A Human Salivary Protein Which Promotes Adhesion ofStreptococcus mutans Serotype c Strains to Hydroxyapatite
E. KISHIMOTO,1 D. I. HAY,2* AND R. J. GIBBONS3
Department of Preventive Dentistry, Okayama University Dental School, Shikata-cho, Okayama 700, Japan,' andDepartments of Biochemistr2 and Microbiology,3 The Forsyth Dental Center, 140 The Fenway,
Boston, Massachusetts 02115
Received 15 May 1989/Accepted 29 August 1989
The aim of this study was to investigate the nature of one of the factors in human submandibular-sublingual(SMSL) saliva which promotes the adhesion of Streptococcus mutans serotype c strains to hydroxyapatite (HA)surfaces. Gel filtration chromatography of SMSL saliva on Trisacryl GF2000 gave a void volume peak whichcontained the major fraction of adhesion-promoting activity for S. mutans JBP to HA. Maximum adhesion-promoting activity, however, eluted slightly later than the maximum 220-nm absorbance of the void volumepeak. Gel filtration of the void volume material after treatment with sodium dodecyl sulfate (SDS) gave an
early-eluting larger peak followed by a smaller peak with which the adhesion-promoting activity wasassociated. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) showed the presence of relatively slowlymigrating material associated with the larger inactive peak, presumably mucin, and a faster-migrating band(s)associated with the smaller active peak. SDS-PAGE indicated molecular weights in the range of 300,000 to350,000 by extrapolation from size standards. Comparison of SMSI, from five individuals showed the presenceof single bands or double bands associated with adhesion-promoting activity, indicating genetic polymorphism.The active material did not resemble either secretory immunoglobulin A, based on SDS-PAGE andimmunoassay, or fibronectin, based on SDS-PAGE, and also differed in molecular weight from salivary mucinsand salivary constituents previously reported to promote aggregation of certain oral bacteria, but a relationshipto these materials cannot be excluded. This adhesion-promoting material may play a significant role in theinitial colonization of tooth surfaces by S. mutans strains.
Since strains of Streptococcus mutans are associated withthe etiology of dental caries in humans (7, 22, 30, 32, 50),factors which modulate colonization of the oral cavity bythis organism are of considerable interest. Several investi-gators have shown that S. mutans and other oral strepto-cocci interact in specific ways with some salivary constitu-ents and that such interactions have the potential to affectthe colonization of the teeth by these organisms. Thus,saliva can cause aggregation of suspensions of many oralstreptococci (21), an activity which may lead to deletion ofaggregated organisms from the mouth. Alternatively, if theactive salivary constituents are adsorbed onto oral surfaces,bacterial adhesion onto these surfaces may be promoted.Several salivary constituents with such activities have beenreported (2, 10-12, 15, 16, 24, 25, 28, 29, 31, 37, 43-45, 48).Recently, we examined fractions from gel filtration chroma-tography of human submandibular-sublingual (SMSL) salivafor their ability to adsorb to hydroxyapatite (HA) surfacesand promote adhesion of S. mutans JBP cells (17). Major andminor regions of adhesion-promoting activity were detected.The major activity was associated with the mucin-containingfraction of the saliva, while the minor activity was associatedwith the acidic proline-rich proteins (18a). The purpose ofthe present study was to examine further the materialresponsible for the major adhesion-promoting activitypresent in the mucin fraction.
MATERIALS AND METHODSCollection of stimulated human salivary secretions. Human
SMSL saliva samples were obtained from five subjects(three males, two females; age range, 23 to 53 years) with
* Corresponding author.
custom-made collecting devices (38). Salivary flow wasstimulated by using acid candy. After collection, the salivawas passed through a fine-tipped pipette several times tohomogenize the serous and mucous phases and then dia-lyzed for 24 h at 4°C against 0.1 M NH4HCO3 (pH 8) bufferwhich contained 0.5% CHCl3 to prevent microbial growth.Freshly dialyzed saliva was used for chromatography, andthe remainder was stored frozen at -20°C.
Microbial cultures and culture conditions. Streptococcusmutans JBP (serotype c) was obtained from the culturecollection of the Forsyth Dental Center. The stock organ-isms were stored in 50% glycerol at -20°C until use. Inoculafrom the stock culture were grown anaerobically in Todd-Hewitt broth (BBL Microbiology Systems, Cockeysville,Md.) in Brewer jars filled with 80% N2, 10% H2, and 10%CO2 at 35°C. For adhesion studies, the organisms wereradiolabeled by growth in Todd-Hewitt broth which con-tained [3H]thymidine (10 ,uCi/ml) (New England NuclearCorp., Boston, Mass.) as previously described (20). Early-stationary-phase cells were used for assays. Cells fromovernight cultures were washed three times and suspendedin buffered KCI (0.05 M KCl, 1 mM CaCl2, 1 mM potassiumphosphate, 0.1 mM MgCl2, pH 6.0) which contained bovineserum albumin (BSA) (5 mg/ml) (Sigma Chemical Co., St.Louis, Mo.).Chromatography of saliva. Saliva was fractionated by gel
filtration on a column (2.6 by 91 cm) of Trisacryl GF2000(LKB, Gaithersburg, Md.) equilibrated with 0.1 MNH4HCO3 (pH 8) buffer at 4°C. Twenty milliliters of freshlydialyzed saliva was applied to the column, which was elutedat 20 ml/h. The eluant was monitored at 220 nm, and 10-mlfractions were collected. Fractions were kept at 4°C until useor stored frozen at -20°C for longer-term storage.
PROMOTION OF S. MUTANS ADHESION BY A SALIVARY PROTEIN 3703
Preparation of experimental pellicles. Pellicles were formedon spheroidal HA beads (B.D.H. Chemicals Ltd., Poole,England). Five milligrams of HA beads were equilibrated inbuffered KCI at room temperature overnight. Experimentalpellicles were prepared by treating the beads with 125 pd ofsaliva or salivary fraction for 1 h with continuous inversionof the mixtures at 6 rpm at room temperature. After washingtwice with buffered KCI, the HA beads were treated for 30min with buffered KCl which contained BSA (5 mg/ml) toblock any uncoated regions of the HA (18). The treatedbeads were then washed twice with buffered KCl andincubated for 1 h with [3H]thymidine-labeled streptococcalcells (6.25 x 106 cells) suspended in 125 ,ul of buffered KClwhich contained BSA (5 mg/ml). The beads were thenwashed three times with buffered KCl, and the number ofstreptococcal cells which attached to the beads was deter-mined by direct scintillation counting. All values werecorrected for quench due to the HA beads. The results wereanalyzed by Student's t test.
Electrophoretic methods. Native polyacrylamide gel elec-trophoresis (PAGE) was done with 7% gels at pH 9.0 (8, 39).Sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) was done in a vertical slab gel unit (SE600series; Hoefer Scientific Instruments, San Francisco, Calif.)cooled with water at 22°C, with 5 or 7.5% gels (27). Fiftymicroliters of sample was heated with 25 ,ul of sample bufferat 100°C for 5 min. After application of samples, voltage wasapplied to give a current of 7.5 mA per gel until the trackingdye entered the separating gel, when the current was in-creased to 15 mA per gel.
Gels were stained with either Coomassie blue or a silverstain. For the former, gels were treated with 0.05%Coomassie brilliant blue in 12% trichloroacetic acid (TCA)overnight at room temperature and destained in 12.5% TCA.Silver staining was accomplished with the Bio-Rad silverstaining kit (Bio-Rad Laboratories, Richmond, Calif.) exceptthat Cleland reagent (Sigma Chemical Co., St. Louis, Mo.)was substituted for the oxidizer in the kit (36). The molecularweights of the adhesion-promoting proteins were calculatedfrom plots of the logarithm of the molecular weights ofstandards (Bio-Rad Laboratories, Richmond, Calif.) versustheir relative mobilities. The molecular weights of the stan-dards used were: myosin, 200,000; P-galactosidase, 116,250;phosphorylase B, 92,500; BSA, 66,200; and ovalbumin,45,000. Fibronectin from human plasma was purchased fromBiomedical Technologies Inc., Stoughton, Mass.Immunoassay for secretory IgA. The enzyme-linked immu-
noassay for secretory immunoglobulin A (IgA) was done asdescribed previously (47). Human colostral secretory IgAused as a standard was obtained from Sigma Chemical Co.
Separation of bacterial adhesion-promoting molecules frommucin. Mucin-containing fractions from chromatography ofsaliva on a Trisacryl GF2000 column were treated with SDS(electrophoresis purity grade; Bio-Rad Laboratories, Rich-mond, Calif.) at a final concentration of 1.4% at 35°C fortimes ranging from 2 h to overnight. The product wasfractionated on a Trisacryl GF2000 column (2.6 by 91 cm) orSuperose 6 column (Pharmacia, Inc., Piscataway, N.J.) byelution with 0.05 M Tris chloride buffer at pH 8.0 containing0.1 M NaCl and 0.1% SDS at 25°C.
RESULTS
Adhesion-promoting activity of fractions obtained from gelfiltration of human SMSL saliva. The results of fractionatinga 10-ml sample of SMSL saliva on a Trisacryl GF2000
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FIG. 1. Adhesion-promoting activity (vertical bars) of adsorbedsalivary proteins prepared from fractions of SMSL saliva obtainedby gel filtration chromatography on Trisacryl GF2000 for S. mutansJBP cells to HA. The continuous line is the A220 of the columneluant.
column and assay of the resulting fractions for adhesion-promoting activity for S. mutans JBP to HA are shown inFig. 1. Typically, these chromatograms showed a high-molecular-weight mucin-containing void volume peak (frac-tions 15 to 18), a peak or shoulder containing secretory IgA(fractions 20 to 22), and other peaks representing the major-ity of the salivary proteins, identified by native PAGE (datanot shown). One major and one minor region of adhesion-promoting activity were found. The major region was asso-ciated with the mucin peak, the minor one with a proteinpeak which contained several salivary proteins, includingthe acidic proline-rich proteins, which have been shown tobe responsible for the second region of adhesion-promotingactivity (18a).
It was consistently observed that the maximum adhesion-promoting activity associated with the void volume peak didnot coincide exactly with the maximum optical density of thevoid volume material at 220 nm (Fig. 1). Instead, the activitytended to be displaced towards the lower-molecular-weightside of the void volume peak. To examine this more pre-cisely, an SMSL saliva sample was fractionated, and smallerfractions (2 instead of 10 ml) were collected and assayed.The results are shown in Fig. 2.The displacement of the adhesion-promoting activity to
the lower-molecular-weight side of the mucin peak wasclearly seen. Examination of these fractions by SDS-PAGEand silver staining gave the results shown in the inset. Therelatively slowly migrating material, presumably mucin, didnot form well-defined bands and was stained poorly with thesilver stain. A pair of well-defined, more rapidly migratingbands were also seen which coincided exactly with theadhesion-promoting activity.Because mucins form complexes with several proteins,
including secretory IgA, which might be expected to interactwith bacterial cells, the fractions were analyzed by anenzyme-linked immunoassay for secretory IgA (47). Theresults are shown in Fig. 3.No secretory IgA was detected in fractions 20 to 35, which
contained the major part of the adhesion-promoting activity,but was found in fractions 40 to 60, consistent with theelution position of secretory IgA under the conditions used.Also, the adhesion-promoting proteins were electrophoreti-cally distinct from secretory IgA and also distinct fromfibronectin (data not shown), which is known to influencemicrobial adhesion (4).
Separation of the adhesion-promoting molecules. Becausethe proteins apparently responsible for the adhesion-pro-
0Li12FIG. 2. Detailed examination of the adhesion-promoting activity
of experimental pellicles formed from void volume fractions ofSMSL saliva from a Trisacryl GF2000 column. Conditions wereidentical to those for Fig. 1 except that 2 ml fractions were collected.The fractions were examined by SDS-PAGE and stained with asilver stain. The adhesion-promoting activity (vertical bars) did notcoincide with total protein as determined by A220 (continuous line),but did coincide closely with the pair of bands revealed by electro-phoresis. The more slowly migrating higher-molecular-weight mate-rial, presumably mucin, stained relatively poorly with the silverstain used.
moting activity could be separated from mucin by SDS-PAGE, the void volume material obtained from TrisacrylGF2000 chromatography (Fig. 1) was examined by gelfiltration on a column of Superose 6 (0.79 cm2 by 30 cm)(Pharmacia, Inc., Piscataway, N.J.) with or without SDStreatment. In the latter case, a single well-defined peak wasobtained at the void volume of the column (Fig. 4).
After treatment with SDS, however, the Trisacryl GF2000void volume material was separated into several constitu-ents. Although the same amount of material was loaded, thevoid volume peak was considerably smaller, and at least two
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FIG. 3. Results of immunoassay for secretory IgA (S-IgA) of thefractions shown in Fig. 2. Note that no secretory IgA was detectedin fractions 20 to 35, which contained the major part of theadhesion-promoting activity.
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FIG. 4. Gel filtration chromatography on a Superose 6 column ofthe mucin-containing fraction from SMSL saliva (pooled fractions14 to 17, Fig. 1) under native conditions (top) and with SDStreatment (bottom). A single high-molecular-weight peak was ob-tained under native conditions, while separation of several constit-uents was seen after SDS treatment. Silver-stained SDS-PAGE gelsof these fractions showed separation of the slowly migrating mucinfrom the adhesion-promoting proteins after treatment with SDS. VO,Void volume; Vt, column volume.
lower-molecular-weight fractions were detected. Examina-tion of the early-eluting fractions from the Superose columnby SDS-PAGE (insets) showed that a substantial degree ofseparation of the material assumed to be mucin, and the twobands associated with the adhesion-promoting activity,could be obtained by gel filtration in the presence of SDS.
This separation was scaled up by using a Trisacryl GF2000column (5.31 cm2 by 91 cm) equilibrated with 0.05 M Trischloride buffer (pH 8) containing 0.1 M NaCl and 0.1% SDS.Mucin fractions from the saliva fractionation describedabove (Fig. 1) were applied to the SDS-Trisacryl column.
Figure 5 shows that good separation of the adhesion-promoting molecules (the smaller, later-eluting peak) fromthe higher-molecular-weight mucin (the larger, void volumepeak) was obtained. A key point in Fig. 5 is the closerelationship between absorption at 220 nm, adhesion-pro-moting activity, and electrophoretic patterns. It is alsoimportant to note that only trace amounts of other proteinswere detected in the adhesion-promoting fractions, withinthe limitations of the high sensitivity of silver staining fortrace contaminants and the high background associated withthis technique. It should be noted that, because of thedifferent concentrations used, these data cannot be com-pared directly with those in Fig. 2.
Heterogeneity of the proteins responsible for adhesion-promoting activity in different individuals. To confirm theexistence of the adhesion-promoting proteins in more thanone person, dialyzed SMSL saliva from five subjects wasexamined by SDS-PAGE (Fig. 6).
PROMOTION OF S. MUTANS ADHESION BY A SALIVARY PROTEIN
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FIG. 5. Elution profile from the chromatography of pooled mucin fractions on Trisacryl GF2000 equilibrated and eluted with 0.05 M Trischloride (pH 8) buffer containing 0.1% SDS (dashed line), adhesion-promoting activity (vertical bars) of each fraction, and silver-stainedSDS-PAGE gels of 10-fold-concentrated fractions 20 to 24.
The bands associated with adhesion-promoting activityshowed marked heterogeneity. Two individuals (subjects 2and 3) showed a pair of bands, similar to those alreadydetected, another two (subjects 4 and 5) showed only a singleband, while a further individual (subject 1) showed a pair ofbands, of which one appeared to correspond to one of thebands already observed, while the second band had a lowermobility. It seems worth noting that both SDS-PAGE andchromatography on Superose 6 after treatment with SDS(Fig. 4) showed differences in molecular weights for the twobands. The adhesion-promoting activity of the material froman individual showing only one band (subject 4; Fig. 6) wassimilar to that possessed by an individual (subject 2; Fig. 6)showing a pair of bands, suggesting a similarity of biologicalproperties in spite of the observed heterogeneity. It is not yetknown, however, whether both proteins possess adhesion-promoting activity in those subjects who possess the twobands. Approximate molecular weights for the adhesion-promoting material were obtained by extrapolation from the
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FIG. 6. Silver-stained SDS-PAGE gels of dialyzed SMSL salivaobtained from five subjects. Note the heterogeneity of the bands inthe 300,000-molecular-weight region. STD, Size standards (molecu-lar weight in thousands). 0, Origin. The arrow indicates the direc-tion of migration.
standards used, which indicated values close to 300,000 forthese molecules. It should be noted that these results wereobtained from unfractionated saliva samples, so more bandsare seen than in the results obtained by using the mucinfraction.
DISCUSSIONThis study has extended previous observations (17, 18a)
that the mucin-containing fraction of human SMSL salivastrongly promotes adhesion of S. mutans JBP cells to HAand has shown that this activity is associated with specificsalivary macromolecules with molecular weights close to300,000 under denaturing conditions. The results show thatof the numerous proteins present in human saliva, relativelyfew have the ability to promote adhesion of S. mutans JBPcells to HA. Quantitatively, the proteins described in thisstudy form a minor fraction of both the total salivary proteinand the mucin fraction. Thus, their activities are highlyspecific, and presumably, they possess very high specificactivities. In addition, results from electrophoretic analysisof the active material from different subjects suggest thatthese proteins exhibit genetic polymorphism, though infor-mation on the relative activities of the genetic variants hasnot yet been obtained.
Several specific salivary macromolecules have been re-ported to interact with oral streptococci in such a way thatthey have the potential to modulate colonization of themouth by these organisms. The only material showing anydegree of similarity to that identified in the present study wasa fucose-rich glycoprotein which aggregated cell suspensionsof many oral streptococci including S. mutans strains, iso-lated from parotid saliva by Ericson and co-workers (15).This protein, however, had a higher molecular weight(440,000 under denaturing conditions) than that found in thepresent study. Also, it has been shown (41) that saliva-mediated adhesion of bacterial cells to HA and saliva-induced aggregation of the same cells are unrelated pro-cesses, presumably mediated by different molecules. Thebiological importance of these effects is emphasized by thefinding (42) in caries-resistant and caries-susceptible subjects
that saliva from the former group was much less effective inpromoting adhesion of an S. sanguis strain than that from thelatter group, while saliva from the caries-resistant groupcaused more aggregation of an S. mutans strain than salivafrom the caries-susceptible group.
Other salivary macromolecules and proteins of nonsali-vary origin present in saliva reported to interact with oralstreptococci include 55- and 60-kilodalton salivary glycopro-teins (2, 3), amylase (9), fibronectin (4, 5, 46), high- andlow-molecular-weight salivary mucins (28, 40, 48), secretoryagglutinins from saliva and other biological fluids (1, 10-12),agglutinins from serum and crevicular fluid (35), secretoryIgA (51), and the parotid saliva basic glycoprotein (45). All ofthe characterized or defined molecules are distinct from theproteins described in the present study. Also, the multiplic-ity of factors which have been reported previously as inter-acting with oral streptococcal cells emphasizes the speci-ficity with which the adhesion of S. mutans JBP cells to HAis shown to be promoted in these studies, by rather few ofthe salivary macromolecules. It is interesting that the acidicproline-rich proteins have been considered to be amongthose salivary proteins which selectively adsorb from salivaonto HA (24), exhibit high affinities for HA (33, 34), and arepresent in the early stages of the formation of the enamelpellicle (6, 26). Also, mucins form adherent films on solidsurfaces and are considered to be involved in formation ofthe acquired pellicle (13, 14, 49).
In considering the specificity of these effects, it seemssignificant that our previous studies have shown that adhe-sion of strains of Actinomyces viscosus, Streptococcus so-brinus, and Bacteroides gingivalis to apatitic surfaces is notpromoted by these proteins which actively promote theadhesion of S. mutans JBP, emphasizing the high specificitywith which S. mutans JBP cells attach to salivary constitu-ents adsorbed onto HA surfaces. It is also important to notethat although varying contributions of electrostatic, hydro-gen, and hydrophobic bonding must underlie adhesion reac-tions, the high specificities shown in the present and relatedstudies of bacterial adhesion to biological surfaces showsthat the basic but nonspecific physical binding forces mustbe strongly modulated by the stereochemical properties ofthe specific macromolecules involved.
ACKNOWLEDGMENTS
These studies were supported by Public Health Service grantsDE-2847, DE-3915, DE-7009, and DE-8601 from the National Insti-tute for Dental Research. E.K. was the recipient of a grant from theMinistry of Education, Science and Culture, Japan.We gratefully acknowledge the technical assistance and artwork
preparation provided by Susan Schluckebier, the contribution fromF. Dewhirst to refinements of the silver staining technique, and helpfrom D. Smith with the secretory IgA immunoassay.
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