INFECTION

Vol. 60, No. 7

AND IMMUNITY, JUlY 1992, p. 2598-2604 0019-9567/92/072598-07$02.00/0 Copyright © 1992, American Society for Microbiology

Inhibition of Porphyromonas gingivalis Adhesion to Streptococcus gordonii by Human Submandibular-Sublingual Saliva M. W. STINSON,l,2* G. G. HARASZTHY,2 X. L. ZHANG,' AND M. J. LEVINE2

Department of Microbiology, School of Medicine and Biomedical Sciences and School of Dental Medicine, and Department of Oral Biology and Dental Research Institute, School of Dental Medicine,2 State University of New York at Buffalo, Buffalo, New York 14214 Received 23 July 1991/Accepted 28 February 1992

Porphyromonas gingivalis W50 adheres in vitro to biofilms of Streptococcus gordonii G9B. This phenomenon is believed to facilitate the initial colonization of the oral cavity by P. gingivalis and to contribute to the maturation of dental plaque. In this report, we describe the modulating effects of human submandibularsublingual saliva (HSMSL) on this in vitro model of intergeneric bacterial adhesion (coaggregation). HSMSL inhibited P. gingivalis adhesion to S. gordonii by 50% at a concentration of 57 t.g of protein per ml. Maximum inhibitory activity was associated with a 43-kDa protein obtained by sequential Sephadex G200 gel filtration and CM52 ion-exchange chromatography of HSMSL. Pools of other column fractions of HSMSL showed no effect or were slightly stimulatory for bacterial adhesion. The binding of radioiodinated column fractions containing the 43-kDa protein by P. gingivalis was accompanied by their rapid enzymatic degradation. Treating P. gingivalis at 60°C for 30 min or with protease inhibitors (phenylmethylsulfonyl fluoride and sodium iodoacetate) reduced adherence to streptococcal biofilms. These treatments did not prevent P. gingivalis from binding soluble HSMSL saliva components, although subsequent proteolysis was nearly eliminated. These observations indicate that surface-associated proteases of P. gingivalis, either independently or in concert with adjacent surface adhesins, interact with surfaces of oral streptococci to facilitate interbacterial adhesion. The adhesion-blocking properties of HSMSL, particularly the 43-kDa protein, may represent an important host defense mechanism in the oral cavity. Porphyromonas gingivalis (25) has been reported to adhere to oral streptococci in vivo (28) and in vitro (17, 28, 30). This intergeneric bacterial adhesion (coaggregation) contributes to the formation and maturation of human dental plaque (8) and may lead to the initial colonization of the mouth by P. gingivalis (28, 30). This bacterium has also been reported to adhere in vitro to experimental salivary pellicles immobilized on mineral surfaces similar to those of teeth (2, 28). Purified proline-rich proteins (PRPs) from saliva appear to enhance the attachment of P. gingivalis to hydroxyapatite surfaces (2). We previously reported (30) that human whole saliva (HWS) inhibited the adherence of planktonic P. gingivalis to a biofilm of Streptococcus gordonii G9B in vitro by more than 95%. Nagata et al. (17) also found that human saliva inhibited the adherence of P. gingivalis to Streptococcus mitis cells in a turbidimetric assay. These observations indicate that salivary components can be important host defense factors for controlling colonization of supragingival plaque by P. gingivalis. Identification and purification of the active salivary component(s) are necessary for a better understanding of bacterial adherence mechanisms in vivo and the modulating effects of saliva in the development of supragingival plaque and possibly periodontal infections. For example, Nishikata et al. (20) recently reported that a histidine-rich protein (histatin 5) from human parotid saliva was a potent inhibitor of a trypsinlike protease of P. gingivalis. In this report, we describe the effects of human subman-

*

dibular-sublingual saliva (HSMSL) on intergeneric bacterial adherence and identify an inhibitory component in HSMSL. The possible contribution of surface proteases of P. gingivalis to the adhesion process is also examined. MATERIALS AND METHODS Chemicals. Human fibrinogen, human serum albumin, and bovine serum albumin were purchased from Sigma Chemical Co., St. Louis, Mo. All other reagents were analytical grade and were obtained from local vendors. Bacteria and culture conditions. S. gordonii G9B, previously called S. sanguis (6, 7, 23), and P. gingivalis W50 (derived from ATCC 53978) were grown as described previously (30). Growth was measured turbidimetrically, and bacteria were harvested by centrifugation during the late logarithmic phase and washed with phosphate-buffered saline (PBS; 0.01 M sodium phosphate [pH 7.2], 0.15 M sodium chloride). P. gingivalis was radiolabeled intrinsically with [methyl-3H]thymidine (ICN Biomedicals, Costa Mesa, Calif.) as described previously (30). The specific activity for 108 P. gingivalis cells was approximately 25,000 cpm. Adhesion assay. The attachment of planktonic P. gingivalis to biofilms of S. gordonii was assayed as described previously (30). Viable streptococci are coated onto CNBr-activated agarose beads (Strepbeads), which are then incubated with radiolabeled late-logarithmic-phase P. gingivalis cells in 10 mM sodium phosphate (pH 7.2)-50 mM potassium chloride-0.1 mM magnesium chloride-0.1 mM calcium chloride (adherence buffer). After 1 h at 22°C, the Strepbeads are collected by differential filtration on polycarbonate mem-

Corresponding author. 2598

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INHIBITION OF P. GINGIVALIS ADHESION TO S. GORDONII

branes with 12-,um pores, and the number of adherent porphyromonads is measured by scintillation spectrometry. Collection of saliva. HSMSL from a 30-year-old female donor was collected and processed as reported previously (27). HWS was collected by expectoration and processed as described before (30). Purification of salivary components. HSMSL was reconstituted in 0.1 M Tris-HCI (pH 7.5)-6 M guanidine-HCI and fractionated by gel filtration chromatography on a column of Sephadex G200 (2.6 by 95 cm) as described by Rammasubbu et al. (22); four pools (A to D) were obtained. Materials in pool B were further fractionated by ion-exchange chromatography on a column (1 by 10 cm) of CM52 (Whatman Ltd., Maidstone, Kent, England) equilibrated with 5 mM sodium acetate at pH 4.2. Proteins, 25 mg in 10 ml of the equilibrating buffer, were applied to the column and eluted with an additional 30 ml of buffer. The column was then eluted with a linear gradient of 40 ml each of equilibrating buffer and 150 mM sodium acetate (pH 4.2). The gradient was followed by 90 ml of equilibrating buffer containing 1 M NaCl. Fractions were monitored for A280, pooled as indicated, dialyzed against distilled water, and lyophilized. Radioisotope labeling of protein. Salivary proteins were radiolabeled with Na125I (ICN Radiochemicals, Irvine, Calif.) by using Iodo-Beads (Pierce Chemical Co., Rockford, Ill.). The protein solution (1 mg in 1 ml of PBS) was added to four Iodo-Beads that had been preincubated with 1 mCi of Na125I in 0.2 ml of PBS for 15 min. After 10 min, the protein solution was removed from the beads and loaded on a column (0.8 by 20 cm) of Sephadex G25 (Pharmacia). The radioactive proteins that eluted in the void volume were collected and dialyzed against adherence buffer. The 1251I labeled proteins had a specific radioactivity of 1.4 x 105 cpm per ,ug of protein. The ability of bacteria to bind the radiolabeled saliva components was determined in a centrifugation assay. Washed log-phase P. gingivalis cells were suspended to 2.4 x 108 cells per ml in 0.3 ml of adherence buffer and mixed with selected amounts of 1251I-protein. After being mixed for 15 min at room temperature, the bacteria were collected by centrifugation (15,000 x g for 5 min) and washed five times with 1% bovine serum albumin in PBS. The bacteria were suspended in PBS, transferred quantitatively to a clean vial, and analyzed in a Beckman gamma radiation counter. In some experiments, bacteria were incubated with radiolabeled proteins, washed as described above, and boiled for 2 min in 2% sodium dodecyl sulfate (SDS) to solubilize any bound saliva components. The detergent extracts were collected after centrifugation and analyzed by SDS-polyacrylamide gel electrophoresis (PAGE). The separated radiolabeled proteins were detected by autoradiography of the dried gel on Kodak X-OMAT film. Electrophoresis. SDS-PAGE was performed with 10% acrylamide gels by the method of Laemmli (9). Egg white lysozyme (14.4 kDa), soybean trypsin inhibitor (20 kDa),

bovine carbonic anhydrase (30 kDa), ovalbumin (45 kDa), bovine serum albumin (67 kDa), and rabbit muscle phosphorylase b (94 kDa) were used as molecular mass standards. Proteins were stained with Coomassie brilliant blue R250

(23).

General chemical methods. Protein determinations and amino acid analyses were performed after hydrolysis of samples in 6 N HCI for 24 h at 110°C. Samples were analyzed with a Beckman model 6300 amino acid analyzer (Beckman Instruments, Palo Alto, Calif.). Protein was also quantified

2599

z 60-

40 20

0

0.01

1 10 pg SALIVA/ml

0.1

100

1000

FIG. 1. Inhibition of adhesion of P. gingivalis W50 to S. gordonii G9B by HWS (i) and HSMSL (0) in vitro. Amounts indicate dry weight; protein comprised 35 and 62% of the salivas, respectively. Bars signify the SD of three separate determinations. Adhesion is normalized to that in the absence of saliva components, which was 2.8 x 107 bacteria, or 7,400 cpm.

by the bicinchoninic acid assay procedure (Pierce Chemical) with bovine serum albumin as a standard. RESULTS

Inhibitory effects of saliva. The effects of human saliva secretions on the adherence of P. gingivalis to a biofilm of S. gordonii G9B are shown in Fig. 1. At 1 mg (dry weight) per ml, HWS and HSMSL inhibited bacterial adhesion by approximately 90%. The 50% inhibitory concentrations of HWS and HSMSL were 50 jig (26 jig of protein) and 165 jig (57 ,ug of protein) per ml, respectively. The slightly better inhibitory activity of HWS may be due to the presence of other salivary components, such as those secreted by the parotid glands. Purification of saliva components. Pools A to D were obtained from gel filtration chromatography of HSMSL and tested for inhibitory activity against intergeneric bacterial adherence (Fig. 2). Pool B inhibited adherence by 76% at 73 ,ug of protein per ml (100 ,ug [dry weight]), whereas the remaining HSMSL pools were either ineffective (pool C) or slightly stimulatory for adherence at equivalent dry weights. SDS-PAGE of pool B revealed two major bands (43 and 37 kDa) and two minor bands (31 and 27 kDa) (Fig. 3). The latter two components were also present in pool C, which had minimal inhibitory activity on bacterial adherence. The 31- and 27-kDa components were identified as acidic PRPs by Western immunoblotting with rabbit anti-PRP serum

(data not shown). To more precisely identify the salivary component(s) that inhibits intergeneric bacterial adherence, pool B was fractionated by ion-exchange chromatography on carboxymethyl cellulose (Fig. 4). Two peaks were obtained and designated pools Bi and B2. SDS-PAGE indicated that pool Bi contained the 37-, 31-, and 27-kDa components, whereas pool B2 contained only the 43-kDa protein (Fig. 4, right). Both preparations inhibited bacterial adherence when included in

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INFECT. IMMUN.

STINSON ET AL.

kDa (lane 1); the 37-kDa and 31-kDa components were not labeled by this radioiodination procedure. Lane 2 contains the radioactive components that were recovered from live S. gordonii cells; only a small band was visible at the dye front, which is consistent with observations of weak binding to intact cells shown in Fig. 5. A large quantity of radioactive components were recovered from an equivalent number of live P. gingivalis cells which also migrated with the tracking dye (lane 3). Increased recoveries of the 43- and 27-kDa proteins and reduction of low-molecular-mass material at the dye front were accomplished by pretreating P. gingivalis at 60°C for 30 min (lane 4); incorporating phenylmethylsulfonyl fluoride, a serine protease inhibitor, and sodium iodoacetate, a thiol protease inhibitor, into the assay mixture (lane 5); or maintaining the assay temperature at 4°C (lane 6). Similar treatments of S. gordonii did not alter its ability to bind radiolabeled pool B components (data not shown). Effects of protease inhibitors on saliva binding. In quantitative binding assays similar to that shown in Fig. 5, P. gingivalis bound 40% more radiolabeled salivary proteins at 4°C (810 + 14 ng) than at room temperature (577 ± 6 ng) (mean ± SD). Inclusion of the protease inhibitors phenylmethylsulfonyl fluoride and sodium iodoacetate in the assay mixture or preheating the bacteria at 60°C for 30 min resulted in a 121 and 116% increase in the binding of radioactive salivary proteins, respectively, compared with untreated controls at room temperature. These results indicate that surface proteases either are located close to the adhesins on the P. gingivalis cell surface or are participating directly in the binding of saliva molecules. At room temperature and

125 LU

100 -

z

LU

C

LU

75.

0 4

50 -

z o-A

25 -

B

175

150 LU

A D

125

z 100 LUI

75 0o

50 o-

25 0

0

.1

1

10

100

gg Saliva/ml FIG. 2. Effects of HSMSL pools A to D

on

adhesion of P.

gingivalis W50 to S. gordonii G9B. Radiolabeled P. gingivalis

was

suspended in the indicated concentrations (dry weight) of pools A to D and assayed for adhesion to Strepbeads. The resulting adhesion is normalized to that in the absence of any added inhibitors, which was 2.8 x 107 bacteria, or 8,360 cpm. Bars indicate the SD for three assays. The percentage of protein in pools A, B, C, and D was 60, 73, 57, and 82%, respectively.

the

assay

mixture. At 100 ,ug of protein

per

37°C, binding was accompanied by rapid proteolysis. Binding specificity. Experiments were also conducted to determine binding specificity. Selected proteins were tested for their abilities to competitively inhibit the binding of 125I-pool B components to P. gingivalis (Table 2). At competitor/radiolabeled protein ratios of 5:1 and 50:1, only homologous protein (unlabeled pool B) was consistently inhibitory (47%). Bovine and human serum albumin and human fibrinogen were weakly inhibitory. These observations indicate that these proteins do not bind to the same P. gingivalis component(s) as the HSMSL pool B components. Effects of protease inhibitors on intergeneric bacterial adhesion. To determine the role of surface proteases in the adherence of P. gingivalis to the streptococci, porphyromonads were treated with phenylmethylsulfonyl fluoride

ml, the 43-kDa

component (pool B2) inhibited adherence 45% + 2% (mean

standard deviation [SD]), whereas pool Bi inhibited it 26% 14% (mean SD). In a parallel assay, the unfractionated pool B preparation inhibited adhesion by 56% + 10% at this protein concentration. The amino acid composition of the 43-kDa protein is shown in Table 1. It is a glycoprotein that contains approximately 18 mol of N-acetylglucosamine per mol of protein. Binding of purified saliva components. To further define the interactions between bacteria and salivary components, radioiodinated pool B components were incubated with P. gingivalis or S. gordonii cells (Fig. 5). Large quantities of radioactive material were bound to P. gingivalis, whereas little was accumulated by an equivalent number of S. gordonii cells. In a parallel experiment, the treated bacteria were washed extensively with buffer, and the bound radioactive components were extracted with 1% SDS. The extract was fractionated by SDS-PAGE and analyzed by autoradiography (Fig. 6). The stock solution of radiolabeled pool B contained two major radioactive components at 43 and 27

94. 67kDa

_

|

43.

29u20

14 ----__

1

2

3

4

5

6

FIG. 3. SDS-PAGE of HSMSL and pools A to D. Lane 1, protein size standards; lane 2, HSMSL; lane 3, pool A; lane 4, pool B; lane 5, pool C; lane 6, pool D. Gels were loaded with 50 ,ug of protein per lane and stained with Coomassie brilliant blue.

INHIBITION OF P. GINGIVALIS ADHESION TO S. GORDONII

VOL. 60, 1992

2601

0.6

E

C~

B1

B2

=1 E

C3

l

i.

43 kEDa ->

ir

0.4 0

cl:

0.2

.0

0

150 mM NaAc, pH 4.2, 1 M NaCi

150 mM NaAc, pH 4.2

Co

.0 0 0

-.

V

du

20

i4l "It,

40

60

80

100

Fraction No. (2.0 ml) B

B2

FIG. 4. Fractionation of pool B components on a column of CM52-cellulose. Absorbance peaks at 280 nm were pooled as indicated. SDS-PAGE (right) shows the composition of pool B and pool B2. NaAc, sodium acetate.

or iodoacetate or both and incubated with Strepbeads. The data in Table 3 indicate that adhesion is partially inhibited by each reagent and that the effects of these reagents are additive. In contrast, pretreatment of Strepbeads with the protease inhibitors caused less than 10% reduction in subsequent adhesion to P. gingivalis. Adhesion of P. gingivalis W50 to Strepbeads was also heat sensitive, as 95% of the activity was lost after pretreating the porphyromonads at 60°C for 30 min. Similar treatment of Strepbeads had no effect on subsequent adhesion of P. gingivalis.

DISCUSSION

The data presented in this report show that the adhesion of P. gingivalis W50 to S. gordonii G9B is strongly inhibited by HWS and HSMSL. The in vitro assay utilizes viable S. TABLE 1. Amino acid composition of the 43-kDa component of HSMSL No. of residues/ Amino acid

Asx ........................................ Thr ....................................... Ser ........................................ Glx ....................................... Pro .......................................

Gly ........................................

Ala .......................................

Cys .......................................

Val ....................................... Met ........................................ Ile ....................................... Leu .......................................

Tyr ........................................ Phe ....................................... His ........................................

Lys ....................................... Arg ........................................

gordonii cells chemically coupled to agarose beads as a simulated dental plaque. The homogeneous streptococcal biofilm is stable, and the adhesion of other bacteria to it can be quantified accurately (30). Moreover, because of their size and density, the Strepbeads are easily and quickly separated from planktonic P. gingivalis by differential filtration in the presence of saliva. Importantly, the adherence of P. gingivalis to the Strepbeads can be distinguished from saliva-induced agglutination of P. gingivalis, since the 12-p.m pores of the filter membrane do not retain agglutinated porphyromonads. P. gingivalis does not adhere to Streptococcus-free agarose control beads in either the presence or absence of saliva (30). Therefore, apparent enhancement of intergeneric bacterial adherence by HSMSL column pools A and D may reflect bridging of bacterial surfaces by salivary components. Pool A contains mucin glycoproteins 1 and 2, immunoglobulin A, and amylase, whereas pool D contains acidic proline-rich peptides and statherin (22). The adherence-inhibitory activity of HSMSL was associated with a Sephadex G200 column fraction (pool B) which

100 residues

11.2 7.0 6.1 11.9 4.2 7.8 6.6 1.1 7.0 0.8 4.6 9.3 5.5 4.3 4.6 4.6 3.6

z 0 z

0.4

0 = 0.2

-

-

1

e

gtg

2 PROTEIN

4 3 ADDED

FIG. 5. Binding of radiolabeled pool B components to viable P.

gingivalis W50 (V) and S. gordonii G9B (-). Bacteria (2.4 x 108 in 0.3 ml) were incubated with the indicated amounts of labeled protein for 15 min and washed with buffer, and the radioactivity was counted. Bars show the SD of three determinations.

2602

INFECT. IMMUN.

STINSON ET AL.

TABLE 3. Effect of protease inhibitors on the adhesion of

P. gingivalis to S. gordoniia 67

.Concn Inhibitr Inhibitor (M)

-

Phenylmethylsulfonyl fluoride (PMSF) lodoacetate (IA)

20,S

Mixture of PMSF and IA

1

2

3

4

5

6

FIG. 6. SDS-PAGE autoradiographs of radioiodinated pool B components after interaction with bacteria. Lane 1, pool B components alone; lane 2, pool B components recovered from viable S. gordonii; lane 3, pool B components recovered from viable P. gingivalis; lane 4, pool B components recovered from heat-killed P. gingivalis; lane 5, pool B components recovered from P. gingivalis treated with phenylmethylsulfonyl fluoride and iodoacetate; lane 6, pool B components recovered from P. gingivalis cells treated at 4°C. Positions of molecular mass markers are shown to the left.

separated into four stainable proteins at 43, 37, 31, and 27 kDa during SDS-PAGE. The two smaller proteins are acidic PRPs and do not appear to be significant inhibitors of adherence, since they were also present in column pool C, which was ineffective in the adherence assay. Further purification and assay showed that the 43-kDa protein possessed the greatest inhibitory activity (80% of pool B activity). The remaining proteins in pool B were only 46% as active as the unfractionated pool B. These observations indicate either that some denaturation, with concomitant loss of activity, occurred during the purification of the 43-kDa protein or that the inhibitory activity of HSMSL involves the synergism or complexing of two or more proteins. For example, both the radioiodinated 43- and 27-kDa components in pool B bound readily to P.1 gingivalis. It can be speculated that physical separation of these components may result in loss of inhibitory activity of the protein in the interbacterial adhesion assay. Also, the inability of unlabeled homologous protein to competitively inhibit binding of radiolabeled HSMSL protein to P. gingivalis indicates that approximately 50% of the binding is nonspecific (5). The molecular mass and amino acid composition of the 43-kDa protein are similar to those reported for human TABLE 2. Effects of selected proteins and HSMSL on the binding of 125I-pool B components to P. gingivalis W50a .Protein Inhibitr

Relative adherence

Inhibitor

(p.g)

(% of control)

Bovine serum albumin

10 100 10 100 10 100 10 100

114 ±14 82 ± 6 86 ± 6 103 ± 8 94 ± 7 78 ± 18 79 ± 4 53 ± 8

Human serum albumin Human

fibrinogen

HSMSL pool B

The indicated amount of inhibitor was mixed with 2 ,ug of radiolabeled pool B and then added to 1.5 x 108 washed P. gingivalis cells. After 15 min at room temperature, the cells were isolated, washed with adherence buffer, and counted for radioactivity. The amount of radioactivity bound to P. gingivalis in the absence of competitor was 7,080 470 cpm (mean ± SD) (control, 100% binding), or 221 ng of protein. Values are means SD. ±

±

Relative adherence (% of control)

0.5

81 ± 4

5.0 0.5 5.0 0.5 each 5.0 each

65±11 36 ± 2 38 5 37 ± 6 15 + 4

a Bacterial adhesion was normalized to that observed in the absence of inhibitors. The amount of radioactivity bound to Strepbeads after 1 h at room temperature was 8,567 + 382 cpm (control, 100% adhesion). This radioactivity corresponded to 3.4 x 10' bacteria. The indicated amounts of inhibitors were incubated with P. gingivalis cells for 30 min at room temperature before being combined with Strepbeads. Values are means ± SD.

salivary carbonic anhydrase (16); however, the proteins are immunologically dissimilar, because antibodies specific for the 43-kDa HSMSL component did not bind to carbonic anhydrase of human or bovine erythrocytes in Western blot assays in our experiments. Also, human and bovine erythrocyte carbonic anhydrase showed weak inhibitory activity for interbacterial adhesion (30% ± 3% [SD] at 100 ug/ml). The presence of N-acetylglucosamine and the interaction of the protein with concanavalin A on SDS-PAGE blots indicate the presence of N-linked saccharide units. Further characterization of the glycoprotein and its mechanism of interaction with P. gingivalis surface components will be a focus of future studies. The binding of salivary components to P. gingivalis in the present study did not exhibit saturation kinetics; at the highest concentrations, more than 50,000 molecules of salivary protein were present per bacterium. One possible explanation is that a significant amount of the binding is by nonspecific mechanisms and is thus unsaturable. Another plausible explanation is that enzymatic degradation of the bound protein leads to rapid turnover at the cell surface, with internalization of the resulting polypeptides in the periplasm or cytoplasm. Intact saliva proteins could not be recovered from P. gingivalis cells unless the bacterial surface proteases were inactivated by heat, cold, or chemical inhibitors. Interestingly, inactivation of proteases destroyed interbacterial adherence but enhanced binding of soluble salivary proteins. These observations are consistent with recent reports by other investigators. For example, Lantz et al. (11) demonstrated that binding of fibrinogen to P. gingivalis is associated with fibrinolytic activity. The addition of thiol-protease inhibitors inactivates proteolysis and appears to stimulate binding of fibrinogen to the bacteria. More recently, they (10) reported on a cell surface component (150 kDa) of P. gingivalis that binds to fibrinogen and on two separate thiol-dependent proteases (120 and 150 kDa) that can degrade fibrinogen. Naito and Gibbons (18) found that the adherence of P. gingivalis to a collagenous substrate is inhibited by both thiol protease and serum protease inhibitors. Adhesion of P. gingivalis to salivary PRP-treated hydroxyapatite was inhibited by serum protease inhibitors but not by phenylmethylsulfonyl fluoride or thiol protease inhibitors (3). More recently, Li et al. (13) reported that the surface proteases of P. gingivalis appear to mediate bacterial adherence toActinomyces viscosus. Mutant strains lacking a trypsinlike protease showed diminished adherence to A. viscosus. Serine protease inhibitors, such as phenylmethyl-

VOL. 60, 1992

INHIBITION OF P. GINGIVALIS ADHESION TO S. GORDONII

sulfonyl fluoride, decreased intergeneric bacterial adhesion by 30 to 50% (13). Several types of proteases are produced by P. gingivalis, including trypsinlike protease (21, 29, 32, 33), collagenase (1, 14), and diaminopeptidases (4, 15, 19, 31). The role of these proteases in the physiology of the bacterium and in disease pathogenesis has not been clearly resolved. The primary function of surface proteases is assumed to be for nutrient acquisition. Several investigators (12, 24, 26) have reported that P. gingivalis preferentially derives carbon, nitrogen, and energy from exogenous polypeptides. P. gingivalis grows with difficulty when a mixture of free amino acids is substituted for the peptides. The different proteases on P. gingivalis are expected to allow the bacterium to hydrolyze a multitude of proteins encountered in the human host, including those located on the surfaces of other oral bacteria. It is possible that the adherence of P. gingivalis to streptococci and other gram-positive bacteria in dental plaque represents an example of intergeneric bacterial parasitism, in which one symbiont is metabolizing the surface proteins of the other. It can also be hypothesized that the enzymes have evolved as a defense mechanism against fouling of the bacterial surface by host proteins, such as immunoglobulins, salivary proteins, and fibrinogen, which it encounters in the mouth or the periodontal pocket. Adsorbed proteins might mask the adhesins necessary for colonization of the host by the bacterium or opsonize the bacteria for phagocytosis by neutrophils. From the behavior of protease-deficient mutants, Li et al. (13) propose that the cell-associated trypsinlike proteases of P. gingivalis facilitate bacterial adherence to oral surfaces either by enzymatically unmasking binding sites on oral surfaces to which other surface proteins of P. gingivalis can bind or, alternatively, by acting directly as adhesins through stereochemical interactions. Both roles can be expected to contribute to disease pathogenesis by enabling the bacterium to attach to host cells and penetrate connective tissues. For example, Naito and Gibbons (18) reported that mild trypsin treatment of fibronectin-collagen complexes removes fibronectin and permits P. gingivalis to adhere to the underlying collagen. Direct adherence to its nutrient source enables the bacterium to feed on the host cell surface. Further resolution of the proteolysis and adherence activities of P. gingivalis cell surfaces, however, must await purification and biochemical characterization of the relevant bacterial components. ACKNOWLEDGMENTS This work was supported by Public Health Service grant DE08240 from the National Institute of Dental Research. We thank Frank Scannapieco and Nour Amir-Mozafari for careful review of the manuscript and helpful criticisms and Nancy Hurley for editorial assistance.

REFERENCES 1. Birkedal-Hansen, H., R. E. Taylor, J. J. Zambon, P. K. Barua, and M. E. Neiders. 1988. Characterization of collagenolytic activity from strains of Bacteroides gingivalis. J. Periodont. Res. 23:258-264. 2. Brissette, R., R. J. Gibbons, D. I. Hay, and S. Schuckebier. 1986. Attachment of Bacteroides gingivalis 381 to salivary prolinerich proteins on hydroxyapatite surfaces. J. Dent. Res. 66:286289. 3. Gibbons, R. J., and D. I. Hay. 1988. Adsorbed salivary prolinerich proteins as bacterial receptors on apatitic surfaces, p. 143-169. In L. M. Switalski, M. Hook, and E. Beachey (ed.),

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