Vol. 50, No. 3INFECTION AND IMMUNITY, Dec. 1985, p. 833-8430019-9567/85/120833-11$02.00/0Copyright © 1985, American Society for Microbiology

Inhibition of Plaque and Caries Formation by a Glucan Produced byStreptococcus muttans Mutant UAB108

KAZUKO TAKADA,t TETSUO SHIOTA,t ROY CURTISS III,§ AND SUZANNE M. MICHALEK*Department of Microbiology and Institute of Dental Research, The University ofAlabama at Birmingham,

Birmingham, Alabama 35294

Received 28 May 1985/Accepted 10 September 1985

A mutant (UAB108) derived from Streptococcus mutans UAB66, a spectinomycin-resistant (Spcr) isolate ofstrain 6715, inhibited plaque formation when grown with strain 6715 in a sucrose medium and also inhibitedcaries formation in gnotobiotic rats infected with both strain UAB108 and 6715. A substance obtained fromUA8108 culture supernatant fluid after ethanol precipitation and DEAE-cellulose treatment, designated glucan108, inhibited S. mutans 6715 virulence and was shown to be a water-soluble glucan. In the presence of sucroseand increasing concentrations of glucan 108, the activity of a glucosyltransferase (GTase) preparation from S.mutans 6715 to synthesize adhesive water-insoluble glucan (ad-WIG) was inhibited, and the activity tosynthesize non-ad-WIG was stimulated. Glucan 108 similarly inhibited sucrose-dependent adherence ofheat-treated cells, was a poor inducer of cell aggregation, and inhibited S. mutans 6715-induced dental cariesin gnotobiotic rats. In the presence of GTase, glucan 108, and sucrose, the glucose moiety of sucrose was foundto be incorporated into glucan 108, and most of this glucose-incorporated glucan 108 was found in thenon-ad-WIG fraction. The mode of inhibition of plaque formation by S. mutans 6715 appears to involve a shiftfrom ad-WIG to non-ad-WIG formation. The water-soluble glucan 108 was found to have an approximatemolecular weight of 2 x 106 and was hydrolyzed by fungal dextranase to yield glucans with an averagemolecular weight of about 1.2 x 104. This glucan (designated glucan 12k) was further hydrolyzed by bacterialdextranase to yield smaller glucans and oligosaccharides, but was refractile to at(1->3) glucanase. These resultssuggest that glucan 108 is a branched at(1-6) glucan, and it is proposed that UAB108 is defective in its abilityto polymerize glucan 12k with a(1-*3)-linked glucosyl residues.

The production of glucans from sucrose by gluco-syltransferase (GTase; EC 2.4.1.5) is one of the virulencetraits of Streptococcus mutans. It is generally held thatglucans are the major constituent of dental plaque, which isa potential site for carious lesion formation (14, 34, 37). Theenzymatic properties of GTase (2, 8, 12, 22, 23, 31) and thechemical-physical properties of glucans (5, 19, 24) have beenwell studied. Exogenously supplied glucans with variousmolecular weights have been reported to stimulate GTaseactivity (3, 8, 12, 22, 23), and these added glucans act asprimers for additional glucan synthesis. Exogenous glucanshave also been reported to inhibit GTase activity (3, 16, 17,19), adherence of cells to smooth surfaces (17, 35), andplaque formation and caries incidence in experimental ani-mals (16). The mechanism of inhibition by glucans, however,is poorly understood.Because of our longstanding interest in genetically de-

fining virulence determinants of S. mutans, a large numberof mutants were isolated and characterized (32). One ap-proach employed to characterize the genetic lesion resultingfrom mutation involved complementation studies. Mutantsdefective in their ability to adhere to glass were grownpairwise to screen for complementation, and several mutant

* Corresponding author.t Present address: Department of Bacteriology, Nihon University

of Matsudo, Matsudo, Japan.t Present address: 17018 Brentwood P1., N.E., Seattle, WA

98155.§ Present address: Department of Biology, Washington Univer-

sity, St. Louis, MO 63130.

combinations were found to complement, resulting in adher-ence (25). In addition, certain adherence-defective mutantswere identified which inhibited plaque formation by theparent strain and by certain plaque-producing mutants (25).Further investigation showed that the inhibiting factor wasan ethanol-precipitable polymer present in the supernatantfluids of sucrose-grown cells. In the case of UAB108, thisstrain was shown to produce a water-soluble glucan (WSG)which was effective in inhibiting the adherence of wild-typestrains (33). The present study describes in detail inhibitionby UAB108 of in vitro plaque and experimental dental cariesformation by S. mutans 6715. Evidence is presented that theinhibitor is a high-molecular-weight WSG produced byUAB108. The possible mode of inhibition of S. mutansvirulence by this glucan is discussed.

MATERIALS AND METHODS

Maintenance of stock cultures. Strains of S. mutans weremaintained as stab cultures in a brain heart infusion agarsupplemented with a few milligrams of solid CaCO3. Thecultures were transferred monthly and stored at 5°C. S.mutans UAB108 was obtained from S. mutans UAB66 aftertreatment with ethyl methanesulfonate (32). Strain UAB66 isa spectinomycin-resistant mutant of streptomycin-resistantS. mutans 6715 (which is designated UAB631 in ourcollection) (32).

Preparation of glucan 108. One liter of an 18-h culture of S.mutans UAB108 grown in partially defined (PD) mediumwith glucose (PD-glucose) (20) was centrifuged (10,000 x g,20 min), and the supernatant fluid was passed through a filter

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(0.45-,um pore diameter; Millipore Corp., Bedford, Mass.).The supernatant fluid was adjusted to 1% sucrose and 0.02%sodium azide, and the mixture was incubated at 37°C for 24h. Three volumes of ethanol were then added, and after 24 hat 5°C, the precipitate was collected by centrifugation(10,000 x g, 20 min) and suspended in 300 ml of distilledwater with stirring, and the small amount of undissolvedmaterial was discarded after centrifugation. The supernatantfluid was applied to a DEAE-cellulose column (3 by 40 cm),and the column was washed with 150 ml of water. Fractions(10 ml) containing carbohydrate (4) were pooled, heated at100°C for 10 min, and centrifuged, and the small amount ofprecipitate found was discarded. Three volumes of ethanolwere added to the supernatant fluid, and the precipitate wascollected as before and then dissolved in water (200 ml).Portions of the solution (20 ml, 10 mg/ml) were added to anUltrogel AcA-34 (linear fractionation range of 4.0 x 105 to2.0 x 104 daltons; LKB Instruments, Inc., Rockville, Md.)column (3 by 40 cm). The column was washed with water,and fractions (10 ml) containing carbohydrate were pooled.The glucan (designated glucan 108) which emerged in thevoid volume was lyophilized (yield, 2.2 g).For in vivo rat studies (see below), 6 liters of a filtered

culture supernatant fluid containing 2.5% sucrose and 0.02%sodium azide was incubated for 2 days at 37°C. The super-natant fluid was dialyzed against water at 5°C for 4 days witheight changes of water. The solution of glucan was adjustedto 4 mg/ml of water, sterilized, and provided to rats in theirdrinking water (see below).To prepare 14C-labeled glucan 108, 30 ,uCi of [U-glucosyl-

14C]sucrose (New England Nuclear Corp., Boston, Mass.),200 ,umol of sucrose, 0.05 M sodium acetate (pH 5.5), 0.02%sodium azide, and a dialyzed supernatant fluid of PD-glucose-grown S. mutans UAB108 (10 p.g of protein) wereincubated together in a volume of 20 ml at 37°C for 18 h. Themixture was heated at 100°C for 5 min and centrifuged, andthe supernatant fluid was applied to an Ultrogel AcA-34column (3 by 40 cm). The column was washed with water,and the radioactive material that emerged in the void volumewas applied to a DEAE-cellulose column (1.7 by 35 cm).Fractions containing radioactive material were pooled andconcentrated by lyophilization. This material was dilutedwith nonradioactive glucan 108 as described below.GTase preparations. S. mutans 6715 and UAB108 were

each grown in PD-glucose at 37°C for 18 h, and the super-natant fluids obtained after centrifugation were filtered(0.45-,um pore diameter; Millipore) and concentrated 10-foldwith polyethylene glycol (approximate molecular weight of20,000; Sigma Chemical Company, St. Louis, Mo.). Theconcentrated fluids were dialyzed against five changes ofwater containing 0.02% sodium azide over a 2-day period at50C. After dialysis, sodium acetate buffer (pH 5.5) was addedto make the concentrated fluid 0.05 M with respect to thebuffer, and the fluid was dispensed in 5-ml portions andstored at -120C.Assay for plaque formation. The production of plaque was

measured by the amount of adherence to glass walls by S.mutans cells grown in PD-sucrose medium. In some exper-iments, the PD-sucrose was supplemented with variousconcentrations of either glucan 108 or dextran T2000. To 3ml of appropriately prepared medium, 10 V1I of PD-glucose-grown cultures (absorbance, 1.0) of UAB108, 6715, orUAB108 and 6715, depending on the experiment, was addedto inoculate the tubes. The tubes were incubated at 37°C for18 h, at which time the culture medium including nonadher-ent material was discarded, and the tubes were washed three

times with 0.01 M Tris (pH 7) buffer, To each tube, 2 ml of1 N NaOH was added to suspend the adhered cells. The cellswere collected by centrifugation, washed twice with 0.01 MTris (pH 7) buffer, and suspended in 2 ml of the same buffer,and the A540 was determined.

Assay for synthesis of glucans. Reaction mixtures forassessing GTase activity consisted of dialyzed concentratedculture supernatant fluid, 0.05 M sodium acetate buffer (pH5.5), 0.02% sodium azide, and 0.29 M sucrose. The amountof specific radioactive sucrose varied and is indicated foreach experiment. The reaction mixture (3 ml) was placed inscrew-cap tubes (13 by 100 mm; Kimax), and the tubes wereincubated in a slanted (100) position at 37°C for the timeindicated. The fluid was carefully removed with a Pasteurpipette, and 1 ml of water was added to each tube. The tubewas then carefully rotated several times in a horizontalposition. This washing procedure was performed five times,and the tube content was then assessed for adhesive water-insoluble glucan (ad-WIG). The fluid and washings werecombined and centrifuged at 10,000 x g for 15 min, and theprecipitated non-ad-WIG was washed three times with wa-ter. The supernatant fluid and washings were combined anddesignated the WSG fraction, and 3 volumes of cold ethanolwere added. After 1 h at 5°C, the precipitate (WSG) wascollected by centrifugation and washed twice with 75%ethanol. The adherent material on the walls of the glass tubes(ad-WIG), the non-ad-WIG, and the alcohol precipitate(WSG) were each dissolved in 3 ml of 1 N NaOH. Theamount of glucan present in each sample was determined bya phenol-sulfuric acid method (4) or by radioactivity withBray scintillation fluid (1) or Aquasol (New England Nu-clear) and a scintillation counter or by both methods. Whenthe phenol-sulfuric acid method was employed, glucose wasused as the standard, and the results are reported as theamount of glucan found in terms of glucose equivalents.When [U-glucosyl-14C]sucrose was used, the specific radio-activity of the radioactive moiety of sucrose employed wasused to calculate the amount of polymer synthesized. Oneunit of enzyme activity is defined as the amount of enzymethat synthesizes 1 ,ug of total glucan (glucose equivalent) permin at 37°C from sucrose.

Effect of glucan 108 and dextran T2000 on glucan synthesis.The experiment on the effect of glucan 108 and dextranT2000 on glucan synthesis was performed as describedabove under Assay for synthesis of glucans, except thesupernatant fluid contained 13 ,ug of protein and variousamounts of either glucan 108 or dextran T2000.

Effect of glucan 108 and dextran T2000 on sucrose andGTase-dependent adherence to glass by heat-treated cells.Cells obtain from PD-glucose-grown cultures of strains 6715and UAB108 were washed twice with saline by centrifuga-tion, heated at 100°C for 10 min, washed twice with saline,and suspended in saline to give an A540 of 3.0. Heat-treatedcells (0.5 ml) and a supernatant fluid of strain 6715 (20 ,ug ofprotein) were added to screw-cap tubes containing buffer,sucrose, and sodium azide, and the tubes were incubated at37°C for 16 h in a slanted position as described above for theassay for glucan synthesis. The fluid was carefully removedfrom each tube, the tubes were washed, and the adheredcells were suspended in 0.5 N NaOH. The amount of cells ineach tube was determined by reading the A540.

Aggregation of strain 6715 and UAB108 cells. PD-glucose-grown 6715 and LJAB108 cells were tested for aggregation bythe method of Gibbons and Fitzgerald (15). The reactionmixtures (1.4 ml, total volume) contained a suspension (0.4ml; absorbance, 5.0) of 6715, UAB108, or an equal mixture

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of 6715 and UAB108 cells; 0.2 mmol of Tris hydrochloridebuffer (pH 8.2); and sucrose, dextran T2000, or glucan 108 atthe concentrations indicated. The mixtures were incubatedat 37°C for 2 h, and 0.3 ml of 20% formaldehyde was added.The number of cells that aggregated was scored from 0 (noaggregation) to 6 (maximum aggregation).Treatment of glucan 108 with fungal dextranase and esti-

mation of molecular weight. ['4C]glucan 108 (5.4 mg, 8.7 x104 dpm) was incubated (37°C, 72 h) with 0.25 U ofdextranase (fungal; Sigma), 0.12 mg of sodium azide, and 30p.mol of potassium phosphate buffer (pH 6.0) in a totalvolume of 0.6 ml. The mixture was then heated at 100°C for5 min, and the supernatant fluid obtained after centrifugationwas applied to an Ultrogel A4 (linear fractionation range of9.0 x 106 to 5.5 x 104 daltons; LKB) column (1.7 by 45 cm).The column was washed with water, and 3-ml fractions were

collected. The fractions were then assayed for radioactivity.Radioactive glucan without enzyme (control) was also incu-bated and treated in a similar manner. The same Ultrogel A4column was used to determine the peak elution volumes forblue dextran, dextran T500, dextran T110, and dextran T40,and the molecular weight calibration curve thus obtainedwas used to estimate the molecular weight of glucan 108.The product of fungal dextranase treatment of glucan 108

obtained from Ultrogel A4 filtration chromatography was

subjected to an Ultrogel AcA-202 (linear fractionation range

of 1.5 x 104 to 1.0 x 103 daltons; LKB) gel filtration column(1.7 by 45 cm), and the molecular weight of the product(designated glucan 12k) was estimated with a calibrationcurve generated by determining the peak volumes of bluedextran, dextran T10, and maltosylsucrose (HayashibaraCo., Japan).Treatment of glucan 12k with glucanases. [14C]glucan 12k

(1 mg, 1.7 x 104 dpm; obtained from procedure describedabove) was treated with a(1-*3) glucanase (Cladosporiumresinae; 13 ,ug of protein), dextranase (16 ,ug of protein;Merck Sharp & Dohme, West Point, Pa.), fungal dextranase(0.01 U), cariogenanase (10 ,ug of protein; Merck Sharp &Dohme), or bacterial dextranase CB (32 ,ug of protein;Calbiochem-Behring, San Diego, Calif.) in the presence of0.02 M potassium phosphate buffer (pH 6.0) and 0.02%sodium azide in a total volume of 0.2 ml at 37°C for 72 h. Thereactions were terminated by heating (100°C, 5 min), and themixtures were individually applied to an Ultrogel AcA-202column (1.7 by 45 cm). The column was washed with water,and 3-ml fractions were collected and assayed for radioac-tivity. Dextran T2000 and nigeran (Sigma), which is rich ina(1-*3) glucosidic linkages, were also treated with theseenzymes and chromatographed on the Ultrogel A4 column.

Incorporation of the glucose moiety of sucrose into glucan108. To determine whether the glucose moiety of sucrose isincorporated into glucan 108 by a GTase preparation of S.mutans 6715, three sets of reaction mixtures were prepared.The first set was the control and contained [U-glucosyl-14C]sucrose (30 mg, 2.3 x 106 dpm); the second contained[U-glucosyl-14C]sucrose (30 mg) and glucan 108 (5 mg), andthe third contained sucrose (30 mg) and ['4C]glucan 108 (5mg, 4.4 x 105 dpm). To each tube, supernatant fluid fromPD-glucose-grown 6715 cells (24 ,ug of protein), 0.15 nmol ofsodium acetate buffer (pH 5.5), and 0.6 mg of sodium azidewere added to a final volume of 3 ml, and the mixtures were

incubated at 37°C for 16 h. Radioactivity and glucan deter-minations of ad-WIG, non-ad-WIG, and WSG were per-

formed as described above.Glucanase treatment of WIG fractions synthesized in the

presence of glucan 108 by GTase preparation of 6715. WIG

fractions (0.1 to 0.5 mg) were incubated with fungaldextranase (0.01 U), C. resinae oa(1-*3) glucanase (6 p.g ofprotein), or no enzyme in a reaction mixture containing0.02% sodium azide and 0.02 M potassium phosphate buffer(pH 5.5) in a total volume of 0.5 ml at 37°C for 72 h. Themixtures were heated at 100°C for 5 min and centrifuged, andthe supernatant fluids were assayed for soluble glucans.

Miscellaneous. Protein determination was carried out by aprotein-binding assay (Bio-Rad Laboratories, Richmond,Calif.), with crystallized bovine serum albumin as the stan-dard. Merck dextranase was kindly provided by J. Birnbaumof Merck Sharp & Dohme; a(1--3) glucanase from C.resinae was provided by E. T. Reese of U.S. Army NatickLaboratories, Natick, Mass.; and purified dextran prepara-tions, originally from Pharmacia Fine Chemicals, Pis-cataway, N.J., were provided by H. Hidaka, Meiji Labora-tory, Kanagawa, Japan.

In vivo gnotobiotic-rat studies. The virulence of UAB108and the ability of UAB108 and its glucan to inhibit thevirulence of S. mutans 6715 were determined in the younggnotobiotic-rat model (28). Weanling germ-free Fischer CDF(344)GN rats (age, 20 days) were transferred to experimen-tal Trexler plastic isolators and challenged with an 18-h brainheart infusion broth culture of S. mutans 6715, UAB108, ora mixture of 6715 and UAB108 consisting of equal numbersof CFU. Rats were provided sterile diet 305 (28) and waterad libitum throughout the experiment. Two days after infec-tion, colonization was assessed by collecting oral swabsamples and culturing them on mitis salivarius (MS) agar(28). In a second series of experiments, weanling rats werechallenged with S. mutans 6715 and provided sterile watercontaining dextran T2000 (Pharmacia), dextran T10(Pharmacia), or glucan 108 at 4 mg/ml. Experiments wereterminated when rats reached 45 days of age, and thenumber of S. mutans present in plaque was assessed (29) byplating samples on MS and MS-containing spectinomycin(500 mg/ml) agar, as previously described (25). Finally, thelevel of caries was determined on mandibular molars fromindividual animals (28).

RESULTS

Certain properties of strains 6715 and UAB108. We previ-ously reported that, when grown with a wild-type strain thatproduces plaque in a sucrose-containing medium, UAB108inhibited plaque formation. UAB108 and other inhibitingstrains when grown in a sucrose medium produced a factorthat inhibited adherence. The factor was identified as aWSG. A GTase preparation from a supernatant fluid ofglucose-grown UAB108 was found to produce the WSGwhich inhibited adherence (33).The current study showed that the supernatant fluids from

glucose-grown 6715 and UAB108 contain the following threetypes of GTase activity: (i) ad-WIG, (ii) non-ad-WIG, and(iii) WSG syntheses. The amount of ad-WIG produced by asupernatant fluid of 6715 increased in a linear manner withrespect both to the amount of supernatant fluid used and totime. Non-ad-WIG and WSG were produced in minoramounts. The amount of WSG produced by a supernatantfluid of UAB108 increased in curvilinear fashion with respectto the amount of supernatant fluid used and was linear withrespect to time. Ad-WIG and non-ad-WIG were produced inminor amounts. The specific activity of GTase from the 6715fluid employed was 0.34 U/,ug of protein, and approximately78, 9, and 13% of the total glucan synthesized was ad-WIG,non-ad-WIG, and WSG, respectively. The specific activity

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TABLE 1. Inhibition of S. mutans 6715 virulence in gnotobiotic rats by mutant UAB108"

Caries scoreshNo. of Buccal Sulcal Proximal Mean body Mean no. of S. mitt(ins

s. mutans strain rats per '___g'_CFUgroup Enamel Dentinal Dentinal Dentinal Dentinal per mandible"slight slight extensive Enamel slight

UAB108 21 10.3 ± 0.6 8.3 ± 0.5 12.4 ± 0.3 2.8 ± 0.4 4.5 ± 0.7 2.0 ± 0.7 98.6 ± 5.4 2.1 x 1066715 12 20.0 ± 0.2 16.6 + 0.3 16.8 ± 0.4 6.3 ± 0.6 8.0 ± 0.0 4.8 ± 0.5 108.8 ± 3.1 4.1 x 106UAB108 + 6715 9 14.7 ± 0.3 10.1 ± 0.3 13.2 + 0.5 4.1 ± 0.3 4.4 ± 0.3 1.3 ± 0.5 102.4 ± 5.2 1.8 x 106 (UAB108)

3.5 x 106 (6715)a Weanling germ-free rats (age, 20 days) were infected with an overnight log-phase culture of the test S. Iniitans; for mixed infection, approximately equal

numbers of CFU (5 x 107) were combiped in the inoculating suspension. Rats were sacrificed at 45 days of age.b Values are the mean ± standard error of the mean as determined by the method of Keyes, as previously described (28).C Determined on MS and MS-plus-spectinomycin agar.

for the supernatant fluid of UAB108 was 0.4 tJ/,ug of protein,and about 87, 12, and 1% of the total gltican synthesized wasWSG, non-ad-WIG, and WIG, respectively (data notshown).Because supernatant fluids from glucose-grown 6715 and

UAB108 were employed in this 5ttidy, it was critical todetermine whether such fluids had fructosyltransferase andglucanase activities in addition to GTase activity. Withfructose-labeled sucrose as the substrate, both supernatantfluids synthesized polysaccharides (glucans); however, fewor no fructosyl moieties of sucrose were incorporated intothe polysaccharide. The supernatant fluids from 24- and 50-hcultures exhibited no activity to degrade dextran T2000 orglucan 108 as determined by Ultrogel A4 gel filtration (datanot shown).

1.0

- Glucan 108

0.8 \\ * Dextran T2000E0'v 0.6-U,)

04

eo.

0~~~~~

.0

0 2.5 5 7.5 10

Glucan 108 or Dextran T2000, mgFIG. 1. Inhibition of plaque formation. S. mutans 6715 was

grown in 3 ml of PD-sucrose medium containing the indicatedamnounts of glucan 108 or dextran T200Q at 37°C. At 18 h, thecultures were processed as described in Materials and Methods foradherence. The A540 of the cells was determined.

The DEAE-cellulose-purified glucan (5 mg) prepared froma supernatant fluid from UAB108 (see Materials and Meth-ods) was hydrolyzed in 1 N HCl at 100°C for 2 h; thehydrolysate was treated with Dowex-1 (OH-), the effluentwas concentrated by lyophilization, and the amount ofglucose was determined by Glucostat (Worthington Diagnos-tics, Freehold, N.J.). Dextran T2000 and glucose weresimilarly treated. By comparing the amount of glucose foundwith the dry weight used for the hydrolysis procedure, therecoveries of glucose from glucan 108 and dextran T2000were 97 and 94%, respectively. The protein content ofglucan 108 was 0.06%.UAB108 inhibition of S. mutans 6715 virulence. The viru-

lence of UAB108 and the ability of this mutant to inhibit S.mutans 6715 virulence was assessed in gnotobiotic rats(Table 1). UAB108 induced significantly (P < 0.01) fewercaries on buccal, sulcal, and proximal molar surfaces ofmonoinfected rats than S. miutans 6715. Of interest was thedemonstration that rats infected with an equal number of S.mutans 6715 and UAB108 organisms exhibited caries activ-ity which was similar to that obtained in UAB108-mono-infected rats. The numbers of S. muitans UAB108 and 6715organisms in and mean body weight of diassociated rats weresimilar to the values obtained with hnonoinfected animals,and thus these parameters did not contribute to the resultantcaries activity. These results indicate that UAB108 waseffective in inhibiting S. mutans 6715-induced dental cariesformation in gnotobiotic rats infected with both strains. Itshould be pointed out that the glucan used in this studyinhibited in vitro adherence of heat-treated cells of 6715(data not presented).

Inhibition of plaque formation. The amount of plaqueaccumulated on walls of glass tubes by strain 6715 decreasedwith the addition of increasing concentrations of glucan 108(Fig. 1). Dextran T2000 also reduced the amount of accumu-lation of plaque by 6715, and maximum inhibition wasobtained with 5 mg of dextran T2000. Higher levels ofdextran T2000 resulted in a small but steady increase inplaque production. Glucan 108 was more efficient thandextran T2000 in reducing plaque formation.

Effect of glucan 108 on certain virulence traits of S. mutans6715 and UAB108. The following experiments were per-formed to test the ability of glucan 108 and in some instancesof dextran T2000 to (i) affect the types of glucan synthesizedby supernatant fluids of 6715, (ii) inhibit the sucrose-mediated adherence of 6715 and UAB108 cells to glasssurfaces, (iii) affect the aggregation of 6715 and UAB108cells, and (iv) inhibit caries formation in gnotobiotic ratsmonoinfected with S. mutans 6715.

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Effect of glucan 108 and dextran T2000 on glucan synthesisby a supernatant fluid from a glucose-grown culture of 6715. Asupernatant fluid from a glucose-grown culture of 6715 wasincubated with a constant amount of sucrose and increasingamounts of glucan 108, and the increasing concentrations ofglucan 108 resulted in a steady increase in the production ofnon-ad-WIG (Fig. 2A). In the case of ad-WIG, there was aninitial increase in the production of this type of glucan as theconcentration of the exogenously supplied glucan was in-creased to about 0.7 mg in a 3-ml reaction mixture. Higherconcentrations of glucan 108 resulted in a decrease in thead-WIG formation. In general, the amounts of WSG re-mained unchanged.When dextran T2000 was substituted for glucan 108, the

amounts of non-ad-WIG increased with increasing levels ofdextran T2000 (Fig. 2B). However, this increase ceased at adextran T2000 concentration of 2 mg. The maximal amountof non-ad-WIG produced in the presence of dextran T2000was about one-third the amount of non-ad-WIG produced inthe presence of glucan 108 (Fig. 2A and B). The amounts ofeither ad-WIG or WSG produced were not greatly affectedby increasing the concentrations of dextran T2000.

In separate experiments (data not shown), glucan 108obtained after a short incubation (15 to 60 min) of UAB108GTase preparation and sucrose was 75% less effective thanglucan 108 preparations obtained after a longer incubation (1to 18 h) in inhibiting the formation of ad-WIG.

Inhibition by glucan 108 and dextran T2000 on sucrose andGTase-dependent adherence by heat-treated cells. Glucan 108(Fig. 3A) and dextran T2000 (Fig. 3B) were tested for theireffect on adherence to glass surfaces by heat-treated cells of6715, UAB108, and a mixture of both types of cells in thepresence of GTase from 6715 and sucrose. Glucan 108 at alevel of 0.5 mg stimulated cell adherence, but with theaddition of more glucan 108, there was a steady decrease inadherence to about 20% of the adherence obtained in theabsence of glucan 108 (Fig. 3A).When dextran T2000 was used in place of glucan 108 (Fig.

3B), there was an initial decrease in cell adherence; how-ever, at a dextran T2000 concentration of 1.5 mg, there wasa reversal in the decrease. At low concentrations of dextranT2000, 6715 cells were most sensitive and showed about a50% decrease in adherence.

Effect of glucan 108 on aggregation of 6715 and UAB108cells. Aggregation of 6715 and UAB108 cells increased as theconcentration of either sucrose or dextran T2000 was in-creased (Table 2). UAB108 cells were somewhat aggregationdefective compared with 6715 cells. On the other hand, theactivity of glucan 108 to initiate cell aggregation was poor.Aggregation of cells occurred when the two types of cellswere mixed in the absence of substrates.

Inhibition of S. mutans 6715 virulence in gnotobiotic rats byglucan 108. The effectiveness of glucan 108 to inhibit S.mutans 6715 virulence was assessed in gnotobiotic rats(Table 3). Rats provided with glucan 108 in their drinkingwater exhibited significantly (P < 0.01) lower caries activityand fewer S. mutans organisms in plaque than controlanimals. Rats provided with dextran T2000 in their drinkingwater were not protected against S. mutans 6715-induceddental caries, whereas dextran T10 exerted some protection.However, dextran T10 was not as effective as glucan 108 ininhibiting S. mutans 6715-induced dental caries.

Effect of fungal dextranase and estimation of molecularweight. Various glucan hydrolases were employed to obtaininformation on the structure of glucan 108. When glucan 108was subjected to fungal dextranase treatment for 72 h, all of

E 15EIC

2 10 _

5

0 1 2 3 4 5 6

Glucan 108,mg

B * Ad-WIG10 0 Non-ad-WIG

2 AWSGSSA

0 ' '0 1 2 3 4 5 6

Dextran T2000, mgFIG. 2. Effect of glucan 108 (A) and dextran T2000 (B) on glucan

synthesis by a supernatant fluid from a glucose-grown culture ofstrain 6715 (13 pug of protein) in reaction mixtures containingsucrose. The indicated amounts of glucan 108 and dextran T2000were employed, and the incubation period was 18 h.

the glucan 108 was converted to a smaller form of glucan(Fig. 4A). The untreated glucan 108 peaked at 27 ml, and theproduct of enzyme hydrolysis peaked at about 70 ml inUltrogel A4 gel filtration chromatography. The recovery ofthe product was 102%, based on the amounts of radioactivityadded to the column and found in the 70-ml peak fraction.Glucan 108 emerged in the same volume as blue dextran(Fig. 4B), and thus the molecular weight of glucan 108 isestimated at 2 x 106. The product was applied to an UltrogelAcA-202 gel filtration column (Fig. 4C), and the principalproduct emerged in 30 ml, which corresponded to a glucanwith an approximate molecular weight of 1.2 x 104 (Fig. 4D)and thus was designated glucan 12k.Treatment of glucan 12k with glucanases. Glucan 12k

obtained by the treatment of glucan 108 by the fungaldextranase (Fig. 4A and C) was resistant to a secondtreatment with the same dextranase, as determined byUltrogel AcA-202 column filtration. Glucan 12k was onlyslightly susceptible to a(1-33) glucanase (C. resinae), as seenby a major peak (tubes 10 through 13) of the refractile glucan12k and a very minor peak (tube 28), suggesting the produc-tion of a very small amount of oligosaccharide (Fig. 5).Simultaneous treatment of glucan 12k with both the fungaldextranase and the C. resinae glucanase gave the sameresults as with the ct(1-*3) glucanase alone. A bacterialdextranase CB, on the other hand, was found to almostcompletely hydrolyze glucan 12k to smaller glucans andoligosaccharides (Fig. 5). Fractions containing the smaller

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110

1004

80

a)) 60

a)a1)V 40

20

a)

SC.)a2)'a)

Glucan 108, mg Dextran T2000, mgFIG. 3. Effect of glucan 108 (A) and dextran T2000 (B) on sucrose-dependent adherence to glass by heat-treated cells in the presence of

GTase (strain 6715).

glucans and oligosaccharides were pooled, concentrated,and treated with ac(1->3) glucanase, and when this mixturewas subjected to Ultrogel AcA-202 gel filtration, an elutionprofile nearly identical to that shown in Fig. 5 for dextranaseCB was again obtained. These results indicated that theproducts of dextranase CB were refractile to ot(1-3)glucanase.

Nigeran, which gave an Ultrogel filtration pattern similarto that of dextran T2000, was tested along with dextranT2000 as substrate for the various glucanases. The fungaldextranase which was inactive with glucan 12k was alsoinactive with nigeran but completely hydrolyzed dextranT2000. The ot(1-*3) glucanase which was also inactive with

TABLE 2. Aggregation of strain 6715 and UAB108 cells'

Substrate and Aggregation (arbitrary score)amt (g) 6715 UAB108 6715 + UAB108

Sucrose0 0 0 30.0004 1 0 30.0008 4 0 30.0020 5 1 30.0040 6 3 30.0080 6 3 3

Dextran T20000.4 1 1 20.20 2 2 20.50 3 2 21.00 4 2 25.00 6 2 2

Glucan 1080.40 0 0 20.20 0 0 20.50 0 0 31.00 0 0 45.00 1 0 4a Assay performed as described in Materials and Methods.

glucan 12k was inactive with dextran T2000 but active withnigeran. The bacterial dextranase which completely hydro-lyzed glucan 12k also hydrolyzed dextran T2000 but wasinactive with nigeran. Both cariogenanase and Merckdextranase hydrolyzed both dextran T2000 and nigeran,indicating that these two enzyme preparations have activi-ties to hydrolyze a(1-+6) and a(1--3) bonds.

Incorporation of glucosyl moiety of sucrose into glucan 108.Experiments were performed to determine the incorporationof the glucose moiety of sucrose into glucan 108 and the fateof exogenous glucan 108 under conditions in which glucan108 inhibited cell adherence. Three types of reaction mix-tures were prepared. The GTase from 6715 (supernatant fluidof a glucose-grown 6715 culture) was incubated with (i)[U-glucosyl-14C]sucrose, (ii) [U-glucosyl-14C]sucrose and 5mg of glucan 108, and (iii) sucrose and [14C]glucan 108. Theresults (Table 4) indicate that in the absence of added glucan108, the GTase preparation catalyzed the incorporation ofthe glucosyl moiety of sucrose into ad-WIG (37%), non-ad-WIG (5%), and WSG (22%). The specific radioactivity ofthese glucans synthesized by GTase from 6715 was similar tothe specific radioactivity of the [U-glucosyl-14C]sucrose used(1.5 x 105 dpm/mg of glucose). In the presence of 5 mg ofglucan 108, the pattern of the amounts of the three forms ofthe glucan produced by GTase from 6715 was altered. Therewas a reduction in the amount of ad-WIG and a largeincrease in the amount of non-ad-WIG, as shown previously(Fig. 2A). The ranking of specific activities (in an increasingorder when labeled sucrose and nonlabeled glucan 108 wereused and in a decreasing order when nonlabeled sucrose andlabeled glucan 108 were used) suggests that the sequence ofthe formation of the three forms of glucan is from WSG toad-WIG and finally to non-ad-WIG. A large proportion of theglucose from sucrose was found to reside in the non-ad-WIGfraction. Similarly, half or more of the exogenous glucan 108appeared in the non-ad-WIG fractions. The recovery of 5 mgof the added glucan was 94 and 84%.Attempts were made to obtain information on the molec-

ular masses of the radioactive WIG fractions (Table 4).

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TABLE 3. Inhibition of S. mutans 6715 virulence in gnotobiotic rats by purified glucan 108"Caries scores" Mean no

of S.No. of Buccal Sulcal Proximal Mean body ofuansType of dextranb rats per wt (g) CFU

group Enamel Dentinal Dentinal Dentinal Dentinal wt pg)CFUslight slight extensive slight mandibler

Glucan 108 14 13.6 ± 0.6 12.6 ± 0.4 12.4 ± 0.3 2.1 ± 0.4 2.5 ± 0.6 1.5 ± 0.6 97.8 ± 3.6 7.1 x 105Dextran T2000 12 18.5 ± 0.8 14.3 ± 0.4 16.8 ± 0.4 5.3 ± 0.7 6.7 ± 0.9 3.8 ± 0.6 127.5 ± 7.9 3.2 x 106Dextran T10 15 16.4 + 0.6 13.5 ± 0.5 14.9 ± 0.6 4.6 ± 0.3 4.3 ± 0.4 2.9 ± 0.3 122.6 ± 4.0 1.8 x 106None 13 19.9 ± 0.6 17.3 ± 0.4 17.1 ± 0.5 5.9 + 0.4 8.0 ± 0.0 5.2 ± 0.6 108.1 ± 5.9 3.7 x 106

a All rats were infected with an overnight log-phase culture of S. mutans 6715.b Glucan 108 and dextran T2000 and T10 were provided for rats in their drinking water (4 mg/ml).c See Table 1, footnote b.d Determined on MS agar.

Samples from ad-WIG, non-ad-WIG, and blue dextran as acomparative standard were subjected to CsCl density gradi-ent centrifugation as described by Germaine et al. (12).Although blue dextran banded in the fraction as reportedelsewhere (12), all of the WIG fractions fell to the bottom ofthe tube. Thus, this procedure was abandoned, and anotherprocedure was used. WIG fractions were sonicated andapplied to molecular sieve columns as described by Inoueand Koga (21). This 2-min sonication allowed an approxi-mately 90% recovery of the applied samples, but, unfortu-nately, about 20% emerged as smaller fragments. The frac-

0

x

a.

0

1 x10'

0

*a

co

E x 10O

0

0

I x 10'

5 10 15 20 25

Tube number (3ml/tube)

0 20 40 60 80 100

ml

tions containing 70% of the larger fragments showed approx-imate molecular weights of 2 x 106, whereas the meanmolecular weight in the fraction containing the smallerfragments was about 2 x 105. When the duration of sonica-tion was reduced to 15 s, few or no smaller fragments wereseen; however, only about 40% of the applied WIG wasrecovered. This peak fraction also represented a glucan withan estimated molecular weight of 2 x 106. Thus, the sonica-tion procedure was also unsuitable as a method to obtaininformation on the molecular mass of the WIG fractions. Theremaining fractions were then subjected to ct(1->3) or

x

0

30 0 5 10 15 20 25 30

Tube number (3ml/tube)

E-W0

-1 x 104

0

1o

I X 1030 20 30 40 50 60 70

mlFIG. 4. Treatment of glucan 108 with fungal dextranase and estimation of molecular weight. (A) Ultrogel A4 gel filtration of fungal

dextranase-treated and untreated glucan 108. (B) Molecular weight estimate of glucan 108 by a calibration curve obtained by using theindicated glucans as standards in Ultrogel A4 gel filtration. (C) Ultrogel AcA-202 gel filtration of the product of fungal dextranase treatmentof glucan 108. (D) Molecular weight estimate of the product of fungal dextranase treatment of glucan 108 by a calibration curve obtained withthe indicated polymers as standards by Ultrogel AcA-202 gel filtration.

A 9ct - (1-6) Glucanase treatment12 A o w/o a- (1-6) Glucanase

treatment

8 b

4

C1aulii 0 00 I,I,00 -aa1

12 -C

8

4-

0.3033a03e33C3ZCGee9ee0e

BBlue dextran, Glucan 108

Dextran T500

Dextran T110

Dextran T40

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12s0* Dextranase CB

O -0 Q

0 5 10 15 20 25 30

Tube number (3ml/tube)FIG. 5. Ultrogel AcA-202 gel filtration of glucan 12k treated with

a(1-3) glucanase or bacterial dextranase CB.

at(1- 6) glucanase treatment, and after 72 h of incubation,the mixtures were centrifuged, and the supernatant fluidswere assayed for carbohydrates to determine the percentsolubility. Sigma dextranase [a(1-*6)] solubilized 33 to 59%of the ad-WIG fractions and 40 to 51% of the non-ad-WIGfractions. Similar ranges of solubilization for these glucanfractions were found after cx(1->3) glucanase treatment.Although we were unable to determine the molecular massof the two WIG fractions, it appears that in the presence ofsucrose, GTase from 6715 modified glucan 108 by incorpo-rating the glucosyl moiety of sucrose into glucan 108 ina(1-+3) linkages.

DISCUSSION

When a non-plaque-forming mutant, S. mutans UAB108,was grown with its plaque-forming parental strain, UAB66,or with 6715 in a sucrose-containing medium, little or noplaque was produced (32, 33). Likewise, when gnotobioticrats were monoassociated with strain UAB108 or diassoci-ated with strains UAB108 and 6715, low caries incidencewas found. This reduction in in vitro plaque formation and invivo caries incidence by mixtures of UAB108 and 6715 didnot appear to be due to a faster growth of one strain over theother (data not presented). Also, little or no in vitro plaquewas noted when strain UAB66 or 6715 was grown in sucrosemedium supplemented with a supernatant fluid from a glu-cose-grown culture of UAB108 or with an alcohol precipitateobtained from a supernatant fluid of UAB108 that wasincubated with sucrose (33). In the present study, thesupernatant fluids from glucose-grown 6715 and UAB108

were employed to determine their activities to producead-WIG, non-ad-WIG, and WSG. Strain 6715 produced largequantities of ad-WIG and relatively little WSG. UAB108, onthe other hand, produced very little ad-WIG and a largequantity of WSG, designated glucan 108. This water-solubleproduct synthesized by strain 108 was found to consist ofglucose units.A detailed study of the pattern of the synthesis of the three

types of glucans in the presence of increasing amounts ofeither glucan 108 (Fig. 2A) or dextran T2000 (Fig. 2B) bysupernatant fluids of 6715 cultures revealed that there was aninitial increase and then a decrease of ad-WIG formationwith increasing amounts of glucan 108. With increasedconcentration of glucan 108, there was a steady increase innon-ad-WIG, but no effect on WSG production. With theexception of a limited increase in the amounts of non-ad-WIG with increasing dextran T2000 concentration, therewere few changes in the formation of ad-WIG. Because thesupernatant fluid from glucose-grown strain 6715 (and alsoUAB108) was free of glucanase activity for dextran T2000and glucan 108 and of fructosyltransferase activity, thealteration of the pattern of glucan synthesis is believed not tobe due to these enzyme activities. Previous investigationshave reported that dextrans stimulate WIG synthesis andthat at high concentrations of dextran, WIG synthesis isinhibited (18, 19, 22). Moreover, at high inhibitory concen-trations of dextrans, there was a shift from WIG synthesis toWSG synthesis (19, 26, 36). Results obtained from similarexperiments with heat-treated cells of 6715 or UAB108 orboth in the presence of increasing amounts of glucan 108(Fig. 3A) indicated that the adherence by such cells corre-lated directly with synthesis of ad-WIG and indirectly (at aglucan 108 concentration greater than 0.5 mg) with synthesisof non-ad-WIG. Dextran T2000 also inhibited cell adher-ence; however, at concentrations higher than 1.5 mg, thisinhibition was reversed. These results suggest that inhibitionof adherence is related indirectly to synthesis of ad-WIG anddirectly to synthesis of non-ad-WIG.There is a general belief that plaque formation and cell

adherence are related to WIG synthesis (14, 17, 34, 37).However, our results suggest that high levels of non-ad-WIGproduction correlate with a decrease in cell adherence. Littleattention has been given to specific study of the synthesis ofad-WIG and its importance in plaque formation and celladherence. Gibbons and Nygaard (17) reported that adherentdeposits from sucrose-grown cultures contained bothglucans and cells. Mukasa and Slade (30) then demonstratedthat the incubation of a GTase preparation and sucroseresulted in the formation of two types of glucans, one thatadheres to glass surfaces and another that sediments (WIG)to the bottom of tubes. A GTase preparation from S. mutans

TABLE 4. Incorporation of glucosyl moiety of sucrose and glucan 108 into the three fractions of glucan by strain 6715 GTasea

Results for:

Reactants (amt) Ad-WIG Non-ad-WIG WSGmg dpm dpm/mg mg dpm dpm/mg mg dpm dpm/mg

[U_glUCOSyl-14C]sucrose (30 mg) 5.82 844,259 145,062 0.39 61,050 154,945 1.71 273,296 160,291[U_glUCOSyl-14C]sucrose (30 mg) 3.32 355,988 107,226 14.44 1,678,581 116,245 0.46 39,345 86,204+ glucan 108 (5 mg)

Sucrose (30 mg) + [14C]glucan 108 (5 mg) 2.04 62,750 30,730 10.48 221,984 21,182 1.46 90,345 62,092a The incorporation experiment was carried out as described in Materials and Methods.

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PLAQUE-INHlBITING GLUCAN FROM S. MUTANS 841

TABLE 5. Glucanase treatment of WIG fractions synthesized inthe presence of glucan 108 by a GTase preparation of strain 6715'

C/c Solubilized

Condition o-16)3)Glusanase Glucanase

[U-glucosy'-'4C]sucroseAd-WIG 33 56Non-ad-WIG 49 41

[U-glucosyl-'4C]sucrose + glucan 108Ad-WIG 59 41Non-ad-WIG 51 50

Sucrose + ['4C]glucan 108Ad-WIG 48 34Non-ad-WIG 40 42

" Ad-WIG and non-ad-WIG obtained from the experiment described inTable 4 were treated with either ot(1-3) glucanase (C. resinc) or fungalox(1-6) glucanase (Sigma dextranase). See Materials and Methods for details.

GS-5 was shown by Kuramitsu (24) to synthesize a glucanthat adhered to glass surfaces to which cells were found toadhere. Recently, Fukushima et al. (10) separated and puri-fied GTase from S. mutans B-13 into two components. Thefirst component, GT-S, synthesized about 96% WSG andminor amounts of ad-WIG and non-ad-WIG. The secondcomponent, GT-I, on the other hand, produced no WSG,and the amount of ad-WIG and non-ad-WIG produced was0.2 and 1%, respectively, of the WSG produced by GT-S.Despite the very low activity of GT-I to produce glucans,about 92% of the added cells adhered when incubated withGT-I and sucrose. When dextran T10 was added to a mixturecontaining GT-I, sucrose, and cells, there was a 20-foldincrease in the amounts of non-ad-WIG production andabout a 15% decrease in cell adherence. These authors madethe suggestion that WIG synthesis per se is not required foradherence. Results with glucan 108 support the notion thatthe ad-WIG is most important for plaque formation.Another trait related to virulence of S. miatans is cell

aggregation (15). Glucan 108, which was shown to bind bothheat-treated and untreated cells (data not presented), causedlow or no aggregation of 6715 and UAB108 cells, whilesucrose or dextran T2000 induced aggregation. Heat treat-ment of cells of S. mutans apparently does not affect thebinding of glucans, as previously demonstrated by others (7,38), but the rclationship between glucan binding and aggre-gation is not clear. Gibbons and Fitzgerald (15) demon-strated that polymer-induced aggregation was limited tohigh-molecular-weight dextrans (>2 x 105) and that levans,starch, and dextran were without activity. Wu-Yuan et al.(38) found that low-molecular-weight dextrans (dextrans T20and T70) bind cells of S. mutans, and McCabe and Smith (27)found that a branched low-molecular-weight dextran wasmore effective than a linear dextran of similar molecularweight to induce aggregation. The low activity of glucan 108to induce cell aggregation appears to reside in the structuralnature of glucan 108.Glucan 108, which was found to be more effective than

dextran T2000 in inhibiting in vitro plaque formation, ad-WIG synthesis, and adherence of cells to glass surfaces andto be less effective than dextran T2000 in promoting cellaggregation, was also found to be more effective in inhibitingplaque and caries formation in gnotobiotic rats infected withstrain 6715. Thus, it would appear that glucan 108 is effectivein inhibiting virulence properties of S. mutans in both invitro and in vivo model systems.

The mutant, S. munttan1s UAB108, produced mainly WSGand negligible amounts of WIG. The molecular weight ofglucan 108 was estimated to be about 2 x 106 (Fig. 4A andB). When this glucan was treated with the fungal dextranase,only glucans of molecular weights of the order of 1.2 x 104(glucan 12k) were produced. The fact that smaller glucansand oligosaccharides were not produced suggested thatglucan 108 may contain many branch points with a periodic-ity of sites sensitive for fungal dextranase (Sigma) to yieldglucan 12k. Glucan 12k was found to be sensitive to bacterialdextranase (Calbiochem) but was refractile to cx(1--3)glucanase (C. resinae). This fact does not necessarily pre-clude the absence of branch points, since it was previouslyreported that this latter enzyme is unable to hydrolyzecx(1->3) linkages that are in juxtaposition to (x(1-*6) gluco-sidic linkages (19). Dextranase CB, on the other hand, maynot be sterically hindered by branch points as long as thereare sites of at least six glucose units in sequence with ox(1->6)glucosidic linkages (19). The fact that the treatment of glucan12k by dextranase CB yielded glucans of molecular weightsranging from 2,000 to 9,000 (Fig. 4D and Fig. 5) and notsmaller oligosaccharides (19) also suggests that the partialhydrolysis of glucan 12k is due to the presence of manybranch points and that the substrate sites for the enzyme arenot found with regular periodicity. Recently it was reportedthat mutants of strain 6715 which are defective in synthesiz-ing WIG synthesize soluble, branched ot(1->6) glucans (6).These results, taken together, suggest that the synthesis ofglucan 108 by mutant UAB108 probably involves the assem-bling of about 200 glucan 12k units with many branch points.The defect in mutant UAB108 appears to be its inability topolymerize the branched points of glucan 12k with chains ofco(l1-3) glucosidic units.

Stimulation of and dependency for exogenous dextrans bydifferent GTase preparations have been investigated bynumerous investigators (8, 12, 19, 23). Although the mech-anism for incorporation of the glucosyl moiety of sucroseinto glucan is not settled (36), there is evidence that incor-poration does occur (9, 12, 13, 27). Recently, Fukui et al. (9)concluded from their study that the glucose of sucrose isincorporated into position C-3 of the glucosyl residue ofdextran TIO. The molecular weight of primers appears to beimportant, since values of degree of polymerization ofisomalto-oligosaccharides greater than eight are required(11, 22). Inhibition of glucan production and cell adherenceby dextrans of low and high molecular weights has beenreported elsewhere (3, 5, 16, 17, 19, 35).The results from isotope studies indicated that a GTase

prepar4tion from strain 6715 catalyzed the incorporation ofglucan 108 and the glucosyl moiety of sucrose into threetypes of glucans. The presence of glucan 108 altered the typeof glucan produced from one that was mainly ad-WIG(absence of glucan 108) to one that was non-ad-WIG. Theinhibition of plaque formation in sucrose-containing mixedcultures of strains 6715 and UAB108 probably also occuredby a similar mechanism; i.e., glucan 108 synthesized by themutant diverted synthesis of ad-WIG by the former of plaqueto synthesis of non-ad-WIG, resulting in a failure to produceplaque.The data presented here and previously (33) indicate that

glucan 108 is potentially an effective antiplaque and -cariesreagent. This product of UAB108 is water soluble andappears to be suitable to a variety of oral delivery systems.In the oral cavity, glucan 108 can act to prevent the initialformation of plaque and thus interrupt the sequence of

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842 TAKADA ET AL.

events leading to caries. It is envisioned that in the presenceof glucan 108, the synthesis of ad-WIG by GTase of cario-genic streptococci is aborted by diverting the synthesistowards a presumably benign non-ad-WIG. To put this beliefon a firmer ground, further work is necessary to obtainadditional information concerning the physical-chemical andtoxic properties of glucan 108 and to perform clinical inves-tigations on the prophylactic property of the glucan.

ACKNOWLEDGMENTS

We greatly appreciate the help of Cecily C. Harmon and GloriaRichardson for in vivo animal studies, of Helene M. Thedford forlaboratory assistance, and of Yvonne Noll for editorial assistance.

This work was supported by U.S. Public Health Service contractDE 62491 and grants DE 02670, DE 06432, and DE 06801.

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13. Germaine, G. R., and C. F. Schachtele. 1976. Streptococcusmutans dextransucrase: mode of interaction with high-molecular-weight dextran and role in cellular aggregation. In-fect. Immun. 13:365-372.

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20. 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 cariogenic-ity. Infect. Immun. 19:477-480.

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22. Koga, T., S. Sato, M. Inoue, K. Takeuchi, T. Furuta, and S.Hamada. 1983. Role of primers in glucan synthesis by glucosyl-transferases from Streptococcus mutans strain OMZ176. J.Gen. Microbiol. 129:751-754.

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26. McCabe, M. M., and E. E. Smith. 1973. Origin of the cell-associated dextransucrase of Streptococcus mutans. Infect.Immun. 7:829-838.

27. McCabe, M. M., and E. E. Smith. 1975. Relationship betweencell-bound dextransucrase and the agglutination of Streptococ-cus mutans. Infect. Immun. 12:512-520.

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31. Mukasa, H., and H. D. Slade. 1974. Mechanism of the adherenceof Streptococcus mutans to smooth surfaces. III. Purificationand properties of the enzyme complex responsible for adher-ence. Infect. Immun. 10:1135-1145.

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