INFECTlON AND IMMUNITy, Feb. 1978, p. 4024100019-9567/78/0019-0402$02.00/0Copyright i 1978 American Society for Microbiology

Vol. 19, No. 2

Printed in U.S.A.

Cellular Adherence, Glucosyltransferase Adsorption, andGlucan Synthesis of Streptococcus mutans AHT Mutants

TOSHIHIKO KOGA AND MASAKAZU INOUE*Department ofPreventive Dentistry, Kyushu University, School of Dentistry, 3-1-1, Maidashi, Higashi-ku,

Fukuoka, 812, JapanReceived for publication 7 June 1977

Streptococcus mutans AHT mutants Ml, M2, and M13 failed to adhere to aglass surface, whereas mutants M9 and M35 exhibited decreased and increasedadherence, respectively, as compared with the parent strain, when grown insucrose broth. Extracellular glucosyltransferase prepared from glucose-growncultures of the adherent strains (wild type, M9, and M35) induced adherence ofheat-killed cells of the homologous and heterologous streptococcal strains aswell as of Escherichia coli K-12 and uncoated resin particles. The glucosyltrans-ferase was adsorbed on all the streptococcal cells and glucan-coated resins, butnot on E. coli cells and the uncoated resins. Glucosyltransferase from thenonadhering mutants (Ml, M2, M13) neither was significantly adsorbed on norinduced adherence of any of the cells and resins. Cell-free enzymes from theglucose-grown adherent strains produced water-soluble and water-insoluble glu-cans, whereas those from the nonadhering mutants produced only water-solubleglucans. Small amounts of alkali-soluble, cell-associated glucan were recoveredfrom the sucrose-grown nonadhering mutants. Thus, the relative proportions ofglucosyltransferase isozymes elaborated by the S. mutans mutants, insofar asthey affect the physico-chemical properties of the glucans produced, seem todetermine the adherence abilities of the cells. The adsorption of glucosyltransfer-ase on glucan molecules on the cell surface is not required for the adherence ofS. mutans, but de novo glucan synthesis is important in the adherence process.

Streptococcus mutans forms adhesive depos-its on teeth and induces dental caries in experi-mental animals reared on a sucrose-containingdiet (1, 9, 13, 50). Evidence has been accumulat-ing that supports the concept that this micro-organism is also a prime etiological agent in thedevelopment of dental caries in humans (5, 20,31, 44). One of the characteristic properties ofS. mutans is an ability to produce extracellularglucans from sucrose by the action of constitu-tive glucosyltransferase (GTF, EC 2.4.1.5; 14).The glucans are highly branched and resistantto enzymatic hydrolysis by dextranase (EC3.2.1.11; 2, 16, 40). Several isozymes of the extra-cellular GTF are probably responsible for syn-thesis of the glucans (3, 11, 16, 21, 39), and theirtypes and relative amounts probably controlphysico-chemical properties of the glucans (2,6, 17, 29). Water-insoluble glucans are supposedto be of major importance in adherence of S.mutans to tooth surfaces (26, 30, 37, 38, 43, 49).Mutants of cariogenic S. mutans that differ

in properties from the parent strain would beuseful in elucidation of the virulence factor(s)of S. mutans. Many investigators have reported

several types of S. mutans mutants (4, 7, 10, 19,22, 23, 28, 35, 36, 42, 47) and have studied theirGTF activity, glucan synthesis, adherence orartificial plaque formation, and cell aggregationin relation to cariogenicity. However, the precisemechanism of development of dental caries stillremains obscure. Adherence seems to be a majorfactor in the cariogenicity of S. mutans, sinceestablishment and persistence of the cariogenicmicroorganisms on tooth surfaces are a primarystep for subsequent onset of tooth decay. Wehave recently isolated several types of mutantsfrom S. mutans AHT possessing different abili-ties to adhere to glass surfaces when grown insucrose broth (24). A comparative study ofthesemutants would facilitate better understandingof the mechanism of adherence of S. mutansand hopefully of the subsequent developmentof dental caries. The present study describescellular adherence to a glass surface, adsorptionof GTF on cells, and glucan synthesis from su-crose by representative strains of the mutantsand the cariogenic wild-type strain. We includeunder the term "adherence" the entire processof sucrose-induced attachment of S. mutans to

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ADHERENCE ABILITY OF S. MUTANS AHT MUTANTS

a smooth surface, i.e., both the initial cell-to-surface adherence and the subsequent cell-to-cell adherence.

MATERIALS AND METHODSBacterial strains and culture conditions. S. mu-

tans AHT (originally clasified as serotype a but morerecently as type g, S. Hamada, personal communica-tion) was kindly supplied by D. D. Zinner of theInstitute of Oral Biology, University of Miami, Miami,Fla. The wild-type strain (designated WT) and itsmutant strains, Ml, M2, M13, M9, and M35, were

used in this study. These mutants differed from WTin their colonial morphology and their ability to adhereto a glass surface when grown in sucrose medium. Adetailed description of the isolation procedures andsome properties of the mutants has previously beenreported (24). For all studies, cultures of the parentand mutant strains were grown in Todd-Hewitt broth(Difco Laboratories, Detroit, Mich.) supplementedwith glucose at a final concentration of 1% or inTrypticase-salt medium (14) containing 5% sucrose at370C under an atmosphere of 90% N2 + 5% C02 + 5%H2 in a Te-Her Anaero-Box (Hirasawa Works, Tokyo,Japan). Either glucose or sucrose was added asepti-cally before inoculation. Escherichia coli K-12 waskindly supplied by Nozomu Otsuji of the Faculty ofPharmaceutical Sciences, Kyushu University, Fuku-oka, Japan. The culture was grown in Todd-Hewittbroth with 1% glucose aerobically at 370C for 6 h.Resin particles. Polyacrylic resin particles (O; 0.5A,

Bofors Co., Sweden) were kindly supplied by Tatsu-hiko Tstisui of the Basic Research Laboratories,LION Dentifrice Co. Ltd., Kanagawa, Japan.Adherence ofgrowing cells. S. mutans cells were

grown in 5 ml of sucrose broth in a glass test tube (18by 180 mm) for 2 days. The tube was kept uprightduring the incubation. After the culture was disardedcarefully, the microbial deposits on the tube wall werewashed twice with deionized water and then sus-pended in 20 ml of water by vigorous vibration witha mixer and scraping with a rubber-coated rod. Thesuspensions were homogenized by sonic oscillation.Optical density at 550 nm (ODm6) of the suspensionswas measured with a Shimadzu-Baush & Lomb Spec-tronic 20 Colorimeter (Sbimadzu Seisakusho Ltd.,Kyoto, Japan).

Distribution of GTF activity of glucose-growncells. Culture of S. mutans strains was grown inglucose broth to the early stationary phase. A portionof the culture was saved for the determination of totalactivity of cell-bound and -free GTF in the wholeculture. Cells were harvested from remaining culture,washed by centrifugation, and suspended in an originalvolume of 0.01 M citrate buffer (pH 5.5). GTF activityin the whole culture and the washed ceU suspensionwas determined by the method described below. Cell-bound enzyme activity was expressed as percentageof the total activity in the whole culture.

Preparations of crude enzyme, heat-killedcells, and resin particles. Crude enzyme prepara-tions and heat-killed cells of S. mutans strains usedin these experiments were obtained as follows. Cultureof the strains was grown in glucose broth for 16 h.

The culture supernatant (1 liter) was obtained bycentrifugation (6,000 x g, 20 min, 400) and precipitatedwith (NH2)4SO4 at 50% saturation overnight at 4°C.The resulting sediment was collected by centrifugation(12,000 x g, 60 min, 4°C), dissolved in 25 ml of 0.05 Mphosphate buffer (pH 6.0) containing 0.02% merthio-late, and dialyzed against the same buffer. It was thencentrifuged again to remove undissolved materials,and the supernatant was stored frozen. GTF activityin the enzyme preparation from each strain is sum-marized in Table 1. No fructosyltransferase (EC2.4.1.10) activity was detected in any preparation. Thecells harvested from the culture were washed by cen-trifugation, suspended in a small volume of the buffer,and heated at 1000C for 20 min to inactivate cell-bound enzyme activity. They were then washed twiceand suspended in the buffer at a concentration of 0.5%(wet weight/volume). Heat-killed cells of E. coli K-12 were prepared similarly. Polyacrylic particles werewashed extensively and suspended in the phosphatebuffer at a concentration of ODMo = 0.6 x 6. Thewashed resins were coated with glucan in a reactionmixture containing 16.6 mU of GTF from WT strainand 1 g of sucrose in 40 ml of 0.05 M phosphate buffer(pH 6.0) at 370C. After 16 h, the reaction mixturewas heated at 1000C for 20 min to inactivate enzymeactivity, and the glucan-coated resins were washed bycentrifugation, suspended in the buffer, and subjectedto sonic treatment to obtain a homogeneous disper-sion. ODsso of the suspension was adjusted to 0.6 x 6.Cell and resin suspensions were stored at 40C untilrequired.Adherence of heat-killed cells. Adherence of

heat-killed cells to a glas surface was measured ac-cording to a slightly modified procedure of Mukasaand Slade (37). The appropriate amount of enzyme, 5mg of heat-killed cells (or 1 ml ofthe resin suspension),and 50 mg of sucrose were allowed to react in 6 ml of0.05 M phosphate buffer (pH 6.0) containing 0.02%merthiolate. After incubation at 370C for 16 h in atest tube held at an angle of 300, the deposits formedon the glass surface were homogeneously suspendedin 6 ml of the buffer by the same manner as describedabove. Turbidity of the suspension was measured at550 nm.Adherence of heat-killed cells to preformed

glucan film. To prepare a glucan film on glass surface,50 id (0.83 mU) of enzyme from WT strain wereincubated with 50 mg of sucrose in 6 ml of 0.05 Mphosphate buffer (pH 6.0) at 370C for 16 h in test

TABLE 1. GTF activity in ceU-free enzymesobtained from WT and mutant strains grown in 1%

glucose broth

Enzyme from GTF activity Protein Sp actstrain (mU/mi) (mg/ml) (mU/mg ofprotein)WT 17.1 19.9 0.86Ml 31.5 8.8 3.58M2 9.8 9.3 1.05M13 7.8 13.8 0.57M9 11.0 16.8 0.65M35 20.8 10.0 2.08

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404 KOGA AND INOUE

tubes kept at a 300 angle. After the reaction fluid wasdiscarded carefully, the film was washed twice withthe buffer by rotating the tubes by hand. The film inhalf of the tubes (at 30° angle) was treated at 37°Cfor 6 h with 2 mg of invertase (EC 3.2.1.26; 11 U/mg;bakers' yeast; Sigma Chemical Co., St. Louis, Mo.) in6 ml of the buffer to remove residual sucrose. Thefilm in the other tubes was treated in the same waywith buffer without invertase. Half of each set of tubesthat had been treated with buffer or with invertasewere then heated at 100°C for 20 min to inactivateGTF and/or invertase entrapped in the film, and thenthe mixture was discarded carefully. Active WT en-zyme was added in a volume of 50 ,lI to the tubes,and finally 6 ml of buffer containing 5 mg of heat-killed WT cells was added gently. In a second seriesof tubes, active WT enzyme was replaced by 50 il ofheat-inactivated enzyme. All the tubes were then in-cubated at 37°C for 16 h at an angle of 300. Thedeposits on the films were suspended in 6 ml of thebuffer as described above, and OD5ws of the homoge-neous suspensions was read.Adsorption ofextracellular GTF on heat-killed

cells. Crude enzyme (4.2 mU) and 2.5 mg of the cells(or 0.5 ml of the resin suspension) were preincubatedin 1 ml of 0.01 M citrate buffer (pH 3.0 to 6.0) orphosphate buffer (pH 6.0 to 8.0) at 4 or 37°C. Imme-diately after an appropriate time of incubation, a por-tion of the mixture was saved, and the remainingsuspension was centrifuged. Precipitated cells werewashed twice and suspended in the same volume ofthe buffer used for preincubation. GTF activity in thewhole and the washed cell suspension was assayed bythe method described below. Adsorbed activity re-covered in the washed cell suspension was expressedas percentage ofthe total activity in the whole mixture.Assay for GTF activity. GTF activity in the crude

enzyme preparations was determined by the methodof H. Suginaka (personal communication) modifiedby Inoue et al. (21). Assay mixture consisted of 5 tlof [U-14C]sucrose (0.01 ,uCi/,tl, 10 mCi/mmol; DaiichiPure Chemicals Co., Tokyo, Japan), 10 id of enzyme,and 10 pl of 0.2 M citrate buffer (pH 6.0). Afterincubation at 37°C for 60 min, 10 ,ul of the digest wasspotted onto a filter paper (no. 51; Toyo-roshi, Tokyo,Japan). The paper was developed by ascending chro-matography with a solvent of n-butanol-pyridine-water (6:4:3) for 4 h. This separates the residual su-crose and released monosaccharides from the synthe-sized polysaccharides that remain at the origin. Radio-activity recovered in the polysaccharides was mea-sured with a liquid scintilation counter (DPO-100,Beckman Instruments Inc., FuUerton, Calif.). One unitof GTF activity was defined as the amount of enzymethat would transform 1 ymol of sucrose to glucan permin under the conditions specified.

Polysaccharide synthesis in sucrose broth. S.mutans cells were grown to early stationary phase insucrose broth. CeUs and three classes of glucans, (i)extraceUlular, water-soluble, (ii) 1 N NaOH-soluble,cell-associated, and (iii) 1 N NaOH-insoluble, cell-as-sociated, were prepared from the culture according tothe procedure of Freedman and Tanzer (10). Thepolysaccharide preparations were hydrolyzed in 4 NHCI at 100°C for 3 h, dried in vacuo, and dissolved in

INFECT. IMMUN.

0.3 M phosphate buffer (pH 7.0). Glucose content ofthe preparations was determined by glucose-oxidasekit (Fujisawa Pharmaceutical Co., Osaka, Japan). Theamount of glucans was normalized to protein contentof the cells, which was determined by the method ofLowry et al. (32).

Cell-free synthesis of water-soluble and -in-soluble glucans. S. mutans was grown in glucosebroth to early stationary phase. Supernatant fluidobtained by centrifugation was filtered through a filterpaper (no. 101, Toyo-roshi). The filtrate was adjustedto pH 7.0 with 1 N NaOH, a few drops of toluenewere added, and then the solution was incubated at37°C for 16 h in the presence of 5% sucrose. Water-insoluble polysaccharides synthesized were harvestedfrom the mixture by centrifugation (20,000 x g, 15min, 4°C), washed three times with water, and thensuspended in a small volume of deionized water. Wa-ter-soluble polysaccharides were precipitated from thesupernatant solution by the addition of 2.5 volumesof ethanol and harvested by centrifugation. After sev-eral ethanol precipitations, they were redissolved in asmall volume of water. The polysaccharide prepara-tions were dialyzed extensively against deionized waterand lyophilized. Amount of polysaccharide in eachfraction was determined by the Anthrone method (48),using glucose as a standard.Smith degradation of polysaccharide and

analysis of the degraded products by gas-liquidchromatography. Degradation of polysaccharideswas performed by the method of Hamilton and Smith(18). Native polysaccharides (300 mg) were oxidizedwith 6 mmol of NaIO4 in 300 ml of 0.05 M citratebuffer (pH 4.0) at 4°C in the dark. After 6 days,oxidation products of the insoluble polysaccharideswere recovered, washed by centrifugation, and sus-pended in the original volume of deionized water.Those of the soluble polysaccharides were dialyzedagainst water at 4°C for 2 days. Both of the oxidizedpreparations were then reduced with NaBH4 (300 mg)at room temperature overnight. Excess borohydridewas decomposed by neutralization with 1 N HCI andremoved by centrifugation or dialysis. The resultantend products were lyophilized. The polyalcohols werehydrolyzed with 2 M H2SO4 at 100°C for 4 h and thenpassed through a small column of Dowex 2 x 4 (bicar-bonate form; Dow Chemical Co., Midland, Mich.) toremove acid. Eluted fractions were pooled and driedin vacuo, and the dried residues were trimethylsily-lated in the usual way and analyzed by gas-liquidchromatography.

RESULTS

Adherence of cells to glass surface. Ad-herence of S. mutans cells grown in sucrosebroth was tested quantitatively (Table 2). Mu-tants Ml, M2, and M13 failed to form adhesivemicrobial deposits. Mutant M9 produced a verysmall quantity of strongly adhering deposits,whereas M35 formed a large amount of looselyadhering deposits.The adherence abilities of growing cells of

WT and mutant strains were reproduced in an

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TABLE 2. Adherence to glass surface ofgrowingcells of WT and mutant strains in 5% sucrose broth

Strain Adherence (OD5e)WT 0.53Ml 0.02M2 0.02M13 0.03M9 0.08M35 0.80

TABLE 3. Adherence to glass surface ofheat-killedcells and resin particles in the presence of cell-free

GTF and sucroseAdherence (OD,a)

Cells GTF (0.8 mU/tube) from strain:Buffer

WT Ml M2 M13 M9 M35

WT 0.03 0.40 0.03 0.03 0.02 0.07 0.17Ml 0.03 0.49 0.02 0.01 0.03 0.20 0.32M2 0.01 0.30 0.02 0.01 0.06 0.18 0.13M13 0.03 0.41 0.03 0.01 0.05 0.18 0.17M9 0.02 0.41 0.03 0.02 0.03 0.14 0.13M35 0.02 0.25 0.01 0.00 0.01 0.03 0.14E. coli 0.06 0.31 0.03 0.04 0.03 0.09 0.16Resin 0.04 0.44 0.04 0.04 0.05 0.09 0.38

assay system for the adherence of nongrowingcells (Table 3). When heat-killed, glucose-growncells of the strains were incubated with the ho-mologous enzymes (0.83 mU) in the presence ofsucrose, the cells of WT, M9, and M35 adheredto the glass surface, and those of nonadheringmutants Ml, M2, and M13 did not. Furthermore,the enzymes from WT strain and mutant M35induced adherence of the heterologous cells ofadherent and nonadhering mutants, E. coli, anduncoated resin particles. The adherence of cellsinduced by M35 enzyme was always in a smallerquantity than that byWT enzyme at equivalentGTF activities. The enzyme from mutant M9also adhered the cells of almost all strains andresins. In contrast, the enzymes from nonadher-ing mutants Ml, M2, and M13 did not induceadherence ofany kind of cells and resin particles.No adherence of heat-killed WT cells was ob-served over a wide range of activities of theenzymes from nonadhering mutants (Fig. 1).This suggests that it is not cell surface compo-nents but the nature of GTF that determinesthe adherence ability of a mutant strain.Demonstration of importance of the de

novo glucan synthesis for adherence. Someinconsistencies among the previous results on

the requirement of de novo synthesis of water-insoluble glucans for adherence of S. mutanscells prompted us to examine binding of heat-killed WT cells to glucan film preformed on a

gls surface and subjected to various treat-ments (Table 4). The celLs adhered to the glucan

: L0.4C=

C.3

0 0.5 1.0G T F (mU/t ube )

FIG. 1. Adherence of heat-kiled WT cells incu-bated with various amounts of cell-free GTF frommutant strains in the presence of sucrose. Enzymesfrom WT (0), Ml (A), M2 (A), M13 (U), M9 (0),and M35 (0).

film even after gentle washings with buffer. Ad-dition of active GTF to the assay mixture didnot increase the adhesion of cells to untreatedfilm. There was no cell adhesion to glucan filmsthat had been heated, but significant adhesionwas induced by the addition of active GTF. Cellsdid not adhere to invertase-treated films eitherwith or without addition of active GTF. Thus,sufficient amounts of active GTF and sucroseto cause adherence remained on or in the buffer-washed film. The ODss of the invertase-treatedand/or heated glucan films was slightly higherthan that of the control glucan film which wasnot exposed to cells, but was much lower thanthat of untreated glucan films or heated glucanfilms to which active GTF was added. Theseresults indicate that de novo synthesis of water-insoluble glucans is an important process in theadherence of S. mutans to a smooth surface.Adsorption of GTF on ceils. Distributions

of cell-free and -bound activity ofGTF producedby the strains grown in glucose broth were tested(Table 5). More than 80% of the enzyme activityof strains WT, M9, and M35 was cell bound,whereas less than 60% was bound to nonadher-ing mutant cells.

Before the following adsorption experiment,we established optimal conditions for the assayand adsorption of cell-free GTF activity on heat-killed cells using the enzyme and cells of WTstrain. GTF (4.2 mU) was incubated with 2.5mg of the cells under various conditions (Fig.2). Fifty to sixty percent of GTF activity wasadsorbed on the cells at pH values below 5.0,

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TABLE 4. Adhesion of heat-killed WT cells to heated and/or invertase-treated glucan film preforned onglass surface

Washing with Invertase treat- Heat treatment Addition of active Addition of WT Adhesion of cellsbuffer ment (mg/tube) (1000C, 20 min) GTF (0.86 mU) cells (5 mg) (OD5w) (A)I+ b - + 0.33 (0.26)+ - _ _ _ 0.07 (0.00)+ oc _ d + 0.38 (0.31)+ 0C - + + 0.36 (0.29)+ 2 - _d + 0.12 (0.05)+ 2 - + + 0.13 (0.06)+ OC + _d + 0.16 (0.09)+ 0c + + + 0.25 (0.18)+ 2 + _d + 0.12 (0.05)+ 2 + + + 0.14 (0.07)

a The numbers in parentheses equal the OD given to the left of the column minus the OD of 0.07 attainedwithout cells added.

b Treatment skipped or supplement not added.c Treated with buffer without invertase.d Heat-inactivated (100°C, 20 min) GTF used.

TABLE 5. CeUl bound GTF activity of WT andmutant strains grown in 1% glucose broth

GTF activity

Strain Whole culture Cell-bound(mU/ml) (% of total)

WT 1.3 82.6Ml 3.7 57.2M2 1.2 59.2M13 0.6 34.1M9 1.8 91.5M35 4.1 99.6

but only approximately 10% of total activity wasadsorbed at pH 8.0. Adsorption rates at pH 4.5remained around 50% over a wide range of en-zyme amount (0.17 to 5.3 mU) (data not shown).Adsorption reached maximum value after 60min of incubation and was temperature inde-pendent. Based on these results, 4.2 mU of en-zyme was incubated with 2.5 mg of cells in 0.01M citrate buffer (pH 4.5) at 40C for 60 min inthe following experiments.As shown in Table 6, GTF activity in the

enzymes from adherent strains (WT, M9, M35)was adsorbed on the cells of all streptococcalstrains and the glucan-coated resin particles, butnot E. coli cells and the uncoated resins. Ad-sorption of the GTFs from nonadhering mutantswas much !-s than that of those from adherentstrains. These results indicate that, as in celladherence to glass, the nature of GTF plays adecisive role in the adsorption of GTF on cellsand that there is no apparent alteration of sur-face structures of the mutant cells from parentWT strain with respect to GTF adsorption.Glucans produced by WT and mutant

strains. Three classes ofglucans produced whenthe strains were grown in 5% sucrose broth were

Ilc

-.

0 37°C

30 60 90 120pH Time(min.)

FIG. 2. Temperature and pH dependency of theadsorption of cell-free GTF on heat-killed cells. (a)Crude WT enzyme (4.2 mU) and 2.5 mg ofheat-killedWT ceUs were preincubated in 1 ml of 0.01 M citratebuffer,pH 3.0 to 6.0 (A, A, V, V), orphosphate buffer,pH 6.0 to 8.0 (0, 0, O, U, at 4°C (solid symbols) or37°C (open symbols) for I h. Broken lines show un-adsorbed activity remaining in the supernatant ofthe reaction mixture. (b) Preincubation was carriedout in 0.01 M citrate buffer, pH 4.5, at 4°C (0) or37°C (0). In both (a) and (b), GTF activity wasassayed atpH 6.0 as described in the text.

fractionated and quantitated (Table 7). Produc-tion of extracellular, water-soluble glucan by allmutant strains, particularly by mutant Ml, dra-matically increased as compared withWT strain.In addition, 1 N NaOH-soluble, cell-associatedglucan synthesis was much less for the non-adhering mutants Ml, M2, and M13 than forthe adherent strains. Mutants M9 and M35 weresiilar to WT in their abilities to produce thisclass of glucan. No marked differences amongall the strains were detected in 1 N NaOH-insoluble, cell-associated glucan synthesis.

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Difference between the adherent and nonad-hering strains was also clearly demonstrated inwater-soluble and -insoluble glucan synthesisfrom sucrose by ceil-free enzyme from the strains(Table 8). The enzymes from nonadhering mu-tants synthesized only water-soluble glucans.The production of soluble glucans by mutant

TABLE 6. Adsorption of ceU-free GTF on heat-killedcells and resin particlesAdsorbed activity (%) ofGTF (4.2 mU/tube)

Cels from strain:aWT Ml M2 M13 M9 M35

WT 39.4 8.9 15.6 13.9 50.8 38.6Ml 25.4 9.7 18.8 16.6 55.0 39.5M2 33.7 6.5 15.4 27.3 58.2 32.8M13 33.1 8.9 17.4 21.2 47.4 31.8M9 38.0 8.5 21.2 20.3 53.2 34.5M35 49.6 7.3 23.6 16.7 45.9 28.0E. coli 6.2 7.5 11.7 -a _ -

Uncoated 5.3 4.9 14.0 - - -resins

Glucan- 28.7 4.6 16.5 - - -coatedresins

a Not tested.

Ml was much higher than that by WT strain.The enzymes from the adherent strains synthe-sized water-soluble and -insoluble glucans.Quantities of both types of glucans produced byenzyme from mutants M9 and M35 were four-to sevenfold greater than those produced byenzyme from WT strain.These glucans were subjected to Smith deg-

radation, and the degraded products were ana-lyzed by gas-liquid chromatography. The resultsindicate that a large number of glucose residuesof water-soluble glucans produced by both ad-

TABLE 7. Extracelular and cell-associated glucansproduced by WT and mutant strauis grown in 5%

sucrose brothGlucan synthesized (ugJpg of protein)

Strain Extracellu- 1 N NaOH- IlNNuabHllar, water- soluble, cell- ,,soluble aociated ated

WT 0.6 2.1 0.3Ml 15.2 0.2 0.2M2 1.1 0.2 0.1M13 6.2 0.1 0.1M9 8.1 1.9 0.3M35 4.6 1.5 0.5

TABLE 8. Water-soluble and -insoluble glucans synthesized from sucrose by cell-free GTF and analysis oftheir Smith degradation products by gas-liquid chromatography

Smith degradation product

Cell-free enzyme Water solubility of Amount of glu- % soluble andfrom strain glucan cauisynthesize insoluble Glycerol Erythritol gcose(mg/liter) (% (% g(%)o

WT Soluble 500 51.0 79.3 9.3 11.4Insoluble 481 49.0 31.7 0.6 67.8Total 981 56.0 5.0 39.0

Ml Soluble 11,738 100.0 95.4 0.9 3.8Insoluble 0 0.0Total 11,738 95.4 0.9 3.8

M2 Soluble 1,038 100.0 77.1 0.3 22.6Insoluble 0 0.0Total 1,038 77.1 0.3 22.6

M13 Soluble 1,863 99.6 86.7 0.9 12.4Insoluble 8 0.4Total 1,871 86.7 0.9 12.4

M9 Soluble 2,450 55.6 66.2 0.2 33.6Insoluble 1,960 44.4 24.3 0.0 75.7Total 4,410 47.6 0.1 52.3

M35 Soluble 1,988 36.2 54.0 0.5 45.6Insoluble 3,500 63.8 33.3 0.3 66.4Total 5,488 40.8 0.4 58.8

Dexan T250 92.7 3.1 4.1

aPharmacia Fine Chemicals AB, Uppsala, Sweden.

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herent and nonadhering strains were a-1,6linked. The water-soluble glucans of nonadher-ing mutants did not always show higher contentsof a-1,6 glucosidic linkage than those ofadherentstrains. On the other hand, more than half ofthe glucose residues of water-insoluble glucansproduced by adherent strains were a-1,3 linked.Total contents of a-1,3-linked glucose residuescontained in both water-soluble and water-in-soluble glucans were distinctly higher for theadherent strains than for nonadhering mutants.The a-1,3-linked glucose contents of the water-insoluble glucans and of total glucans producedby adherent strains did not correlate with theadherence ability of the growing cells.

DISCUSSIONMechanisms of adherence of S. mutans to

smooth surfaces have been studied by manyinvestigators. Mukasa and Slade (37, 38) haveproposed that the adherence requires adsorptionof glucan synthetase on specific binding sites oncell surfaces and subsequent synthesis of water-insoluble glucans in situ. As demonstrated inthe present study, however, heat-killed cells ofE. coli and uncoated resins, on which the GTFfrom WT strain was not adsorbed, were alsoadhered when incubated with WT enzyme andsucrose. These results clearly indicate that ad-herence of S. mutans does not necessarily re-quire the adsorption of GTF on a cell surface.S. Hamada (personal communication) and Slade(45) have observed that heat-killed cells of bac-terial species other than S. mutans, includingS. sanguis, S. salivarius, and group E strepto-cocci, fail to bind GTF from S. mutans B13 (d),but adhere to glass surfaces in the presence ofthe GTF and sucrose.Requirement ofde novo synthesis of adhesive,

water-insoluble glucans for adherence of S. mu-tans has been proposed by several investigators(37, 49). Recently, Kuramitsu (26) has shownthat heat-killed cells of several strains of S.mutans could adhere to glucan film preformedon test tube walls in the absence of sucrose andclaimed that in situ synthesis of glucans is notnecessarily required for the adherence process.However, the possibility has not been eliminatedin his study that active GTF and sucrose wouldstill remain on the film after washing with bufferand induce new glucan synthesis during the as-say for cell adhesion. The failure of heat-killedS. mutans AHT WT cells to adhere to theheated and/or invertase-treated glucan film con-firmed importance of de novo glucan synthesisin the adherence process, although the presentresults cannot exclude the possibility that thepresence of sucrose-free and GTF-free glucan

can somewhat enhance cell attachment to glasssurfaces. More recently, however, a result incon-sistent with our present observation has beenpresented by other investigators: that the heat-killed (1000C, 10 min) cells of S. mutans 6715-49 irreversibly bind to the protease-treated,GTF-free dextran layers preformed on a glasssurface (34). The cause(s) of the inconsistencypresently remains unresolved, but it might benoteworthy in this connection that, after heatingat 100°C for 10 min, S. mutans AHT WT cellsstill retained sufficient cell-bound GTF activityto induce cell adherence to a glass surface inthe presence of sucrose (T. Koga and M. Inoue,unpublished data).

Adsorption ofGTF on cells is of great interest,even though it may not be related to adherenceof S. mutans to a smooth surface. Todd-Hewittand Trypticase soy broth contain a very smallquantity of contaminant sucrose (30, 38, 46),and, therefore, the cells and GTF obtained fromculture of S. mutans in these media must carryglucan molecules. Our present study showedthat E. coli cells and uncoated resin particles,in contrast to glucan-coated resins, did not ad-sorb the cell-free GTF of WT strain. Further-more, we have observed that cell-bound GTFactivity was negligibly low when S. mutansAHTWT was grown in invertase-treated Todd-Hew-itt broth, and that heat-killed cells obtainedfrom culture in the broth did not adsorb extra-cellular GTF (M. Inoue, K. Shibuya, and T.Koga, unpublished data). These results are con-sistent with the reports of several investigatorswho have shown that a binding site for GTF isglucan molecules located on the cell surface (33,38, 41, 46). However, the types of glucans donot seem to determine the rate of GTF adsorp-tion, because the adsorption rates of a GTFpreparation on the cells of adherent and non-adhering strains were almost the same. Thisresult and the observation that relatively smalleramounts of the GTF from nonadhering mutantsbound to both WT and mutant cells would alsoindicate that the state of GTF molecules is an-other important factor affectingGTF adsorptionrate. These results could be explained in thefollowing way. Molecules of cell-free GTF pro-duced by the adherent strains may make a largeraggregate with highly branched glucans synthe-sized by itself, and thus one aggregate producedby the adherent strains might contain more GTFmolecules than one produced by the nonadher-ing mutants, which synthesize only water-solu-ble (less-branched) glucans. The existing results(12, 27, 39) support this assumption.From our present results and the above-men-

tioned studies reported by others, it can be con-

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cluded that the production of water-insolubleglucans would promote the concentration of wa-ter-soluble and -insoluble glucan synthetasesand consequently the accumulation of adhesiveglucans preferentially on the cell surface, andthus facilitate the adherence of S. mutans cellsto a smooth surface. The adhesive property ofthe glucans, i.e., their physico-chemical proper-ties, would be determined by the relative pro-portions ofGTF isozymes. The possible presenceof multiple isozymes of GTF produced by S.mutans has been indicated by many investiga-tors (3, 11, 16, 21, 40). Unfortunately, all of theexisting methods tested have proven to be un-satisfactory to analyze relative proportions ofGTF isozymes. Development of a rapid and re-liable method with high recovery of enzymeactivity is currently under investigation.Some possible causes of variations in the ad-

herence ability of the mutants of S. mutansAHT can be elucidated by the observations ob-tained in the present study. The failure of mu-tant strains Ml, M2, and M13 to form depositson a glass surface can be attributed to theirinabilities to produce water-insoluble, alkali-sol-uble, cell-associated glucans. Freedman andTanzer (10) have reported that production ofthis class of glucan is decreased for the mutantsincapable of forming adherent plaque on wires,in contrast to their parent strain S. mutans 6715-13 (g). Meager adherence of growing cells ofmutant M9 might most likely be due to itsrelatively poor growth in sucrose broth, resultingin unavailability of enough cells to induce ampleadherence. This assumption can be supportedby the results that adherence of the mutant cellswas evidently increased to a great extent whensufficient amounts of homologous heat-killedcells and enzyme were incubated with sucrose.The nature of glucans produced by the mutantwould be suitable for inducing strong cell adher-ence. Mutant M35 produced large amounts ofloosely adhering deposits when grown in sucrosebroth. It is uncertain whether this loose attach-ment ofthe mutant is due to its rather decreasedproduction of alkali-soluble, cell-associated glu-cans as compared with adherent strainsWT andM9. The amounts and a-1,3 glucosidic linkagecontents of water-insoluble glucans produced bycell-free GTF cannot account for the increasedadherence ability of this mutant. Therefore, re-spective extents of adherence of strains WT,M9, and M35 cannot be elucidated either bythe quantity or by the relative proportions ofglucosidic linkages of water-soluble and -insolu-ble glucans produced by them. Michalek et al.(35, 36) have recently presented the result thatGTF activity, cell adherence, and cariogenic vir-

ulence of different mutants of S. mutans 6715(g) are closely related to each other. The resultsobtained in the present study, however, suggestthat relations among them might not be so sim-ple. Our preliminary experiment (E. Kishimotoand T. Koga, unpublished data) has shown thatnone of the five mutants, even adherent mutantsM9 and M35, induces significant carious lesionsin hamsters. Further detailed studies will berequired to conclude the precise relations amongglucans, adherence, and cariogenicity of S. mu-tans.

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