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