Archs ord
Biol. Vol. 20. pp. 50!%615.
PergamonPress 1975.Printed
I” Great Bntam.
STUD:[ES ON THE BACTERIAL COMPONENTS WHICH BIND STREPTOCOCCUS SANGUZS AND STREPTOCOCCUS MUTANS TO HYDROXYAPATITE W. F. LILJEMARKand S. V. SCHAUER &hool of Dentistry and Department of Microbiology, University of Minnesota, Minneapolis, Minnesota 55455, U.S.A. Summary-Experiments utilizing radio labelled oral streptococci in an in-vitro assay system have demonstrated the selective ability of certain species of these bacteria preferentially to adhere to saliva-coated and dextran-coated hydroxyapatite (HA). Pretreatment of Streptococcus sangnis and Streptococcus mutans with the proteolyic enzymes trypsin and pronase greatly reduced their ability to adhere to saliva or dextran-coated HA respectively. Pretreatment of Strep. sanguis with lipase increased its adsorption to saliva-coated HA and stearic acid-blocked adsorption, Cell walls isolated from Strep. sanguis proved effective in blocking Strep. sanguis adsorption to salivacoated apatite. Cells of Strep. mutans after treatment with dextranase or low molecular weight dextrans did not adhere well to dextran-coated HA.
INTRODUCTION Selective bacterial localization in the human oral cavity has been most studied with Streptococcus species (Krasse, 1954; Carlsson, 1967) and is purportedly related to their ability to attach to different oral surfaces (van Houte, Gibbons and Pulkkinin, 1971). Furthermore, it has also been proposed that bacterial adherence to different surfaces is a major ecological determinant governing the colonization of bacteria in various sites in the mouth (van Houte rt al., 1971; Gibbons and van Haute, 1971). Because much evidence has accumulated which indicates that certain oral streptococci are highly cariogenic (Jordan, Englander and Lim, 1969) it is important to understand the mechanisms responsible for their numerical proportional differences as they relate to adherence, specifically to the tooth surface and dental plaque. The adherence of bacteria to the tooth surface and the production of dental plaque has been shown to be a very complex process involving a number of specific components (Gibbons and van Houte, 1973). The research described in this report evaluates the interaction of specific stretptococcal surface components with salivary proteins and dextrans in their adherence to hydroxyapatite (HA). MATICRIALSAND Cultures and cultural
METHODS
conditions
Laboratory strains of Strep. mutans strain 6715, Strep. saliuarius strain 9GS2, Strep. sanguis strain H7P and Strep. miteor strain 26 were obtained from the culture collection of the Forsyth Dental Center. Strains 3 and C-l of Strep. xdiuarius were isolated from human saliva, and strains 7, 2 and 156 of Strep. sanguis were isolated from tooth surfaces. All organisms were grown in Trypticase Soy Broth (Baltimore Biol. Lab. BBL) and maintained by weekly transfer of mitis-salivarius agar (BBL). Bacteria were incubated anaerobically at 37°C in Brewer jars containing gas packs (BBL). All strains of Strep. sanguis and Strep. miteor were selected
for their ability to aggregate with saliva (Gibbons and Spinell, 1969) or. in the case of Strep. mutans, with dextran (Gibbons and Fitzgerald, 1969). Radio-labelled bacteria were prepared in a trypticase salts medium described by Gibbons, van Houte and Liljemark (1972). Uniformly r4C-labelled amino acids in the form of acid hydrolysed algal protein (New England Nuclear Corp.) were added to give a final activity of 2.0 /ICI/ml. Glucose was autoclaved separately and added to the medium to give a concentration of @2 per cent. Assay qj’adhrrencr titr (HA)
qf oral streptococci
to hydroxyupu-
Cell suspensions were prepared from 24 to 48 hr cultures. The organisms were harvested by centrifugation. washed twice with 0.067 M phosphate buffer (pH 6.0) and resuspended in buffer at a concentration of IO”/ml. These suspensions were routinely sonicated for I5 set in a Branson sonifier, Model S75, at a number 2 setting which virtually eliminated bacterial chains. Cells were then washed once in phosphate buffer (pH 6.0) to remove any labelled components released by sonication. Cell numbers were determined by direct microscopic count in a Petroff-Hauser counting chamber. Prior to use in the assay system, the HA (Bio-Gel HTP, Bio-Rad Richmond, California) was subjected to extensive washing procedures for removal of very small apatite “fines”. Two and a half grams of HA were allowed to settle for 5 min in 500 ml of 0.067 M phosphate buffer (pH 6.0) and the supernatant decanted. This procedure was repeated 3 times and the HA resuspended in lOOOm1 of the buffer. Again the HA was allowed to settle for 5 min and the supernatant decanted. This 5 min settling in 1000 ml of buffer was repeated twice. The HA was again resuspended in 500 ml of the buffer and dispensed in 10 ml aliquots into 15 ml conical centrifuge tubes. The apatite was centrifugated at full velocity in an International Clinical centrifuge Model L5655 for 5 sec. The supernatant was decanted and the procedure repeated 15 times. This extensive washing was necessary to ensure quanti609
610
W. F. Liljemark and S. V. Schauer
tative recovery of the HA in the washing procedure following incubation with bacteria. Collection and preparation of whole saliva for the coating of HA followed the method of Gibbons and Spine11 (1969), and the HA was then coated with this saliva by the method of Hay (1967). Dextran-coated HA was prepared by the method of Rolla and Mathiesen (1969). Pre-weighed dried samples of washed HA weighing from l-50 mg were suspended in 0.067 M phosphate buffer (pH 6.0) and a standard curve derived relating optical density to weight with a Klett-Summerson calorimeter at 440 nm. Neither saliva-coating nor dextrdn-coating of HA significantly altered the weight determinations of apatite samples as measured by the Klett-Summerson calorimeter. To test for the adsorption of bacteria to HA, 1.0 ml of 0.067 M phosphate buffer (pH 6.0) containing 5 mg of the dried-washed HA preparation was mixed with 1.0 ml of a washed radio-labelled bacterial cell suspension (I O”/ml) and incubated for 60 min at 37°C. During this period, the mixture was sufficiently agitated to keep the HA particles in suspension. The HA bacterial mixture was then washed 4 times with phosphate buffer by the centrifugation procedure described above. This procedure was found to remove 99 per cent ofall bacteria which had not adsorbed to the HA. To recover the bacteria which remained adsorbed to the HA after washing, the HA was solubilized by susin 0.2 M ethylenedinitrilotetraacetic acid pension (EDTA) at pH 8.0 for 3@60 min. After the HA was dissolved, the remaining bacteria were collected on 0.45 pm Millipore filters. The filters were dried, placed in liquid scintillation vials, and radioactivity was Packard liquid scintillation monitored in a spcctromcter. The number of bacteria adsorbed to HA in each experiment was expressed as a percentage of the total number of bacteria in the reaction mixtures. Eflkct qf’pH on the adherence Strep. miteor to HA
qf Strep. sanguis
and
To test the effect of pH. a series of reaction mixtures with a range in pH from 4.5 to 9.0 were prepared by combining 0067 M di-basic sodium and mono-basic potassium phosphate solutions in appropriate proportions. Cells from each bacterial species were washed and suspended separately in the test solutions with saliva-coated HA. Prctreattnmt
qf‘hactrriul
crlls with etnymrs,
lipids and
low molecular weight destrans Samples of “C-labelled bacterial suspensions were incubated with various enzyme preparations at 37°C for 60min. Controls consisted of bacterial cells incubated with buffer or with heat-inactivated enzymes. After incubation, the treated bacteria without subsequent washing were assayed for this ability to adhere lo saliva-coated or dextran-coated HA. Enzyme treatments which influenced bacterial adherence were repeated. After incubation the bacteria were washed twice by centrifugation to remove residual enzyme. The organisms were restandardized in buffer, and assayed for their ability to adhere to HA. Their adherence was compared to cells treated with buffer or with heat-inactivated enzymes in a comparable manner. Sources of’ ettzytws. Trypsin (3 x crystallized, salt free. Worthington Biochemicals), pronase (California
Biochemicals. nuclease free). dextranase (Worthington Biochemicals, BGC purified grade) and hyaluronidase (Worthington Biochemicals, chromatographically prepared) were used in 0.067 M phosphate buffer (pH 6.X). Wheat germ Lipase (Nutritional Biochemical Corp.. salt free) was dissolved in 0.067 M phosphate buffer (pH 5.0). The effect of pretreating Sfrrp. sanguis cells with lipids and Strep. muram with low molecular weight dextrans was also determined. Oleyl alcohol (25Opg, mg). stearic acid (1 mg/ml) and hyaluronic acid (1 mg/ ml) were prepared in ethanol and subsequently diluted with water. Cell suspensions of radio-labelled Strep. sanguis strain 7 were incubated with each lipid for 60min. The lipid-treated cell suspensions were then mixed with HA and incubated for 60 min and their ability to adhere to saliva-coated HA was determined. Cell suspensions of radio-labelled Strep. mutans were pretreated with Pharamacia dextrans of mol. wt 10.000 and 40,000 at a concentration of 1 mg/ml and assayed for their ability to adhere to dextran-coated HA. &j&-t
qf’culturejkctiotzs
to saliva-coated
qf Strep. sanguis on udherence
H,4
Strc~ptoc0cc1t.ssunguis strain 7 was grown to 68 x lo* cells/ml in predialysed trypticase soy broth. The bacterial cells were removed by centrifugation, and the culture liquor dialysed against 0.067 M phosphate buffer at 4°C. The dialysate was concentrated 50-fold by dialysis against polyethylene glycol, and stored at - 20°C. The streptococcal cells from the culture were washed once with phosphate buffer (pH 6.8), and suspended in 20ml of the same buffer. The suspension was mixed with 5 g of glass beads (62-88 pm, Lapine Scientific) and the bacteria ruptured by exposure to sonic oscillation 4 times for 5 min intervals using a Branson sonifier model S75 at maximum amplitude in an ice bath. The mixture was centrifuged at x 2000 g for 5 min to remove intact cells and beads. The supernatant was centrifuged at x 87009 for 20min. The supernatant was discarded the pellet resuspended and centrifuged at x 10,800g for 20min. After resuspending this pellet, a final centrifugation at x 2500 g for 5 min to remove any remaining whole cells was performed and the supernatant saved. The supernatant was examined by electron microscopy to assure that only cell walls remained. The supernatant from the original culture and cell wall preparations were tested for their ability to block the adherence of Strep. sunguis to saliva-coated HA. Five milligrams of saliva-coated HA were incubated with 2 ml of each preparation for 60 min. The HA was washed once. the radio-labelled streptococcal cells were added, and the mixture was incubated as previously described. RESULTS
Adsorption qforal coated aputite
streptococci
to saliva and destran-
The adsorption of several strains of Strep. sanguis, Strep. sulivarius, Strep. miteor and Strep. mutans to saliva-coated, dextran-coated and uncoated apatite was tested using the conditions described above, and the results are reported in Table 1. Note that Strep. sattguis and Strep. miteor adhered best to saliva-coated
salivarius 9GS.F 3*
Stwp. Strrp. Stwp.
SW/~. II~U~~III.C 67 IS5
Strep.wireor M29
.sccli~arius rniteor 26”
17
14* 8 16_+9 58 _+ 7 56
49 17_+ 12
smguis smguis
Strep. Strep.
2 I56+
61 40 2 11
“/, Adsorption of cells to saliva-coated HA
of Strep. sulicarius,
Stwp. sunguis H7p Strep. stlnguis 7*
Bacterial strains
1. Adsorption
1
1
6 5 7
2 8
92
Number of experiments
Strep. sanguis,
and
Strep.
46 _t 9
15
”i0 Adsorption of cells to dextran-coated HA
Strep. miteor
_
_ -
_ _
_
_. ,,,
_._.,
-
,-.
.,.--
Bacteria (I.0 x 109) and 5 mg HA suspended in 2 mls of 0.067 M phosphate buffer was used in each experiment. * @ < 0.005) Comparison of adsorption to saliva-coated HA and adsorption to uncoated HA. 10, < OGOS) Comparison of adsorption to dextran-coated HA and adsorption to uncoated HA.
Plaque
Cheek
Tongue dorsum
Plaque
Primary ecological niche
Table
to
5
2
Number of experiments
mutans
13 f 8
45 f 13 30 + 6 24 4 f 34
28 41 * 1
21 34 * 8
‘:i Adsorption of cells to uncoated HA
hydroxyapatite
3
ET “a 0’ $ 3 z io”
3 0”
I
4 3 4
53. Y, w R 4 m 5
2 6
27
Number of experiments
9 0 7 5’ ? FJ 5’ 09 P
2
W. F. Liljemark and S. V. Schauer
612
Strep. matans 6715 adsorption to dextran-coated HA did not vary significantly (data not shown). Under the optimum conditions as described for this system. i.e. (pH 6.0) and a cell concentration of 5.0 x IO8 bacteria per ml in 2 ml with 5 mg HA, adsorption of Strep. sunguis 7 and Strep. miteor 26 continued for 45-60min before reaching a maximum as shown in Fig. 3. The adsorption of Strep. mutans 6715 to dextran-coated HA reached a maximum level in 5 min (data not shown). All subsequent experiments used a mixture of 5.0 x JO8 bacteria per ml in 2 ml of 0.067 M phosphate buffer (pH 60), containing 5 mg HA which was incubated at 37°C for I hr unless indicated otherwise.
01 Cell numbers
IO10
IO9
IO8
IO'
pw
reaction
mix
Fig. I. Effect of cell concentration on the ability of Strep. str~gr~i 7 to adhere to saliva-coated hydroxyapatite; 9.1 x IO3 counts/min would correspond to 100 per cent adsorption.
HA. while Strep. saliaarius adhered best to uncoated HA, Streptococcus mutans adhered better to dextrancoated HA than uncoated or saliva-coated HA, while the adsorption of Strep. sanguis was not enhanced by dextran-coated HA. Eflect ~$cell concentration und pH on the udherence qf Strep. sanguis, Strep. miteor and Strep. mutans to saliva and dextran-coated HA The effect of cell concentration of the ability of Strep. sanguis 7 to adhere to saliva-coated apatite is shown in Fig. 1. A concentration of IO9 bacteria suspended in 2 ml of 0.067 M phosphate buffer (pH 6.0) with 5 mg of saliva-coated HA yielded the highest per cent adsorption. Figure 2 illustrates the effect of pH on the ability of pure cultures of Strep. suryuis 7 and Strep. miteor 26 to adhere to saliva-coated HA. Each organism adsorbed maximally at a pH of 6.0, which is within the 57-7.0 pH range of normal human saliva (Burnett and Scherp. 1957). However, in the pH range from 4.5-9.0.
Effect qfpretreatment with enzymes. lipids and low molecular weight dextran on adherence qf‘strep. sanguis and Strep. mutans to saliva or destran-coated HA Pretreatment of Strep. saryuis with trypsin (I mg/ml) or pronase (1 mg/ml) reduced its ability to adhere to saliva-coated HA eight- and three-fold respectively. Pretreatment with lipase (1 mg/ml) or hyaluronidase (I 50 units/ml), however, increased Strep. sanguis adsorption to saliva-coated HA (Table 2). Pretreating Strep. sanguis with stearic acid (250 ,ug/ml) significantly decreased its adsorption to saliva-coated apatite. Dextranase (20 units/ml). trypsin. or pronase pretreatment of S~,‘CJ~I. wtrrm reduced its adsorption to dcxtrancoated HA 8- and 20-fold respectively. Pretreatment with lipase increased Strep. mutans adsorption only slightly. Addition of low molecular weight dextran (10,000 or 40,OOOmol. wt at 1 m&ml) resulted in a three-fold decrease in adsorption (Table 2). Blocking effect qf culture jxction qf’ Strep. sanguis. Preincubation of unlabelled Strep. sanguis with salivacoated HA resulted in a 2-fold decrease in per cent adsorption to HA when compared to the control (Table 3). Preincubation of boiled unlabelled Strep. sanguis did not affect the susequent adsorption of radio labelled Strep. saf7gui.s. In a similar experiment. preincubation with the culture liquor from Strep. sanguis had no inhibitory effect on the adsorption of radiolabelled Strep. sanguis unless it was dialysed and concentrated more than 50-fold. Only then was the inhibition ofadherence by the culture liquor substantial. Cell walls isolated from Strep. sanguis by sonic oscillation with small glass beads and differential centrifugation
“V
A
LA-
4 50 E ‘6 8
“0
40
‘0 E = b
,/
/
*‘\ h
/I
30
S
\
9
.
S.
s.
mitmr
* S. songuis
w
ae Fig. 2. The effect of pH on the ability of Srrep. sanguis 7 and Strc’p. n~iteor 26 to adhere to saliva-coated hydroxyapatite; X.2 x 10’ counts/min would correspond to 100 per cent adsorption.
I
0
I 20
I
I 40
I
Time.
III 60
I 60
I loo
min
Fig. 3. Rate of adsorption of Strep. sanguis 156 and Srrrp. rnitror. 26 to saliva-coated hydroxyapatite: 4.3 x IO3 counts/min would correspond to 100 per cent adsorption.
613
Factors influencing bacterial adsorption to hydroxyapatite Table 2. Effect of enzyme, lipid and low mol. wt dextran pretreatment Strep. mutans to HA % Adsorption of Strep. sanguis 2 to saliva-coated HA Control Trypsin Pronase Lipase Hyduronidase Dextranase Hyaluronic acid Stearic acid Oleyl alcohol Low mol. wt dextran
of Strep. sunguis and
on the adsorption
of Strep. mutans 6715 to dextran-coated HA
% Adsorption
40*9 5 * 4* 15+6* 55 + 77 44+5 4017 30f 13 17$ 11* 44+ 13
42 _t 8 5 -+_5* 2 * 4* 47+ 11 2 * 3*
14 * 14 * *
* (p < ON)l) Comparison of adsorption to coated HA of the control and experimentals. t (b < 0.01) Comparison of adsorption to coated HA of the control and experimentals. produced an inhibitory effect as great as the whole cells. Boiled cell walls did not produce an inhibitory effect. The untreated HA control included in Table 3 demonstrates that any residual adherence activity after treatment of the HA may be due, in part, to saliva-independent adsorption to HA. This observation may also be extended to include the experiments presented in Table 2 which show residual adherence activities exist after enzyme treatment of the bacteria. DISCUSSION
This report has described a new assay system for the detection of bacterial adsorption to hydroxyapatite. In addition, the assay has been utilized to study some of the parameters which may be involved in the selective colonization of tooth surfaces by oral streptococci, and has concentrated on determining the characteristics of certain surface components of Strep. mutans and Strep. sanguis and how they effect bacterial adherence to hydroxyapatite. AS shown in Table 1 this in-vitro assay system demonstrates the selective adsorption of several species of oral streptococci to HA. These in-aitro observations are consistent with the selective adsorption of oral bacteria to saliva-coated and uncoated HA as reported by Hillman, van Houte and Gibbons (1970). Table
3. Effect of pretreatment
Also the rate of adsorption of Strep. miteor strain 26 to saliva-coated HA, shown in Fig. 3, was similar to that described by Hillman et al. (1970). However, Hillman rt al. (1970) showed that adsorption of Strep. miteor strain 26 to coated-apatite is considerable at pH 6.0 and that the difference between adsorption to saliva-coated and uncoated HA is minimal at pH 6.0, and greater at 7.0 and 7.5. In our experiments we observed maximal adsorption to saliva-coated apatite at pH 6.0 and did not observe the dramatic pH effect on uncoated HA, at least at pH 6.0 and 6.8, as described by Hillman et al. (1970). Our data on the adsorption of Strep. mitcor strain 26 to uncoated HA at pH 60 and 68 show a difference in the same direction observed by Hillman et al. (1970) but not nearly as marked. In addition, no significant pH effect of this nature was observed in experiments conducted with Strep. sanguis or Strep. mutans. Treatment of Strep. mutans with a variety of enzymes and low molecular weight dextran has supplied information concerning the nature of the binding components present on the surface of this organism. These experiments have shown that Strep. mutans has a trypsin and pronase-sensitive surface component. When this organism is pretreated with either of these proteolytic enzymes, its ability to adhere to dextran-coated HA is significantly reduced. This is consistent with the observations of Mukasa and Slade (1974) and Gibbons
of saliva-coated adhesion
Pretreatment No pretreament Stwp. sanguis culture liquor Stnup. sanguis concentration culture liquor Stnrp. sunguis Boiled Strep. sanguis Strep. saryuis cell walls Boiled Strep. satyuis cell walls
apatite
on Strep. sanguis 7
‘A Adsorption 56 f 49-J 28 + 28 f 56 f 28 + 62f
8* 14 9* 5* IO 4” 11
Adsorption of Strep. sanguis 7 to uncoated HA with no pretreatment 31 per cent. * (p < 0.001) Comparison of adsorption to saliva-coated HA of no pretreatment to the experimentals.
614
W. F. Liljemark and S. V. Schauer
and Spine11 (1974). Secondly, dextranase pretreatment produced a marked reduction in the ability of Strup. mmn~.s to adsorb to dcxtran-coated HA. Since the cells arc glucose-grown, the small account of sucrose in the yeast extract and glucose (Mukasa and Slade, 1974) present in the growth medium has probably initiated the formation of significant quantities of cell-bound dextran. It has been shown (Mukasa and Slade. 1973) that the production of cell-bound dcxtran by Strc/~. ~mrtc~~,sis required for its adherence to a glass surface. Thus our observation supports this concept and. in addition. that cell-bound dextran may also be necessary for Strep. ~UTLUISbinding to dextran-coated HA. However, addition of low mol. wt dextran (40,000) to Strrp. rnutar~s cells prior to incubation with dextrdncoated HA produced a significant inhibition of Strrp. mtuns adsorption to the dextran-coated HA. This inhibition of adsorption implies that somehow addition of low molecular weight dextran blocks the binding sites involved in the adherence process. This effect is similar to the observation of Gibbons and Nygaard (1968) that low mol. wt dextrans could prevent aggregation of Strep. mm~~s cells by high mol. wt dextrdns, and is consistent with the mechanism of dextran-sucrose induced aggregation of Strep. mutam proposed by Olson. Guggenheim and Small (1974). Proteolytic enzyme treatment of Strep. smguis, particularly with trypsin. significantly reduced its ability to adsorb to saliva-coated HA. Similar treatment of Strep. sa/iuarius, Strep. rniteor and group A streptococci with trypsin has been shown to be effective in reducing their ability to adhere to epithelial cells (Gibbons rt al.. 1972; Liljemark and Gibbons, 1972). In addition the adherence to epithelial cells by these organisms as observed in the electron microscope appears to be mediated by a trypsin-sensitive fibrillar “fuzzy coat” (Gibbons tar al., 1972; Liljemark and Gibbons, 1972). Furthermore, Strrp. saliuurius and the group A streptococci also release components into the culture liquor of in-citro grown cells which block these organisms’ adherence to epithelial cells (Gibbons et al., 1972; Liljemark and Gibbons. 1972; Ellen and Gibbons. 1972; Strrptococcus
Swanson, smguis,
Hsu
and
Gotschlich,
1969).
however. does not appear to have a “fuzzy coat” (Skobe, Gibbons and van Houte, 1973) and. as shown in our experiments. releases very little adherence-blocking components into the culture liquor. Although very little adherence-blocking components are found in the Stwp. smguis culture liquor. it contains relatively high dextran and levan sucrase activity. Thus it is unlikely that these enzymes are the components which bind Stwp. sarxguis to saliva-coated HA. It appears that even though the binding components of these organisms are protein in nature. they differ from Strrp. salirarius and the group A streptococci not only in the specificity of their binding components. but in the nature of the association of the binding component to the bacterial cell surface. Use of cell-wall preparations of Strep. suyuis confirmed the presence of a wall-associated binding compound in the blocking experiments. Kashket and Donaldson (1972) suggested that the salivary-induced aggregation of Strcjp. sarzguis, which is similar to the salivary-mediated adherence of Strep. srr,~guis to HA (Hillman et ul.. 1970: van Houte, Gibbons and Banghart, 1970). is mediated by its peptido-
glycan. Our data do not support the hypothesis that adsorption is mediated by peptidoglycan because trypsin causes a significant inhibition of adsorption to saliva-coated HA. and trypsin is not active against the 1/’amino or u-alanine bonds from lysine in the pentapeptide of the peptidoglycan (Park and Hancock, 1960). Pretreatment of Stwp. sunguis cells with lipase enhanced adsorption to saliva-coated apatite, and stearicacid-blocking adsorption. Thus it is possible that a lipid component may also be involved in the adherence of Srrrp. srrugui.s to saliva-coated HA. Acknorrlr,ll~/~,?l~,flt~This study was supported research contract NIH-NIDR 72-2407.
in part
by
REFERENCES
Carlsson J. 1967. Presence of various types of non-haemolytic streptococci in dental plaque and in other sites of the oral cavity in man. Odom. Reoy 18, 55~.74.
Ellen R. P. and Gibbons R. J. 1972. M protein-associated adherence of Strrptococcus p,m~mrs to epithelial surfaces: prerequisite for virulence. Inflcr. I,nmunol. 5, 826& x30. Gibbons R. J. and Fitzgerald R. J. 1969. Dextran-induced agglutination of Streptococcus mms, and its potential role in the formation of microbial dental plaques. J. Bact. 98, 341-346. Gibbons R. J. and Nygaard M. 1968. Synthesis of insoluble dextran and its significance in the formation of gelatinous deposits by plaque-forming streptococci. Archs oral Biol. 13, 1249-1262.
Gibbons R. J. and Spine11 D. M. 1969. Salivary-induced aggregation of plaque bacteria. In: DYUTUIPlauue (Edited by McHugh W. D.i. pp. 207-215. Livingstone.’ Gibbons R. J. and Sninell D. M. 1974. Studies of the binding of dextran and glycosyl transfcrases to Strip. ~U~NIU. Internat. Ass. for Dent. Res. Preprinted abstracts. 52nd General Meeting. Abstract 757. Gibbons R. J. and van Houte J. 1971. Selective bacterial adherence to oral epithelial surfaces and its role as an ecological determinant. I+YY. I!ntnunol. 3, 567-573. Gibbons R. J., van Houte J. and Liljemark W. F. 1972. Parameters that effect the adherence of Strrpmwccus sulfrtrrius to oral epithelial surfaces. J. drrlt. RL’s. 51, 424-435. Gibbons R. J. and van Houte J. 1973. On the formation of dental plaques. J. Prriodmt. 44, 347-360. Hay D. 1. 1967. The adsorption of salivary protems by hydroxylapatite and enamel. Archs oral Biol. 12, 937.-946. Hillman J. D., van Houte J. and Gibbons R. J. 1970. Sorption of bacteria to human enamel powder. Archs orul Biol. 15, X99--903. Jordan H. V., Englander H. R. and Lim S. 1969. Potentially cariogenic streptococci in selected population groups in the Western Hemisphere. J. Am. drrlt. Asa. 78, 1331&1335. Kashket S. and Donaldson C. G. 1972. Saliva-induced aggregation of oral streptococci. J. Boer. 112, I127- 1133. Krasse B. 1954. The proportional distribution of St,rptococcus ,suliwriu.s and other streptococci in various parts of the mouth. Odmt. RcJL’\~5, 203-21 1. Liljemark W. F. and Gibbdns R. J. 1972. Proportional distribution and relative adherence of Streptococcus rnitror (mitis) on various surfaces in the human oral cavity. Iu,f>cr. /rnr,luriol. 6, X52 859. Mukasa H. and Slade H. D. 1973. Mechanism of adherence of Sm~ptococcus mutms to smooth surfaces-l: Roles of insoluble dextran-levan synthetase enzymes and cell wall polysaccharide antigen in plaque formation. Infect. I/t?mutiol. 8, 555-562. Mukasa H. and Slade H. D. 1974. Mechanism of adherence of Stwptococcus muram to smooth surfaces-II: Nature of the binding sltc and the adsorption of dextran-levan
Factors
influencing
bacterial
synthetase enzymes an the cell-wall surface of the Streptococcus, 1974. Infect. Immunol. 9, 419-429. Olson G. A., Guggenheim B. and Small P. A. Jr. 1974. Antibody-mediated inhibition of dextran/sucrose-induced agglutination of StrcJptococcus muraw Infect. Immunol. 9. 273-278. Park J. T. and Hancoc:k R. 1960. A fractionation procedure for studies of the synthesis of cell mucopeptide and other polymers in cells Staphylococcus awws. J. yrrz. Microhiol. 22, 249-258. Rolla G. and Mathiesen P. 1969. The adsorption of salivary proteins and dextrans to hydroxylapatite. In: Drrltul Plaque (Edited by McHugh W. D.). pp. 129~141. Livingstone.
adsorption
to hydroxyapatite
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Skobe Z., Gibbons R. J. and van Houte J. 1973. Ultrastructural differences of surface components of oral streptococci. Internat. Ass. for Dent. Res. Preprinted abstracts. 52nd General Meeting. Abstract 747. Swanson J., Hsu K. C. and Gotschlich E. C. 1969. Electron microscopic studies on streptococci&I: M antigen. J. L’Y~.Med. 130, 1063%1091. van Houte J., Gibbons R. J. and Banghart S. B. 1970. Adherence as a determinant of the presence of Streptococcus salivarius and Streptococcus sarquis on the human tooth surface. Arch 04 Biol. IS. 102551034. van Houte J.. Gibbons R. J. and Pulkkinen A. J. 1971. Adherence as an ecological determinant for streptococci in the human mouth. Archs orai Bid. 16. 1131~1141.
Biol. Vol. 20. pp. 50!%615.
PergamonPress 1975.Printed
I” Great Bntam.
STUD:[ES ON THE BACTERIAL COMPONENTS WHICH BIND STREPTOCOCCUS SANGUZS AND STREPTOCOCCUS MUTANS TO HYDROXYAPATITE W. F. LILJEMARKand S. V. SCHAUER &hool of Dentistry and Department of Microbiology, University of Minnesota, Minneapolis, Minnesota 55455, U.S.A. Summary-Experiments utilizing radio labelled oral streptococci in an in-vitro assay system have demonstrated the selective ability of certain species of these bacteria preferentially to adhere to saliva-coated and dextran-coated hydroxyapatite (HA). Pretreatment of Streptococcus sangnis and Streptococcus mutans with the proteolyic enzymes trypsin and pronase greatly reduced their ability to adhere to saliva or dextran-coated HA respectively. Pretreatment of Strep. sanguis with lipase increased its adsorption to saliva-coated HA and stearic acid-blocked adsorption, Cell walls isolated from Strep. sanguis proved effective in blocking Strep. sanguis adsorption to salivacoated apatite. Cells of Strep. mutans after treatment with dextranase or low molecular weight dextrans did not adhere well to dextran-coated HA.
INTRODUCTION Selective bacterial localization in the human oral cavity has been most studied with Streptococcus species (Krasse, 1954; Carlsson, 1967) and is purportedly related to their ability to attach to different oral surfaces (van Houte, Gibbons and Pulkkinin, 1971). Furthermore, it has also been proposed that bacterial adherence to different surfaces is a major ecological determinant governing the colonization of bacteria in various sites in the mouth (van Houte rt al., 1971; Gibbons and van Haute, 1971). Because much evidence has accumulated which indicates that certain oral streptococci are highly cariogenic (Jordan, Englander and Lim, 1969) it is important to understand the mechanisms responsible for their numerical proportional differences as they relate to adherence, specifically to the tooth surface and dental plaque. The adherence of bacteria to the tooth surface and the production of dental plaque has been shown to be a very complex process involving a number of specific components (Gibbons and van Houte, 1973). The research described in this report evaluates the interaction of specific stretptococcal surface components with salivary proteins and dextrans in their adherence to hydroxyapatite (HA). MATICRIALSAND Cultures and cultural
METHODS
conditions
Laboratory strains of Strep. mutans strain 6715, Strep. saliuarius strain 9GS2, Strep. sanguis strain H7P and Strep. miteor strain 26 were obtained from the culture collection of the Forsyth Dental Center. Strains 3 and C-l of Strep. xdiuarius were isolated from human saliva, and strains 7, 2 and 156 of Strep. sanguis were isolated from tooth surfaces. All organisms were grown in Trypticase Soy Broth (Baltimore Biol. Lab. BBL) and maintained by weekly transfer of mitis-salivarius agar (BBL). Bacteria were incubated anaerobically at 37°C in Brewer jars containing gas packs (BBL). All strains of Strep. sanguis and Strep. miteor were selected
for their ability to aggregate with saliva (Gibbons and Spinell, 1969) or. in the case of Strep. mutans, with dextran (Gibbons and Fitzgerald, 1969). Radio-labelled bacteria were prepared in a trypticase salts medium described by Gibbons, van Houte and Liljemark (1972). Uniformly r4C-labelled amino acids in the form of acid hydrolysed algal protein (New England Nuclear Corp.) were added to give a final activity of 2.0 /ICI/ml. Glucose was autoclaved separately and added to the medium to give a concentration of @2 per cent. Assay qj’adhrrencr titr (HA)
qf oral streptococci
to hydroxyupu-
Cell suspensions were prepared from 24 to 48 hr cultures. The organisms were harvested by centrifugation. washed twice with 0.067 M phosphate buffer (pH 6.0) and resuspended in buffer at a concentration of IO”/ml. These suspensions were routinely sonicated for I5 set in a Branson sonifier, Model S75, at a number 2 setting which virtually eliminated bacterial chains. Cells were then washed once in phosphate buffer (pH 6.0) to remove any labelled components released by sonication. Cell numbers were determined by direct microscopic count in a Petroff-Hauser counting chamber. Prior to use in the assay system, the HA (Bio-Gel HTP, Bio-Rad Richmond, California) was subjected to extensive washing procedures for removal of very small apatite “fines”. Two and a half grams of HA were allowed to settle for 5 min in 500 ml of 0.067 M phosphate buffer (pH 6.0) and the supernatant decanted. This procedure was repeated 3 times and the HA resuspended in lOOOm1 of the buffer. Again the HA was allowed to settle for 5 min and the supernatant decanted. This 5 min settling in 1000 ml of buffer was repeated twice. The HA was again resuspended in 500 ml of the buffer and dispensed in 10 ml aliquots into 15 ml conical centrifuge tubes. The apatite was centrifugated at full velocity in an International Clinical centrifuge Model L5655 for 5 sec. The supernatant was decanted and the procedure repeated 15 times. This extensive washing was necessary to ensure quanti609
610
W. F. Liljemark and S. V. Schauer
tative recovery of the HA in the washing procedure following incubation with bacteria. Collection and preparation of whole saliva for the coating of HA followed the method of Gibbons and Spine11 (1969), and the HA was then coated with this saliva by the method of Hay (1967). Dextran-coated HA was prepared by the method of Rolla and Mathiesen (1969). Pre-weighed dried samples of washed HA weighing from l-50 mg were suspended in 0.067 M phosphate buffer (pH 6.0) and a standard curve derived relating optical density to weight with a Klett-Summerson calorimeter at 440 nm. Neither saliva-coating nor dextrdn-coating of HA significantly altered the weight determinations of apatite samples as measured by the Klett-Summerson calorimeter. To test for the adsorption of bacteria to HA, 1.0 ml of 0.067 M phosphate buffer (pH 6.0) containing 5 mg of the dried-washed HA preparation was mixed with 1.0 ml of a washed radio-labelled bacterial cell suspension (I O”/ml) and incubated for 60 min at 37°C. During this period, the mixture was sufficiently agitated to keep the HA particles in suspension. The HA bacterial mixture was then washed 4 times with phosphate buffer by the centrifugation procedure described above. This procedure was found to remove 99 per cent ofall bacteria which had not adsorbed to the HA. To recover the bacteria which remained adsorbed to the HA after washing, the HA was solubilized by susin 0.2 M ethylenedinitrilotetraacetic acid pension (EDTA) at pH 8.0 for 3@60 min. After the HA was dissolved, the remaining bacteria were collected on 0.45 pm Millipore filters. The filters were dried, placed in liquid scintillation vials, and radioactivity was Packard liquid scintillation monitored in a spcctromcter. The number of bacteria adsorbed to HA in each experiment was expressed as a percentage of the total number of bacteria in the reaction mixtures. Eflkct qf’pH on the adherence Strep. miteor to HA
qf Strep. sanguis
and
To test the effect of pH. a series of reaction mixtures with a range in pH from 4.5 to 9.0 were prepared by combining 0067 M di-basic sodium and mono-basic potassium phosphate solutions in appropriate proportions. Cells from each bacterial species were washed and suspended separately in the test solutions with saliva-coated HA. Prctreattnmt
qf‘hactrriul
crlls with etnymrs,
lipids and
low molecular weight destrans Samples of “C-labelled bacterial suspensions were incubated with various enzyme preparations at 37°C for 60min. Controls consisted of bacterial cells incubated with buffer or with heat-inactivated enzymes. After incubation, the treated bacteria without subsequent washing were assayed for this ability to adhere lo saliva-coated or dextran-coated HA. Enzyme treatments which influenced bacterial adherence were repeated. After incubation the bacteria were washed twice by centrifugation to remove residual enzyme. The organisms were restandardized in buffer, and assayed for their ability to adhere to HA. Their adherence was compared to cells treated with buffer or with heat-inactivated enzymes in a comparable manner. Sources of’ ettzytws. Trypsin (3 x crystallized, salt free. Worthington Biochemicals), pronase (California
Biochemicals. nuclease free). dextranase (Worthington Biochemicals, BGC purified grade) and hyaluronidase (Worthington Biochemicals, chromatographically prepared) were used in 0.067 M phosphate buffer (pH 6.X). Wheat germ Lipase (Nutritional Biochemical Corp.. salt free) was dissolved in 0.067 M phosphate buffer (pH 5.0). The effect of pretreating Sfrrp. sanguis cells with lipids and Strep. muram with low molecular weight dextrans was also determined. Oleyl alcohol (25Opg, mg). stearic acid (1 mg/ml) and hyaluronic acid (1 mg/ ml) were prepared in ethanol and subsequently diluted with water. Cell suspensions of radio-labelled Strep. sanguis strain 7 were incubated with each lipid for 60min. The lipid-treated cell suspensions were then mixed with HA and incubated for 60 min and their ability to adhere to saliva-coated HA was determined. Cell suspensions of radio-labelled Strep. mutans were pretreated with Pharamacia dextrans of mol. wt 10.000 and 40,000 at a concentration of 1 mg/ml and assayed for their ability to adhere to dextran-coated HA. &j&-t
qf’culturejkctiotzs
to saliva-coated
qf Strep. sanguis on udherence
H,4
Strc~ptoc0cc1t.ssunguis strain 7 was grown to 68 x lo* cells/ml in predialysed trypticase soy broth. The bacterial cells were removed by centrifugation, and the culture liquor dialysed against 0.067 M phosphate buffer at 4°C. The dialysate was concentrated 50-fold by dialysis against polyethylene glycol, and stored at - 20°C. The streptococcal cells from the culture were washed once with phosphate buffer (pH 6.8), and suspended in 20ml of the same buffer. The suspension was mixed with 5 g of glass beads (62-88 pm, Lapine Scientific) and the bacteria ruptured by exposure to sonic oscillation 4 times for 5 min intervals using a Branson sonifier model S75 at maximum amplitude in an ice bath. The mixture was centrifuged at x 2000 g for 5 min to remove intact cells and beads. The supernatant was centrifuged at x 87009 for 20min. The supernatant was discarded the pellet resuspended and centrifuged at x 10,800g for 20min. After resuspending this pellet, a final centrifugation at x 2500 g for 5 min to remove any remaining whole cells was performed and the supernatant saved. The supernatant was examined by electron microscopy to assure that only cell walls remained. The supernatant from the original culture and cell wall preparations were tested for their ability to block the adherence of Strep. sunguis to saliva-coated HA. Five milligrams of saliva-coated HA were incubated with 2 ml of each preparation for 60 min. The HA was washed once. the radio-labelled streptococcal cells were added, and the mixture was incubated as previously described. RESULTS
Adsorption qforal coated aputite
streptococci
to saliva and destran-
The adsorption of several strains of Strep. sanguis, Strep. sulivarius, Strep. miteor and Strep. mutans to saliva-coated, dextran-coated and uncoated apatite was tested using the conditions described above, and the results are reported in Table 1. Note that Strep. sattguis and Strep. miteor adhered best to saliva-coated
salivarius 9GS.F 3*
Stwp. Strrp. Stwp.
SW/~. II~U~~III.C 67 IS5
Strep.wireor M29
.sccli~arius rniteor 26”
17
14* 8 16_+9 58 _+ 7 56
49 17_+ 12
smguis smguis
Strep. Strep.
2 I56+
61 40 2 11
“/, Adsorption of cells to saliva-coated HA
of Strep. sulicarius,
Stwp. sunguis H7p Strep. stlnguis 7*
Bacterial strains
1. Adsorption
1
1
6 5 7
2 8
92
Number of experiments
Strep. sanguis,
and
Strep.
46 _t 9
15
”i0 Adsorption of cells to dextran-coated HA
Strep. miteor
_
_ -
_ _
_
_. ,,,
_._.,
-
,-.
.,.--
Bacteria (I.0 x 109) and 5 mg HA suspended in 2 mls of 0.067 M phosphate buffer was used in each experiment. * @ < 0.005) Comparison of adsorption to saliva-coated HA and adsorption to uncoated HA. 10, < OGOS) Comparison of adsorption to dextran-coated HA and adsorption to uncoated HA.
Plaque
Cheek
Tongue dorsum
Plaque
Primary ecological niche
Table
to
5
2
Number of experiments
mutans
13 f 8
45 f 13 30 + 6 24 4 f 34
28 41 * 1
21 34 * 8
‘:i Adsorption of cells to uncoated HA
hydroxyapatite
3
ET “a 0’ $ 3 z io”
3 0”
I
4 3 4
53. Y, w R 4 m 5
2 6
27
Number of experiments
9 0 7 5’ ? FJ 5’ 09 P
2
W. F. Liljemark and S. V. Schauer
612
Strep. matans 6715 adsorption to dextran-coated HA did not vary significantly (data not shown). Under the optimum conditions as described for this system. i.e. (pH 6.0) and a cell concentration of 5.0 x IO8 bacteria per ml in 2 ml with 5 mg HA, adsorption of Strep. sunguis 7 and Strep. miteor 26 continued for 45-60min before reaching a maximum as shown in Fig. 3. The adsorption of Strep. mutans 6715 to dextran-coated HA reached a maximum level in 5 min (data not shown). All subsequent experiments used a mixture of 5.0 x JO8 bacteria per ml in 2 ml of 0.067 M phosphate buffer (pH 60), containing 5 mg HA which was incubated at 37°C for I hr unless indicated otherwise.
01 Cell numbers
IO10
IO9
IO8
IO'
pw
reaction
mix
Fig. I. Effect of cell concentration on the ability of Strep. str~gr~i 7 to adhere to saliva-coated hydroxyapatite; 9.1 x IO3 counts/min would correspond to 100 per cent adsorption.
HA. while Strep. saliaarius adhered best to uncoated HA, Streptococcus mutans adhered better to dextrancoated HA than uncoated or saliva-coated HA, while the adsorption of Strep. sanguis was not enhanced by dextran-coated HA. Eflect ~$cell concentration und pH on the udherence qf Strep. sanguis, Strep. miteor and Strep. mutans to saliva and dextran-coated HA The effect of cell concentration of the ability of Strep. sanguis 7 to adhere to saliva-coated apatite is shown in Fig. 1. A concentration of IO9 bacteria suspended in 2 ml of 0.067 M phosphate buffer (pH 6.0) with 5 mg of saliva-coated HA yielded the highest per cent adsorption. Figure 2 illustrates the effect of pH on the ability of pure cultures of Strep. suryuis 7 and Strep. miteor 26 to adhere to saliva-coated HA. Each organism adsorbed maximally at a pH of 6.0, which is within the 57-7.0 pH range of normal human saliva (Burnett and Scherp. 1957). However, in the pH range from 4.5-9.0.
Effect qfpretreatment with enzymes. lipids and low molecular weight dextran on adherence qf‘strep. sanguis and Strep. mutans to saliva or destran-coated HA Pretreatment of Strep. saryuis with trypsin (I mg/ml) or pronase (1 mg/ml) reduced its ability to adhere to saliva-coated HA eight- and three-fold respectively. Pretreatment with lipase (1 mg/ml) or hyaluronidase (I 50 units/ml), however, increased Strep. sanguis adsorption to saliva-coated HA (Table 2). Pretreating Strep. sanguis with stearic acid (250 ,ug/ml) significantly decreased its adsorption to saliva-coated apatite. Dextranase (20 units/ml). trypsin. or pronase pretreatment of S~,‘CJ~I. wtrrm reduced its adsorption to dcxtrancoated HA 8- and 20-fold respectively. Pretreatment with lipase increased Strep. mutans adsorption only slightly. Addition of low molecular weight dextran (10,000 or 40,OOOmol. wt at 1 m&ml) resulted in a three-fold decrease in adsorption (Table 2). Blocking effect qf culture jxction qf’ Strep. sanguis. Preincubation of unlabelled Strep. sanguis with salivacoated HA resulted in a 2-fold decrease in per cent adsorption to HA when compared to the control (Table 3). Preincubation of boiled unlabelled Strep. sanguis did not affect the susequent adsorption of radio labelled Strep. saf7gui.s. In a similar experiment. preincubation with the culture liquor from Strep. sanguis had no inhibitory effect on the adsorption of radiolabelled Strep. sanguis unless it was dialysed and concentrated more than 50-fold. Only then was the inhibition ofadherence by the culture liquor substantial. Cell walls isolated from Strep. sanguis by sonic oscillation with small glass beads and differential centrifugation
“V
A
LA-
4 50 E ‘6 8
“0
40
‘0 E = b
,/
/
*‘\ h
/I
30
S
\
9
.
S.
s.
mitmr
* S. songuis
w
ae Fig. 2. The effect of pH on the ability of Srrep. sanguis 7 and Strc’p. n~iteor 26 to adhere to saliva-coated hydroxyapatite; X.2 x 10’ counts/min would correspond to 100 per cent adsorption.
I
0
I 20
I
I 40
I
Time.
III 60
I 60
I loo
min
Fig. 3. Rate of adsorption of Strep. sanguis 156 and Srrrp. rnitror. 26 to saliva-coated hydroxyapatite: 4.3 x IO3 counts/min would correspond to 100 per cent adsorption.
613
Factors influencing bacterial adsorption to hydroxyapatite Table 2. Effect of enzyme, lipid and low mol. wt dextran pretreatment Strep. mutans to HA % Adsorption of Strep. sanguis 2 to saliva-coated HA Control Trypsin Pronase Lipase Hyduronidase Dextranase Hyaluronic acid Stearic acid Oleyl alcohol Low mol. wt dextran
of Strep. sunguis and
on the adsorption
of Strep. mutans 6715 to dextran-coated HA
% Adsorption
40*9 5 * 4* 15+6* 55 + 77 44+5 4017 30f 13 17$ 11* 44+ 13
42 _t 8 5 -+_5* 2 * 4* 47+ 11 2 * 3*
14 * 14 * *
* (p < ON)l) Comparison of adsorption to coated HA of the control and experimentals. t (b < 0.01) Comparison of adsorption to coated HA of the control and experimentals. produced an inhibitory effect as great as the whole cells. Boiled cell walls did not produce an inhibitory effect. The untreated HA control included in Table 3 demonstrates that any residual adherence activity after treatment of the HA may be due, in part, to saliva-independent adsorption to HA. This observation may also be extended to include the experiments presented in Table 2 which show residual adherence activities exist after enzyme treatment of the bacteria. DISCUSSION
This report has described a new assay system for the detection of bacterial adsorption to hydroxyapatite. In addition, the assay has been utilized to study some of the parameters which may be involved in the selective colonization of tooth surfaces by oral streptococci, and has concentrated on determining the characteristics of certain surface components of Strep. mutans and Strep. sanguis and how they effect bacterial adherence to hydroxyapatite. AS shown in Table 1 this in-vitro assay system demonstrates the selective adsorption of several species of oral streptococci to HA. These in-aitro observations are consistent with the selective adsorption of oral bacteria to saliva-coated and uncoated HA as reported by Hillman, van Houte and Gibbons (1970). Table
3. Effect of pretreatment
Also the rate of adsorption of Strep. miteor strain 26 to saliva-coated HA, shown in Fig. 3, was similar to that described by Hillman et al. (1970). However, Hillman rt al. (1970) showed that adsorption of Strep. miteor strain 26 to coated-apatite is considerable at pH 6.0 and that the difference between adsorption to saliva-coated and uncoated HA is minimal at pH 6.0, and greater at 7.0 and 7.5. In our experiments we observed maximal adsorption to saliva-coated apatite at pH 6.0 and did not observe the dramatic pH effect on uncoated HA, at least at pH 6.0 and 6.8, as described by Hillman et al. (1970). Our data on the adsorption of Strep. mitcor strain 26 to uncoated HA at pH 60 and 68 show a difference in the same direction observed by Hillman et al. (1970) but not nearly as marked. In addition, no significant pH effect of this nature was observed in experiments conducted with Strep. sanguis or Strep. mutans. Treatment of Strep. mutans with a variety of enzymes and low molecular weight dextran has supplied information concerning the nature of the binding components present on the surface of this organism. These experiments have shown that Strep. mutans has a trypsin and pronase-sensitive surface component. When this organism is pretreated with either of these proteolytic enzymes, its ability to adhere to dextran-coated HA is significantly reduced. This is consistent with the observations of Mukasa and Slade (1974) and Gibbons
of saliva-coated adhesion
Pretreatment No pretreament Stwp. sanguis culture liquor Stnup. sanguis concentration culture liquor Stnrp. sunguis Boiled Strep. sanguis Strep. saryuis cell walls Boiled Strep. satyuis cell walls
apatite
on Strep. sanguis 7
‘A Adsorption 56 f 49-J 28 + 28 f 56 f 28 + 62f
8* 14 9* 5* IO 4” 11
Adsorption of Strep. sanguis 7 to uncoated HA with no pretreatment 31 per cent. * (p < 0.001) Comparison of adsorption to saliva-coated HA of no pretreatment to the experimentals.
614
W. F. Liljemark and S. V. Schauer
and Spine11 (1974). Secondly, dextranase pretreatment produced a marked reduction in the ability of Strup. mmn~.s to adsorb to dcxtran-coated HA. Since the cells arc glucose-grown, the small account of sucrose in the yeast extract and glucose (Mukasa and Slade, 1974) present in the growth medium has probably initiated the formation of significant quantities of cell-bound dextran. It has been shown (Mukasa and Slade. 1973) that the production of cell-bound dcxtran by Strc/~. ~mrtc~~,sis required for its adherence to a glass surface. Thus our observation supports this concept and. in addition. that cell-bound dextran may also be necessary for Strep. ~UTLUISbinding to dextran-coated HA. However, addition of low mol. wt dextran (40,000) to Strrp. rnutar~s cells prior to incubation with dextrdncoated HA produced a significant inhibition of Strrp. mtuns adsorption to the dextran-coated HA. This inhibition of adsorption implies that somehow addition of low molecular weight dextran blocks the binding sites involved in the adherence process. This effect is similar to the observation of Gibbons and Nygaard (1968) that low mol. wt dextrans could prevent aggregation of Strep. mm~~s cells by high mol. wt dextrdns, and is consistent with the mechanism of dextran-sucrose induced aggregation of Strep. mutam proposed by Olson. Guggenheim and Small (1974). Proteolytic enzyme treatment of Strep. smguis, particularly with trypsin. significantly reduced its ability to adsorb to saliva-coated HA. Similar treatment of Strep. sa/iuarius, Strep. rniteor and group A streptococci with trypsin has been shown to be effective in reducing their ability to adhere to epithelial cells (Gibbons rt al.. 1972; Liljemark and Gibbons, 1972). In addition the adherence to epithelial cells by these organisms as observed in the electron microscope appears to be mediated by a trypsin-sensitive fibrillar “fuzzy coat” (Gibbons tar al., 1972; Liljemark and Gibbons, 1972). Furthermore, Strrp. saliuurius and the group A streptococci also release components into the culture liquor of in-citro grown cells which block these organisms’ adherence to epithelial cells (Gibbons et al., 1972; Liljemark and Gibbons. 1972; Ellen and Gibbons. 1972; Strrptococcus
Swanson, smguis,
Hsu
and
Gotschlich,
1969).
however. does not appear to have a “fuzzy coat” (Skobe, Gibbons and van Houte, 1973) and. as shown in our experiments. releases very little adherence-blocking components into the culture liquor. Although very little adherence-blocking components are found in the Stwp. smguis culture liquor. it contains relatively high dextran and levan sucrase activity. Thus it is unlikely that these enzymes are the components which bind Stwp. sarxguis to saliva-coated HA. It appears that even though the binding components of these organisms are protein in nature. they differ from Strrp. salirarius and the group A streptococci not only in the specificity of their binding components. but in the nature of the association of the binding component to the bacterial cell surface. Use of cell-wall preparations of Strep. suyuis confirmed the presence of a wall-associated binding compound in the blocking experiments. Kashket and Donaldson (1972) suggested that the salivary-induced aggregation of Strcjp. sarzguis, which is similar to the salivary-mediated adherence of Strep. srr,~guis to HA (Hillman et ul.. 1970: van Houte, Gibbons and Banghart, 1970). is mediated by its peptido-
glycan. Our data do not support the hypothesis that adsorption is mediated by peptidoglycan because trypsin causes a significant inhibition of adsorption to saliva-coated HA. and trypsin is not active against the 1/’amino or u-alanine bonds from lysine in the pentapeptide of the peptidoglycan (Park and Hancock, 1960). Pretreatment of Stwp. sunguis cells with lipase enhanced adsorption to saliva-coated apatite, and stearicacid-blocking adsorption. Thus it is possible that a lipid component may also be involved in the adherence of Srrrp. srrugui.s to saliva-coated HA. Acknorrlr,ll~/~,?l~,flt~This study was supported research contract NIH-NIDR 72-2407.
in part
by
REFERENCES
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Ellen R. P. and Gibbons R. J. 1972. M protein-associated adherence of Strrptococcus p,m~mrs to epithelial surfaces: prerequisite for virulence. Inflcr. I,nmunol. 5, 826& x30. Gibbons R. J. and Fitzgerald R. J. 1969. Dextran-induced agglutination of Streptococcus mms, and its potential role in the formation of microbial dental plaques. J. Bact. 98, 341-346. Gibbons R. J. and Nygaard M. 1968. Synthesis of insoluble dextran and its significance in the formation of gelatinous deposits by plaque-forming streptococci. Archs oral Biol. 13, 1249-1262.
Gibbons R. J. and Spine11 D. M. 1969. Salivary-induced aggregation of plaque bacteria. In: DYUTUIPlauue (Edited by McHugh W. D.i. pp. 207-215. Livingstone.’ Gibbons R. J. and Sninell D. M. 1974. Studies of the binding of dextran and glycosyl transfcrases to Strip. ~U~NIU. Internat. Ass. for Dent. Res. Preprinted abstracts. 52nd General Meeting. Abstract 757. Gibbons R. J. and van Houte J. 1971. Selective bacterial adherence to oral epithelial surfaces and its role as an ecological determinant. I+YY. I!ntnunol. 3, 567-573. Gibbons R. J., van Houte J. and Liljemark W. F. 1972. Parameters that effect the adherence of Strrpmwccus sulfrtrrius to oral epithelial surfaces. J. drrlt. RL’s. 51, 424-435. Gibbons R. J. and van Houte J. 1973. On the formation of dental plaques. J. Prriodmt. 44, 347-360. Hay D. 1. 1967. The adsorption of salivary protems by hydroxylapatite and enamel. Archs oral Biol. 12, 937.-946. Hillman J. D., van Houte J. and Gibbons R. J. 1970. Sorption of bacteria to human enamel powder. Archs orul Biol. 15, X99--903. Jordan H. V., Englander H. R. and Lim S. 1969. Potentially cariogenic streptococci in selected population groups in the Western Hemisphere. J. Am. drrlt. Asa. 78, 1331&1335. Kashket S. and Donaldson C. G. 1972. Saliva-induced aggregation of oral streptococci. J. Boer. 112, I127- 1133. Krasse B. 1954. The proportional distribution of St,rptococcus ,suliwriu.s and other streptococci in various parts of the mouth. Odmt. RcJL’\~5, 203-21 1. Liljemark W. F. and Gibbdns R. J. 1972. Proportional distribution and relative adherence of Streptococcus rnitror (mitis) on various surfaces in the human oral cavity. Iu,f>cr. /rnr,luriol. 6, X52 859. Mukasa H. and Slade H. D. 1973. Mechanism of adherence of Sm~ptococcus mutms to smooth surfaces-l: Roles of insoluble dextran-levan synthetase enzymes and cell wall polysaccharide antigen in plaque formation. Infect. I/t?mutiol. 8, 555-562. Mukasa H. and Slade H. D. 1974. Mechanism of adherence of Stwptococcus muram to smooth surfaces-II: Nature of the binding sltc and the adsorption of dextran-levan
Factors
influencing
bacterial
synthetase enzymes an the cell-wall surface of the Streptococcus, 1974. Infect. Immunol. 9, 419-429. Olson G. A., Guggenheim B. and Small P. A. Jr. 1974. Antibody-mediated inhibition of dextran/sucrose-induced agglutination of StrcJptococcus muraw Infect. Immunol. 9. 273-278. Park J. T. and Hancoc:k R. 1960. A fractionation procedure for studies of the synthesis of cell mucopeptide and other polymers in cells Staphylococcus awws. J. yrrz. Microhiol. 22, 249-258. Rolla G. and Mathiesen P. 1969. The adsorption of salivary proteins and dextrans to hydroxylapatite. In: Drrltul Plaque (Edited by McHugh W. D.). pp. 129~141. Livingstone.
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Skobe Z., Gibbons R. J. and van Houte J. 1973. Ultrastructural differences of surface components of oral streptococci. Internat. Ass. for Dent. Res. Preprinted abstracts. 52nd General Meeting. Abstract 747. Swanson J., Hsu K. C. and Gotschlich E. C. 1969. Electron microscopic studies on streptococci&I: M antigen. J. L’Y~.Med. 130, 1063%1091. van Houte J., Gibbons R. J. and Banghart S. B. 1970. Adherence as a determinant of the presence of Streptococcus salivarius and Streptococcus sarquis on the human tooth surface. Arch 04 Biol. IS. 102551034. van Houte J.. Gibbons R. J. and Pulkkinen A. J. 1971. Adherence as an ecological determinant for streptococci in the human mouth. Archs orai Bid. 16. 1131~1141.
