INFECrION AND IMMUNITY, OCt. 1992, p. 4301-4308
Vol. 60, No. 10
0019-9567/92/104301-08$02.00/0 Copyright © 1992, American Society for Microbiology
Adhesion of Glucosyltransferase Phase Variants to Streptococcus gordonii Bacterium-Glucan Substrata May Involve Lipoteichoic Acid M. M. VICKERMAN1 AND G. W. JONES2* Department of Cariology and General Dentistry, School of Dentistry, 1 and Department of Microbiology and Immunology, School of Medicine,2 University ofMichigan, Ann Arbor, Michigan 48109 Received 6 May 1992/Accepted 9 July 1992
Growing Streptococcus gordonii Spp+ phase variants, which have normal levels of glucosyltransferase (GTF) activity, use sucrose to promote their accumulation on surfaces by forming a cohesive bacterium-insoluble glucan polymer mass (BPM). Spp- phase variants, which have lower levels of GTF activity, do not form BPMs and do not remain in BPMs formed by Spp+ cells when grown in mixed cultures. To test the hypothesis that segregation of attached Spp+ and unattached Spp- cells was due to differences in adhesiveness, adhesion between washed, [3lHlthymidine-labeled cells and preformed BPM substrata was measured. Unexpectedly, the results showed that cells of both phenotypes, as well as GTF-negative cells, attached equally well to preformed BPMs, indicating that attachment to BPMs was independent of cell surface GTF activity. Initial characterization of this binding interaction suggested that a protease-sensitive component on the washed cells may be binding to lipoteichoic acids sequestered in the BPM, since exogenous lipoteichoic acid inhibited adhesion. Surprisingly, the adhesion of both Spp' and Spp- cells was markedly inhibited in the presence of sucrose, which also released lipoteichoic acid from the BPM. These in vitro findings suggest that, in vivo, sucrose and lipoteichoic acid may modify dental plaque development by enhancing or inhibiting the attachment of additional bacteria.
Streptococcus gordonii, previously classified as Streptococcus sanguis (19), is found primarily on the tooth surface in dental plaque (10, 14). The initial adhesion of S. gordonii and S. sanguis to hydroxyapatite (HA) surfaces with adsorbed salivary components has been investigated at the physicochemical level (1, 6), and several bacterial adhesins (8, 11, 31) and salivary receptors (13, 28) have been identified. Following the initial adhesion of S. gordonii to HA or glass beads, the bacteria can continue to grow and accumulate on these surfaces (37). This accumulation may be promoted in the presence of sucrose. S. gordonii has an extracellular glucosyltransferase (GTF) enzyme which splits sucrose to make both water-soluble and water-insoluble glucan polymers (15, 25). Phase variation in the activity of this GTF (35) has recently been shown to influence bacterial accumulation (37). In the presence of sucrose, growing Spp+ cells, which have normal levels of GTF activity (35), show additional accumulation on the bead surfaces due to the production of insoluble glucan polymers (37). These glucans envelop the growing attached bacteria and their products and enmesh the bead substratum to form a single, cohesive bacterium-polymer mass (BPM). A complete biochemical analysis of the BPM has not yet been performed, but it is known to be composed of viable bacteria at a concentration of >5 x 1010 cells cm-3 in a matrix of water-soluble and water-insoluble glucans (37). Reversible phase variation gives rise to Spp- cells (35), which show both quantitative and qualitative differences in GTF activity. Spp- cells have about 20% of the GTF activity of Spp+ cells (35), make ca. 30-fold-less insoluble glucan, and hence do not show sucrose-promoted accumulation (37). The significance of phase variation is apparent during *
Corresponding author. 4301
growth in medium supplemented with sucrose as a substrate for the GTF and with beads as a substratum for attachment. Under these conditions, GTF phase variants preferentially segregate as attached and unattached cells. Spp+ cells that arise as revertants from Spp- populations or that are grown in mixed cultures with Spp- cells preferentially attach to the substratum and accumulate to form BPMs. Conversely, Spp- cells that arise from Spp+ cells or that are grown with Spp+ cells in mixed cultures tend to be excluded from the BPM formed by the Spp+ cells and remain as unattached cells in the culture supernatants. The segregation of the two phenotypes as attached or unattached cells does not occur in the absence of sucrose (38). These results raised questions about the relative abilities of Spp+ and Spp- cells to attach to and remain in BPMs formed by Spp+ cells and the potential role of these bacteria in the ecology of the oral cavity. It has been proposed (37, 38) that the insoluble glucans synthesized by S. gordonii Spp+ cells may contribute to the accumulation of Spp+ cells on tooth surfaces. However, since Spp- phase variants are not retained in BPMs but show increased adhesion to saliva-coated HA (39), it is possible that, in vivo, these phase variants are dispersed from the developing plaque to other oral sites. To better understand the significance of sucrose-promoted accumulation and differential segregation of phase variants in the colonization of the oral cavity by S. gordonii, the present studies on the adhesion of Spp+ and Spp- cells to preformed BPMs were initiated. Because of the differential partitioning of growing phase variants (38), it was hypothesized that Spp+ cells would show greater adhesion to BPMs than Spp- cells and that sucrose might play a role in promoting this adhesion. However, the results do not support this hypothesis, but suggest that adhesion of both phenotypes may be modified by sucrose and by lipoteichoic acid (LTA) sequestered in the BPM substratum.
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MATERIALS AND METHODS Bacterial strains. Bacterial strains were stored in 50% glycerol at -70°C. All strains were derivatives of the Spp+ parental strain Challis CH1 (35, 37, 38). Strains CHlCl and CH97 are independently derived Spp- variants. Strain CHD2 is an Spp+ revertant of strain CHlCl (35). Strain CHAl is a spontaneous GTF-negative mutant which appears to have a mutation in the GTF structural gene. Strains CH319 and CH321 carry independent insertions of plasmid pVA891 (23) in the GTF structural gene (33). The last two strains were grown in the presence of erythromycin (5 ,g/ml, Sigma, St. Louis, Mo.) to maintain the integrated plasmid. Strains CHAl, CH319, and CH321 showed no GTF band on activity gels (35). Strain CH107 is a spontaneous mutant which lacks 0.6 kbp in the gtf structural gene, encoding the C-terminal repeat portion of the protein, and produces a truncated GTF. Strains CHAl, CH319, CH321, and CH107 produce soft colonies on sucrose-agar plates similar to those of the Spp- variants. Bacterium-BPM adhesion tests. The media and culture conditions have been described previously in detail (37). The bacteria to be used for attachment to the substrata in the adhesion tests were grown anaerobically with 2 p,Ci of [3H]thymidine (Amersham) per ml in chemically defined FMC (36) medium. At late log phase, the cells were washed twice by centrifugation at 1,000 x g and resuspended to 108 cells per ml in buffered KCl (2 mM KH2PO4, 2 mM K2HPO4, 1 mM CaCl2, 50 mM KCl [pH 6.8] [1]). To prepare BPMs, late-log-phase strain Challis cultures grown in FMC medium were diluted to approximately 5 x 105 cells per ml in fresh FMC medium supplemented with 1% (wt/vol) sucrose. One-milliliter amounts were dispensed into 1-dram (ca. 3.7-ml) glass vials (Fisher Scientific) containing 15 mg of sterile glass beads (0.2 mm average diameter; Thomas Scientific). The cultures then were grown anaerobically on a rotating drum (10 rpm; New Brunswick Scientific Co., Edison, N.J.) until the late log stage. By this phase of growth, a single, cohesive BPM formed which consisted of glass beads covered with bacteria and their products enmeshed in an insoluble glucan polymer matrix. The BPMs contained >2 x 109 viable cells and had a volume of 40 pl (37). Culture supernatants were removed, and the BPMs were rinsed twice in 2 ml of buffered KCI and used immediately as substrata unless otherwise noted. BPMs remained macroscopically intact throughout the adhesion tests. To control for adhesion of the radiolabeled bacteria to the bacteria in the BPM by mechanisms such as coaggregation (20), bacterium-coated beads were also used as substrata. These were prepared by growing strain Challis cells with glass beads in FMC medium without sucrose to the late log stage as described above; ca. 1.5 x 108 cells attached to the beads under these conditions (37). Finally, to control for bacterial adhesion to possible unsaturated sites on the beads within the BPM and to monitor the previously described adhesion of washed cells to glass beads (37), sterile glass beads were used as substrata. To establish the test conditions initially, some preparations of BPMs and cell-covered beads were heat-treated at 60°C for 30 min to kill the bacteria and to prevent their active contribution to binding interactions. Because the polymer matrix could potentially sequester molecules, such as sucrose or soluble dextran, that might enhance or inhibit binding, other BPM preparations were washed extensively for 24 h at 4°C in buffered KCl until an anthrone reaction (42) indicated the absence of detectable hexoses in the wash.
INFECr. IMMUN.
Aliquots (1 ml) of the washed, radiolabeled cells were added to the vials containing the substrata and rotated at 10 rpm at 36°C anaerobically for 1 to 3 h. After incubation, the supernatants containing the unattached cells were removed, and the substrata were rinsed twice with 2 ml of buffered KCl. The radioactivity in the supernatants and substrata was counted in 4 ml of Biosafe II (Research Products International Co., Mount Prospect, Ill.) scintillation cocktail in a Beckman scintillation counter. Adhesion was expressed as the percentage of the total counts that were associated with the substrata. Total recovery of radiolabeled bacteria from the substrata and supernatants was >90%. Tests were done in duplicate and repeated at least twice. Differences between pairs of strains and treatments were analyzed by Student's t test for small sample means. Adhesion inhibition studies. In some cases, potential inhibitors or enhancers were added to the adhesion test system. All chemicais were from Sigma unless specified. The carbohydrates used were sucrose, lactose, arabinose, water-soluble a-1,6-dextran (72 kDa), and water-insoluble a-1,3-nigeran (384 kDa); nigeran was dispersed by sonication (Heat Systems Ultrasonics, Inc.) before use. Adhesion inhibition tests were also done in the presence of commercially available Enterococcusfaecalis or Streptococcus mutans LTA or in the presence of column-purified strain Challis LTA (a gift from R. E. Kessler [17]). Each test substance was added with the washed cells to the vials containing the substrata and remained in the system throughout the adhesion tests. Controls in buffered KCl alone were included in all experiments. Pretreatment of BPMs and radiolabeled bacteria. The nature of the bacterium-BPM binding interaction was investi-
gated by pretreatment of washed [3H]thymidine-labeled cells
or BPMs for 30 min at 37°C on a rotating drum (10 rpm) with 1-ml volumes of the following reagents: proteinase K (200
,ug/ml), pronase E (500 p,g/ml), and trypsin (120 U/ml), all in 0.1 M potassium phosphate buffer (pH 7.5), and mutanolysin (50 U/ml) and 1% (vol/vol) Triton X-100 detergent, both in buffered KCl (pH 6.8). Buffer-treated controls were used in all tests. Pretreated cells were then washed twice with buffered KCl by centrifugation (1,000 x g) and resuspended in buffered KCl. Chemically pretreated BPMs were washed in six changes of 4-ml volumes of buffered KCl overnight at 4°C. Pretreated bacteria were tested for their ability to attach to untreated BPMs, and pretreated BPMs were used as substrata for untreated, washed, radiolabeled bacteria. Analysis of BPMs and bacteria for LTA. LTA was extracted from BPMs and bacteria with hot (68°C) 45% aqueous phenol (nucleic acid grade; BRL) for 30 min with vigorous stirring (17). The phases were separated by centrifugation at 1,000 x g at 4°C for 5 min, and the aqueous phase was removed. The organic phase was reextracted with an equal volume of deionized water. The aqueous phases were pooled, dialyzed against four 10Ox volumes of deionized water at 4°C, and assayed by the enzyme-linked immunosorbent assay (ELISA). Dry weights were determined by drying the bacteria or BPMs on a Savant Speedvac concentrator until the weights remained constant. The weight of the enmeshed glass beads was subtracted from the dry weight of BPMs. For the ELISA, LTA extracts were serially diluted in phosphate-buffered saline (PBS; Oxoid), and 100-p,l volumes were adsorbed to wells of polyvinyl plates (Dynatech Laboratories, Inc., Alexandria, Va.) for 72 h at 4°C. The wells were then washed three times with PBS and blocked with 5% (wt/vol) fat-free dried milk (Carnation) in PBS for 60 min at
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S. GORDONII ADHESION TO BACTERIUM-POLYMER MASSES
room temperature. Rabbit antiserum (a gift from R. E. Kessler [18]) specific for the polyglycerol phosphate backbone of streptococcal LTA was diluted in 5% dried milkPBS; 100-pl volumes were added to each well and incubated overnight at 4°C. The wells were washed with PBS, and horseradish peroxide-conjugated, goat anti-rabbit immunoglobulin G (IgG) antiserum (Bio-Rad Laboratories) in 5% dried milk-PBS was added. The plates were incubated overnight at 4°C. Color reactions were developed with a 3,3',5,5'-tetramethylbenzidine substrate-hydrogen peroxide mixture (Kirkegaard & Perry Laboratories, Inc., Gaithersburg, Md.) and stopped with 1 M H2SO4, and the A450 was read. Standard curves were constructed from the results obtained with purified strain Challis LTA at concentrations of 1.5 to 50 ng/ml. Standard curves were analyzed and values for unknowns were determined by least-squares regression; the coefficients of determination (r2) for these assays were consistently >0.98. The specificity of the reaction for LTA was demonstrated by preincubation of the antiserum at 37°C for 60 min with deacylated (41) cardiolipin (Sigma) prior to its addition to the LTA-coated wells. RESULTS Strain Challis cells attach to BPM substrata. Initial experiments were designed to determine whether washed radiolabeled cells of strain Challis attached to the BPM formed by strain Challis and whether adhesion to the bacteria and/or glass beads within the BPM played a significant role in this attachment. The results of experiments done to define the conditions of the adhesion test with untreated, washed strain Challis cells and various substrata (see Materials and Methods) are shown in Fig. 1. Adhesion to the substrata was time dependent. In buffer alone (Fig. IA), radiolabeled bacteria attached to both rinsed and extensively washed BPMs, suggesting that the sites of adhesion on the BPM were not readily eluted. Comparison of heated (60°C) and unheated BPMs showed that heating significantly reduced adhesion (P < 0.001). Extensive washing of the heat-treated BPM, however, partially restored adhesion, implying that heat treatment did not irreversibly denature bacterial adhesion sites on the BPM, but rather may have released an inhibitor(s) of adhesion. These results showed that BPMs of strain Challis could serve as effective substrata for the attachment of additional strain Challis cells and that viable bacteria within the BPMs did not contribute substantially to adhesion.
Bacterium-coated beads, whether heat-treated or not, did not serve as effective substrata (Fig. 1A), indicating that bacterial cells within the BPM were not serving as significant
binding sites. Although there were more total cells present in the BPM (>2 x 109) than on the bacterium-coated beads (ca. 1.5 x 108), most of the bacteria in the BPM are occluded within the insoluble glucan mass (37) and are probably not available to serve as attachment sites. As expected (37), washed cells attached effectively to glass beads. Sucrose inhibits binding of strain Challis cells to BPMs. The addition of 29 mM sucrose as a substrate for the GTF on the strain Challis cell surface resulted in significant inhibition of adhesion of strain Challis cells to all substrata except glass beads (P c 0.02, Fig. 1B). The inhibition of adhesion to the BPM by sucrose (P c 0.004) suggested that the increased adhesion seen with the rinsed BPM compared with the adhesion to the extensively washed BPM was not due to residual sucrose in the rinsed BPM. These results also suggested that the mechanism of adhesion of washed strain
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1.5 2.5 2.0 3.0 Incubation (hours) FIG. 1. Adhesion of parental S. gordonu strain Challis to BPMs, bacterium-coated beads, and glass beads. Substrata were prepared 0.5
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as described in Materials and Methods to define the adhesion test conditions. BPMs were heat-treated (-) or unheated (E) and washed overnight in buffered KCI. Heat-treated BPMs (0), unheated BPMs (0), heat-treated bacterium-coated beads (A), unheated bacterium-coated beads (A), and glass beads (x) were rinsed in buffered KCl before the addition of 108 ['H]thymidine-labeled strain Challis cells. The percentages of the total washed radiolabeled bacteria that were associated with each substratum at 1, 2, and 3 h of anaerobic incubation at 36°C were calculated and expressed as percent adhesion. Total recovery of radiolabel counts was .90%. (A) Adhesion of radiolabeled cells suspended in buffered KCl alone. (B) Adhesion of radiolabeled cells suspended in buffered KCl with 29 mM sucrose.
Challis cells to the BPM was different from the mechanism of adhesion to the glass beads and that unsaturated bead surfaces in the BPM were not major attachment sites. pH changes over the range found throughout the tests (pH 5.0 to 6.8) had no effect on relative adhesion to any of the substrata (data not shown), and no coaggregation of the washed cells was observed microscopically or macroscopically. On the basis of these results, rinsed, unheated BPMs were used as the standard substratum for subsequent adhesion studies. Experiments were carried out at 36°C under anaerobic conditions for 2 h. Bacterium-coated beads and glass beads were included as controls in all tests.
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VICKERMAN AND JONES
INF'Ecr. IMMUN.
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Substrata FIG. 2. Inhibition of adhesion of washed cells of strain Challis to BPMs in the presence of sucrose. Percentage of 10' washed radiolabeled strain Challis cells attached to each substratum after 2 h of incubation in the presence of buffer alone (solid bars) or buffer containing 29 mM sucrose (open bars), 29 mM lactose (shaded bars), or 29 mM arabinose (hatched bars). Substrata were buffer-rinsed BPMs (BPM), glass beads with adsorbed bacteria (Bacteria), and glass beads without adsorbed bacteria (Beads). Error bars indicate standard deviations.
Carbohydrate effects on adhesion. In contrast to the adhesion inhibition seen with sucrose, no inhibition of adhesion to BPMs or to the control substrata was observed in the presence of equimolar (29 mM) concentrations of the irrelevant, nonmetabolizable carbohydrate arabinose or the metabolizable carbohydrate lactose (Fig. 2), suggesting that inhibition was sucrose specific. Higher concentrations of lactose (60 mM), which Wave been shown to inhibit lectinmediated coaggregation between some streptococcal cells (20), also failed to inhibit binding to any of the substrata (data not shown). This latter finding supported the conclusions drawn from tests with bacterium-coated bead substrata (Fig. 1A) that streptococcal cells in the BPM are not serving as significant binding sites. Equivalent binding of Spp+ and Spp- cells to BPMs. When tested in buffer alone, the attachment of washed Sppvariants CHlCl and CH97 and the Spp+ revertant CH1D2 showed patterns of adhesion to all substrata similar to those of the parental strain Challis (Table 1). Sucrose at 29 mM similarly inhibited the adhesion of both Spp+ and Spp- cells
to BPMs (P < 0.01) but tended to increase adhesion to glass beads (Table 1). The attachment of Spp- and Spp+ cells to heated (60°C) BPMs and heated bacterium-coated beads was also similar to that of strain Challis (data not shown). These results suggested that the cell surface GTF, levels of which are ca. sixfold higher on washed Spp+ cells than on washed Spp- cells (39), did not play a role in the adhesion of the washed cells to the BPMs. The absence of a role for GTF in the adhesion of washed cells to BPMs was confirmed with three different GTFnegative strains (Table 2). All three strains showed the same pattern of adhesion to the substrata as that of strain Challis, as well as the same inhibition of adhesion by sucrose (P < 0.01, Table 2). In addition, strain CH107, which is missing 0.6 kbp encoding the C-terminal repeat portion of the GTF protein, thought to be involved in glucan binding by the GTE of other oral streptococci (9, 27), showed a pattern of binding to all substrata similar to that of strain Challis (data not shown). These results suggested either that strain Challis and its derivatives had glucan-binding moieties independent of GTF or that the component in the BPM to which the radiolabeled cells attach was not glucan. The inhibition of binding to the BPM by the GTF-negative strains in the presence of sucrose also confirmed that the sucrose inhibition was not caused by occlusion of binding sites by glucans formed by the attaching cells. Evidence that adhesion to BPMs does not involve glucans. Competitive binding tests done in the presence of soluble dextrans, which contain primarily a-1,6-glucosidic linkages, or in the presence of insoluble nigeran, which contains primarily a-1,3-glucosidic linkages, lent supportive evidence to the conclusion that the components in the BPM to which the washed cells bound were not glucans. These polymers contain the same major glucosidic linkages as the glucans of S. gordonu strains 10558 and Challis (15, 25). No significant inhibition of adhesion to the BPM or control substrata was seen in the presence of up to 0.3 mg of dextran or 1 mg of nigeran per ml (data not shown). Viscosity effects precluded the use of higher dextran concentrations. However, the concentrations used appear to be appropriate for inhibition studies, as presumably only the surface glucan of the BPM is available as a binding site. Calculations based on the total glucan present in the BPM (37) suggest that the 1-,um-deep surface layer contains about 2 ,g of glucan, which is 50- and 150-fold less than the amounts of nigeran and dextran used, respectively.
TABLE 1. Similar adhesion of Spp+ and Spp- strains in the absence and presence of sucrose Mean % adhesione + SD Buffer and substratama Challis CH1D2 CHlCl
Buffer alone BPM Bacteria Beads Buffer + sucrose (29 mM) BPM Bacteria Beads
CH97
(Spp+)
(Spp-)
(Spp+)
(Spp-)
28.5 ± 2.3 5.7 ± 0.2 25.8 ± 0.4
24.6 ± 1.9 3.7 ± 0.1 21.2 ± 1.8
29.2 ± 0.2 5.8 ± 0.2 28.8 + 1.7
33.1 ± 4.3
6.3 ± 1.4 4.5 ± 0.3
4.2 ± 0.1 2.9 ± 0.1
6.1 ± 0.8 4.0 ± 0.1
5.3 ± 4.3 4.6 ± 0.8
31.7 ± 0.3
28.0 ± 3.3
35.4 ± 0.9
34.9 ± 2.6
6.6 ± 0.1
29.3 ± 0.2
The substrata tested were BPM, composed of strain Challis bacterium-glucan polymer mass; bacteria, composed of strain Challis bacteria adsorbed to glass beads; and beads, which were sterile glass beads without adsorbed bacteria. All substrata were rinsed twice in sterile buffered KCI before radiolabeled cells suspended in buffer or buffer containing 29 mM sucrose were added. b Percentage of total radiolabeled bacteria of each strain attached to the substratum at 2 h. a
S. GORDONII ADHESION TO BACTERIUM-POLYMER MASSES
VOL. 60, 1992
TABLE 2. Similar adhesion of parental strain Challis and GTFnegative strains in the absence and presence of sucrose Buffer and substratumn
Mean % adhesionb ± SD CH319 CHAl
Challis
Buffer alone BPM 27.6 ± 1.5 35.4 Beads 21.2 ± 1.5 25.5 Buffer + sucrose (29 mM) BPM 7.6 ± 1.3 11.9 Beads 27.6 + 3.1 29.5
CH321
± 2.4 36.3 ± 2.8 34.0 ± 3.4 ± 0.3 31.3 ± 3.0 27.0 ± 1.9
+ 1.0 10.7 ± 1.4 9.9 ± 0.8 ± 0.5 30.8 ± 0.3 27.3 ± 1.2
a The substrata used were BPM, composed of strain Challis bacteriumpolymer mass, and beads, which were sterile glass beads without adsorbed bacteria. All substrata were rinsed twice in sterile buffered KCI, and then radiolabeled bacteria in buffer or buffer containing 29 mM sucrose were added. b Percentage of total radiolabeled bacteria attached to the substratum at 2 h.
Effects of chemical and enzymatic treatment of BPMs and bacteria on adhesion. In order to gain insight into the nature of the components of the BPM and the washed bacteria involved in the adhesive interaction, BPMs and washed bacteria were pretreated with proteases, mutanolysin, and Triton X-100 as described in Materials and Methods. None of these treatments had any effect on the macroscopic structure of the BPMs. Pretreatment of the BPM with various proteases did not reduce the ability of the untreated washed cells to attach to it, but adhesion was significantly inhibited by detergent extraction (Table 3). Reduced adhesion following mutanolysin treatment was statistically insignificant (P > 0.05, Table 3). Thus, the BPM factor(s) involved in the washed-cell-BPM binding interaction appeared to be detergent sensitive or detergent extractable and protease resistant. In the converse experiment, treatment of the washed bacteria with proteases decreased the washed cells' ability to attach to the BPM (Table 3). However, Triton X-100 and heat treatment of the washed cells did not significantly reduce their ability to attach. These results suggested that the washed-cell surface moiety involved in the washed-cellBPM binding interaction either is a relatively heat-resistant
TABLE 3. Adhesion of strain Challis to BPM after various treatments of the BPM or the attaching radiolabeled cells Mean % adhesione ± SD after treatment of:
Treatment'
BPM
None Heat (60°C) Proteinase K Pronase E Trypsin
Mutanolysin Triton X-100
15.3 13.2 16.3 16.3 16.0 13.4 8.0
± 1.3 ± 0.8 ± 0.7 ± 1.8 ± 0.6 ± 0.7
± 1.4**
Attaching cells 14.9 ± 1.2 16.0 ± 1.1 2.6 ± 0.3* 2.4 ± 0.3* 4.6 ± 0.2* NDC 21.7 ± 0.8
a Radiolabeled attaching cells were treated prior to the adhesion test with untreated BPMs, and BPMs were treated prior to the adhesion test with untreated radiolabeled cells, as described in the text. b Percentage of total radiolabeled bacteria attached to the substratum after 2 h of incubation at 37°C. Significance: different from untreated control at P < 0.001 (*) or at P < 0.01 (**), Student's t test. c ND, not done.
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protein or is dependent on a protein linkage for its stabilization on the bacterial surface. Sucrose releases LTA from BPMs. Because LTA is protease resistant and detergent extractable and has been reported to be associated with insoluble glucans formed by S. gordonii and other oral streptococci (29), LTA was considered a likely candidate for the BPM substance involved in binding. ELISA showed that the average BPM contained about 4.4 + 0.6 ,ug of LTA (0.8 + 0.1 p,g/mg [mean + standard error of the mean] [dry weight]). An equivalent number of washed cells (ca. 2 x 10') grown in parallel cultures without sucrose yielded 1.8 ± 0.4 p,g of LTA. Since sucrose specifically inhibited the adhesion of washed cells to BPMs (Fig. 2) and LTA is known to be released from bacteria and from saliva-glucan complexes by 20 mM sucrose (4, 25), BPMs were analyzed for the release of LTA under the conditions of the adhesion test, i.e., after 2 h of anaerobic incubation at 36°C in buffer containing 29 mM sucrose. After incubation in 29 mM sucrose, the BPM released up to 200 ng of LTA, whereas 108 washed cells released about 50 ng of LTA. After incubation in buffer alone, the LTA released from the BPM or the washed cells was insignificant (
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0019-9567/92/104301-08$02.00/0 Copyright © 1992, American Society for Microbiology
Adhesion of Glucosyltransferase Phase Variants to Streptococcus gordonii Bacterium-Glucan Substrata May Involve Lipoteichoic Acid M. M. VICKERMAN1 AND G. W. JONES2* Department of Cariology and General Dentistry, School of Dentistry, 1 and Department of Microbiology and Immunology, School of Medicine,2 University ofMichigan, Ann Arbor, Michigan 48109 Received 6 May 1992/Accepted 9 July 1992
Growing Streptococcus gordonii Spp+ phase variants, which have normal levels of glucosyltransferase (GTF) activity, use sucrose to promote their accumulation on surfaces by forming a cohesive bacterium-insoluble glucan polymer mass (BPM). Spp- phase variants, which have lower levels of GTF activity, do not form BPMs and do not remain in BPMs formed by Spp+ cells when grown in mixed cultures. To test the hypothesis that segregation of attached Spp+ and unattached Spp- cells was due to differences in adhesiveness, adhesion between washed, [3lHlthymidine-labeled cells and preformed BPM substrata was measured. Unexpectedly, the results showed that cells of both phenotypes, as well as GTF-negative cells, attached equally well to preformed BPMs, indicating that attachment to BPMs was independent of cell surface GTF activity. Initial characterization of this binding interaction suggested that a protease-sensitive component on the washed cells may be binding to lipoteichoic acids sequestered in the BPM, since exogenous lipoteichoic acid inhibited adhesion. Surprisingly, the adhesion of both Spp' and Spp- cells was markedly inhibited in the presence of sucrose, which also released lipoteichoic acid from the BPM. These in vitro findings suggest that, in vivo, sucrose and lipoteichoic acid may modify dental plaque development by enhancing or inhibiting the attachment of additional bacteria.
Streptococcus gordonii, previously classified as Streptococcus sanguis (19), is found primarily on the tooth surface in dental plaque (10, 14). The initial adhesion of S. gordonii and S. sanguis to hydroxyapatite (HA) surfaces with adsorbed salivary components has been investigated at the physicochemical level (1, 6), and several bacterial adhesins (8, 11, 31) and salivary receptors (13, 28) have been identified. Following the initial adhesion of S. gordonii to HA or glass beads, the bacteria can continue to grow and accumulate on these surfaces (37). This accumulation may be promoted in the presence of sucrose. S. gordonii has an extracellular glucosyltransferase (GTF) enzyme which splits sucrose to make both water-soluble and water-insoluble glucan polymers (15, 25). Phase variation in the activity of this GTF (35) has recently been shown to influence bacterial accumulation (37). In the presence of sucrose, growing Spp+ cells, which have normal levels of GTF activity (35), show additional accumulation on the bead surfaces due to the production of insoluble glucan polymers (37). These glucans envelop the growing attached bacteria and their products and enmesh the bead substratum to form a single, cohesive bacterium-polymer mass (BPM). A complete biochemical analysis of the BPM has not yet been performed, but it is known to be composed of viable bacteria at a concentration of >5 x 1010 cells cm-3 in a matrix of water-soluble and water-insoluble glucans (37). Reversible phase variation gives rise to Spp- cells (35), which show both quantitative and qualitative differences in GTF activity. Spp- cells have about 20% of the GTF activity of Spp+ cells (35), make ca. 30-fold-less insoluble glucan, and hence do not show sucrose-promoted accumulation (37). The significance of phase variation is apparent during *
Corresponding author. 4301
growth in medium supplemented with sucrose as a substrate for the GTF and with beads as a substratum for attachment. Under these conditions, GTF phase variants preferentially segregate as attached and unattached cells. Spp+ cells that arise as revertants from Spp- populations or that are grown in mixed cultures with Spp- cells preferentially attach to the substratum and accumulate to form BPMs. Conversely, Spp- cells that arise from Spp+ cells or that are grown with Spp+ cells in mixed cultures tend to be excluded from the BPM formed by the Spp+ cells and remain as unattached cells in the culture supernatants. The segregation of the two phenotypes as attached or unattached cells does not occur in the absence of sucrose (38). These results raised questions about the relative abilities of Spp+ and Spp- cells to attach to and remain in BPMs formed by Spp+ cells and the potential role of these bacteria in the ecology of the oral cavity. It has been proposed (37, 38) that the insoluble glucans synthesized by S. gordonii Spp+ cells may contribute to the accumulation of Spp+ cells on tooth surfaces. However, since Spp- phase variants are not retained in BPMs but show increased adhesion to saliva-coated HA (39), it is possible that, in vivo, these phase variants are dispersed from the developing plaque to other oral sites. To better understand the significance of sucrose-promoted accumulation and differential segregation of phase variants in the colonization of the oral cavity by S. gordonii, the present studies on the adhesion of Spp+ and Spp- cells to preformed BPMs were initiated. Because of the differential partitioning of growing phase variants (38), it was hypothesized that Spp+ cells would show greater adhesion to BPMs than Spp- cells and that sucrose might play a role in promoting this adhesion. However, the results do not support this hypothesis, but suggest that adhesion of both phenotypes may be modified by sucrose and by lipoteichoic acid (LTA) sequestered in the BPM substratum.
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MATERIALS AND METHODS Bacterial strains. Bacterial strains were stored in 50% glycerol at -70°C. All strains were derivatives of the Spp+ parental strain Challis CH1 (35, 37, 38). Strains CHlCl and CH97 are independently derived Spp- variants. Strain CHD2 is an Spp+ revertant of strain CHlCl (35). Strain CHAl is a spontaneous GTF-negative mutant which appears to have a mutation in the GTF structural gene. Strains CH319 and CH321 carry independent insertions of plasmid pVA891 (23) in the GTF structural gene (33). The last two strains were grown in the presence of erythromycin (5 ,g/ml, Sigma, St. Louis, Mo.) to maintain the integrated plasmid. Strains CHAl, CH319, and CH321 showed no GTF band on activity gels (35). Strain CH107 is a spontaneous mutant which lacks 0.6 kbp in the gtf structural gene, encoding the C-terminal repeat portion of the protein, and produces a truncated GTF. Strains CHAl, CH319, CH321, and CH107 produce soft colonies on sucrose-agar plates similar to those of the Spp- variants. Bacterium-BPM adhesion tests. The media and culture conditions have been described previously in detail (37). The bacteria to be used for attachment to the substrata in the adhesion tests were grown anaerobically with 2 p,Ci of [3H]thymidine (Amersham) per ml in chemically defined FMC (36) medium. At late log phase, the cells were washed twice by centrifugation at 1,000 x g and resuspended to 108 cells per ml in buffered KCl (2 mM KH2PO4, 2 mM K2HPO4, 1 mM CaCl2, 50 mM KCl [pH 6.8] [1]). To prepare BPMs, late-log-phase strain Challis cultures grown in FMC medium were diluted to approximately 5 x 105 cells per ml in fresh FMC medium supplemented with 1% (wt/vol) sucrose. One-milliliter amounts were dispensed into 1-dram (ca. 3.7-ml) glass vials (Fisher Scientific) containing 15 mg of sterile glass beads (0.2 mm average diameter; Thomas Scientific). The cultures then were grown anaerobically on a rotating drum (10 rpm; New Brunswick Scientific Co., Edison, N.J.) until the late log stage. By this phase of growth, a single, cohesive BPM formed which consisted of glass beads covered with bacteria and their products enmeshed in an insoluble glucan polymer matrix. The BPMs contained >2 x 109 viable cells and had a volume of 40 pl (37). Culture supernatants were removed, and the BPMs were rinsed twice in 2 ml of buffered KCI and used immediately as substrata unless otherwise noted. BPMs remained macroscopically intact throughout the adhesion tests. To control for adhesion of the radiolabeled bacteria to the bacteria in the BPM by mechanisms such as coaggregation (20), bacterium-coated beads were also used as substrata. These were prepared by growing strain Challis cells with glass beads in FMC medium without sucrose to the late log stage as described above; ca. 1.5 x 108 cells attached to the beads under these conditions (37). Finally, to control for bacterial adhesion to possible unsaturated sites on the beads within the BPM and to monitor the previously described adhesion of washed cells to glass beads (37), sterile glass beads were used as substrata. To establish the test conditions initially, some preparations of BPMs and cell-covered beads were heat-treated at 60°C for 30 min to kill the bacteria and to prevent their active contribution to binding interactions. Because the polymer matrix could potentially sequester molecules, such as sucrose or soluble dextran, that might enhance or inhibit binding, other BPM preparations were washed extensively for 24 h at 4°C in buffered KCl until an anthrone reaction (42) indicated the absence of detectable hexoses in the wash.
INFECr. IMMUN.
Aliquots (1 ml) of the washed, radiolabeled cells were added to the vials containing the substrata and rotated at 10 rpm at 36°C anaerobically for 1 to 3 h. After incubation, the supernatants containing the unattached cells were removed, and the substrata were rinsed twice with 2 ml of buffered KCl. The radioactivity in the supernatants and substrata was counted in 4 ml of Biosafe II (Research Products International Co., Mount Prospect, Ill.) scintillation cocktail in a Beckman scintillation counter. Adhesion was expressed as the percentage of the total counts that were associated with the substrata. Total recovery of radiolabeled bacteria from the substrata and supernatants was >90%. Tests were done in duplicate and repeated at least twice. Differences between pairs of strains and treatments were analyzed by Student's t test for small sample means. Adhesion inhibition studies. In some cases, potential inhibitors or enhancers were added to the adhesion test system. All chemicais were from Sigma unless specified. The carbohydrates used were sucrose, lactose, arabinose, water-soluble a-1,6-dextran (72 kDa), and water-insoluble a-1,3-nigeran (384 kDa); nigeran was dispersed by sonication (Heat Systems Ultrasonics, Inc.) before use. Adhesion inhibition tests were also done in the presence of commercially available Enterococcusfaecalis or Streptococcus mutans LTA or in the presence of column-purified strain Challis LTA (a gift from R. E. Kessler [17]). Each test substance was added with the washed cells to the vials containing the substrata and remained in the system throughout the adhesion tests. Controls in buffered KCl alone were included in all experiments. Pretreatment of BPMs and radiolabeled bacteria. The nature of the bacterium-BPM binding interaction was investi-
gated by pretreatment of washed [3H]thymidine-labeled cells
or BPMs for 30 min at 37°C on a rotating drum (10 rpm) with 1-ml volumes of the following reagents: proteinase K (200
,ug/ml), pronase E (500 p,g/ml), and trypsin (120 U/ml), all in 0.1 M potassium phosphate buffer (pH 7.5), and mutanolysin (50 U/ml) and 1% (vol/vol) Triton X-100 detergent, both in buffered KCl (pH 6.8). Buffer-treated controls were used in all tests. Pretreated cells were then washed twice with buffered KCl by centrifugation (1,000 x g) and resuspended in buffered KCl. Chemically pretreated BPMs were washed in six changes of 4-ml volumes of buffered KCl overnight at 4°C. Pretreated bacteria were tested for their ability to attach to untreated BPMs, and pretreated BPMs were used as substrata for untreated, washed, radiolabeled bacteria. Analysis of BPMs and bacteria for LTA. LTA was extracted from BPMs and bacteria with hot (68°C) 45% aqueous phenol (nucleic acid grade; BRL) for 30 min with vigorous stirring (17). The phases were separated by centrifugation at 1,000 x g at 4°C for 5 min, and the aqueous phase was removed. The organic phase was reextracted with an equal volume of deionized water. The aqueous phases were pooled, dialyzed against four 10Ox volumes of deionized water at 4°C, and assayed by the enzyme-linked immunosorbent assay (ELISA). Dry weights were determined by drying the bacteria or BPMs on a Savant Speedvac concentrator until the weights remained constant. The weight of the enmeshed glass beads was subtracted from the dry weight of BPMs. For the ELISA, LTA extracts were serially diluted in phosphate-buffered saline (PBS; Oxoid), and 100-p,l volumes were adsorbed to wells of polyvinyl plates (Dynatech Laboratories, Inc., Alexandria, Va.) for 72 h at 4°C. The wells were then washed three times with PBS and blocked with 5% (wt/vol) fat-free dried milk (Carnation) in PBS for 60 min at
VOL. 60, 1992
S. GORDONII ADHESION TO BACTERIUM-POLYMER MASSES
room temperature. Rabbit antiserum (a gift from R. E. Kessler [18]) specific for the polyglycerol phosphate backbone of streptococcal LTA was diluted in 5% dried milkPBS; 100-pl volumes were added to each well and incubated overnight at 4°C. The wells were washed with PBS, and horseradish peroxide-conjugated, goat anti-rabbit immunoglobulin G (IgG) antiserum (Bio-Rad Laboratories) in 5% dried milk-PBS was added. The plates were incubated overnight at 4°C. Color reactions were developed with a 3,3',5,5'-tetramethylbenzidine substrate-hydrogen peroxide mixture (Kirkegaard & Perry Laboratories, Inc., Gaithersburg, Md.) and stopped with 1 M H2SO4, and the A450 was read. Standard curves were constructed from the results obtained with purified strain Challis LTA at concentrations of 1.5 to 50 ng/ml. Standard curves were analyzed and values for unknowns were determined by least-squares regression; the coefficients of determination (r2) for these assays were consistently >0.98. The specificity of the reaction for LTA was demonstrated by preincubation of the antiserum at 37°C for 60 min with deacylated (41) cardiolipin (Sigma) prior to its addition to the LTA-coated wells. RESULTS Strain Challis cells attach to BPM substrata. Initial experiments were designed to determine whether washed radiolabeled cells of strain Challis attached to the BPM formed by strain Challis and whether adhesion to the bacteria and/or glass beads within the BPM played a significant role in this attachment. The results of experiments done to define the conditions of the adhesion test with untreated, washed strain Challis cells and various substrata (see Materials and Methods) are shown in Fig. 1. Adhesion to the substrata was time dependent. In buffer alone (Fig. IA), radiolabeled bacteria attached to both rinsed and extensively washed BPMs, suggesting that the sites of adhesion on the BPM were not readily eluted. Comparison of heated (60°C) and unheated BPMs showed that heating significantly reduced adhesion (P < 0.001). Extensive washing of the heat-treated BPM, however, partially restored adhesion, implying that heat treatment did not irreversibly denature bacterial adhesion sites on the BPM, but rather may have released an inhibitor(s) of adhesion. These results showed that BPMs of strain Challis could serve as effective substrata for the attachment of additional strain Challis cells and that viable bacteria within the BPMs did not contribute substantially to adhesion.
Bacterium-coated beads, whether heat-treated or not, did not serve as effective substrata (Fig. 1A), indicating that bacterial cells within the BPM were not serving as significant
binding sites. Although there were more total cells present in the BPM (>2 x 109) than on the bacterium-coated beads (ca. 1.5 x 108), most of the bacteria in the BPM are occluded within the insoluble glucan mass (37) and are probably not available to serve as attachment sites. As expected (37), washed cells attached effectively to glass beads. Sucrose inhibits binding of strain Challis cells to BPMs. The addition of 29 mM sucrose as a substrate for the GTF on the strain Challis cell surface resulted in significant inhibition of adhesion of strain Challis cells to all substrata except glass beads (P c 0.02, Fig. 1B). The inhibition of adhesion to the BPM by sucrose (P c 0.004) suggested that the increased adhesion seen with the rinsed BPM compared with the adhesion to the extensively washed BPM was not due to residual sucrose in the rinsed BPM. These results also suggested that the mechanism of adhesion of washed strain
40
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4303
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2.5
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-
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10
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X-
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1.5 2.5 2.0 3.0 Incubation (hours) FIG. 1. Adhesion of parental S. gordonu strain Challis to BPMs, bacterium-coated beads, and glass beads. Substrata were prepared 0.5
1.0
as described in Materials and Methods to define the adhesion test conditions. BPMs were heat-treated (-) or unheated (E) and washed overnight in buffered KCI. Heat-treated BPMs (0), unheated BPMs (0), heat-treated bacterium-coated beads (A), unheated bacterium-coated beads (A), and glass beads (x) were rinsed in buffered KCl before the addition of 108 ['H]thymidine-labeled strain Challis cells. The percentages of the total washed radiolabeled bacteria that were associated with each substratum at 1, 2, and 3 h of anaerobic incubation at 36°C were calculated and expressed as percent adhesion. Total recovery of radiolabel counts was .90%. (A) Adhesion of radiolabeled cells suspended in buffered KCl alone. (B) Adhesion of radiolabeled cells suspended in buffered KCl with 29 mM sucrose.
Challis cells to the BPM was different from the mechanism of adhesion to the glass beads and that unsaturated bead surfaces in the BPM were not major attachment sites. pH changes over the range found throughout the tests (pH 5.0 to 6.8) had no effect on relative adhesion to any of the substrata (data not shown), and no coaggregation of the washed cells was observed microscopically or macroscopically. On the basis of these results, rinsed, unheated BPMs were used as the standard substratum for subsequent adhesion studies. Experiments were carried out at 36°C under anaerobic conditions for 2 h. Bacterium-coated beads and glass beads were included as controls in all tests.
4304
VICKERMAN AND JONES
INF'Ecr. IMMUN.
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Substrata FIG. 2. Inhibition of adhesion of washed cells of strain Challis to BPMs in the presence of sucrose. Percentage of 10' washed radiolabeled strain Challis cells attached to each substratum after 2 h of incubation in the presence of buffer alone (solid bars) or buffer containing 29 mM sucrose (open bars), 29 mM lactose (shaded bars), or 29 mM arabinose (hatched bars). Substrata were buffer-rinsed BPMs (BPM), glass beads with adsorbed bacteria (Bacteria), and glass beads without adsorbed bacteria (Beads). Error bars indicate standard deviations.
Carbohydrate effects on adhesion. In contrast to the adhesion inhibition seen with sucrose, no inhibition of adhesion to BPMs or to the control substrata was observed in the presence of equimolar (29 mM) concentrations of the irrelevant, nonmetabolizable carbohydrate arabinose or the metabolizable carbohydrate lactose (Fig. 2), suggesting that inhibition was sucrose specific. Higher concentrations of lactose (60 mM), which Wave been shown to inhibit lectinmediated coaggregation between some streptococcal cells (20), also failed to inhibit binding to any of the substrata (data not shown). This latter finding supported the conclusions drawn from tests with bacterium-coated bead substrata (Fig. 1A) that streptococcal cells in the BPM are not serving as significant binding sites. Equivalent binding of Spp+ and Spp- cells to BPMs. When tested in buffer alone, the attachment of washed Sppvariants CHlCl and CH97 and the Spp+ revertant CH1D2 showed patterns of adhesion to all substrata similar to those of the parental strain Challis (Table 1). Sucrose at 29 mM similarly inhibited the adhesion of both Spp+ and Spp- cells
to BPMs (P < 0.01) but tended to increase adhesion to glass beads (Table 1). The attachment of Spp- and Spp+ cells to heated (60°C) BPMs and heated bacterium-coated beads was also similar to that of strain Challis (data not shown). These results suggested that the cell surface GTF, levels of which are ca. sixfold higher on washed Spp+ cells than on washed Spp- cells (39), did not play a role in the adhesion of the washed cells to the BPMs. The absence of a role for GTF in the adhesion of washed cells to BPMs was confirmed with three different GTFnegative strains (Table 2). All three strains showed the same pattern of adhesion to the substrata as that of strain Challis, as well as the same inhibition of adhesion by sucrose (P < 0.01, Table 2). In addition, strain CH107, which is missing 0.6 kbp encoding the C-terminal repeat portion of the GTF protein, thought to be involved in glucan binding by the GTE of other oral streptococci (9, 27), showed a pattern of binding to all substrata similar to that of strain Challis (data not shown). These results suggested either that strain Challis and its derivatives had glucan-binding moieties independent of GTF or that the component in the BPM to which the radiolabeled cells attach was not glucan. The inhibition of binding to the BPM by the GTF-negative strains in the presence of sucrose also confirmed that the sucrose inhibition was not caused by occlusion of binding sites by glucans formed by the attaching cells. Evidence that adhesion to BPMs does not involve glucans. Competitive binding tests done in the presence of soluble dextrans, which contain primarily a-1,6-glucosidic linkages, or in the presence of insoluble nigeran, which contains primarily a-1,3-glucosidic linkages, lent supportive evidence to the conclusion that the components in the BPM to which the washed cells bound were not glucans. These polymers contain the same major glucosidic linkages as the glucans of S. gordonu strains 10558 and Challis (15, 25). No significant inhibition of adhesion to the BPM or control substrata was seen in the presence of up to 0.3 mg of dextran or 1 mg of nigeran per ml (data not shown). Viscosity effects precluded the use of higher dextran concentrations. However, the concentrations used appear to be appropriate for inhibition studies, as presumably only the surface glucan of the BPM is available as a binding site. Calculations based on the total glucan present in the BPM (37) suggest that the 1-,um-deep surface layer contains about 2 ,g of glucan, which is 50- and 150-fold less than the amounts of nigeran and dextran used, respectively.
TABLE 1. Similar adhesion of Spp+ and Spp- strains in the absence and presence of sucrose Mean % adhesione + SD Buffer and substratama Challis CH1D2 CHlCl
Buffer alone BPM Bacteria Beads Buffer + sucrose (29 mM) BPM Bacteria Beads
CH97
(Spp+)
(Spp-)
(Spp+)
(Spp-)
28.5 ± 2.3 5.7 ± 0.2 25.8 ± 0.4
24.6 ± 1.9 3.7 ± 0.1 21.2 ± 1.8
29.2 ± 0.2 5.8 ± 0.2 28.8 + 1.7
33.1 ± 4.3
6.3 ± 1.4 4.5 ± 0.3
4.2 ± 0.1 2.9 ± 0.1
6.1 ± 0.8 4.0 ± 0.1
5.3 ± 4.3 4.6 ± 0.8
31.7 ± 0.3
28.0 ± 3.3
35.4 ± 0.9
34.9 ± 2.6
6.6 ± 0.1
29.3 ± 0.2
The substrata tested were BPM, composed of strain Challis bacterium-glucan polymer mass; bacteria, composed of strain Challis bacteria adsorbed to glass beads; and beads, which were sterile glass beads without adsorbed bacteria. All substrata were rinsed twice in sterile buffered KCI before radiolabeled cells suspended in buffer or buffer containing 29 mM sucrose were added. b Percentage of total radiolabeled bacteria of each strain attached to the substratum at 2 h. a
S. GORDONII ADHESION TO BACTERIUM-POLYMER MASSES
VOL. 60, 1992
TABLE 2. Similar adhesion of parental strain Challis and GTFnegative strains in the absence and presence of sucrose Buffer and substratumn
Mean % adhesionb ± SD CH319 CHAl
Challis
Buffer alone BPM 27.6 ± 1.5 35.4 Beads 21.2 ± 1.5 25.5 Buffer + sucrose (29 mM) BPM 7.6 ± 1.3 11.9 Beads 27.6 + 3.1 29.5
CH321
± 2.4 36.3 ± 2.8 34.0 ± 3.4 ± 0.3 31.3 ± 3.0 27.0 ± 1.9
+ 1.0 10.7 ± 1.4 9.9 ± 0.8 ± 0.5 30.8 ± 0.3 27.3 ± 1.2
a The substrata used were BPM, composed of strain Challis bacteriumpolymer mass, and beads, which were sterile glass beads without adsorbed bacteria. All substrata were rinsed twice in sterile buffered KCI, and then radiolabeled bacteria in buffer or buffer containing 29 mM sucrose were added. b Percentage of total radiolabeled bacteria attached to the substratum at 2 h.
Effects of chemical and enzymatic treatment of BPMs and bacteria on adhesion. In order to gain insight into the nature of the components of the BPM and the washed bacteria involved in the adhesive interaction, BPMs and washed bacteria were pretreated with proteases, mutanolysin, and Triton X-100 as described in Materials and Methods. None of these treatments had any effect on the macroscopic structure of the BPMs. Pretreatment of the BPM with various proteases did not reduce the ability of the untreated washed cells to attach to it, but adhesion was significantly inhibited by detergent extraction (Table 3). Reduced adhesion following mutanolysin treatment was statistically insignificant (P > 0.05, Table 3). Thus, the BPM factor(s) involved in the washed-cell-BPM binding interaction appeared to be detergent sensitive or detergent extractable and protease resistant. In the converse experiment, treatment of the washed bacteria with proteases decreased the washed cells' ability to attach to the BPM (Table 3). However, Triton X-100 and heat treatment of the washed cells did not significantly reduce their ability to attach. These results suggested that the washed-cell surface moiety involved in the washed-cellBPM binding interaction either is a relatively heat-resistant
TABLE 3. Adhesion of strain Challis to BPM after various treatments of the BPM or the attaching radiolabeled cells Mean % adhesione ± SD after treatment of:
Treatment'
BPM
None Heat (60°C) Proteinase K Pronase E Trypsin
Mutanolysin Triton X-100
15.3 13.2 16.3 16.3 16.0 13.4 8.0
± 1.3 ± 0.8 ± 0.7 ± 1.8 ± 0.6 ± 0.7
± 1.4**
Attaching cells 14.9 ± 1.2 16.0 ± 1.1 2.6 ± 0.3* 2.4 ± 0.3* 4.6 ± 0.2* NDC 21.7 ± 0.8
a Radiolabeled attaching cells were treated prior to the adhesion test with untreated BPMs, and BPMs were treated prior to the adhesion test with untreated radiolabeled cells, as described in the text. b Percentage of total radiolabeled bacteria attached to the substratum after 2 h of incubation at 37°C. Significance: different from untreated control at P < 0.001 (*) or at P < 0.01 (**), Student's t test. c ND, not done.
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protein or is dependent on a protein linkage for its stabilization on the bacterial surface. Sucrose releases LTA from BPMs. Because LTA is protease resistant and detergent extractable and has been reported to be associated with insoluble glucans formed by S. gordonii and other oral streptococci (29), LTA was considered a likely candidate for the BPM substance involved in binding. ELISA showed that the average BPM contained about 4.4 + 0.6 ,ug of LTA (0.8 + 0.1 p,g/mg [mean + standard error of the mean] [dry weight]). An equivalent number of washed cells (ca. 2 x 10') grown in parallel cultures without sucrose yielded 1.8 ± 0.4 p,g of LTA. Since sucrose specifically inhibited the adhesion of washed cells to BPMs (Fig. 2) and LTA is known to be released from bacteria and from saliva-glucan complexes by 20 mM sucrose (4, 25), BPMs were analyzed for the release of LTA under the conditions of the adhesion test, i.e., after 2 h of anaerobic incubation at 36°C in buffer containing 29 mM sucrose. After incubation in 29 mM sucrose, the BPM released up to 200 ng of LTA, whereas 108 washed cells released about 50 ng of LTA. After incubation in buffer alone, the LTA released from the BPM or the washed cells was insignificant (
