Vol. 20, No. 3
INFECTION AND IMMUNITY, June 1978, p. 592-599
0019-9567/78/0020-0592$02.00/0 Copyright © 1978 American Society for Microbiology
Printed in U.S.A.
Effect of Sucrose in Culture Media on the Location of Glucosyltransferase of Streptococcus mutans and Cell Adherence to Glass Surfaces SHIGEYUKI HAMADA* AND MITSUO TORII Department of Oral Microbiology, Osaka University Dental School, Kita-ku, Osaka, 530 Japan Received for publication 20 January 1978
Streptococcus mutans strain B13 (serotype d) almost exclusively produced free glucosyltransferase (GTase) in the culture supernatant when grown in sucrosefree TTY broth medium, which was composed of Trypticase (Baltimore Biological Laboratory [BBL] Cockeysville, Md.), tryptose (Difco Laboratories, Detroit, Mich.), yeast extract (BBL), salts, and 1% glucose. Organisms grown in sucrosefree TTY broth retained very weak cell-associated GTase activity and did not adhere significantly to glass surfaces in the presence of exogenous sucrose. If sucrose was added to TTY broth, however, GTase was found on the cell surface where cell-bound, water-insoluble glucans were synthesized. Most commercially available products of Todd-Hewitt broth were found to contain trace amounts of sucrose, as did Trypticase soy broth (BBL), whereas brain heart infusion broth (Difco and BBL) was found to be essentially free of sucrose. Almost all detectable GTase activity was cell associated when S. mutans B13 was grown in ToddHewitt or trypticase soy broth. Heat-treated B13 cells grown in Todd-Hewitt broth and cell-free, water-insoluble glucans bound free GTase and produced marked adherence in the presence of sucrose. Experiments strongly suggest that the binding sites for free GTase are the surface glucans, and cell-associated and extracellular GTases are most likely alternate states of the same enzyme protein. The strong cariogenic activity of Streptococcus mutans has been ascribed to the ability of this bacterium to synthesize water-insoluble glucans from sucrose that adhere to various smooth surfaces (7, 25-27). Synthesis of the glucans is mediated by the enzymatic action of cell-free (extracellular) and/or cell-bound glucosyltransferase(s) (GTase[s]). The location of S. mutans GTase appears to be complicated (1, 3, 11, 16, 20, 22, 23, 28) and remains to be elucidated. It has been reported that extracellular GTase of S. mutans B13 was bound to the cell surface of both viable and heat-treated S. mutans but not to the cells of other species of bacteria, with a few exceptions (26; S. Hamada, Y. Kobayashi, and H. D. Slade, Microbiol. Immunol., in press). After the free, unbound GTase was removed from the reaction mixture, S. mutans cells produced significant adherence to glass surfaces in the presence of sucrose and synthesized cellbound ['4C]glucan from ["4C]sucrose. Todd-Hewitt (TH) broth is preferentially used for cultivation of various streptococci; therefore, we have used this broth medium for cultivation of S. mutans and for the production of extracellular GTase from the culture supernatant. In our experience, the free GTase activ-
ity in the centrifuged supernatant of TH broth cultures of S. mutans varied from lot to lot, but the activity was usually significantly less than that of brain heart infusion (BHI) broth cultures. The aim of this study is to investigate the differences in the distribution of free and cellassociated GTase in various cultures of S. mutans. The results are discussed in terms of adherence and cell-bound glucan synthesis of S. mutans. MATERIALS AND METHODS Bacterial strains. S. mutans strain B13 (serotype d) (14) was used in the present study. This strain was kindly supplied by S. Edwardsson (University of Lund, Malmo, Sweden). Culture media. To prepare extracellular GTase of S. mutans, TH broths (Difco Laboratories, Detroit, Mich., and Baltimore Biological Laboratory [BBL], Cockeysville, Md.), and Trypticase soy (TS) broth (BBL), tryptic soy (TS) broth (Difco), and BHI broths (BBL and Difco) were used. A broth medium designated TTY was devised as a medium essentially free of sucrose. TTY broth contained (per liter): trypticase (BBL), 15 g; tryptose (Difco), 4 g; yeast extract, 4 g; K2HPO4, 2 g; KH2PO4, 5 g; Na2CO3, 2 g; NaCl, 2 g; and glucose, 10 g. Glucose was autoclaved separately and combined with the broth aseptically. Final reaction of 592
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the medium was at pH 7.4 ± 0.2 at 250C. Media were routinely inoculated (1%, vol/vol) from a stationaryphase culture, and cells were grown for 18 h at 370C. Cell growth and distribution of GTases. S. mutans B13 was serially transferred several times in each medium to adapt the bacterial cells to the medium in which they would be propagated. Cells were grown in broth medium for 18 h at 370C in 10-ml volumes after inoculation with 0.5 ml of an overnight culture of the cells grown in the same medium. At the end of growth, the culture was vigorously vibrated, using a Vortex mixer for a few minutes, and sample (500 ILI) of the broth culture was pipetted out. The culture was mixed with 2.0 ml of 0.05 M potassium phosphate buffer, pH 6.8 (KPB), followed by centrifugation. The centrifuged cells were resuspended in 3.0 ml of KPB by sonic oscillation at a maximum amplitude for 10 s, using a Tomy UR-150P ultrasonicator (Tomy Seiko Co., Ltd., Tokyo), and the optical density at 550 nm was read to compare the cell growth in the various media used. To assess the distribution of GTase in the broth culture of S. mutans, a sample of the broth culture (5.0 ml) was centrifuged in the cold. After the collected cells were washed two times with 5.0 ml of KPB, the cells were mixed with 900 Ad of KPB containing 0.02% Merthiolate and 100 pl of ["4C]glucose-labeled sucrose solution (59,000 dpm) and incubated at 370C for various time intervals to measure cell-associated GTase activity. The radioactive sucrose solution (hereafter called ['4C]sucrose) was prepared by mixing 100 pi (2.0 ,uCi) of ['4C]glucose-labeled sucrose (New England Nuclear, Boston, Mass.; specific activity, 275 ,iCi/mmol) and 342 mg of unlabeled sucrose in 7.9 ml of KPB. The cells were collected by centrifugation and washed two times with 2.5 ml of KPB. The final cell pellet was transferred to a scintillation vial, using 2.5 ml of scintillation fluid, and the tube was rinsed three times (final volume, 10 ml). The scintillation fluid contained 4.0 g of 2,5-diphenyloxazole (Dotite DPO, Wako Pure Chemicals, Osaka), 0.1 g of 1,4-bis-[2]-(5phenyloxazolyl)benzene (Dotite POPOP, Wako Pure Chemicals) in 600 ml of xylene, 400 ml of Nonion NS210 (Nihon Yushi Ltd., Tokyo), and 100 ml of 10% ethanol. Samples were counted for radioactivity due to cell-associated GTase with an Aloka model LSC673 liquid scintillation spectrometer (Aloka Ltd., Tokyo) (count I). Cell-free GTase synthesizing water-insoluble glucan was assayed as follows. The culture supernatant (500 above, was pl), obtained by centrifugation100as described mixed with 400 PIl of KPB, pl of the ['4C]sucrose solution, and Merthiolate to a final concentration of 0.02%. After the reaction mixture was incubated at 371C for each desired time interval, the mixture was filtered by vacuum onto 2.5-cm glass filter disks (Whatman GF/A) held in a glass filter holder (Millipore Corp., Bedford, Mass.). The sample tube was washed twice with 1.5 ml of KPB, and each washing was filtered over the sample. In some experiments, total glucan was assayed after an equal amount of ethanol was added to the sample to precipitate watersoluble glucans, as described previously (10, 15, 22). The air-dried filter disks were placed in vials with 10 ml of the scintillation fluid, and the radioactivity was measured (count II) as described above. The relative
593
ratio of activity of cell-free GTase versus cell-associated GTase was calculated as follows: (10 x disintegrations per minute of count II)/disintegrations per minute of count I. As a matter of convenience, the disintegrations per minute after 18 h of incubation were counted as an indicator of GTase activity of a sample in the present study. Comparison of cell-associated GTase activities in cells grown in TTIY broth containing different concentrations of sucrose or in TH broth containing dextranase. S. mutans B13 was grown at 370C for 18 h in TTY broth containing 0, 0.005, 0.01, 0.05, 0.1, or 0.5% (final concentration) sucrose plus 1% glucose. B13 cells were also grown in TH broth containing filter-sterilized dextranase AD17 (0.1 to 0.5 U/ml of culture, final concentration) (9). Cells were collected by centrifugation, washed three times with water, and lyophilized. Cells were resuspended in KPB at a concentration of 5.0 mg (dry weight)/ml. For assay of cell-associated GTase, a sample (400 1A) of the suspension was mixed with 500 gil of KPB and 100 gil of the ['4C]sucrose solution and incubated at 370C for 18 h. Measurement of the radioactivity was performed as described above. Preparation of cell-free GTase. Cell-free GTase was obtained from the culture supernatant of S. mutans B13 grown in TTY broth by 50% ammonium sulfate precipitation, as previously described (10, 15). The GTase preparation produced water-insoluble product almost exclusively, and no significant watersoluble glucan was detected by the method used in this study. The protein content of the preparation was 13.8 mg/ml, and the activity was 4.1 dextransucrase units/mg of protein (29). No detectable fructosyltransferase activity was demonstrated as determined by the incorporation of radioactivity into ethanol-insoluble polysaccharides from ['4C]fructose-labeled sucrose. A single preparation of GTase was used throughout the study. Preparation of water-insoluble glucans. A 5% sucrose solution in KPB (400 ml) was mixed with cellfree GTase (8.0 ml) of S. mutans B13 and incubated at 371C for 18 h. The mixture was then centrifuged at 10,000 x g for 30 min to collect water-insoluble glucan, washed twice with water (200 ml each), and lyophilized. Binding ability of GTase to S. mutans cells and cell-free water-insoluble glucan. Lyophilized cell and water-insoluble glucan suspensions (5.0 mg [dry weight]/ml of water) were held at 100'C for 10 min to inactivate cell-associated GTase, washed two times with water, and lyophilized. Heated cells and glucans were resuspended in KPB (5.0 mg [dry weight]/ml of KPB) by sonic oscillation. Desired amounts of GTase were added to 0.4 ml of the suspension, and the final volume was adjusted to 2.0 ml with KPB. After standing at room temperature for 10 min, the mixture was centrifuged, and the cells were washed twice with 2.0 ml of KPB. KPB (900 gl) and 100 gl of the ['4C]sucrose solution were then added to the cell pellet. After the mixture was incubated at 370C for 18 h, it was centrifuged. The pellet was washed twice with 2.0 ml of KPB, mixed with the scintillation fluid, and counted as described above. Binding of GTase (20 gl) to other polysaccharides,
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e.g., starch (Merck AG, Darmstadt, Germany), cellulose (average particle size, 20 jum; Sigma Chemical Co., St. Louis, Mo.), Sephadex G-25 (fine, Pharmacia Fine Chemicals Inc., Piscataway, N. J.), and inulin (Wako Pure Chemicals, Osaka), was also determined as described above. Starch and inulin were essentially insoluble in cold KPB. Adherence of S. mutans cells and water-insoluble glucans. Determination of the adherence of heat-treated cells and cell-free, water-insoluble glucans to a glass surface in the presence of sucrose was performed according to the procedure previously reported (10, 19), with slight modification. Heat-treated cells or water-insoluble glucan (1.0 mg, dry weight) and increasing amounts of GTase were first incubated in KPB (2.0 ml) for 10 min at room temperature. Cells were collected by centrifugation and washed two times with KPB to remove unbound GTase. Buffered sucrose (2.4 ml) was added and stirred. After incubation overnight at a 300 angle, cell and glucan adherences to the glass surface were determined by reading the optical density at 550 nm. Effect of various treatments on the binding of GTase and subsequent adherence. Heat-treated cells, grown in TH broth containing trace amounts of sucrose, were pretreated at 370C for 60 min with various reagents as follows: Spicaria violacea dextranase (9), Penicillium sp. dextranase (Sigma), protease (subtilisin, Sigma), and lysozyme (Sigma) were added in a final concentration of 0.2% (wt/vol); NaOH and HCl in a concentration of 1.0 N; sodium dodecyl sulfate, Brij 35 (Sigma), Triton X-100 (Wako Pure Chemicals), and concanavalin A (Pharmacia) at 1.0%; and Ricinus communes agglutinin I (8, 21) at 0.5%. Type d antiserum (anti-S. mutans B13 whole-cell serum) and antiglucan serum were prepared as previously described (10), and 40 ,ul of the sera was used in the experiment. After the reaction mixture (2.0, final volume) was centrifuged and washed three times with KPB, it was reconstituted as described, and the binding of GTase and subsequent adherence were assayed. For determining the cell adherence and binding of GTase to the cell surface, 1.0- and 2.0-mg (dry weight) samples of heat-treated cells respectively, were used.
RESULTS Rate of cell-free and cell-bound glucan synthesis. Figure 1 shows typical curves of glucan formation by a cell-free culture supernatant and a washed cell suspension from overnight cultures of S. mutans B13 which had been grown in TH (Difco) and TTY broths as a function of time. Cell-bound ['4C]glucan production increased almost linearly up to 4 h with THgrown cells and up to 6 h with TTY-grown cells, after which little increase was observed. On the other hand, water-insoluble glucan production by the cell-free culture supernatant reached plateau levels at 8 to 10 h and again at 18 to 22 h, indicating the presence of multiple forms of GTases in S. mutans cultures. In this connection, there was no substantial difference in the amounts of water- and ethanol-insoluble (total)
INFECT. IMMUN.
Incubation (h) FIG. 1. Water-insoluble ['4Clglucan synthesis from ['4Clglucose-labeled sucrose by cell-free culture liquor and washed cell suspension as a function of time. Cells of S. mutans B13 were grown overnight in TH and TTY broths. After samples of the culture (5.0 ml) were centrifuged in the cold, the collected cells were washed twice with KPB and assayed for cellbound, water-insoluble ['4CJglucan. Samples (0.5 ml) of the centrifuged supernatant were assayed for total and water-insoluble glucan as described in the text. Both cell-free and cell-associated GTase synthesized primarily water-insoluble glucans. Therefore, only water-insoluble glucan production is depicted in this figure. Symbols: (------) cell-free, water-insoluble glucan synthesis by extracellular culture liquor; (-) cell-bound, water-insoluble glucan synthesis by cellassociated GTase; (0) TTY-grown culture; (0) THgrown culture.
glucans synthesized for each incubation interval. Therefore, for simplicity, each water-insoluble glucan synthesized (disintegrations per minute) after incubation with ["4C]sucrose for 18 h was referred to as GTase activity of a test sample. Comparison of growth and GTase production of S. mutans in various culture media. The degree of growth and GTase production of S. mutans B13 in TTY broth were defined as 100%, because preliminary experiments revealed that TTY broth cultures of various S. mutans strains always gave luxuriant growth and high GTase production in the culture supernatant. The relative growth and GTase production in different culture media and different lots of a medium of the same commercial brand are shown in Table 1. Considerably less cell growth (ca. 40%) was obtained in TH, BHI, and TS broths as compared with that in TTY broth. Strong or moderate GTase activity was demonstrated in TTY or BHI broth cultures of S. mutans B13, respectively, whereas TS or TH broth cultures generally produced relatively
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595
little GTase in the culture liquor. Only one lot of TH broth medium examined gave significant GTase activity in the culture liquor. When TH and TS broths were fortified with 0.8% glucose (1.0%, final concentration) as a supplemental carbon source, cell growth increased to nearly 70% of that of the TTY culture. However, extracellular GTase activity decreased to less than 10% of that in the TTY culture. Similar findings were also obtained when S. mutans MT703 (type e) was used as a test strain instead of strain B13. Ratio of cell-free and cell-associated GTases of S. mutans in various culture media. Table 2 shows that when S. mutans B13 was grown in TTY or BHI broth, most GTase activity was released from the growing cells into
the culture supernatant. On the other hand, TS broth- or TH broth-grown cells retained significant cell-associated GTase activity, and the ratio of cell-free GTase to cell-associated GTase was far smaller as compared with that in TTY or BHI broth cultures. However, the culture grown in one lot of TH broth (lot 525121) produced mainly free GTase, with activity comparable to those in the TTY and BHI broth cultures. The fact that S. mutans grown in TH broth had properties similar to those in TS broth with respect to distribution of GTase indicated that the TH broth may contain some sucrose, as does the TS broth (28). To determine the effect of sucrose on the distribution of GTase, B13 cells were grown in TTY broths containing various amounts of sucrose. Table 3 shows a direct reTABLE 1. Cell growth and GTase production by S. lationship between the recovery of GTase assomutans B13 grown in various culture media ciated with the cell surface and the increase of sucrose in TTY media. When sucrose was added Cell Extracellugrowth lar GTase to BHI broth, a similar shift in GTase recovery Medium was obtained. ODss)a activity)' Effect of dextranase on cell-associated TTYC 100 100 GTase activity. S. mutans was grown in TH BHI (Difco) 44 39 broth, which resulted in the production of sig35 BHI (BBL) 61 nificant cell-associated GTase (cf. Table 2). Ta29 Tryptic soy broth (Difco) 13 ble 4 shows that incorporation of dextranase into 36 Trypticase soy broth 14 the TH broth markedly prevented localization (BBL) of GTase on the cell surface but did not inhibit TH (Difco) growth of-the cells. The GTase activity in the 525121id40 61 culture supernatant was not estimated because 617841 36 18 34 476715 24 of the presence of the added dextranase. 621966 36 20 Cell-associated GTase activity of su625032 NDe 26 crose-grown S. mutans cells and binding of TH (BBL) 36 38 GTase to the heat-treated cells. Washed cells a OD550, Optical density at 550 nm. (2.0 mg, dry weight) of S. mutans B13 grown in Incorporation of 14C into water-insoluble glucan sucrose-free TTY broth did not produce signififrom ['4C]glucose-labeled sucrose was assayed. cant cell-bound and water-insoluble glucans See text for composition. when incubated with sucrose (Table 5) and d Lot number. caused no sucrose-induced agglutination during e ND, Not determined. overnight incubation. However, cells grown in TTY broth containing increasing amounts of TABLE 2. Localization of GTase activity of S. sucrose retained cell-associated GTase and synmutans grown in various culture media thesized glucans. The enzyme activities inRatio of GTase activcreased concomitant with the sucrose added to Medium ity: extracellular/cellthe TTY broth. Table 5 also shows that heatassociated '
TTY BHI (Difco) BHI (BBL) Tryptic soy broth (Difco) Trypticase soy broth (BBL) TH (Difco) 525121a 617841 476715 621966 625032 630836 Lot number. a
35.6 29.5 9.2
1.8 1.4
28.6 1.5 2.0 1.6 2.0 1.9
TABLE 3. Effect of sucrose on the localization of GTase in TTY-grown S. mutans B13 Sucrose added in TTY broth (% final concn) 0 0.0005
0.001 0.005 0.01 0.05 0.1
Ratio of GTase activity:
extracellular/cell-associated 37.8 30.9 19.5 13.8 9.90 1.53 1.29
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HAMADA AND TORII
TABLE 4. Relative activity of cell-associated GTase of S. mutans grown in TH broth containing dextranasea Dextranase added (U/ml of TH broth)
Cell growth (%
OD)b
Cell-associated GTase
(% activity) 100 25.6 17.4 13.0
100 None 100 0.10 97 0.25 97 0.50 a S. violacea dextranase (500 U/mg of protein) was used. See reference 9. b OD550, Optical density at 550 nm.
TABLE 5. Cell-associated GTase activity and GTase-binding ability of sucrose-grown S. mutans B13 cells Glucan synthesized (dpm)b Sucrose added
Cell-assoofino
(%)a GTase of nontreated cells
Binding of GTase to heated cells
679 0 1,151 1,258 3,557 0.005 1,746 0.01 5,061 4,936 0.05 8,768 0.1 7,357 9,756 9,648 0.5 10,306 a TTY broth was used as a base medium. b Per 2.0 mg (dry weight) of cells, or per test tube.
treated cells (2.0 mg, dry weight) grown in TTY broth containing sucrose bound cell-free GTase and produced cell-bound glucan. The degree of binding of GTase increased with increasing amounts of cell-bound glucan synthesized during growth in TTY-sucrose broths. Inhibition of cell adherence and GTase binding by chemical/enzymatic treatments. Treatment ofheat-treated B13 cells with various chemicals, enzymes, antisera, and lectins (Table 6) has supplied significant data concerning the nature ofthe GTase-binding components present on the cell surface of S. mutans. Treatment with NaOH and dextranases resulted in an almost complete loss of adherence and GTasebinding abilities, whereas protease and sodium dodecyl sulfate treatment removed the adherence ability but not the GTase-binding ability. On the other hand, treatment with glucan antibody significantly reduced GTase binding but not cell adherence. B13 whole-cell antiserum strongly agglutinated cells during incubation, and the cells redispersed poorly after extensive washing and subsequent sonic treatment. Therefore, the agglutinated cells could not adhere to the glass surface because of the heavy weight of the aggregates, even though the cells bound GTase and synthesized a significant amount of
TABLE 6. Effect of various treatments on the binding of GTase from S. mutans B13 and subsequent adherence of heat-treated B13 cellsa AdherTreatment of heat-treated B13 cells (final concn)
Binding of GTase (%)
ence
to
a
glass surface (%)
100 100 Control 10 6.5 NaOH (1 N) 110 120 HCO (1 N) 3.7 8.3 Dextranaseb (0.2%) 15 12 Dextranasec (0.2%) 5.2 130 Proteased (0.2%) 100 110 Lysozyme (0.2%) 3.0 84 SDS' (1.0%) 120 150 Brij 35 (1.0%) 110 86 Triton X-100 (1.0%) 100 37 Antiglucan serum (40 p1) 20 100 Anti-B13 serum (401,l) 100 110 RCAIf (0.5%) 100 120 Concanavalin A (0.5%) a Heat-treated (100'C, 10 min) S. mutans B13 cells grown in TH broth (Difco) were treated by various reagents followed by washing with KPB. The reaction mixture was reconstituted, and the binding of GTase (20 p1) and cell adherence to a glass surface were determined as described in the text. The degree of relative GTase binding and cell adherence is expressed as a percentage of that of heated but nontreated control cells. b From S. violacea (9). 'From Penicillium sp. (Sigma). d Subtilisin (Sigma). eSDS, Sodium dodecyl sulfate. f RCAI, R. communes agglutinin I (21).
cell-bound glucans. R. communes agglutinin I also agglutinated the B13 cells, but the cells redispersed after washing and sonic treatment. The R. communes agglutination I-treated cells bound GTase and produced adherence to an extent similar to that of the nontreated control. Various other treatments exerted no significant inhibitory effects on GTase binding and cell adherence. Binding of GTase to heat-treated S. mutans cells and cell-free, water-insoluble glucans and subsequent glucan synthesis and adherence. Both TH-grown cells of S. mutans B13 and cell-free, water-insoluble glucans that had been synthesized from extracellular GTase and unlabeled sucrose bound the extracellular B13 GTase from the TTY broth culture (Fig. 2). De novo ['4C]glucan synthesis on the surface of heat-treated cells and glucans due to GTase binding increased linearly and then gradually reached plateau levels. Both cells and glucans which had bound extracellular GTase markedly adhered to glass surfaces due to de novo glucan synthesis only in the presence
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is E CL
10 c 0
5
,IIf--4-----*-----24- 50'
9
I
2o 0
20
40
60
GTase (ui)
80
100
FIG. 2. Glucan synthesis from ['4Cjglucose-labeled sucrose by extracellular GTase bound to the surface of heat-treated S. mutans B13 cells and cellfree, water-insoluble glucans and subsequent adherence to glass surfaces. To 1.0 mg of heat-treated cells or insoluble glucans was added 5 to 100 Ml of extracellular GTase (specific activity, 56.6 mU/Ml), and the mixture was incubated for 10 min at room temperature. The suspension was centrifuged, washed twice with KPB, and assayed for ability to adhere and celland glucan-associated GTase activity, as described in the text. Symbols: (-4*) cell-bound ["Ciglucan synthesis; (O-O) glucan-bound [''Ciglucan synthesis; ( ----4) adherence ofcells due to new glucan synthesis; (O----O) adherence of glucans due to new glucan synthesis.
of sucrose. Although larger amounts of GTase were required to obtain adherence by the glucans as compared to the cells, the rate of adherence gradually reached about the same level after a addition of GTase. Other polysaccharides such as starch, cellulose, inulin, and
20-p1
Sephadex G-25 resin did not bind detectable GTase. DISCUSSION In this study, we observed the occurrence of strong cell-associated GTase activity in TH broth cultures of S. mutans. We failed in many cases to recover significant GTase from the culture supernatants by 50% ammonium sulfate precipitation. However, the recovery of GTase fluctuated depending on the lot of TH preparation used. In some cases it was possible to obtain a fraction which had strong GTase activity from TH broth cultures (10, 15, 18, 20; Hamada et al., in press). When S. mutans was grown in sucrosefree TTY broth, significantly higher GTase activity was detected in the culture supernatant as compared with several commercial broth media, except BHI (Table 2). Other investigators (3, 11, 16, 23) also demonstrated strong free GTase activity of S. mutans grown in media similar to TTY broth or in a chemically defined medium. Spinell and Gibbons (28) reported that growth of S. mutans in sucrose-containing medium promoted cell-associated GTase activity as compared to free GTase activity in the culture supernatant. The addition of sucrose to TTY broth
597
resulted in significant GTase activity on the cell surface from the culture supernatant (Table 3). The results suggested that most commercial preparations of TH broth contained trace amounts of sucrose, as does TS broth. This concept was supported by the following findings. (i) If sucrose-containing TH was pretreated with yeast invertase before autoclaving, the resultant S. mutans culture contained increased amounts of cell-free GTase activity (Table 1). Similar abilities of invertase to degrade contaminating sucrose in culture media were observed by M. Inoue and H. Mukasa (personal communications). (ii) If rabbits were immunized with THor TS-grown cells of S. mutans, the rabbits almost always elaborated significant antibodies against glucan/dextran (unpublished data), indicating that the use of TH broth as well as TS broth for preparation of serotype-specific vaccines for immunization should be discouraged. TTY broth is recommended not only for preparations of GTase, but also for mass culture of various species of streptococci. Our experiences showed that recovery of bacterial cells was 1.0 + 0.3 g (dry weight)/liter of TTY broth culture. The presence or addition of sucrose in broth media permitted S. mutans to synthesize waterinsoluble, cell-bound glucans, which should be responsible for the binding site of free GTase. This hypothesis is supported by the following experimental results. (i) Cells grown in TH broth containing S. violacea dextranase retained no significant cell-associated GTase as compared to those grown in TH broth without dextranase (Table 4). Previous studies also revealed that dextranase prevented the formation of cellbound glucans (9, 10, 19, 24). (ii) Lyophilized cells grown in sucrose-containing TTY broth retained increasing cell-associated GTase concomitant with sucrose concentration in the broth (Tables 3 and 5). Furthermore, the heat-treated cells bound free GTase. The degree of binding increased with the concentration of sucrose in TTY broth in which the test strains had been cultivated (Table 5). (iii) Pretreatment of the heat-treated, TH-grown cells with dextranase or NaOH resulted in marked loss of GTase-binding ability by the cells (Tables 4 and 6). (iv) Pretreatment with antiglucan serum, but not with anti-B13 serum or R. communes agglutinin 1 (8), significantly reduced the binding of GTase to the cell surface (Table 6). The anti-B13 serum retained a marked agglutinating or precipitating titer and reacted with hot saline-extracted antigens from serotype a and g S. mutans strains in addition to the homologous B13 antigen, indicating the presence of anti-d and anti-d-a-g antibodies (S. Hamada. N. Masuda, and S. Kotani, Arch Oral Biol., in press). The results shown in
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Table 6 suggest that the type d-specific polysaccharide antigen, which is a polymer of galactose and glucose (14), does not participate in the binding of GTase to the cell surface. The adherence inhibition by anti-B13 serum would be due to the formation of heavy aggregates of the cells, which resulted in the prompt sedimentation of the cells and the physical inability of the cells to be contiguous to the smooth surface, which is a prerequisite for adherence. Although it has been claimed that both glucan and the a-d site of the serotype a polysaccharide antigen play essential roles in the binding of GTase and subsequent adherence of S. mutans HS6 cells (18, 19), glucan may possess a greater affinity for the enzyme (27). (v) Binding of extracellular GTase to cellfree, water-insoluble glucans (Fig. 2) renders definite support to this hypothesis. Several other investigators have reported the binding or adsorption of GTase to insoluble glucans elaborated by extracellular GTase of S. mutans (12, 17, 22), but without special reference to the adherence ability of S. mutans. McCabe and Smith (17) reported that the addition of clinical dextran to an insoluble glucan-crude GTase complex column resulted in desorption of reversibly bound GTase activity. It has also been found that addition of glucan to extracellular GTase resulted in the formation of salt-stable enzyme-glucan complexes, indicating a high affinity of GTase for glucans (4-6). Germaine et al. (5) have emphasized the roles of glucan and enzyme aggregation in enzyme binding to the cell surface. Furthermore, it is of interest to note that many "purified" GTase preparations contain a tightly bound carbohydrate (probably glucan) moiety (2, 4, 20). However, low-molecular-weight GTase free of contaminating glucan may be obtained by growing the bacterium in a chemically defined sucrosefree medium (23). The fact that treatments of S. mutans cells with protease, sodium dodecyl sulfate, and NaOH markedly reduced its ability to adhere to a glass surface suggests that proteinaceous surface components could function as another important factor in adherence (Table 6). The technique used in this experiment would be applicable to a screening test for possible plaquepreventing agents. In view of the above-mentioned data, we believe that the precise binding site of GTase is surface glucan on the cells, which is synthesized during growth in sucrose-containing medium by the action of GTase near the cell surface. The ability of S. mutans cells to colonize various smooth surfaces may be ascribed to the adherent and insoluble glucans synthesized from sucrose by the bound GTase (26; Hamada et al., Micro-
INFECT. IMMUN.
biol. Immunol., in press). In this connection, previous experiments by these authors (M. Torii, S. Hamada, and S. Kotani, Abstr. 19th Annu. Meet. Jpn. Assoc. Oral Biol., Abstr. 2C1336, 1977) revealed that many clinically isolated strains of S. sanguis also bound the cell-free GTase produced by S. mutans B13, synthesized cell-bound glucans, and produced adherence in the presence of sucrose. Adherence of few strains of S. sanguis due to the GTase of S. mutans has been reported by other laboratories (13, 18). The presence of a variety of bacteria in dental plaque, a complex ecosystem, may be explained by these results. ACKNOWLEDGMENTS We are grateful to Rosemary Linzer (State University of New York at Buffalo, Buffalo, N.Y.) for critical reading of the manuscript. This work was supported by a grant (257464) from the Ministry of Education, Science and Culture of Japan.
LITERATURE CITED 1. Bozzola, J. J., M. C. Johnson, and I. L. Shechmeister. 1977. Ultrastructural localization of sucrase in Streptococcus mutans GS-5 and an extracellular polysaccharide mutant: a comparative cytochemical and immunocytochemical study. Infect. Immun. 17:447-457. 2. Ciardi, J. E., A. J. Beaman, and C. L. Wittenberger. 1977. Purification, relation, and interaction of the glucosyltransferases of Streptococcus mutant 6715. Infect. Immun. 18:237-246. 3. Chassy, B. M., J. R. Beall, R. M. Bielawski, E V. Porter, and J. A. Donkersloot. 1976. Occurrence and distribution of sucrose-metabolizing enzymes in oral streptococci. Infect. Immun. 14:408-415. 4. Germaine, G. R., A. M. Chludzinski, and C. F. Schachtele. 1974. Streptococcus mutans dextransucrase: requirement for primer dextran. J. Bacteriol 120:287-294. 5. Germaine, G. R., S. K. Harlander, W. S. Leung, and C. F. Schachtele. 1977. Streptococcus mutans dextransucrase: functioning of primer deztran and endogenous deztranase in water-soluble and water-insoluble glucan synthesis. Infect. Immun. 16:637-648. 6. Germaine, G. R., and C. F. Schachtele. 1976. Streptococcus mutans dextransucrase: mode of interaction with high-molecular-weight dextran and role in cellular agregation. Infect. Immun. 13:365-372. 7. Gibbons, R. J., and J. Van Houte. 1975. Bacterial adherence in oral microbial ecology. Annu. Rev. Microbiol. 29:19-44. 8. Hamada, S., K. Gill, and H. D. Slade. 1977. The binding of lectins to Streptococcus mutans cells and type-specific polysaccharides and effect on adherence. Infect. Immun. 18:708-716. 9. Hamada, S., J. Mizuno, Y. Murayama, T. Ooshima, N. Masuda, and S. Sobue. 1975. Effect of dextranase on the extracellular polysaccharide synthesis of Streptococcus mutans: Chemical and scanning electron microscopy studies. Infect. Immun. 12:1415-1425. 10. Hamada, S., and H. D. Slade. 1Q76. Adherence of serotype e Streptococcus mutans and the inhibitory effect of Lancefield group E and S. mutans type e antiserum. J. Dent. Res. 55:C65-C74. 11. Janda, W., and H. K. Kuramitsu. 1976. Regulation of extracellular glucosyltransferase production and the relationship between extracellular and cell-associated ac-
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tivities in Streptococcus mutans. Infect. Immun. 14:191-202. 12. Koga, T., and M. Inoue. 1977. A possible interaction on variations in adherence ability of Streptococcus mutans AHT mutants. Jpn. J. Oral Biol. 19:311-323. 13. Kuramitsu, H., and IL Ingersoll. 1977. Molecular basis for the different sucrose-dependent adherence properties of Streptococcus mutans and Streptococcus sanguis. Infect. Immun. 17:330-337. 14. Linzer, R., and H. D. Slade. 1974. Purification and characterization of Streptococcus mutans group d cell wall polysaccharide antigen. Infect. Immun. 10:361-368. 15. Linzer, R., and H. D. Slade. 1976. Characterization of an anti-glucosyltransferase serum specific for insoluble glucan synthesis by Streptococcus mutans. Infect. Immun. 13:494-500. 16. McCabe, M. M., and E. E. Smith. 1973. Origin of the cell-associated dextransucrase of Streptococcus mutans. Infect. Immun. 7:829-838. 17. McCabe, M. M., and E. E. Smith. 1977. Specific method for the purification of Streptococcus mutans dextransucrase. Infect. Immun. 16:760-765. 18. Mukasa, H., and H. D. Slade. 1973. Mechanism of adherence of Streptococcus mutans to smooth surfaces. I. Roles of insoluble dextran-levan synthetase enzymes and cell wall polysaccharide antigen in plaque formation. Infect. Immun. 8:555-562. 19. Mukasa, H., and H. D. Slade. 1974. 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. 20. Mukasa, H., and H. D. Slade. 1974. Mechanism of adherence of Streptococcus mutans to smooth surfaces. III. Purification and properties of the enzyme complex responsible for adherence. Infect. Immun. 10:1135-1145. 21. Nicolson, G. L., J. Blaustein, and M. E. Etzler. 1974.
22.
23.
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25.
26.
27.
28.
29.
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Characterization of two plant lectins from Ricinus communis and their quantitative interaction with a murine lymphoma. Biochemistry, 13:196-204. Robrish, S., W. Reid, and M. I. Krichevsky. 1972. Distribution of enzymes forming polysaccharide from sucrose and the composition of extracellular polysaccharide synthesized by Streptococcus mutans. Appl. Microbiol. 24:184-190. Schachtele, C. F., S. K. Harlander, and G. R. Germaine. 1976. Streptococcus mutans dextransucrase: availability of disaggregated enzyme after growth in a chemically defined medium. Infect. Immun. 13:1522-1524. Schachtele, C. F., R. H. Staat, and S. K. Harlander. 1975. Dextranases from oral bacteria: inhibition of water-insoluble glucan production and adherence to smooth surfaces by Streptococcus mutans. Infect. Immun. 12:309-317. Scherp, H. W. 1971. Dental caries: prospect for prevention. Science 173:1199-1205. Slade, H. D. 1976. In vitro models for the study of adherence of oral streptococci, p. 21-38. In W. H. Bowen, R. J. Genco, and J. C. O'Brien (ed.), Immunologic aspects of dental caries (Spec. Suppl. Immunol. Abstr.). Information Retrieval, Inc., Washington, D.C. Slade, H. D. 1977. Cell surface antigenic polymers of Streptococcus mutans and their role in adherence of the microorganism in vitro p. 411-416. In D. Schlessinger (ed.), Microbiology-1977. American Society for Microbiology, Washington, D.C. Spinell, D. M., and R. J. Gibbons. 1974. Influence of culture medium on the glucosyl transferase and dextran-binding capacity of Streptococcus mutans 6715 cells. Infect. Immun. 10:1448-1451. Tsuchiya, H. M., H. J. Koepsell, J. Corman, G. Bryant, M. 0. Bogard, V. H. Feger, and R. W. Jackson. 1952. The effect of certain cultural factors on production of dextransucrase by Leuconostoc mesenteroides. J. Bacteriol. 64:521-526.
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0019-9567/78/0020-0592$02.00/0 Copyright © 1978 American Society for Microbiology
Printed in U.S.A.
Effect of Sucrose in Culture Media on the Location of Glucosyltransferase of Streptococcus mutans and Cell Adherence to Glass Surfaces SHIGEYUKI HAMADA* AND MITSUO TORII Department of Oral Microbiology, Osaka University Dental School, Kita-ku, Osaka, 530 Japan Received for publication 20 January 1978
Streptococcus mutans strain B13 (serotype d) almost exclusively produced free glucosyltransferase (GTase) in the culture supernatant when grown in sucrosefree TTY broth medium, which was composed of Trypticase (Baltimore Biological Laboratory [BBL] Cockeysville, Md.), tryptose (Difco Laboratories, Detroit, Mich.), yeast extract (BBL), salts, and 1% glucose. Organisms grown in sucrosefree TTY broth retained very weak cell-associated GTase activity and did not adhere significantly to glass surfaces in the presence of exogenous sucrose. If sucrose was added to TTY broth, however, GTase was found on the cell surface where cell-bound, water-insoluble glucans were synthesized. Most commercially available products of Todd-Hewitt broth were found to contain trace amounts of sucrose, as did Trypticase soy broth (BBL), whereas brain heart infusion broth (Difco and BBL) was found to be essentially free of sucrose. Almost all detectable GTase activity was cell associated when S. mutans B13 was grown in ToddHewitt or trypticase soy broth. Heat-treated B13 cells grown in Todd-Hewitt broth and cell-free, water-insoluble glucans bound free GTase and produced marked adherence in the presence of sucrose. Experiments strongly suggest that the binding sites for free GTase are the surface glucans, and cell-associated and extracellular GTases are most likely alternate states of the same enzyme protein. The strong cariogenic activity of Streptococcus mutans has been ascribed to the ability of this bacterium to synthesize water-insoluble glucans from sucrose that adhere to various smooth surfaces (7, 25-27). Synthesis of the glucans is mediated by the enzymatic action of cell-free (extracellular) and/or cell-bound glucosyltransferase(s) (GTase[s]). The location of S. mutans GTase appears to be complicated (1, 3, 11, 16, 20, 22, 23, 28) and remains to be elucidated. It has been reported that extracellular GTase of S. mutans B13 was bound to the cell surface of both viable and heat-treated S. mutans but not to the cells of other species of bacteria, with a few exceptions (26; S. Hamada, Y. Kobayashi, and H. D. Slade, Microbiol. Immunol., in press). After the free, unbound GTase was removed from the reaction mixture, S. mutans cells produced significant adherence to glass surfaces in the presence of sucrose and synthesized cellbound ['4C]glucan from ["4C]sucrose. Todd-Hewitt (TH) broth is preferentially used for cultivation of various streptococci; therefore, we have used this broth medium for cultivation of S. mutans and for the production of extracellular GTase from the culture supernatant. In our experience, the free GTase activ-
ity in the centrifuged supernatant of TH broth cultures of S. mutans varied from lot to lot, but the activity was usually significantly less than that of brain heart infusion (BHI) broth cultures. The aim of this study is to investigate the differences in the distribution of free and cellassociated GTase in various cultures of S. mutans. The results are discussed in terms of adherence and cell-bound glucan synthesis of S. mutans. MATERIALS AND METHODS Bacterial strains. S. mutans strain B13 (serotype d) (14) was used in the present study. This strain was kindly supplied by S. Edwardsson (University of Lund, Malmo, Sweden). Culture media. To prepare extracellular GTase of S. mutans, TH broths (Difco Laboratories, Detroit, Mich., and Baltimore Biological Laboratory [BBL], Cockeysville, Md.), and Trypticase soy (TS) broth (BBL), tryptic soy (TS) broth (Difco), and BHI broths (BBL and Difco) were used. A broth medium designated TTY was devised as a medium essentially free of sucrose. TTY broth contained (per liter): trypticase (BBL), 15 g; tryptose (Difco), 4 g; yeast extract, 4 g; K2HPO4, 2 g; KH2PO4, 5 g; Na2CO3, 2 g; NaCl, 2 g; and glucose, 10 g. Glucose was autoclaved separately and combined with the broth aseptically. Final reaction of 592
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the medium was at pH 7.4 ± 0.2 at 250C. Media were routinely inoculated (1%, vol/vol) from a stationaryphase culture, and cells were grown for 18 h at 370C. Cell growth and distribution of GTases. S. mutans B13 was serially transferred several times in each medium to adapt the bacterial cells to the medium in which they would be propagated. Cells were grown in broth medium for 18 h at 370C in 10-ml volumes after inoculation with 0.5 ml of an overnight culture of the cells grown in the same medium. At the end of growth, the culture was vigorously vibrated, using a Vortex mixer for a few minutes, and sample (500 ILI) of the broth culture was pipetted out. The culture was mixed with 2.0 ml of 0.05 M potassium phosphate buffer, pH 6.8 (KPB), followed by centrifugation. The centrifuged cells were resuspended in 3.0 ml of KPB by sonic oscillation at a maximum amplitude for 10 s, using a Tomy UR-150P ultrasonicator (Tomy Seiko Co., Ltd., Tokyo), and the optical density at 550 nm was read to compare the cell growth in the various media used. To assess the distribution of GTase in the broth culture of S. mutans, a sample of the broth culture (5.0 ml) was centrifuged in the cold. After the collected cells were washed two times with 5.0 ml of KPB, the cells were mixed with 900 Ad of KPB containing 0.02% Merthiolate and 100 pl of ["4C]glucose-labeled sucrose solution (59,000 dpm) and incubated at 370C for various time intervals to measure cell-associated GTase activity. The radioactive sucrose solution (hereafter called ['4C]sucrose) was prepared by mixing 100 pi (2.0 ,uCi) of ['4C]glucose-labeled sucrose (New England Nuclear, Boston, Mass.; specific activity, 275 ,iCi/mmol) and 342 mg of unlabeled sucrose in 7.9 ml of KPB. The cells were collected by centrifugation and washed two times with 2.5 ml of KPB. The final cell pellet was transferred to a scintillation vial, using 2.5 ml of scintillation fluid, and the tube was rinsed three times (final volume, 10 ml). The scintillation fluid contained 4.0 g of 2,5-diphenyloxazole (Dotite DPO, Wako Pure Chemicals, Osaka), 0.1 g of 1,4-bis-[2]-(5phenyloxazolyl)benzene (Dotite POPOP, Wako Pure Chemicals) in 600 ml of xylene, 400 ml of Nonion NS210 (Nihon Yushi Ltd., Tokyo), and 100 ml of 10% ethanol. Samples were counted for radioactivity due to cell-associated GTase with an Aloka model LSC673 liquid scintillation spectrometer (Aloka Ltd., Tokyo) (count I). Cell-free GTase synthesizing water-insoluble glucan was assayed as follows. The culture supernatant (500 above, was pl), obtained by centrifugation100as described mixed with 400 PIl of KPB, pl of the ['4C]sucrose solution, and Merthiolate to a final concentration of 0.02%. After the reaction mixture was incubated at 371C for each desired time interval, the mixture was filtered by vacuum onto 2.5-cm glass filter disks (Whatman GF/A) held in a glass filter holder (Millipore Corp., Bedford, Mass.). The sample tube was washed twice with 1.5 ml of KPB, and each washing was filtered over the sample. In some experiments, total glucan was assayed after an equal amount of ethanol was added to the sample to precipitate watersoluble glucans, as described previously (10, 15, 22). The air-dried filter disks were placed in vials with 10 ml of the scintillation fluid, and the radioactivity was measured (count II) as described above. The relative
593
ratio of activity of cell-free GTase versus cell-associated GTase was calculated as follows: (10 x disintegrations per minute of count II)/disintegrations per minute of count I. As a matter of convenience, the disintegrations per minute after 18 h of incubation were counted as an indicator of GTase activity of a sample in the present study. Comparison of cell-associated GTase activities in cells grown in TTIY broth containing different concentrations of sucrose or in TH broth containing dextranase. S. mutans B13 was grown at 370C for 18 h in TTY broth containing 0, 0.005, 0.01, 0.05, 0.1, or 0.5% (final concentration) sucrose plus 1% glucose. B13 cells were also grown in TH broth containing filter-sterilized dextranase AD17 (0.1 to 0.5 U/ml of culture, final concentration) (9). Cells were collected by centrifugation, washed three times with water, and lyophilized. Cells were resuspended in KPB at a concentration of 5.0 mg (dry weight)/ml. For assay of cell-associated GTase, a sample (400 1A) of the suspension was mixed with 500 gil of KPB and 100 gil of the ['4C]sucrose solution and incubated at 370C for 18 h. Measurement of the radioactivity was performed as described above. Preparation of cell-free GTase. Cell-free GTase was obtained from the culture supernatant of S. mutans B13 grown in TTY broth by 50% ammonium sulfate precipitation, as previously described (10, 15). The GTase preparation produced water-insoluble product almost exclusively, and no significant watersoluble glucan was detected by the method used in this study. The protein content of the preparation was 13.8 mg/ml, and the activity was 4.1 dextransucrase units/mg of protein (29). No detectable fructosyltransferase activity was demonstrated as determined by the incorporation of radioactivity into ethanol-insoluble polysaccharides from ['4C]fructose-labeled sucrose. A single preparation of GTase was used throughout the study. Preparation of water-insoluble glucans. A 5% sucrose solution in KPB (400 ml) was mixed with cellfree GTase (8.0 ml) of S. mutans B13 and incubated at 371C for 18 h. The mixture was then centrifuged at 10,000 x g for 30 min to collect water-insoluble glucan, washed twice with water (200 ml each), and lyophilized. Binding ability of GTase to S. mutans cells and cell-free water-insoluble glucan. Lyophilized cell and water-insoluble glucan suspensions (5.0 mg [dry weight]/ml of water) were held at 100'C for 10 min to inactivate cell-associated GTase, washed two times with water, and lyophilized. Heated cells and glucans were resuspended in KPB (5.0 mg [dry weight]/ml of KPB) by sonic oscillation. Desired amounts of GTase were added to 0.4 ml of the suspension, and the final volume was adjusted to 2.0 ml with KPB. After standing at room temperature for 10 min, the mixture was centrifuged, and the cells were washed twice with 2.0 ml of KPB. KPB (900 gl) and 100 gl of the ['4C]sucrose solution were then added to the cell pellet. After the mixture was incubated at 370C for 18 h, it was centrifuged. The pellet was washed twice with 2.0 ml of KPB, mixed with the scintillation fluid, and counted as described above. Binding of GTase (20 gl) to other polysaccharides,
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HAMADA AND TORII
e.g., starch (Merck AG, Darmstadt, Germany), cellulose (average particle size, 20 jum; Sigma Chemical Co., St. Louis, Mo.), Sephadex G-25 (fine, Pharmacia Fine Chemicals Inc., Piscataway, N. J.), and inulin (Wako Pure Chemicals, Osaka), was also determined as described above. Starch and inulin were essentially insoluble in cold KPB. Adherence of S. mutans cells and water-insoluble glucans. Determination of the adherence of heat-treated cells and cell-free, water-insoluble glucans to a glass surface in the presence of sucrose was performed according to the procedure previously reported (10, 19), with slight modification. Heat-treated cells or water-insoluble glucan (1.0 mg, dry weight) and increasing amounts of GTase were first incubated in KPB (2.0 ml) for 10 min at room temperature. Cells were collected by centrifugation and washed two times with KPB to remove unbound GTase. Buffered sucrose (2.4 ml) was added and stirred. After incubation overnight at a 300 angle, cell and glucan adherences to the glass surface were determined by reading the optical density at 550 nm. Effect of various treatments on the binding of GTase and subsequent adherence. Heat-treated cells, grown in TH broth containing trace amounts of sucrose, were pretreated at 370C for 60 min with various reagents as follows: Spicaria violacea dextranase (9), Penicillium sp. dextranase (Sigma), protease (subtilisin, Sigma), and lysozyme (Sigma) were added in a final concentration of 0.2% (wt/vol); NaOH and HCl in a concentration of 1.0 N; sodium dodecyl sulfate, Brij 35 (Sigma), Triton X-100 (Wako Pure Chemicals), and concanavalin A (Pharmacia) at 1.0%; and Ricinus communes agglutinin I (8, 21) at 0.5%. Type d antiserum (anti-S. mutans B13 whole-cell serum) and antiglucan serum were prepared as previously described (10), and 40 ,ul of the sera was used in the experiment. After the reaction mixture (2.0, final volume) was centrifuged and washed three times with KPB, it was reconstituted as described, and the binding of GTase and subsequent adherence were assayed. For determining the cell adherence and binding of GTase to the cell surface, 1.0- and 2.0-mg (dry weight) samples of heat-treated cells respectively, were used.
RESULTS Rate of cell-free and cell-bound glucan synthesis. Figure 1 shows typical curves of glucan formation by a cell-free culture supernatant and a washed cell suspension from overnight cultures of S. mutans B13 which had been grown in TH (Difco) and TTY broths as a function of time. Cell-bound ['4C]glucan production increased almost linearly up to 4 h with THgrown cells and up to 6 h with TTY-grown cells, after which little increase was observed. On the other hand, water-insoluble glucan production by the cell-free culture supernatant reached plateau levels at 8 to 10 h and again at 18 to 22 h, indicating the presence of multiple forms of GTases in S. mutans cultures. In this connection, there was no substantial difference in the amounts of water- and ethanol-insoluble (total)
INFECT. IMMUN.
Incubation (h) FIG. 1. Water-insoluble ['4Clglucan synthesis from ['4Clglucose-labeled sucrose by cell-free culture liquor and washed cell suspension as a function of time. Cells of S. mutans B13 were grown overnight in TH and TTY broths. After samples of the culture (5.0 ml) were centrifuged in the cold, the collected cells were washed twice with KPB and assayed for cellbound, water-insoluble ['4CJglucan. Samples (0.5 ml) of the centrifuged supernatant were assayed for total and water-insoluble glucan as described in the text. Both cell-free and cell-associated GTase synthesized primarily water-insoluble glucans. Therefore, only water-insoluble glucan production is depicted in this figure. Symbols: (------) cell-free, water-insoluble glucan synthesis by extracellular culture liquor; (-) cell-bound, water-insoluble glucan synthesis by cellassociated GTase; (0) TTY-grown culture; (0) THgrown culture.
glucans synthesized for each incubation interval. Therefore, for simplicity, each water-insoluble glucan synthesized (disintegrations per minute) after incubation with ["4C]sucrose for 18 h was referred to as GTase activity of a test sample. Comparison of growth and GTase production of S. mutans in various culture media. The degree of growth and GTase production of S. mutans B13 in TTY broth were defined as 100%, because preliminary experiments revealed that TTY broth cultures of various S. mutans strains always gave luxuriant growth and high GTase production in the culture supernatant. The relative growth and GTase production in different culture media and different lots of a medium of the same commercial brand are shown in Table 1. Considerably less cell growth (ca. 40%) was obtained in TH, BHI, and TS broths as compared with that in TTY broth. Strong or moderate GTase activity was demonstrated in TTY or BHI broth cultures of S. mutans B13, respectively, whereas TS or TH broth cultures generally produced relatively
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595
little GTase in the culture liquor. Only one lot of TH broth medium examined gave significant GTase activity in the culture liquor. When TH and TS broths were fortified with 0.8% glucose (1.0%, final concentration) as a supplemental carbon source, cell growth increased to nearly 70% of that of the TTY culture. However, extracellular GTase activity decreased to less than 10% of that in the TTY culture. Similar findings were also obtained when S. mutans MT703 (type e) was used as a test strain instead of strain B13. Ratio of cell-free and cell-associated GTases of S. mutans in various culture media. Table 2 shows that when S. mutans B13 was grown in TTY or BHI broth, most GTase activity was released from the growing cells into
the culture supernatant. On the other hand, TS broth- or TH broth-grown cells retained significant cell-associated GTase activity, and the ratio of cell-free GTase to cell-associated GTase was far smaller as compared with that in TTY or BHI broth cultures. However, the culture grown in one lot of TH broth (lot 525121) produced mainly free GTase, with activity comparable to those in the TTY and BHI broth cultures. The fact that S. mutans grown in TH broth had properties similar to those in TS broth with respect to distribution of GTase indicated that the TH broth may contain some sucrose, as does the TS broth (28). To determine the effect of sucrose on the distribution of GTase, B13 cells were grown in TTY broths containing various amounts of sucrose. Table 3 shows a direct reTABLE 1. Cell growth and GTase production by S. lationship between the recovery of GTase assomutans B13 grown in various culture media ciated with the cell surface and the increase of sucrose in TTY media. When sucrose was added Cell Extracellugrowth lar GTase to BHI broth, a similar shift in GTase recovery Medium was obtained. ODss)a activity)' Effect of dextranase on cell-associated TTYC 100 100 GTase activity. S. mutans was grown in TH BHI (Difco) 44 39 broth, which resulted in the production of sig35 BHI (BBL) 61 nificant cell-associated GTase (cf. Table 2). Ta29 Tryptic soy broth (Difco) 13 ble 4 shows that incorporation of dextranase into 36 Trypticase soy broth 14 the TH broth markedly prevented localization (BBL) of GTase on the cell surface but did not inhibit TH (Difco) growth of-the cells. The GTase activity in the 525121id40 61 culture supernatant was not estimated because 617841 36 18 34 476715 24 of the presence of the added dextranase. 621966 36 20 Cell-associated GTase activity of su625032 NDe 26 crose-grown S. mutans cells and binding of TH (BBL) 36 38 GTase to the heat-treated cells. Washed cells a OD550, Optical density at 550 nm. (2.0 mg, dry weight) of S. mutans B13 grown in Incorporation of 14C into water-insoluble glucan sucrose-free TTY broth did not produce signififrom ['4C]glucose-labeled sucrose was assayed. cant cell-bound and water-insoluble glucans See text for composition. when incubated with sucrose (Table 5) and d Lot number. caused no sucrose-induced agglutination during e ND, Not determined. overnight incubation. However, cells grown in TTY broth containing increasing amounts of TABLE 2. Localization of GTase activity of S. sucrose retained cell-associated GTase and synmutans grown in various culture media thesized glucans. The enzyme activities inRatio of GTase activcreased concomitant with the sucrose added to Medium ity: extracellular/cellthe TTY broth. Table 5 also shows that heatassociated '
TTY BHI (Difco) BHI (BBL) Tryptic soy broth (Difco) Trypticase soy broth (BBL) TH (Difco) 525121a 617841 476715 621966 625032 630836 Lot number. a
35.6 29.5 9.2
1.8 1.4
28.6 1.5 2.0 1.6 2.0 1.9
TABLE 3. Effect of sucrose on the localization of GTase in TTY-grown S. mutans B13 Sucrose added in TTY broth (% final concn) 0 0.0005
0.001 0.005 0.01 0.05 0.1
Ratio of GTase activity:
extracellular/cell-associated 37.8 30.9 19.5 13.8 9.90 1.53 1.29
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HAMADA AND TORII
TABLE 4. Relative activity of cell-associated GTase of S. mutans grown in TH broth containing dextranasea Dextranase added (U/ml of TH broth)
Cell growth (%
OD)b
Cell-associated GTase
(% activity) 100 25.6 17.4 13.0
100 None 100 0.10 97 0.25 97 0.50 a S. violacea dextranase (500 U/mg of protein) was used. See reference 9. b OD550, Optical density at 550 nm.
TABLE 5. Cell-associated GTase activity and GTase-binding ability of sucrose-grown S. mutans B13 cells Glucan synthesized (dpm)b Sucrose added
Cell-assoofino
(%)a GTase of nontreated cells
Binding of GTase to heated cells
679 0 1,151 1,258 3,557 0.005 1,746 0.01 5,061 4,936 0.05 8,768 0.1 7,357 9,756 9,648 0.5 10,306 a TTY broth was used as a base medium. b Per 2.0 mg (dry weight) of cells, or per test tube.
treated cells (2.0 mg, dry weight) grown in TTY broth containing sucrose bound cell-free GTase and produced cell-bound glucan. The degree of binding of GTase increased with increasing amounts of cell-bound glucan synthesized during growth in TTY-sucrose broths. Inhibition of cell adherence and GTase binding by chemical/enzymatic treatments. Treatment ofheat-treated B13 cells with various chemicals, enzymes, antisera, and lectins (Table 6) has supplied significant data concerning the nature ofthe GTase-binding components present on the cell surface of S. mutans. Treatment with NaOH and dextranases resulted in an almost complete loss of adherence and GTasebinding abilities, whereas protease and sodium dodecyl sulfate treatment removed the adherence ability but not the GTase-binding ability. On the other hand, treatment with glucan antibody significantly reduced GTase binding but not cell adherence. B13 whole-cell antiserum strongly agglutinated cells during incubation, and the cells redispersed poorly after extensive washing and subsequent sonic treatment. Therefore, the agglutinated cells could not adhere to the glass surface because of the heavy weight of the aggregates, even though the cells bound GTase and synthesized a significant amount of
TABLE 6. Effect of various treatments on the binding of GTase from S. mutans B13 and subsequent adherence of heat-treated B13 cellsa AdherTreatment of heat-treated B13 cells (final concn)
Binding of GTase (%)
ence
to
a
glass surface (%)
100 100 Control 10 6.5 NaOH (1 N) 110 120 HCO (1 N) 3.7 8.3 Dextranaseb (0.2%) 15 12 Dextranasec (0.2%) 5.2 130 Proteased (0.2%) 100 110 Lysozyme (0.2%) 3.0 84 SDS' (1.0%) 120 150 Brij 35 (1.0%) 110 86 Triton X-100 (1.0%) 100 37 Antiglucan serum (40 p1) 20 100 Anti-B13 serum (401,l) 100 110 RCAIf (0.5%) 100 120 Concanavalin A (0.5%) a Heat-treated (100'C, 10 min) S. mutans B13 cells grown in TH broth (Difco) were treated by various reagents followed by washing with KPB. The reaction mixture was reconstituted, and the binding of GTase (20 p1) and cell adherence to a glass surface were determined as described in the text. The degree of relative GTase binding and cell adherence is expressed as a percentage of that of heated but nontreated control cells. b From S. violacea (9). 'From Penicillium sp. (Sigma). d Subtilisin (Sigma). eSDS, Sodium dodecyl sulfate. f RCAI, R. communes agglutinin I (21).
cell-bound glucans. R. communes agglutinin I also agglutinated the B13 cells, but the cells redispersed after washing and sonic treatment. The R. communes agglutination I-treated cells bound GTase and produced adherence to an extent similar to that of the nontreated control. Various other treatments exerted no significant inhibitory effects on GTase binding and cell adherence. Binding of GTase to heat-treated S. mutans cells and cell-free, water-insoluble glucans and subsequent glucan synthesis and adherence. Both TH-grown cells of S. mutans B13 and cell-free, water-insoluble glucans that had been synthesized from extracellular GTase and unlabeled sucrose bound the extracellular B13 GTase from the TTY broth culture (Fig. 2). De novo ['4C]glucan synthesis on the surface of heat-treated cells and glucans due to GTase binding increased linearly and then gradually reached plateau levels. Both cells and glucans which had bound extracellular GTase markedly adhered to glass surfaces due to de novo glucan synthesis only in the presence
VOL. 20, 1978
SUCROSE AND ENZYME LOCATION OF S. MUTANS
is E CL
10 c 0
5
,IIf--4-----*-----24- 50'
9
I
2o 0
20
40
60
GTase (ui)
80
100
FIG. 2. Glucan synthesis from ['4Cjglucose-labeled sucrose by extracellular GTase bound to the surface of heat-treated S. mutans B13 cells and cellfree, water-insoluble glucans and subsequent adherence to glass surfaces. To 1.0 mg of heat-treated cells or insoluble glucans was added 5 to 100 Ml of extracellular GTase (specific activity, 56.6 mU/Ml), and the mixture was incubated for 10 min at room temperature. The suspension was centrifuged, washed twice with KPB, and assayed for ability to adhere and celland glucan-associated GTase activity, as described in the text. Symbols: (-4*) cell-bound ["Ciglucan synthesis; (O-O) glucan-bound [''Ciglucan synthesis; ( ----4) adherence ofcells due to new glucan synthesis; (O----O) adherence of glucans due to new glucan synthesis.
of sucrose. Although larger amounts of GTase were required to obtain adherence by the glucans as compared to the cells, the rate of adherence gradually reached about the same level after a addition of GTase. Other polysaccharides such as starch, cellulose, inulin, and
20-p1
Sephadex G-25 resin did not bind detectable GTase. DISCUSSION In this study, we observed the occurrence of strong cell-associated GTase activity in TH broth cultures of S. mutans. We failed in many cases to recover significant GTase from the culture supernatants by 50% ammonium sulfate precipitation. However, the recovery of GTase fluctuated depending on the lot of TH preparation used. In some cases it was possible to obtain a fraction which had strong GTase activity from TH broth cultures (10, 15, 18, 20; Hamada et al., in press). When S. mutans was grown in sucrosefree TTY broth, significantly higher GTase activity was detected in the culture supernatant as compared with several commercial broth media, except BHI (Table 2). Other investigators (3, 11, 16, 23) also demonstrated strong free GTase activity of S. mutans grown in media similar to TTY broth or in a chemically defined medium. Spinell and Gibbons (28) reported that growth of S. mutans in sucrose-containing medium promoted cell-associated GTase activity as compared to free GTase activity in the culture supernatant. The addition of sucrose to TTY broth
597
resulted in significant GTase activity on the cell surface from the culture supernatant (Table 3). The results suggested that most commercial preparations of TH broth contained trace amounts of sucrose, as does TS broth. This concept was supported by the following findings. (i) If sucrose-containing TH was pretreated with yeast invertase before autoclaving, the resultant S. mutans culture contained increased amounts of cell-free GTase activity (Table 1). Similar abilities of invertase to degrade contaminating sucrose in culture media were observed by M. Inoue and H. Mukasa (personal communications). (ii) If rabbits were immunized with THor TS-grown cells of S. mutans, the rabbits almost always elaborated significant antibodies against glucan/dextran (unpublished data), indicating that the use of TH broth as well as TS broth for preparation of serotype-specific vaccines for immunization should be discouraged. TTY broth is recommended not only for preparations of GTase, but also for mass culture of various species of streptococci. Our experiences showed that recovery of bacterial cells was 1.0 + 0.3 g (dry weight)/liter of TTY broth culture. The presence or addition of sucrose in broth media permitted S. mutans to synthesize waterinsoluble, cell-bound glucans, which should be responsible for the binding site of free GTase. This hypothesis is supported by the following experimental results. (i) Cells grown in TH broth containing S. violacea dextranase retained no significant cell-associated GTase as compared to those grown in TH broth without dextranase (Table 4). Previous studies also revealed that dextranase prevented the formation of cellbound glucans (9, 10, 19, 24). (ii) Lyophilized cells grown in sucrose-containing TTY broth retained increasing cell-associated GTase concomitant with sucrose concentration in the broth (Tables 3 and 5). Furthermore, the heat-treated cells bound free GTase. The degree of binding increased with the concentration of sucrose in TTY broth in which the test strains had been cultivated (Table 5). (iii) Pretreatment of the heat-treated, TH-grown cells with dextranase or NaOH resulted in marked loss of GTase-binding ability by the cells (Tables 4 and 6). (iv) Pretreatment with antiglucan serum, but not with anti-B13 serum or R. communes agglutinin 1 (8), significantly reduced the binding of GTase to the cell surface (Table 6). The anti-B13 serum retained a marked agglutinating or precipitating titer and reacted with hot saline-extracted antigens from serotype a and g S. mutans strains in addition to the homologous B13 antigen, indicating the presence of anti-d and anti-d-a-g antibodies (S. Hamada. N. Masuda, and S. Kotani, Arch Oral Biol., in press). The results shown in
598
HAMADA AND TORII
Table 6 suggest that the type d-specific polysaccharide antigen, which is a polymer of galactose and glucose (14), does not participate in the binding of GTase to the cell surface. The adherence inhibition by anti-B13 serum would be due to the formation of heavy aggregates of the cells, which resulted in the prompt sedimentation of the cells and the physical inability of the cells to be contiguous to the smooth surface, which is a prerequisite for adherence. Although it has been claimed that both glucan and the a-d site of the serotype a polysaccharide antigen play essential roles in the binding of GTase and subsequent adherence of S. mutans HS6 cells (18, 19), glucan may possess a greater affinity for the enzyme (27). (v) Binding of extracellular GTase to cellfree, water-insoluble glucans (Fig. 2) renders definite support to this hypothesis. Several other investigators have reported the binding or adsorption of GTase to insoluble glucans elaborated by extracellular GTase of S. mutans (12, 17, 22), but without special reference to the adherence ability of S. mutans. McCabe and Smith (17) reported that the addition of clinical dextran to an insoluble glucan-crude GTase complex column resulted in desorption of reversibly bound GTase activity. It has also been found that addition of glucan to extracellular GTase resulted in the formation of salt-stable enzyme-glucan complexes, indicating a high affinity of GTase for glucans (4-6). Germaine et al. (5) have emphasized the roles of glucan and enzyme aggregation in enzyme binding to the cell surface. Furthermore, it is of interest to note that many "purified" GTase preparations contain a tightly bound carbohydrate (probably glucan) moiety (2, 4, 20). However, low-molecular-weight GTase free of contaminating glucan may be obtained by growing the bacterium in a chemically defined sucrosefree medium (23). The fact that treatments of S. mutans cells with protease, sodium dodecyl sulfate, and NaOH markedly reduced its ability to adhere to a glass surface suggests that proteinaceous surface components could function as another important factor in adherence (Table 6). The technique used in this experiment would be applicable to a screening test for possible plaquepreventing agents. In view of the above-mentioned data, we believe that the precise binding site of GTase is surface glucan on the cells, which is synthesized during growth in sucrose-containing medium by the action of GTase near the cell surface. The ability of S. mutans cells to colonize various smooth surfaces may be ascribed to the adherent and insoluble glucans synthesized from sucrose by the bound GTase (26; Hamada et al., Micro-
INFECT. IMMUN.
biol. Immunol., in press). In this connection, previous experiments by these authors (M. Torii, S. Hamada, and S. Kotani, Abstr. 19th Annu. Meet. Jpn. Assoc. Oral Biol., Abstr. 2C1336, 1977) revealed that many clinically isolated strains of S. sanguis also bound the cell-free GTase produced by S. mutans B13, synthesized cell-bound glucans, and produced adherence in the presence of sucrose. Adherence of few strains of S. sanguis due to the GTase of S. mutans has been reported by other laboratories (13, 18). The presence of a variety of bacteria in dental plaque, a complex ecosystem, may be explained by these results. ACKNOWLEDGMENTS We are grateful to Rosemary Linzer (State University of New York at Buffalo, Buffalo, N.Y.) for critical reading of the manuscript. This work was supported by a grant (257464) from the Ministry of Education, Science and Culture of Japan.
LITERATURE CITED 1. Bozzola, J. J., M. C. Johnson, and I. L. Shechmeister. 1977. Ultrastructural localization of sucrase in Streptococcus mutans GS-5 and an extracellular polysaccharide mutant: a comparative cytochemical and immunocytochemical study. Infect. Immun. 17:447-457. 2. Ciardi, J. E., A. J. Beaman, and C. L. Wittenberger. 1977. Purification, relation, and interaction of the glucosyltransferases of Streptococcus mutant 6715. Infect. Immun. 18:237-246. 3. Chassy, B. M., J. R. Beall, R. M. Bielawski, E V. Porter, and J. A. Donkersloot. 1976. Occurrence and distribution of sucrose-metabolizing enzymes in oral streptococci. Infect. Immun. 14:408-415. 4. Germaine, G. R., A. M. Chludzinski, and C. F. Schachtele. 1974. Streptococcus mutans dextransucrase: requirement for primer dextran. J. Bacteriol 120:287-294. 5. Germaine, G. R., S. K. Harlander, W. S. Leung, and C. F. Schachtele. 1977. Streptococcus mutans dextransucrase: functioning of primer deztran and endogenous deztranase in water-soluble and water-insoluble glucan synthesis. Infect. Immun. 16:637-648. 6. Germaine, G. R., and C. F. Schachtele. 1976. Streptococcus mutans dextransucrase: mode of interaction with high-molecular-weight dextran and role in cellular agregation. Infect. Immun. 13:365-372. 7. Gibbons, R. J., and J. Van Houte. 1975. Bacterial adherence in oral microbial ecology. Annu. Rev. Microbiol. 29:19-44. 8. Hamada, S., K. Gill, and H. D. Slade. 1977. The binding of lectins to Streptococcus mutans cells and type-specific polysaccharides and effect on adherence. Infect. Immun. 18:708-716. 9. Hamada, S., J. Mizuno, Y. Murayama, T. Ooshima, N. Masuda, and S. Sobue. 1975. Effect of dextranase on the extracellular polysaccharide synthesis of Streptococcus mutans: Chemical and scanning electron microscopy studies. Infect. Immun. 12:1415-1425. 10. Hamada, S., and H. D. Slade. 1Q76. Adherence of serotype e Streptococcus mutans and the inhibitory effect of Lancefield group E and S. mutans type e antiserum. J. Dent. Res. 55:C65-C74. 11. Janda, W., and H. K. Kuramitsu. 1976. Regulation of extracellular glucosyltransferase production and the relationship between extracellular and cell-associated ac-
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tivities in Streptococcus mutans. Infect. Immun. 14:191-202. 12. Koga, T., and M. Inoue. 1977. A possible interaction on variations in adherence ability of Streptococcus mutans AHT mutants. Jpn. J. Oral Biol. 19:311-323. 13. Kuramitsu, H., and IL Ingersoll. 1977. Molecular basis for the different sucrose-dependent adherence properties of Streptococcus mutans and Streptococcus sanguis. Infect. Immun. 17:330-337. 14. Linzer, R., and H. D. Slade. 1974. Purification and characterization of Streptococcus mutans group d cell wall polysaccharide antigen. Infect. Immun. 10:361-368. 15. Linzer, R., and H. D. Slade. 1976. Characterization of an anti-glucosyltransferase serum specific for insoluble glucan synthesis by Streptococcus mutans. Infect. Immun. 13:494-500. 16. McCabe, M. M., and E. E. Smith. 1973. Origin of the cell-associated dextransucrase of Streptococcus mutans. Infect. Immun. 7:829-838. 17. McCabe, M. M., and E. E. Smith. 1977. Specific method for the purification of Streptococcus mutans dextransucrase. Infect. Immun. 16:760-765. 18. Mukasa, H., and H. D. Slade. 1973. Mechanism of adherence of Streptococcus mutans to smooth surfaces. I. Roles of insoluble dextran-levan synthetase enzymes and cell wall polysaccharide antigen in plaque formation. Infect. Immun. 8:555-562. 19. Mukasa, H., and H. D. Slade. 1974. 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. 20. Mukasa, H., and H. D. Slade. 1974. Mechanism of adherence of Streptococcus mutans to smooth surfaces. III. Purification and properties of the enzyme complex responsible for adherence. Infect. Immun. 10:1135-1145. 21. Nicolson, G. L., J. Blaustein, and M. E. Etzler. 1974.
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