(Bacteroides) Gingivalis Fimbriae Function in Adhesion to Actinomyces Viscosus P

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JOURNAL OF BACTERIOLOGY, Sept. 1991, p. 5266-5274 Vol. 173, No. 17 0021-9193/91/175266-09$02.00/0 Copyright X 1991, American Society for Microbiology Evidence that Porphyromonas (Bacteroides) gingivalis Fimbriae Function in Adhesion to Actinomyces viscosus P. ANDREW GOULBOURNE AND RICHARD P. ELLEN* Faculty ofDentistry, 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 ofP. 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 phosphate- buffered 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.

Several species of bacteria bear long surface appendages teroides gingivalis from J. Slots, State University of New which have been shown to mediate their adhesion to host York at Buffalo) were maintained by weekly transfer on surfaces. The fimbriae of the periodontal pathogen Porphy- laked blood agar. The medium contained blood agar base no. romonas (Bacteroides) gingivalis have been well character- 2 (Oxoid Ltd., Basingstoke, Hampshire, England) supple- ized in terms of their morphological, biochemical, and im- mented with 7% laked sheep's blood, 0.5 mg of L-cysteine munological properties (10, 15, 27, 28), but their function in per ml, and 1 ,ug each of filter-sterilized hemin and menadi- adhesion remains unclear. Recently, Isogai and coworkers one per ml. The plates were incubated at 37°C for 7 days in reported the ability of purified immunoglobulin G (IgG) and anaerobic jars containing palladium catalyst and a gas mix- Fab fragments of monoclonal antibodies raised against iso- ture of 80% N2, 10% H2, and 10% CO2 (anaerobic condi- lated P. gingivalis fimbriae to block adherence of the bacte- tions). ria to buccal epithelial cells (12). This observation impli- Cultures of P. gingivalis 2561 used in experiments were cates, albeit indirectly, P. gingivalis fimbriae in mediating grown in Trypticase yeast extract broth containing Trypti- bacterial adhesion to cells from the oral cavity, but mucosal case peptone (BBL Microbiology Systems, Becton Dickin- surfaces do not appear to be the site initially colonized by P. son and Co., Cockeysville, Md.) supplemented with 3 mg of gingivalis (22). yeast extract (Difco Laboratories, Detroit, Mich.), 5 mg of The preferential localization of P. gingivalis in mixed NaCl, 2.5 mg of K2HPO4, 2.5 mg of dextrose, 5 ,ug of communities on and around teeth, coupled with its coaggre- filter-sterilized hemin, 0.5 jxg of filter-sterilized menadione, gation with gram-positive tooth colonizers like A. viscosus, and 1 mg ofNaHCO3 per ml (8). The cultures were incubated suggest this to be the site of initial colonization in the oral at 37°C for 36 to 40 h (early stationary phase) in a Coy cavity. In vitro experiments have also demonstrated the avid anaerobic chamber (Ann Arbor, Mich.) containing a gas adhesion of P. gingivalis to A. viscosus monolayers on mixture similar to that described above. saliva-coated hydroxyapatite (16, 20). Little is known about Stock cultures of A. viscosus WVU627 (obtained orig- the bacterial adhesins responsible for this interaction. Evi- inally from M. A. Gerencser, West Virginia University) were dence compiled in this study implicates P. gingivalis fim- maintained by monthly transfer on brain-heart infusion agar briae as one ofthe structures which mediate coadhesion with slants (Difco Laboratories). The cultures were incubated at A. viscosus. To our knowledge, this is the first demonstra- 37°C for 48 h in jars under anaerobic conditions and then tion of direct adhesion of these surface structures to bacte- stored aerobically at 4°C. Cultures of A. viscosus WVU627 rial or any other cells associated with the oral cavity. used in experiments were cultivated in tryptic soy broth (Difco Laboratories) in the anaerobic chamber at 37°C for 48 MATERIALS AND METHODS h. Cultures and cultural conditions. Stock cultures of P. Isolation of P. gingivalis fimbriae. Fimbriae were isolated gingivalis 2561 (ATCC 33277) (obtained originally as Bac- 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 * Corresponding author. menadione. Bacteria harvested from fresh cultures were 5266 VOL. 173, 1991 ADHESION OF P. GINGIVALIS FIMBRIAE TO A. VISCOSUS 5267 suspended in 20 mM Tris-HCl containing 0.15 M NaCl and lated fimbriae (iFm; see Fig. 5, peak A) mixed with Freund's 10 mM MgCl2, pH 7.4, by repeated pipetting. The total complete adjuvant. Three weeks later, 50 ,ug of the same volume of resuspending buffer represented 10o of the cul- preparation, mixed with Freund's incomplete adjuvant, was ture medium volume. The suspension was stirred magneti- inoculated at a similar site. Boosters of 50 ,ug of isolated cally for 30 min and then centrifuged at 8,000 x g for 20 min fimbriae were administered to the rabbits 3 weeks after the (Beckman J2-21M induction drive refrigerated centrifuge; second injection, and they were bled for their antisera 2 Beckman Instruments Inc., Fullerton, Calif.), and the super- weeks later. Preimmune serum (PI) was collected 1 week natant containing fimbriae was retained for further use. prior to and on the day of the first inoculation. Ammonium sulfate was added to the bacterial wash to 40% Determination of serospecificity to fimbrial antigens by saturation. The precipitation reaction mixture was stirred at immunoblotting. A crude fimbrial preparation (fimbrial prep- room temperature (RT) for 45 min and allowed to stand aration prior to anion-exchange chromatography) was overnight at 4°C. Precipitated proteins were collected by loaded onto a 14% polyacrylamide minigel, each well con- centrifugation at 25,000 x g at 4°C for 30 min and resus- taining 0.5 ,ug of protein, and run under the SDS-PAGE pended in a small volume of 20 mM Tris-HCl, pH 8.0 (Tris conditions described above. The prestained molecular mass buffer). The suspension was dialyzed against Tris buffer for markers run concurrently included phosphorylase b, 110 48 h at 4°C. Afterwards, the dialysate was clarified by kDa; bovine serum albumin, 84 kDa; ovalbumin, 47 kDa; centrifugation at 10,000 x g for 15 min at 4°C, and the sample carbonic anhydrase, 33 kDa; soybean trypsin inhibitor, 24 was applied to a column of DEAE-Sepharose CL-6B (Phar- kDa; and lysozyme, 16 kDa. The separated preparation macia, Uppsala, Sweden) equilibrated with Tris buffer. The components as well as the molecular weight markers were column was washed with Tris buffer and eluted with a linear transferred to nitrocellulose with a Bio-Rad transblotting cell gradient of 0 to 0.3 M NaCl, followed by a stepwise gradient (Bio-Rad Laboratories) under a constant voltage of 60 V for of 0.3 to 1 M NaCl. Fractions were monitored spectropho- 2 h. The following day, air-dried nitrocellulose blots were tometrically at 280 nm, and those containing protein were wetted in Tris-buffered saline (TBS), pH 7.4, transferred to a further analyzed by sodium dodecyl sulfate-polyacrylamide blocking solution of TBS containing 0.05% Tween 20 and 3% gel electrophoresis (SDS-PAGE). Fractions with similar bovine serum albumin (BSA), and then exposed to diluted protein profiles were pooled, and the samples were desalted antifimbrial antiserum. Immunoreactivity was identified by and concentrated with Centricon-30 microconcentrators or horseradish peroxidase (HRP) conjugated to goat anti-rabbit Diaflo YM30 ultrafiltration membranes (Amicon, Amicon (GAR) IgG in the presence of 4-chloro-1-naphthol, the HRP Division, W. R. Grace and Co., Danvers, Mass.). Samples color substrate (Bio-Rad Instructional Manual; Bio-Rad containing fimbriae were prepared for observation by trans- Laboratories). mission electron microscopy with the negative stain methyl- Preparation of P. gingivalis cells for antiserum absorption. amine tungstate by the method of Handley and Tipler (10). The antiserum was submitted to a series of absorptions to SDS-PAGE. Protein fractions were analyzed by the SDS- remove antibodies against heat-resistant antigens to which PAGE procedure of Laemmli (14). Each sample was added the rabbits evidently reacted. P. gingivalis 2561 was har- to an equal volume of dissociating sample buffer and heated vested from broth cultures by centrifugation at 8,500 x g for at 100°C for 10 min. Polyacrylamide (12.5%) gels were 15 min at 20°C. The pellet was washed twice in 0.01 M loaded with 20 ,ul, containing approximately 0.23 p.g of phosphate-buffered saline (PBS), pH 7.2, and its cell con- protein, for each sample, and a constant voltage of 175 V centration was adjusted to an OD550 of 2.0. The bacterial was applied for 45 min (Bio-Rad mini gel system; Bio-Rad suspension was sonicated to disperse the cells and auto- Laboratories, Richmond, Calif.). The molecular mass mark- claved at 126°C under 20 kPa of pressure for 20 min. The ers included phosphorylase b, 94 kDa; bovine serum albu- cells were washed once in PBS and resuspended in PBS to min, 67 kDa; ovalbumin, 43 kDa; carbonic anhydrase, 30 one-sixth the volume of the previously adjusted bacterial kDa; soybean trypsin inhibitor, 20 kDa; and a-lactalbumin, suspension. Nine milliliters of P. gingivalis 2561 antifimbrial 14.4 kDa (Pharmacia). Gels were stained for protein with antiserum was mixed with an equal volume of the autoclaved Bio-Rad silver stain (Bio-Rad Laboratories) or for lipopoly- P. gingivalis 2561 cell suspension and incubated for 1 h at RT saccharide (LPS) by the method described by Tsai and under constant rotation. The antiserum was separated from Frasch (24). the bacteria by centrifugation at 8,000 x g for 20 min at 4°C, Transmission electron microscopy. Ten microliters of the and the absorption was repeated. The antiserum was passed fimbrial preparation, suspended in deionized H20, was through a Millex-GV 0.22-p.m filter unit (Millipore, Bedford, mixed with 5 p.l of 0.05% bacitracin as a wetting agent. Mass.) and stored in sterile tubes. Fifteen microliters of 1% methylamine tungstate (Emscope Purification of IgG fraction from AFAS. IgG was purified Laboratories Ltd., Ashford, Kent, England) was then added from absorbed antifimbrial antiserum (AFAS) by using the (10). Formvar-carbon-coated nickel grids were floated on the Bio-Rad Affi-Gel Protein A MAPS II kit (Monoclonal Anti- mixed droplet for 30 s, blotted dry, and observed with a body Purification System; Bio-Rad Laboratories). Eluent Phillips 400T electron microscope. from the protein A-agarose column containing IgG was Biochemical assays. Total protein was estimated with the dialyzed against PBS, and the purified preparation was Bio-Rad protein assay kit (Bio-Rad Laboratories) with passed through a Millex-GV 0.22-p.m filter unit before stor- gamma globulin as a protein standard or by measuring A280. age at -20°C in sterile tubes. (spectrophotometer model 350; G. K. Turner Associates, EEM. P. gingivalis 2561 was washed and suspended in Tris Palo Alto, Calif.). The phenol-sulfuric acid colorimetric buffer. The bacterial concentration was adjusted to an OD550 method described by Hodge and Hofreiter was used to of 1.0, and the suspension was sonicated for 40 s as de- estimate the carbohydrate concentration of the samples (11). scribed below. Methods for indirect immunoelectron micros- Hexoses and methylated hexoses were measured at A490. copy (IEM) were based on those of Ellen and coworkers (5). Glucose was used as a carbohydrate standard. Formvar-carbon-coated nickel grids were floated on droplets Antifimbrial antisera. Two female New Zealand White of the P. gingivalis suspension for 5 min, blotted with filter rabbits were inoculated intramuscularly with 50 p.g of iso- paper, and then exposed for 30 min to Tris buffer containing 5268 GOULBOURNE AND ELLEN J. BACTERIOL.

1.0% (vol/vol) Tween 20 and 1.0% (wt/vol) BSA. The grids immunoblotting assay. After being washed, the bacteria were exposed for 60 min to AFAS-IgG (3.4 g.ig/ml), followed were air dried and gently heat-fixed on glass slides, exposed by three washes in Tris-Tween-BSA buffer. A 30-min expo- to AFAS, washed, and exposed to fluorescein isothiocy- sure to a GAR IgG-gold probe (GAR-IgG gold; Janssen anate-conjugated GAR antibodies. Auroprobe EM GAR G10), diluted 1/20, localized antibody ELISA for quantifying fimbrial adhesion. A. viscosus reactions to the bacterial surface. Grids were washed three WVU627 was suspended in PBS to a cell density of4.5 x 108 times in deionized distilled water to remove buffer salts as cells per ml, and 100-,u aliquots were pipetted into wells of well as excess gold probe, and the specimens were nega- Dynatech Immulon plates (Fischer Scientific Ltd., Ottawa, tively stained with 1% methylamine tungstate. Controls Canada). Wells not containing bacteria were filled with a consisted of specimens treated identically but with the similar volume ofPBS. The plates were incubated at 37°C for substitution of the first antibody by either preimmune IgG or 3 h and then at 4°C overnight. The unbound bacteria were Tris buffer. removed by washing three times with PBS containing 0.05% Antibody inhibition ofP. gingivalis and A. viscosus coaggre- (vol/vol) Tween 20. Plates were washed in a similar manner gation. P. gingivalis 2561 and A. viscosus WVU627 har- after each incubation with a new reagent. All incubations vested from broth cultures were washed twice and resus- were done at 37°C. PBS containing 5% (wt/vol) BSA was pended in PBS. Clumped cells were dispersed by passage incubated for 30 min in the wells to block nonspecific binding through a 25-gauge syringe needle 15 times, and the optical of iFm or antibodies to the plates. After washing, iFm in densities of both bacterial suspensions were adjusted to an dilutions ranging from 10 to 0.04 Fg of protein per ml was OD550 of 1.0. Immediately prior to the assay, the suspen- added in 100-p,l volumes across the rows of the plates and sions were sonicated for 35 s at a setting of 4 by a Kontes incubated for 90 min. Rows to which PBS was added served Micro-Ultrasonic Cell Disrupter (Kontes, Vineland, N.J.) to as controls. disperse smaller bacterial aggregates. Examination of P. Since antibodies might bind nonspecifically to A. viscosus, gingivalis by electron microscopy after dispersion confirmed PBS containing 2% (vol/vol) horse serum was added to the the presence of fimbriae. wells to block this reaction and plates were reincubated for AFAS-IgG and PI-IgG (3.4 mg/ml) were serially diluted in 30 min. AFAS-IgG diluted 1:100 and 1:1,000 was added in a microtiter plate ip twofold steps in 10 IlI of PBS. One 100-,lI aliquots down the columns of the plates, one dilution hundred microliters of P. gingivalis suspension, containing per plate, and incubated for 1 h. PI-IgG diluted 1:100 and approximately 4.25 x 109 cells per ml, was then added to 1:1,000 and PBS were run in parallel to control for antibody each well and to control wells containing buffer only. Plates specificity. HRP-GAR-IgG diluted 1:200 was added to all containing the mixtures of antibody and bacteria were incu- wells and incubated for 30 min. The substrate o-phenylene- bated for 45 min at RT on a shaker platform and observed for diamine was then added, and the reaction was stopped by agglutination. Aggregates were dispersed with constant pi- the addition of 2 N H2SO4. Plates were read at a wavelength petting, and 100 RI of A. viscosus suspension, containing of 492 nm with an enzyme-linked immunosorbent assay approximately 1.25 x 109 cells per ml, was added to the (ELISA) plate reader (Titertek Multiscan Plus; Flow Labo- wells. The plates were reincubated under the same condi- ratories Inc., Mississauga, Ontario, Canada). tions and observed for coaggregation inhibition. Results Bacterial agglutination assay. An iFm preparation with a were considered valid only when control wells monitoring protein concentration of 21 Lg/ml was diluted in twofold autoaggregation and antibody-mediated agglutination were steps in 50-1l aliquots of PBS across the rows of wells of a negative. Wells close to the inhibition endpoint were also microtiter plate. Rows containing PBS served as controls. monitored by Gram stain to determine the presence ofmixed Fifty microliters of A. viscosus suspension, OD550 of 1.5, coaggregates. was added to each well, and the plate was allowed to stand Immunoblotting assay for fimbrial adhesion to A. viscosus. at RT for 45 min before being observed for aggregation by A. viscosus WVU627 was harvested and washed by centrif- microscopy. Tenfold serial dilutions of AFAS in 50-,ul ali- ugation at 700 x g for 10 min at RT and resuspended in PBS. quots were then added down the columns of the plate, and in Clumped cells were dispersed by 10 passages through a one column PBS was substituted for the antiserum. The 25-gauge syringe needle. The final bacterial concentration plate was reincubated and again observed microscopically was adjusted to an OD550 of 1.4. Equal volumes (0.5 ml) of for aggregation. A. viscosus suspension and P. gingivalis 2561 iFm (80 pLg/ml) Hemagglutination assay. An iFm preparation (20 ,ug/ml) were mixed in microtest tubes and incubated at RT for 1 h was serially diluted in twofold steps in 50 ,lI of PBS, pH 7.4, under constant rotation. Control tubes in which buffer was in the wells of a microtiter plate. A similar volume of a substituted for iFm were also included. Duplicate sets of the washed 2% sheep erythrocyte (SRBC) suspension was mixture were then washed twice in PBS, one set being added to each of these wells and to control wells containing sonicated for 15 s between washes. After each wash, the only PBS. The plates were rotated for 1 h at RT and supernatants were retained, as was the final pellet of A. observed for direct hemagglutination. Fifty microliters of a viscosus. The supernatants and pellets were subjected to 100-fold dilution of either unabsorbed antifimbrial antiserum SDS-PAGE, transferred to nitrocellulose, and probed with or AFAS was then added, and the plate was observed 1 h P. gingivalis unabsorbed antifimbrial antiserum to detect the later for indirect hemagglutination. broadest possible group of immunoreactive proteins. A parallel control experiment in which porcine serum albumin (PSA) was substituted for the iFm preparation was con- RESULTS ducted. Nitrocellulose blots with PSA were probed with Isolation ofP. gingivalis fimbriae. Fimbriae were separated anti-PSA, kindly provided by H. Limeback (University of from outer membrane components by anion-exchange chro- Toronto). matography (Fig. 1). Most of the fimbriae were eluted with The association of iFm with the A. viscosus cell surface 0.15 M NaCl (Fig. 1A, peak A). They were identified was also observed by immunofluorescence microscopy. The throughout the isolation procedure by their structural mono- experimental conditions were identical to those used in the mer, fimbrillin, an obvious 40-kDa band on 12.5% polyacryl- VOL. 173, 1991 ADHESION OF P. GINGIVALIS FIMBRIAE TO A. VISCOSUS 5269

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peak A peak B FIG. 1. Isolation of fimbriae and SDS-PAGE of fimbrial proteins. (A) Elution profile from fractionation on DEAE-Sepharose CL-6B FIG. 2. Pooled fractions of peak A negatively stained with meth- column by linear (0 to 0.3 M) and stepwise (0.3 to 1.0 M) NaCl ylamine tungstate. The iFm preparation contained long, helical gradients. Most of the fimbrial protein was eluted at 0.15 M NaCl fimbriae which tended to clump longitudinally. Bar, 0.1 ,um. (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. 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 com- amide-SDS gels (Fig. 1B). Those fractions containing only ponents, including LPS. Preliminary absorption studies had fimbrillin (peak A) were pooled, concentrated, and used in demonstrated LPS and autoclaved P. gingivalis cells to be 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. 75kDa_ z Fimbriae isolated from P. gingivalis 2561 were helical 5okDa_- 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% 39kDae- protein and 6% carbohydrate. The fimbrillin subunit of 40 27 kDa.- was kDa the major protein clearly identifiable by SDS- 17kDaw- PAGE. Proteins of 51 and 75 kDa were also present but barely detectable (Fig. 1B, peak A). LPS was not detected A B on LPS silver-stained gels of the same preparation (data not shown). FIG. 3. Immunoblots of the crude fimbrial preparation probed Antibody preparations specific for P. gingivalis fimbriae. with antisera raised against isolated fimbriae. Approximately 0.5 ,ug of well was SDS-PAGE Rabbit antisera fimbriae were not protein per separated by (14% separating raised against isolated gel) and transferred to nitrocellulose. (A) Unabsorbed antifimbrial monospecific for the fimbrillin subunit (Fig. 3). Western antiserum; (B) antifimbrial antiserum absorbed twice with boiled P. immunoblots of crude fimbrial preparations (from 14.0% gingivalis cells (AFAS). The protein concentration for both unab- polyacrylamide-SDS gels) overlaid with the antisera showed sorbed and absorbed antisera was 96 ,ug/ml. Duplicates are included a steplike banding pattern characteristic of LPS as well as a in the figure to illustrate the range of reactions, which were large band of 44 kDa (Fig. 3A), corresponding to fimbrillin. sometimes not identical. 5270 GOULBOURNE AND ELLEN J. BACTERIOL. equally effective in achieving a more fimbrillin-specific anti- SRBC. P. gingivalis fimbriae, as entities separate from whole serum; the latter was the method of choice for absorption of cells, appeared to possess a single, or monovalent, adhesin large volumes of antiserum. In addition to antifimbrillin accessible for their attachment to A. viscosus receptors, as antibodies, AFAS contained antibodies to the copurifying A. viscosus cells exposed to P. gingivalis fimbriae, even at 75-kDa and 51-kDa proteins as well as to components of 52 the highest concentration tested, were aggregated only after and 58 kDa. The most obvious reaction with transferred the addition of antifimbrial antibodies. AFAS diluted 1:100 proteins in Western blots was with fimbrillin (Fig. 3B). agglutinated iFm-exposed A. viscosus cells. Although P. IEM of P. gingivalis fimbriae. Immunogold probes bound gingivalis cells and some P. gingivalis proteins are able to along the fimbriae, which appeared to be associated with, agglutinate SRBC, purified P. gingivalis fimbriae are char- and partially obliterated by, an amorphous material (Fig. 4A acteristically unable to hemagglutinate (27). The iFm prepa- and C). A recent communication by Sojar and coworkers ration demonstrated weak hemagglutinating activity at a (22b) suggests that this material is lipid complexed with concentration of 10.5 ,ug/ml. This may be attributed to either fimbrillin. Virtually no label was detected when PI-IgG was nonspecific hemagglutination of SRBC or the presence of a substituted for AFAS (Fig. 4B). P. gingivalis vesicles, the small amount of P. gingivalis hemagglutinin in the pooled membranes of which are similar to the bacterial outer fractions. Fimbriae were apparently not responsible, as membrane, were not labeled. Therefore, labeling observed addition of AFAS did not amplify the weak hemagglutination on or close to the bacterial cell body may be attributed to beyond that observed with the PBS control. In contrast, fimbriae lying on its surface. The pattern of labeling with addition of unabsorbed antifimbrial antiserum increased the AFAS-IgG confirms the immunoblotting results that it con- hemagglutination titer 16-fold, probably due to antibodies tains antibodies almost exclusively directed to fimbriae- directed against heat-resistant antigens like LPS, which associated antigens. would have been absorbed out in the preparation of AFAS. Coaggregation inhibition. AFAS inhibited coaggregation of P. gingivalis and A. viscosus at a much higher dilution DISCUSSION (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 Adhesive interactions with bacteria already bound to the crude antifimbrial antiserum (1:64 to 1:128). Purified AFAS surfaces of teeth are thought to foster colonization of the IgG (3.4 pg/ml) inhibited coaggregation at a dilution of 1:16; gingival crevice by some pathogenic microorganisms such as PI-IgG did not inhibit coaggregation. Coaggregation inhibi- P. gingivalis. The function of attaching to such surfaces tion by antibodies raised to isolated fimbriae supports the involves multiple interactions, both specific and nonspecific, hypothesis that these structures are important in adhesion of which are often mediated by adhesins borne on extracellular P. gingivalis to A. viscosus. structures. Among the coaggregating oral bacteria studied Immunoblot analysis of isolated fimbriae bound to A. visco- previously, fimbriae appear to be important in the coadhe- sus. Western blots of washed mixtures of A. viscosus and sion of A. viscosus and Actinomyces naeslundii with Strep- iFm, developed with unabsorbed antifimbrial antiserum, tococcus sanguis (3) and Bacteroides loescheii with Actino- were used to determine the ability of fimbriae to adhere to A. myces israelii and S. sanguis (25). Evidence in this study viscosus. The iFm preparation chosen for these experiments demonstrates that the adhesion of the periodontal pathogen contained fimbrillin as well as many other immunodetectable P. gingivalis with A. viscosus, a prominent tooth colonizer components which copurified with it (Fig. 5A, lane 1). which is often cocultivated from dental plaque with P. Although much of the excess iFm was partitioned into the gingivalis, may also involve fimbriae on the P. gingivalis supernatant after the first wash and centrifugation cycle (Fig. cells. 5A, lane 2), A. viscosus pellets still retained detectable Inhibition of P. gingivalis-A. viscosus coaggregation by amounts of fimbrillin after two buffer washes (Fig. 5A, lane antifimbrial antibodies and direct adhesion of iFm to A. 5). Components of 51, 52, and 75 kDa, identified as proteins viscosus, studied by immunoblotting, immunofluorescence, by protein silver staining, were also present but not as indirect AFAS-mediated agglutination, and ELISA, provide distinguishable or as consistently detectable as fimbrillin. evidence supporting the hypothesis that these structures The association of fimbrillin with A. viscosus provides one carry some of the adhesins mediating P. gingivalis adhesion line of direct evidence that P. gingivalis fimbriae are able to to A. viscosus. A similar approach was taken by Tempro and bind to the bacteria. The control protein, PSA, was unable to coworkers (23) to characterize the lectinlike adhesin on bind to A. viscosus (Fig. 5B), suggesting that the association Capnocytophaga gingivalis DR2001. Monoclonal antibodies of fimbrial proteins with A. viscosus is specific. reactive with a 140-kDa polypeptide found in the outer Indirect immunofluorescent labeling of A. viscosus ex- membrane of the bacteria inhibited the interaction of P. posed to iFm corroborated the observations of the immuno- gingivalis with carbohydrate receptors on its partner A. blot experiments. The bacteria fluoresced only when ex- israelii. The adhesins were arranged nonuniformly on the posed to iFm and AFAS. No fluorescence was observed in bacterial surface, as observed by IEM. controls in which the fimbrial incubation step was deleted. Fimbrial preparations isolated from P. gingivalis 2561 Quantitation of fimbrial adhesion by ELISA. Isolated fim- were composed mostly of fimbria-associated proteins. SDS- briae demonstrated concentration-dependent adherence to polyacrylamide gels identified the structural fimbrial mono- whole A. viscosus cells (Fig. 6). Although fimbriae also mer fimbrillin as the major protein present in these prepara- showed a minor degree of binding to BSA-coated wells, tions. Variation in the molecular mass of this protein within binding to A. viscosus-coated wells was significantly greater. the range of 40 to 44 kDa, as noted by others (4, 15), can be The reproducibility of the assay between replicates (as attributed to differences in SDS-PAGE running conditions reflected by the small standard deviations) and between for our different assays as well as differences in the migration assays done on different days demonstrated its effectiveness of fimbrillin compared with that of globular molecular mass in quantitative studies as a second line of direct evidence of markers. fimbrial adhesion. Besides fimbrillin, barely detectable copurifying proteins Inability of isolated fimbriae to agglutinate A. viscosus and of 51 and 75 kDa were also observed by SDS-PAGE of iFm VOL. 173, 1991 ADHESION OF P. GINGIVALIS FIMBRIAE TO A. VISCOSUS 5271 ..S.. .s. I .o. .. 4

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. .*,1_

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.'1 Seventy-five-kilodalton proteins often copu- evidence has suggested that both are proteolipids (22b). rify with fimbrillin (15, 27) and have been reported to be Degradation of the 75-kDa protein gives rise to fragments difficult'",i_Ito separate from the fimbrial monomer (29). Analy- which are approximately 20 kDa smaller in size (22a); thus, ses of purified 75-kDa protein have found it to be structurally the 51-kDa protein observed in SDS-polyacrylamide gels of and antigenically different from fimbrillin (29). More recent iFm preparations might represent such a degradation prod- 5272 GOULBOURNE AND ELLEN J. BACTERIOL.

A B Absorption of antisera with fimbria-deficient mutants has been used to effectively remove nonfimbrial antibodies. For example, Salit and coworkers removed anti-LPS antibodies 75kDa_- 75k Da _ from antisera raised to denatured Escherichia coli Pap pilin 5OkDam.- ... by repeated absorption with Pap pilin mutants (19). Fimbria- 5OkDamP 39kDamm-...._ deficient mutants of P. gingivalis have not been described. _.1 .. Instead, the sensitivity of proteins and, conversely, the 39kDa- higher resistance of LPS to heat treatment were used as a strategy to mimic fimbria-deficient bacteria for the purpose of antiserum absorption. The final absorbed antibody preparation used for direct ELISAs and coaggregation inhibition 2 3 experiments was evidently specific for the fimbrial protein, FIG. 5. Immunoblots of A. viscosus cell pellets and wash super- with no evidence that it reacted with nonproteinaceous natants developed with unabsorbed antifimbrial antiserum and HRP- components. Even when crude antiserum was used to max- GAR-IgG to determine whether fimbrial proteins bound to A. imize the opportunity to detect reactions between nonfim- viscosus cells. (A) Lane 1, iFm, 0.4 ,ug per well; lane 2, supernatant brial components and A. viscosus cells (Fig. 5), only one of initial mixture; lane 3, supernatant of first wash; lane 4, supernatant of second wash; lane 5, A. viscosus cells plus iFm washed twice prominent band corresponding to that of the fimbrillin pro- in PBS. (B) Control: mixtures of A. viscosus and PSA. Blots were tein was detected. developed with anti-PSA antiserum. Lane 1, PSA, 0.4 ,ug per well; In coaggregation inhibition assays, exposure of one of the lane 2, supernatant of initial mixture; lane 3, supernatant of first coaggregating pair to adhesin-specific antibodies has often wash; lane 4, supernatant of second wash; lane 5, A. viscosus plus been used to identify surface structures involved in attach- PSA washed twice in PBS. A. viscosus cells did not react with ment. For example, monoclonal antibodies raised to B. antifimbrial antiserum or with anti-PSA antiserum (not shown). 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 uct. It is unclear whether degradation products of fimbrillin this adhesin to its carbohydrate receptor on S. sanguis, are also common. Examination of fimbrial preparations by while monoclonal antibodies directed to a 45-kDa fimbrial transmission electron microscopy demonstrated the pres- protein appeared to be responsible for inhibition of binding ence of fimbriae similar to those described by Yoshimura to A. israelii (26). The first line of evidence suggesting that P. and coworkers (27). As this was the only structure identified, gingivalis fimbriae mediate adhesion to A. viscosus was it added some assurance that the preparations contained derived from coaggregation inhibition assays in which anti- mostly, if not exclusively, fimbria-associated components. sera against P. gingivalis iFm as well as AFAS-IgG inhibited Antisera raised to the iFm preparation were not monospe- coaggregation of P. gingivalis and A. viscosus. Both P. cific for fimbrial proteins. Antibodies to other bacterial gingivalis and A. viscosus autoagglutinate, making it neces- components, probably LPS, were also detected on Western sary to disperse bacterial aggregates prior to the assay. blots of the crude fimbrial preparation. SDS-polyacrylamide Treatment of P. gingivalis by syringe passage and sonication gels of the iFm preparation which had been stained for LPS evidently diminished but did not eliminate fimbriation. Bac- did not detect it, which suggests that LPS was present only teria dispersed by syringing and sonication demonstrated a in a concentration sufficient to be immunogenic. However, reduction in the amount of detectable fimbrillin on Western the fimbrial preparation did contain some carbohydrate. 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, E we cannot be sure that none of the inhibition was due to cmJC subtle antibody-mediated agglutination of one of the part- 0 le ners. 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 -W in adhesion functions were demonstrated in experiments direct adhesion of the fimbriae to A. viscosus. co assessing 10 Binding curves generated by an ELISA showed that isolated fimbriae bound in a concentration-dependent man- 0. ner to A. viscosus. These structures can evidently remain 0. bound after vigorous washing, as shown by the presence of 0 40-kDa fimbrillin and copurifying 75-kDa proteins on antifimbrial antibody-developed Western blots of electropho- Conc. of Isolated flmbriae (ug/mi) retic gels of A. viscosus cells which had been mixed with P. gingivalis fimbriae and then washed. Even sonication be- FIG. 6. Binding curve for ELISA determination of P. gingivalis tween washes did not completely remove detectable fim- adhesion to A. viscosus cells. Values represent the mean of three brillin from the cell pellet, suggesting a rather avid interac- replicates ± standard deviation. Standard deviations for some mean values were small enough to be within the height of the symbols and tion between P. gingivalis fimbriae and A. viscosus cells. are not evident in the figure. Symbols: C, A. viscosus plus iFm plus Boyd and McBride also described a fraction of Bacteroides AFAS-IgG; *, A. viscosus plus iFm plus PI-IgG; *, BSA plus iFm gingivalis W12 extracts which contained a 41.5-kDa protein plus AFAS-IgG. with affinity for gram-positive bacteria, including A. visco- VOL. 173, 1991 ADHESION OF P. GINGIVALIS FIMBRIAE TO A. VISCOSUS 5273 sus, and which they considered a major outer membrane fimbrial adhesins; they might all function in concert. The protein (2). Their method of isolating outer membranes ability ofP. gingivalis fimbriae to bind directly to A. viscosus would not have excluded fimbriae. in combination with the ability of antifimbrial antibodies to The molecular interactions involved in the adhesion of P. inhibit P. gingivalis and A. viscosus coaggregation provides gingivalis fimbriae are still not known. They appear to be several lines of evidence that fimbrial structures function in specific, as supported by our findings that iFm binds to A. P. gingivalis adhesion to A. viscosus. viscosus cells but not to SRBC, priming the former but not the latter for AFAS-mediated agglutination. Previous studies ACKNOWLEDGMENTS in our laboratory have attempted to characterize the nature of the reactions involved in P. gingivalis-A. viscosus coad- We thank Meja Song for her technical assistance. hesion. The interaction is not lectinlike, as are many other This study was supported by grant MT-5619 from the Medical bacterial coaggregation interactions (7), including the lac- Research Council of Canada. tose-inhibitable coaggregation of P. gingivalis and Fusobac- terium nucleatum recently reported by Kinder and Holt (13). REFERENCES Evidence supporting a nonlectin interaction with A. viscosus 1. Bourgeau, G., and D. Mayrand. 1990. Aggregation of Actino- has been extended by Bourgeau and Mayrand (1). The P. myces strains by extracellular vesicles produced by Bacteroides gingivalis. Can. J. Microbiol. 36:362-365. gingivalis component is apparently heat sensitive, while the 2. Boyd, J., and B. C. McBride. 1984. Fractionation of hemagglu- A. viscosus component is heat stable (7). Because it is heat tinating and bacterial binding adhesins of Bacteroides gingiva- sensitive, it is unlikely that LPS present in the fimbrial lis. Infect. Immun. 45:403-409. preparation acts as a P. gingivalis adhesin. Indeed, work in 3. Cisar, J. 0. 1982. Coaggregation reactions between oral bacte- our lab (7) showed previously that purified P. gingivalis LPS ria: studies of specific cell-to-cell adherence mediated by micro- did not interfere with coadhesion with A. viscosus at con- bial lectins, p. 121-131. In R. J. Genco and S. E. Mergenhagen centrations which differed from those in controls. Moreover, (ed.), Host-parasite interactions in periodontal diseases. Amer- coating erythrocytes with purified P. gingivalis LPS did not ican Society for Microbiology, Washington, D.C. cause hemagglutination by A. viscosus. 4. Dickinson, D. P., M. A. Kubiniec, F. Yoshimura, and R. J. More Genco. 1988. Molecular cloning and sequencing of the gene recently, Rosenberg and coworkers have demon- encoding the fimbrial subunit protein of Bacteroides gingivalis. strated the specificity of A. viscosus-P. gingivalis coadhe- J. 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