Vol. 21, No. 1
INFECTION AND IMMUNrry, July 1978, p. 345-347 0019-9567/78/0021-0345$02.00/0 Copyright © 1978 American Society for Microbiology
Printed in U.S.A.
Enhanced Glucosyltransferase Activity in Penicillin-Treated Cultures of Streptococcus mutans WILLIAM M. JANDA AND HOWARD K. KURAMITSU* Department of Microbiology-Immunology, Northwestern University Medical-Dental Schools, Chicago, Illinois 60611 Received for publication 15 February 1978
Penicillin-treated cultures of Streptococcus mutants GS-5 produced elevated levels of extracellular glucosyltransferase activity. A variety of experimental approaches have suggested the important role of glucosyltransferase (GTF) (EC 2.4.1.5) activity in the cariogenicity of Streptococcus mutans (1). Recently, Weld and Sandham (7) reported that low concentrations of penicillin inhibited plaque formation on wires by S. mutans growing in sucrose-containing media. The levels of penicillin utilized (0.5 ng/ml) had little effect on either the synthesis or activity of the GTF enzymes. Since penicillin acts on the cell wall of growing bacteria and the surface properties of S. mutans are intimately associated with its pathogenicity (5), it was of interest to examine the effects of higher concentrations of this antibiotic on the cariogenic properties of the organism. S. mutans GS-5 cells were grown in a chemically defined (CD-1% glucose) medium as described previously (3). Penicillin G (1,650 U/mg) (Sigma Biochemical Corp.) was added to the medium at concentrations up to 250 tug/ml. Cell growth, production of extracellular and cell-associated GTF activity, and ["4C]leucine incorporation into cellular protein were determined as described earlier (3). Penicillin at concentrations of 100 and 250 ,ug/ml did not affect cell growth (Fig. 1) until the control cultures had grown for approximately 45 min. When total extracellular GTF production was examined (Fig. 2), we observed that organisms growing in penicillin-containing medium produced extracellular GTF activity at a significantly faster rate than cells grown in the absence of the antibiotic. This increased rate was reflected in both insoluble (data not shown) and total glucan synthetic activity (Fig. 2). Penicillin in the nanogram concentrations utilized by Weld and Sandham (7) had no effect on extracellular GTF synthesis. The elevated GTF activities observed in the presence of penicillin was probably not the result of cell lysis and release of preformed enzyme since previous results suggested no intracellular pools of GTF activity when cells were grown in the same medium without peni-
cillin (3). Furthermore, penicillin at concentrations up to 500 ,ug/ml had no effect on either soluble or insoluble glucan-synthesizing activity when added directly to assay mixtures. Examination of the cell-associated GTF activities during growth in the presence of penicillin (Fig. 3) demonstrated an initial increase in activity over that of control cells during the first 60 mi, followed by an abrupt decrease to about onethird of the control activity. Since it has been demonstrated that extracellular GTF production is dependent upon ongoing protein synthesis (3), it was possible that the elevated appearance of GTF activity observed in the presence of penicillin may reflect a transient stimulation of protein synthesis by the antibiotic. In this regard, ["4C]leucine incorporation into cellular protein was measured in the absence and presence of penicillin (Fig. 4). The demonstrated inhibition of protein synthesis in the presence of penicillin indicates that extracellular GTF activity was accumulating at a faster rate relative to control cultures when total protein synthesis was declining. Furthermore, the increased rate of appearance of extracellular GTF activity in the presence of penicillin required de novo protein synthesis since chloramphenicol prevented the appearance of extracellular activity (data not shown). This further indicates that the penicillin effect on GTF activity is not the result of cell lysis. Although the mechanism of penicillin-mediated stimulation of extracellular GTF activity is unknown, recent results from several laboratories suggest a possible explanation. Horne et al. (2) have demonstrated that the addition of penicillin to growing cultures of several streptococci results in the release of large amounts of lipid material into the medium. In addition, Harlander and Schachtele (la) have recently demonstrated that several different phospholipids stimulated S. mutans GTF activity. Furthermore, the addition of Tween 80 to growing cultures of S. mutans resulted in a marked elevation 345
346
NOTES
INFECT. IMMUN. D
LL 0
2.0
a
0 K
280
m
1.0
0
.0 W 4.
0
230
60
120
Time (min) FIG. 3. Effects ofpenicillin on cell-associated GTF activity. Total GTF activity was determined for cells grown in CD-glucose medium with no penicillin (0); penicillin (100 pg/ml) (0).
0.
10
130_
.
60
0
120 CM l
Time (min) FIG. 1. Effects of penicillin on cell growth. Cells in CD-glucose medium as described previously (3), in the presence of no penicillin (-); penicillin (100 pg/ml) (A); penicillin (250 pg/ml) (U). were grown
8
UL. a
6
0
t-o
4 8.0
2 U.
6.0
-
0
30 60 Time (min) FIG. 4. Effects ofpenicillin on ['4Clleucine incorporation. ['4Clleucine (50.6 mCi/mmol) incorporation into total cellular protein was determined as previously described (3). No penicillin ();penicillin (100 pg/ml) (A); penicillin (250 pg/ml) (U). 0
en4.0C
U.2.0
0
60
120
Time (min) FIG. 2. Effects ofpenicillin on extracellular GTF activity. Total GTF activity released was measured for cells grown in CD-glucose medium containing: no penicillin (0); penicillin (100 pg/ml) (A); penicillin (250 pg/ml) (U). CFU, Colony-forming units (3 x 108 CFU is equivalent to 100 Klett units).
of extracellular GTF activity (6, 8) and an alteration in the fatty acid composition of the cellular membrane (6). Thus, the observed penicillin stimulatory effects on GTF activity may result from the release of lipid from the cell surface of S. mutans GS-5 and the subsequent interaction of lipid components with the enzymes resulting in an elevation of enzyme activity. Alternatively, the action of penicillin may release highly active
NOTES
VOL. 21, 1978
GTF-membrane lipid complexes involved in extracellular protein synthesis and extrusion from the cell surface. Experiments are currently in progress to examine the interaction of purified GTF preparations with membrane components of strain GS-5. This investigation was supported by Public Health Service grant DE-03258 from the National Institute of Dental Research. LITERATURE CITED van Houte. 1975. Dental caries. Annu. Rev. Med. 26:121-136. laliarlander, S. K., and C. F. Schachtele. (1978). Streptococcus mutans dextransucrase: stimulation of glucan formation by phosphoglycerides. Infect. Immun. 19:450-456. 2. Horne, D., R. Hakenbeck, and A. Tomasz. 1977. Secretion of lipids induced by inhibition of peptidoglycan synthesis in streptococci. J. Bacteriol. 132:704-717. 1. Gibbons, R. J., and J.
347
3. Janda, W. M., and H. K. Kuramitsu. 1976. Regulation of extracellular glucosyltransferase production and the relationship between extracellular and cell-associated activities in Streptococcus mutans. Infect. Immun. 14:191-202. 4. Janda, W. M., and H. K. Kuramitsu. 1977. Properties of a variant of Streptococcus mutans altered in its ability to interact with glucans. Infect. Immun. 16:575-586. 5. Mukasa, H., and H. D. Slade. 1973. Mechanism of adherence of Streptococcus mutans to smooth surfaces. II. Nature of the binding site and the adsorption of dextran-levan synthetase enzymes on the cell-wall surface of the streptococcus. Infect. Immun. 9:419-429. 6. Umesaki, Y., Y. Kawai, and M. Mutai. 1977. Effect of Tween 80 on glucosyltransferase production in Streptococcus mutans. Appl. Environ. Microbiol. 34:115-119. 7. Weld, H. G., and H. J. Sandham. 1976. In vitro effects of low concentrations of penicillin and sulfadiazine on Streptococcus mutans. Antimicrob. Agents Chemother. 10:196-199.
8. Wittenberger, C. L, A. J. Beaman, and L N. Lee. 1978. Tween 80 effect on glucosyltransferase synthesis by Streptococcus salivarius. J. Bacteriol. 133:231-239.
INFECTION AND IMMUNrry, July 1978, p. 345-347 0019-9567/78/0021-0345$02.00/0 Copyright © 1978 American Society for Microbiology
Printed in U.S.A.
Enhanced Glucosyltransferase Activity in Penicillin-Treated Cultures of Streptococcus mutans WILLIAM M. JANDA AND HOWARD K. KURAMITSU* Department of Microbiology-Immunology, Northwestern University Medical-Dental Schools, Chicago, Illinois 60611 Received for publication 15 February 1978
Penicillin-treated cultures of Streptococcus mutants GS-5 produced elevated levels of extracellular glucosyltransferase activity. A variety of experimental approaches have suggested the important role of glucosyltransferase (GTF) (EC 2.4.1.5) activity in the cariogenicity of Streptococcus mutans (1). Recently, Weld and Sandham (7) reported that low concentrations of penicillin inhibited plaque formation on wires by S. mutans growing in sucrose-containing media. The levels of penicillin utilized (0.5 ng/ml) had little effect on either the synthesis or activity of the GTF enzymes. Since penicillin acts on the cell wall of growing bacteria and the surface properties of S. mutans are intimately associated with its pathogenicity (5), it was of interest to examine the effects of higher concentrations of this antibiotic on the cariogenic properties of the organism. S. mutans GS-5 cells were grown in a chemically defined (CD-1% glucose) medium as described previously (3). Penicillin G (1,650 U/mg) (Sigma Biochemical Corp.) was added to the medium at concentrations up to 250 tug/ml. Cell growth, production of extracellular and cell-associated GTF activity, and ["4C]leucine incorporation into cellular protein were determined as described earlier (3). Penicillin at concentrations of 100 and 250 ,ug/ml did not affect cell growth (Fig. 1) until the control cultures had grown for approximately 45 min. When total extracellular GTF production was examined (Fig. 2), we observed that organisms growing in penicillin-containing medium produced extracellular GTF activity at a significantly faster rate than cells grown in the absence of the antibiotic. This increased rate was reflected in both insoluble (data not shown) and total glucan synthetic activity (Fig. 2). Penicillin in the nanogram concentrations utilized by Weld and Sandham (7) had no effect on extracellular GTF synthesis. The elevated GTF activities observed in the presence of penicillin was probably not the result of cell lysis and release of preformed enzyme since previous results suggested no intracellular pools of GTF activity when cells were grown in the same medium without peni-
cillin (3). Furthermore, penicillin at concentrations up to 500 ,ug/ml had no effect on either soluble or insoluble glucan-synthesizing activity when added directly to assay mixtures. Examination of the cell-associated GTF activities during growth in the presence of penicillin (Fig. 3) demonstrated an initial increase in activity over that of control cells during the first 60 mi, followed by an abrupt decrease to about onethird of the control activity. Since it has been demonstrated that extracellular GTF production is dependent upon ongoing protein synthesis (3), it was possible that the elevated appearance of GTF activity observed in the presence of penicillin may reflect a transient stimulation of protein synthesis by the antibiotic. In this regard, ["4C]leucine incorporation into cellular protein was measured in the absence and presence of penicillin (Fig. 4). The demonstrated inhibition of protein synthesis in the presence of penicillin indicates that extracellular GTF activity was accumulating at a faster rate relative to control cultures when total protein synthesis was declining. Furthermore, the increased rate of appearance of extracellular GTF activity in the presence of penicillin required de novo protein synthesis since chloramphenicol prevented the appearance of extracellular activity (data not shown). This further indicates that the penicillin effect on GTF activity is not the result of cell lysis. Although the mechanism of penicillin-mediated stimulation of extracellular GTF activity is unknown, recent results from several laboratories suggest a possible explanation. Horne et al. (2) have demonstrated that the addition of penicillin to growing cultures of several streptococci results in the release of large amounts of lipid material into the medium. In addition, Harlander and Schachtele (la) have recently demonstrated that several different phospholipids stimulated S. mutans GTF activity. Furthermore, the addition of Tween 80 to growing cultures of S. mutans resulted in a marked elevation 345
346
NOTES
INFECT. IMMUN. D
LL 0
2.0
a
0 K
280
m
1.0
0
.0 W 4.
0
230
60
120
Time (min) FIG. 3. Effects ofpenicillin on cell-associated GTF activity. Total GTF activity was determined for cells grown in CD-glucose medium with no penicillin (0); penicillin (100 pg/ml) (0).
0.
10
130_
.
60
0
120 CM l
Time (min) FIG. 1. Effects of penicillin on cell growth. Cells in CD-glucose medium as described previously (3), in the presence of no penicillin (-); penicillin (100 pg/ml) (A); penicillin (250 pg/ml) (U). were grown
8
UL. a
6
0
t-o
4 8.0
2 U.
6.0
-
0
30 60 Time (min) FIG. 4. Effects ofpenicillin on ['4Clleucine incorporation. ['4Clleucine (50.6 mCi/mmol) incorporation into total cellular protein was determined as previously described (3). No penicillin ();penicillin (100 pg/ml) (A); penicillin (250 pg/ml) (U). 0
en4.0C
U.2.0
0
60
120
Time (min) FIG. 2. Effects ofpenicillin on extracellular GTF activity. Total GTF activity released was measured for cells grown in CD-glucose medium containing: no penicillin (0); penicillin (100 pg/ml) (A); penicillin (250 pg/ml) (U). CFU, Colony-forming units (3 x 108 CFU is equivalent to 100 Klett units).
of extracellular GTF activity (6, 8) and an alteration in the fatty acid composition of the cellular membrane (6). Thus, the observed penicillin stimulatory effects on GTF activity may result from the release of lipid from the cell surface of S. mutans GS-5 and the subsequent interaction of lipid components with the enzymes resulting in an elevation of enzyme activity. Alternatively, the action of penicillin may release highly active
NOTES
VOL. 21, 1978
GTF-membrane lipid complexes involved in extracellular protein synthesis and extrusion from the cell surface. Experiments are currently in progress to examine the interaction of purified GTF preparations with membrane components of strain GS-5. This investigation was supported by Public Health Service grant DE-03258 from the National Institute of Dental Research. LITERATURE CITED van Houte. 1975. Dental caries. Annu. Rev. Med. 26:121-136. laliarlander, S. K., and C. F. Schachtele. (1978). Streptococcus mutans dextransucrase: stimulation of glucan formation by phosphoglycerides. Infect. Immun. 19:450-456. 2. Horne, D., R. Hakenbeck, and A. Tomasz. 1977. Secretion of lipids induced by inhibition of peptidoglycan synthesis in streptococci. J. Bacteriol. 132:704-717. 1. Gibbons, R. J., and J.
347
3. Janda, W. M., and H. K. Kuramitsu. 1976. Regulation of extracellular glucosyltransferase production and the relationship between extracellular and cell-associated activities in Streptococcus mutans. Infect. Immun. 14:191-202. 4. Janda, W. M., and H. K. Kuramitsu. 1977. Properties of a variant of Streptococcus mutans altered in its ability to interact with glucans. Infect. Immun. 16:575-586. 5. Mukasa, H., and H. D. Slade. 1973. Mechanism of adherence of Streptococcus mutans to smooth surfaces. II. Nature of the binding site and the adsorption of dextran-levan synthetase enzymes on the cell-wall surface of the streptococcus. Infect. Immun. 9:419-429. 6. Umesaki, Y., Y. Kawai, and M. Mutai. 1977. Effect of Tween 80 on glucosyltransferase production in Streptococcus mutans. Appl. Environ. Microbiol. 34:115-119. 7. Weld, H. G., and H. J. Sandham. 1976. In vitro effects of low concentrations of penicillin and sulfadiazine on Streptococcus mutans. Antimicrob. Agents Chemother. 10:196-199.
8. Wittenberger, C. L, A. J. Beaman, and L N. Lee. 1978. Tween 80 effect on glucosyltransferase synthesis by Streptococcus salivarius. J. Bacteriol. 133:231-239.
