Caseinolytic and Glyoprotein Hydrolase Activity of Streptococcus mutans R. A. COWMAN, M. M. PERRELLA, and R. J. FITZGERALD Dental Research Unit, Veterans Administration Hospital, and Division of Oral Biology, Department of Family Medicine, University of Miami, Miami, Florida 33125, USA
After the sonic rupture of Streptococcus mutans cells, two fractions were obtained. The soluble cell contents hydrolyzed casein but not porcine gastric glycoprotein. The celldebris fraction showed proteolytic activity on both casein and gastric glycoprotein. Glycoprotein hydrolase activity also was demonstrated in the medium from which the cells were harvested.
It has recently been reported that incubation of Streptococcus mutans with human saliva produced substantial alterations in the electrophoretic patterns of the anionic salivary proteins.' Although S mutans is not classified among the proteolytic streptococci,2,3 it has been reported to possess proteolytic activity when grown on skim milk agar plates.4,5 Neuraminidase activity6,7 and relatively low levels of arylaminopeptidase activity8 also have been detected in S mutans. In the present study, we show that S mutans elaborates to the medium's extracellular glycoprotein hydrolase enzyme during growth, and that sonically rupttured cells possess hydrolytic enzyme activity for casein and glycoprotein in the insoluble cell-debris fraction, whereas the soluble cell contents possess only caseinolytic activity.
Materials and Methods S mutans strains AHT-12, BHT-1, FA-1, GS-5, VA-29, SL-1, KI-R, and substrains of KI-R (6715, 6715-19, 6715-49) derived from passage in hamsters and representing antigenic types a, b, c, and d as designated by Bratthall9'10 were selected for study. The This investigation was supported, in part, by Grant No. DEO-2552-07-08 from the National Institute of Dental Research, Bethesda, Md. Received for publication May 1, 1975. Accepted for publication November 25, 1975.
cultures were propagated in Trypticasea Glucose Broth, with incubation under 90% N210% CO, for 16 hours at 35 C. Stock cultures were maintained by weekly transfer in this medium with 0.5% calcium carbonate added. Between transfers, the cultures were stored at 5 C. PREPARATION OF CASEIN AND GLYCOPROTEIN SUBSTRATES.-A 10-gm amount of casein (Hammarsten) b was solubilized in 0.1 N NaOH by stirring overnight in the cold, whereas a 10-gm amount of crude porcine gastric glycoprotein, Type Ilc was solubilized in 0.01 N NaOH by stirring for 48 hours in the cold. The substrate solutions were neutralized to a pH of 6.6 or a pH of 7.4 by the slow addition of 0.1 N HCl and diluted with 0.2 M sodium phosphate buffer of the desired pH to give a final concentration of 1 % casein or glycoprotein, respectively. Toluene was added as a preservative and the substrate solutions were stored at 3 C until needed. PREPARATION OF CELL FRACTIONS.-Cells were grown in 200-ml amounts of the Trypticase medium using a 1% (v/v) inoculum, with incubation under 90% N2-10% CO2 for 16 hours at 35 C. After incubation, the cells were collected by centrifugation (10,000 X g) and in certain experiments the culture fluid was saved for the assay of proteolytic activity. The cell pellet was washed three times using 0.04 M sodium phosphate buffer (pH, 7.0), after which the cells were resuspended in the same buffer to a volume of 10 ml. The suspension was then chilled in an ice bath. The cell suspensions were sonically disrupted for ten minutes at 50 w using a a
Baltimore Biological Laboratory, Cockeysville, Md.
b
Nutritional Biochemicals, Inc., Cleveland, Ohio. Sigma Chemical Co., St. Louis, Mo.
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sonifierd During cellular disruption, the suspensions were kept in an ice bath that limited the temperature from rising higher than 7 C. The cell lysate was then centrifuged at 27,000 X g for ten minutes in the cold after which the supernatant fluid consisting of soluble intracellular components was decanted. This was designated the soluble fraction. The remaining sediment was resuspended in 5 ml of buffer and will be referred to as the cell-debris fraction. STANDARDIZED ENZYNIE ASSAYS.-Protein hydrolysis was followed by measuring the increase in absorbancy at 280 nm of trichloroacetic acid (TCA) filtrates after precipitation of the protein. The reaction mixture for casein hydrolysis contained I ml of substrate in 8 ml of 0.2 M phosphate buffer and 1 ml of enzyme solution (undiluted or diluted cell fractions containing 2 to 3 mg/ml as soluble fraction; 2 to 4 mg/ml as cell-debris fraction, or 1 ml of culture fluid) that were tempered separately at 37 + I C. At zero hour and subsequent intervals, samples were removed from incubation and mixed with an equal volume of 11% TCA while being shaken to stop the reaction. After ten minutes, the mixture was filtered througlh Whatman no. 4 filter paper, and the absorbance of the filtrates was measured at 280 nm. After subtraction of the zero-hour value, one unit of proteinase activity was defined as that amount of enzyme that caused a 0.01optical density increase at 280 nm in three hours. During this incubation period, the increase in absorbance as a result of enzyme action was linear. Difficulty in measuring the protein content of the cell-debris fractions was encountered with the Lowry protein procedure; therefore, aliquots of the cell fractions, in triplicate, were dried to constant weight, in vacuo, and the enzymatic activity was expressed as units of proteinase per milligram of residue weight. Appropriate controls were included in all experiments. Glycoprotein hydrolase activity was measured in a similar manner, except portions of the reaction mixture were mixed with 24% TCA and centrifuged at 27,000 x g for 20 minutes before filtering. ELECTROPHORETIC ASSAY STUDIES.-In these
studies, a-caseine and bovine submaxillary d Branson Sonifier, model W-140, Heat Systems-Ultrasonics, Inc., Plainview, Long Island, NY. eNutritional Biochemicals, Inc., Cleveland, Ohio.
j Dent Res May-June 1976
glycoprotein, Type L,f served as substrates for the electrophoretic analysis of the proteinase activity of the cell fractions of S mutans strain KI-R. A 10-mg amount of
a-casein or bovine submaxillary glycoprotein was solubilized in 10 ml of 0.2 M sodium phosphate buffer (pH, 6.6). Then, 1.0 ml of enzyme solution (soluble or cell-debris fraction) was mixed with 4-ml amounts of a-casein or bovine submaxillary glycoprotein and incubated at 37 + 1 C for 0, 3, and 24 hours. At each time interval, 300-1l portions of assay mixtures were removed and centrifuged (21,000 x g). Controls consisting of substrate, buffer, and water were incubated and treated simultaneously with the experimental preparations. After centrifugation, the density of each sample supernatant was increased by adding solid sucrose, and 100-/A portions were layered on the surface of polyacrylamide disc gel columns containing both a separation and stacking gel." Electrophoretic analysis of the a-casein digests was performed in 7.0%-1 acrylamideg gel columns at a pH of 8.3. The continuous electrode buffer was Tris-glycine, with a pH of 8.3 (2.88 gm glycine-0.6 gm Tris per liter). After the samples had been layered, 2 ml of 0.1 %, bromophenol blue dye was added to the upper electrode chamber. Electrophoresis was begun and maintained at 1 ma/ column until the tracking dye had migrated through the stacking gel. The amperage was then increased and maintained at 4.0 ma/ column until the dye had migrated a distance of 10 cm toward the anodal end of the gel column. The bovine submaxillary glycoprotein digests were similarly analyzed, excepting that the separations were performed using 5.2% acrylamide columns containing 1 M urea (recrystallized from warm ethanol). Duplicate columns were run for separate staining of protein and carbohydrate. After electrophoretic separation, the gels were stained for 30 minutes with 0.25% Coomassie Brilliant Blue in 12% TCA. The carbohydrate in bovine submaxillary glycoprotein was visualized using the procedure described by Holden et al.'2 After destaining, the gels were scanned at 560 nm using a spectrophotometer equipped with a gel f Sigma Chemical Co., St. Louis, Mo. g Cyanogum, E. C. Apparatus Corp., St. Petersburg, Fla.
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0
0.5
PROTEINASES OF ORAL STREPTOCOCCI
1.0
1.5 2.0 215 3.0 3.5 4:0
INCUBATION TIME 1N HOURS
FIG 1.-Hydrolysis of casein by soluble and cell-debris fractions of S mutans strain Kl-R as function of incubation time at 37 C and at pH of 6.6. Symbols: solid circles, cell-debris fraction; open circles, soluble fraction; connected solid anzd open circles, boiled soluble and celldebris fractions.
scanning attachment and linear recorder.h The distance that any protein or carbohydrate-containing bands migrated from the origin was measured in centimeters. Results
393
zymatic activity of the cell-debris fraction compared to that of the soluble fraction in these tests was attributable to differences in dilution of the original preparations. To examine the quantitative aspects of the hydrolytic activity of the soluble and celldebris fractions from S mutans strains K1-R, 6715, 6715-19, and 6715-49, enzyme assays were performed at both a pH of 6.6 and a pH of 7.4. However, since the results were comparable, only data for a pH of 7.4 will be presented. On the basis of relative specific activity (units of proteinase per milligram of fraction residue weight), the soluble fraction of each of the four strains tested appeared to be more active toward casein than the respective cell-debris fraction (Fig 2). For example, S mutans strain Kl-R soluble fraction had a relative specific activity of 115 as compared to 60 for the cell-debris fraction. The activity of each fraction of each culture responded linearly with respect to the increasing enzyme concentration, suggesting that the fractions contained neither an activator nor inhibitor for enzyme activity. However, it was noted that the curves for S mutans strain KI-R cell-debris fraction and the soluble fraction from S mutans strain 6715
CASEIN HYDROLYSIS.-Preliminary studies with S mutans strains KI-R and 6715 showed
that whole cells of these organisms possessed hydrolytic activity toward casein and hemoglobin at a pH of 7.0. Also, the soluble and cell-debris fractions, obtained after sonic rupture of the cultures, possessed enzymatic activity toward casein, with the soluble fractions showing greater activity at a pH of 6.6, but the cell-debris fractions were more active at a pH of 7.4. Linearity of the hydrolysis of casein by cellular fractions of S mutans strain KI-R was measured as a function of time at a pH of 6.6. Boiled enzyme fractions possessed no hydrolytic activity toward casein during the four-hour test period (Fig 1). Slight curvilinearity in the activity of the soluble fraction was observed in the first 1.5 hours of incubation; thereafter, hydrolysis of casein by this fraction was linear with time. The hydrolysis of casein by the cell-debris fraction indicated that the enzymatic activity was essentially linear throughout the experimental incubation period. The apparent greater enh
Gilford Spectrophotometer, model 240, Gilford In-
struments, Inc., Oberlin, Ohio.
A. STRAIN KI-R
B. STRAIN 6715 7.0
5.0
CO*3.0
5.0 -
1.0 0 Z _7.0
-
2 4 6 MG RESIDUE WEIGHT C. STRAIN 6715-19
1.0 o
2 4 6 MG RESIDUE WEIGHT 0. STRAIN 6715-49
7.0
-
5.0/. /
C.25.0
-
3.0 3.0
3.0 3.0
1.0
1.0
-
.30
0 2 4 6 2 4 6 21 o MG RESIDUE WEIGHT MG RESIDUE WEIGHT FIG 2.-Hydrolysis of casein at pH of 7.4 by S muttans strains KI-R, 6715, 6715-19, and 6715-49 soluble and cell-debris fractions as function of cnzyme concentration. Open circles, soluble fractions; solid circles, cell-debris fractions.
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COWMAN, PERRELLA, AND FITZGERALD
TABLE 1 HYDROLYSIS OF CASEIN BY CELLULAR FRACTIONS OF S MUTANS AT A PH OF 6.6 AND OF 7.4 Units Proteinase Per mg Residue wt S mutans Strain
Antigenic Type
AHT-12 BHT-1 FA-1 VA-29 GS-5 KI-R 6715 6715-19 6715-49 SL-1
a
Cell-Debris Fraction
Soluble Fraction
b b C c
d d d d d
pH, 6.6
pH, 7.4
pH, 6.6
pH, 7.4
81 ±990+15 120 ± 12 59± 3 58±7 74 + 17
63+10
112+22 74+23 112±4-10
97 ± 12
90±7 70 ± 15 51 +±16 44±13 65 + 20 137±15 92 21 154 12 95 ± 1.0
53±12 126± 10 97 ± 20 106±20
83 24 76+9 76 17 84±25 33 10 42 7 60 ±3
87±6 78 ±+14 158 + 5.0 122+13 104 ± 18 68+ 13 64 12 65 17 39 +7.0
* Each value represents mean ± standard error of the mean for data from 15 replicated experiments.
did not pass through the origin when extrapolated to zero time. These observations suggest that the initial attack on the casein substrate by the S mutans strain Kl-R and 6715 fractions is different from the subsequent hydrolysis.13,14 The relative specific proteinase activity of additional strains of S mutans, representing different antigenic types, was then compared using casein as the substrate. All of the strains of S mutans tested, regardless of antigenic type, possessed proteinase activity in both the soluble and cell-debris fractions. The soluble fraction of S mutans strain AHT-12 (type a) had a lower specific activity than the cell-debris fraction at both a pH of 6.6 and of 7.4 (Table 1) The specific .
activity of the soluble fractions of S mutans type b strains, BHT-1 and FA-1, was not greatly different from that of the cell-debris fraction at either a pH of 6.6 or of 7.4. The soluble fractions of type c S mutans strains VA-29 and GS-5 had a much lower specific activity than the respective cell-debris fractions, particularly at a pH of 7.4, where the cell-debris fractions had a specific activity three times that of the soluble fractions. Among the cultures representing type d S mutans, there was some variability, but a majority of the strains had a higher soluble fraction activity at both a pH of 6.6 and of 7.4 relative to that observed for the respective cell-debris fractions. Since some enterococci are known to re-
TABLE 2 HYDROLYSIS OF CRUDE PORCINE GASTRIC GLYCOPROTEIN BY CELLULAR FRACTIONS OF S MUTANS Units of Hydrolase per mg Fraction S mutans
Antigenic
Strain
Type
AHT-12
a
BHT-1
b b C
FA-1 VA-29 GS-5 K1-R 6715 6715-19 6715-49 SL-1
c
d d d d d
* Each value represents
Soluble Fraction pH, 6.6
3 # 3*
Cell-Debris Fraction pH, 7.4
pH, 7.4
pH, 6.6
4±3
154±+10
0+0
39 ±+21 13+13
3 2 0+0
3
43 ±+16 27+5 100 ± 24
3±5
31±16
I11±+7 0
0
0± 0 0± 0
5±2 0±0
mean +
6
12 13 0±0
0±0 0±0 16±10
81 + 18
30+13 47±+10 29+3 39+5
160±14 64 +±10 70+18 144 ± 17 74+17 96 ±+19
62+20
24±5 0±0 86±18
standard error of the mean of eight replicate
ex-
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lease an extracellular proteinase to the medium, an effort was made to ascertain whether any of the S mutans strains produced an extracellular enzyme capable of hydrolyzing casein. Proteinase activity in the culture fluid was assayed using casein as a substrate, but none of the cultures tested in Table 1 was found to possess an extracellular proteinase activity. GLYCOPROTEIN HYDROLYSIS.-In the initial phases of this study, crude porcine gastric glycoprotein served as the substrate, and the potential enzymatic activity was assayed at a pH of 6.6 and a pH of 7.4. With S mutans strain AHT-12, extremely low activity was present in the soluble fraction regardless of the pH of the assay. However, a very high
.480 A. .420
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.300 0 1 2 3 4 5 6 7 8 9 CENTIMETERS MIGRATION TOWARD ANODE
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1
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.180
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CENTIMETERS MIGRATION TOWARD ANODE FIG 3.-Tracing of densitometric scans of acasein incubated with soluble fraction of S mutans strain K1-R at 37 C. A, solid line, zerohour profile of casein-fraction mixture; dashed line, three-hour incubation of casein-fraction mixture at pH of 6.6 and 37 C. B, 24-hour incubation of casein-fraction mixture at pH of 6.6 and 37 C. Electrophoretic separations were performed at constant current of 4 ma/tube in 7.0% acrylamide columns at pH of 8.3.
FIG 4.-Tracing of densitometric scans of axcasein incubated with cell-debris fraction of S mutans strain KI-R at 37 C. A, solid line, zerohour profile of casein-fraction mixture; dashed line, three-hour incubation of casein-fraction mixture at pH of 6.6 and 37 C. B, 24-hour incubation of casein-fraction mixture at pH of 6.6 and 37 C. Electrophoretic separations were performed at constant current of 4 ma/tube in 7.0% acrylamide columns at pH of 8.3.
relative specific activity toward the glycoprotein by the cell-debris fraction of this culture was observed (Table 2). Similar, low-tononexistent soluble fraction relative specific activities of other strains representing antigenic types b, c, and d were found. Generally, higher specific activities were found in the cell-debris fraction of most strains particularly when assayed at a pH of 7.4. Exceptions were noted in S mutans strains 6715-19 and 6715-49 which showed lower activities at a pH of 7.4 than at a pH of 6.6. The spent growth medium from all cultures showed high glycoprotein hydrolase activity at both a pH of 6.6 or a pH of 7.4, with the exception of S mutans strain BHT-1 which possessed no extracellular activity.
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COWMAN, PERRELLA, AND FITZGERALD
ELECTROPHORETIC STUDIES WITH CELL FRACTIONS OF S MUTANS STRAIN Kl-R.-Cell fractions and a-casein were incubated in 0.1 M sodium phosphate buffer at a pH of 6.6 for 0, 3, and 24 hours. At each interval, aliquots were removed, centrifuged to remove particulate matter, and analyzed by disc gel electrophoresis at a pH of 8.3. Electrophoresis of soluble fraction plus casein mixture at zero hour showed the presence of two distinct protein zones, one at 5.5 cm and a second at 8.9 cm from the origin (Fig 3, A). Both zones were attributable to the a-casein substrate since electrophoresis of the protein alone gave identical zones. After three hours of incubation, the zone originally present at 5.5 cm was found to migrate to a distance of 5.9 cm. Although the zone originally present at 8.9 cm did not shift in position, an increase in stainability was observed when compared with the zero-hour gel that was stained simultaneously. After incubation for 24 hours, the zones originally present at 5.5 or 5.9 and 8.9 cm had largely disappeared (Fig 3, B). Concomittant with the disappearance of these zones, new and different major protein-staining zones were found to occur at 0.5 and 7.5 cm. In addition, less intense zones were observable at 1.1, 1.7, 4.9, 6.7, 8.5, and 9.0 cm. Assay of the cell-debris fraction of S mutans strain KI-R with a-casein under similar conditions showed only minor changes in the electrophoretic profile of a-casein after three hours of incubation (Fig 4, A). However, after 24 hours of incubation, a major protein-staining band migrating 8 cm from the origin was observed. Several additional zones, particularly in the region of I to 4 cm were also detected (Fig 4, B). Electrophoretic studies were performed to ascertain whether hydrolysis of bovine submaxillary glycoprotein by the cell fractions was limited to the carbohydrate moiety or included hydrolysis of the protein core as well. Electrophoresis of the submaxillary glycoprotein at zero hour, followed by staining of duplicate gel columns for protein and for carbohydrate showed that two distinct bands were present within the major protein zone (Fig 5, A). No carbohydrate was associated with the protein zone detected at 6.0 cm. After incubation of submaxillary glycoprotein with S mutans strain KI-R soluble fraction for three hours, no major changes in the protein or carbohydrate moieties were
J Dent Res May-June 1976
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vine submaxillary glycoprotein incubated with soluble fraction of S mutans in 0.1 M sodium phosphate buffer (pH, 6.6) at 37 C. A, zerohour profile; B, three-hour incubation of glycoprotein-fraction mixture at 37QC C, 24-hour incubation of glycoprotein-fraction mixture at 37 C. Electrophoretic separations were performed at 4 ma/tube in 5.2%,, acrylamide-1.0 M urea columns at pH of 8.3. Protein was stained using Coomassie Brilliant Blue and carbohydrate by method of Holden et al.1
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PRO TEINASES OF ORAL STREPTOCOCCI
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detected (Fig 5, B). However, a carbohydrate-staining zone associated with weak protein staining was detected at approximately 8.0 cm from the origin. This zone was not present in the zero-hour profile. After 24 hours of incubation, the major protein occurring at 1.5 cm in both the zero- and threehour profile migrated more slowly, moving only about 1.1 cm from the origin in the .0904-1 C..090 24-hour profile (Fig 5, C). However, this ~ ~ ~ ~ ~ ~ ~ ~ zone did not contain significant carbohydrate, suggesting that the soluble fraction of .060 .060 S mutans strain Kl-R split off only the carbohydrate moiety. The zone occurring at 6.0 was unaffected. When bovine submaxilcm C .030t to .030 to U~~~~~~~~ lary glycoprotein was incubated with the cell~~~~~E debris fraction of S mutans strain KI-R, the E 1 2 3 4 5 6 7 8 9 zero-hour electrophoretogram showed the i~~~~~~~~~n presence of a major zone between 1.0 to 1.5 cm and a second minor zone at 6.3 cm (Fig 6, A). Carbohydrate was associated only C z.120w with the major zone of protein staining. HrJi Hydrolysis o 32ri 1 oS After three hours of incubation, new carbo.20. .090 hydrate-containing protein zones were de.1 6 tected at 3.9 and 8.3 cm (Fig 6, B) . Howr 12 ever, after 24 hours of incubation, these zones ..060 were not detectable, but increased amounts of protein-staining material were detected 04 .030Ocn in the region between 7 and 9 cm (Fig 6, C) In addition, the major carbohydrate-protein U)FC .0 6.Taigotesioercsasofo j zone at 3.0 cm had decreased considerably 7 2 3 4 5 9 c1 and represented a loss of approximately 75% 090u C. of the protein and 67% of the carbohydrate zd ~24 H r. Hydrolysis formerly present in the region. .020 0 '- .060 c 0 Discussion CL ~~~~~~~~cc The data presented in this study have in.010 0 .030 dicated that strains representing four different serotypes of S mutans can hydrolyze a tie It f83 rti p ura olun casein and porcine gastric glycoprotein, two structurally different substrates. Based on CM MIGRATION TOWARD ANODE the electrophoretic analyses of the casein of boFIG 6.-Tracing of densitometric digests after incubation with the soluble convine submaxillary glycoprotein incubated with tents or insoluble cell-debris fractions from strain Kl-R in cell-debris fraction of S S mutans, as well as plots of relative specific 37 0.1 sodium phosphate buffer (pH, 6.6) activities, it is suggested that two different C. A, zero-hour profile; B, three-hour incubaproteolytic enzymes may be responsible for tion of glycoprotein-fraction mixture 37 C; C, the activity against casein. However, the pos24-hour incubation of glycoprotein-fraction mnixsibility of a single enzyme possessing different 37 C. Electrophoretic separations of dukinetic properties in the soluble and bound plicate columns performed of 4.0 ma/tube in 5.2% acrylamide-0.1 M states cannot be excluded. stained columns pH of 8.3. Protein Proteolytic activity against porcine gastric using Coomassie Brilliant Blue and carbohyglycoprotein and bovine submaxillary glycodrate by method of Holden al.' protein was demonstrated in the cell-debris fraction but not in the soluble cell contents of S mutans. However, the soluble cell frac4-0
-
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scans
mutans
at
at
ture
at
at
were
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cur-
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398
COWMAN, PERRELLA, AND FITZGERALD
tion did hydrolyze the carbohydrate moiety of the submaxillary glycoprotein substrate, a finding that is consistent with the previous report of carbohydrate hydrolase activity in these organisms.15 A number of streptococci are known to elaborate extracellular proteinases.14'16-18 In the present study, we have shown that an extracellular enzyme elaborated by S mutans can attack porcine gastric glycoprotein but not casein. This suggests that the extracellular glycoprotein hydrolase is different from the enzyme (s) found in the soluble cell contents or associated with the cellular debris after the sonic rupture of S mutans. The carbohydrate metabolism of S mutans hias been well studied,19-24 but knowledge of the nitrogen metabolism of these organisms had been confined mainly to their amino acid requirements.25-27 Although S mu tans can survive and grow in the absence of added carbohydrates,28 it cannot do so without a source of essential amino acids. Our findings indicate that these organisms have the capacity to attack both dietary proteins, such as casein, and salivary proteins of human' and animal origin. It is possible that required amino acids might become available to S mutans through the metabolic activities of other plaque microorganisms, in vivo. However, the ability of S mutans to colonize and cause caries in the teeth of gnotobiotic animals in the absence of any other microorganisms lends further support to the concept that S mutans possesses the necessary complement of proteolytic enzymes whereby the organism can obtain needed amino acids for growth from dietary or salivary proteins. We are continuing to investigate the implications of these findings in relation to the oral ecology of S mutans and its role in the origin of dental caries.
Conclusions Proteolytic hydrolysis of casein and porcine or bovine glycoproteins by S mutans was demonstrated in the present study. Caseinolytic activity was found in both the soluble contents of the cells and the cellular debris after rupture of the cells. However, caseinolytic activity could not be demonstrated after growth in the culture fluid from any of the strains of S mutans tested. The soluble fractions of S mutans did not possess glycoprotein hydrolase activity toward porcine or bovine glycoprotein, but
j Dent Res May-June 1976
glycoprotein hydrolase activity was present in both the cell-debris preparations from cells and in the culture fluid after growth of the cultures. Based on these observations, S mutans may possess several different types of proteinase enyzmes with differing specificities for protein substrates. References 1. COWMAN, R.A., and FITZGERALD, R.J.: Effects of Oral Streptococci on Electrophoretic Properties of Human Salivary Anionic Proteins, J Dent Res 54: 298-303, 1975. 2. FITZGERALD, R.J., and KEYES, P.H.: Demonstration of the Etiologic Role of Streptococci in Experimental Caries in the Hamster, JADA 61: 23/9-33/19, 1960. 3. FITZGERALD, R.J.; JORDAN, H.V.; and STANLEY, H.P.: Experimental Caries and Gin-
4.
5.
6.
7.
8.
gival Pathologic Changes in the Gnotobiotic Rat, 1 Dent Res 39: 923-935, 1960. KRISCHER, K.N., and ZINNER, D.D.: Proteolytic Activity of Cariogenic Streptococci, abstracted, IADR Program and Abstracts of Papers, No. 61, 1969. KRISCHER, K.N., and ZINNER, D.D.: Proteolytic Activity of Oral Streptococci, abstracted, IADR Program and Abstracts of Papers, No. 18, 1970. LEACH, S.A:. Plaque Chemistry and Caries, Ala J Med Sci 5: 247-255, 1968. LEACH, S.A., and MELVILLE, T.: Investigation of Some Human Oral Organisms Capable of Releasing the Carbohydrates from Salivary Glycoproteins, Arch Oral Biol 15: 87-88,1970. OYA, H.; NAGATSu, T.; KOBAYASHI, Y.; and TAKEI, M.: Arylaminopeptidase Activities in Human Cariogenic and Non-Cariogenic Oral Bacteria, Arch Oral Biol 16: 675-680,
1971. 9. BRATrHALL, D.: Demonstration of Five Serological Groups of Streptococcal Strains Resembling Streptococcus mutans, Odontol Rev
21: 143-152, 1970. 10. BRATTHALL, D.: Immunofluorescent Identification of Streptococcus mutans, Odontol Rev 23 (suppl 23): 11-16, 1972. 11. COOKSEY, K.E.: Disc Electrophoresis, in NORRIS, J.R., and RIBBONS, D.W. (eds): Methods in Microbiology, Vol 5B, New York: Academic Press Inc., 1971, pp 573-595. 12. HOLDEN, K.G.; YIM, N.C.F.; GRICGS, LJ.; and WEISBACH, J.A.: Gel Electrophoresis of Mucous Glycoproteins: I. Effect of Gel Porosity, Biochemistry 10: 3105-3109, 1971. 13. BENSUSAN, H.B.; DEROW, M.V.; and WALKER, B.S.: The Proteolytic Enzymes of Proteus vulgaris, Arch Biochem Biophys 49: 293-302,
1954.
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PROTEINASES OF ORAL STREPTOCOCCI
14. GRUTTER, F.H., and ZIMMERMAN, L.N.: A Proteolytic Enzyme of Streptococcus zymogenes, J Bacteriol 69: 728-732, 1955. 15. PINTER, J.K.; HAYASHI, J.A.; and BAHN, A.N.: Carbohydrate Hydrolases of Oral Streptococci, Arch Oral Biol 14: 735-744, 1969. 16. BLEIWEIS, A.S., and ZIMMERMAN, L.N.: Properties of Proteinase from Streptococcus faecalis var. liquefaciens, J Bacteriol 88: 653-
659, 1964. 17. Liu, T.Y., and ELLIOTT, S.D.: Activation of Streptococcal Proteinase and Its Zymogen by Bacterial Cell Walls, Nature 206: 33-34, 1965. 18. WILLIAMSON, W.T.; TOVE, S.B.; and SPECK, M.L.: Extracellular Proteinase of Streptococcus lactis, J Bacteriol 87: 49-53, 1964. 19. GIBBONs, R.J., and BANGHART, S.B.: Synthesis of Extracellular Dextran by Cariogenic Bacteria and Its Presence in Human Dental Plaque, Arch Oral Biol 12: 11-24, 1967. 20. ROBRISH, S.A., and KRICHEVSKY, M.I.: Acid Production from Glucose and Sucrose by Growing Cultures of Caries-Conducive Streptococci, J Dent Res 51: 734-739, 1972. 21. SCHACHTELE, C.F.; LOKEN, A.E.; and KNUDSON, D.J.: Preferential Utilization of the Glucosyl Moiety of Sucrose by a Cariogenic Sttain of Streptococcus mutans, Infect Immun 5: 531-536, 1972.
399
22. TANZER, J.M.; BROWN, A.T.; and MCINERNEY, M.F.: Identification, Preliminary Characterization and Evidence for Regulation of Invertase in Streptococcus mutans, J Bacteriol 116: 192-202, 1973. 23. TANZER, J.M.: Studies on the Fate of the Glucosyl Moiety of Sucrose Metabolized by Streptococcus mutans, J Dent Res 51: 415523, 1972. 24. TANZER, J.M.; CHASSY, B.M.; and KRICHEVSKY, M.I.: Sucrose Metabolism by Strepto-
coccUs mutans, SL-1, Biochim Biophys Acta 261: 379-387, 1972. 25. COWMAN, R.A.; PERRELLA, M.M.; and FITZGERALD, R.J.: Influence of Incubation Atmosphere on Growth and Amino Acid Requirements of Streptococcus muttans, Appl MAicrobiol 27: 86-92, 1974. 26. CARLSSON, J.: Nutritional Requirements of Streptococcus mrutans, Caries Res 4: 305-320, 1970. 27. INWARD, P.W.; UPSTONE, D.; and VAN HOUTE, J.: Nutritional Requirements of Oral Streptococci, in McHuGH, W.D. (ed): Dental Plaque, Dundee, Eng: Livingston, 1970, pp
217-224. 28. KNUTTILA, M.L.E., and MAKINEN, K.K.: Effect of Xylitol on the Growth and Metabolism of Streptococcus mutans, Caries Res 9: 177-189, 1975.
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After the sonic rupture of Streptococcus mutans cells, two fractions were obtained. The soluble cell contents hydrolyzed casein but not porcine gastric glycoprotein. The celldebris fraction showed proteolytic activity on both casein and gastric glycoprotein. Glycoprotein hydrolase activity also was demonstrated in the medium from which the cells were harvested.
It has recently been reported that incubation of Streptococcus mutans with human saliva produced substantial alterations in the electrophoretic patterns of the anionic salivary proteins.' Although S mutans is not classified among the proteolytic streptococci,2,3 it has been reported to possess proteolytic activity when grown on skim milk agar plates.4,5 Neuraminidase activity6,7 and relatively low levels of arylaminopeptidase activity8 also have been detected in S mutans. In the present study, we show that S mutans elaborates to the medium's extracellular glycoprotein hydrolase enzyme during growth, and that sonically rupttured cells possess hydrolytic enzyme activity for casein and glycoprotein in the insoluble cell-debris fraction, whereas the soluble cell contents possess only caseinolytic activity.
Materials and Methods S mutans strains AHT-12, BHT-1, FA-1, GS-5, VA-29, SL-1, KI-R, and substrains of KI-R (6715, 6715-19, 6715-49) derived from passage in hamsters and representing antigenic types a, b, c, and d as designated by Bratthall9'10 were selected for study. The This investigation was supported, in part, by Grant No. DEO-2552-07-08 from the National Institute of Dental Research, Bethesda, Md. Received for publication May 1, 1975. Accepted for publication November 25, 1975.
cultures were propagated in Trypticasea Glucose Broth, with incubation under 90% N210% CO, for 16 hours at 35 C. Stock cultures were maintained by weekly transfer in this medium with 0.5% calcium carbonate added. Between transfers, the cultures were stored at 5 C. PREPARATION OF CASEIN AND GLYCOPROTEIN SUBSTRATES.-A 10-gm amount of casein (Hammarsten) b was solubilized in 0.1 N NaOH by stirring overnight in the cold, whereas a 10-gm amount of crude porcine gastric glycoprotein, Type Ilc was solubilized in 0.01 N NaOH by stirring for 48 hours in the cold. The substrate solutions were neutralized to a pH of 6.6 or a pH of 7.4 by the slow addition of 0.1 N HCl and diluted with 0.2 M sodium phosphate buffer of the desired pH to give a final concentration of 1 % casein or glycoprotein, respectively. Toluene was added as a preservative and the substrate solutions were stored at 3 C until needed. PREPARATION OF CELL FRACTIONS.-Cells were grown in 200-ml amounts of the Trypticase medium using a 1% (v/v) inoculum, with incubation under 90% N2-10% CO2 for 16 hours at 35 C. After incubation, the cells were collected by centrifugation (10,000 X g) and in certain experiments the culture fluid was saved for the assay of proteolytic activity. The cell pellet was washed three times using 0.04 M sodium phosphate buffer (pH, 7.0), after which the cells were resuspended in the same buffer to a volume of 10 ml. The suspension was then chilled in an ice bath. The cell suspensions were sonically disrupted for ten minutes at 50 w using a a
Baltimore Biological Laboratory, Cockeysville, Md.
b
Nutritional Biochemicals, Inc., Cleveland, Ohio. Sigma Chemical Co., St. Louis, Mo.
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392
COWMAN, PERRELLA, AND FITZGERALD
sonifierd During cellular disruption, the suspensions were kept in an ice bath that limited the temperature from rising higher than 7 C. The cell lysate was then centrifuged at 27,000 X g for ten minutes in the cold after which the supernatant fluid consisting of soluble intracellular components was decanted. This was designated the soluble fraction. The remaining sediment was resuspended in 5 ml of buffer and will be referred to as the cell-debris fraction. STANDARDIZED ENZYNIE ASSAYS.-Protein hydrolysis was followed by measuring the increase in absorbancy at 280 nm of trichloroacetic acid (TCA) filtrates after precipitation of the protein. The reaction mixture for casein hydrolysis contained I ml of substrate in 8 ml of 0.2 M phosphate buffer and 1 ml of enzyme solution (undiluted or diluted cell fractions containing 2 to 3 mg/ml as soluble fraction; 2 to 4 mg/ml as cell-debris fraction, or 1 ml of culture fluid) that were tempered separately at 37 + I C. At zero hour and subsequent intervals, samples were removed from incubation and mixed with an equal volume of 11% TCA while being shaken to stop the reaction. After ten minutes, the mixture was filtered througlh Whatman no. 4 filter paper, and the absorbance of the filtrates was measured at 280 nm. After subtraction of the zero-hour value, one unit of proteinase activity was defined as that amount of enzyme that caused a 0.01optical density increase at 280 nm in three hours. During this incubation period, the increase in absorbance as a result of enzyme action was linear. Difficulty in measuring the protein content of the cell-debris fractions was encountered with the Lowry protein procedure; therefore, aliquots of the cell fractions, in triplicate, were dried to constant weight, in vacuo, and the enzymatic activity was expressed as units of proteinase per milligram of residue weight. Appropriate controls were included in all experiments. Glycoprotein hydrolase activity was measured in a similar manner, except portions of the reaction mixture were mixed with 24% TCA and centrifuged at 27,000 x g for 20 minutes before filtering. ELECTROPHORETIC ASSAY STUDIES.-In these
studies, a-caseine and bovine submaxillary d Branson Sonifier, model W-140, Heat Systems-Ultrasonics, Inc., Plainview, Long Island, NY. eNutritional Biochemicals, Inc., Cleveland, Ohio.
j Dent Res May-June 1976
glycoprotein, Type L,f served as substrates for the electrophoretic analysis of the proteinase activity of the cell fractions of S mutans strain KI-R. A 10-mg amount of
a-casein or bovine submaxillary glycoprotein was solubilized in 10 ml of 0.2 M sodium phosphate buffer (pH, 6.6). Then, 1.0 ml of enzyme solution (soluble or cell-debris fraction) was mixed with 4-ml amounts of a-casein or bovine submaxillary glycoprotein and incubated at 37 + 1 C for 0, 3, and 24 hours. At each time interval, 300-1l portions of assay mixtures were removed and centrifuged (21,000 x g). Controls consisting of substrate, buffer, and water were incubated and treated simultaneously with the experimental preparations. After centrifugation, the density of each sample supernatant was increased by adding solid sucrose, and 100-/A portions were layered on the surface of polyacrylamide disc gel columns containing both a separation and stacking gel." Electrophoretic analysis of the a-casein digests was performed in 7.0%-1 acrylamideg gel columns at a pH of 8.3. The continuous electrode buffer was Tris-glycine, with a pH of 8.3 (2.88 gm glycine-0.6 gm Tris per liter). After the samples had been layered, 2 ml of 0.1 %, bromophenol blue dye was added to the upper electrode chamber. Electrophoresis was begun and maintained at 1 ma/ column until the tracking dye had migrated through the stacking gel. The amperage was then increased and maintained at 4.0 ma/ column until the dye had migrated a distance of 10 cm toward the anodal end of the gel column. The bovine submaxillary glycoprotein digests were similarly analyzed, excepting that the separations were performed using 5.2% acrylamide columns containing 1 M urea (recrystallized from warm ethanol). Duplicate columns were run for separate staining of protein and carbohydrate. After electrophoretic separation, the gels were stained for 30 minutes with 0.25% Coomassie Brilliant Blue in 12% TCA. The carbohydrate in bovine submaxillary glycoprotein was visualized using the procedure described by Holden et al.'2 After destaining, the gels were scanned at 560 nm using a spectrophotometer equipped with a gel f Sigma Chemical Co., St. Louis, Mo. g Cyanogum, E. C. Apparatus Corp., St. Petersburg, Fla.
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0
0.5
PROTEINASES OF ORAL STREPTOCOCCI
1.0
1.5 2.0 215 3.0 3.5 4:0
INCUBATION TIME 1N HOURS
FIG 1.-Hydrolysis of casein by soluble and cell-debris fractions of S mutans strain Kl-R as function of incubation time at 37 C and at pH of 6.6. Symbols: solid circles, cell-debris fraction; open circles, soluble fraction; connected solid anzd open circles, boiled soluble and celldebris fractions.
scanning attachment and linear recorder.h The distance that any protein or carbohydrate-containing bands migrated from the origin was measured in centimeters. Results
393
zymatic activity of the cell-debris fraction compared to that of the soluble fraction in these tests was attributable to differences in dilution of the original preparations. To examine the quantitative aspects of the hydrolytic activity of the soluble and celldebris fractions from S mutans strains K1-R, 6715, 6715-19, and 6715-49, enzyme assays were performed at both a pH of 6.6 and a pH of 7.4. However, since the results were comparable, only data for a pH of 7.4 will be presented. On the basis of relative specific activity (units of proteinase per milligram of fraction residue weight), the soluble fraction of each of the four strains tested appeared to be more active toward casein than the respective cell-debris fraction (Fig 2). For example, S mutans strain Kl-R soluble fraction had a relative specific activity of 115 as compared to 60 for the cell-debris fraction. The activity of each fraction of each culture responded linearly with respect to the increasing enzyme concentration, suggesting that the fractions contained neither an activator nor inhibitor for enzyme activity. However, it was noted that the curves for S mutans strain KI-R cell-debris fraction and the soluble fraction from S mutans strain 6715
CASEIN HYDROLYSIS.-Preliminary studies with S mutans strains KI-R and 6715 showed
that whole cells of these organisms possessed hydrolytic activity toward casein and hemoglobin at a pH of 7.0. Also, the soluble and cell-debris fractions, obtained after sonic rupture of the cultures, possessed enzymatic activity toward casein, with the soluble fractions showing greater activity at a pH of 6.6, but the cell-debris fractions were more active at a pH of 7.4. Linearity of the hydrolysis of casein by cellular fractions of S mutans strain KI-R was measured as a function of time at a pH of 6.6. Boiled enzyme fractions possessed no hydrolytic activity toward casein during the four-hour test period (Fig 1). Slight curvilinearity in the activity of the soluble fraction was observed in the first 1.5 hours of incubation; thereafter, hydrolysis of casein by this fraction was linear with time. The hydrolysis of casein by the cell-debris fraction indicated that the enzymatic activity was essentially linear throughout the experimental incubation period. The apparent greater enh
Gilford Spectrophotometer, model 240, Gilford In-
struments, Inc., Oberlin, Ohio.
A. STRAIN KI-R
B. STRAIN 6715 7.0
5.0
CO*3.0
5.0 -
1.0 0 Z _7.0
-
2 4 6 MG RESIDUE WEIGHT C. STRAIN 6715-19
1.0 o
2 4 6 MG RESIDUE WEIGHT 0. STRAIN 6715-49
7.0
-
5.0/. /
C.25.0
-
3.0 3.0
3.0 3.0
1.0
1.0
-
.30
0 2 4 6 2 4 6 21 o MG RESIDUE WEIGHT MG RESIDUE WEIGHT FIG 2.-Hydrolysis of casein at pH of 7.4 by S muttans strains KI-R, 6715, 6715-19, and 6715-49 soluble and cell-debris fractions as function of cnzyme concentration. Open circles, soluble fractions; solid circles, cell-debris fractions.
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394
J Dent Res May-June 1976
COWMAN, PERRELLA, AND FITZGERALD
TABLE 1 HYDROLYSIS OF CASEIN BY CELLULAR FRACTIONS OF S MUTANS AT A PH OF 6.6 AND OF 7.4 Units Proteinase Per mg Residue wt S mutans Strain
Antigenic Type
AHT-12 BHT-1 FA-1 VA-29 GS-5 KI-R 6715 6715-19 6715-49 SL-1
a
Cell-Debris Fraction
Soluble Fraction
b b C c
d d d d d
pH, 6.6
pH, 7.4
pH, 6.6
pH, 7.4
81 ±990+15 120 ± 12 59± 3 58±7 74 + 17
63+10
112+22 74+23 112±4-10
97 ± 12
90±7 70 ± 15 51 +±16 44±13 65 + 20 137±15 92 21 154 12 95 ± 1.0
53±12 126± 10 97 ± 20 106±20
83 24 76+9 76 17 84±25 33 10 42 7 60 ±3
87±6 78 ±+14 158 + 5.0 122+13 104 ± 18 68+ 13 64 12 65 17 39 +7.0
* Each value represents mean ± standard error of the mean for data from 15 replicated experiments.
did not pass through the origin when extrapolated to zero time. These observations suggest that the initial attack on the casein substrate by the S mutans strain Kl-R and 6715 fractions is different from the subsequent hydrolysis.13,14 The relative specific proteinase activity of additional strains of S mutans, representing different antigenic types, was then compared using casein as the substrate. All of the strains of S mutans tested, regardless of antigenic type, possessed proteinase activity in both the soluble and cell-debris fractions. The soluble fraction of S mutans strain AHT-12 (type a) had a lower specific activity than the cell-debris fraction at both a pH of 6.6 and of 7.4 (Table 1) The specific .
activity of the soluble fractions of S mutans type b strains, BHT-1 and FA-1, was not greatly different from that of the cell-debris fraction at either a pH of 6.6 or of 7.4. The soluble fractions of type c S mutans strains VA-29 and GS-5 had a much lower specific activity than the respective cell-debris fractions, particularly at a pH of 7.4, where the cell-debris fractions had a specific activity three times that of the soluble fractions. Among the cultures representing type d S mutans, there was some variability, but a majority of the strains had a higher soluble fraction activity at both a pH of 6.6 and of 7.4 relative to that observed for the respective cell-debris fractions. Since some enterococci are known to re-
TABLE 2 HYDROLYSIS OF CRUDE PORCINE GASTRIC GLYCOPROTEIN BY CELLULAR FRACTIONS OF S MUTANS Units of Hydrolase per mg Fraction S mutans
Antigenic
Strain
Type
AHT-12
a
BHT-1
b b C
FA-1 VA-29 GS-5 K1-R 6715 6715-19 6715-49 SL-1
c
d d d d d
* Each value represents
Soluble Fraction pH, 6.6
3 # 3*
Cell-Debris Fraction pH, 7.4
pH, 7.4
pH, 6.6
4±3
154±+10
0+0
39 ±+21 13+13
3 2 0+0
3
43 ±+16 27+5 100 ± 24
3±5
31±16
I11±+7 0
0
0± 0 0± 0
5±2 0±0
mean +
6
12 13 0±0
0±0 0±0 16±10
81 + 18
30+13 47±+10 29+3 39+5
160±14 64 +±10 70+18 144 ± 17 74+17 96 ±+19
62+20
24±5 0±0 86±18
standard error of the mean of eight replicate
ex-
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lease an extracellular proteinase to the medium, an effort was made to ascertain whether any of the S mutans strains produced an extracellular enzyme capable of hydrolyzing casein. Proteinase activity in the culture fluid was assayed using casein as a substrate, but none of the cultures tested in Table 1 was found to possess an extracellular proteinase activity. GLYCOPROTEIN HYDROLYSIS.-In the initial phases of this study, crude porcine gastric glycoprotein served as the substrate, and the potential enzymatic activity was assayed at a pH of 6.6 and a pH of 7.4. With S mutans strain AHT-12, extremely low activity was present in the soluble fraction regardless of the pH of the assay. However, a very high
.480 A. .420
l
e2.360-
.300 0 1 2 3 4 5 6 7 8 9 CENTIMETERS MIGRATION TOWARD ANODE
°.180 06.120
1
2
3 4
5 6 1
8 9
C..
e
.180
C=
.040-
CENTIMETERS MIGRATION TOWARD ANODE FIG 3.-Tracing of densitometric scans of acasein incubated with soluble fraction of S mutans strain K1-R at 37 C. A, solid line, zerohour profile of casein-fraction mixture; dashed line, three-hour incubation of casein-fraction mixture at pH of 6.6 and 37 C. B, 24-hour incubation of casein-fraction mixture at pH of 6.6 and 37 C. Electrophoretic separations were performed at constant current of 4 ma/tube in 7.0% acrylamide columns at pH of 8.3.
FIG 4.-Tracing of densitometric scans of axcasein incubated with cell-debris fraction of S mutans strain KI-R at 37 C. A, solid line, zerohour profile of casein-fraction mixture; dashed line, three-hour incubation of casein-fraction mixture at pH of 6.6 and 37 C. B, 24-hour incubation of casein-fraction mixture at pH of 6.6 and 37 C. Electrophoretic separations were performed at constant current of 4 ma/tube in 7.0% acrylamide columns at pH of 8.3.
relative specific activity toward the glycoprotein by the cell-debris fraction of this culture was observed (Table 2). Similar, low-tononexistent soluble fraction relative specific activities of other strains representing antigenic types b, c, and d were found. Generally, higher specific activities were found in the cell-debris fraction of most strains particularly when assayed at a pH of 7.4. Exceptions were noted in S mutans strains 6715-19 and 6715-49 which showed lower activities at a pH of 7.4 than at a pH of 6.6. The spent growth medium from all cultures showed high glycoprotein hydrolase activity at both a pH of 6.6 or a pH of 7.4, with the exception of S mutans strain BHT-1 which possessed no extracellular activity.
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396
COWMAN, PERRELLA, AND FITZGERALD
ELECTROPHORETIC STUDIES WITH CELL FRACTIONS OF S MUTANS STRAIN Kl-R.-Cell fractions and a-casein were incubated in 0.1 M sodium phosphate buffer at a pH of 6.6 for 0, 3, and 24 hours. At each interval, aliquots were removed, centrifuged to remove particulate matter, and analyzed by disc gel electrophoresis at a pH of 8.3. Electrophoresis of soluble fraction plus casein mixture at zero hour showed the presence of two distinct protein zones, one at 5.5 cm and a second at 8.9 cm from the origin (Fig 3, A). Both zones were attributable to the a-casein substrate since electrophoresis of the protein alone gave identical zones. After three hours of incubation, the zone originally present at 5.5 cm was found to migrate to a distance of 5.9 cm. Although the zone originally present at 8.9 cm did not shift in position, an increase in stainability was observed when compared with the zero-hour gel that was stained simultaneously. After incubation for 24 hours, the zones originally present at 5.5 or 5.9 and 8.9 cm had largely disappeared (Fig 3, B). Concomittant with the disappearance of these zones, new and different major protein-staining zones were found to occur at 0.5 and 7.5 cm. In addition, less intense zones were observable at 1.1, 1.7, 4.9, 6.7, 8.5, and 9.0 cm. Assay of the cell-debris fraction of S mutans strain KI-R with a-casein under similar conditions showed only minor changes in the electrophoretic profile of a-casein after three hours of incubation (Fig 4, A). However, after 24 hours of incubation, a major protein-staining band migrating 8 cm from the origin was observed. Several additional zones, particularly in the region of I to 4 cm were also detected (Fig 4, B). Electrophoretic studies were performed to ascertain whether hydrolysis of bovine submaxillary glycoprotein by the cell fractions was limited to the carbohydrate moiety or included hydrolysis of the protein core as well. Electrophoresis of the submaxillary glycoprotein at zero hour, followed by staining of duplicate gel columns for protein and for carbohydrate showed that two distinct bands were present within the major protein zone (Fig 5, A). No carbohydrate was associated with the protein zone detected at 6.0 cm. After incubation of submaxillary glycoprotein with S mutans strain KI-R soluble fraction for three hours, no major changes in the protein or carbohydrate moieties were
J Dent Res May-June 1976
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CM MIGRATION TOWARD ANODE FIG 5.-Tracing of densitometric scans of bo-
vine submaxillary glycoprotein incubated with soluble fraction of S mutans in 0.1 M sodium phosphate buffer (pH, 6.6) at 37 C. A, zerohour profile; B, three-hour incubation of glycoprotein-fraction mixture at 37QC C, 24-hour incubation of glycoprotein-fraction mixture at 37 C. Electrophoretic separations were performed at 4 ma/tube in 5.2%,, acrylamide-1.0 M urea columns at pH of 8.3. Protein was stained using Coomassie Brilliant Blue and carbohydrate by method of Holden et al.1
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PRO TEINASES OF ORAL STREPTOCOCCI
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397
detected (Fig 5, B). However, a carbohydrate-staining zone associated with weak protein staining was detected at approximately 8.0 cm from the origin. This zone was not present in the zero-hour profile. After 24 hours of incubation, the major protein occurring at 1.5 cm in both the zero- and threehour profile migrated more slowly, moving only about 1.1 cm from the origin in the .0904-1 C..090 24-hour profile (Fig 5, C). However, this ~ ~ ~ ~ ~ ~ ~ ~ zone did not contain significant carbohydrate, suggesting that the soluble fraction of .060 .060 S mutans strain Kl-R split off only the carbohydrate moiety. The zone occurring at 6.0 was unaffected. When bovine submaxilcm C .030t to .030 to U~~~~~~~~ lary glycoprotein was incubated with the cell~~~~~E debris fraction of S mutans strain KI-R, the E 1 2 3 4 5 6 7 8 9 zero-hour electrophoretogram showed the i~~~~~~~~~n presence of a major zone between 1.0 to 1.5 cm and a second minor zone at 6.3 cm (Fig 6, A). Carbohydrate was associated only C z.120w with the major zone of protein staining. HrJi Hydrolysis o 32ri 1 oS After three hours of incubation, new carbo.20. .090 hydrate-containing protein zones were de.1 6 tected at 3.9 and 8.3 cm (Fig 6, B) . Howr 12 ever, after 24 hours of incubation, these zones ..060 were not detectable, but increased amounts of protein-staining material were detected 04 .030Ocn in the region between 7 and 9 cm (Fig 6, C) In addition, the major carbohydrate-protein U)FC .0 6.Taigotesioercsasofo j zone at 3.0 cm had decreased considerably 7 2 3 4 5 9 c1 and represented a loss of approximately 75% 090u C. of the protein and 67% of the carbohydrate zd ~24 H r. Hydrolysis formerly present in the region. .020 0 '- .060 c 0 Discussion CL ~~~~~~~~cc The data presented in this study have in.010 0 .030 dicated that strains representing four different serotypes of S mutans can hydrolyze a tie It f83 rti p ura olun casein and porcine gastric glycoprotein, two structurally different substrates. Based on CM MIGRATION TOWARD ANODE the electrophoretic analyses of the casein of boFIG 6.-Tracing of densitometric digests after incubation with the soluble convine submaxillary glycoprotein incubated with tents or insoluble cell-debris fractions from strain Kl-R in cell-debris fraction of S S mutans, as well as plots of relative specific 37 0.1 sodium phosphate buffer (pH, 6.6) activities, it is suggested that two different C. A, zero-hour profile; B, three-hour incubaproteolytic enzymes may be responsible for tion of glycoprotein-fraction mixture 37 C; C, the activity against casein. However, the pos24-hour incubation of glycoprotein-fraction mnixsibility of a single enzyme possessing different 37 C. Electrophoretic separations of dukinetic properties in the soluble and bound plicate columns performed of 4.0 ma/tube in 5.2% acrylamide-0.1 M states cannot be excluded. stained columns pH of 8.3. Protein Proteolytic activity against porcine gastric using Coomassie Brilliant Blue and carbohyglycoprotein and bovine submaxillary glycodrate by method of Holden al.' protein was demonstrated in the cell-debris fraction but not in the soluble cell contents of S mutans. However, the soluble cell frac4-0
-
-D
scans
mutans
at
at
ture
at
at
were
constant
cur-
rent
urea
at
was
et
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398
COWMAN, PERRELLA, AND FITZGERALD
tion did hydrolyze the carbohydrate moiety of the submaxillary glycoprotein substrate, a finding that is consistent with the previous report of carbohydrate hydrolase activity in these organisms.15 A number of streptococci are known to elaborate extracellular proteinases.14'16-18 In the present study, we have shown that an extracellular enzyme elaborated by S mutans can attack porcine gastric glycoprotein but not casein. This suggests that the extracellular glycoprotein hydrolase is different from the enzyme (s) found in the soluble cell contents or associated with the cellular debris after the sonic rupture of S mutans. The carbohydrate metabolism of S mutans hias been well studied,19-24 but knowledge of the nitrogen metabolism of these organisms had been confined mainly to their amino acid requirements.25-27 Although S mu tans can survive and grow in the absence of added carbohydrates,28 it cannot do so without a source of essential amino acids. Our findings indicate that these organisms have the capacity to attack both dietary proteins, such as casein, and salivary proteins of human' and animal origin. It is possible that required amino acids might become available to S mutans through the metabolic activities of other plaque microorganisms, in vivo. However, the ability of S mutans to colonize and cause caries in the teeth of gnotobiotic animals in the absence of any other microorganisms lends further support to the concept that S mutans possesses the necessary complement of proteolytic enzymes whereby the organism can obtain needed amino acids for growth from dietary or salivary proteins. We are continuing to investigate the implications of these findings in relation to the oral ecology of S mutans and its role in the origin of dental caries.
Conclusions Proteolytic hydrolysis of casein and porcine or bovine glycoproteins by S mutans was demonstrated in the present study. Caseinolytic activity was found in both the soluble contents of the cells and the cellular debris after rupture of the cells. However, caseinolytic activity could not be demonstrated after growth in the culture fluid from any of the strains of S mutans tested. The soluble fractions of S mutans did not possess glycoprotein hydrolase activity toward porcine or bovine glycoprotein, but
j Dent Res May-June 1976
glycoprotein hydrolase activity was present in both the cell-debris preparations from cells and in the culture fluid after growth of the cultures. Based on these observations, S mutans may possess several different types of proteinase enyzmes with differing specificities for protein substrates. References 1. COWMAN, R.A., and FITZGERALD, R.J.: Effects of Oral Streptococci on Electrophoretic Properties of Human Salivary Anionic Proteins, J Dent Res 54: 298-303, 1975. 2. FITZGERALD, R.J., and KEYES, P.H.: Demonstration of the Etiologic Role of Streptococci in Experimental Caries in the Hamster, JADA 61: 23/9-33/19, 1960. 3. FITZGERALD, R.J.; JORDAN, H.V.; and STANLEY, H.P.: Experimental Caries and Gin-
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coccUs mutans, SL-1, Biochim Biophys Acta 261: 379-387, 1972. 25. COWMAN, R.A.; PERRELLA, M.M.; and FITZGERALD, R.J.: Influence of Incubation Atmosphere on Growth and Amino Acid Requirements of Streptococcus muttans, Appl MAicrobiol 27: 86-92, 1974. 26. CARLSSON, J.: Nutritional Requirements of Streptococcus mrutans, Caries Res 4: 305-320, 1970. 27. INWARD, P.W.; UPSTONE, D.; and VAN HOUTE, J.: Nutritional Requirements of Oral Streptococci, in McHuGH, W.D. (ed): Dental Plaque, Dundee, Eng: Livingston, 1970, pp
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