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Infection and Immunity, May 2007, p. 2484-2492, Vol. 75, No. 5
0019-9567/07/$08.00+0     doi:10.1128/IAI.02004-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Flow Cytometric Analysis of Adherence of Porphyromonas gingivalis to Oral Epithelial Cells{triangledown}

Rishi D. Pathirana,1 Neil M. O'Brien-Simpson,1 Kumar Visvanathan,2 John A. Hamilton,2 and Eric C. Reynolds1*

Cooperative Research Centre for Oral Health Science, School of Dental Science, and the Bio21 Institute of Molecular Science and Biotechnology, The University of Melbourne,1 Cooperative Research Centre for Chronic Inflammatory Diseases, Department of Medicine, Royal Melbourne Hospital, The University of Melbourne, Melbourne, Victoria, Australia2

Received 21 December 2006/ Returned for modification 24 January 2007/ Accepted 27 February 2007


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
By using fluorescence microscopy, fluorescently labeled Porphyromonas gingivalis W50 was shown to adhere to oral epithelial (KB) cells as discrete cells or small cell aggregates, whereas P. gingivalis ATCC 33277 bound as large cell aggregates. Flow cytometric analysis showed that for P. gingivalis W50 there was a logarithmic relationship between the bacterial cell ratio (BCR), that is the number of bacterial cells to KB cells, and the percentage of KB cells with W50 cells attached. This percentage of KB cells with W50 attached reached a plateau of ~84% cells at a BCR of 500:1. In contrast, a quadratic relationship was observed between BCR and the percentage of KB cells with P. gingivalis ATCC 33277 attached, reaching a maximum of 47% at a BCR of 100:1 but decreasing to 7% at a BCR of 1,000:1. The lower binding of ATCC 33277 at high cell concentrations was attributed to autoaggregation. P. gingivalis W50 cells treated with an inhibitor (N{alpha}-p-tosyl-L-lysine chloromethyl ketone [TLCK]) of its RgpA-Kgp proteinase-adhesin complex exhibited significantly reduced binding to KB cells than to untreated cells, suggesting a role for proteinase activity in binding to KB cells. Competitive inhibition with purified proteinase-active and TLCK-inactivated RgpA-Kgp complex significantly decreased the adherence of P. gingivalis W50 cells to KB cells. Furthermore, isogenic mutants of P. gingivalis W50 lacking the kgp gene product, but not the rgpA or rgpB gene products, exhibited significantly decreased adherence to KB cells compared to the wild type.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Chronic periodontitis is an inflammatory disease of the supporting tissues of the teeth associated with specific bacteria. In the United States, it has been estimated that chronic periodontitis affects around 30% of the adult dentate population (24) with an economic burden of $14.3 billion (4). Furthermore, chronic periodontitis has been linked with other health problems, such as cardiovascular diseases (3, 16, 41), diabetes (28), spontaneous preterm birth and low birth weight (23), rheumatoid arthritis (15), and respiratory infections (32).

A consortium of oral pathogens, including Tanerella forsythia (formerly Bacteroides forsythus), Treponema denticola, and Porphyromonas gingivalis, has been strongly associated with chronic periodontitis (34). Among these, P. gingivalis has been the most studied (13). P. gingivalis has been shown to induce disease in animal models of periodontitis (2, 7, 26), and the pathogenicity of the bacterium has been attributed to a number of virulence factors, among them the Arg- and Lys-specific proteinases and their associated adhesins, designated the RgpA-Kgp proteinase-adhesin complex (RgpA-Kgp complex). The proteinases of the complex have been reported to be major virulence factors, as they are able to degrade a range of host proteins and dysregulate host defense mechanisms (22). The RgpA-Kgp complex has also been reported to play a role in the adherence of P. gingivalis to a variety of host surfaces, including extracellular matrix proteins and hemoglobin (21, 27).

The specific adherence of pathogenic bacteria, such as P. gingivalis, to oral epithelial cells may be an important step in the colonization of the periodontal pocket and in the initiation and progression of chronic periodontitis (14). A positive correlation has been reported between the number of bacteria attached to epithelial cells obtained from the marginal gingiva and the severity of inflammation (37). Previous studies analyzing the adherence of P. gingivalis to cultured oral epithelial cells have reported heterogeneity in the adhesive and invasive abilities of different strains of P. gingivalis (6, 31, 40). By using radiolabeled bacteria, strains of P. gingivalis that tend to autoaggregate in cell suspension, such as ATCC 33277 and 381, were shown to bind well to oral epithelial cells, whereas W50- or W83-like strains, which have a low ability to autoaggregate, bound poorly (31, 40). Watanabe et al. (40) reported that radiolabeled cells of P. gingivalis strains 381 and ATCC 33277 bound to epithelial cells, whereas cells of W50 and W83 strains did not show significant binding. Sandros et al. (31) also reported that radiolabeled cells of P. gingivalis strain 381 exhibited significantly higher binding (28.8% retained radioactivity) to cultured and epithelial cells compared with cells of strain W50 (2.6% retained radioactivity). Furthermore, using a colony counting assay, Duncan et al. (6) reported that approximately 10% of P. gingivalis ATCC 33277 cells (CFU) bound to the epithelial cell monolayer, whereas less than 0.024% of W50 cells adhered. These findings have led to strains ATCC 33277 and 381 being classified as adherent, whereas strains W50 and W83 were described as nonadherent. This classification does not appear to be consistent with the virulence of these strains, as P. gingivalis strains W50 and W83 have been described as virulent/invasive strains and strains 381 and ATCC 33277 have been described as noninvasive/less virulent on the basis of their ability to cause ulcerative spreading lesions distant from the injection site in animal models (17, 38). Furthermore, analysis of plaque samples from 311 patients revealed that W50/W83-like strains were more closely associated with clinical measures of chronic periodontitis than other strains, including 381-like strains (9). This could suggest that adherence to oral epithelial cells is not a virulence-associated characteristic or that the methodologies used to measure adherence underestimated the binding of the W50 and W83 strains.

In this study we describe a method based on dual-fluorescence flow cytometry for the accurate measurement of the adherence of P. gingivalis strains W50 and ATCC 33277 to oral epithelial (KB) cell monolayers in vitro. Further, we evaluated the roles played by the RgpA and Kgp proteinases and their associated adhesins in the adherence of P. gingivalis W50 to KB cells. The results presented indicate that P. gingivalis W50 does bind to cultured oral epithelial cells and that the Kgp proteinase plays a major role in this process.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Bacterial strains and growth conditions. Lyophilized cultures of P. gingivalis ATCC 33277 and W50 wild-type strain and isogenic mutants rgpA (W501), kgp (W50KIA), rgpB (W50D7) and rgpA rgpB (W50AB) were obtained from the culture collection of the Cooperative Research Centre for Oral Health Science, The University of Melbourne, Melbourne, Victoria, Australia, and have been described before (20, 39). The rgpA rgpB kgp triple mutant (W50ABK) was generated for this study (see below). Bacteria were maintained in an anaerobic chamber (MK3 anaerobic workstation; Don Whitley Scientific Ltd., Shipley, England) at 37°C on horse blood agar plates supplemented with 10% (vol/vol) lysed horse blood. Bacterial colonies were used to inoculate brain heart infusion medium containing 5 µg/ml of hemin and 0.5 µg/ml of cysteine; for growth of the rgpA (W501), rgpB (W50D7) and kgp (W50KIA) P. gingivalis W50 isogenic mutants, the medium also contained 10 µg/ml erythromycin (20). For the rgpA rgpB (W50AB) isogenic mutant, the medium also contained 1 µg/ml tetracycline and 10 µg/ml chloramphenicol. Escherichia coli strain JM109 was grown aerobically in Luria broth at 37°C. Batch culture growth was monitored at 650 nm using a spectrophotometer (model 295E; Perkin Elmer). Bacterial cells were enumerated by plating on horse blood agar, and this was performed for every individual strain. Culture purity was routinely checked by Gram staining and by colony morphology.

Generation of the P. gingivalis rgpA rgpB kgp isogenic mutant (W50ABK). The P. gingivalis W50 isogenic mutant (W50ABK) lacking RgpA, RgpB, and Kgp was generated using the rgpA rgpB isogenic mutant (W50AB) and pNS1 (1) containing kgp insertionally disrupted with an ErmF/AM cassette. The kgp::ermA insert of pNS1 was amplified by PCR and electroporated into P. gingivalis W50AB to generate W50ABK. Disruption of kgp in strain W50ABK was confirmed by Southern blot analysis whereby chromosomal DNA was probed with a 2.1-kb KpnI-BamHI ErmF/AM cassette and with a 3.3-kb BamHI fragment from pNS1 encoding the catalytic domain of Kgp (33). Whole-cell assays of W50ABK using Arg and Lys chromogenic substrates showed that the mutant was devoid of RgpA/B and Kgp proteolytic activity (20).

P. gingivalis whole-cell Arg-specific and Lys-specific proteinase assays. The Arg-specific and Lys-specific proteolytic activity of P. gingivalis W50 and ATCC 33277 whole cells were measured as described previously by O'Brien-Simpson et al. (20). P. gingivalis cells were harvested at mid-exponential phase under anaerobic conditions by centrifuging the cells at 7,000 x g for 20 min at 4°C, washing the cells, and resuspending them in 1 ml of PG buffer (50 mM Tris-HCl, 150 mM NaCl, 5 mM CaCl2, and 5 mM cysteine, pH 8.0). The cells were immediately analyzed for Arg-specific and Lys-specific proteolytic activity using N-{alpha}-benzoyl-L-Arg-p-nitroanilide (Bz-Arg-pNA) (Sigma, New South Wales, Australia) and benzyloxycarbonyl-L-Lys-p-nitroanilide (Bz-Lys-pNA) (Novabiochem, New South Wales, Australia), respectively, as previously described (20). Amidolytic activity is expressed as micromoles of substrate converted per minute at 37°C.

Oral epithelial (KB) cell culture. The human oral epithelial cell line KB (ATCC CCL 17; American Type Culture Collection), derived from a human oral epidermoid carcinoma was maintained as frozen stocks and cultured in Earl's minimum essential medium (EMEM) (JRH Biosciences, Victoria, Australia) supplemented with 25 mM L-glutamine, 10% (vol/vol) heat-inactivated fetal calf serum (JRH Biosciences, Victoria, Australia), 100 IU/ml penicillin/streptomycin (JRH Biosciences, Victoria, Australia), and 30 µg/ml gentamicin (JRH Biosciences, Victoria, Australia) in a 5% CO2 atmosphere at 37°C. Cell passage was performed twice a week, and the KB cells used in experiments were never older than 12 passages.

FITC labeling of P. gingivalis and E. coli. P. gingivalis strains ATCC 33277 and W50 and the rgpA, kgp, rgpB, rgpA rgpB, and rgpA rgpB kgp proteinase isogenic mutants and E. coli were grown to mid-exponential phase as described above. The bacterial concentrations were determined spectrophotometrically according to a specific standard curve and confirmed retrospectively by counting viable cell colonies plated on blood agar for P. gingivalis and on LB plates for E. coli. The bacteria were harvested by centrifugation at 7,000 x g for 20 min at 4°C and washed once in phosphate-buffered saline (PBS), pH 7.4. In order to select an optimal concentration for bacterial labeling, P. gingivalis strains ATCC 33277 and W50 and E. coli (2.5 x 109/ml) were resuspended in 0.5 M NaHCO3, pH 8.0, containing different concentrations of fluorescein isothiocyanate (FITC) (Invitrogen, New South Wales, Australia) and incubated for 30 min at 37°C in an anaerobic chamber with constant stirring. FITC concentrations of 0.15 mg/ml for wild-type P. gingivalis W50 and the proteinase mutants as well as E. coli and 0.015 mg/ml for P. gingivalis ATCC 33277 were determined as optimal, as they yielded homogenous labeling and a suitable staining intensity (i.e., no significant difference in fluorescence intensity between each bacterial strain). These FITC concentrations were used in all subsequent experiments. After incubation with FITC, the bacteria were pelleted at 7,000 x g for 5 min, washed three times with PBS to remove unbound FITC, and resuspended in EMEM supplemented with 1% (vol/vol) L-glutamine and 25 mM HEPES. FITC labeling of P. gingivalis cells was found not to reduce cell viability or the Arg-X-specific or Lys-X-specific proteinase activity (data not shown).

Adherence of FITC-labeled P. gingivalis to KB cells. KB cells were transferred to 24-well plates and were grown to near confluence (~95%) at a density of approximately 106 cells per well using the culture conditions described above. Immediately prior to incubation with P. gingivalis strains ATCC 33277, W50, proteinase isogenic mutants, or E. coli, the cell culture medium was removed, and the KB cell monolayers were washed twice with sterile PBS. FITC-labeled bacteria were prepared as described above. Immediately prior to incubation with epithelial cells, the optical density of the bacterial solution was monitored at 650 nm, and the bacterial concentration was adjusted to 2.5 x 109 bacteria/ml by diluting the bacteria in EMEM supplemented with 1% (vol/vol) glutamine and 25 mM HEPES. To determine the incubation period needed to obtain maximal adherence, 200-µl aliquots of FITC-labeled bacteria (5 x 108 bacterial cells; bacterial cell ratio [BCR] of 500:1 [this ratio represents the ratio of the number of bacterial cells to the number of KB cells]) were added to wells containing KB cell monolayers. The cell culture plates were then centrifuged at 800 x g for 5 min at room temperature and incubated for 15 min, 30 min, 60 min, and 90 min at 37°C in the anaerobic chamber (Don Whitley Scientific Ltd., Shipley, England). A 90-min incubation period was sufficient to obtain maximal adherence and used in all subsequent experiments. To compare the abilities of P. gingivalis strains W50 and ATCC 33277 to bind to KB cells, FITC-labeled P. gingivalis W50 and ATCC 33277 were incubated with KB cell monolayers at BCRs of 10:1, 50:1, 100:1, 500:1, and 1,000:1 for 30 min at 37°C. To determine the roles of Arg-specific and Lys-specific proteinases and their associated adhesins in adherence of P. gingivalis W50 to KB cells, isogenic mutants of P. gingivalis W50 lacking either rgpA, rgpB, kgp, rgpA rgpB, or rgpA rgpB kgp genes were incubated with epithelial cell monolayers at a BCR of 500:1 for 30 min at 37°C. Following incubation, the supernatants containing unbound bacteria and detached KB cells were removed from the wells and collected into 1-ml tubes. The remaining KB cells were then detached from the wells by incubating with a trypsin-EDTA mixture (300 µl/well) (JRH Biosciences, Victoria, Australia) for 5 min at 37°C. The detached KB cells were collected and pooled with the corresponding collected supernatants, which contained the previously detached KB cells and the unbound bacteria. The KB cells were then centrifuged at 400 x g for 5 min at room temperature and washed twice in PBS to remove the unbound bacteria. After the KB cells were washed, they were resuspended in 300 µl of PBS and 5 µl of either phycoerythrin-conjugated anti-CD29 immunoglobulin G (IgG) antibodies (CD29-PE) (BD Pharmingen) or the phycoerythrin-conjugated isotype-matched antibody control (PE-mouse IgG1 {kappa} chain; BD Pharmingen) and incubated for 30 min on ice. CD29 (ß-integrin) was chosen, as it is found on epithelial cells and is not efficiently cleaved by P. gingivalis Arg- and Lys-specific proteolytic activities (35). Following incubation, the KB cells were centrifuged at 800 x g for 5 min at room temperature, washed twice in PBS, and resuspended in 500 µl of fixative solution (PBS containing 1% [vol/vol] formalin) and stored at 4°C until analyzed by flow cytometry within 24 h.

Competitive inhibition of P. gingivalis adherence to KB cells by RgpA-Kgp proteinase-adhesin complex. The abilities of both proteinase-active and inactivated RgpA-Kgp complexes to inhibit FITC-labeled P. gingivalis W50 adherence to KB cells were analyzed. The RgpA-Kgp proteinase-adhesin complex was purified from P. gingivalis W50 by the method of Pathirana et al. (25). The Arg- and Lys-specific proteinase activity of the purified complex was inactivated by incubating with 5 mM of the proteinase inhibitor N{alpha}-p-tosyl-L-lysine chloromethyl ketone (TLCK) (Sigma, New South Wales, Australia) for 30 min at 37°C. The excess TLCK was removed by using a PD-10 desalting column and equilibrated with TC50 buffer (50 mM NaCl, 5 mM CaCl2, 5 mM Tris-HCl, pH 7.4), and the proteinase-inhibited RgpA-Kgp complex was eluted with TC50 buffer according to the manufacturer's instructions (Amersham Pharmacia Biotech, Uppsala, Sweden). FITC-labeled P. gingivalis W50 cells (5 x 108 cells) were mixed with either 6.25, 12.5, 25, or 50 µg/ml of proteinase-active or inactivated RgpA-Kgp complex and immediately added to wells containing KB cell monolayers at a BCR of 500:1, and the adherence assay was performed as described above.

Treatment of P. gingivalis W50 cells with TLCK. The adherence of TLCK-treated P. gingivalis W50 whole cells to KB cell monolayers was also studied. FITC-labeled P. gingivalis W50 cells were incubated with 5 mM TLCK for 30 min at 37°C in the anaerobic chamber with consistent mixing. After the incubation, the bacteria were centrifuged at 7,000 x g for 5 min at room temperature and the excess TLCK was removed by washing the bacteria twice in PBS (7,000 x g, 5 min, room temperature). The bacterial pellet was resuspended in EMEM containing 1% (vol/vol) glutamine and 25 mM HEPES at a concentration of 2.5 x 109 P. gingivalis/ml, and the adherence assay was performed as described above.

Fluorescence microscopy. The adherence of P. gingivalis strains W50 and ATCC 33277 to KB cells was visualized using a fluorescence microscope (model L5; Leica, New South Wales Australia). A 10-µl drop of the sample was added onto mounting fluid (Aqua PolyMount; PolySciences, PA) placed on a glass slide and overlaid with a coverslip. Adherence of FITC-labeled bacteria was examined using a blue excitation filter (excitation wavelength, 450 to 464 nm; emission wavelength, 550 to 554 nm) with an oil immersion objective (x100 magnification).

Flow cytometric analysis of FITC-labeled P. gingivalis and E. coli adherence to KB cells. The adherence of FITC-labeled P. gingivalis to KB cells stained with CD29-PE was analyzed using a FACSCaliber flow cytometer (Becton Dickinson, San Jose, CA) equipped with an argon laser operating at an excitation wavelength of 488 to 610 nm. The fluorescence from PE was measured through a 575-nm filter (FL2), and the green emission of FITC was measured with a 525-nm filter (FL1). The multiparametric data were analyzed using CellQuest software (Becton Dickinson, San Jose, CA). Forward and side scatter properties were used to acquire a total of 10,000 KB cells and to gate out the cell debris. KB cells were then specifically identified by gating for PE fluorescence (FL2). KB cells that had FITC-labeled P. gingivalis attached were identified by gating on the FITC fluorescence (FL1) within the gated PE region. All measurements were done in duplicate, and for quantitation of FITC fluorescence, mean fluorescence intensity (MFI) values were used.

Adherence of [3H]thymidine-labeled P. gingivalis cells to oral epithelial cells. The adherence of P. gingivalis to epithelial cells was also studied by using the [3H]thymidine-labeled P. gingivalis protocol described previously by Sandros et al. (30). Briefly, P. gingivalis W50 and ATCC 33277 colonies grown on blood agar plates were used to inoculate 5 ml of brain heart infusion medium containing 5 µg/ml of hemin, 0.5 µg/ml of cysteine, 5 µg/ml vitamin K (for strain ATCC 33277), and 15 µCi/ml [3H]thymidine (Amersham Biosciences, New South Wales, Australia). The bacteria were grown overnight to late exponential phase. The radiolabeled bacteria were harvested by centrifugation at 7,000 x g for 5 min at 4°C, washed three times with PBS, and resuspended in adhesion buffer (50 mM K2HPO4, 1 mM CaCl2, 0.1 mM MgCl2, pH 6.5) at a concentration of 2.5 x 109/ml. KB cells grown to near confluence (~95%) in a 24-well plate as described above were washed three times in sterile PBS and incubated with 200 µl of the bacterial suspension (BCR of 500:1). The plates were incubated for 30 min at 37°C in an anaerobic chamber (Don Whitley Scientific Ltd., Shipley, England). Following the incubation, the bacterial suspension was removed, and the KB cell monolayers were washed three times with sterile PBS. The remaining attached epithelial cells were then detached from the wells after incubating with 500 µl/well of tissue solubilizer (NCS-II; Amersham Biosciences, New South Wales, Australia) for 20 min. The tissue solubilizer was neutralized by the addition of glacial acetic acid (24 µl/well) and transferred into vials containing 5 ml of counting scintillant (Amersham Biosciences, New South Wales, Australia). The level of [3H]thymidine was measured on a Wallac microbeta scintillation counter (Perkin Elmer, New South Wales, Australia), and the data are presented as percentages (mean and standard deviation) of retained radioactivity from two independent experiments.

Statistical analysis. The Arg- and Lys-specific proteolytic activities of P gingivalis W50 and ATCC 33277 cells and the retained radioactivity of [3H]thymidine-labeled P gingivalis W50 and ATCC 33277 bound to KB cells were analyzed by t test using SPSS software. The FITC MFI values of KB cells incubated with FITC-labeled wild-type P. gingivalis or isogenic mutants lacking either rgpA, rgpB, kgp, rgpA rgpB, or rgpA rgpB kgp genes were analyzed using a one-way classification analysis of variance and SPSS software (19). Regression analysis of the percentage of KB cells with P. gingivalis strains W50 and ATCC 33277 attached and inhibition of P. gingivalis W50 binding to KB cells by RgpA-Kgp complex was performed using SPSS software. Effect sizes, represented as Cohen's d were calculated using the effect size calculator provided online by the Evidence-Based Education UK website at http://www.cemcenter.org/renderpage.asp?linkID=30325017 and were used for post hoc comparisons. According to Cohen (5), a small effect size is 0.2 ≤ d < 0.5, a moderate effect size is 0.5 ≤ d < 0.8, and a large effect size is d ≥ 0.8.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Adherence of [3H]thymidine-labeled P. gingivalis to oral epithelial (KB) cells. The adherence of P. gingivalis W50 and ATCC 33277 to KB cell monolayers was initially analyzed using the radiolabeled cell method originally described by Sandros et al. (30). The percentage of retained radioactivity found using this method was significantly (P < 0.001) higher for P. gingivalis ATCC 33277 (65.898% ± 5.285%) than for W50 (0.348% ± 0.098%). However, it was observed during the assay that KB cell monolayers incubated with P. gingivalis strain W50 became detached from the plate surface, whereas the majority of the KB cell monolayers incubated with P. gingivalis strain ATCC 33277 remained adherent. We found that removing the supernatant and washing the remaining adherent cells resulted in the loss of >95% of the KB cells incubated with P. gingivalis W50, whereas there was little loss of epithelial cells from the plate incubated with P. gingivalis ATCC 33277.

As KB cell monolayers are typically detached from tissue culture plates by the addition of trypsin, the Arg- and Lys-specific trypsin-like proteolytic activities of whole cells of P. gingivalis strains ATCC 33277 and W50 were determined. P. gingivalis W50 had a significantly (P < 0.01) higher (7.8-fold) Lys-X-specific activity (1.25 ± 0.1 µmol of substrate converted per min at 37°C per 1011 cells) than ATCC 33277 did (0.16 ± 0.08 µmol of substrate converted/min at 37°C/1011 cells), whereas P. gingivalis ATCC 33277 strain had a significantly (P < 0.05) higher (~twofold) Arg-X-specific activity (17.04 ± 1.41 µmol of substrate converted/min at 37°C/1011 cells) than W50 did (8.39 ± 0.16 µmol of substrate converted/min at 37°C/1011 cells). Further, as the W50 kgp isogenic mutant did not detach the KB cells, we attributed the loss of the KB cells from the culture plate to the greater Lys-specific extracellular proteolytic activity of strain W50. However, regardless of the cause of the KB cell detachment, it was clear that this method of measuring bacterial cell adherence relying on KB cell attachment was not appropriate for investigating adherence of P. gingivalis W50. We therefore developed a method based on flow cytometry to measure bacterial adherence to KB cells. All subsequent assays were based on this method.

Flow cytometric analysis of bacterial adherence to KB cells. Initially, fluorescence microscopy was used to visualize the adherence of P. gingivalis W50 and ATCC 33277 to KB cells (Fig. 1). FITC-labeled P. gingivalis W50 was found to adhere to KB cells either as discrete cells or small aggregates of cells (Fig. 1A and C), whereas P. gingivalis strain ATCC 33277 bound to KB cells, predominantly as large aggregates of cells (Fig. 1B and D). FITC-labeled E. coli was found not to adhere to KB cells (data not shown).


Figure 1
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  		FIG. 1. Fluorescence microscopy of FITC-labeled P. gingivalis binding to oral epithelial (KB) cells. (A and B) Fluorescence microscopic images of KB cells with FITC-labeled P. gingivalis W50 (A) and FITC-labeled P. gingivalis ATCC 33277 (B) attached. (C and D) Light microscopic images of the same epithelial cell with bound P. gingivalis W50 (C) and P. gingivalis ATCC 33277 (D). The magnification in all panels is x100.

 
The quantification of adherence of FITC-labeled P. gingivalis to KB cells was analyzed by flow cytometry. After the incubation of KB cells with FITC-labeled bacteria, supernatants consisting of nonadhered bacteria and detached KB cells were collected and then combined with the remaining KB cells (from the respective wells) detached from the plate using trypsin-EDTA. The KB cells were then washed three times using centrifugation to remove the nonadhering bacteria. The KB cells were then labeled with PE-conjugated anti-CD29 antibody or isotype-matched IgG-PE control, and after the cells were washed, bacterial adherence was analyzed by flow cytometry. Figure 2 shows the adherence of P. gingivalis strain W50 to KB cells as determined by flow cytometry. Cell debris, unbound bacteria, and cell clumps were excluded from analysis by forward and side scatter gating (gate 1 [G1] [Fig. 2A]). KB cells labeled with CD29-PE antibody were identified by gating on the PE fluorescence (G2 [Fig. 2B]) that was above the PE fluorescence obtained from KB cells stained with the isotype-matched IgG-PE control (peak 1 [Fig. 2B]). It was found that 93.4% of the KB cell population was labeled with CD29-PE antibody (peak 2 [Fig. 2B]) and 5.6% of the KB cell population was positive for IgG-PE isotype control (peak 1 [Fig. 2B]). Only CD29-PE-positive KB cells were used to determine bacterial adherence, both in terms of the percentage of KB cells with FITC-labeled P. gingivalis attached and the relative amount of P. gingivalis attached as determined by the mean fluorescence intensity of FITC fluorescence. The KB cells with FITC-labeled P. gingivalis attached were identified by gating on FITC fluorescence (G3 [Fig. 2C]) above the limit of autofluorescence of CD29-PE-positive KB cells incubated with non-FITC-labeled P. gingivalis (peak 3 [Fig. 2C]) and FITC fluorescence of nonadherent FITC-labeled E. coli coeluting with CD29-PE-positive KB cells (peak 4 [Fig. 2C]). It was found that 96.6% of CD29-PE-positive KB cells had FITC-labeled P. gingivalis attached (peak 5 [Fig. 2C]) and that there was 1.8% coelution of CD29-PE-positive KB cells with the nonadherent FITC-labeled E. coli. These flow cytometric controls and settings were used for all subsequent experiments to determine P. gingivalis adherence to epithelial cells.


Figure 2
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  		FIG. 2. Development of a flow cytometric method to analyze bacterial adherence to oral epithelial (KB) cells. FITC-labeled bacteria were incubated with KB cells for 30 min at a BCR of 500:1, harvested, washed, and stained with CD29-PE. (A) Live epithelial cells were gated according to forward and side scatter properties (G1). (B) KB cells were stained with negative isotype IgG1-PE control (peak 1) or CD29-PE (peak 2). KB cells with bacteria attached were analyzed by gating on the FITC fluorescence (G3) within the gated PE region (G2). (C) Peak 3 represents autofluorescence of KB cells incubated with non-FITC-labeled P. gingivalis W50. Peak 4 represents FITC fluorescence of KB cells with E. coli strain JM109, a nonadherent gram-negative bacteria control. Peak 5 represents FITC fluorescence of KB cells with P. gingivalis W50 attached. A total of 10,000 CD29-PE-positive events were obtained, and experiments were performed in duplicate.

 
Adherence of P. gingivalis strains W50 and ATCC 33277 to oral epithelial (KB) cells. The adherence of P. gingivalis strain W50 to KB cells was compared to that of strain ATCC 33277 by flow cytometry as described above. Figure 3A shows that there is an increase in FITC MFI of KB cells incubated with increasing numbers of P. gingivalis W50 cells. Regression analysis demonstrated that there was a significant (P < 0.0002) positive linear relationship (r = 0.995) between FITC MFI values and the cell numbers of P. gingivalis W50 incubated with KB cells (Fig. 3A). Furthermore, the percentage of KB cells with P. gingivalis W50 attached increased with a corresponding increase in the bacterial cell ratio (Fig. 3A). The percentage of KB cells with P. gingivalis W50 attached reached a maximum of 84.4% ± 5.6% at a BCR of 500:1 and continued to plateau at a BCR of 1,000:1. Regression analysis showed that there was a significant (P < 0.007) logarithmic relationship (r = 0.937) between the percentage of KB cells with P. gingivalis W50 attached and BCR.


Figure 3
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  		FIG. 3. Adherence of FITC-labeled P. gingivalis to oral epithelial (KB) cell monolayers. Confluent KB cell monolayers were incubated with P. gingivalis W50 (A) and P. gingivalis ATCC 33277 (B) at BCRs of 10:1, 50:1,100:1, 500:1, and 1,000:1 for 90 min at 37°C, and adherence was analyzed by flow cytometry. The results are presented as the percentages of KB cells with attached P. gingivalis (black bars) and the FITC fluorescence emitted from the attached P. gingivalis as indicated by the MFI ± standard deviation (white bars). A total of 10,000 CD29-PE-positive events were obtained, and experiments were performed in duplicate.

 
Figure 3B shows that there is a corresponding increase in FITC MFI values of KB cells incubated with increasing numbers of FITC-labeled P. gingivalis ATCC 33277 cells. Regression analysis showed that there was a significant (P < 0.012) positive linear relationship (r = 0.908) between FITC MFI values and BCR. A dose-dependent increase in the percentage of KB cells with P. gingivalis ATCC 33277 attached was observed until reaching a maximum of 47.3% ± 7.1% at a BCR of 100:1. However, in contrast to strain W50, the percentage of KB cells with P. gingivalis ATCC 33277 attached decreased at a BCR of 500:1 and continued to decrease, reaching 7.5% ± 3.5% at a BCR of 1,000:1. Regression analysis showed that there was a significant (P < 0.041) quadratic (inverted U) relationship (r = 0.959) between the percentage of KB cells with P. gingivalis ATCC 33277 attached and BCR (Fig. 3B).

Adherence of P. gingivalis W50 and rgpA, rgpB, kgp, rgpA rgpB, and rgpA rgpB kgp isogenic mutants to oral epithelial (KB) cells. The ability of wild-type P. gingivalis W50 to adhere to KB cells was compared to those of P. gingivalis W50 isogenic mutants lacking a proteinase gene(s) (rgpA, rgpB, kgp, rgpA rgpB, and rgpA rgpB kgp). Figure 4 shows that 97.5% ± 0.6% of KB cells had wild-type P. gingivalis W50 attached with a FITC MFI value of 117.2 ± 5.3. Both the percentage of KB cells with bacteria attached (99.6% ± 0.1%; P < 0.05) and the FITC MFI value (185.8 ± 6.0; P < 0.01) were significantly higher in KB cells incubated with rgpA isogenic mutant than in wild-type P. gingivalis W50. In contrast, the percentages of KB cells with the kgp (84.4% ± 2.6%) and rgpA rgpB kgp (88.8% ± 2.7%) isogenic mutants attached were significantly (P < 0.05) decreased compared to percentages with wild-type P. gingivalis W50. Further, the FITC MFI values were significantly lower (P < 0.01) in KB cells incubated with either the kgp (64.2 ± 4.3) or rgpA rgpB kgp (62.9 ± 6.2) isogenic mutants than with wild-type P. gingivalis W50. No significant difference was observed between the rgpB and rgpA rgpB isogenic mutants and wild-type P. gingivalis W50 with respect to either the percentage of epithelial cells with bacteria attached or FITC MFI value.


Figure 4
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  		FIG. 4. Adherence of wild-type P. gingivalis W50 and rgpA, rgpB, kgp, rgpA rgpB, and rgpA rgpB kgp isogenic mutants to oral epithelial (KB) cell monolayers. KB cell monolayers were incubated with wild-type P. gingivalis W50 and each of the isogenic mutant strains at a BCR of 500:1, and adherence was measured by flow cytometry. Values that are significantly different from the value for wild-type P. gingivalis are indicated by asterisks. The results are presented as a percentage of KB cells with attached P. gingivalis (black bars) and the FITC fluorescence emitted from the attached P. gingivalis as indicated by the MFI ± standard deviation (white bars). A total of 10,000 CD29-PE-positive events were obtained, and experiments were performed in duplicate.

 
Role of RgpA-Kgp proteinase-adhesin complex in adherence of P. gingivalis W50 to oral epithelial (KB) cells. The contribution of the Arg- and Lys-specific proteinase activity to binding of P. gingivalis strain W50 to KB cells was investigated by pretreatment of P. gingivalis W50 whole cells with the proteinase inhibitor TLCK. The FITC MFI value of KB cells incubated with TLCK-treated P. gingivalis W50 was significantly smaller (P < 0.01) than the value of KB cells incubated with non-TLCK-treated P. gingivalis W50 cells (Fig. 5A).


Figure 5
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  		FIG. 5. Inhibition of binding of P. gingivalis W50 to oral epithelial (KB) cells. (A) Adherence of proteinase-active (Figure 5) and inactivated (Figure 5) P. gingivalis W50 (BCR 500:1) to KB cells as measured by flow cytometry. The value for the TLCK-treated group was significantly different (P < 0.01) from the value for the control group (proteinase-active P. gingivalis W50). (B) Inhibition of P. gingivalis W50 adherence to KB cells by the RgpA-Kgp complex. Confluent KB cell monolayers were incubated with FITC-labeled P. gingivalis W50 (BCR of 500:1) in the presence of either 50 µg/ml, 25 µg/ml, 12.5 µg/ml, or 6.25 µg/ml of either TLCK-treated (white bars) or non-TLCK-treated (black bars) RgpA-Kgp complex. The MFI values were statistically analyzed using effect size (Cohen's d). Values that were significantly different from the value for the control group incubated with no RgpA-Kgp complex are indicated by asterisks. All results are expressed as FITC fluorescence as indicated by the geometric mean plus standard deviation of duplicate samples. A total of 10,000 CD29-PE-positive events were obtained for each experiment.

 
To further investigate the role of the RgpA-Kgp complex in P. gingivalis binding to KB cells, assays were performed where KB cell monolayers were incubated with P. gingivalis W50 (BCR of 500:1) in the presence of increasing concentrations of either proteinase-active or proteinase-inactive (TLCK-treated) purified RgpA-Kgp complex (Fig. 5B). Increasing concentrations of both proteinase-active and -inactive RgpA-Kgp complex were found to significantly (P < 0.05 and P < 0.01, respectively) reduce the number of P. gingivalis W50 cells bound to KB cells. The proteinase-active RgpA-Kgp complex significantly (P < 0.05, P < 0.01) inhibited binding of P. gingivalis W50 cells to KB cells at concentrations of ≥12.5 µg/ml, whereas proteinase-inactive RgpA-Kgp complex significantly (P < 0.05) inhibited binding only at a concentration of 50 µg/ml. Regression analysis showed that there was a significant (P < 0.0349) logarithmic relationship (r = 0.931) between the concentration of proteinase-active RgpA-Kgp complex used to inhibit binding and the number of P. gingivalis W50 cells bound to KB cells. Similarly, regression analysis of the inhibition of binding induced by increasing concentrations of proteinase-inactive RgpA-Kgp complex also showed a significant (P < 0.0334) logarithmic relationship (r = 0.934).


   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
A number of studies have investigated the adherence of P. gingivalis to cultured oral epithelial cells using radiolabeled bacteria or colony counting assays (10, 12, 18, 29, 30, 40). Results from these studies suggest heterogeneity in the ability of different strains of P. gingivalis to adhere to cultured oral epithelial cells, where ATCC 33277-like strains have been reported to bind strongly and W50-like strains are described as nonadherent (6, 31, 40). In the current study, the adherence of P. gingivalis strains W50 and ATCC 33277 to cultured oral epithelial (KB) cells was initially studied using a radioactivity assay as described by Sandros et al. (31). Consistent with earlier findings reported by Sandros et al. (31), we observed a significantly (P < 0.001) greater level of retained radioactivity for P. gingivalis ATCC 33277 (65.898% ± 5.285%) than for P. gingivalis W50 (0.348% ± 0.098%), suggesting that P. gingivalis W50 strain does not adhere to KB cells. However, this radioactive method underestimates the adherence of P. gingivalis W50-like strains to cultured oral epithelial cells, as we observed that incubation of KB cells with P. gingivalis strain W50 resulted in detachment of the cell monolayers from the culture plates with more than 95% of the KB cells being lost through the method's washing steps. This detachment may be the result of P. gingivalis W50 possessing very high extracellular Lys-specific proteolytic activity, as we have shown that P. gingivalis W50 exhibits an 7.8-fold-greater Lys-specific proteolytic activity than that of the ATCC 33277 strain and that the W50 kgp isogenic mutant did not detach the KB cells.

In an approach to investigate adherence of P. gingivalis W50 to cultured epithelial cells, we developed a flow cytometric method. Initial experiments using fluorescence microscopy showed that P. gingivalis strain W50 binds to KB cells as either discrete cells or very small aggregates of cells, whereas strain ATCC 33277 bound only as large aggregates of cells. Using the developed flow cytometry method, the percentage of KB cells with adherent bacteria and the relative amount of bacteria attached to KB cells (FITC MFI value) were determined. For both W50 and ATCC 33277 strains, a significant positive relationship was observed between BCR and FITC MFI values. However, significant differences were observed between the two P. gingivalis strains with regards to the percentage of KB cells with bacteria attached. The binding of P. gingivalis W50 to KB cells was found to follow a logarithmic relationship where the percentage of KB cells with bacteria attached reached a maximum of 84% at a BCR of 500:1 and remained constant at higher BCRs. This plateauing effect of the percentage of KB cells with P. gingivalis W50 attached is most likely attributable to the number of KB cell binding sites (receptors) available for bacterial binding being limiting and unavailable on a proportion of the KB cell population. The relationship observed for binding to KB cells may be a result of P. gingivalis W50 binding to KB cells as either very small aggregates or as discrete cells due to its low autoaggregation ability. In contrast to P. gingivalis W50, binding of strain ATCC 33277 was found to have a quadratic (inverted U) relationship possibly as a consequence of this strain having a high autoaggregation ability. The percentage of KB cells with P. gingivalis ATCC 33277 attached was found to reach a maximum of 47% at a BCR of 100:1 and then decreased to 7% at a BCR of 1,000:1. This quadratic relationship in binding of P. gingivalis ATCC 33277 to KB cells may in part be explained by the tendency of this strain to autoaggregate such that ATCC 33277 at high cell concentrations autoaggregate in preference to binding to the KB cells. Notwithstanding this tendency to autoaggregate and the binding to a smaller number of KB cells, incubation with strain ATCC 33277 resulted in higher FITC MFI values than incubation with strain W50 did. This would have resulted from large aggregates of P. gingivalis ATCC 33277 cells attached to epithelial cells emitting a stronger FITC fluorescence than the FITC fluorescence emitted from either discrete cells or small aggregates of P. gingivalis strain W50 cells. These results therefore show that P. gingivalis W50 binding as discrete cells or small cell aggregates adheres to a significantly greater percentage of epithelial cells (>80%) than strain ATCC 33277 does (<50%) and that autoaggregation may be a characteristic that could limit adherence of ATCC 33277 at high bacterial cell concentrations.

The ability of P. gingivalis W50 to adhere to a greater percentage of oral epithelial cells than strain ATCC 33277 may have implications in virulence. Previous studies in the mouse lesion model have shown that P. gingivalis ATCC 33277 is noninvasive, inducing localized abscesses around the inoculation site, whereas P. gingivalis strains W50 and W83 at the same infective dose are considered invasive, inducing secondary lesions distal from the injection site (17). Furthermore, it has been reported that P. gingivalis W50/W83-like strains, but not ATCC 33277-like strains, are more closely associated with severe periodontitis in humans (9). Considered together, these results suggest that P. gingivalis W50 and W50-like strains may be more virulent because they do not autoaggregate and possess the ability to adhere more effectively to host cells and invade deeper into the subjacent tissues.

The RgpA-Kgp proteinase-adhesin complex is a major virulence factor of P. gingivalis, and in this study we have shown it to play a major role in the adherence of P. gingivalis W50 to KB cells. Inhibition of Arg- and Lys-specific proteinase activity on whole cells by using a proteinase inhibitor (TLCK) significantly reduced the adherence of P. gingivalis W50 cells to KB cells. This suggests that the Arg- and/or Lys-specific proteinase activity plays a critical role in effective adherence of P. gingivalis W50 to KB cells. Furthermore, proteinase-active RgpA-Kgp complex inhibited P. gingivalis W50 binding to KB cells in a dose-dependent manner, whereas the proteinase-inactive (TLCK-treated) RgpA-Kgp complex, while still inhibiting binding at high concentrations, displayed less of an inhibitory effect. Considered together, this suggests that the proteinase(s) mediates adherence through the exposure of hidden or latent binding sites on the KB cell surface by proteolytic cleavage to which the RgpA-Kgp complex can then adhere through the adhesin domains, which are in close association with the proteinases in the form of a noncovalently associated complex. This concept of latent binding sites is similar to that originally described by Gibbons et al. (8) and then later by Kontani et al. (11) who showed that proteolytic cleavage of human matrix proteins increased the binding of P. gingivalis cells. Competitive inhibition of binding of strain W50 to KB cells with the proteinase-inactive (TLCK-treated) RgpA-Kgp complex clearly showed that binding was not via the proteinase active site of the complex and was consistent with binding being mediated by the adhesin domains of the RgpA-Kgp complex. However, the inhibition of binding of W50 to KB cells by the proteinase-active (nontreated) RgpA-Kgp complex was significantly greater than that observed with the same concentration of the TLCK-treated complex (Fig. 5). This again supports the close association between proteolytic activity and adhesin binding by the complex and the hypothesis that the proteolytic activity exposes latent binding sites by cleavage of surface molecules on KB cells. The greater inhibition by the proteinase-active complex at the very high concentrations of proteinase (20 to 50 µg/ml) may also be consistent with the proteolytic activity under these conditions extensively modifying the surfaces of the KB cells and therefore inhibiting binding by destroying the binding sites (receptors) for W50 cell adherence.

The roles of Arg- and Lys-specific proteinases and their associated adhesins in the adherence to KB cells were further studied using isogenic mutants of P. gingivalis W50 lacking rgpA, rgpB, and kgp gene products. Isogenic mutants lacking either rgpA rgpB or rgpB gene products exhibited levels of binding to oral epithelial cells similar to that of wild-type P. gingivalis W50, suggesting that RgpB and the RgpA proteinase-adhesin complex have little role in adherence of P. gingivalis W50 to oral epithelial cells. Interestingly, the rgpA isogenic mutant of P. gingivalis W50 displayed increased binding to oral epithelial cells relative to the wild type. This is consistent with the results reported by Tokuda et al. (36), where an rgpA mutant of P. gingivalis strain 381 exhibited increased binding to oral epithelial cells compared with the parental strain. This result may be attributable to a change in cell surface composition of P. gingivalis, as RgpA is a major surface component of the bacterium (22) and therefore its absence may increase the availability or specific activity of other surface molecules involved in adherence, (e.g., Kgp). Isogenic mutants lacking either kgp or rgpA rgpB kgp gene products were shown to exhibit significantly reduced binding to KB cells compared with the parental strain. No significant difference was observed between the adherence of kgp and rgpA rgpB kgp isogenic mutants to KB cells. These data in combination with the TLCK inhibition data suggest that the Kgp proteinase's catalytic activity is important for effective adherence of P. gingivalis W50 to oral epithelial cells. Kgp appears to mediate adherence by cleaving surface proteins/receptors on the epithelial cells, exposing latent binding sites for the adhesins in close association with the proteolytic activity in the proteinase-adhesin complexes on the bacterial cell surface (25). The residual binding to KB cells by the rgpA rgpB kgp isogenic mutant is possibly mediated by other binding proteins on the P. gingivalis W50 cell surface, such as HagA, which exhibits high sequence similarity to the adhesins of Kgp (39). Presumably, this residual binding is direct and does not appear to require activation by proteolytic activity.

In conclusion, this study presents a method based on flow cytometry to quantify the adherence of different strains of P. gingivalis to oral epithelial cells that can be applied to a variety of host cells and bacteria. Both P. gingivalis strains W50 and ATCC 33277 were shown to bind to epithelial cells, but the pattern of binding was strain dependent. This study further showed that the Kgp proteinase of P. gingivalis W50 plays an important role in adherence to epithelial cells.


   ACKNOWLEDGMENTS
 
We thank Narelle Skinner for her assistance with fluorescence microscopy.

This project is supported by the Australian National Health and Medical Research Council (project 251708) and the National Institutes of Health (grant 1R01DE14198-01). Neil O'Brien-Simpson is a C. R. Roper Fellow, and John Hamilton is a Senior Principal Research Fellow with the National Health and Medical Research Council.


   FOOTNOTES
 
* Corresponding author. Mailing address: Centre for Oral Health Science, School of Dental Science, Faculty of Medicine, Dentistry and Health Sciences, The University of Melbourne, 720 Swanston Street, Melbourne, Victoria 3010, Australia. Phone: 61 3 9341 1547. Fax: 61 3 9341 1596. E-mail: e.reynolds{at}unimelb.edu.au Back

{triangledown} Published ahead of print on 5 March 2007. Back

Editor: A. D. O'Brien


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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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Infection and Immunity, May 2007, p. 2484-2492, Vol. 75, No. 5
0019-9567/07/$08.00+0     doi:10.1128/IAI.02004-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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