Variations in Surface Polymers of Streptococcus mutans D. C. ELLWOOD, J. K. BAIRD, J. R. HUNTER, and V. M. C. LONGYEAR Microbiological Research Establishment, Porton Down, Salisbury, Wiltshire, SP4 OJG, England
Streptococcus mutans is an etiologic agent in experimental dental caries in animals.',2 Various strains of this organism have been isolated and classified into four types on the basis of their genetic, antigenic, and biochemical characteristics.3 The classification based on the chemical composition of the cell wall of S mutans strains gives three types.4 However, on the basis of serological tests, Bratthall5 was able to classify strains of this organism into five groups. The cell surface of S mutans is of course the bacterial cell wall. When the organism is grown in the presence of sucrose, the polysaccharides formed from the sucrose by the glycosyltransferase secreted by the organism may also be found at the cell surface. The bacterial component first "seen" by the immunologic defense systems of the invaded host is the cell surface of an organism. Hence, it would be expected that immunization with surface components, or the enzymes that formed them, would give protection against that organism. Much work has been done on the polysaccharide formed from sucrose by the glycosyltransferase of S mutans. Initially, it was shown that the water-soluble polysaccharide I 6 glucan was a mixture of a dextran and a levan.6 The water-insoluble polysaccharide formed by this reaction was found I -e 3 to be a glucan with predominantly linkages.7 The quantitative analysis of the cell wall components of several strains of S mutans and S sanguis has also been reported.8 More recently, the cell surface antigens that characterize the five serogroups of S mutans have been investigated. Van de Rijn and Bleiweis9 suggested that a membrane-associated glycerol teichoic acid with /3-galactose and probably glucose residues was the major antigen of the group a strain, AHT. Vaught and Bleiweisl' also reported the presence of a
-*
a
a cell wall glycerol teichoic acid in the b
strain, BHT, the specificity of the antigen residing in a P-galactose substituent of the teichoic acid. In contrast, Mukasa and Slade" characterized the group a antigen of HS6 as a cell wall polysaccharide containing glucose, galactose, glucosamine, and galactosamine. These workers also showed that in the b strain FAI the group antigen was a polysaccharide, the antigenicity of which resided in D-galactose and galactosamine residues.12 Iacono et al'3 showed that the antigen from S mutans 6715 was a carbohydrate material with a 3-galactoside as the immunodeterminant residue. The cell wall composition of bacteria varies considerably with changes in growth conditions.14 In view of this and the contradictory reports in the literature, it seemed desirable to study the cell surface components (cell wall and exocellular polysaccharides) of representative strains of the five serotypes of S mutans when they were all grown by the same standard method. This article reports the results of such a study.
Materials and Methods The following bacterial strains were used: Z/AHT, OMZ 51, OMZ 176, Ingbritt LH, B 13, LM 7, Ingbritt B, and Ingbritt L. GROWVTH MEDIUm.-The medium was the modified'5 7d medium of Cybulska and Pakula'6 supplemented with 4% glucose. In the initial stages of this investigation, the results from duplicate cultures often varied. The reason for this was traced to the use of different batches of medium, so a 60-liter batch of medium was prepared and samples taken from this were used for all subsequent growth experiments. GROWVTH OF CULTURES.-The various strains were grown under identical conditions in a Porton-type stirred fermenter at 37 C and a
C42 Downloaded from jdr.sagepub.com at SIMON FRASER LIBRARY on June 26, 2015 For personal use only. No other uses without permission.
Vol 55 1976
VARIATIONS IN SURFACE POLYMERS OF S MUTANS
pH of 6.5. The culture was kept anaerobic with 5% CO2 and 95% N2 which was sterilized by filtration, passed down the hollow stirrer shaft, and sparged by the impeller blades. The medium (1,200 ml) was brought to the 37 C temperature and a pH of 6.5 and was inoculated with the heavy deposit from a 100-ml, 18- to 24-hour seed culture grown in the same medium. Two normal NaOH was supplied automatically on demand to maintain the pH at 6.5, and the demand recorded continuously. Silicone antifoam was added when required. Samples were taken at hourly intervals, examined microscopically, and the optical density measured at 540 nm. At the end of the exponential growth phase, as shown by the leveling off of the OD curve and the reduction in the rate of alkali demand, the culture was harvested and centrifuged. The supernatant was stored under toluene for subsequent examination and the cell paste stored at -20 C. All strains were grown at least twice to check on the reproducibility of the system. GLYCOSYLTRANSFERASE ACTIVITY.-A sample at a pH of 6.5 (1 ml) was incubated with sucrose (0.1 ml of 50% w/v aqueous solution) at 37 C for two hours; sodium acetate solution (1.0 ml of 10% w/v aqueous solution) was then added followed by ethanol (6.0 ml), and the sample thoroughly mixed. The mixture was allowed to stand for 16 hours at 4 C before centrifugation (3,000 X g for 20 minutes). The precipitate was washed three times with a mixture of ethanol: 10% sodium acetate (4:1 v/v) by centrifugation. It was then suspended in water, and an aliquot (containing between 20 to 100 yg of glucan) made up to 1.0 ml with water and analyzed for total hexose by the phenol-sulfuric acid method.'7 The activity is expressed as micrograms of polymer formed per hour per milliliter of culture supernatant, under the aforementioned conditions. PREPARATION OF POLYMERS.-The culture supernatants (1,000 ml) were incubated at 37 C under toluene with sucrose solution (200 ml of 50% w/v). Samples (5 ml) were removed at intervals and plunged into boiling water for one minute before assaying for polysaccharides. These were differentiated by first removing the water-insoluble material, precipitating with 55% ethanol, and removing the precipitate by centrifugation. Finally, the ethanol concentration was adjusted to 75% and this precipitate col-
C43
lected by centrifugation. The precipitates were washed with alcohol and the polysaccharides present estimated by the phenolsulfuric acid method.'7 At the end of 24 hours, the pH was still 6.5 + 0.1, showing that no acid had been produced during the incubation. After 24 hours, the incubation mixture was centrifuged (5,000 X g for 30 minutes) to collect the water-insoluble polymer. The precipitate was washed three times with water by centrifugation, freezedried, and weighed. Ethanol was added to the supernatant to give 75% concentration and the mixture allowed to stand for 24 hours at 4 C. The precipitate was collected as just described and washed three times with 98% ethanol. It was then dissolved in water and dialyzed against changes of distilled water for three days, and finally the dialyzate was freeze-dried and weighed. The analytical methods, the methylation, methanolysis, acid hydrolysis, and subsequent analysis of the products by gas chromatography were done as described by Baird, Longyear, and Ellwood.15 Organisms were broken in a Braun M.S.K. homogenizer and the cell walls were isolated quantitatively, as described by Ell-
wood.18 The analyses for total carbohydrate and phosphorus were as described by Ellwood.18 The quantitative analyses of the cell wall sugars were done as they were for the alditol acetates19 or the acetylated aldononitriles20 by gas liquid chromatography. The proteins in the cell wall were solubilized by treatment with sodium dodecyl sulfate (SDS) and mercaptoethanol and then subjected to SDS polyacrylamide gel electrophoresis. Gels were stained with Coomassie blue and after removal of excess stain were scanned at 540 nm in a Chromoscan. The methods used were as described by Spencer and Guest.2'
Results and Discussion It seemed desirable to study the surface polymers of strains of S mutans that represented the five serological groups of this organism. Cultures of the various strains were grown in medium taken from a standard batch to obviate any variation from different media, all the other growth factors were also controlled, and reproducible results were obtained. The growth curves for the strains studied are shown in Figure 1 and although there is some spread in the type of curve, no significant differences can
Downloaded from jdr.sagepub.com at SIMON FRASER LIBRARY on June 26, 2015 For personal use only. No other uses without permission.
C44
ELLWOOD ET AL
J Dent Res Special Issue
C
OMZ 176
OPTICAL DENSITY
GROWTH (HOURS)
FIG 1.-Growth of five serotypes of S mutans. be seen. Duplicate experiments on each strain gave similar curves, and all three Ingbritt strains were identical. The most interesting feature was that for all strains during the latter part of the exponential growth phase, agglomerates of organisms appeared that were similar to those seen when Ingbritt B was grown in continuous cul-
ture.15 The glycosyltransferase activity, defined by the amount of total polysaccharide that was insoluble when the ethanol concentration was adjusted to 75%, was determined on the culture supernatant when the organisms were harvested. The results are given in Table I and indicate that variation in the glycosyltransferase activities occurs between strains of the same serotype.
The formation of the water-insoluble polymer by the culture supernatants on incubation with sucrose as a function of time is shown in Figure 2. The culture supernatants of Ingbritt LH and LM 7 did not form any polymer under these conditions, and that from OMZ 51 formed only small amounts. The two strains Z/AHT and OMZ 176 both gave supernatants that formed water-insoluble polymer at approximately a steady rate, but the glycosyltransferase activity in the culture supernatant from Ingbritt B showed a slow steady rate up to 600 minutes and then rapid formation of polymer took place. Methylation analysis of the water-insoluble polysaccharide isolated from the various strains at the end of the incubation + INGbRITT
6
3000r
Z/AIHT
0W4~ ~ ~ ~ 176~OZ 0/
X INGfIRITT LH Si
*O 0M
O LtA 7
INCUbATION TIME (Mims)
FIG 2.-Rate of formation of polysaccharide insoluble in water. Downloaded from jdr.sagepub.com at SIMON FRASER LIBRARY on June 26, 2015 For personal use only. No other uses without permission.
VARIATIONS IN SURFACE POLYMERS OF S MUTANS
Vol 55 1976
sults
TABLE 1 GLYCOSYLTRANSFERASE ACriVITY OF STRAINS OF STREPTOCOCCUS MUTANS
C4$
shown in Figures 3 and 4. The notable feature of Figure 3 is that a considerable change in the rate of polymer formation can be seen for several strains, Strain Serotype Units/ml OMZ 51 giving the most pronounced effect. The results for the 75%-ethanol-insoluble a 266 Z/AHT b 119 OMZ 51 polymer shown in Figure 4 are somewhat b BHT 465 complex and suggest that rapid conversion C Ingbritt LH 190 of this kind of polymer to the other types c Ingbritt B 367 was taking place. ND IngbrittL c The water-soluble polysaccharides were d OMZ 176 180 collected at the end of incubation after pred B13 380 cipitation with 75% ethanol because in preE LM7 82 liminary experiments no clear fractionation Note: ND, not determined. was obtained by the different alcohol concentrations. The yields of total water-soluble showed that only glucans were present. The polysaccharides are given in Table 3 and yields of water-insoluble polymer produced again noticeable variations in the amount of and the relative percentages of the 1 e 6 polysaccharides formed by strains were ob1 -e 3 linkages present (calculated from to served; BHT, Ingbritt LH, and Ingbritt B the results of methylation analyses) are gave high yields of material. The ratio of given in Table 2. There did not appear to glucose to fructose in these polysaccharides be any correlation between serological classi- was determined by colorimetry and by methfication and the ability to produce the same ylation analysis; the results are also given in amount of water-insoluble polymer, but Table 3. The feature of these results is the there is an apparent relation between sero- very high fructan values found for the Inggroups and the types of linkage. The ratio britt B and L materials. These fructans 6 to of a 3 linkages is approx- were examined after methylation and methimately 50:50 in the two b strains; approx- anolysis by gas liquid chromatography unimately 70 to 80% 1 -e 3 in the two Ing- der conditions that allowed separation and britt strains that did form this polysaccha- identification of all the methyl-O-methyl ride; and predominantly a 1 3 linked in a-/3-D-fructofuranosides. The principal prodthe two d strains. ucts were 3:4:6 tri-O-methyl and 1:3:4:6 The formation of water-soluble polysac- tetra-O-methyl fructose identified by their charides differentiated by their insolubility retention times and by comparison with the in 55 and 75% ethanol was followed as a products obtained, using the same condifunction of time on the incubation of cul- tions, from a levan derived from Bacillus sub tilis and inulin. ture supernatants with sucrose, and the reTABLE 2 are
most
a
a
->
a
->
a
->
WATER-INSOLUBLE POLYSACCHARIDES PRODUCED FROM SUCROSE BY THE GLYCOSYLTRANSFERASES OF STRAINS OF STREPTOCOCCUS MUTANS Yield of Polysaccharide Strain
Serotype
Z/AHT OMZ l BHT Ingbritt LH Ingbritt B Ingbritt L OMZ 176 B 13 LM 7
a
(mg/ml)* 2.8
Percentages of al-*6 to al-)3 Linkages
al->3 80 49 43
al-*6
20 51 57
b
0.11
b
2.9
Streptococcus mutans is an etiologic agent in experimental dental caries in animals.',2 Various strains of this organism have been isolated and classified into four types on the basis of their genetic, antigenic, and biochemical characteristics.3 The classification based on the chemical composition of the cell wall of S mutans strains gives three types.4 However, on the basis of serological tests, Bratthall5 was able to classify strains of this organism into five groups. The cell surface of S mutans is of course the bacterial cell wall. When the organism is grown in the presence of sucrose, the polysaccharides formed from the sucrose by the glycosyltransferase secreted by the organism may also be found at the cell surface. The bacterial component first "seen" by the immunologic defense systems of the invaded host is the cell surface of an organism. Hence, it would be expected that immunization with surface components, or the enzymes that formed them, would give protection against that organism. Much work has been done on the polysaccharide formed from sucrose by the glycosyltransferase of S mutans. Initially, it was shown that the water-soluble polysaccharide I 6 glucan was a mixture of a dextran and a levan.6 The water-insoluble polysaccharide formed by this reaction was found I -e 3 to be a glucan with predominantly linkages.7 The quantitative analysis of the cell wall components of several strains of S mutans and S sanguis has also been reported.8 More recently, the cell surface antigens that characterize the five serogroups of S mutans have been investigated. Van de Rijn and Bleiweis9 suggested that a membrane-associated glycerol teichoic acid with /3-galactose and probably glucose residues was the major antigen of the group a strain, AHT. Vaught and Bleiweisl' also reported the presence of a
-*
a
a cell wall glycerol teichoic acid in the b
strain, BHT, the specificity of the antigen residing in a P-galactose substituent of the teichoic acid. In contrast, Mukasa and Slade" characterized the group a antigen of HS6 as a cell wall polysaccharide containing glucose, galactose, glucosamine, and galactosamine. These workers also showed that in the b strain FAI the group antigen was a polysaccharide, the antigenicity of which resided in D-galactose and galactosamine residues.12 Iacono et al'3 showed that the antigen from S mutans 6715 was a carbohydrate material with a 3-galactoside as the immunodeterminant residue. The cell wall composition of bacteria varies considerably with changes in growth conditions.14 In view of this and the contradictory reports in the literature, it seemed desirable to study the cell surface components (cell wall and exocellular polysaccharides) of representative strains of the five serotypes of S mutans when they were all grown by the same standard method. This article reports the results of such a study.
Materials and Methods The following bacterial strains were used: Z/AHT, OMZ 51, OMZ 176, Ingbritt LH, B 13, LM 7, Ingbritt B, and Ingbritt L. GROWVTH MEDIUm.-The medium was the modified'5 7d medium of Cybulska and Pakula'6 supplemented with 4% glucose. In the initial stages of this investigation, the results from duplicate cultures often varied. The reason for this was traced to the use of different batches of medium, so a 60-liter batch of medium was prepared and samples taken from this were used for all subsequent growth experiments. GROWVTH OF CULTURES.-The various strains were grown under identical conditions in a Porton-type stirred fermenter at 37 C and a
C42 Downloaded from jdr.sagepub.com at SIMON FRASER LIBRARY on June 26, 2015 For personal use only. No other uses without permission.
Vol 55 1976
VARIATIONS IN SURFACE POLYMERS OF S MUTANS
pH of 6.5. The culture was kept anaerobic with 5% CO2 and 95% N2 which was sterilized by filtration, passed down the hollow stirrer shaft, and sparged by the impeller blades. The medium (1,200 ml) was brought to the 37 C temperature and a pH of 6.5 and was inoculated with the heavy deposit from a 100-ml, 18- to 24-hour seed culture grown in the same medium. Two normal NaOH was supplied automatically on demand to maintain the pH at 6.5, and the demand recorded continuously. Silicone antifoam was added when required. Samples were taken at hourly intervals, examined microscopically, and the optical density measured at 540 nm. At the end of the exponential growth phase, as shown by the leveling off of the OD curve and the reduction in the rate of alkali demand, the culture was harvested and centrifuged. The supernatant was stored under toluene for subsequent examination and the cell paste stored at -20 C. All strains were grown at least twice to check on the reproducibility of the system. GLYCOSYLTRANSFERASE ACTIVITY.-A sample at a pH of 6.5 (1 ml) was incubated with sucrose (0.1 ml of 50% w/v aqueous solution) at 37 C for two hours; sodium acetate solution (1.0 ml of 10% w/v aqueous solution) was then added followed by ethanol (6.0 ml), and the sample thoroughly mixed. The mixture was allowed to stand for 16 hours at 4 C before centrifugation (3,000 X g for 20 minutes). The precipitate was washed three times with a mixture of ethanol: 10% sodium acetate (4:1 v/v) by centrifugation. It was then suspended in water, and an aliquot (containing between 20 to 100 yg of glucan) made up to 1.0 ml with water and analyzed for total hexose by the phenol-sulfuric acid method.'7 The activity is expressed as micrograms of polymer formed per hour per milliliter of culture supernatant, under the aforementioned conditions. PREPARATION OF POLYMERS.-The culture supernatants (1,000 ml) were incubated at 37 C under toluene with sucrose solution (200 ml of 50% w/v). Samples (5 ml) were removed at intervals and plunged into boiling water for one minute before assaying for polysaccharides. These were differentiated by first removing the water-insoluble material, precipitating with 55% ethanol, and removing the precipitate by centrifugation. Finally, the ethanol concentration was adjusted to 75% and this precipitate col-
C43
lected by centrifugation. The precipitates were washed with alcohol and the polysaccharides present estimated by the phenolsulfuric acid method.'7 At the end of 24 hours, the pH was still 6.5 + 0.1, showing that no acid had been produced during the incubation. After 24 hours, the incubation mixture was centrifuged (5,000 X g for 30 minutes) to collect the water-insoluble polymer. The precipitate was washed three times with water by centrifugation, freezedried, and weighed. Ethanol was added to the supernatant to give 75% concentration and the mixture allowed to stand for 24 hours at 4 C. The precipitate was collected as just described and washed three times with 98% ethanol. It was then dissolved in water and dialyzed against changes of distilled water for three days, and finally the dialyzate was freeze-dried and weighed. The analytical methods, the methylation, methanolysis, acid hydrolysis, and subsequent analysis of the products by gas chromatography were done as described by Baird, Longyear, and Ellwood.15 Organisms were broken in a Braun M.S.K. homogenizer and the cell walls were isolated quantitatively, as described by Ell-
wood.18 The analyses for total carbohydrate and phosphorus were as described by Ellwood.18 The quantitative analyses of the cell wall sugars were done as they were for the alditol acetates19 or the acetylated aldononitriles20 by gas liquid chromatography. The proteins in the cell wall were solubilized by treatment with sodium dodecyl sulfate (SDS) and mercaptoethanol and then subjected to SDS polyacrylamide gel electrophoresis. Gels were stained with Coomassie blue and after removal of excess stain were scanned at 540 nm in a Chromoscan. The methods used were as described by Spencer and Guest.2'
Results and Discussion It seemed desirable to study the surface polymers of strains of S mutans that represented the five serological groups of this organism. Cultures of the various strains were grown in medium taken from a standard batch to obviate any variation from different media, all the other growth factors were also controlled, and reproducible results were obtained. The growth curves for the strains studied are shown in Figure 1 and although there is some spread in the type of curve, no significant differences can
Downloaded from jdr.sagepub.com at SIMON FRASER LIBRARY on June 26, 2015 For personal use only. No other uses without permission.
C44
ELLWOOD ET AL
J Dent Res Special Issue
C
OMZ 176
OPTICAL DENSITY
GROWTH (HOURS)
FIG 1.-Growth of five serotypes of S mutans. be seen. Duplicate experiments on each strain gave similar curves, and all three Ingbritt strains were identical. The most interesting feature was that for all strains during the latter part of the exponential growth phase, agglomerates of organisms appeared that were similar to those seen when Ingbritt B was grown in continuous cul-
ture.15 The glycosyltransferase activity, defined by the amount of total polysaccharide that was insoluble when the ethanol concentration was adjusted to 75%, was determined on the culture supernatant when the organisms were harvested. The results are given in Table I and indicate that variation in the glycosyltransferase activities occurs between strains of the same serotype.
The formation of the water-insoluble polymer by the culture supernatants on incubation with sucrose as a function of time is shown in Figure 2. The culture supernatants of Ingbritt LH and LM 7 did not form any polymer under these conditions, and that from OMZ 51 formed only small amounts. The two strains Z/AHT and OMZ 176 both gave supernatants that formed water-insoluble polymer at approximately a steady rate, but the glycosyltransferase activity in the culture supernatant from Ingbritt B showed a slow steady rate up to 600 minutes and then rapid formation of polymer took place. Methylation analysis of the water-insoluble polysaccharide isolated from the various strains at the end of the incubation + INGbRITT
6
3000r
Z/AIHT
0W4~ ~ ~ ~ 176~OZ 0/
X INGfIRITT LH Si
*O 0M
O LtA 7
INCUbATION TIME (Mims)
FIG 2.-Rate of formation of polysaccharide insoluble in water. Downloaded from jdr.sagepub.com at SIMON FRASER LIBRARY on June 26, 2015 For personal use only. No other uses without permission.
VARIATIONS IN SURFACE POLYMERS OF S MUTANS
Vol 55 1976
sults
TABLE 1 GLYCOSYLTRANSFERASE ACriVITY OF STRAINS OF STREPTOCOCCUS MUTANS
C4$
shown in Figures 3 and 4. The notable feature of Figure 3 is that a considerable change in the rate of polymer formation can be seen for several strains, Strain Serotype Units/ml OMZ 51 giving the most pronounced effect. The results for the 75%-ethanol-insoluble a 266 Z/AHT b 119 OMZ 51 polymer shown in Figure 4 are somewhat b BHT 465 complex and suggest that rapid conversion C Ingbritt LH 190 of this kind of polymer to the other types c Ingbritt B 367 was taking place. ND IngbrittL c The water-soluble polysaccharides were d OMZ 176 180 collected at the end of incubation after pred B13 380 cipitation with 75% ethanol because in preE LM7 82 liminary experiments no clear fractionation Note: ND, not determined. was obtained by the different alcohol concentrations. The yields of total water-soluble showed that only glucans were present. The polysaccharides are given in Table 3 and yields of water-insoluble polymer produced again noticeable variations in the amount of and the relative percentages of the 1 e 6 polysaccharides formed by strains were ob1 -e 3 linkages present (calculated from to served; BHT, Ingbritt LH, and Ingbritt B the results of methylation analyses) are gave high yields of material. The ratio of given in Table 2. There did not appear to glucose to fructose in these polysaccharides be any correlation between serological classi- was determined by colorimetry and by methfication and the ability to produce the same ylation analysis; the results are also given in amount of water-insoluble polymer, but Table 3. The feature of these results is the there is an apparent relation between sero- very high fructan values found for the Inggroups and the types of linkage. The ratio britt B and L materials. These fructans 6 to of a 3 linkages is approx- were examined after methylation and methimately 50:50 in the two b strains; approx- anolysis by gas liquid chromatography unimately 70 to 80% 1 -e 3 in the two Ing- der conditions that allowed separation and britt strains that did form this polysaccha- identification of all the methyl-O-methyl ride; and predominantly a 1 3 linked in a-/3-D-fructofuranosides. The principal prodthe two d strains. ucts were 3:4:6 tri-O-methyl and 1:3:4:6 The formation of water-soluble polysac- tetra-O-methyl fructose identified by their charides differentiated by their insolubility retention times and by comparison with the in 55 and 75% ethanol was followed as a products obtained, using the same condifunction of time on the incubation of cul- tions, from a levan derived from Bacillus sub tilis and inulin. ture supernatants with sucrose, and the reTABLE 2 are
most
a
a
->
a
->
a
->
WATER-INSOLUBLE POLYSACCHARIDES PRODUCED FROM SUCROSE BY THE GLYCOSYLTRANSFERASES OF STRAINS OF STREPTOCOCCUS MUTANS Yield of Polysaccharide Strain
Serotype
Z/AHT OMZ l BHT Ingbritt LH Ingbritt B Ingbritt L OMZ 176 B 13 LM 7
a
(mg/ml)* 2.8
Percentages of al-*6 to al-)3 Linkages
al->3 80 49 43
al-*6
20 51 57
b
0.11
b
2.9
