Synthesis of extracellular dextran by cariogenic bacteria and its presence in human dental plaque

Arch oral Biol. Vo1.12,

pp.1l-24,

1967. Pergamon Press Ltd. Rimed in Ct. Britain.

SYNTHESIS OF EXTRACELLULAR DEXTRAN BY CARIOGENIC BACTERIA AND ITS PRESENCE IN HUMAN DENTAL PLAQUE R. J. GIBBONSand S. B. BANGHART Forsyth Dental Center and Harvard School of Dental Medicine, Boston, Massachusetts, U.S.A. Summary-The extracellular polysaccharide synthesized primarily from sucrose by certain human and rodent cariogenic bacteria has been found to be a dextran-like polymer. Maximum quantities of this polysaccharide were synthesized in 10% sucrose broth, and the presence of free glucose or fructose did not markedly repress synthesis of dextran from sucrose. The dextrans synthesized by rat, hamster, and human cariogenic streptococci, and by a cariogenic strain ofLactobacillus acidophilus proved immunologically similar. The extracellular dextran was found to be relatively resistant to attack by mixed oral bacterial growth; to form insoluble precipitates with serum, clarified saliva, and various protein solutions; and to adhere to powdered hydroxyapatite. Samples of pooled human dental plaque were found to contain a constituent which was immunologically similar to dextran, and which comprised almost 2% of its dry weight. It is proposed that dextran synthesis by cariogenic but not non-cariogenic bacteria, enables these organisms to form dental plaque which is required for the production of dental caries.

INTRODUCTION

with conventional hamsters and gnotobiotic rats have indicated that the bacteria required to initiate caries are more specific than heretofore was recognized (FITZGERALDand KEYES,1960; FITZGERALD,1963). Recently data have been obtained which suggest that these studies with rodents may have direct application to man, for certain strains of human streptococci have proved to be cariogenic when tested in conventional hamsters or gnotobiotic rats, whereas other strains were ineffective (ZINNERet al., 1965 ; KRASE, 1966; GIBBONSet al., 1966). The biochemical and serological characteristics of the human cariogenic streptococci were similar, if not identical to the rodent cariogenic organisms. Both rodent and human cariogenic streptococci were found to synthesize large quantities of extracellular polysaccharide, particularly from sucrose, whereas strains of non-cariogenic bacteria formed only trace amounts. The production of this polysaccharide in sucrose broth cultures resulted in gelatinous masses of bacteria which adhered to the sides or bottom of culture vessels, suggesting that polysaccharide formation could be responsible for bacterial plaque accumulations which occur in animals inoculated with cariogenic, but not non-cariogenic, bacteria (GIBBONSet al., 1966). The present investigation describes the chemical and serological properties of the extracellular polysaccharide synthesized by cariogenic bacteria and demonstrates its presence as a component of the matrix of human dental plaque. STUDIES

12

R. J. GIBBONS ANDS. B. BANWART METHODS

Cultures and cultural conditions

Rodent cariogenic bacteria were obtained through the generosity of Dr. R. J. Fitzgerald of the National Institute for Dental Research. Rat streptococcus strain GF71 and Luctobacillus acidophilus strain 108 TR have been shown to be cariogenic in gnotobiotic rats, while hamster streptococcus strains HS 6 and E 49 induced dental caries in conventional hamsters (FITZGERALDand KEYES, 1960; FITZGERALD,1963; FITZGERALD,JORDANand ARCHARD, 1966). Human streptococcus strains PK 1, LM 7, GS 5, and 120 were found to induce caries in gnotobiotic rats (GIBBONSet al., 1966; GIBBONSand LOESCHE,unpublished data). All strains were maintained by biweekly transfer in tubes of Trypticase soy broth or on plates of Trypticase soy glucose agar. These were incubated anaerobically in Brewer jars filled with 95% hydrogen, 5% carbon dioxide at 35°C for all experiments reported. Serological studies

Antisera were prepared in rabbits immunized with vaccines prepared from glucose grown organisms as previously described (GIBBONSet al., 1966). Gel diffusion plates were prepared using 0.85% Ionagar no. 2 (Oxoid) in buffered saline at pH 7.2, using the general procedures described by CROWLE(1961). Polysaccharide estimation and extraction

For polysaccharide estimation and extraction, the organisms were grown in a medium consisting of Trypticase (B.B.L.), 2% ; NaCl, 0.2% ; K,HPO,, 0.4% ; and KH,P04, 0.2% supplemented with the desired carbohydrate. The quantity of extracellular polysaccharide synthesized by growing cultures was estimated as previously described (GIBBONSet al., 1966). Purified polysaccharide was isolated from 10% sucrose broth cultures of the organisms, which had been incubated anaerobically for 48 hr. The cultures were vigorously blended in a Servall omnimixer and then centrifuged to remove the cells. One and one half volumes of ethanol were added to the supernatant to precipitate the polysaccharide. In instances where considerable polysaccharide was still adherent to the cells, the organisms were extracted with 0.1 N KOH and the extracted material then precipitated with ethanol. Precipitated polysaccharide was harvested by centrifugation and washed with ethanol. For further purification, it was dissolved in water and sufficient trichloroacetic acid added to give a final concentration of 10%. The mixture was centrifuged to remove precipitated proteins, and the supernatant dialysed to remove the trichloroacetic acid. The polysaccharide finally was harvested by lyophilization. Degradation of dextran and levan by mixed oral bacteria

Samples of dental plaque and saliva, and swabbings from the tongue were obtained from nine individuals. These were inoculated into a medium consisting of 1% trypticase (B.B.L.), 0*05% yeast extract (Difco), and 0.5% NaCl adjusted to pH 7.2 and supplemented with O*15o/oof either dextran or levan previously prepared from

SYNTHFSIS

OF EXTRACELLULAR

DEXTRAN

13

Leuconostoc mesenteroides or Streptococcus salivarius respectively.

The cultures were incubated anaerobically for 48 hr, and then centrifuged to remove the mixed bacterial growth. An equal volume of ethanol was added to the culture supernatant to precipitate residual dextran or levan. The failure to obtain a precipitate with ethanol indicated that the mixed bacterial growth had hydrolysed the added polysaccharide. Uninoculated control tubes of either dextran or levan broth always gave a precipitate after ethanol addition. Determination of polysaccharide in human dental plaque

Plaque samples were collected several hours after eating from twelve caries active individuals 8-17 years old. The samples were pooled and lyophilized. Lyophilized plaque was suspended in 1N KOH and heated at 100°C for 20 min. The suspension was clarified by centrifugation and 2 vol. of ethanol added to the supernatant to precipitate extracted polysaccharides. The precipitate was harvested by centrifugation and washed 4 times with ethanol. It was dissolved in buffered saline and tested for precipitation with antisera to cariogenic streptococci in gel diffusion plates. An attempt was made to estimate the quantity of material serologically similar to the extracellular polysaccharide in human dental plaque. Extracts of plaque were prepared by suspending 12 mg of lyophilized plaque in O-5ml of 1N KOH. Uniformly labelled Cl* extracellular polysaccharide (7Opg) isolated from human streptococcus strain GS5, having a specific activity of 230 counts/min per pg was added to the plaque suspension and the mixture heated at 100°C for 30 min. The mixture was neutralized by the addition of HCl and the volume adjusted to 6 ml to make it isotonic. This suspension was centrifuged and the supernatant filtered through a membrane filter (pore size 0.45~) to remove particulate matter. A polysaccharide absorbed antiserum was prepared by incubating 3 mg of polysaccharide with 3 ml of antiserum at 37” for 2 hr, followed by refrigeration overnight. The precipitate was removed by centrifugation and the absorbed serum clarified by membrane filtration. One ml of a 1:2 dilution of either antiserum, absorbed antiserum, or normal rabbit serum was incubated with 0.5 ml of either plaque extract containing added Cl* extra-cellular polysaccharide or Cl* extracellular polysaccharide (100 pg) alone. The reaction mixtures were incubated at 35°C for 2 hr and then refrigerated overnight. These were then centrifuged, and the precipitates washed 3 times with 3-ml portions of chilled saline. Precipitates were dissolved in dilute alkali and analysed for total The quantity of material antigenically carbohydrate and counted for radioactivity. related to the bacterial extracellular polysaccharide was estimated in plaque by determining the decrease in specific activity of the added radioactive polysaccharide in the plaque precipitate. Chemical methods

Total carbohydrate was determined using anthrone reagent (SCOTTand MELVIN, 1953) and reducing sugars by the method of FOLINand MALMROS (1929). Glucose was analysed in hydrolysed samples of the polysaccharide using glucose oxidase (Glucostat,

R. J.

14

GIBBONS AND

S. B. BANGHART

Worthington Biochem. Corp., New Jersey). Ketohexoses were determined using cysteine, carbazole, H,SOI (DISCHE, 1955) and protein was measured by the method of LOWRY et al. (1951). Samples of polysaccharide were hydrolysed with 2N H,SO, at 100°C for 1, 3, and 5 hr for chromatographic analysis. The hydrolysates were neutralized with barium carbonate and clarified by centrifugation. Samples of the hydrolysates were applied to paper and cellulose thin layer plates and developed with solvents consisting of ethyl acetate, pyridine, water (12 :5 :4), ethyl acetate, acetic acid, water (3 :3 : I), and phenol, water (4:l). Spots were located using aniline-diphenylamine, aniline hydrogen phthalate, and naphthoresoresorcinol reagents as described by SMITH (1960) and HOUGH and JONES (1962).

RESULTS

Production and composition qf extracellular polysaccharide It was found previously that human and rodent cariogenic streptococci produced

large quantities of extracellular non-dialysable carbohydrate, particularly from sucrose, whereas non-cariogenic bacteria failed to do so (GIBBONS et al., 1966). In order to determine the sucrose concentration promoting maximal synthesis of extracellular polysaccharide, human cariogenic streptococcus strain GS 5 was grown in broth containing varying amounts of sucrose and the amount of polysaccharide formed was determined after 48 hr incubation. It was found (Fig. I) that maximal growth was attained in broth containing 2% sucrose, and higher concentrations of sucrose tended to be inhibitory as measured by the yield of dry cells per ml of culture medium. However, the quantity of extracellular polysaccharide synthesized by the cultures was found to be proportional to the sucrose concentration, up to 10%. Although growth

FIG. 1. Growth (broken line) and extracellular polysaceharide synthesis (solid line) by streptococcus strain GS 5 in broth containing various concentrations of suerose.

SYN'I'HESlSOFEXTRACELLULARDEXTRAN

15

of the organisms was impaired by high concentrations of sucrose, more polysaccharide was formed on a per cell basis, and organisms grown in 15% sucrose broth synthesized 70 times their dry weight as extracellular polysaccharide. Since glucose has been found to generally repress the formation of catabolic enzymes involved in the metabolism of other sugars, it was of interest to determine if the presence of glucose or fructose could inhibit the formation of polysaccharide from sucrose. Therefore, the quantity of polysaccharide synthesized by cultures of human streptococcus strain GS 5 grown in 5% sucrose broth with and without glucose and fructose was determined. It was found (Table 1) that the presence of 2% glucose in TABLE

1.

EFFECT OF GLUCOSE AND FRUCTOSE ON EXTRACELLULAR SUCROSE BY HUMAN CARKNJBMC STREPT-CCUS

Carbohydrate in medium

Extracellular polysaccharide (pg/ml)

POLYSACCHARIDE STRAIN GS 5

Growth (pg dry wt. cells/ml)

SYNTHESIS FROM

pg polysaccharide pg dry cells

1. 5 % sucrose

4080

428

9.5

2. 2 % glucose 2 % fructose

362

424

0.9

3. 2% glucose 5 % sucrose

3020

380

7,9

4. 2% fructose 5 % sucrose

2170

140

15.5

2030

376

5.4

5. 2 % glucose 2 % fructose 5 % sucrose

the culture medium did not markedly reduce the quantity of polysaccharide synthesized from sucrose on a cell dry weight basis. The addition of 2% fructose to sucrose broth was found to impair growth of the organisms, but did not prevent the synthesis of polysaccharide from sucrose. Under these inhibitory conditions, the organisms actually synthesized more polysaccharide on a cell dry weight basis than did control sucrose cultures. In addition, only small quantities of extracellular polysaccharide were produced from cultures containing a combination of glucose and fructose indicating that the intact sucrose molecule is necessary as a substrate for polysaccharide production. These data indicate that glucose or fructose are unable to repress markedly the formation of enzymes necessary for synthesis of the extracellular polysaccharide from sucrose. Chemical and serological characteristics of polysaccharide

Purified extracellular polysaccharides isolated from human streptococcus strains GS 5, PK 1, and 120, Rat strain GF 71, and hamster strains E 49 and HS 6 have been found to assay between 95 and 102% carbohydrate when analysed with anthrone reagent. Each polysaccharide was found to contain a small amount of ketohexose

16

R. J. GIBBONS ANDS. B. BANCXURT

varying between 1.7 and 4.6% and less than 1% protein. Preparations of the polysaccharide from human strains PK 1, 120 and GS5 were found to contain between 0.07 and 0.19% of organic phosphorus, and negligible amounts of inorganic phosphorus. Phosphorus analyses were not performed on the remaining polysaccharide preparations. The extracellular polysaccharide was comparatively resistant to acid hydrolysis for maximum liberation of reducing sugars was attained only after samples were hydrolysed with 2N H,SOI for 5 hr at 100°C. Thin layer and paper chromatographic analysis of 5 hr hydrolysates of the polysaccharide indicated it contained one major and at least one minor component. The major component was identified as glucose by its Rf values in the solvents used, and it gave a positive reaction with glucose oxidase. The minor component migrated comparably to fructose and gave a positive ketohexose reaction with naphthoresorcinol. Its colour reaction with aniline diphenylamine was identical to fructose. The results of these analyses indicate the extracellular polysaccharide is primarly a glucose polymer containing small amounts of fructose. Solutions of polysaccharide did not give a colour reaction with iodine solution, indicating it was not similar to polysaccharides of the starch-amylopectinglycogen group. Its chemical composition and its synthesis from sucrose suggested it was a dextran-like polymer. This was substantiated using enzymatic and serological techniques. Culture filtrates of Penicillium funiculosum (N.R.R.L. 1768) containing dextranase activity were prepared by growing the mold in 1y. trypticase broth adjusted to pH 5 and supplemented with l*O”hdextran isolated from Leuconostoc mesenteroides using a procedure similar to that described by TSUCHIYA et al. (1952). Filtrates prepared from these cultures were found to rapidly hydrolyze dextran obtained from Leuconostoc mesenteroides and also extracellular polysaccharide isolated from streptococcus strain GS 5, as judged by their inability to be precipitated with ethanol after enzyme treatment. These filtrates showed little or no activity upon soluble starch. When solutions of purified polysaccharide were tested in gel diffusion plates against antisera to either human or rodent cariogenic streptococci, precipitin bands developed with each polysaccharide studied (Fig. 2) but not with saline or uninoculated culture medium. The antigenic similarity of the polysaccharide extended across bacterial genera, as evidenced by the precipitin band formed by the polysaccharide isolated from cariogenic lactobacillus strain 108 TR. Solutions (1 mg/ml) of authentic dextran obtained from Leuconostoc mesenteroides formed precipitin bands with antisera to the cariogenic organisms which appeared identical to those of the extracellular polysaccharides. No precipitin bands developed with either normal rabbit serum or dextran absorbed antiserum, further indicating that the extracellular polysaccharide is a dextran. Interactions of extracellular polysaccharide

It was found previously that solutions of the bacterial extracellular polysaccharide formed non-specific precipitates with normal rabbit serum when added in high concentrations (10 mg per ml or higher, GIBBONSet al., 1966). It was therefore of interest to

17

SYNTHESISOFEXTRACELLlJLARDEXntAN

determine if non-specific precipitates developed with other protein solutions. Saline solutions of gelatine, casein, blood albumin, egg albumin and hog gastric mucin of various concentrations were prepared. These were layered over saline solutions of the polysaccharide isolated from human streptococcus strain GS 5 at concentrations of 10, 25, and 50 mg per ml in capillary tubes. Tubes were incubated at 35°C for 2 hr, followed by overnight refrigeration. Non-specific precipitates were found to develop with all protein solutions tested (Table 2). Precipitation seemed dependent upon both TABLET. PRECIP~~A~ONOEM~ACELLULARPOLYSACCHARIDE\KITHPRO~~SOLUTIONS,RABB~~SERUM, AND CLARIFIED SALIVA

Gelatine Polysaccharide (%) concentration ~ 10 5 2 (mg/ml)

Casein (%) ~ 10 5 2

Blood albumin (“A ~10 5 2

gastric mucin (“A 10 5 2

J3& albumin

Human Normal clarified rabbit Saline saliva serum control

50

+ffN.D.+-+--

+i-

+fi

+

-

25 10

N.D.---_-+ +*--NN.D.-----

+ f+--

+ &&--

+ 4

-

+

N.D. Not determined; +, large precipitate; f, slight precipitate; -, no precipitate. the concentration of polysaceharide as well as the concentration of protein used. Precipitates also developed with normal rabbit serum and whole human clarified saliva at the three concentrations of polysaccharide tested. The addition of calcium

chloride at a concentration of 1O-3 M to solutions of the proteins did not enhance precipitation. When saline solutions of polysaccharide were added to whole saliva which had been clartied by centrifugation to give a concentration of 5 mg of polysaccharide per ml, a generalized turbidity developed after 15-30 min incubation. Upon continued incubation for 30-90 min, a flocculent insoluble precipitate usually, though not always, resulted. No such precipitation occurred when saline alone was added to saliva. The ability to form flocculent precipitates with saliva did not appear to be a general characteristic of polysaccharides, for levan isolated from Streptococcus salivurius, and soluble starch did not form precipitates when tested in concentrations up to 10 mg per ml. The flocculent polysaccharide-saliva precipitates could not be dissolved in water, saline. or 1N NaOH. The material could be solubilized by hydrolysing it with 2N HCl at 100°C or it could be dissolved with O*lM solutions of ethylene diamine tetraacetic acid adjusted to pH 8.6. Washed precipitates formed from 5 ml samples of saliva incubated with 5 mg per ml of bacterial polysaccharide were found to contain approximately 560 pg of carbohydrate, as determined with anthrone reagent. Signifkant quantities of protein (141 pg), calcium (102 pg), and phosphorus (60 pg) were also present. In order to determine if the bacterial polysaccharide could adhere to tooth mineral, suspensions of 2 mg per ml of powdered hydroxyapatite were incubated with 1 mg per ml of polysaccharide isolated from streptococcus strain GS 5 at 35°C for 30 min. B

R. J. GIBBONS ANDS. B. BANGIURT

18

The suspension was then washed 3 times with saline to remove excess polysaccharide and the coated apatite was tested for agglutination with an equal volume of a 1:4 dilution of normal rabbit serum or antiserum to human strain GS 5. Marked agglutination occurred with the antiserum, whereas no agglutination occurred with normal rabbit serum or with uncoated apatite controls. Identical results were obtained when apatite powder was first coated with saliva, according to the method of HAY (1966), prior to exposure to the bacterial polysaccharide. These data indicate that the dextran-like polysaccharide formed by certain cariogenic bacteria is able to become tenaciously bound to both untreated and saliva treated hydroxyapatite. Hydrolysis of levan and dextran by mixed oral bacterial growth

Dextran was found to be relatively resistant to bacterial attack for mixed growth derived from plaque or saliva samples obtained from nine individuals failed to hydrolyse it (Table 3). Some hydrolysis did occur from growth derived from tongue swabs, however. In contrast, levan was found to be highly susceptible to bacterial attack; and the mixed growth from all of twenty-seven samples studied was found to hydrolyse it. TABLE3. HYDROLYSISOFDEXTRANAND DERIVED FROM SAWA,

LEVAN BYMD(ED BACTERIALGROWTH PLAQUE, AND TONGUE SWABS

Hydrolysis LeVZlll

Inoculum Saliva Plaque Tongue swab

None 0 0 0

Dextran

Partial Complete 2 0 0

9’ 9

None

9’ 6

Partial Complete

8 1

8 2

Numbers indicate the number of individuals. Qualitative and quantitive estimation of dextran in human dental plaque

Extracts of human dental plaque were found to form a precipitin band with antiserum to human cariogenic streptococci when tested in gel diffusion plates (Fig. 3). The band appeared identical to those produced by the extracellular polysaccharides of cariogenic streptococci, and also authentic dextran, suggesting their identity. No precipitin bands developed when normal rabbit serum was substituted for antiserum, indicating the immunologic nature of the reaction. The antigenic specificity was determined using gel diffusion plates which incorporated known dextran at a concentration of 1 mg per ml into the gel to specifically absorb anti-dextran antibodies. No precipitin bonds developed in such plates with either plaque extracts or solutions of extracellular polysaccharide, indicating that the component present in the plaque extract was antigenically similar to dextran. An attempt was made to estimate the quantity of dextran-like material in pooled samples of human dental plaque by taking advantage of the ability of the immunologic

19

-0FExTRACELLULARDExTRAN

precipitin reaction to selectively remove dextran from plaque extracts, and of isotope dilution techniques to measure the quantity of this material. Precipitates developed when antiserum was incubated with either plaque extract or with purified uniformly labelled Cl4 extracellular polysaccharide but not with controls consisting of normal rabbit serum or polysaccharide absorbed antiserum (Table 4). The specific activity TABLE

4. ESTIMATION OF DEXTRAN

IN PLAQUE _____

Immunologic precipitation

Reaction mixture

_.___

._ Plaque extract containing + GS 5 antiserum

Cl4 polysaccharide

Plaque extract containing -+ normal rabbit serum

Cl4 polysaccharide

-t-

Specific activity of carbohydrate in precipitate (counts/min per Pg)

59

-

Plaque extract containing Cl4 polysaccharide + dextran absorbed antiserum Plaque extract containing Cl4 polysaccharide + saline C’” polysaccharide

BY ISOTOPE DILUTION

+ GS 5 antiserum

Cl4 Polysaccharide

+ normal rabbit serum

CL4polysaccharide

+ saline

+-

225

of the carbohydrate present in the precipitate which developed with plaque extracts was approximately 1/4th that present in precipitates from purified Cl4 polysaccharide alone. Assuming that the decrease in specific activity of the plaque precipitates was due to the presence of unlabelled dextran from the plaque, and since 70 pg of Cl4 polysaccharide was added to 12 mg of plaque, it may be calculated that approximately 1.8% of the dry weight of plaque consists of a dextran-like polysaccharide. DISCUSSION

has been found that cultures of rodent and human cariogenic bacteria synthesize large quantities of extracellular polysaccharide, particularly from sucrose, whereas non-cariogenic bacteria form little or none (GIBBONS el al., 1966). The present investigation has found that this extracellular polysaccharide is a dextran-like polymer, for it is composed mainly of glucose accompanied by small amounts of fructose. It proved susceptible to the hydrolytic action of dextranase preparations which had little effect upon starch, and the polysaccharide appeared to be antigenically identical with authentic dextran isolated from Leuconostoc mesenteroides. The production of dextran by the cariogenic strains of bacteria studied was found to occur principally from sucrose, and the quantity of dextran synthesized by cultures was proportional to the sucrose concentration up to 10%. However, it is clear that It

20

R. J. GIBBONS ANDS. B. BAN~~ART

other sugars can promote at least limited synthesis of dextran by these organisms, for antisera prepared against glucose grown cells have been found to contain appreciable quantities of antidextran antibodies. In addition, studies with certain strains of human cariogenic streptococci have indicated that significant quantities of dextran may also be synthesized from maltose under certain experimental conditions (GIBBONSand BANGHART,unpublished data). The synthesis of dextran from sucrose by other microorganisms occurs by the enzyme dextran sucrase (reviewed by NEELY, 1960). This enzyme transfers glucose residues directly from sucrose to an existing primer molecule, liberating free fructose from the substrate. Thus organisms synthesizing dextran from sucrose are constantly exposed to large quantities of free reducing sugars in their environment. Because of this, it is interesting that the presence of 2% glucose or fructose in sucrose broth culture was not found to markedly repress the formation of the enzymes necessary for dextran synthesis from sucrose by cariogenic bacteria. It is generally recognized that cariogenic bacteria are able to initiate the formation of dental plaque for it has been a consistent finding that animals harbouring cariogenie bacteria develop bacterial plaque whereas little or none accumulates in animals harbouring non-cariogenic bacteria (KEYES, 1960; FITZGERALDand KEYES, 1961; GIBBONS et al., 1966). As a result of extracellular dextran formation, sucrose broth cultures of cariogenic bacteria have been found to produce gelatinous masses of bacteria which frequently adhere to the walls of culture vessels suggesting that dextran formation may enable these bacteria to form plaque in vivo (GIBBONSet al., 1966). Thus, the high cariogenicity of sucrose in certain but not all caries test systems (SHAFER,1949; KRASSE,1965) could be related to its ability to enhance dextran synthesis and thus plaque formation by cariogenic bacteria. In this regard, it should be noted that CARLSSONand EGELBERG(1965) have observed that sucrose stimulates plaque formation in man, and CARLSSON (1965) has further suggested that the bacterial synthesis of extracellular polysaccharides from sucrose may be significant in this process. The extracellular dextran synthesized by cariogenic bacteria has been found to have several characteristics which could be important in enabling it to function as a matrix for plaque formation. First, it has been found to adhere to powdered hydroxyapatite or apatite which had been coated with saliva. HAY (1966) has obtained data which indicates that the surface of teeth in the oral cavity are coated with certain salivary proteins. This coating can be mimicked in vitro by exposing apatite powder to clarified whole human saliva. The ability of the dextran to adhere to these apatite suspensions suggests that it could also adhere to the tooth surface in the mouth. The second characteristic of the dextran which would seem to have significance in plaque formation is that it is able to form an insoluble complex when incubated with saliva. These complexes were found to consist at least in part of carbohydrate, protein, calcium and phosphorus, and could not readily be redissolved. The formation of such an insoluble complex would seem to be an important characteristic of a substance serving as a matrix for plaque for it would prevent the plaque from being easily washed away. The comparatively large amounts of calcium and phosphorus found in these complexes

SYNTHESIS OF EXTRACELLULAR

DEXTRAN

21

could well explain the high concentrations of these elements reported present in human plaque by DAWES and JENKINS(1962). Finally, dextran has been found to be relatively resistant to hydrolysis by mixed bacterial growth derived from samples of plaque and saliva. This suggests that it would be biologically stable in the mouth, and thus well suited to function as a matrix for plaque. In contrast, levan isolated from S. salivarim was found highly susceptible to bacterial attack. Although this polysaccharide has been shown to be synthesized from sucrose by plaque bacteria (MCDOUGALL,1964), its biological instability suggests it is not functioning as a matrix component, but rather as a carbohydrate storage compound as suggested by MANLY(1961). R. S. MANLY(Tufts University, Boston, Massachusetts, personal communication) has further found that fasting plaque contained little or none of this polysaccharide, thus supporting this view. In the present study, human dental plaque was found to contain a constituent which was antigenically identical to the dextran synthesized by certain cariogenic bacteria. This indicates that at least one component of the matrix of human dental plaque consists of extracellular polysaccharide produced by cariogenic bacteria. Using a combination of serologic and isotope dilution techniques, it was estimated that dextran comprised almost 2% of the total dry weight of pooled plaque samples. Since the plaque matrix constitutes only a portion of the total dry weight of plaque, dextran would comprise a considerably higher percentage of the matrix of plaque. The concept emerging from the present and previous studies (GIBBONSet al., 1966) is that the formation of extracellular dextran, particularly from sucrose, by cariogenic bacteria appears to enable these organisms to form plaque which is necessary for the production of dental caries. In contrast, non-cariogenic bacteria are unable to synthesize significant quantities of dextran and therefore cannot form plaque. Thus synthesis of certain extracellular polysaccharides would appear to be one of several characteristics required by a bacterium in order to be cariogenic. This hypothesis could explain adequately the unusual specificity which cariogenic bacteria seem to possess in caries test systems in use today. The extracellular polysaccharides formed by all cariogenic bacteria studied in the present investigation proved to be dextran-like compounds. However, the data obtained do not preclude the possibility that other types of extracellular polysaccharides may function in an analogous manner, and enable bacteria to form plaque. This question can only be resolved when larger numbers of cariogenic bacteria have been isolated and characterized. Acknowledgement-The authors are indebted to I. SHAPIROand H. MCCANNof the Forsyth Dental Center for performing the phosphorus and calcium analyses. This investigation was supported in part by grant DE-01471 from the National Institute for Dental Research, and in part by a grant from the Colgate-Palmolive Co. The senior author was supported by a Career Development award from the U.S. Public Health Service.

R. J. GIBBONSANL-J S. B. BANGHART

22

R&utn&-Les polysaccharides extra-cellulaires, synthetists prhnitivement a partir du saccharose par certaines batteries cariogenes humaines et de rongeurs, semblent etre un polymtre analogue au dextrane. Des quantites maximales de ce polysaccharide sont synthetisees dans des bouillons saccharods a 10% et la presence de glucose ou de fructose libre n’inhibe pas de facon notable la synthese de dextrane par le saccharose. Les dextranes, synthetises par des streptocoques cariogenes de rats, de hamsters et de 1’Homme et par une espece cariogene de Luctobucilfus acidophilus, paraissent immunologiquement identiques. Le dextrane extra-cellulaire est relativement resistant a la destruction par la croissance bactbienne mixte de la bouche: il produit des precipites insolubles avec le serum, de la salive Claire et differentes solutions proteiques: il adhere a de l’hydroxyapatite en poudre. Des echantillons de plaques dentaires humanies melang& contiennent un constituant immunologiquement identique au dextrane et qui comporte presque 2% de son poids sec. Les auteurs emettent l’hypothese que la synthese de dextrane par des batteries cariogenes, mais pas con cariogenes, permet a ses organismes de former des plaques dentaires necessaires a la production des caries dentaires. Zusammenfassung-Es

wurde gefunden, dass die von bestimmten kariogenen Bakterien des Menschen und der Nager vorzugsweise aus Rohrzucker synthetisierten extrazellul&en Polysaccharide ein Dextran-Lhnliches Polymer darstellen. Die grossten Mengen dieses Polysaccharides wurden in 10% Rohrzucker-Nahrboden synthetisiert; die Gegenwart freier Glukose oder Fruktose verminderte die Synthese von Dextran aus Rohrzucker nicht nachhaltig. Die von kariogenen Streptokokken der Ratte, des Hamsters und des Menschen und von einem kariogenen Stamm von Lactobaciflus ncidophilus synthetisierten Dextrane verhielten sich immunologisch Ihnlich. Es wurde gefunden, dass das extrazellullre Dextran gegeniiber dem Angriff gemischter Mundbakterien relativ resistent ist, dass es mit Serum, klarem Speichel und verschiedenen Eiweisslosungen unlosliche Niederschlage bildet und dass es an gepulvertem Hydroxylapatit anhaftet. Proben gemischter menschlicher Zahnplaques enthielten einen Bestandteil, der dem Dextran immunologisch Lhnlich war und der nahezu 2% seines Trockengewichtes ausmachte. Es wird angenommen, dass die Dextran-Synthese durch kariogene, jedoch nicht von nichtkariogenen Bakterien diese Organismen in die Lage versetzt, Zahnplaques zu entwickeln, die ftir die Entwicklung der Zahnkaries beniitigt

werden. REFERENCES CARL.WN, J. 1965. Zooglea-forming streptococci resembling Strepfococcus sanguis isolated from dental plaque in man. Odont. Revy 16, 349-358. CARLSSON,J., and EGELBERG,J. 1965. Effect of diet on early plaque formation in man. Odont. Revy 16, 112-125. CROWLE,A. J. 1961. Zmmunodiffision, pp. 181-264. Academic Press, New York (1961). DAWES,C. and JENKINS,G. N. 1962. Some inorganic constituents of dental plaque and their relationship to early calculus formation and caries. Archs oral Biol. 7, 161-172. DISCHE,Z. 1955. New color reactions for determination of sugars in polysaccharides. Meth. biochem. Analysis 2, 313-358. FITZGERALD,R. 5. 1963. Gnotobiotic contribution to oral microbiology. J. dent. Res. 42, 548-552. FITZGERALD,R. J., JORDAN, H. V. and ARCHARD, H. 0. 1966. Dental caries in gnotobiotic rats infected with a variety of Lactobacillus acidophilus. Archs orul Biol. 11,473-476. FITZGERALD,R. J., and KEYES, P. H. 1960. Demonstration of the etiologic role of streptococci in experimental caries in the hamster. J. Am. dent. Ass. dent. Cosmos 61,9-31. FOLIN, 0. and MALMROS,H. 1929. An improved form of Folin’s micro method for blood sugar determinations. J. biol. Chem. 83, 115-120. GIBBONS,R. J., BERMAN,K. S., KNOETTNER,P. and KAPSIMALIS,B. 1966. Dental caries and alveolar bone loss in gnotobiotic rats infected with capsule forming streptococci of human origin. Archs oral Biol. 11, 549-560. HAY, D. I. 1966. The absorption of saliva proteins onto apatite and enamel powder. International Association for Dental Research, 44th General Meeting, Abstract. p. 154. HOUGH,L. and JONES,J. K. N. 1962. Methods in Carbohydrate Chemistry, Vol. 1, pp. 21-3 1. Academic Press, New York.

St’NTHFsIsOF EXTEACELLULAR DEXTEAN

23

KEYES, P. H. 1960. Infectious and transmissible nature of experimental dental caries. Archs oral Eiol. 1.304-320. KEASSE, B. 1965. The effect of caries-inducing streptococci in hamsters fed diets with sucrose or glucose. Archs oral Biol. 10,223-226. KRASSE,B. 1966. Human streptococci and experimental caries in hamsters. Archs oral Biol. 11, 429-436. LOWRY,0. H., ROSEBROUGH, N. J., FARR, A. L. and RANDALL,R. J. 1951. Protein measurement with the Folin phenol reagent. J. biol. Chem. 193,265-275. MANLY, R. S. 1961. Retention of carbohydrate from sugar solutions by salivary sediment. J. dent. Res. 40,319. MCDOUGALL,W. A. 1964. Studies on the dental plaque. IV. Levans and the dental plaque. Auf. dent. J. 9, I-5. NEELY,W. B. 1960. Dextran: structure and synthesis. Adv. Curbohyd. Chem. 15,341-369. Scorr, T. A. and MELVIN, E. H. 1953. Determination of dextran with anthrone. Al&t. Chem. 25, 1656-1661.

SHAFER,W. G. 1949. The caries-producing capacity of starch, glucose, and sucrose diets in the Syrian hamster. Science, N. Y. 110,143-144. SMITH, I. 1960. Chronlatographic and Electrophoretic Techniques, Vol. 1I, pp. 246-260. Interscience, New York. TSUCHIYA,H. M., JEANES,A., BRICKER,H. M. and WILHAM,C. A. 1952. Dextran degrading enzymes from molds. J. But. 64,513-519. ZINNEE, D. D., JABLON,J. M., ARAN, A. P. and SASLOW,M. S. 1965. Experimental caries induced in animals by streptococci of human origin. Proc. Sot. exp. Biol. Med. U&766-770.

PLATE 1 OVERLEAP

R. J.

GIBBONS AND

S. B.

BANGHART

FIG. 2. Gel diffusion plate showing precipitin bands developing with antiserum to streotococcus strain GS 5 in centre. well. and solutions (1 rnglrnl) of dextran. and extra&lular polysaccharide from rodent and human caribgen:; bacteria pla&d in surrounding wells. Similar precipitin bands also developed with extracellular polysaccharide isolated from hamster streptococcus strain E 49, and human streptococcus strains PK 1, LM 7, and 120. Antigen controls consisting of saline or uninoculated culture medium did not form precipitin bands when similarly tested.

FIO. 3. Gel diffusion plate containing antiserum to human streptococcus strain GS 5 in centre well showing precipitin bands which developed with plaque extract, extracellular polysaccharides from cariogenic bacteria, and dextran solutions contained in surrounding wells.

SYNTHESIS

OF EXTRACELLULAR

DEXTRAN

PLATE

A.0.n.

1

f.p 24