Porphyromonas salivosa ATCC 49407 fimbriae induced osteoclast differentiation and cytokine production

Porphyromonas salivosa ATCC 49407 is a black-pigmented, anaerobic, Gram-negative, and rod-shaped organism. P. salivosa is isolated from the gingival sulcus of various animals including dogs and cats. Fimbriae are filamentous components on the cell surface and are thought to play an important role in the colonization of periodontal tissues. We examined the involvement of the fimbrial protein in osteoclast differentiation and cytokine production in murine macrophages. Furthermore, alveolar bone resorption induced by P. salivosa infection in rats was evaluated. Fimbrial protein was purified from P. salivosa by selective protein precipitation and chromatography on a DEAE CL-6B anion exchange column. Western blotting analysis was performed with PAbs against fimbrial protein from P. salivosa. Expression of fimbriae on the surface of P. salivosa was investigated using transmission electron microscopy. To estimate osteoclast differentiation, bone marrow cells and MC3T3-G2/PA6 cells were cultured with or without the purified fimbrial protein. BALB/c mouse peritoneal macrophages were stimulated with the purified fimbrial protein, and cytokine production was determined by ELISA. Sprague-Dawley rats were infected with P. salivosa. Forty-five days after the last infection, the periodontal bone levels were determined by a morphometric measurement. We determined P. salivosa had fimbrial structure on the cell surface, and purified 60-kDa fimbrial protein. Osteoclast differentiation was significantly enhanced with the treatment of the 60-kDa fimbrial protein. The purified fimblial protein induced IL-1β and TNF-α production. Rats orally infected with P. salivosa exhibited significant bone loss compared with that of sham-infected rats. These results suggest that P. salivosa 60-kDa fimbriae may provoke an inflammatory response in host and be involved in periodontal tissue breakdown. *Corresponding author: Dr. Nobushiro HAMADA 82 Inaoka-cho, Yokosuka, Kanagawa, 238-8580 Japan 2019 年 12 月 Role of P. salivosa fimbriae 93 were found to be the most frequently isolated blackpigmented anaerobic bacteria associated with canine periodontitis7). Norris et al.8) reported that the strong positive correlation between P. salivosa and the grade of periodontal disease at canine sites strongly supported P. salivosa playing an important role in canine periodontal disease. Furthermore, P. salivosa is the same species as P. macacae isolated from monkeys9, 10). P. gingivalis is a pathogen that causes periodontal disease, a typical chronic inflammatory disease11-14). P. gingivalis fimbriae are important cell structures that contribute to adherence to and the invasion of host cells15-18), and induce inflammatory processes in periodontal tissues through a number of mechanisms19-21). Furthermore, Ozaki et al.22) reported that fimbriae function as virulence factors in inflammatory reactions because they stimulated the production of inflammatory cytokines by macrophages and fibroblasts. Cytokines such as interleukin-1 (IL-1), IL-6, and tumor necrosis factor-α (TNF-α) called bone resorptive cytokines because of their ability to promote bone resorption23). These cytokines may induce the expression and promote the solubilization of RANKL through osteoclast-supporting cells, osteoblasts, and stromal cells. Previous studies suggested that soluble RANKL affects not only osteoclast differentiation, but also cell migration and survival extension24-26). Moreover, Hamada et al.27) reported that P. gingivalis 67-kDa fimbriae induced the expression of IL-1α, IL-1β, IL-6, and TNF-α in mouse peritoneal macrophages. These findings indicate that fimbriae play an important role in the induction of inflammatory responses in periodontal disease and alveolar bone loss. Koyata et al.28) showed that 60-kDa fimbriae were present on the surface of P. salivosa and also that the N-terminal amino acid sequence of the 60-kDa fimbrial protein of P. salivosa clearly differed from previously reported fimbrial proteins. However, the role of P. salivosa fimbriae in periodontal disease remains unclear. In the present study, we investigated the effects of the fimbrial protein from P. salivosa on the induction of osteoclast differentiation and cytokine production in murine macrophages. Materials and Methods 1. Strains and cultivation conditions P. salivosa ATCC 49407 was incubated anaerobically (15% CO2, 15% H2, and 70% N2) (ANX-1; HIRASAWA, Tokyo, Japan) at 37°C in brain heart infusion (BHI) broth (Becton Dickinson Co., NJ, U.S.A.) supplemented with 0.5% yeast extract (Becton Dickinson Co.), 5 μg/ml hemin (Wako, Osaka, Japan), and 0.5 μg/ml vitamin K1 (Wako). 2. Isolation and purification of 60-kDa fimbriae from P. salivosa ATCC 49407 The method of Yoshimura et al.29) was employed to purify 60-kDa fimbriae. P. salivosa was incubated anaerobically for 18 hr in BHI broth. The bacterial cell pellet was harvested by centrifugation at 8,000 rpm at 4°C for 20 min and washed twice with 20 mM Tris-HCl buffer (pH 8.0) by repeated pipetting. The suspension was subjected to ultrasonication with a 3-mm microtip (Branson Ultrasonics Corporation, Danbury, CT, U.S.A.) and an output power of 25 W on the pulse setting with 3 cycles of 5 min in an icebox. The suspension of the sonic extract was centrifuged at 16,000 rpm at 4°C for 15 min and subjected to 40% ammonium sulfate saturation by the stepwise addition of ammonium sulfate. The precipitated protein was collected by centrifugation at 16,000 rpm at 4°C for 8 min, suspended in a minimum volume of 20 mM Tris-HCl buffer (pH 8.0), and dialyzed against the same buffer. The dialysate sample containing most of the fimbriae was subjected to further purification on a diethylaminoethyl (DEAE)-Sepharose CL-6B (GE Healthcare Bio-Sciences, Pittsburgh, PA, U.S.A.) column equilibrated with 20 mM Tris-HCl buffer (pH 8.0). The column was washed with 20 mM Tris-HCl buffer and then eluted with a linear gradient of 0 to 0.3 M NaCl (Wako). The protein contents of the fractions were measured by ultraviolet light adsorption at 280 nm. Lipopolysaccharide (LPS) was not detected on silver-stained gels of the same preparation by a Silver Stain II kit (Wako). The endotoxicity of the fimbrial protein was not detected by a colorimetric Limulus amoebocyte lysate assay (GenScript, Tokyo, Japan). 3. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) Protein extracts were heated at 100°C for 10 min in loading buffer. Samples were applied to 12% polyacrylamide slab gels with a 4% stacking gel and electrophoresed at a constant current of 30 mA for 1 hr. Proteins were stained with Coomassie brilliant blue R-250 (Wako). A precision plus proteinTM standards dual color (Bio-Rad Laboratories, Inc., Hercules, CA, U.S.A.) was used for molecular weight calibrations. 94 神 奈 川 歯 学 第 54 巻第 2 号 4. Polyclonal antibodies Polyclonal antibodies (PAbs) against the 60-kDa fimbrial protein were prepared using the purified protein described above as an immunogen. BALB/c mice (Nihon SLC, Inc., Shizuoka, Japan) were subcutaneously injected at multiple sites with 50 μg of the 60-kDa fimbrial protein emulsified with Freund’s incomplete adjuvant (Becton Dickinson Co.). After 2 weeks, mice were injected weekly for 4 weeks with the immunogen. Each mouse was bled after the last booster injection, and antibodies were tested against the corresponding antigen by Western blotting. After an adequate antibody titer was obtained, mice were bled by cardiac puncture and sera were prepared and stored at −20°C. The experimental procedures of the present study were reviewed and approved by the Committee of Ethics on Animal Experiments of Kanagawa Dental University (Nos. 051, 17-026, and 17-051). 5. Western blotting In the immunoblot analysis, proteins separated by 12% SDS-PAGE were transferred to a polyvinylidene difluoride membrane (Immun-Blot® PVDF membrane; Bio-Rad Laboratory) at 200 mA for 1 hr. Membranes were then treated with Tris-Buffer saline (TBS; 20 mM Tris-HCl pH 7.4, 0.5 M NaCl) containing 1% bovine serum albumin (BSA; Sigma-Aldrich,St. Louis,MO,U.S.A.) to block unoccupied proteinbinding sites. They were then incubated with PAbs specific for the 60-kDa fimbrial protein of P. salivosa ATCC 49407 at 37°C for 1 hr, washed in TBS-Tween (TBS with 0.5% Tween 20), incubated for 1 hr with goat anti-mouse immunoglobulin G conjugated with horseradish peroxidase (GE Healthcare), and then immersed in 4-chloro-1-naphthol (Sigma-Aldrich) solution to develop the color. The reaction was stopped by immersing the membranes in distilled water, and the membranes were then dried. 6. Electron microscopy The fimbriae of P. salivosa cells were examined with a transmission electron microscope. Bacterial cells from an 18 hr anaerobic culture were collected by centrifugation (10,000 rpm for 1 min), washed, and resuspended (5 × 108 cells/ml) in phosphate-buffered saline (Nissui Pharmaceutical Co., Tokyo, Japan) (pH 7.4). Ten microliters of the cell suspension or purified fimbriae was applied to a copper grid coated with a thin Formvar film and air-dried. Samples were then negatively stained with 2% (wt./vol) uranyl acetate for 1 min, air-dried, examined, and photographed with a JEM-1220 electron microscope (JEOL Ltd., Tokyo, Japan) operating at 80 kV. 7. Detection of antibodies against 60-kDa fimbriae in cat serum Twenty-five cats ranging in age 4 months to 16 years, both sexes (12 males, 13 females) and various breeds were included in the study. Cat serum was provided by Ogawa Animal Hospital in Yokosuka. Crude fimbrial proteins were separated by 12% SDS-PAGE and transferred to a PVDF membrane at 200 mA for 1 hr. The membranes were then treated with TBS containing 1% BSA (Sigma-Aldrich) to block unoccupied proteinbinding sites. Serum collected from cats acted on this membrane at 4°C overnight. After washing with TBS, membranes were incubated with goat anti-cat immunoglobulin G conjugated with horseradish peroxidase (Invitro