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J Dent Res 84(5):462-467, 2005
© 2005 International and American Associations for Dental Research

RESEARCH REPORT
Biological

Tannerella forsythia-induced Alveolar Bone Loss in Mice Involves Leucine-rich-repeat BspA Protein

A. Sharma1,*, S. Inagaki1, K. Honma2, C. Sfintescu1, P.J. Baker3, and R.T. Evans1

1 Department of Oral Biology, School of Dental Medicine, State University of New York, 3435 Main Street, Buffalo, NY 14214, USA;
2 Department of Microbiology, Oral Health Science Center, Tokyo Dental College, Chiba 261-8502, Japan; and
3 Department of Biology, Bates College, Lewiston, ME 04240, USA;

* corresponding author, sharmaa{at}buffalo.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Tannerella forsythia (formerly Bacteroides forsythus) is one of the periodontal pathogens recently implicated in the development of periodontal disease. The cell-surface-associated, as well as the secreted, leucine-rich-repeat protein (BspA) of this bacterium have been suggested to play roles in bacterial adherence, and also in inflammation, by triggering release of pro-inflammatory cytokines from monocytes and chemokines from osteoblasts, leading to inflammation and bone resorption. In this study, we sought to determine the pathogenic potential of T. forsythia and the in vivo role of the BspA protein in pathogenesis in the mouse model of infection-induced alveolar bone loss. The results showed alveolar bone loss in mice infected with the T. forsythia wild-type strain, whereas the BspA mutant was impaired. In conclusion, evidence is presented in support of T. forsythia as an important organism involved in inducing alveolar bone loss, and the BspA protein is an important virulence factor of this bacterium.

KEY WORDS: Tannerella forsythia • alveolar bone loss • BspA protein • leucine-rich-repeat protein


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Tannerella forsythia (formerly Bacteroides forsythus), a Gram-negative bacterium, has recently emerged as an important periodontal pathogen (Grossi et al., 1994, 1995; Tanner et al., 1998). It belongs to the ‘red-complex’ bacteria, an association of Bacteroides forsythus, Porphyromonas gingivalis, and Treponema denticola that appears to be a major etiologic contributor to common adult forms of periodontitis (Socransky et al., 1998). Although the role of P. gingivalis is well-established in disease development, evidence for the role of T. forsythia and T. denticola is scant. A few putative T. forsythia virulence factors that have been identified to date include: a trypsin-like protease (Saito et al., 1997), a sialidase (Braham and Moncla, 1992; Ishikura et al., 2003), a cell-surface-associated and secreted BspA protein belonging to the leucine-rich-repeat protein family (Sharma et al., 1998), an apoptosis-inducing activity (Arakawa et al., 2000), alpha-D-glucosidase and N-acetyl-beta-glucosaminidase-D-glucosidase (Hughes et al., 2003), a hemagglutinin (Murakami et al., 2002), components of the bacterial S-layer (Sabet et al., 2003), and methylglyoxal production (Maiden et al., 2004). The BspA binds the extracellular matrix components fibronectin and fibrinogen (Sharma et al., 1998). The BspA protein also induces the release of bone-resorbing pro-inflammatory cytokines from monocytic cells via the TLR-2-dependent pathway (Hajishengallis et al., 2002) and stimulates the expression of the CXC chemokine in pre-osteoblastic murine cells (Ruddy et al., 2004). These activities may have implications in neutrophil recruitment, inflammation, and tissue destruction.

The present study was undertaken to determine the pathogenic potential of T. forsythia in the mouse model of infection-induced alveolar bone loss. Further, since in vitro studies have suggested the role of BspA protein in inflammation, we sought to determine its potential in pathogenesis in the mouse model.


   MATERIALS & METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Bacterial Strains and Culture Conditions
T. forsythia (B. forsythus ATCC 43037) was grown in TF broth or on TF-agar plates (1.5% agar in TF broth) under anaerobic conditions as described previously (Sharma et al., 1998). A T. forsythia mutant (BFM571) defective in the expression of BspA protein (Honma et al., 2001) was grown in broth or agar plates containing tetracycline at 5 µg/mL concentration.

Inoculation of Mice
Specific-pathogen-free BALB/cByJ male mice (Jackson Laboratory, Bar Harbor, ME, USA) were maintained in the Laboratory Animal Facility of the University at Buffalo. The animal protocols were approved by the Institutional Animal Care and Use Committee. Animals within the group were age-matched (3 wks old at the start of the experiment; 8 mice per group) and quarantined for 1 wk prior to the experiment. Mice were first treated with sulfamethoxazole-trimethoprim (10 mL/L water; Sulfatrim, Goldline Laboratories, Ft. Lauderdale, FL, USA) for 10 days ad libitum, followed by a five-day antibiotic-free period (Baker et al., 1994). Mice were infected by gavage with 109 cfu/mL of live bacteria (T. forsythia wild-type Tf43037, or the mutant strain BFM571) in 100 µL of PBS with 2% carboxymethyl cellulose (CMC) 3 times at 48-hour intervals (Baker et al., 1994). Control (sham-infected) mice received antibiotic pre-treatment and the CMC gavage without the bacteria.

rBspA Purification and Immunization
Animals were immunized with full-length recombinant BspA protein (rBspA), prepared according to our previously described procedure for rBsp70, a truncated derivative expressing the leucine-rich-repeat domain of BspA (Sharma et al., 1998). The BspA-encoding DNA fragment was amplified from Tf430377 genomic DNA by PCR. The forward (5'-GCGCGGATCCTTGA CGACCCTGGGCGCTACGGC-3') and reverse (5'-CGCGGAA TTCTCACTTTATAAGAATTTTGGTTACCCG-3') primers contained BamHI and EcoRI restriction sites, respectively, to facilitate cloning into the corresponding sites of the pGEX-4T expression vector (Amersham Pharmacia Biotech, Piscataway, NJ, USA). For immunization, each animal received 0.1 mL of 20 µg/mL rBspA in TiterMax Gold adjuvant (CytRx Corporation, Norcross, GA, USA) divided into 2 portions injected subcutaneously into the scapular region. Three weeks later, the mice received the same antigen concentration subcutaneously without the adjuvant as a booster.

PCR Detection to Assess Infection
Subgingival plaque samples were obtained from the molars of each mouse by means of sterile paper points (Johnson & Johnson, Piscataway, NJ, USA). Briefly, paper points were placed subgingivally for 5 sec and then transferred into 1 mL T. forsythia growth medium supplemented with 100 µg/mL gentamycin, and vortexed for 10 sec. Following one week’s incubation, medium was spun at 12,000 x g for 10 min, and the total bacterial genomic DNA was isolated with use of the PureGene genomic DNA isolation kit (Gentra, Minneapolis, MN, USA). We performed PCR by taking 250 ng of genomic DNA using T. forsythia-specific primers as described previously (Sakamoto et al., 2001). The T. forsythia-specific primers (forward primer, 5'-GCGTATGTAACCTGCCCGCA-3'; reverse primer, 5'-TGCTTCAGTTCAGTTATACCT-3') amplify a 641-bp amplicon from the 16 rRNA gene. Similarly, a pair of ubiquitous bacterial primers that match almost all bacterial 16S RNA genes (forward, 5'-GTGCTGCAGAGAGTTTGATCATGCCTCAG-3'; reverse, 5'-CACGGATCCTACGGGTACCTTGTTACGACTT-3') was used as a positive control (amplicon length, 1.4 kb; Marchesi et al., 1998). This also served to indicate the presence of most bacteria in plaque samples. PCR products were analyzed by agarose (1%) gel electrophoresis and ethidium bromide staining.

Antibody Response
Forty-two days following the last infection, the mice were killed, and serum was collected and stored at –70°C for assessment of specific IgG responses. The amounts of specific IgG or IgA antibodies were determined by mouse IgG or IgA ELISA quantification kits (Bethyl Laboratories, Montgomery, TX, USA). Briefly, microtiter wells were coated with either formalin-fixed whole T. forsythia cells in 0.1 M NaHCO3, pH 9.6, at an absorbance (650 nm) of 0.10, or with the rBspA protein (1 µg/100 µL/well) as antigens. We measured the amount of IgG (or IgA) by establishing a mouse IgG (or IgA) standard curve on each plate in a capture ELISA format. Wells were incubated with serial dilutions of mouse serum for 1 hr at room temperature, washed with PBS containing 0.05% Tween-20 (PBST), and detected by horseradish-peroxidase-conjugated goat anti-mouse IgG (or goat anti-mouse IgA) and the TMB enzyme substrate reagent. We used the dilution of the serum that falls on the linear range of the antibody standard curve to determine the amount of specific antibody.

Lymphocyte Proliferative Response
The lymphoid cells were obtained from the spleens of mice (Kruisbeek, 1998). Briefly, we prepared single-cell suspensions by mincing and passing spleen tissue through a 30-µm mesh filter (Miltenyi Biotec, Auburn, CA, USA). Erythrocytes were subjected to lysis with ammonium chloride, and the dead cells were removed by centrifugation on a Lymphopaque density gradient medium (Accurate Chemicals, Westbury, NY, USA). Lymphocytes were cultured in 96-well flat-bottomed plates (Falcon) in quadruplicate at 5 x 105 cells/well in RPMI 1640 supplemented with 2 mM L-glutamine, 50 µg/mL gentamycin, and 10% FBS (complete medium). Cultures were incubated (37°C, 5% CO2) alone or with various concentrations of concanavalin A (ConA; 5 µg or 1 µg/mL) or rBspA (10 µg or 1 µg/mL) for 48 hrs (ConA) or 96 hrs (rBspA). Cultures were pulsed with 0.5 µCi/well [3H]thymidine (Amersham Corp., Arlington Heights, IL, USA) during the last 18 hrs of incubation. Cells were harvested onto glass fiber filters with a cell harvester, and the amount of tritiated thymidine incorporation was measured by liquid scintillation counting. The proliferative stimulation index (SI) was calculated as the mean level of radioactive thymidine uptake by cultures incubated with the stimulant, minus the mean level of uptake by the respective control culture with no stimulant, divided by the control uptake.

Alveolar Bone Measurements
Horizontal bone loss around the maxillary molars was assessed by a morphometric method (Evans et al., 1992; Baker et al., 1994). Boiled, de-fleshed skulls were immersed overnight in 3% hydrogen peroxide, pulsed for 1 min in bleach, and stained with 1% methylene blue. The distance from the cemento-enamel junction to the alveolar bone crest (referred to as CEJ:ABC) was measured in a blinded fashion by at least two individuals, using a calibrated dissecting microscope (x30), at 14 buccal sites per mouse.

SDS-PAGE and Western Blotting
The specific reactivity of serum IgG and IgA from mice to rBspA protein was assessed by Western immunoblotting as described previously (Sharma et al., 1998). The rBspA protein electroblotted onto nitrocellulose membrane was used as the antigen.

Statistics
Differences between groups were analyzed by the Student t test, and the one-way analysis of variance with multiple-group comparisons was done with Tukey’s test. A P value of less than 0.05 was considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Immune Response to T. forsythia and BspA Protein
We determined the serum antibody response against bacteria and the BspA protein, to confirm infection and determine if T. forsythia elicited an immune response. Both the wild-type (Group B) and the BFM571 BspA-mutant (Group C) strains induced significant levels of anti-whole-cell-specific antibodies following oral infection, compared with sham-infected controls (Fig. 1AGo). Only the wild-type T. forsythia strain induced anti-BspA antibodies (Group B; Fig. 1BGo), and, as expected, the animals infected with the BspA mutant (Group C; Fig. 1BGo) did not produce anti-BspA antibodies, confirming that the animals were successfully infected with the wild-type and the mutant strains, respectively, and that the anti-BspA response was seen only in animals infected with the wild-type strain. Subcutaneous immunization with rBspA protein induced a strong serum anti-rBspA IgG response (Fig. 1BGo). The serum IgG anti-rBspA response seen in the rBspA-immunized and T. forsythia 43037-infected group (Group D) was slightly higher than that in the rBspA-immunized group (Group E; Fig. 1BGo), although this increase was not statistically significant. In addition, anti-rBspA serum IgA was detected in the animals immunized with rBspA protein (groups D and E; Fig. 1CGo). This was also confirmed by Western immunoblotting (Fig. 2Go). BspA-specific serum IgA response was not detectable in animals that were infected with T. forsythia alone. A robust rBspA-specific serum antibody response following immunization suggests that BspA is a strong immunogen.



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  		Figure 1. Serum antibody levels in mice following infection/immunization. (A) Anti-whole-specific IgG, (B) rBspA-specific IgG, and (C) rBspA-specific serum IgA. Group A, control (sham-infected and sham-immunized); group B, T. forsythia 43037-infected; group C, BFM571-infected; group D, BspA-immunized and T. forsythia 43037-infected; group E, BspA-immunized. Bars represent mean ± standard error (n = 12).

 


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  		Figure 2. Detection of serum IgA by Western immunoblotting. The individual nitrocellulose strips electroblotted with rBspA protein following SDS-PAGE were probed with respective pooled mouse sera (1:100 dilution), followed by goat anti-IgA and color developed with TMB membrane reagent. 1, Group A, control; 2, Group B, T. forsythia- infected; 3, Group C, BFM-571-infected; 4, Group D, rBspA-immunized and T. forsythia-infected; and 5, Group E, rBspA-immunized.

 
Antigen-induced Proliferative Response
Proliferative responses to rBspA were detected in splenic lymphoid cell cultures from mice immunized with the rBspA protein (Fig. 3AGo). Maximum stimulation was seen with 10 µg/mL rBspA protein. Proliferative response was also seen in mice infected with T. forsythia. This response, however, was not as robust as that seen in rBspA-immunized mice. In comparison, no stimulation was observed in mice infected with BFM571 mutant strain or in mice that were sham-infected. Cells from each of the groups of mice responded to polyclonal T-cell activator ConA (Fig. 3BGo).



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  		Figure 3. Proliferative responses of splenic lymphoid cells derived from mice (12 per group) to rBspA (A) and to ConA (B). Cultures were performed in quadruplicate, and the results are expressed as means of the stimulation index, determined as described in the text. Group A, control (sham-infected and sham-immunized); group B, T. forsythia-43037 infected; group C, BFM571-infected; group D, rBspA-immunized and T. forsythia 43037-infected; and group E, rBspA-immunized. **P < 0.005 and *P < 0.05, values significantly higher compared with group A.

 
T. forsythia Colonization
All mice in each group gave a predicted 1.4-kb PCR product with the ubiquitous primer pair, confirming the presence of most bacteria in these conventional animals. In contrast, mice that gave a 620-bp product with the T. forsythia-specific primer pair were considered positively infected. Based on PCR results (data not shown), T. forsythia was recovered from the oral cavities of 58% of the mice infected with the wild-type strain, 60% of mice infected with the mutant strain, and 57% of mice immunized with rBspA and infected with the wild-type strain. T. forsythia was not recovered from sham-infected (group A) or sham-infected/BspA-immunized mice.

Assessment of Alveolar Bone Loss
Alveolar bone loss was seen at all sites in animals infected with T. forsythia 43037 (Fig. 4AGo; sites 1, 2, & 3 on first molars, sites 4 & 5 on second molars, and sites 6 & 7 on third molars) when compared with the same sites in sham-infected animals. No significant bone loss was observed at any of the same sites in mice infected with BspA-mutant BFM 571 (Fig. 4BGo) compared with sham-infected animals. For comparison of the total bone resorption in each group, the average of 14-site total CEJ:ABC distance of animals in each group was determined. The bone loss per site for each group was then calculated and plotted. The results of this transformation are summarized in Fig. 4CGo. The mean CEJ:ABC distance, a measure of bone loss, was greater in the T. forsythia-infected group as compared with the sham-infected group. No bone loss was seen in the BFM571 BspA-mutant-infected group, nor was there significant alveolar bone loss in the rBspA-immunized and T. forsythia 43037-infected group compared with sham-infected controls, indicating that anti-BspA response is protective against T. forsythia-induced alveolar bone loss.



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  		Figure 4. (A,B) Bone loss after infection with T. forsythia in BALB/cByJ mice. Sites 1, 2, & 3 are on first molars, sites 4 & 5 are on second molars, and sites 6 & 7 are on third molars. L, left; R, right. Data points represent the mean ± SEM from 8 mice. (A) Comparison between the T. forsythia 43037 (Tf43037)-infected and sham-infected mice. The CEJ:ABC was greater in Tf43037-infected mice than in sham-infected mice at every site, indicating bone loss. (B) Comparison between the mutant BFM571- and sham-infected mice. (C) Comparison of total horizontal bone loss between groups calculated as the average of 14-site total CEJ-ABC distance for each group. Data represented as the millimeter bone loss per site per group. Significant T. forsythia 43037-induced alveolar bone loss was observed as compared with that in sham-infected controls. Bone loss in mice infected with the mutant strain BFM571 was not significantly different from that in sham-infected mice. Immunization with rBspA protein significantly reduced T. forsythia 43037-induced bone loss in mice. *Values significantly greater than in sham-infected controls (P < 0.05). P = NS, not significant compared with controls.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
We sought to assess the pathogenesis of T. forsythia in an animal model and to determine the in vivo pathogenic potential of its BspA protein, a cell-surface-associated and a secreted leucine-rich-repeat protein, by utilizing a BspA mutant recently constructed in our laboratory. Our results showed that the mutant was significantly impaired in its ability to induce alveolar bone loss in mice, as compared with the wild-type strain. The putative role of BspA in pathogenesis was further corroborated from results showing protection from T. forsythia-induced alveolar bone loss by rBspA immunization, which resulted in BspA-specific serum IgG and IgA antibodies. We found similar percentages of mice to be positive for T. forsythia by PCR when infected with either the wild-type or the mutant strain, supporting the notion that the BspA protein might not be crucial in adherence. Moreover, in each group of mice, neither the antibody levels nor the bone levels were significantly different in those infected mice from which T. forsythia was detected by PCR, compared with infected mice from which it was not. In addition, T. forsythia was recovered from mice that were protected from T. forsythia-induced alveolar bone loss following the rBspA immunization. Therefore, BspA might be an important virulence factor specifically involved in inflammation by triggering release of bone-resorbing cytokines, IL-ß and TNF-{alpha}, and/or the activation of chemokines involved in neutrophil recruitment, leading to inflammation and bone resorption. Additionally, the BspA protein may promote osteolysis via its other (yet to be identified) virulence functions, especially since proteins belonging to the leucine-rich-repeat family have diverse functions. Future studies, such as in TLR-2 knock-out mice, could assist in determining if, indeed, in vivo cytokine-inducing activity of the BspA is responsible for T. forsythia-induced alveolar bone loss. Our results also showed that BspA is an immunogenic protein, since a robust antibody response was elicited following immunization. In support of this notion, periodontitis patients harboring T. forsythia also elicit BspA-specific serum antibodies (Sharma et al., 1998). We also noted that, in gnotobiotic rats, T. forsythia did not colonize or induce alveolar bone loss (unpublished results). Studies in a wound chamber model in rabbits (Takemoto et al., 1997) and in the murine skin abscess model (Bird et al., 2001; Yoneda et al., 2001) have demonstrated that the pathogenic potential of T. forsythia is dependent on other bacteria. Therefore, it appears that the resident flora of the mouse oral cavity in conventional mice likely aid in pathogenesis via promoting colonization/growth and virulence of T. forsythia.

In conclusion, these studies have demonstrated, for the first time, the virulence of T. forsythia in the mouse model of periodontal disease and further support the notion that the BspA protein plays an important role in bacterial virulence.


   ACKNOWLEDGMENTS
 
This study was supported by Public Health Service grant DE014749 from the National Institute of Dental and Craniofacial Research.

Received June 15, 2004; Last revision December 9, 2004; Accepted February 11, 2005


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 ABSTRACT
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 MATERIALS & METHODS
 RESULTS
 DISCUSSION
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