) showed that enzymes from serotypes a (strain AHT) and g (strain 6715) migrated to similar, although not identical, positions when supernatant-derived GTF preparations from several strains of S. mutans were subjected to electrophoresis in 5% polyacrylamide gels. Enzymes from serotypes c (strain 10449) and e (strain LM7) also showed comparable migration, but to areas different from a and g. Other evidence for the similarity of GTF from serotypes a and d is derived from a study (35) of the susceptibility of polysaccharides synthesized by GTF of several S. mutans strains to enzymatic hydrolytic cleavage. Newbrun was able to show that glucans synthesized by GTF from serotypes a (strains HS6 and AHT) and d (strain 0MZ176) were hydrolyzed at similar rates when he used any of three different dextranase preparations (35). Therefore, these studies suggest that GTF from serotypes a, d, and g and serotypes c and e may be chemically and/or functionally similar within each subset. This could be the result of structural similarities, which in our study are manifested as the sharing of common antigenic determinants

VOL. 15, 1977

ANTIGENIC RELATEDNESS OF GLUCOSYLTRANSFERASE

within the respective enzyme subsets giving the patterns of inhibition observed. That the assignment of GTF into such subsets may also reflect additional (and perhaps more fundamental) differences among these two or three broad groupings of S. mutans is suggested by the partial cross-reactivity seen among the serotype carbohydrate antigens of groups a, d, andg (25, 27, 30) and groups c and e (22) and, in part, by the similarities in deoxyribonucleic acid content (guanine plus cytosine) among these groups (8, 10). Linzer and Slade (31) have reported that an antiserum directed against an enzyme complex from serotype a (strain HS6), which synthesized 96% insoluble glucan, inhibited ["4C]glucose incorporation into soluble glucan of all representative S. mutans serotypes. Total glucan synthesis was inhibited in all but one strain (serotype d, strain B13), in which the inhibition of insoluble glucan was compensated by the synthesis of soluble glucan. However, in our studies of inhibition of total glucan synthesis, antisera to a GTF preparation from serotype g (strain 6715), which also predominantly formed water-insoluble polymer, selectively inhibited enzyme from serotypes a, d, andg only. Consideration was given to the fact that primer was not routinely used in the assays of the antiserotype a GTF antiserum (31), as it was in the present investigation. However, the elimination of dextran T-20 from our system did not significantly alter the pattern of cross-reactivity seen in its presence (unpublished observations), although there wag a general reduction in glucan synthesis, which has been reported to occur under such conditions (15, 17). The possibility that our enzyme preparation had associated primer has not been ruled out. It is of interest that other workers (15) have been unable to detect antibody against serotype b, c, or e in antisera directed to purified dextransucrase which had also been prepared from strain HS6. However, the GTF preparation used in the former study (31) may have been enriched in a more cross-reactive enzyme component, since somewhat different preparative techniques were used (34). In any case, these observations merit confirmation using GTF prepared from other serotype a strains and using additional antisera. GTF inhibitory activity was clearly present in the salivas of rats immunized with cell-associated antigen, as previously reported (13), as well as being present in rats immunized with

supernatant-derived GTF. Secretory IgA,

which can predominate among salivary immunoglobulins of rats (1, 39) and hamsters (21), has been suggested to have a broader specificity

101

than serum IgG (43). However, the general pattern of salivary inhibition was quite similar to that of serum inhibition in the present study, supporting the observations of Richman et al. (37). These workers demonstrated that the specificity of secretary antibody of the respiratory tract for influenza A virus was virtually identical to the specificity found in serum antibody. The low level of salivary inhibition precluded more detailed comparison of the antigenic relatedness among serotypes a, d, and g in the present work. The actual amount of salivary anti-GTF inhibitory antibody may be significantly higher in vivo than that measured in our studies because of the lower concentration of immunoglobulin in pilocarpine-stimulated versus unstimulated saliva. We have recently demonstrated that CEA or DEA-1 or DEA-2 from serotype g, when administered in complete Freund adjuvant in the salivary gland region, can markedly reduce the cariogenic effects of S. mutans strain 6715 in conventional rats, gnotobiotic rats, and hamsters. The apparent effectiveness of GTF as an antigen in these models, together with the ever-increasing demonstration in various rodent (33, 40) and primate (2, 11, 29) models that whole-cell antigens (which usually bear GTF as surface antigens, Fig. 1) can also confer on the host a degree of protection against caries, suggests that antigens of S. mutans in general, and GTF in particular, may become important as agents for immunization to inhibit the development of dental caries. The isolation of cariogenic S. mutans of every serotype from humans indicates the breadth of protection required. The results of the present study suggest that the potential formulation of a GTF-containing vaccine that might elicit a protective immune response to all serotypes of S . mutans might include representative GTF from two or three serotypes. ACKNOWLEDGMENTS This research was performed pursuant to Public Health Service contract no. DE-42438 with the National Institute of Dental Research and was supported also by Public Health Service Research Career Development awards K04 DE 70122 (to M.A.T.) and K04-0024 (to D.J.S.) from the National Institute of Dental Resesrch. We would like to thank M. Dhond and C. Kereakoglow for expert technical assistance. Also, we are grateful to J. Bulkacz, Medical College of Georgia, for GTF from S. mutans strain E49 and to T. Myoda, Dupont Institute, for antisera to teichoic acid. LITERATURE CITED 1. Bistany, T. S., and T. B. Tomasi, Jr. 1970. Serum and secretary immunoglobulins of the rat. Immunochemistry 7:453-460. 2. Bowen, W. H., B. Cohen, M. F. Cole, and G. Colman. 1975. Immunization against dental caries. Br. Dent. J. 139:45-58.

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3. Bratthall, D. 1970. Demonstration of five serological groups of streptococcal strains resembling Streptococcus mutans. Odontol. Revy 21:143-152. 4. Burckhardt, J. J., and B. Guggenheim. 1976. Interactions of antisera, sera, and oral fluid with glucosyltransferases. Infect. Immun. 13:1009-1022. 5. Carlsson, J., E. Newbrun, and B. Krasse. 1969. Purification and properties of dextransucrase from Streptococcus sanguis. Arch. Oral Biol. 14:469-478. 6. Ceska, M., K. Granath, B. Norman, and B. Guggenheim. 1972. Structural and enzymatic studies on glucans synthesized with glucosyltranferases of some strains of oral streptococci. Acta Chem. Scand. 26:2223-2230. 7. Ciardi, J. E., G. J. Hageage, and C. L. Wittenberger. 1976. The multicomponent nature of the glucosyltransferase system of Streptococcus mutans. J. Dent. Res. 55 (Suppl.):C87-C96. 8. Coykendall, A. L. 1974. Four types of Streptococcus mutans based on their genetic, antigenic and biochemical characteristics. J. Gen. Microbiol. 83:327338. 9. Dubois, M., K. A. Gilles, J. K. Hamilton, P. A. Rebers, and F. Smith. 1956. Colorimetric method for determination of sugars and related substances. Anal. Chem. 28:350-356. 10. Dunny, G. M., T. Hausner, and D. B. Clewell. 1972. Bouyant densities of DNA from various strains of Streptococcus mutans. Arch. Oral Biol. 17:1001-1003. 11. Evans, R. T., F. G. Emmings, and R. J. Genco. 1975. Prevention of Streptococcus mutans infection of tooth surfaces by salivary antibody in irus monkeys (Macaca fascicularis). Infect. Immun. 12:293-302. 12. Evans, R. T., and R. J. Genco. 1973. Inhibition of glucosyltransferase activity by antisera to known serotypes of Streptococcus mutans. Infect. Immun. 7:237-241. 13. Evans, R. T., R. J. Genco, D. J. Smith, and M. A. Taubman. 1974. S. mutans glucosyltransferase inhibition by serum and saliva from immunized rats. J. Dent. Res. 53 (Suppl.):183. 14. Fukui, K., Y. Fukui, and T. Moriyama. 1974. Purification and properties of dextransucrase and invertase from Streptococcus mutans HS6. J. Bacteriol. 118:796-804. 15. Fukui, K., Y. Fukui, and T. Moriyama. 1974. Some immunochemical properties of dextransucrase and invertase from Streptococcus mutans. Infect. Immun. 10:985-990. 16. Genco, R. J., R. T. Evans, and M. A. Taubman. 1974. Specificity of antibodies to Streptococcus mutans; significance in inhibition of adherence. Adv. Exp. Med. Biol. 45:327-336. 17. Germaine, G. R., A. M. Chludzinski, and C. F. Schachtele. 1974. Streptococcus mutans dextransucrase: requirement for primer dextran. J. Bacteriol. 120:287294. 18. Gibbons, R. J., and J. van Houte. 1975. Bacterial adherence in oral microbial ecology. Annu. Rev. Microbiol. 29:1944. 19. Guggenheim, B. H., H. Muhlemann, B. Regolati, and R. Schmid. 1970. The effect of immunization against streptococci or glucosyl-transferases on plaque formation and dental caries in rats, p. 287-296. In W. D. McHugh (ed.), Dental plaque. D. C. Thompson and Co., Dundee, Scotland. 20. Guggenheim, B., and B. Newbrun. 1969. Extracellular glucosyltransferase activity of an HS strain of Streptococcus mutans. Helv. Odontol. Acta 13:84-97. 21. Haakenstad, A. O., and J. E. Coe. 1975. The immune response in the hamster. IV. Studies on IgA. J. Immunol. 106:1026-1034. 22. Hamada, S., and H. D. Slade. 1976. The adherence of serotype e Streptococcus mutans and the inhibitory

effect of Lancefield group E and S. mutans type e antiserum. J. Dent. Res. 55 (Suppl.):C65-C74. 23. Hardie, J. M., and G. H. Bowden. 1974. Cell wall and serological studies on Streptococcus mutans. Caries Res. 8:301-316. 24. Hayashi, J. A., I. L. Shklair, and A. N. Bahn. 1972. Immunization with dextransucrases and glycosidic hydrolases. J. Dent. Res. 51:436-442. 25. Iacono, V. J., M. A. Taubman, D. J. Smith, P. G. Garant, and J. J. Pollack. 1976. Structure and function of the type-specific polysaccharide of Streptococcus mutans 6715, p. 75-90. In W. H. Bowen, R. J. Genco, and T. C. O'Brien (ed.), Immunology abstracts. Information Retrieval Inc., Arlington, Va. 26. Iacono, V. J., M. A. Taubman, D. J. Smith, and M. J. Levine. 1975. Isolation and immunochemical characterization of the group-specific antigen of Streptococcus mutans 6715. Infect. Immun. 11:117-128. 27. Iacono, V. J., M. A. Taubman, D. J. Smith, and E. C. Moreno. 1976. A spectrophotometric procedure for quantitation of antibody directed to bacterial antigens. Immunochemistry 13:235-243. 28. Kuramitsu, H. K. 1975. Characterization of extracellular glucosyltransferase activity of Streptococcus mutans. Infect. Immun. 12:738-749. 29. Lehner, T., S. J. Challacombe, and J. Caldwell. 1975. Immunological and bacteriological basis for vaccination against dental caries in rhesus monkeys. Nature (London) 254:517-520. 30. Linzer, R., H. Mukasa, and H. D. Slade. 1975. Serological purification of polysaccharide antigens from Streptococcus mutans serotypes a and d: characterization of multiple antigenic determinants. Infect. Immun. 12:791-798. 31. Linzer, R., and H. D. Slade. 1976. Characterization of an anti-glucosyltransferase serum specific for insoluble glucan synthesis by Streptococcus mutans. Infect. Immun. 13:494-500. 32. McCabe, M. M., and E. E. Smith. 1973. Origin of the cell-associated dextransucrase of Streptococcus mutans. Infect Immun. 7:829-838. 33. McGhee, J. R., S. M. Michalek, J. Webb, J. M. Navia, A. F. R. Rahman, and D. W. Legler. 1975. Effective immunity to dental caries: protection of gnotobiotic rats by local immunization with Streptococcus mutans. J. Immunol. 114:300-305. 34. Mukasa, H., and H. Slade. 1974. Mechanisms of adherence of S. mutans to smooth surfaces. III. Purification and properties of the enzyme complex responsible for adherence. Infect Immun. 10:1135-1145. 35. Newbrun, E. 1972. Extracellular polysaccharides synthesized by glucosyltransferases of oral streptococi. Composition and susceptibility to hydrolysis. Caries Res. 6:132-147. 36. Perch, B., E. Kjems, and T. Ravn. 1974. Biochemical and serological properties of Streptococcus mutans from various human and animal sources. Acta Pathol. Microbiol. Scand. 82:357-370. 37. Richman, D. D., B. R. Murphy, E. L. Tierney, and R. M. Chanock. 1974. Specificity of the local secretary antibody to influenza A virus infection. J. Immunol. 113:1654-1656. 38. Russell, M. W., S. J. Challacombe, and T. Lehner. 1976. Serum glucosyltransferase-inhibiting antibodies ahd dental caries in Rhesus monkeys immunized

against Streptococcus mutans. Immunology 30:619. 39. Taubman, M. A., and D. J. Smith. 1973. Induction of salivary IgA antibody in rats and hamsters. J. Dent. Res. 52(Suppl.):276. 40. Taubman, M. A., and D. J. Smith. 1974. Effects of local

immunization with Streptococcus mutans on induc-

tion of salivary immunoglobulin A antibody and experimental dental caries in rats. Infect. Immun. 9:

1079-1091.

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41. Taubman, M. A., D. J. Smith, and D. Cox. 1976. Immunization with glucosyltransferases (GTF): effects on experimental dental caries. J. Dent. Res. 55(Suppl.):B82. 42. Thomson, L. A., W. Little, and G. J. Hageage. 1976. Application of fluorescent antibody methods in the analysis of plaque samples. J. Dent. Res. 52(Suppl.): A80-A86. 43. Waldman, R. H., R. M. Wigley, and P. A. Small, Jr.

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1970. Specificity of respiratory secretion antibody against influenza virus. J. Immunol. 105:1477-1483. 44. Wicken, A. J., and K. W. Knox. 1970. Studies on the group f antigen of lactobacilli: isolation of a teichoic acid-lipid complex from Lactobacillus ferment. J. Gen. Microbiol. 60:293-301. 45. Wood, J. M. 1967. A dextransucrase activity from Streptococcus FA-1. Arch. Oral Biol. 12:1659-1660.