INFECTION AND IMMUNITY, Dec. 1979, p. 1079-1087 0019-9567/79/12-1079/09$02.00/0

Vol. 26, No. 3

Effect of Carbohydrate Source and Growth Conditions on the Production of Lipoteichoic Acid by Streptococcus mutans Ingbritt NICHOLAS A. JACQUES,It* LYN HARDY,' LINDY K. CAMPBELL,' KENNETH W. KNOX,' JUDY D. EVANS,2 AND ANTHONY J. WICKEN2 Institute of Dental Research, United Dental Hospital, Sydney, New South Wales 2010,1 and School of Microbiology, University of New South Wales, Kensington, New South Wales 2033,2 Australia Received for publication 12 July 1979

Streptococcus mutans Ingbritt was grown in a chemostat at defined dilution rates and pH values and under carbohydrate limitation. At a constant dilution rate of D = 0.1 h-' and with either 0.5% glucose or 0.5% sucrose, the amounts of both cellular and extracellular lipoteichoic acid increased as the culture pH increased from 5.0 to 7.5. At a constant pH of 6.0, the amount of cellular lipoteichoic acid formed by cultures growing in 0.2% or 0.5% glucose was relatively constant over a range of dilution rates, although the amount of extracellular lipoteichoic acid formed in 0.2% glucose at intermediate dilution rates was less than that formed in 0.5% glucose. Organisms grown in 0.5% sucrose at pH 6.0 contained increasing amounts of cellular lipoteichoic acid as the dilution rate was increased. A comparison of the amounts of cellular lipoteichoic acid formed by organisms growing at D = 0.5 h-' and pH 6.0 in glucose, sucrose, fructose, or mixtures of glucose and fructose in limiting amounts suggested that the enhanced production of lipoteichoic acid by sucrose-grown organisms was due to the fructose component. The culture fluids from both glucose- and sucrose-grown organisms contained detectable amounts of serotype c antigen, whereas glucosegrown cultures also contained significant amounts of an extracellular hexosecontaining polymer. Streptococcus mutans is regarded as an important component of the dental plaque because of its ability to cause dental caries (10), and there is therefore considerable interest in its physiological and serological properties. Seven serological types, a through g, have been defined (1, 3, 21), though serotype c strains generally predominate in human plaque (2, 21, 25). The typing antigen is a cell wall polysaccharide (19, 30), but there are also antigens that are not typespecific, including acylated lipoteichoic acid (LTA) (16, 20) and protein components (23). LTAs display a wide variety of biological properties that are of potential importance when considering the interaction of plaque bacteria with oral tissues. The amphipathic nature of LTA, which derives from the presence of covalently linked glycolipid, accounts for a number of these properties (18, 32, 34). LTA also carries a negative charge because of the glycerol teichoic acid moiety, and it has been proposed that

this component contributes to the formation of dental plaque by its interaction with the tooth surface (22). In view of the many studies that have implicated sucrose in plaque formation, presumably because of the formation of adhesive glucans (10, 11, 13), it is relevant that recent studies have shown that plaque from humans and monkeys ingesting sucrose had a higher concentration of LTA than did plaque obtained after glucose or xylitol ingestion (G. Rolla, P. Bonesvoll, R. V. Oppermann, B. Melson, T. E. Ciardi, and W. H. Bowen, Annu. Meet. Am. Assoc. Dent. Res. 1979, abstr. 1339, p. 425; G. Rolla, T. E. Ciardi, W. H. Bowen and K. W. Knox, Eur. Organ. Caries Res. Meet. 1979, in press). A variety of streptococci (14, 20) and lactobacilli (20) produce significant amounts of extracellular LTA when grown in batch cultures, the greatest amounts being formed by S. mutans strains, particularly the type b strain BHT (20). Subsequently (12), an examination was undertaken of the production of LTA and its deacylated monomeric form (deacylated LTA) by cul-

t Present address: Microbiology Section, Laboratory of Microbiology and Immunology, National Institute of Dental Research, Bethesda, MD 20205. 1079

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INFECT. IMMUN.

JACQUES ET AL.

tures of strain BHT grown in continuous culture with glucose limitation, and it was shown that the amounts detectable were influenced by both pH of growth and generation time. Because of the wide variety of biological properties displayed by LTA and because of the greater prevalence of type c strains of S. mutans, it was relevant to seek verification of these results by examining a strain of this serotype, namely strain Ingbritt. In addition to showing that S. mutans Ingbritt does produce extracellular LTA when grown in continuous culture, the study was extended by examining the influence of differences in glucose concentration and the effect of replacing glucose by sucrose and fructose. Culture fluids were also examined for the presence of the type c-specific antigen. As described subsequently, organisms obtained from cultures grown with limited glucose were examined for possible variations in composition and immunogenicity of cell surface components (15). MATERIALS AND METHODS Organism. S. mutans Ingbritt was available from previous studies (20) and was kindly supplied by B. Krasse, University of Goteborg, Sweden. Growth conditions. The medium for growth consisted of the low-molecular-weight components of the complex medium described by van Houte and Saxton (29), which was essentially free of sucrose (12). Whereas the previous study (12) employed the dialyzable fraction of the medium, the present study used that fraction of the biological components of the medium that passed through an Amicon hollow fiber filter HlP10 (Amicon Corp., Lexington, Mass.). The appropriate salts and carbohydrate (analytical reagent grade) were added to the diffusate from the hollow fiber filtration, and the complete medium was then sterilized by filtration (Sartorius filter, 0.2 Mm; 100-mm diameter; Sartorius Membranfilter, Gottingen, Germany). Preliminary studies showed that growth was limited at a glucose or sucrose concentration of 0.5%. Continuous culture was carried out under the same conditions as employed previously for S. mutans BHT (12), namely, under anaerobic conditions (95% N2-5% C02) at 370C and at constant pH in a BioFlo chemostat of 325-ml capacity (model C30; New Brunswick Scientific Co. Inc., New Brunswick, N.J.). The same procedures were also followed for the routine monitoring of cultures, the collection of the culture in a cooling bath, and the subsequent separation of organisms from culture fluid. Under all chemostat conditions with glucose or sucrose, some adherence to the glass walls of the vessel occurred after 4 to 7 days of culturing. When sucrose was used as the carbon source, cells also adhered to the stainless-steel baffles in the vessel. The build up of clumped cells between the baffles and vessel wall became so marked that these cells were preferentially washed by the inflowing culture medium, exacerbating the adherence problem to the detriment of the cells growing in the medium below. To overcome this dif-

ficulty, the culture vessel was prepared without stainless-steel baffles. The gas flow rate was also reduced to 60 to 100 ml/min to prevent the inflowing medium from spraying over the walls of the vessel. By taking these precautions, adherence was minimized to a level similar to that for cells grown on glucose. No adherence to the vessel wall was observed below the meniscus of the culture medium. Glucose utilization, as determined by the glucose oxidase method (5), exceeded 99.7%. Sucrose utilization exceeded 98% as determined by the hot anthrone method (27) and exceeded 99.6% as determined by the cold anthrone method, which detects only fructose

(28). Cell fractions. Cell wall polysaccharide from S. mutans Ingbritt was available from previous studies

(4). Culture fluids were examined for the presence of the polysaccharide by determining total rhamnose and total hexose as described previously (4). LTA from L. casei NCTC 6375 was available from previous studies (20). LTA was isolated from batch-grown S. mutans Ingbritt by extraction with hot aqueous phenol (31) followed by chromatography on AcA22 (LKB-Produkter; Bromma, Sweden) gel columns (40 by 2.6 cm) in 0.2 M ammonium acetate, pH 6.9. Fractions containing LTA, which were eluted with Ka,, = 0.12, were dialyzed and freeze dried. The structure of the teichoic acid moiety was studied by examining the products obtained on hydrolysis with dilute acid, dilute alkali, and hydrofluoric acid followed by subsequent hydrolysis with a- andf/-glucosidase (31, 33). Serological procedures. For comparing the relative amounts of LTA in organisms grown under different conditions, cell suspensions (approximately 10 mg/ ml) were extracted with hot aqueous phenol (12). Culture fluid was freed from residual bacteria by passing through a membrane filter (0.22 ,um; Millipore Corp.), dialyzed against 0.85% NaCl, and either examined immediately for serological properties or frozen until required. Concentration and fractionation of dialyzed culture fluid was achieved by ultrafiltration in an Amicon stirred ultrafiltration cell no. 12 containing either a Diaflo XM 300 or an XM 50 filter (12). Antiserum against L. casei LTA (rabbit 409) (12) was employed for detecting LTA by immunoelectrophoresis, rocket immunoelectrophoresis, and hemagglutination and for determining the culture fluid titer (12, 20). Rocket heights were calculated in centimeters per 50Mug of cell equivalent. The type-specific antigen was detected with homologous antiserum. Antiserum to S. mutans Ingbritt was prepared by injecting rabbits intravenously with a suspension of heat-killed organisms in 0.85% NaCl at 3- to 4-day intervals by the following schedule: 0.2, 0.5, 1.0, and 1.5 ml of suspension with absorbance of 1.0 at 600 nm in a 1-cm cell, followed by 1.0 and 1.25 ml of suspension of absorbance 2.0. The antiserum employed in the present study (rabbit 532) had been characterized previously (4) and was prepared against organisms grown at pH 6.0 and D = 0.5 h-'.

RESULTS Growth characteristics. Cells of S. mutans Ingbritt grown with limiting glucose generally

VOL. 26, 1979

LIPOTEICHOIC ACID OF S. MUTANS INGBRITT

consisted of chains of 2 to approximately 12 cells. The only significant differences in chain length occurred with cultures grown with 0.2% glucose, where shorter chains of 2 to 6 cells predominated at all dilution rates, and at pH 7.0 and above with 0.5% glucose, where chains varied from 2 to 30 cells in length. No clumping of cells was observed under any of the conditions employed. Sucrose-grown organisms possessed chains of 2 to approximately 12 cells, irrespective of growth conditions. Batch cultures grown with sucrose (0.5%) showed visible signs of clumping. No such clumping was apparent, even microscopically, under any of the chemostat conditions once sucrose became the limiting factor for growth. The yield of organisms obtained under various growth conditions (Table 1) increased on increasing the glucose content of the medium from 0.2 to 0.5%, but 0.5% glucose and 0.5% sucrose gave similar results. The yield at a particular pH was less for slower growing organisms (D = 0.05 and 0.1 h-'), and the yield at a particular dilution rate was greatest at pH 6.5. Properties of cellular LTA. Quantitative analyses gave a molar ratio of phosphorus/glucose/rhamnose of 1.0:0.29:0.01, indicative of a glucosyl-substituted LTA contaminated with a small proportion of wall polysaccharide. Acid degradation products were glycerol, glycerol mono- and diphosphate, glucose, and fatty acids. Degradation with alkali was incomplete, which is indicative of glucosidic substitution on the 2 position of glycerol in the polyglycerophosphate chain. Hydrolysis with hydrofluoric acid at 00C gave glycerol and a product with the chromatographic mobility of 2-o-D-glucosylglycerol as water-soluble products. The latter was isolated preparatively by paper chromatography and hydrolyzed by acid to give equimolar glucose and glycerol. The glucoside was hydrolyzed by figlucosidase but not a-glucosidase, indicating that it was 2-o-/3-D-glucosylglycerol. Quantitative serological studies showed that TABLE 1. Effect ofpH, dilution rate, and carbohydrate on the yield of organisms obtained on continuous culture under anaerobic conditions Dry wt (mg/ml)

pH 6.0 6.0 6.0 5.0 6.5 7.5 7.5 7.5 a

D (h-')

Glucose

0.05

(0.2%) 0.43 0.60 0.51

0.30 0.50 0.10 0.10 0.10 0.30 0.50

-, Not determined.

-a

-

-

Glucose (0.5%) 0.81 0.98 0.99 0.67 1.22 0.92 1.15 1.00

Sucrose (0.5%) 0.88 1.07 1.09 0.64

1.03 0.98 -

1081

the reaction between the LTA preparation (40 and S. mutans Ingbritt antiserum (0.1 ml) was inhibited 13% by 100 ,umol of methyl-a-Dglucoside and 8% by methyl-fi-D-glucoside. This antiserum cross-reacted strongly with L. casei LTA, 0.66 mg of antibody being precipitated from 1 ml of serum compared with 0.83 mg in the homologous reaction. These results indicate that antibody specificity is primarily directed against the glycerol phosphate backbone of the LTA. The S. mutans Ingbritt LTA also reacted strongly with L. casei antiserum which is specific for this glycerol phosphate backbone; by rocket immunoelectrophoresis, a straight-line relationship was obtained between S. mutans Ingbritt LTA and L. casei antiserum up to 0.1 ,tg of LTA where the rocket height was 2.8 cm. Serologically active components in cell extracts and culture fluid. Aqueous phenol extracts of organisms and culture fluid were examined by immunoelectrophoresis for the presence of components reacting with antisera to L. casei LTA and to S. mutans Ingbritt. The concentration of components in dialyzed culture fluid was generally too low for detection, and accordingly, culture fluids were concentrated 10fold by ultrafiltration on an Amicon XM 50 membrane. The XM 50 diffusate was retained, dialyzed against water, then freeze-dried and reconstituted in an appropriate volume. A portion of the XM 50 retentate was also fractionated on an XM 300 membrane. An examination of typical cellular and extracellular fractions for their reactivity with antiserum to L. casei LTA showed the presence in cell extracts of a component with the same mobility as L. casei LTA (Fig. 1). The XM 50 retentate of culture fluid contained LTA and an additional faster-moving component with the characteristics of deacylated LTA (12), whereas only LTA was present in the XM 300 retentate (Fig. 1); the XM 50 diffusate did not contain detectable amounts of reactive material. An examination of the extracellular fraction for reactivity with antiserum to S. mutans Ingbritt confirmed these results and also showed that a component with the properties of the type c-specific antigen was present in the XM 50 retentate but not the XM 300 retentate (Fig. 2). Effect of glucose concentration and dilution rate on formation of acylated and deacylated LTA. The total amounts of LTA and deacylated LTA in cell extracts and culture fluids were estimated by rocket immunoelectrophoresis against antiserum to L. casei LTA. Rocket heights were standardized on 50 ug (dry weight) of S. mutans Ingbritt cells and the amount of culture fluid corresponding to 50 ug of cells.

Isg)

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JACQUES ET AL.

...........

:. . . ....,..., ~~~~~~~~~~~.....

slightly under these conditions. The amounts of extracellular material only equalled or exceeded the cellular content at intermediate dilution rates with 0.5% glucose. An examination of cultures grown in 0.5% glucose at pH 7.5 and at various dilution rates provided evidence of a greater variation in the amounts of cellular and extracellular material (Fig. 4). At this pH, there was a considerable increase in the absolute amounts of detectable material, and the amount of extracellular material exceeded the cellular content at most dilution rates. The XM 300 retentate of culture fluid contained LTA but not deacylated LTA (Fig. 1). Accordingly, representative culture fluids that

FIG. 1. Detection of acylated and deacylated L TA by immunoelectrophoresis against antiserum, to L. casei LTA. Fractions from organisms grown at D = 0.1 h -' and pH 7.5 and LTA preparations were electrophoresed for 10 min at 15 V cm -': (1) L. casei LTA (500 uglml); (2) retentate from an Amicon XM 50 membrane of a phenol extract of whole cells; (3) retentate from an Amicon XM 50 membrane of dialyzed culture fluid (x10); (4) retentate from an Amicon XM 300 membrane of dialyzed culture fluid (x 10); (5) column-purified LTA from S. mutans Ingbritt (500

0.2 % GLUCOSE

e 2.0

.. ~

E

_U I- 1.0.

Pg/ml).

0.5 % GLUCOSE

s,2.0

E

5

LU

...... ...

1.000

0

. ....

0

0.05

0.2

0.1

0.3

0.5

0.4

DILUTION RATE

FIG. 3. Effect of dilution rate on the amount of material detected by rocket immunoelectrophoresis against LTA antiserum for organisms grown in 0.2 or 0.5% glucose at pH 6.0. Solid bars, cellular material; hatched bars, extracellular material. 7.0

FIG. 2. Detection of antigenic components of S. Ingbritt by immunoelectrophoresis against antiserum to S. mutans Ingbritt. Fractions from or0.1 and pH 7w5 and ganisms grown at D =hLT standard fractions were electrophoresed for 10 min at 15 V cm-': () retentate from an XM 50 membrane from an of dialyzed culture fluid (x10); (2) retentate XM 300 membrane of dialyzed culture fluid (x10); (3) S. mutans Ingbritt polysaccharide; (4) column-purified LTA from S. mutans Ingbritt (500 ug/ml); (5) L. case LTA (500 )g/ml). mutants

A comparison was made of the amounts of reactive cellular and extracellular material produced by organisms grown in 0.2 or 0.5% glucose at pH 6.0 and at various dilution rates (Fig. 3). The amounts of cellular material differed only

E

6.0

o4.0 I3.0 W

2.

U 0

1.0 0

0.05

0.05

0.1

0.2

0.3

0.4

0.5

DILUTION RATE

FIG. 4. Effect of dilution rate on the amount of material detected by rocket immunoelectrophoresis against LTA antiserum for organisms grown in 0.5% glucose at pH 7.5. Solid bars, cellular material; hatched bars, extracellular material.

LIPOTEICHOIC ACID OF S. MUTANS INGBRITT

VOL. 26, 1979

had been concentrated 10-fold on an XM 50 membrane were further fractionated on an XM 300 membrane. Fractions were then examined by rocket immunoelectrophoresis, and the rocket height of the XM 300 retentate was calculated as a percentage of the rocket height for the original XM 50 retentate (12). The results (Table 2) indicated that all the culture fluids examined contained approximately the same proportion of high-molecular-weight material (LTA). A direct measure of the relative amounts of LTA in dialyzed culture fluids can be obtained by determining the culture fluid titer, which is the greatest dilution that will fully sensitize erythrocytes for agglutination by antiserum to L. case LTA. For most of the cultures grown at pH 6.0 and at various dilution rates, the titers were very low (Table 3), but the results did confirm that greater amounts of extracellular material were generally formed by organisms grown in 0.5% glucose. Considerably higher values were obtained at all dilution rates for cultures grown at pH 7.5 (Table 3), confirming the results of rocket immunoelectrophoresis. Effect of various carbohydrates on the formation of LTA and deacylated LTA. The experiment performed at pH 6.0 and at various dilution rates was varied by replacing 0.5% glucose with 0.5% sucrose (Fig. 5). Stable growth TABLE 2. Percentage of extracellular LTA fraction from glucose-grown cells that was retained by an XAM 300 membrane D (h-')

pH

XM 300 retentate

0.05 0.05 0.10 0.10 0.20 0.20 0.50 0.50

6.0 7.5 6.0 7.5 6.0 7.5 6.0 7.5

33 36 46 41 33 30 43 30

TABLE 3. Comparison of the relative amounts of extracellular acylated LTA produced by S. mutans Ingbritt grown at different dilution rates as shown by the culture fluid titers D (h-')

0.05 0.10 0.20 0.30 0.40 0.50

Culture fluid titer in: Glucose Glucose Glucose (0.2%) pH 6.0 (0.5%) pH 6.0 (0.5%) pH 7.5 27 5 2 18 5 2 18 5 2 18 5 2 18 2 2 12 2 2

1083

3.0

E 2.0

Uj 0

1.0

0 0.05

0.1

0.2

0.3

DILUTION

0.4

0.5

0.69

RATE

FIG. 5. Effect of dilution rate on the amount of material detected by rocket immunoelectrophoresis against LTA antiserum for organisms grown in 0.5% sucrose at pH 6.0. Solid bars, cellular material; hatched bars, extracellular material.

conditions could be achieved at dilution rates up to 0.69 h-', whereas with glucose, "wash-out" of cells occurred at this high dilution rate (equivalent to a 1-h generation time). In contrast to the results obtained with glucose (Fig. 3), the amounts of detectable cellular material increased almost fourfold as the dilution rate increased from between 0.05 and 0.2 h-' to between 0.50 and 0.69 h-', whereas the amount of extracellular material was greatest for slowgrowing organisms, particularly at D = 0.05 h-1. The presence of relatively high amounts of LTA in the culture fluid of slow-growing organisms was confirmed by determining the culture fluid titer, which was 18 at D = 0.05 h1-, 7.5 at D = 0.1 h-1, and 2 for faster growing organisms. This apparent effect of sucrose on LTA production was examined further by growing organisms at pH 6.0 and D = 0.5 h-' and changing the medium reservoir every 24 h to provide a different source of carbohydrate. Cultures were equilibrated overnight with each medium (approximately 12 generations) and then collected for examination. Table 4 summarizes the results of rocket immunoelectrophoresis of cultures grown in 30 mM (0.54%) glucose and fructose, 15 mM fructose, 15 mM fructose + 15 mM glucose, and 15 mM sucrose (0.51%). Repeating the experiment confirmed that the enhancement of LTA production by sucrose-grown cells is also obtained with an equimolar mixture of glucose and fructose and that this effect is apparently due to the fructose component. Effect of pH on the formation of LTA and deacylated LTA. Organisms were grown at a constant dilution rate (0.1 h-1) and at various

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TABLE 4. Effect of carbohydrate source on LTA formation by S. mutans Ingbritt grown at pH 6.0 and D = 0.5 h-'

Rocket height (cm/50,Lg of

cells)

Carbohydrate source

Glucose (30 mM) Sucrose (15 mM) Glucose (15 mM) + fructose (15 mM) Fructose (15 mM) Fructose (30 mM)

LTA Cellular CelllarLTA

Extracellular LTA

1.5 3.3 3.6

0.4 0.4 0.3

7.5 7.5

0.8 0.7

pH values to determine the effect of pH on the total amounts of reactive material. With 0.5% glucose as the limiting nutrient (Fig. 6), the amounts of material in aqueous phenol extracts of cells that could be detected by rocket immunoelectrophoresis remained relatively constant between pH 5.0 and 6.5 but then increased markedly as the pH of growth was increased to 7.0 and 7.5. The amount of material in the culture fluid that was present in the concentrate from XM 50 membrane filtration showed a similar trend, with an even greater increase at pH 7.0 and 7.5. This effect of pH was confirmed by repeating the experiment with 0.5% sucrose as the growthlimiting nutrient (Fig. 6). Although some differences are apparent between the results for the two experiments at the different pH values, the major effect was again obtained by increasing the pH from 6.5 to 7.5 when the amount of extractable and detectable cellular material increased nearly fivefold and the amount of extracellular material increased fourfold. Culture fluids were also examined by the hemagglutination procedure to detect acylated LTA. The results in Table 5 provide both the values for the maximum hemagglutination titer obtained with the antiserum and the maximum dilutions of culture fluid (culture fluid titer) that gave this value. The culture fluid titers confirm the results of rocket immunoelectrophoresis, namely, that greater amounts of reactive material are present when organisms are grown at the higher pH values. Generally the hemagglutination titers for a series of experiments are the same, but in the experiments reported in Table 5 there was a trend toward an increasing hemagglutination titer with rise in pH. In the case of the experiments with 0.5% glucose, this trend was also observed when cells were sensitized with 10-fold-concentrated culture fluid (XM 50 retentate); the hemagglutination titer increased from 400 (pH 5.0, 5.5) to 800 (pH 6.0) and then 1,600 (pH > 6.5). In contrast, the hemagglutination titer of extracts obtained from cells was

800 under all conditions of growth. Comparison of cellular and extracellular carbohydrate components. The XM 50 retentates of culture fluid contained a component with the properties of type c antigen (Fig. 2). This antigen has rhamnose as its major component, and accordingly, fractions obtained by membrane ultrafiltration were examined for their rhamnose content. By comparing the results with the rhamnose content of the organisms derived from the corresponding medium and taking into account the blank value given by uninoculated medium, the amount of extracellular rhamnose could be calculated as a per6.0 5.0

E

0.5% GLUCOSE

4.0 3.0.

2.0E w

1.0

0 0

6.; 0

0.5 % SUCROSE

e, 5.i.o

0

Lo E E

4

3 I

2. U'

0I

1.0

1. 0

E 5.0

5.5

6.0

6.5

7.0

7.5

pH

FIG. 6. Effect of pH on the amount of material detected by rocket immunoelectrophoresis against LTA antiserum for organisms grown in 0.5% glucose or 0.5% sucrose at a dilution rate of 0.1 h-1. Solid bars, cellular material; hatched bars, extracellular material. TABLE 5. Application of the hemagglutination procedure to the examination of culture fluids from organisms grown at various pH values in 0.5% carbohydrate H titera

CF titerb

pH Glucose

Sucrose

Glucose

Sucrose

2 2 5.0 400 400 2 5.5 400 400 5 800 2 7.5 6.0 800 12 6.5 800 800 7.5 800 12 7.5 7.0 1,600 800 7.5 18 18 1,600 a H titer, Hemagglutination titer, i.e., maximum dilution of antiserum that agglutinated fully sensitized

erythrocytes. b CF titer, Culture fluid titer, i.e., dilution of culture fluid that would fully sensitize erythrocytes.

LIPOTEICHOIC ACID OF S. MUTANS INGBRITT

VOL. 26, 1979

centage of the cellular value. The results for the XM 50 retentate from the culture fluid from organisms grown with glucose (0.5%) and sucrose (0.5%) at various dilution rates are given in Table 6; other fractions, namely, the XM 50 diffusates and the XM 300 retentates, did not contain detectable amounts of rhamnose. The results indicate the presence of a variable but low amount of a rhamnose-containing polysaccharide fraction. Although the ratio of rhamnose to hexose in all the tested organisms approximated that for the type antigen, the culture fluid from glucose-grown organisms contained a considerably greater amount of nondiffusible hexose than could be accounted for by the presence of the type antigen. An examination of cultures of organisms grown with glucose (0.5%) and sucrose (0.5%) at D = 0.1 h-' and at pH 5.5, 6.5, and 7.5 gave similar results, namely, the presence of a low amount of rhamnose (1 to 7% of the cellular content), whereas extracellular hexose was only present in significant amounts in the cultures of glucose-grown organisms (24 to 82% of cellular content).

DISCUSSION The results of serological and chemical studies on the culture fluid of chemostat-grown S. mutans Ingbritt indicate the presence of various amounts of LTA together with small amounts of the typing antigen and, under certain circumstances, an additional presumptive carbohydrate polymer. Evidence for the formation of the carbohydrate polymer by glucose-grown cells comes from analyses showing that the total hexose content of fractionated culture fluid exceeds that expected for the typing antigen. Although considerable emphasis has been placed on the role of glucan synthesis from sucrose as a factor in plaque formation (10, 11, 13), a recent study has shown that type c strains differ from most other S. mutans strains by forming adherent

1085

colonies when grown in glucose, where there is the concomitant production of an extracellular, glucose-containing polysaccharide (26). Observations on the cultures growing in the chemostat indicated very little adherence of organisms to surfaces, provided that conditions were properly controlled, and there was no evidence for aggregation of organisms. Employing different growth conditions and a different strain of Ingbritt, namely one that had been "reisolated from a carious lesion in a monkey" (8, 9), Ellwood and co-workers showed that organisms growing in 1% glucose at a high dilution rate (0.5 h-') formed conglomerates of 200 to 500 cells (7, 9) and that similar conglomerates were formed by sucrose-grown organisms (7). In the present study, no such aggregation occurred regardless of whether glucose or sucrose was used as the carbohydrate source. Furthermore, the results of quantitative analysis on organisms and culture fluid gave no evidence for glucan synthesis. Because of the biological potential of LTA, studies were primarily concerned with the effect of growth conditions on the formation and excretion of this component. There was no direct relationship between dilution rate and production of extracellular LTA at pH 6.0 as observed with strain BHT, where the greatest amounts were found in cultures grown at D = 0.05 h-' (12). However, by varying the pH, dilution rate and carbohydrate content of the medium the extent of the variation in the amounts of extracellular material exceeded the range for strain BHT (12). Furthermore, variations in the growth conditions had a much greater effect on the relative amounts of cellular LTA than was observed with strain BHT (12). To illustrate the extent of these variations, Table 7 presents a comparison of the results of rocket immunoelectrophoresis of cell extracts TABLE 7. Relative proportions of cellular and extracellular material detectable by rocket

immunoelectrophoresis

TABLE 6. Cellular and extracellular carbohydrate components of organisms grown at pH 6.0 Carbohydrate (h-') (0.5%) D

Cellular carbohydrate (,ug/mg)

Extracellular car-

Rham-

Rham-

Hexose

Sucrose

Hexose

nose

nose

Glucose

bohydratea

0.05

92

68

3

12

0.20 0.50

94 88 98

7 5 5 4 1

61 68

0.05

51 36 65 57 43

0

6 102 0.20 0 106 0.50 a The extracellular carbohydrate (expressed as percent cellular carbohydrate) was determined from an XM 50 retentate (X10) of dialyzed culture fluid.

Carbohydrate

(h h) pH

Rocket height Extracel Cellular lular

Exularl

1.0 1.0 0.10 6.0 Glucose (0.5%) 1.5 0.8 0.20 6.0 Glucose (0.5%) 0.4 1.0 0.20 6.0 Glucose (0.2%) 5.3 2.4 0.05 7.5 Glucose (0.5%) 4.2 2.2 0.10 7.5 Glucose (0.5%) 4.3 4.6 0.50 7.5 Glucose (0.5%) 2.6 1.0 0.05 6.0 Sucrose (0.5%) 0.6 0.8 0.10 6.0 Sucrose (0.5%) 0.3 3.3 0.50 6.0 Sucrose (0.5%) 0.6 6.0 0.50 6.0 Fructose (30 mM) a Expressed as a proportion of the values for organisms grown in 0.5% glucose (D = 0.1 h-'; pH 6.0).

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and culture fluid, in which the values are expressed relative to those for the culture grown in 0.5% glucose at D = 0.1 h-' and pH 6.0. As the rocket heights for the cellular and extracellular fractions from this culture were very similar (1.25 and 1.20 cm, respectively), the figures also enable a comparison of the relative amounts of cellular and extracellular material under the different conditions. The results of rocket immunoelectrophoresis may be influenced by the relative amounts of LTA and deacylated LTA (12). However, under the conditions studied there was very little difference in the relative amounts of high-molecular-weight extracellular material retained by an XM 300 membrane. Furthermore, the results for culture fluid titer, which only detects LTA, generally reflected the results obtained by rocket immunoelectrophoresis. The proportion of the extracellular fraction retained by the XM 300 membrane was generally less from strain Ingbritt than for strain BHT (12). However, for strain BHT, the XM 300 retentate contained deacylated LTA in association with LTA (12), whereas with strain Ingbritt this association was not found. Similarly LTA from strain BHT forms complexes with polysaccharide (12, 20), whereas membrane filtration of Ingbritt culture fluid separated the polysaccharide component from LTA. The reactivity of different LTAs with antiserum to L. case LTA is influenced by the degree of carbohydrate substitution, as shown by the quantitative precipitin method (17) and rocket immunoelectrophoresis (unpublished data). Thus the results of rocket immunoelectrophoresis in the present study could vary if the LTA component varied in composition under different growth conditions. The LTA from batchgrown S. mutans Ingbritt has a low degree of glucose substitution, but this seems to have little influence on its serological properties. Furthermore, preliminary studies on the cellular LTA of chemostat-grown organisms indicate that growth conditions have little effect on the degree of glucose substitution (unpublished data). The constant hemagglutination titer (as distinct from culture fluid titer) of cell extracts also indicates that there are not marked differences in composition. The differences in the hemagglutination titer of extracellular material could indicate a subsequent change in composition, but extracellular LTA has not yet been isolated in sufficiently pure form to test this proposition. The studies on the effect of growth conditions on LTA production were extended by examining the effect of different carbohydrates and their concentration. Lowering the glucose content

INFECT. IMMUN.

from 0.5 to 0.2% decreased the yield of cells. However, the cellular LTA content remained relatively constant and extracellular LTA was still formed in 0.2% glucose, although at intermediate dilution rates the amounts were less than those produced by organisms grown in 0.5%

glucose. Evidence that factors other than the total carbohydrate content of the medium influenced LTA formation was provided by studies with sucrose and fructose. The initial experiment with 0.5% sucrose at pH 6.0 and increasing dilution rates (Fig. 5) gave results that differed from those obtained in a comparable study with 0.5% glucose for both the amounts of cellular and extracellular LTA. Evidence that this enhancement of LTA production by sucrose-grown organisms was a phenotypic property was provided by the subsequent lowering of cellular LTA to the original value by lowering the dilution rate to 0.1 h-1 and the pH to 5.5 (Fig. 6). In the subsequent studies at D = 0.5 h-' (Table 4), the effect of sucrose was not as great. However, these studies did show that the effect of sucrose could be achieved with a mixture of glucose and fructose and that a greater enhancement was achieved by fructose alone. The reasons for the effect of these differences of energy source on LTA production are not apparent. Cultures of S. mutans grown in batch culture with sucrose or glucose grow in a balanced fashion at the same exponential rate (6), although separate inducible phosphoenolpyruvate-dependent uptake systems appear to be required for sucrose, glucose, and fructose (24). The effect of varying the growth conditions on the formation of LTA by organisms grown in sucrose and fructose in the presence and absence of glucose is under further investigation. The results with sucrose and fructose are particularly pertinent to recent studies on the LTA content of plaque (G. Rolla et al., Annu. Meet. Am. Assoc. Dent. Res. 1979, abstr. 1339, p. 425; G. Rolla et al., Eur. Organ. Caries Res. Meet. 1979, in press). In these studies, plaque was collected from individuals who had regularly rinsed with sucrose, glucose, or xylitol. The plaque was extracted with hot aqueous phenol, and LTA was determined by its ability to sensitize erythrocytes that were then agglutinated by anti-LTA antiserum. The results, which were supported by similar studies with monkeys, showed that in relation to the bacterial count, plaque from sucrose rinsing contained a significantly higher LTA content than plaque from glucose rinsing, whereas xylitol plaque contained negligible amounts. It has been proposed that LTA plays a role in plaque formation (22), and

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thus the known effect of sucrose on plaque formation may depend not only on glucan production but also on the concomitant production of increased amounts of LTA. ACKNOWLEDGMENTS This work was supported by the National Health and Medical Research Council of Australia and by Public Health Service grants R01 DE04174 and R01 DE04175 from the National Institute of Dental Research. We thank Rosemary Ann Brown for her excellent technical assistance.

LITERATURE CITED 1. Bratthall, D. 1969. Immunodiffusion studies on the serological specificity of streptococci resembling Streptococcus mutans. Odontol. Revy 20:231-243. 2. Bratthall, D. 1972. Demonstration of Streptococcus mutans strains in some selected areas of the world. Odontol. Revy 23:401-410. 3. Bratthall, D., and B.-M. Pettersson. 1976. Common and unique antigens of Streptococcus mutans. J. Dent. Res. 55:A60-A64. 4. Campbell, L. K., K. W. Knox, and A. J. Wicken. 1978. Extractability of cell wall polysaccharide from lactobacilli and streptococci by autoclaving and by dilute acid. Infect. Immun. 22:842-851. 5. Dahlqvist, A. 1961. Determination of maltase and isomaltase activities with a glucose oxidase reagent. Biochem. J. 80:547-555. 6. Daneo-Moore, L., B. Terleckyj, and G. D. Shockman. 1975. Analysis of growth rate in sucrose-supplemented cultures of Streptococcus mutans. Infect. Immun. 12: 1195-1205. 7. Ellwood, D. C. 1976. Chemostat studies of oral bacteria, p. 785-798. In H. M. Stiles, W. J. Loesche, and T. C. O'Brien (ed.), Proceedings: microbial aspects of dental caries. Special supplement to microbiology abstracts, vol. 3. Information Retrieval Inc., Washington, D.C. 8. Ellwood, D. C., J. K. Baird, J. R. Hunter, and V. M. C. Longyear. 1976. Variations in surface polymers of Streptococcus mutans. J. Dent. Res. 55:C42-C49. 9. Ellwood, D. C., J. R. Hunter, and V. M. C. Longyear. 1974. Growth of Streptococcus mutans in a chemostat. Arch. Oral Biol. 19:659-664. 10. Gibbons, R. J. 1972. Ecology and cariogenic potential of oral streptococci, p. 371-385. In L. W. Wannamaker, and J. M. Matsen (ed.), Streptococci and streptococcal diseases. Academic Press Inc., New York. 11. Gibbons, R. J., and J. van Houte. 1973. On the formation of dental plaque. J. Periodontol. 44:347-360. 12. Jacques, N. A., L. Hardy, K. W. Knox, and A. J. Wicken. 1979. Effect of growth conditions on the formation of extracellular lipoteichoic acid by Streptococcus mutans BHT. Infect. Immun. 25:75-84. 13. Jones, G. W. 1978. Attachment of bacteria to the surfaces of animal cells, p. 139-175. In J. C. Reissig (ed.), Microbial Interactions. Chapman Hall, London. 14. Joseph, R., and G. D. Shockman. 1975. Synthesis and excretion of glycerol teichoic acid during growth of two streptococcal species. Infect. Immun. 12:333-338. 15. Knox, K. W., N. A. Jacques, L. K. Campbell, A. J. Wicken, S. F. Hurst, and A. S. Bleiweis. 1979. Phenotypic stability of the cell wall of Streptococcus mutans Ingbritt grown under various conditions. Infect.

Immun. 26:1071-1078. 16. Knox, K. W., J. L. Markham, and A. J. Wicken. 1976. Formation of cross-reacting antibodies against cellular and extracellular lipoteichoic acid of Streptococcus mutans BHT. Infect. Immun. 13:647-652. 17. Knox, K. W., and A. J. Wicken. 1977. Immunochemistry of lipoteichoic acids, p. 356-359. In D. Schlessinger (ed.), Microbiology-1977. American Society for Microbiology, Washington, D.C. 18. Knox, K. W., and A. J. Wicken. 1973. Immunological properties of teichoic acids. Bacteriol. Rev. 37:215-257. 19. Linzer, R., K. Gill, and H. D. Slade. 1976. Chemical composition of Streptococcus mutans type c antigen: comparison to type a, b and d antigens. J. Dent. Res.

55:A109-A115.

20. Markham, J. L., K. W. Knox, A. J. Wicken, and M. J. Hewett 1975. Formation of extracellular lipoteichoic acid by oral streptococci and lactobacilli. Infect. Immun.

12:378-386.

21. Perch, B., E. Kjems, and T. Ravn. 1974. Biochemical and serological properties of Streptococcus mutans from various human and animal sources. Acta Pathol.

Microbiol. Scand. 82:357-375. 22. Rolla, G. 1977. Formation of dental integuments-some basic chemical considerations. Swed. Dent. J. 1:241251. 23. Russell, M. W., and T. Lehner. 1978. Characterization of antigens extracted from cells and culture fluids of Streptococcus mutans serotype c. Arch. Oral Biol. 23: 7-15. 24. Slee, A. M., and J. M. Tanker. 1979. Phosphoenolpyruvate-dependent sucrose phosphotransferase activity in Streptococcus mutans NCTC 10449. Infect. Immun. 24: 821-828. 25. Thomson, L. A., W. Little, and G. J. Hageage. 1976. Application of fluorescent antibody methods in the analysis of plaque samples. J. Dent. Res. 55:A80-A86. 26. Tinanoff, W., J. M. Tanker, and M. L. Freedman. 1978. In vitro colonization of Streptococcus mutans on enamel. Infect. Immun. 21:1010-1019. 27. van Handel, E. 1965. Microseparation of glycogen, glucose and lipids. Anal. Biochem. 11:226-271. 28. van Handel, E. 1967. Determination of fructose and fructose-yielding carbohydrates with cold anthrone. Anal. Biochem. 19:193-194. 29. van Houte, J., and C. A. Saxton. 1971. Cell wall thickening and intracellular polysaccharide in microorganisms of the dental plaque. Caries Res. 5:30-43. 30. Wetherell, J. R., Jr., and A. S. Bleiweis. 1975. Antigens of Streptococcus mutans. Characterization of a polysaccharide antigen from walls of strain GS-5. Infect. Immun. 12:1341-1348. 31. Wicken, A. J., J. W. Gibbens, and K. W. Knox. 1973. Comparative studies on the isolation of membrane lipoteichoic acid from Lactobacillus fermenti. J. Bacte-

riol. 113:365-372.

32. Wicken, A. J., and K. W. Knox. 1974. Lipoteichoic acids-a new class of bacterial antigens. Science 187: 1161-1167. 33. Wicken, A. J., and K. W. Knox. 1975. Characterization of group N streptococcus lipoteichoic acid. Infect. Immun. 11:973-981. 34. Wicken, A. J., and K. W. Knox. 1977. Biological properties of lipoteichoic acids. p. 360-365. In D. Schlessinger (ed.), Microbiology-1977. American Society for Microbiology, Washington, D.C.

Effect of carbohydrate source and growth conditions on the production of lipoteichoic acid by Streptococcus mutans Ingbritt.

INFECTION AND IMMUNITY, Dec. 1979, p. 1079-1087 0019-9567/79/12-1079/09$02.00/0 Vol. 26, No. 3 Effect of Carbohydrate Source and Growth Conditions o...
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