INFECTION AND IMMUNITY, Sept. 2006, p. 5023–5028 0019-9567/06/$08.00⫹0 doi:10.1128/IAI.00062-06 Copyright © 2006, American Society for Microbiology. All Rights Reserved.
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Porphyromonas gingivalis Vesicles Enhance Attachment, and the Leucine-Rich Repeat BspA Protein Is Required for Invasion of Epithelial Cells by “Tannerella forsythia” Satoru Inagaki, Shinsuke Onishi, Howard K. Kuramitsu, and Ashu Sharma* Department of Oral Biology, School of Dental Medicine, State University of New York at Buffalo, Buffalo, New York 14214 Received 12 January 2006/Returned for modification 13 April 2006/Accepted 13 June 2006
The human oral cavity harbors more than 500 species of bacteria. Periodontitis, a bacterially induced inflammatory disease that leads to tooth loss, is believed to result from infection by a select group of gram-negative periodontopathogens that includes Porphyromonas gingivalis, Treponema denticola, and “Tannerella forsythia” (opinion on name change from Tannerella forsythensis pending; formerly Bacteroides forsythus). Epithelial cell invasion by periodontopathogens is considered to be an important virulence mechanism for evasion of the host defense responses. Further, the epithelial cells with invading bacteria also serve as reservoirs important in recurrent infections. The present study was therefore undertaken to address the epithelial cell adherence and invasion properties of T. forsythia and the role of the cell surface-associated protein BspA in these processes. Further, we were interested in determining if P. gingivalis, one of the pathogens frequently found associated in disease, or its outer membrane vesicles (OMVs) could modulate the epithelial cell adherence and invasion abilities of T. forsythia. Here we show that epithelial cell attachment and invasion by T. forsythia are dependent on the BspA protein. In addition, P. gingivalis or its OMVs enhance the attachment and invasion of T. forsythia to epithelial cells. Thus, interactions between these two bacteria may play important roles in virulence by promoting host cell attachment and invasion. with T. forsythia in disease, or outer membrane vesicles (OMVs) produced by this bacterium could modulate the epithelial cell adherence and invasion ability of T. forsythia. A large number of gram-negative bacteria, including P. gingivalis, have an ability to extracellularly release membrane vesicles (7, 20, 44). These vesicles retain full components of outer membrane constituents of the cell wall including proteins, lipopolysaccharide, muramic acid, capsule, and fimbriae (44). The P. gingivalis OMVs released into the environment are thought to play important roles in periodontal disease by serving as vehicles for toxin and enzyme spreading as well as being involved in the adherence and aggregation of bacteria (1, 6, 20). P. gingivalis OMVs have also been shown to be potent activators of adhesion (16) and aggregation for other oral bacteria (15). P. gingivalis OMVs have been shown to aggregate host platelets (33) as well as to invade epithelial cells in vitro (13, 41). To our knowledge there are no reports on the effects of P. gingivalis or its OMVs on T. forsythia virulence relative to epithelial cell attachment and invasion. The BspA protein has been shown to possess multiple biological activities in vitro, such as fibronectin and fibrinogen binding (34), coaggregation activity with other oral bacteria (12, 32), and the ability to induce secretion of proinflammatory cytokines and chemokines from host cells (9, 28). Recently, we have also shown that the T. forsythia-induced alveolar bone loss in mice requires expression of the BspA protein (31). The BspA protein (34) belongs to the leucine-rich repeat (LRR) protein family (17). In addition, a BLAST search of the BspA protein identified two bacterial immunoglobulin-like (Big_2) domains in the C-terminal region. Both the LRR and Big_2 domains have been shown to be involved in a wide variety of receptor-ligand interactions involving bacterial proteins and their host receptors. For example, the LRR domains of Listeria
Among the more than 500 species identified in the human oral cavity, gram-negative organisms of the “red complex”— Porphyromonas gingivalis, Treponema denticola, and “Tannerella forsythia” (opinion on name change from Tannerella forsythensis pending; formerly Bacteroides forsythus)—are recognized as potential periodontal pathogens in the progression of periodontitis (26, 36). In addition, T. forsythia has been directly implicated in the development of periodontal diseases (7, 8, 36, 37, 39). In spite of clinical evidence for the association of T. forsythia with disease, there has been a lack of progress on the understanding of virulence mechanisms of this organism. This is partly attributed to difficulties in the propagation of the organism as well as to the lack of genetic systems for constructing random mutants of this organism. A few studies have suggested epithelial cell invasion by T. forsythia as a plausible virulence mechanism (10, 29, 30). The mechanisms by which T. forsythia adheres to or invades epithelial cells are poorly understood. Recently, Rudney et al. (29) have identified T. forsythia as one of the members of the polymicrobial flora invading buccal epithelial cells taken directly from the mouth. It has been suggested that buccal epithelial cells provide a protected environment in vivo and a reservoir for recolonization of the gingival crevice. The present study was therefore undertaken to address the epithelial cell adherence and invasion abilities of T. forsythia and the role of the cell surface-associated protein BspA in these properties. Further, we were interested in determining if P. gingivalis, one of the pathogens frequently found associated * Corresponding author. Mailing address: Department of Oral Biology, School of Dental Medicine, 211 Foster Hall, University at Buffalo, State University of New York, Buffalo, NY 14214. Phone: (716) 829-2759. Fax: (716) 829-3942. E-mail:
[email protected]. 5023
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monocytogenes cell surface-associated internalins (InlA and InlB) promote adherence and entry into host cells via binding to their respective receptors (22, 23, 35), the Shigella flexneri IpaH protein containing a LRR domain facilitates bacterial escape from the phagocytic vacuole (3), and Samonella enterica proteins (SlrP, SspHI, and SspH2) with leucine-rich repeat signatures have been shown to be important for host adaptation and colonization (40). Recently we have also shown that the Treponema denticola leucine-rich repeat protein LrrA plays a role in the attachment and penetration of human epithelial cells (12). Likewise, the Big_2 domains are present in bacterial cell surface intimins, the bacterial adhesins of enteropathogenic and enterohemorrhagic strains of Escherichia coli. The E. coli intimins/invasins are responsible for bacterial attachment to epithelial cells (4, 5, 21). So far, analyses of tissue attachment and invasion by periodontal bacteria have focused mainly on P. gingivalis and Actinobacillus actinomycetemcomitans (2, 18, 19, 24, 25, 27). Since periodontal diseases result from complex interactions of multiple microorganisms, it is essential to investigate the interaction between different periodontal bacteria and epithelial cells. In this study, we show that epithelial cell attachment and invasion by T. forsythia are dependent on the BspA protein. In addition, P. gingivalis or its OMVs enhance the attachment of T. forsythia to epithelial cells in a BspA-independent manner. Thus, interactions between these two bacteria may play roles in virulence by promoting host cell attachment and invasion. MATERIALS AND METHODS Bacterial strains and culture conditions. T. forsythia ATCC 43037 was grown anaerobically (5% CO2, 10% H2, 85% N2) in brain heart infusion (Difco Laboratories, Detroit, MI) broth containing 5 g/ml hemin, 0.5 g/ml menadione, 0.001% N-acetylmuramic acid, and 5% fetal bovine serum (Life Technologies, Grand Island, NY). The BspA-deficient mutant BFM571 (11) was grown anaerobically in the same medium containing 5 g/ml tetracycline. Preparation of P. gingivalis OMVs. OMVs were isolated from P. gingivalis 381 essentially as previously described by Kadurugamuwa and Beveridge (14). Briefly, bacterial cultures in early-stationary phase were centrifuged at 6,000 ⫻ g for 15 min. The supernatant fluids were passed through 0.22-m-pore-size filters (Millipore Corp., Bedford, MA) to remove residual bacteria. OMVs were collected from the filtrate by centrifugation in a L8-70M ultracentrifuge (Beckman, Palo Alto, CA) at 150,000 ⫻ g for 3 h at 4°C, resuspended with phosphatebuffered saline (PBS), and stored at ⫺70°C. Aggregation assay. Aggregation was evaluated by the turbidimetric method as described previously (32). The aggregation was monitored at 37°C by measuring the decrease in absorbance at 600 nm in PBS. Epithelial cell attachment and invasion assays. Epithelial cells (KB cell line) were maintained in Dulbecco’s modified Eagle medium (Gibco, Buffalo, NY) supplemented with 10% fetal bovine serum and 50 g/ml of gentamicin. The cultures were incubated at 37°C under 5% CO2. KB cells, originally thought to be derived from an epidermal carcinoma of the mouth, have now been shown to be derived from HeLa cell cultures as a contaminant (American Type Culture Collection, Manassas, VA). KB cells were grown to near-confluence (⬃95%) for the assays. Bacteria were added at a multiplicity of infection of 100 and incubated under 5% CO2 at 37°C. For the studies described here, we added 107 bacteria to 105 KB cells attached as monolayers. For attachment assays, KB cell monolayers were incubated for 4 h with T. forsythia cells alone or in the presence of P. gingivalis OMVs (5 g/ml). The monolayers were then washed gently with PBS four times. The cells were lysed with water, and the amount of total cell-associated T. forsythia was estimated by CFU counts on blood agar plates. Invasion assays were conducted as previously described (10). For invasion assays, the bacteria were incubated with the monolayers for 4 h. For mixed infections of KB cells with P. gingivalis and T. forsythia, each bacterium was used at a multiplicity of infection of 100 and monolayers were incubated for 4 h as described above. Both bacteria were mixed and incubated for 10 min before
INFECT. IMMUN. addition to monolayers. In some experiments, T. forsythia cells were mixed with P. gingivalis vesicles (final concentration, 5 g/ml) and incubated for 15 min at room temperature before addition on to KB cell monolayers. Following incubation, KB cell monolayers were washed twice with sterile PBS, and fresh medium containing gentamicin (300 mg/ml) and metronidazole (200 mg/ml; Sigma) was added for an additional 1 h to kill extracellular bacteria. The monolayers were rinsed again with PBS before being lysed with sterile water. Internalized bacteria were enumerated on agar plates by CFU counting. Control experiments showed that bacterial viability was not affected by the water treatment during cell lysis. Multiplication of the bacteria in the tissue culture medium during the assay was minimal. Therefore, unless otherwise indicated, the level of invasion was expressed as the percentage of bacteria retrieved following cell lysis relative to the total number of bacteria initially added. All experiments were performed in duplicate or triplicate and repeated at least three times. The role of the BspA protein in adherence and invasion was also examined by preincubating T. forsythia cells with heat-inactivated (56°C for 30 min) rabbit antisera raised against the BspA protein prior to the addition to KB cells. T. forsythia cells (109 cells/ml) were resuspended in PBS containing a 1:100 dilution of the anti-BspA serum or a control preimmune serum for 1 h anaerobically. Antibody-bound cells were washed twice with PBS and used for the attachment and invasion assays described above. In addition, purified recombinant BspA (rBspA) protein (31) was used in the assays as a competitive inhibitor. Briefly, the KB cells were preincubated with increasing concentrations of rBspA protein or bovine serum albumin (BSA) (control) at 25, 50, 100, and 200 g/ml for 1 h at 37°C before addition of wild-type T. forsythia. Determination of bacterial entry by a double-labeling technique. To differentiate attached bacteria from internalized bacteria, the double-fluorescence technique described below was used. Briefly, KB cells were grown on coverslips in six-well tissue culture plates and infected with T. forsythia under the conditions described above. Coverslips were fixed in 3% paraformaldehyde (Electron Microscopy Sciences, Washington, PA) for 10 min, washed in PBS, and then incubated with a rabbit anti-T. forsythia antiserum diluted 1:500 in PBS–0.5% BSA for 60 min. Following incubation, coverslips were washed three times with PBS and incubated with Alexa Fluor 488 (green fluorescent dye)-conjugated goat anti-rabbit immunoglobulin G (Molecular Probes, Eugene, OR) diluted 1:500 for 30 min to visualize attached bacteria. Internalized bacteria were then stained by first permeabilizing KB cells by dipping coverslips in 0.4% Triton X-100 solution for 5 min and then staining with the rabbit anti-T. forsythia antiserum followed by Alexa Fluor 568 (red fluorescent dye)-coupled goat anti-rabbit immunoglobulin G (Molecular Probes) diluted 1:500 as described above. Actin filaments were stained with Alexa Fluor 647 (blue fluorescent dye) conjugated to phalloidin (Molecular Probes) for 30 min according to the manufacturer’s recommendations to visualize the cellular cytoskeleton and confirm internalization. Coverslips mounted in a mounting medium were examined by confocal scanning laser microscopy (CSLM) at the Confocal Microscopy and 3-D Imaging Facility (School of Medicine and Biomedical Sciences, University at Buffalo) using a Bio-Rad MRC1024 confocal scanning laser (Kr/Ar) microscope attached to a Nikon Diaphot microscope and a Plan Apo 60⫻ (numerical aperture, 1.4) objective. Statistical analysis. Differences between groups were determined by analysis of variance by using InStat (San Diego, Calif.) software. For all comparisons, a P value of ⬍0.05 was considered significant.
RESULTS Aggregation assay. T. forsythia ATCC 43037 showed increased coaggregation in the presence of P. gingivalis or P. gingivalis OMVs compared to that for T. forsythia ATCC 43037 alone (Fig. 1). There were no significant differences between P. gingivalis- or P. gingivalis OMV-induced aggregations of T. forsythia ATCC 43037 and those of the BspA-defective mutant BFM571. These results thus indicated that the BspA protein is not involved in the aggregation with the P. gingivalis cell surface component mediating coaggregation. Tissue attachment and invasion assays. T. forsythia ATCC 43037 and its BspA-defective mutant BFM571 adhered to KB cells to different degrees. T. forsythia ATCC 43037 showed binding activity as much as threefold higher than that of BFM571 (Fig. 2). This suggests that although BspA is involved
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FIG. 1. Coaggregation of T. forsythia in the presence of P. gingivalis and outer membrane vesicles purified from P. gingivalis. Data are representative of three independent experiments. Data points represent means ⫾ standard errors of triplicate readings. Tf, T. forsythia 43037; Pg, P. gingivalis 381; BFM571, BspA-defective mutant of T. forsythia; Pgv, P. gingivalis 381 OMVs.
in epithelial cell binding, other adhesins may also be involved. The results of attachment assays also demonstrated that P. gingivalis OMVs increased the attachment ability of T. forsythia ATCC 43037 and T. forsythia BFM571 to KB epithelial cells significantly. In the presence of P. gingivalis OMVs, attachment of T. forsythia ATCC 43037 was increased fourfold. The presence of a polyclonal rabbit anti-BspA serum resulted in a statistically significant decrease in the attachment of T. forsythia to KB cells when the antiserum was diluted 1/100 or 1/50 (Fig. 3). A preimmune serum used at similar dilutions as a negative control did not inhibit attachment of bacteria to KB cells (Fig. 3). In the competitive assays, various concentrations (0, 5, 25, and 50 g/ml) of purified rBspA protein were preincubated with KB cells prior to the addition of bacteria. Bovine serum albumin at similar concentrations was used as a control. The results of a competitive inhibition assay showed a statis-
FIG. 2. Attachment of T. forsythia 43037 (Tf) and BFM571 to KB cells in the absence and presence of P. gingivalis OMVs (Pgv). Attachment levels (means ⫾ standard errors) were expressed as the percentage of attached bacteria relative to the total number of input bacteria. ⴱⴱ and ⴱ, P ⬍ 0.01 and P ⬍ 0.05, respectively, for comparison to T. forsythia 43037; #, P ⬍ 0.05 for comparison to BFM571.
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FIG. 3. Inhibition of the attachment of T. forsythia to KB cells by preincubation with various concentrations of a rabbit polyclonal serum raised against recombinant BspA (stippled bars) or with a control preimmune serum (solid bars). Results were reported as mean (⫾ standard error of the mean) bacterial attachment to the host cells. The attachment value for T. forsythia in the absence of antibody was normalized to 100% and was used as the control to derive the other attachment values. ⴱ, P ⬍ 0.05 for comparison between groups.
tically significant inhibition (P ⬍ 0.001) of attachment of T. forsythia when rBspA was used at a concentration of 50 g/ml (32% ⫾ 4.6% inhibition) compared to that with the BSA control at a similar concentration (2.3% ⫾ 1.6% inhibition). The results of invasion assays showed that while T. forsythia 43037 invaded KB cells (approximately 1.5% invasion based on input bacteria), the BFM571 mutant was significantly attenuated (0.022% invasion). In the presence of P. gingivalis OMVs, the apparent invasion by T. forsythia increased significantly (P ⬍ 0.05) compared to invasion in the absence of OMVs (Fig. 4). The invasion ability of the mutant did not increase significantly in the presence of the vesicles (Fig. 4). Taken together, the results demonstrate that invasion is dependent on
FIG. 4. Invasion by T. forsythia (Tf) of KB cells and dependence of invasion on the presence of P. gingivalis vesicles (Pgv). The invasion level was expressed as the percentage of bacteria retrieved following antibiotic killing of external bacteria and KB cell lysis relative to the total number of input bacteria. Results reported as invasion levels (means ⫾ standard errors) are representative of three independent experiments. ⴱ, P ⬍ 0.05 for comparison with T. forsythia 43037 invasion; ns, not significant (P ⬎ 0.05) for comparison with BFM571 invasion.
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the BspA protein and that P. gingivalis vesicles enhance epithelial cell attachment, which then potentiates BspA-dependent invasion processes. The enhanced binding and invasion abilities of T. forsythia observed in the presence of P. gingivalis vesicles were also observed in the presence of P. gingivalis cells (Fig. 4). The role of BspA in invasion was corroborated by results that showed an approximately 98% inhibition of invasion when bacteria were pretreated with polyclonal rabbit antiBspA sera diluted to 1/100 compared to invasion in the absence of antibody. Preimmune serum at identical dilutions, used as a negative control, did not inhibit attachment of T forsythia to KB cells (data not shown). Determination of bacterial entry by the double-labeling technique. CSLM following dual labeling of extracellular and intracellular bacteria was performed to verify the attachment and invasion of bacteria in KB cells. The results (Fig. 5) confirm KB cell invasion by wild-type T. forsythia cells. Extracellular bacteria appear green or yellow, intracellular bacteria are labeled red, and the cell cytoskeleton is labeled blue. The frequency of T. forsythia bacteria attached to or invading epithelial cells increased in the presence of coaggregating P. gingivalis 381 or vesicles prepared from this strain. The invasion observed with CSLM correlated well with the results of the invasion assays (data not shown). Host cell components involved in invasion. To dissect the biochemical pathways involved in T. forsythia invasion, a group of metabolic inhibitors was utilized (Table 1). The results indicated that changes in the F-actin and perhaps microtubule cytoskeletons are needed for efficient entry. In addition, internalization may require activity of one or more host tyrosine or threonine/serine kinases, and de novo host protein synthesis does not appear to be essential. The reasons for partial inhibition with staurosporine are currently unknown, especially since we do not yet know the specific molecules involved and whether this inhibitor at the concentrations used fully or partially impaired their activities. These molecules could be involved in signaling pathways upstream of actin and tubulin polymerization. Alternatively, it is also likely that the staurosporine-sensitive factors could act independently of cytoskeletal changes and may be involved in alternative pathways of bacterial entry. Thus, invasion of KB cells by T. forsythia requires multiple host cellular components involved in cytoskeleton changes, intracellular signaling, and energy metabolism.
FIG. 5. Attachment and invasion of T. forsythia into KB cells visualized by confocal scanning laser microscopy following dual labeling. T. forsythia 43037 organisms invading KB cells were stained red (indicated by red arrows), while outside bacteria were detected as green or yellow cocci. The KB cell cytoskeleton stained with phalloidin appeared blue. (A) T. forsythia 43037; (B) T. forsythia 43037 in the presence of P. gingivalis OMVs; (C) the T. forsythia BspA mutant BFM571.
INFECT. IMMUN. TABLE 1. Effects of metabolic inhibitors on invasion by T. forsythia 43037 Inhibitor (concn)a
Target
None Cytochalasin D (1 g/ml) Nocodazole (2.5 g/ml) Staurosporine (0.2 M) Cycloheximide (100 g/ml) Sodium azide (50 mM)
Actin Microtubule Protein kinase Protein synthesis Energy metabolism
Invasion of KB cells (%)b
100 ⫾ 13.5 7.7 ⫾ 3.3* 27.7 ⫾ 9.9* 42.6 ⫾ 7.7* 98.6 ⫾ 12.6NS 12.7 ⫾ 4.7*
a The inhibitors were preincubated with KB cells at the indicated concentrations. b Expressed as a percentage of the control level without any inhibitor (mean ⫾ standard deviation). *, P ⬍ 0.01 compared to control (no inhibitor); NS, not significantly different from control.
DISCUSSION In this study we have confirmed the role of the BspA protein in attachment and invasion of epithelial cells by T. forsythia. We have also demonstrated that coaggregating partners P. gingivalis 381 and its OMVs promote the ability of T. forsythia to attach to and invade epithelial cells. Further, the BspA protein appeared not to be involved in coaggregation with either the P. gingivalis cells or their OMVs. The wild-type T. forsythia strain ATCC 43037 and its BspA-defective mutant adhered to KB cells to significantly different degrees. The attachment levels of the mutant BFM571 were about two- to threefold lower than those of T. forsythia ATCC 43037. The results, suggesting a potential role for BspA in epithelial cell attachment and colonization, also indicate that additional adhesins may also be involved in adherence. Since the BspA protein belongs to the LRR domain family as well as the Big_2 domain family, involved in receptor-ligand recognition via protein-protein interactions, it is possible that BspA interacts with a specific epithelial cell surface receptor for adherence and invasion. Previous studies have shown that P. gingivalis OMVs are potent activators of adhesion and aggregation (15, 16) for other gram-positive bacteria. Our investigations of the interactions between P. gingivalis and T. forsythia have shown that the attachment abilities of T. forsythia ATCC 43037 and its BspA mutant were increased in the presence of P. gingivalis vesicles, supporting the notion that synergy exists between these two species with respect to adherence and colonization. Previous reports have analyzed synergistic effects of animal infection between these two species (42, 43); however, the mechanisms responsible are poorly understood. Although OMVs released into the environment are thought to play important roles in periodontal disease by serving as vehicles for toxin and enzyme spreading as well as being involved in the adherence of other oral bacteria (6, 7), to our knowledge there are no reports on the effects of P. gingivalis or its vesicles on T. forsythia virulence relative to epithelial cell attachment and invasion. P. gingivalis OMVs have been shown to be potent activators of adhesion (16) and aggregation for other oral bacteria (15). Here we show that in the presence of P. gingivalis OMVs, T. forsythia ATCC 43037 was significantly more invasive. This also appears to be the first report demonstrating the enhancement of epithelial cell invasion by T. forsythia due to coaggregation of T. forsythia with P. gingivalis and its OMVs.
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The BspA protein does not appear to be involved in coaggregation with P. gingivalis. This is in contrast to aggregation of T. forsythia with Fusobacterium nucleatum, which appears to be BspA dependent (32). Therefore, surface proteins of P. gingivalis are likely involved in coaggregation with a T. forsythia surface protein other than BspA and/or with carbohydrate components. This notion is supported by our observation that vesicles prepared from the P. gingivalis MT10 mutant (Arggingipain A protease mutant), showing reduced expression of cell surface proteins (38), showed reduced ability compared to vesicles from the wild-type strain to promote attachment and invasion by T. forsythia cells (S. Inagaki, unpublished results). The vesicles from the parent strain P. gingivalis 381 likely retain a full complement of outer membrane constituents of the cell surface, including proteins, lipopolysaccharide, capsule, and fimbriae, whereas the MT10 vesicles are primarily devoid of RgpA protease and type I fimbriae (38). Further, heat treatment of P. gingivalis vesicles significantly abrogated their activity with respect to T. forsythia attachment (Inagaki, unpublished). The fact that the BspA mutant was defective in invasion of epithelial cells implied that a specific epithelial cell surface receptor might be involved in interaction with the BspA protein, leading to induction of downstream eukaryotic signaling pathways and bacterial invasion. The receptor binding region may involve the N-terminal LRR domain and/or the C-terminal immunoglobulin-like (Ig-SF) domain of BspA protein. Based on studies described here, future studies will now address whether T. forsythia and P. gingivalis exist as coaggregating partners during the invasion process and synergize with respect to invasion or whether invasion by one species occurs independently of the other. In addition, the role of a bspA homolog (gene ID TF1843) identified in the recently completed draft sequence of the T. forsythia genome (www.oralgen .lanl.gov/) in epithelial cell attachment will be determined. The N-terminal region of the homolog shows only weak similarity with the N-terminal LRR region of BspA. On the other hand, the C-terminal regions of the two proteins containing immunoglobulin-like Big_2 domains are identical. Since the BspA mutant is completely attenuated in invasion but retains some attachment activity, we hypothesize that the homolog plays roles in activities other than invasion. In summary, we have demonstrated that T. forsythia is able to adhere to and invade epithelial cells. Invasion by T. forsythia requires expression of the cell surface-associated protein BspA. Further, the invasiveness of T. forsythia ATCC 43037 is significantly enhanced in the presence of P. gingivalis or OMVs derived from it. Therefore, the BspA protein may represent an important virulence factor of T. forsythia with multifunctional activities involved in bacterial pathogenesis, and the mixed P. gingivalis and T. forsythia infections may exacerbate disease pathogenesis. ACKNOWLEDGMENTS We thank Keith Ireton at the University of Toronto, Toronto, Ontario, Canada, for critical reading of the manuscript and valuable advice during the course of the studies. We also thank Wade Sigurdson of the Imaging and Confocal Facility of the University at Buffalo for expert help with confocal microscopy and imaging. This study was supported by National Institutes of Health grant DE014749.
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