JOURNAL OF BACTERIOLOGY, Sept. 1991, p. 5266-5274 0021-9193/91/175266-09$02.00/0 Copyright X 1991, American Society for Microbiology

Vol. 173, No. 17

Evidence that Porphyromonas (Bacteroides) gingivalis Fimbriae Function in Adhesion to Actinomyces viscosus P. ANDREW GOULBOURNE AND RICHARD P. ELLEN* Faculty of Dentistry, University of Toronto, 124 Edward Street, Toronto, Ontario, Canada M5G IG6 Received 29 January 1991/Accepted 17 June 1991

Porphyromonas (Bacteroides) gingivalis adheres to gram-positive bacteria, such as Actinomyces viscosus, when colonizing the tooth surface. However, little is known of the adhesins responsible for this interaction. A series of experiments were performed to determine whether P. gingivalis fimbriae function in its coadhesion with A. viscosus. Fimbriae typical of P. gingivalis were isolated from strain 2561 (ATCC 33277) by the method of Yoshimura et al. (F. Yoshimura, K. Takahashi, Y. Nodasaka, and T. Suzuki, J. Bacteriol. 160:949-957, 1984) in fractions enriched with a 40-kDa subunit, the fimbrillin monomer. P. gingivalis-A. viscosus coaggregation was inhibited by purified rabbit antifimbrial immunoglobulin G (IgG) at dilutions eightfold higher than those of preimmune IgG, providing indirect evidence implicating P. gingivalis fimbriae in coadhesion. Three types of direct binding assays further supported this observation. (i) Mixtures of isolated P. gingivalis fimbriae and A. viscosus WVU627 cells were incubated for 1 h, washed vigorously with phosphatebuffered saline (pH 7.2), and subjected to electrophoresis. Transblots onto nitrocellulose were probed with antifimbrial antiserum. Fimbrillin labeled positively on these blots. No reaction occurred with the control protein, porcine serum albumin, when blots were exposed to anti-porcine serum albumin. (ii) A. viscosus cells incubated with P. gingivalis fimbriae were agglutinated only after the addition of antifimbrial antibodies. (iii) Binding curves generated from an enzyme immunoassay demonstrated concentration-dependent binding of P. gingivalis fimbriae to A. viscosus cells. From these lines of evidence, P. gingivalis fimbriae appear to be capable of binding to A. viscosus and mediating the coadhesion of these species. teroides gingivalis from J. Slots, State University of New York at Buffalo) were maintained by weekly transfer on laked blood agar. The medium contained blood agar base no. 2 (Oxoid Ltd., Basingstoke, Hampshire, England) supplemented with 7% laked sheep's blood, 0.5 mg of L-cysteine per ml, and 1 ,ug each of filter-sterilized hemin and menadione per ml. The plates were incubated at 37°C for 7 days in anaerobic jars containing palladium catalyst and a gas mixture of 80% N2, 10% H2, and 10% CO2 (anaerobic conditions). Cultures of P. gingivalis 2561 used in experiments were grown in Trypticase yeast extract broth containing Trypticase peptone (BBL Microbiology Systems, Becton Dickinson and Co., Cockeysville, Md.) supplemented with 3 mg of yeast extract (Difco Laboratories, Detroit, Mich.), 5 mg of NaCl, 2.5 mg of K2HPO4, 2.5 mg of dextrose, 5 ,ug of filter-sterilized hemin, 0.5 jxg of filter-sterilized menadione, and 1 mg of NaHCO3 per ml (8). The cultures were incubated at 37°C for 36 to 40 h (early stationary phase) in a Coy anaerobic chamber (Ann Arbor, Mich.) containing a gas mixture similar to that described above. Stock cultures of A. viscosus WVU627 (obtained originally from M. A. Gerencser, West Virginia University) were maintained by monthly transfer on brain-heart infusion agar slants (Difco Laboratories). The cultures were incubated at 37°C for 48 h in jars under anaerobic conditions and then stored aerobically at 4°C. Cultures of A. viscosus WVU627 used in experiments were cultivated in tryptic soy broth (Difco Laboratories) in the anaerobic chamber at 37°C for 48 h. Isolation of P. gingivalis fimbriae. Fimbriae were isolated by the method of Yoshimura and coworkers (27), with minor modifications. P. gingivalis 2561 was grown in 6 liters of Trypticase yeast extract broth supplemented with hemin and menadione. Bacteria harvested from fresh cultures were

Several species of bacteria bear long surface appendages which have been shown to mediate their adhesion to host surfaces. The fimbriae of the periodontal pathogen Porphyromonas (Bacteroides) gingivalis have been well characterized in terms of their morphological, biochemical, and immunological properties (10, 15, 27, 28), but their function in adhesion remains unclear. Recently, Isogai and coworkers reported the ability of purified immunoglobulin G (IgG) and Fab fragments of monoclonal antibodies raised against isolated P. gingivalis fimbriae to block adherence of the bacteria to buccal epithelial cells (12). This observation implicates, albeit indirectly, P. gingivalis fimbriae in mediating bacterial adhesion to cells from the oral cavity, but mucosal surfaces do not appear to be the site initially colonized by P. gingivalis (22). The preferential localization of P. gingivalis in mixed communities on and around teeth, coupled with its coaggregation with gram-positive tooth colonizers like A. viscosus, suggest this to be the site of initial colonization in the oral cavity. In vitro experiments have also demonstrated the avid adhesion of P. gingivalis to A. viscosus monolayers on saliva-coated hydroxyapatite (16, 20). Little is known about the bacterial adhesins responsible for this interaction. Evidence compiled in this study implicates P. gingivalis fimbriae as one of the structures which mediate coadhesion with A. viscosus. To our knowledge, this is the first demonstration of direct adhesion of these surface structures to bacterial or any other cells associated with the oral cavity. MATERIALS AND METHODS Cultures and cultural conditions. Stock cultures of P. gingivalis 2561 (ATCC 33277) (obtained originally as Bac*

Corresponding author. 5266

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suspended in 20 mM Tris-HCl containing 0.15 M NaCl and 10 mM MgCl2, pH 7.4, by repeated pipetting. The total volume of resuspending buffer represented 10o of the culture medium volume. The suspension was stirred magnetically for 30 min and then centrifuged at 8,000 x g for 20 min (Beckman J2-21M induction drive refrigerated centrifuge; Beckman Instruments Inc., Fullerton, Calif.), and the supernatant containing fimbriae was retained for further use. Ammonium sulfate was added to the bacterial wash to 40% saturation. The precipitation reaction mixture was stirred at room temperature (RT) for 45 min and allowed to stand overnight at 4°C. Precipitated proteins were collected by centrifugation at 25,000 x g at 4°C for 30 min and resuspended in a small volume of 20 mM Tris-HCl, pH 8.0 (Tris buffer). The suspension was dialyzed against Tris buffer for 48 h at 4°C. Afterwards, the dialysate was clarified by centrifugation at 10,000 x g for 15 min at 4°C, and the sample was applied to a column of DEAE-Sepharose CL-6B (Pharmacia, Uppsala, Sweden) equilibrated with Tris buffer. The column was washed with Tris buffer and eluted with a linear gradient of 0 to 0.3 M NaCl, followed by a stepwise gradient of 0.3 to 1 M NaCl. Fractions were monitored spectrophotometrically at 280 nm, and those containing protein were further analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Fractions with similar protein profiles were pooled, and the samples were desalted and concentrated with Centricon-30 microconcentrators or Diaflo YM30 ultrafiltration membranes (Amicon, Amicon Division, W. R. Grace and Co., Danvers, Mass.). Samples containing fimbriae were prepared for observation by transmission electron microscopy with the negative stain methylamine tungstate by the method of Handley and Tipler (10). SDS-PAGE. Protein fractions were analyzed by the SDSPAGE procedure of Laemmli (14). Each sample was added to an equal volume of dissociating sample buffer and heated at 100°C for 10 min. Polyacrylamide (12.5%) gels were loaded with 20 ,ul, containing approximately 0.23 p.g of protein, for each sample, and a constant voltage of 175 V was applied for 45 min (Bio-Rad mini gel system; Bio-Rad Laboratories, Richmond, Calif.). The molecular mass markers included phosphorylase b, 94 kDa; bovine serum albumin, 67 kDa; ovalbumin, 43 kDa; carbonic anhydrase, 30 kDa; soybean trypsin inhibitor, 20 kDa; and a-lactalbumin, 14.4 kDa (Pharmacia). Gels were stained for protein with Bio-Rad silver stain (Bio-Rad Laboratories) or for lipopolysaccharide (LPS) by the method described by Tsai and Frasch (24). Transmission electron microscopy. Ten microliters of the fimbrial preparation, suspended in deionized H20, was mixed with 5 p.l of 0.05% bacitracin as a wetting agent. Fifteen microliters of 1% methylamine tungstate (Emscope Laboratories Ltd., Ashford, Kent, England) was then added (10). Formvar-carbon-coated nickel grids were floated on the mixed droplet for 30 s, blotted dry, and observed with a Phillips 400T electron microscope. Biochemical assays. Total protein was estimated with the Bio-Rad protein assay kit (Bio-Rad Laboratories) with gamma globulin as a protein standard or by measuring A280. (spectrophotometer model 350; G. K. Turner Associates, Palo Alto, Calif.). The phenol-sulfuric acid colorimetric method described by Hodge and Hofreiter was used to estimate the carbohydrate concentration of the samples (11). Hexoses and methylated hexoses were measured at A490. Glucose was used as a carbohydrate standard. Antifimbrial antisera. Two female New Zealand White rabbits were inoculated intramuscularly with 50 p.g of iso-

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lated fimbriae (iFm; see Fig. 5, peak A) mixed with Freund's complete adjuvant. Three weeks later, 50 ,ug of the same preparation, mixed with Freund's incomplete adjuvant, was inoculated at a similar site. Boosters of 50 ,ug of isolated fimbriae were administered to the rabbits 3 weeks after the second injection, and they were bled for their antisera 2 weeks later. Preimmune serum (PI) was collected 1 week prior to and on the day of the first inoculation. Determination of serospecificity to fimbrial antigens by immunoblotting. A crude fimbrial preparation (fimbrial preparation prior to anion-exchange chromatography) was loaded onto a 14% polyacrylamide minigel, each well containing 0.5 ,ug of protein, and run under the SDS-PAGE conditions described above. The prestained molecular mass markers run concurrently included phosphorylase b, 110 kDa; bovine serum albumin, 84 kDa; ovalbumin, 47 kDa; carbonic anhydrase, 33 kDa; soybean trypsin inhibitor, 24 kDa; and lysozyme, 16 kDa. The separated preparation components as well as the molecular weight markers were transferred to nitrocellulose with a Bio-Rad transblotting cell (Bio-Rad Laboratories) under a constant voltage of 60 V for 2 h. The following day, air-dried nitrocellulose blots were wetted in Tris-buffered saline (TBS), pH 7.4, transferred to a blocking solution of TBS containing 0.05% Tween 20 and 3% bovine serum albumin (BSA), and then exposed to diluted antifimbrial antiserum. Immunoreactivity was identified by horseradish peroxidase (HRP) conjugated to goat anti-rabbit (GAR) IgG in the presence of 4-chloro-1-naphthol, the HRP color substrate (Bio-Rad Instructional Manual; Bio-Rad Laboratories). Preparation of P. gingivalis cells for antiserum absorption. The antiserum was submitted to a series of absorptions to remove antibodies against heat-resistant antigens to which the rabbits evidently reacted. P. gingivalis 2561 was harvested from broth cultures by centrifugation at 8,500 x g for 15 min at 20°C. The pellet was washed twice in 0.01 M phosphate-buffered saline (PBS), pH 7.2, and its cell concentration was adjusted to an OD550 of 2.0. The bacterial suspension was sonicated to disperse the cells and autoclaved at 126°C under 20 kPa of pressure for 20 min. The cells were washed once in PBS and resuspended in PBS to one-sixth the volume of the previously adjusted bacterial suspension. Nine milliliters of P. gingivalis 2561 antifimbrial antiserum was mixed with an equal volume of the autoclaved P. gingivalis 2561 cell suspension and incubated for 1 h at RT under constant rotation. The antiserum was separated from the bacteria by centrifugation at 8,000 x g for 20 min at 4°C, and the absorption was repeated. The antiserum was passed through a Millex-GV 0.22-p.m filter unit (Millipore, Bedford, Mass.) and stored in sterile tubes. Purification of IgG fraction from AFAS. IgG was purified from absorbed antifimbrial antiserum (AFAS) by using the Bio-Rad Affi-Gel Protein A MAPS II kit (Monoclonal Antibody Purification System; Bio-Rad Laboratories). Eluent from the protein A-agarose column containing IgG was dialyzed against PBS, and the purified preparation was passed through a Millex-GV 0.22-p.m filter unit before storage at -20°C in sterile tubes. EEM. P. gingivalis 2561 was washed and suspended in Tris buffer. The bacterial concentration was adjusted to an OD550 of 1.0, and the suspension was sonicated for 40 s as described below. Methods for indirect immunoelectron microscopy (IEM) were based on those of Ellen and coworkers (5). Formvar-carbon-coated nickel grids were floated on droplets of the P. gingivalis suspension for 5 min, blotted with filter paper, and then exposed for 30 min to Tris buffer containing

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1.0% (vol/vol) Tween 20 and 1.0% (wt/vol) BSA. The grids were exposed for 60 min to AFAS-IgG (3.4 g.ig/ml), followed by three washes in Tris-Tween-BSA buffer. A 30-min exposure to a GAR IgG-gold probe (GAR-IgG gold; Janssen Auroprobe EM GAR G10), diluted 1/20, localized antibody reactions to the bacterial surface. Grids were washed three times in deionized distilled water to remove buffer salts as well as excess gold probe, and the specimens were negatively stained with 1% methylamine tungstate. Controls consisted of specimens treated identically but with the substitution of the first antibody by either preimmune IgG or Tris buffer. Antibody inhibition of P. gingivalis and A. viscosus coaggregation. P. gingivalis 2561 and A. viscosus WVU627 harvested from broth cultures were washed twice and resuspended in PBS. Clumped cells were dispersed by passage through a 25-gauge syringe needle 15 times, and the optical densities of both bacterial suspensions were adjusted to an OD550 of 1.0. Immediately prior to the assay, the suspensions were sonicated for 35 s at a setting of 4 by a Kontes Micro-Ultrasonic Cell Disrupter (Kontes, Vineland, N.J.) to disperse smaller bacterial aggregates. Examination of P. gingivalis by electron microscopy after dispersion confirmed the presence of fimbriae. AFAS-IgG and PI-IgG (3.4 mg/ml) were serially diluted in a microtiter plate ip twofold steps in 10 IlI of PBS. One hundred microliters of P. gingivalis suspension, containing approximately 4.25 x 109 cells per ml, was then added to each well and to control wells containing buffer only. Plates containing the mixtures of antibody and bacteria were incubated for 45 min at RT on a shaker platform and observed for agglutination. Aggregates were dispersed with constant pipetting, and 100 RI of A. viscosus suspension, containing approximately 1.25 x 109 cells per ml, was added to the wells. The plates were reincubated under the same conditions and observed for coaggregation inhibition. Results were considered valid only when control wells monitoring autoaggregation and antibody-mediated agglutination were negative. Wells close to the inhibition endpoint were also monitored by Gram stain to determine the presence of mixed coaggregates. Immunoblotting assay for fimbrial adhesion to A. viscosus. A. viscosus WVU627 was harvested and washed by centrifugation at 700 x g for 10 min at RT and resuspended in PBS. Clumped cells were dispersed by 10 passages through a 25-gauge syringe needle. The final bacterial concentration was adjusted to an OD550 of 1.4. Equal volumes (0.5 ml) of A. viscosus suspension and P. gingivalis 2561 iFm (80 pLg/ml) were mixed in microtest tubes and incubated at RT for 1 h under constant rotation. Control tubes in which buffer was substituted for iFm were also included. Duplicate sets of the mixture were then washed twice in PBS, one set being sonicated for 15 s between washes. After each wash, the supernatants were retained, as was the final pellet of A. viscosus. The supernatants and pellets were subjected to SDS-PAGE, transferred to nitrocellulose, and probed with P. gingivalis unabsorbed antifimbrial antiserum to detect the broadest possible group of immunoreactive proteins. A parallel control experiment in which porcine serum albumin (PSA) was substituted for the iFm preparation was conducted. Nitrocellulose blots with PSA were probed with anti-PSA, kindly provided by H. Limeback (University of Toronto). The association of iFm with the A. viscosus cell surface was also observed by immunofluorescence microscopy. The experimental conditions were identical to those used in the

J. BACTERIOL.

immunoblotting assay. After being washed, the bacteria were air dried and gently heat-fixed on glass slides, exposed to AFAS, washed, and exposed to fluorescein isothiocyanate-conjugated GAR antibodies. ELISA for quantifying fimbrial adhesion. A. viscosus WVU627 was suspended in PBS to a cell density of 4.5 x 108 cells per ml, and 100-,u aliquots were pipetted into wells of Dynatech Immulon plates (Fischer Scientific Ltd., Ottawa, Canada). Wells not containing bacteria were filled with a similar volume of PBS. The plates were incubated at 37°C for 3 h and then at 4°C overnight. The unbound bacteria were removed by washing three times with PBS containing 0.05% (vol/vol) Tween 20. Plates were washed in a similar manner after each incubation with a new reagent. All incubations were done at 37°C. PBS containing 5% (wt/vol) BSA was incubated for 30 min in the wells to block nonspecific binding of iFm or antibodies to the plates. After washing, iFm in dilutions ranging from 10 to 0.04 Fg of protein per ml was added in 100-p,l volumes across the rows of the plates and incubated for 90 min. Rows to which PBS was added served as controls. Since antibodies might bind nonspecifically to A. viscosus, PBS containing 2% (vol/vol) horse serum was added to the wells to block this reaction and plates were reincubated for 30 min. AFAS-IgG diluted 1:100 and 1:1,000 was added in 100-,lI aliquots down the columns of the plates, one dilution per plate, and incubated for 1 h. PI-IgG diluted 1:100 and 1:1,000 and PBS were run in parallel to control for antibody specificity. HRP-GAR-IgG diluted 1:200 was added to all wells and incubated for 30 min. The substrate o-phenylenediamine was then added, and the reaction was stopped by the addition of 2 N H2SO4. Plates were read at a wavelength of 492 nm with an enzyme-linked immunosorbent assay (ELISA) plate reader (Titertek Multiscan Plus; Flow Laboratories Inc., Mississauga, Ontario, Canada). Bacterial agglutination assay. An iFm preparation with a protein concentration of 21 Lg/ml was diluted in twofold steps in 50-1l aliquots of PBS across the rows of wells of a microtiter plate. Rows containing PBS served as controls. Fifty microliters of A. viscosus suspension, OD550 of 1.5, was added to each well, and the plate was allowed to stand at RT for 45 min before being observed for aggregation by microscopy. Tenfold serial dilutions of AFAS in 50-,ul aliquots were then added down the columns of the plate, and in one column PBS was substituted for the antiserum. The plate was reincubated and again observed microscopically for aggregation. Hemagglutination assay. An iFm preparation (20 ,ug/ml) was serially diluted in twofold steps in 50 ,lI of PBS, pH 7.4, in the wells of a microtiter plate. A similar volume of a washed 2% sheep erythrocyte (SRBC) suspension was added to each of these wells and to control wells containing only PBS. The plates were rotated for 1 h at RT and observed for direct hemagglutination. Fifty microliters of a 100-fold dilution of either unabsorbed antifimbrial antiserum or AFAS was then added, and the plate was observed 1 h later for indirect hemagglutination. RESULTS Isolation of P. gingivalis fimbriae. Fimbriae were separated from outer membrane components by anion-exchange chromatography (Fig. 1). Most of the fimbriae were eluted with 0.15 M NaCl (Fig. 1A, peak A). They were identified throughout the isolation procedure by their structural monomer, fimbrillin, an obvious 40-kDa band on 12.5% polyacryl-

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i E U 0 z z

a 0

4

z1

VOLUME (ml)

B 94kDap-

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FIG. 1. Isolation of fimbriae and SDS-PAGE of fimbrial proteins. (A) Elution profile from fractionation on DEAE-Sepharose CL-6B column by linear (0 to 0.3 M) and stepwise (0.3 to 1.0 M) NaCl gradients. Most of the fimbrial protein was eluted at 0.15 M NaCl (peak A), with a small amount eluted with 0.3 M NaCl (peak B). Proteins which remained bound to the column were eluted with 1.0 M NaCl (peak C). (B) SDS-PAGE (12.5% polyacrylamide separating gel) of pooled fractions from anion-exchange peaks A and B.

amide-SDS gels (Fig. 1B). Those fractions containing only fimbrillin (peak A) were pooled, concentrated, and used in subsequent assays as the isolated fimbrial preparation (iFm). Fimbriae also eluted with higher salt concentrations (0.3 and 1 M NaCl; Fig. 1A, peaks B and C). SDS-PAGE (Fig. 1B) and IEM detected no differences between fimbriae eluted at different salt concentrations. Fimbriae isolated from P. gingivalis 2561 were helical filaments 5 nm in diameter and up to 600 nm in length (Fig. 2), similar in structure to those described previously (10, 27, 28). The iFm preparation contained approximately 94% protein and 6% carbohydrate. The fimbrillin subunit of 40 kDa was the major protein clearly identifiable by SDSPAGE. Proteins of 51 and 75 kDa were also present but barely detectable (Fig. 1B, peak A). LPS was not detected on LPS silver-stained gels of the same preparation (data not shown). Antibody preparations specific for P. gingivalis fimbriae. Rabbit antisera raised against isolated fimbriae were not monospecific for the fimbrillin subunit (Fig. 3). Western immunoblots of crude fimbrial preparations (from 14.0% polyacrylamide-SDS gels) overlaid with the antisera showed a steplike banding pattern characteristic of LPS as well as a large band of 44 kDa (Fig. 3A), corresponding to fimbrillin.

FIG. 2. Pooled fractions of peak A negatively stained with methylamine tungstate. The iFm preparation contained long, helical fimbriae which tended to clump longitudinally. Bar, 0.1 ,um.

Antibodies to the 51-kDa and 75-kDa proteins were also apparent. The antibodies absorbed from the antifimbrial antiserum appeared to be those against heat-stable P. gingivalis components, including LPS. Preliminary absorption studies had demonstrated LPS and autoclaved P. gingivalis cells to be

z

75kDa_ 5okDa_39kDae27 kDa.-

17kDaw-

A

B

FIG. 3. Immunoblots of the crude fimbrial preparation probed with antisera raised against isolated fimbriae. Approximately 0.5 ,ug of protein per well was separated by SDS-PAGE (14% separating gel) and transferred to nitrocellulose. (A) Unabsorbed antifimbrial antiserum; (B) antifimbrial antiserum absorbed twice with boiled P. gingivalis cells (AFAS). The protein concentration for both unabsorbed and absorbed antisera was 96 ,ug/ml. Duplicates are included in the figure to illustrate the range of reactions, which were sometimes not identical.

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equally effective in achieving a more fimbrillin-specific antiserum; the latter was the method of choice for absorption of large volumes of antiserum. In addition to antifimbrillin antibodies, AFAS contained antibodies to the copurifying 75-kDa and 51-kDa proteins as well as to components of 52 and 58 kDa. The most obvious reaction with transferred proteins in Western blots was with fimbrillin (Fig. 3B). IEM of P. gingivalis fimbriae. Immunogold probes bound along the fimbriae, which appeared to be associated with, and partially obliterated by, an amorphous material (Fig. 4A and C). A recent communication by Sojar and coworkers (22b) suggests that this material is lipid complexed with fimbrillin. Virtually no label was detected when PI-IgG was substituted for AFAS (Fig. 4B). P. gingivalis vesicles, the membranes of which are similar to the bacterial outer membrane, were not labeled. Therefore, labeling observed on or close to the bacterial cell body may be attributed to fimbriae lying on its surface. The pattern of labeling with AFAS-IgG confirms the immunoblotting results that it contains antibodies almost exclusively directed to fimbriaeassociated antigens. Coaggregation inhibition. AFAS inhibited coaggregation of P. gingivalis and A. viscosus at a much higher dilution (range, 1:64 to 1:128) than PI serum (1:8) but at a comparable dilution to anti-whole cell antiserum (1:32 to 1:128) and crude antifimbrial antiserum (1:64 to 1:128). Purified AFAS IgG (3.4 pg/ml) inhibited coaggregation at a dilution of 1:16; PI-IgG did not inhibit coaggregation. Coaggregation inhibition by antibodies raised to isolated fimbriae supports the hypothesis that these structures are important in adhesion of P. gingivalis to A. viscosus. Immunoblot analysis of isolated fimbriae bound to A. viscosus. Western blots of washed mixtures of A. viscosus and iFm, developed with unabsorbed antifimbrial antiserum, were used to determine the ability of fimbriae to adhere to A. viscosus. The iFm preparation chosen for these experiments contained fimbrillin as well as many other immunodetectable components which copurified with it (Fig. 5A, lane 1). Although much of the excess iFm was partitioned into the supernatant after the first wash and centrifugation cycle (Fig. 5A, lane 2), A. viscosus pellets still retained detectable amounts of fimbrillin after two buffer washes (Fig. 5A, lane 5). Components of 51, 52, and 75 kDa, identified as proteins by protein silver staining, were also present but not as distinguishable or as consistently detectable as fimbrillin. The association of fimbrillin with A. viscosus provides one line of direct evidence that P. gingivalis fimbriae are able to bind to the bacteria. The control protein, PSA, was unable to bind to A. viscosus (Fig. 5B), suggesting that the association of fimbrial proteins with A. viscosus is specific. Indirect immunofluorescent labeling of A. viscosus exposed to iFm corroborated the observations of the immunoblot experiments. The bacteria fluoresced only when exposed to iFm and AFAS. No fluorescence was observed in controls in which the fimbrial incubation step was deleted. Quantitation of fimbrial adhesion by ELISA. Isolated fimbriae demonstrated concentration-dependent adherence to whole A. viscosus cells (Fig. 6). Although fimbriae also showed a minor degree of binding to BSA-coated wells, binding to A. viscosus-coated wells was significantly greater. The reproducibility of the assay between replicates (as reflected by the small standard deviations) and between assays done on different days demonstrated its effectiveness in quantitative studies as a second line of direct evidence of fimbrial adhesion. Inability of isolated fimbriae to agglutinate A. viscosus and

J. BACTERIOL.

SRBC. P. gingivalis fimbriae, as entities separate from whole cells, appeared to possess a single, or monovalent, adhesin accessible for their attachment to A. viscosus receptors, as A. viscosus cells exposed to P. gingivalis fimbriae, even at the highest concentration tested, were aggregated only after the addition of antifimbrial antibodies. AFAS diluted 1:100 agglutinated iFm-exposed A. viscosus cells. Although P. gingivalis cells and some P. gingivalis proteins are able to agglutinate SRBC, purified P. gingivalis fimbriae are characteristically unable to hemagglutinate (27). The iFm preparation demonstrated weak hemagglutinating activity at a concentration of 10.5 ,ug/ml. This may be attributed to either nonspecific hemagglutination of SRBC or the presence of a small amount of P. gingivalis hemagglutinin in the pooled fractions. Fimbriae were apparently not responsible, as addition of AFAS did not amplify the weak hemagglutination beyond that observed with the PBS control. In contrast, addition of unabsorbed antifimbrial antiserum increased the hemagglutination titer 16-fold, probably due to antibodies directed against heat-resistant antigens like LPS, which would have been absorbed out in the preparation of AFAS.

DISCUSSION Adhesive interactions with bacteria already bound to the surfaces of teeth are thought to foster colonization of the gingival crevice by some pathogenic microorganisms such as P. gingivalis. The function of attaching to such surfaces involves multiple interactions, both specific and nonspecific, which are often mediated by adhesins borne on extracellular structures. Among the coaggregating oral bacteria studied previously, fimbriae appear to be important in the coadhesion of A. viscosus and Actinomyces naeslundii with Streptococcus sanguis (3) and Bacteroides loescheii with Actinomyces israelii and S. sanguis (25). Evidence in this study demonstrates that the adhesion of the periodontal pathogen P. gingivalis with A. viscosus, a prominent tooth colonizer which is often cocultivated from dental plaque with P. gingivalis, may also involve fimbriae on the P. gingivalis cells. Inhibition of P. gingivalis-A. viscosus coaggregation by antifimbrial antibodies and direct adhesion of iFm to A. viscosus, studied by immunoblotting, immunofluorescence, indirect AFAS-mediated agglutination, and ELISA, provide evidence supporting the hypothesis that these structures carry some of the adhesins mediating P. gingivalis adhesion to A. viscosus. A similar approach was taken by Tempro and coworkers (23) to characterize the lectinlike adhesin on Capnocytophaga gingivalis DR2001. Monoclonal antibodies reactive with a 140-kDa polypeptide found in the outer membrane of the bacteria inhibited the interaction of P. gingivalis with carbohydrate receptors on its partner A. israelii. The adhesins were arranged nonuniformly on the bacterial surface, as observed by IEM. Fimbrial preparations isolated from P. gingivalis 2561 were composed mostly of fimbria-associated proteins. SDSpolyacrylamide gels identified the structural fimbrial monomer fimbrillin as the major protein present in these preparations. Variation in the molecular mass of this protein within the range of 40 to 44 kDa, as noted by others (4, 15), can be attributed to differences in SDS-PAGE running conditions for our different assays as well as differences in the migration of fimbrillin compared with that of globular molecular mass markers. Besides fimbrillin, barely detectable copurifying proteins of 51 and 75 kDa were also observed by SDS-PAGE of iFm

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ADHESION OF P. GINGIVALIS FIMBRIAE TO A. VISCOSUS

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FIG. 4. IEM of P. gingivalis 2561. Samples were exposed to AFAS-IgG or PI-IgG and immunolabeled with GAR-IgG-gold (10 nm). (A) Cell-associated fimbriae (AFAS-IgG); (B) cell-associated fimbriae (PI-IgG); (C) crude fimbrial extract (AFAS-IgG). Bars, 0.25 pm.

preparateions. Seventy-five-kilodalton proteins often copurify with fimbrillin (15, 27) and have been reported to be difficult to separate from the fimbrial monomer (29). Analyses of purified 75-kDa protein have found it to be structurally and antigenically different from fimbrillin (29). More recent

evidence has suggested that both are proteolipids (22b). Degradation of the 75-kDa protein gives rise to fragments which are approximately 20 kDa smaller in size (22a); thus, the 51-kDa protein observed in SDS-polyacrylamide gels of iFm preparations might represent such a degradation prod-

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Absorption of antisera with fimbria-deficient mutants has been used to effectively remove nonfimbrial antibodies. For example, Salit and coworkers removed anti-LPS antibodies from antisera raised to denatured Escherichia coli Pap pilin by repeated absorption with Pap pilin mutants (19). Fimbriadeficient mutants of P. gingivalis have not been described. Instead, the sensitivity of proteins and, conversely, the higher resistance of LPS to heat treatment were used as a

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2

3

FIG. 5. Immunoblots of A. viscosus cell pellets and wash supernatants developed with unabsorbed antifimbrial antiserum and HRPGAR-IgG to determine whether fimbrial proteins bound to A. viscosus cells. (A) Lane 1, iFm, 0.4 ,ug per well; lane 2, supernatant of initial mixture; lane 3, supernatant of first wash; lane 4, supernatant of second wash; lane 5, A. viscosus cells plus iFm washed twice in PBS. (B) Control: mixtures of A. viscosus and PSA. Blots were developed with anti-PSA antiserum. Lane 1, PSA, 0.4 ,ug per well; lane 2, supernatant of initial mixture; lane 3, supernatant of first wash; lane 4, supernatant of second wash; lane 5, A. viscosus plus PSA washed twice in PBS. A. viscosus cells did not react with antifimbrial antiserum or with anti-PSA antiserum (not shown). uct. It is unclear whether degradation products of fimbrillin

also common. Examination of fimbrial preparations by transmission electron microscopy demonstrated the presence of fimbriae similar to those described by Yoshimura and coworkers (27). As this was the only structure identified, it added some assurance that the preparations contained mostly, if not exclusively, fimbria-associated components. Antisera raised to the iFm preparation were not monospecific for fimbrial proteins. Antibodies to other bacterial components, probably LPS, were also detected on Western blots of the crude fimbrial preparation. SDS-polyacrylamide gels of the iFm preparation which had been stained for LPS did not detect it, which suggests that LPS was present only in a concentration sufficient to be immunogenic. However, the fimbrial preparation did contain some carbohydrate. are

E C cmJ

0

le

-W

co 10

0.

0. 0

Conc. of Isolated

flmbriae (ug/mi)

FIG. 6. Binding curve for ELISA determination of P. gingivalis adhesion to A. viscosus cells. Values represent the mean of three replicates ± standard deviation. Standard deviations for some mean values were small enough to be within the height of the symbols and are not evident in the figure. Symbols: C, A. viscosus plus iFm plus AFAS-IgG; *, A. viscosus plus iFm plus PI-IgG; *, BSA plus iFm

plus AFAS-IgG.

strategy to mimic fimbria-deficient bacteria for the purpose of antiserum absorption. The final absorbed antibody preparation used for direct ELISAs and coaggregation inhibition experiments was evidently specific for the fimbrial protein, with no evidence that it reacted with nonproteinaceous components. Even when crude antiserum was used to maximize the opportunity to detect reactions between nonfimbrial components and A. viscosus cells (Fig. 5), only one prominent band corresponding to that of the fimbrillin protein was detected. In coaggregation inhibition assays, exposure of one of the coaggregating pair to adhesin-specific antibodies has often been used to identify surface structures involved in attachment. For example, monoclonal antibodies raised to B. loescheii fimbriae effectively inhibited coaggregation with S. sanguis and with A. israelii. Monoclonal antibodies directed to a 75-kDa fimbrial protein apparently blocked binding of this adhesin to its carbohydrate receptor on S. sanguis, while monoclonal antibodies directed to a 45-kDa fimbrial protein appeared to be responsible for inhibition of binding to A. israelii (26). The first line of evidence suggesting that P. gingivalis fimbriae mediate adhesion to A. viscosus was derived from coaggregation inhibition assays in which antisera against P. gingivalis iFm as well as AFAS-IgG inhibited coaggregation of P. gingivalis and A. viscosus. Both P. gingivalis and A. viscosus autoagglutinate, making it necessary to disperse bacterial aggregates prior to the assay. Treatment of P. gingivalis by syringe passage and sonication evidently diminished but did not eliminate fimbriation. Bacteria dispersed by syringing and sonication demonstrated a reduction in the amount of detectable fimbrillin on Western blots probed with AFAS-IgG compared with those not treated by these techniques (data not shown), yet they still had fimbriae visible by electron microscopy. Coaggregation inhibition assays were carefully controlled for autoaggregation and antibody agglutination, and coaggregation was confirmed by Gram stain. Despite these controls, we cannot be sure that none of the inhibition was due to subtle antibody-mediated agglutination of one of the partners. However, it would seem unlikely that this could have accounted for the elevated AFAS-IgG inhibition titer. More definitive lines of evidence implicating P. gingivalis fimbriae in adhesion functions were demonstrated in experiments assessing direct adhesion of the fimbriae to A. viscosus. Binding curves generated by an ELISA showed that isolated fimbriae bound in a concentration-dependent manner to A. viscosus. These structures can evidently remain bound after vigorous washing, as shown by the presence of 40-kDa fimbrillin and copurifying 75-kDa proteins on antifimbrial antibody-developed Western blots of electrophoretic gels of A. viscosus cells which had been mixed with P. gingivalis fimbriae and then washed. Even sonication between washes did not completely remove detectable fimbrillin from the cell pellet, suggesting a rather avid interaction between P. gingivalis fimbriae and A. viscosus cells. Boyd and McBride also described a fraction of Bacteroides gingivalis W12 extracts which contained a 41.5-kDa protein with affinity for gram-positive bacteria, including A. visco-

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ADHESION OF P. GINGIVALIS FIMBRIAE TO A. VISCOSUS

sus, and which they considered a major outer membrane protein (2). Their method of isolating outer membranes would not have excluded fimbriae. The molecular interactions involved in the adhesion of P. gingivalis fimbriae are still not known. They appear to be specific, as supported by our findings that iFm binds to A. viscosus cells but not to SRBC, priming the former but not the latter for AFAS-mediated agglutination. Previous studies in our laboratory have attempted to characterize the nature of the reactions involved in P. gingivalis-A. viscosus coadhesion. The interaction is not lectinlike, as are many other bacterial coaggregation interactions (7), including the lactose-inhibitable coaggregation of P. gingivalis and Fusobacterium nucleatum recently reported by Kinder and Holt (13). Evidence supporting a nonlectin interaction with A. viscosus has been extended by Bourgeau and Mayrand (1). The P. gingivalis component is apparently heat sensitive, while the A. viscosus component is heat stable (7). Because it is heat sensitive, it is unlikely that LPS present in the fimbrial preparation acts as a P. gingivalis adhesin. Indeed, work in our lab (7) showed previously that purified P. gingivalis LPS did not interfere with coadhesion with A. viscosus at concentrations which differed from those in controls. Moreover, coating erythrocytes with purified P. gingivalis LPS did not cause hemagglutination by A. viscosus. More recently, Rosenberg and coworkers have demonstrated the specificity of A. viscosus-P. gingivalis coadhesion using a new kinetic assay (18). They observed that A. viscosus bound preferentially to P. gingivalis cells coated on hexadecane droplets rather than to exposed hexadecane, even though A. viscosus is known to be hydrophobic. While this implies that specific recognition rather than hydrophobicity is crucial for the initial interaction, they also showed that a known inhibitor of hydrophobic interactions, defatted BSA, could impair adhesion. Coadhesion on hexadecane droplets is also inhibited by a broad range of other proteins and the amino acid arginine (7a). Therefore, P. gingivalis coadhesion with A. viscosus is probably mediated via some kind of specific stereochemical recognition of peptide domains, with binding affinity or stability possibly affected by hydrophobic interactions. Other surface structures which bear P. gingivalis adhesins are the extracellular vesicles. These also contain enzymes associated with virulence. In vitro, P. gingivalis vesicles have been shown to adhere directly to A. viscosus (6) and to foster the adhesion and coaggregation of several other oral bacteria (6, 9, 21), increasing the interest in the nature of their adhesins. SDS-PAGE shows little difference between P. gingivalis outer membranes and vesicle membranes (9). This led to one hypothesis that they constitute extracellular adherence organelles which expose adhesins over an increased surface area (6). Fimbriae can be seen on some but not all P. gingivalis vesicles (not shown). It is possible that both of these structures carry adhesins significant for coadhesion with A. viscosus. Li and coworkers have recently demonstrated the association of P. gingivalis trypsinlike proteases with the coadhesion of P. gingivalis and A. viscosus cells (17). These proteases, which are borne in outer membranes of whole cells and vesicles, may represent direct bacterial adhesins or, alternatively, may be required, through their degradative functions, to expose cryptic domains on P. gingivalis's target substrates, like the A. viscosus surface. Recognition of other structures bearing adhesins, like vesicles, or molecules which may modulate adhesive interactions, like proteases, should in no way diminish the potential importance of

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fimbrial adhesins; they might all function in concert. The ability of P. gingivalis fimbriae to bind directly to A. viscosus in combination with the ability of antifimbrial antibodies to inhibit P. gingivalis and A. viscosus coaggregation provides several lines of evidence that fimbrial structures function in P. gingivalis adhesion to A. viscosus. ACKNOWLEDGMENTS

We thank Meja Song for her technical assistance. This study was supported by grant MT-5619 from the Medical Research Council of Canada. REFERENCES 1. Bourgeau, G., and D. Mayrand. 1990. Aggregation of Actinomyces strains by extracellular vesicles produced by Bacteroides gingivalis. Can. J. Microbiol. 36:362-365. 2. Boyd, J., and B. C. McBride. 1984. Fractionation of hemagglutinating and bacterial binding adhesins of Bacteroides gingivalis. Infect. Immun. 45:403-409. 3. Cisar, J. 0. 1982. Coaggregation reactions between oral bacteria: studies of specific cell-to-cell adherence mediated by microbial lectins, p. 121-131. In R. J. Genco and S. E. Mergenhagen (ed.), Host-parasite interactions in periodontal diseases. American Society for Microbiology, Washington, D.C. 4. Dickinson, D. P., M. A. Kubiniec, F. Yoshimura, and R. J. Genco. 1988. Molecular cloning and sequencing of the gene encoding the fimbrial subunit protein of Bacteroides gingivalis. J. Bacteriol. 170:1658-1665. 5. Ellen, R. P., I. A. Buivids, and J. R. Simardone. 1989. Actinomyces viscosus fibril antigens detected by immunogold electron microscopy. Infect. Immun. 57:1327-1331. 6. Ellen, R. P., and D. A. Grove. 1989. Bacteroides gingivalis vesicles bind to and aggregate Actinomyces viscosus. Infect. Immun. 57:1618-1620. 7. Ellen, R. P., S. Schwarz-Faulkner, and D. A. Grove. 1988. Coaggregation among periodontal pathogens, emphasizing Bacteroides gingivalis-Actinomyces viscosus cohesion on a saliva coated mineral surface. Can. J. Microbiol. 34:299-306. 7a.Ellen, R. P., and M. Song. Unpublished data. 8. Gibbons, R. J., and J. B. MacDonald. 1960. Hemin and vitamin K compounds as required factors for the cultivation of certain strains of Bacteroides melaninogenicus. J. Bacteriol. 80:164170. 9. Grenier, D., and D. Mayrand. 1987. Functional characterization of extracellular vesicles produced by Bacteroides gingivalis. Infect. Immun. 55:111-117. 10. Handley, P. S., and L. S. Tipler. 1986. An electron microscope survey of the surface structure and hydrophobicity of oral and non-oral species of the bacterial genus Bacteroides. Arch. Oral Biol. 31:325-335. 11. Hodge, J. E., and B. T. Hofreiter. 1962. Determination of reducing sugars and carbohydrates, p. 380-394. In R. L. Whistler and M. L. Wolfram (ed.), Methods in carbohydrate chemistry. Academic Press, Inc., New York. 12. Isogai, H., E. Isogai, F. Yoshimura, T. Suzuki, W. Kagota, and K. Takano. 1988. Specific inhibition of adherence of an oral strain of Bacteroides gingivalis 381 to epithelial cells by monoclonal antibodies against the bacterial fimbriae. Arch. Oral Biol. 33:479-485. 13. Kinder, S. A., and S. C. Holt. 1989. Characterization of coaggregation between Bacteroides gingivalis T22 and Fusobacterium nucleatum T18. Infect. Immun. 57:3425-3433. 14. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227:680-685. 15. Lee, J.-Y., H. T. Sojar, G. S. Bedi, and R. J. Genco. 1991. Porphyromonas (Bacteroides) gingivalis fimbrillin: size, aminoterminal sequence, and antigenic heterogeneity. Infect. Immun. 59:383-389. 16. Li, J., and R. P. Ellen. 1989. Relative adherence of Bacteroides species and strains to Actinomyces viscosus on saliva-coated

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