INFECTION AND IMMUNITY, July 1978, p. 17-27 0019-9567/78/0021-0017$02.00/0 Copyright i 1978 American Society for Microbiology

Vol. 21, No. 1

Printed in U.S.A.

Analyses of Glucans from Cariogenic and Mutant Streptococcus mutans M. FREEDMAN,`* D. BIRKEDI AND K. GRANATH2 Departments of Oral Diagnosis' and Restorative Dentistry, University of Connecticut Health Center, School of Dental Medicine, Farmington, Connecticut 06032, and Research Division, Pharmacia AB, Uppsala,

Sweden2

Received for publication 25 January 1978

The extracellular, water-soluble and cell-associated, 1 N NaOH-soluble glucans from cariogenic Streptococcus mutans 6715-13 "wild type" (WT) and glucan synthesis-defective mutants with diminished virulence have been quantitatively and qualitatively analyzed by methylation analysis and gel chromatography. The mutants synthesized more of a highly branched a-(1 -l 6)-rich extracellular polymer than WT, and some of this glucan was also found to be cell associated in all but one case. WT, in distinction to the mutants, also synthesized a highly branched, a-(1 -* 3)-rich, cell-associated polymer. Treatment of these two distinct polymer types with dextranase or an a-(1 -* 3)-hydrolyzing enzyme indicated they were composed of both a-(1 -l 3) and a-(1 -* 6) linkages and of a-(1 6) with branches at the 3-position, rather than of separate a-(1 -. 3) and a-(1 6) homopolymer mixtures. Gel chromatography before enzymatic hydrolysis disclosed a high degree of polydispersity in both glucan classes. After hydrolysis polydispersity was reduced, again without resolution of two glucan populations. These findings suggest that (i) there are two distinct glucan classes, one a-(1 -+ 3) rich and the other a-(1 -- 6) rich in WT, (ii) diminution of virulence in the mutants is probably ascribable to a failure to form the a-(1 -l 3)-rich component, (iii) both a-(1 -. 6)- and a-(1 -* 3)-rich glucans are found in association with the cell, and (iv) both highly branched glucan types are dextranase and a-(1 -* 3)hydrolase sensitive, and methylation analysis and gel chromatography suggest polymers with highly polydisperse molecular weights which contain mixtures of linkage types. The significance of extracellular polysaccharides in the bacterial etiology of caries has been reviewed by several authors (10, 17, 18, 20, 39, 50). Glucans, enzymatically synthesized from the glucosyl moieties of sucrose by various cariogenic streptococci, especially the smooth-surface/sulcal pathogen Streptococcus mutans, are important factors in the stabilization of initial attachment of the bacterium to the tooth and in the accumulation of plaque (15, 16, 18). Structural analyses of glucans have shown that they contain predominantly a-(1 -. 6) and a-(1 -+ 3) linkages (2, 5, 19, 23, 41, 42) and have a tree- or comblike, highly branched form (11, 33). Such glucans are bound to the cell surface by apparently proteinaceous (30) receptors (13, 35) which appear distinct from the synthetic enzymes. Glucan synthesis-defective mutants of S. mutans strain 6715-13, which have lost the ability to form adhesive microbial deposits (plaque) on

wires, formed little plaque on smooth surfaces of teeth and caused diminished smooth surface caries in specific-pathogen-free and gnotobiotic rats (13, 38, 49). When these mutants were cultured in sucrose-containing medium, there was increased synthesis of water-soluble, extracellular glucans and decreased synthesis of alkalisoluble, cell-associated glucans compared to the "wild type" (WT), whereas other screened caries-linked properties, notably exogenously supplied dextran-mediated agglutination, remained unaltered (13). Inasmuch as different glucans have different water solubilities ascribable to, for example, the percentage of a-(1 - 3) linkages, we investigated whether or not the alterations at smooth tooth surfaces represented qualitative as well as the already documented quantitative synthetic alterations. We, therefore, isolated extracellular, water-soluble and cell-associated, alkali-soluble glucans synthesized from sucrose by WT S. mutans strain 6715-13 and glucan synthesis-defective mutants 4, 27, and 33 and determined the

t Present address: Department of Oral Microbiology, University of Lund, School of Dentistry, S-214 21 Malmo, Sweden.

17

18

FREEDMAN, BIRKHED, AND GRANATH

proportions of various linkages in the structure of these polysaccharides by enzymatic analyses, gas-liquid chromatography (GLC) of partially methylated alditol acetates, and gel filtration.

MATERIALS AND METHODS Bacterial strains and culture conditions. S. mutans parental strain 6715-13 WT and mutants 4, 27, and 33 (13) were included in the study. WT and mutants were stored as frozen or lyophilized stationary-phase cultures in 20% skim milk and maintained for in vitro study by monthly transfer in fluid thioglycolate medium (Difco) containing 20% (vol/vol) meat extract and 15 g of CaCO3 powder per liter. For preparation of polysaccharide material, 10 ml of Jordan's medium (29) containing yeast extract and hydrolyzed casein (Difco) and 0.1% glucose, separately added, were inoculated and incubated overnight at 370C in a candle jar. These cultures, in which the pH did not drop below 6.0, were used to inoculate 1 liter of modified Jordan's medium prepared as follows: 10 g each of yeast extract and Trypticase (BBL) were dissolved in 100 ml of water and dialyzed at 40C against 900 ml of deionized water. After 18 h the dialysis bag and its contents were discarded, the volume was readjusted to 900 ml, 5 g of K2HPO4 and 50 mg of Na2CO3 were added, the pH was adjusted to 7.0 with concentrated HCl, and the medium was sterilized by autoclaving. Finally, when cool, 100 ml of sterile 50% sucrose was added, followed by the cell inoculum. The culture at 370C was mechanically stirred, the surface was flushed with a gas mixture of 95% N2 plus 5% C02, and the pH was maintained between 6.8 and 7.0 by means of a pH stat, which governed the addition of 10 N NaOH (12). The cells were harvested by centrifugation (9,000 x g, 15 min, 000) after 18 h.

Isolation and purification of polysaccharide material. Polysaccharides were prepared as summarized in Fig. 1. For DNA determinations, cold distilled

water-suspended cells were extracted at 900C for 20 min in 0.5 N perchloric acid and chilled to 0°C, and the supernatant fluid, after centrifugation (15,000 x g, 10 min, 000), was assayed for DNA by the diphenylamine reaction (4), using calf thymus DNA as standard. To reduce background the absorbance at 650 nm was subtracted from that at 595 nm. Glucose was quantitated by the glucose oxidase reaction (Glucostat, Worthington) on neutralized, buffered (0.3 M KPO4, pH 7), hot acid hydrolysates (4 N H2SO4, 2 h, 100°C) of S. mutans glucan. Protein contents were determined by the procedure of Lowry et al. (34) on samples hydrolyzed for 18 h in 0.1 N NaOH. Linkage and glucose analysis. Linkage analyses of the putative glucans were performed by gas chromatographic separation of partially methylated alditol acetates, using stainless-steel columns (190 by 0.15 cm) containing 3% OV-225 on GasChrom Q (Applied Sciences). A Hewlett-Packard 5710A gas chromatograph with flame ionization detector was used, with nitrogen as carrier gas (20 ml/min) and a column temperature of 170°C. Commercial dextran of 4 x 104 molecular weight (Sigma) was used as standard. These methods are modifications of those described by Birkhed and Rosell (3) and Lindberg (32) and are detailed in Fig.

INFECT. IMMUN. 2a and b. Qualitative determinations and measurement of the percentage of glucose in the samples were performed as described in Fig. 2b; D-arabinose served as internal standard (3). The same carrier gas, column, and detector as above were used. However, the carrier gas flow rate was 30 ml/min and the column temperature was 200°C. All injection volumes were 1 ,ul. Enzymatic treatment of polysaccharide material. Samples containing 15 mg of polysaccharide were

suspended in 1.0 ml -of 0.05 M potassium phosphate buffer containing 0.02% (wt/vol) NaN3 and 38 U of a1,6-glucan 6-glucanohydrolase (EC 3.2.1.11) dextranase from a Penicillium species (Sigma) or 85 U of Cariogenanase (a-1,3-glucan 3-glucanohydrolase; courtesy of T. Stoudt, Merck, Sharpe and Dohme). The glucans were incubated at 370C for 48 h, with samples treated with boiled enzyme serving as controls. After incubation all were dialyzed against cold running water for 24 h and lyophilized. Linkage analyses of enzyme-treated samples and controls were conducted as described above. Commercial dextran and Cariogenan, an a-(l -* 3)-rich glucan from S. mutans strain SL-1 (courtesy of T. Stoudt, Merck, Sharpe, and Dohme), served as standards. Molecular weight distributions. Molecular weight distributions of selected polysaccharides were determined by gel chromatography on Sepharose CL2B (Pharmacia). Experimental conditions and calculations have been described (1, 40). The gel was calibrated with a series of dextran fractions with weightaverage molecular weights (MW), determined by light scattering, of 107 to 104. The molecular weight exclusion limit of the gel for dextran was estimated to be about 30 x 106. The entire molecular weight distributions of the S. mutans polysaccharides were derived from the corresponding elution chromatograms by applying the calibration curve, Kay versus log molecular weight, established with dextrans on the column used (40). Samples (100 mg) of representative S. mutans glucans were treated with dextranase (5 U/ml) in phosphate-azide buffer for 48 h at 37°C. Samples exposed to heat-inactivated enzyme (1000C, 15 min) served as controls. After incubation, the samples were dialyzed, lyophilized, and reweighed. For molecular weight distribution analysis of S. mutans glucans, 5 mg of enzyme-treated or control polysaccharide was dissolved in 0.5 ml of 1 N NaOH and neutralized with 0.5 ml of 1 N HCl, and the volume was brought to 3 ml with 0.25 M NaCl. A 2-ml sample was applied to the gel and eluted with 0.25 M NaCl, and fractions of 3 ml were collected for automated carbohydrate analysis (27) by the anthrone reaction. This was followed by calculation of molecular weight distributions as well as the M. and the number-average molecular weight (Mn).

RESULTS Several changes have been made in the original glucan isolation procedure (13). These are detailed in the flow diagram (Fig. 1) and include culture liquor precipitation with an equal volume as opposed to 2.5 volumes of 100% ethanol to reduce coprecipitation of fructans such as

GLUCANS FROM CARIOGENIC AND MUTANT S. MUTANS

VOL. 21, 1978

t

19

Culture

Centrifugation, 9000

x

g,

DOC, 15

Cell Pellet

Culture Liquor Supernatant

ml H20, by Wash centre fugati on; Supernatants discarded; Resuspend in 100 ml 0.1 N KPO4 1 M KCl pH 7; Incubate 37oC, 120 min

Ethanol Precipitationl

Itwice, 50

mn

Centrifugaton, 9000 OOC, 15 min

+

g,

Pellet resuspended in 100 ml H20;

Supernatant discarded

Centrifugation

x

Dialyzed'

Resuspend in 105 ml H20; Aliquot taken for DNA analysis; NaOH added to 1 N; Incubate 370C, 60 min Centre fugati on KOH added to 4.3 M; Extracted 1000C, 10

Extracted Cells Discarded

I

min

I

Neutralized with HCl;

Dialyzed Centrifuge Supernatant Ethanol Precipitation

Ethanol Precipitation

Centre1fu gation

|Centr fugati on Supernatant Discarded

II l I

Supernatant Discarded

Pellets lyophilized; Weighqd; CH20 and linkages analyzed

AS-I1 AS-I Alkali Soluble 1.

2. 3. 4.

I

WS-I Water Soluble

All Ethanol precipitations 50% (vol/vol) final concentration, All subsequent centrifugations 15,000 x g, 0C, 10 min All dialyses against running tap water. 4C, 48 hr Fig. 2a,b

-lOC,

120 min

FIG. 1. Flow diagram of preparation of extracellular, water-soluble glucan WS-I and cell-associated, water-insoluble, 1 N NaOH-solubilized glucans AS-I and AS-II from S. mutans 6715-13 WT and mutants 4, 27, and 33. The 1-liter culture was maintained at pH 6.8 to 7.0, 370C, for 18 h.

levan or inulin (46). Although it has been shown (12) that S. mutans 6715-13 is a weak synthesizer of intracellular polysaccharide, cells were incubated in phosphate-buffered KCl (2 h, 370C) to diminish the chance of any contamination of cell-associated glucans by intracellular, glycogen-like polymers (24) which contain a-(1 4) linkages. Furthermore, all culture media were predialyzed, and only low-molecular-weight components traversing the membrane, suitably buffered and supplemented with sucrose, were used to grow cell crops at a constant pH. Protein contamination of the glucans arising from coprecipitated medium components or through glucan entrapment of its own synthetic enzymes or medium proteins was reduced by hot alkali ex-.

traction (4.3 M KOH, 1000C, 90 min) as shown in Table 1. After KOH extraction, neutralization, and dialysis (Fig. 1), the 1 N NaOH-soluble, or more precisely solubilized, glucan contained a water-insoluble glucan precipitate, AS-I, and a water-soluble fraction. This water-soluble material, designated AS-II, was precipitated with an equal volume of ethanol, as were the original water-soluble, extracellular glucans, WS-I. Table 2 presents specific glucan quantities normalized to total cellular DNA amounts for S. mutans WT and mutants. The mutants produced from 10 to 80 times more WS-I glucan per mg of DNA and less AS-I and AS-II material compared to WT. Mutant 33 produced no cellassociated glucan, and the AS-I fraction ex-

20

FREEDMAN, BIRKHED, AND GRANATH b

a

10 mg Glucan + 3 ml dimethylsulfoxide; Dissolve and flush with No; Add 30 mg NaH; Vent; Hold at 200C l hr

1 mg Glucan + 1 mg D-Arabinose in 2 ml 0.25M H2S04; Heat at 1000C, 18 hr; Neutralize with BaCO3

I

Freeze; Add 2 ml methyliodide; Mix 201C 3 hr

Evaporate, CH3I; 16 hr,

INFECT. IMMUN.

Add 1 ml

Centrifugation, 15,000 x g, 10 min, 40C; Filtration through Whatman No. 2

20 mg NaBH4 + 5 ml H20; Incubate 180 min, room temperature; Neutralize with Dowex 50W-H+

H20; Dialyze

0mlH0Diyz

Filtration

through Whatman

No. 1

Evaporate1; Extract residue 3 x 5 ml methanol

Evaporate; Add 5 ml 90% formic acid; Heat at 1000C 120 min Evaporate; Add 5 ml 0.25M H2SO4; Heat 100%C 18 hr; Neutralize with BaCO3

Dissolve in 1 ml pyridine; Add 1 ml acetic anhydride; Heat at 1000C, 15 min

Continue as for Carbohydrate Analysis, Fig. 2b

Evaporate; Extract 5 x 2.5 ml Ethanol, 1 x 5 ml toluene

Dissolve residue in chloroform; Filtration through glass wool; Evaporate witb dry N2; Re-dissolve in chloroform' I. All rotary vacuum evaporations at 2. Constant volume 0.15 ml used

40C

FIG. 2. Flow diagrams for preparation of partially methylated alditol acetates for determination of linkages in glucans. (b) Flow diagram for acetylation of hydrolyzed glucans. This procedure is used for the quantitative and qualitative analyses ofglucose in these carbohydrates. D-Arabinose is the internal standard. TABLE 1. Effect of KOH extraction on protein content of extracellular, water-soluble glucan, WS-I, from S. mutans 6715-13 WT and mutants % Protein S. mutans 671513 type

WT 4

27 33 a

b

KOH extracted

Nonextracted control

3.3 0.027 0.13 0.46

24.2 0.8 2.0 6.7

By Lowry colorimetric analysis (34). 4.3 M,

1000C, 90 min.

tracted from mutant 4 contained only 10% glucose after sugar characterization and was not analyzed further. Samples of all fractions which exceeded 10 mg in total weight and which had glucose compositions near or equal to 100% were prepared for methylation analysis by GLC (Fig. 2a and b). Figure 3a and b shows representative chromatograms of S. mutans 6715-13 WT AS-I (Fig. 3a)

and WS-I (Fig. 3b). In this and all other cases where glucanases were not used, there were only four major peaks and recording past 100 min was uneventful. The figure shows inverse relationships between a-(1 -* 3) and a-(l -* 6) linkages in the AS-I and WS-I fractions. Table 3 compares the quantities and types of linkages in all glucan fractions. WS-I fractions generally were highly branched with 2 to 3% a(1 .-* 3) linkages and 26 to 35% a-(1 -. 6) linkages. WS-I from mutant 27 is notably less branched (10%) and more a-(1 -* 6) rich (75%). S. mutans 6715-13 WT AS-I has more a-(1-* 3) than a-(1-* 6) linkages compared to WS-I, although it is not dramatically less branched. In all cases, the AS-II fraction freed from the total cell-associated glucan by KOH extraction, both in its water solubility during dialysis and in its linkages, resembles the WS-I fraction. Selected samples were extensively treated with commercial endohydrolytic dextranase from a Penicillium species and with a glucanase with specificity for a-(1 -l 3) linkages, which was

GLUCANS FROM CARIOGENIC AND MUTANT S. MUTANS

VOL. 21, 1978

21

TABLE 2. Qualitative and quantitative analyses of putative glucans from S. mutans 6715-13 WT and mutants Glucan fractiona

mg of

mg of

mg of Amt (mg)b

AS-II

AS-I

WS-I

S. mutans 6715-13 type

glucan/ mg of DNA

% Glu-

Amt (mg)

cose'

glucan/ mg of DNA

% Glucose

glucan/

Amt (mg)

mg of DNA

% Glucose

100 465 4.4 994 9.5 100 100 100 0.25 17 10 120 1.7 100 100 0.34 70 82 4 0.02 97 27 _d 100 33 a WS-I is water soluble, extracellular; AS-I is 1 N NaOH soluble, cell associated, and does not become soluble during dialysis, whereas AS-II does and is precipitated with 50% (vol/vol) ethanol (Fig. 1). b Dry weight of lyophilized fractons from 1-liter culture at constant pH of 7. 'By methylation analysis with D-arabinose internal standard (Fig. 2). d No 1 N NaOH-soluble, cell-associated material detected, as in reference 13.

WT

0.67 55.0 20.0 7.2

70 3,822 4,720 1,542

4

II

2,3,4,6

2,4,6

a.

3

2 2,3,4

2,4

I I-.

x 0

wo

A.

w

b.

F:3 4K -i

'O. 2-

I.

0.

p

0

i

I

20

40

60

60

100

RETENTION TIME, min. FIG. 3. Recorder responses for GLC separation of partially methylated alditol acetates of S. mutans 671513 WT glucan AS-I (a) and WS-I (b). Both samples were similarly treated and analyzed. The injection volume was 1 IA. The first peak, 2,3,4,6, is 1,5-di-O-acetyl-2,3,4,6-tetra-0-methyl-D-glucitol, which corresponds 6) 3) and a-(1 to glucan end groups. Subsequent peaks, labeled 2,4,6, 2,3,4, and 2,4, correspond to a-(1 linkages and branch residues, respectively.

22

FREEDMAN, BIRKHED, AND GRANATH

INFECT. IMMUN. 1 3. Methylation analysis of glucans (water-soluble, extracellular WS-I and N NaOH-soluble, cellassociated AS-I, AS-II) from S. mutans 6715-13 WT and mutants 4, 27, and 33

TABLE

Mol0a Methyl group position in glucitolb

WT

T

2%

4

33

T-4Od AS-II WS-I AS-II WS-I AS-II WS-I 1.00 32 24 24 13 7 31 34 28 27 2, 3, 4, 6 2 3 2 6 3 7 2 1.83±0.04 35 3 2,4,6 37 2.28±0.04 30 15 24 35 75 77 26 66 2,3,4 36 35 27 37 35 10 9 41 4.37 ± 0.06 5 2, 4 a Determined by integration of areas of the four methylated glucitols. b 2, 3, 4, 6 is 1,5-di-O-acetyl-2,3,4,6-tetra-O-methyl-D-glucitol; other substituted glucitols designated similarly. 2,3,4,6 corresponds to end groups; 2,4,6 corresponds to a-(1 -+ 3) linkages; 2,3,4 corresponds to a-(1 6) linkages; and 2,4 corresponds to glucan branch points. T values are retention times + standard deviations relative to 2,3,4,6-(OMe)4-glucitol. d Commercial dextran M. 40,000 from L. mesenteroides. WS-I

AS-I

2

2,3,4,6

0

2,4,6

0 w .