Filamentous Actin Disruption and Diminished Inositol Phosphate Response in Gingival Fibroblasts Caused byTreponema denticola

Treponemal cultures and cultural conditions.Stock cultures of T. denticola ATCC 35405 (type strain), originally provided by W. J. Loesche, University of Michigan, and ATCC 35404, ATCC 33520, e, and e′, provided by E. C. S. Chan, McGill University, were maintained and grown for experiments in a complex spirochete broth medium containing brain heart infusion, tryptic peptone, yeast extract, and volatile fatty acids and supplemented with 2.0% rabbit serum as previously described (7). Strains ATCC 35405, e, and e′ have been shown to induce actin rearrangement and detachment of cultured HGF from the substratum (2).Treponema vincentii ATCC 35580 and Treponema socranskii ATCC 35536 were obtained from the American Type Culture Collection and maintained similarly. Cells were grown at 37°C and subcultured weekly in an anaerobic chamber (Coy Laboratory Products, Ann Arbor, Mich.) in an atmosphere of 80% N₂, 10% CO₂, and 10% H₂ (Canox, Toronto, Ontario, Canada). For experiments in which bacterial suspensions were used to challenge gingival fibroblasts, 3-day, stationary-phase cultures were harvested by centrifugation at 12,000 × g for 6 min and washed twice in 0.01 M phosphate-buffered saline at pH 7.2 (PBS) prior to resuspension in a CO₂-independent tissue culture medium.

T. denticola OM preparation.Spirochete medium was inoculated with a 3-day culture of T. denticola ATCC 35405 at a ratio of 30:1 (fresh medium to inoculum) and incubated at 37°C for 4 days. Bacteria were harvested by centrifugation at 12,000 × g for 15 min at 4°C and washed twice in PBS. The pellet was weighed, dispersed uniformly, and resuspended at 1.0 g (wet weight) per 10 ml of PBS containing 10 mM MgCl₂. A modification of the detergent extraction method of Penn et al. (5, 31) was used for the initial extraction. Triton X-100 (Surfact-Amps X-100; Pierce, Rockford, Ill.) was added to a final concentration of 0.2% (vol/vol). The suspension was incubated with constant mixing at 37°C for 30 min and then repeatedly centrifuged at 12,000 × g for 6 min until no visible pellet remained. The clear supernatant was dialyzed (molecular mass cutoff, 50 kDa; Spectra/Por, Spectrum, Houston, Tex.) against deionized H₂O at 4°C for several days until precipitates formed. The contents of the dialysis tubing were centrifuged at 25,000 ×g for 45 min. The pellet was resuspended in deionized H₂O to the predialysis volume and stored at −70°C. The dry weight of a lyophilized aliquot of the outer membrane (OM) extract was determined so that the actin-perturbing activity of the extract could be compared with that of whole T. denticola cells on a dry weight basis. Aliquots were also run by standard sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using a 12% acrylamide gel and silver stain to compare the migration of OM polypeptides qualitatively with migration patterns of polypeptides from whole cells that had been boiled in SDS for 10 min.

T. denticola cell suspensions and the OM extract were tested for peptidase activity by using the chromogenic peptidesN-succinyl-l-alanyl-l-alanyl-l-prolyl-l-phenylalanine-p-nitroanilide (SAAPNA) andN-benzoyl-dl-arginine-p-nitroanilide (BAPNA; Sigma) (15). Whole T. denticola cells at the concentration used for the assays described below had the equivalent SAAPNA-degrading activity of 0.2 μg of chymotrypsin per ml and BAPNA-degrading activity of 0.04 μg of trypsin per ml. The undiluted OM extract contained SAAPNA-degrading activity equivalent to 4.0 μg of chymotrypsin per ml and no detectable BAPNA-degrading activity.

Gingival fibroblast cultures.HGF cell cultures were established from primary human tissue explants as described previously (1). The cells were cultured in alpha minimal essential medium (αMEM) containing 400 U of penicillin G per ml and 10% (vol/vol) heat-inactivated fetal bovine serum (FBS; BioWhittaker, Walkersville, Md.) and maintained at 37°C in a humidified atmosphere of 95% air and 5% CO₂. The fibroblasts were subcultured weekly and were used for experiments between passages 14 and 25.

Bacterial perturbation of F-actin in HGF.Two assays were used in time course experiments to measure the degree of actin perturbation in HGF challenged by type strain ATCC 35405 whole cell or OM extract suspensions.

(i) Stress fiber assay.HGF in confluent monolayers were trypsinized, washed in PBS, and adjusted to a density of 10⁵ cells/ml in αMEM containing penicillin G and FBS. The cell suspensions were distributed at 1.0 ml/well in 24-well dishes (Corning Glass Works), each containing one sterilized, circular glass coverslip (no. 1, 12-mm diameter; Fisher Scientific). The HGF were incubated at 37°C in a 5% CO₂ atmosphere for 3 h to allow spreading, and they were nonconfluent for the bacterial challenge experiments. Prior to the addition of bacteria, each well was washed once with CO₂-independent medium (CIM; Gibco). The treponemes were washed, resuspended in serum-free CIM to a cell density of 2 × 10⁹ bacteria/ml, and added to the wells containing the coverslips at 1.0 ml/well in duplicate. Control wells received CIM without bacteria. The dishes were incubated at 37°C. In a time course experiment of 140 min, coverslips were selected at 20-min intervals and the HGF were fixed in 1.0 ml of 3.75% (vol/vol) formaldehyde in PBS. The coverslips were then washed with PBS, and the HGF were permeabilized and labeled for F-actin.

The permeabilizing and labeling solution contained rhodamine-phalloidin (Molecular Probes, Eugene, Oreg.), 0.6 U/ml in 0.1% (vol/vol) Nonidet P-40 (Sigma) in PBS. Five hundred microliters was added per well, and the dishes were incubated at room temperature for 30 min. The coverslips were washed twice in deionized water and mounted on glass slides with an antifade mounting medium containing 0.1% (wt/vol)p-phenylenediamine, 10% (vol/vol) 0.01 M PBS (pH 7.4), and 90% (vol/vol) glycerol, adjusted to pH 8.0 with 0.5 M carbonate-bicarbonate buffer. Single HGF were examined by fluorescence microscopy at a magnification of ×400, using a Leitz Dialux microscope equipped with a Leitz numerical-aperture 1.30, 160/0.17 objective lens, and epifluorescence with an N2 filter block (excitation filter, 530 to 560 nm; suppression filter, 580 nm) for red fluorescence. Two hundred HGF on duplicate coverslips were scored dichotomously according to preset criteria for presenting either a normal or altered stress fiber pattern, and the outcome was expressed as the percentage of cells with altered stress fibers. HGF scored as normal had stress fibers that were abundant in quantity, evenly and brightly fluorescent, long, mostly straight, and detected in well-spread cells. In contrast, HGF scored as altered had stress fibers that were reduced in quantity or absent entirely, fragmented and unevenly fluorescent, and found in cells with retraction of cytoplasmic processes. The samples were coded to obscure their identity. Statistical significance between experimental and control groups at each time point and within-group differences between time points was determined by chi-square analysis.

(ii) Confocal microscopy.The degree of fluorescence in optical sections of control and T. denticola ATCC 35405-challenged HGF was measured by confocal microscopy. The HGF were prepared as for the stress fiber assay. The confocal laser scanning microscope (Leica Lasertechnik GmbH, Heidelberg, Germany) was set with an argon ion laser of 488/514-nm emission, BP568 laser filter, RSP 488/568 beam splitter, pinhole setting at 40, and offset at −30. Fluorescence intensity of rhodamine-phalloidin-labeled F-actin was recorded in serial 1-μm optical sections in the x-y plane at a magnification of ×400 from the dorsal to the ventral (substratum) surface. Thirty cells per group per time point were examined, and fluorescence intensity per section was recorded. The total fluorescence intensity in an individual cell was calculated as the sum of fluorescence in all its optical sections, and the mean fluorescence per cell was derived for the 30 cells counted per time period per experimental group. As exposure to T. denticola caused many of the HGF to undergo progressive changes in shape, including cellular rounding and increased height, the number of optical sections for complete coverage of each single cell was determined, and the sections were categorized into thirds by cell height. The mean fluorescence per ventral, middle, and dorsal third for the 30 cells per group was calculated. For statistical analysis, significant differences in mean fluorescence between experimental groups at each time period and within-group differences at various time periods were determined by Student’s t test.

A comparison of whole T. denticola cells and OM extract for actin-perturbing activity was determined in 90-min assays using the stress fiber assay. The effect of inhibiting the proteolytic activity of the bacteria and OM extracts was also tested. They were pretreated with phenylmethylsulfonyl fluoride (PMSF) at 170 μg/ml, which we had shown previously to inhibit T. denticola’s fibronectin-degrading activity and detachment of HGF from the extracellular matrix (15).

IP assay.IPs generated during the experimental time course were measured by a modification of the radiotracer assays of Ruschkowski et al. (38) and Dean and Beaven (9). HGF in confluent monolayers were harvested by trypsinization and resuspended in αMEM with 10% FBS to a density of 2 × 10⁵ cells/ml. The cell suspensions were dispensed (2.5 ml in 60- by 15-mm petri dishes [Falcon]) and incubated at 37°C to allow spreading. After 2 h, each petri dish was washed twice with warm PBS, and 2.0 ml of αMEM containing penicillin G and 1% (vol/vol) FBS was added. One hundred microliters of 1/10-concentratedmyo-[³H]inositol (1.0 mCi/ml; Amersham, Arlington Heights, Ill.) in 10% (vol/vol) ethanol was added to each dish. The dishes were incubated for at least 18 h, the culture fluids were removed, and the dishes were washed once with warm serum-free CIM. Two milliliters of CIM containing 1.0% (vol/vol) FBS and 10 mM LiCl was added, and the dishes were incubated for 15 min at 37°C in air. The LiCl inhibits endogenous phosphatases and thereby allows the accumulation of ³H-labeled intermediates in the IP pathway.

Three-day cultures of treponemes were harvested by centrifugation, washed once in CIM, and resuspended in CIM containing 1.0% (vol/vol) FBS and 10 mM LiCl to a density of 2 × 10⁹bacteria/ml. Three milliliters of the bacterial suspension was added to each culture dish in the experimental groups. Positive control dishes, for which substantial increases in accumulated ³H-labeled IP intermediates would be anticipated received 3.0 ml of CIM containing 15% (vol/vol) FBS and LiCl, and the background control dishes received 3.0 ml of CIM containing 1.0% (vol/vol) FBS and LiCl as specified by Ruschkowski et al. (38). In some experiments ATP, 100 μM in CIM containing 1.0% FBS, was used as the positive agonist.

Duplicate dishes per treatment group were analyzed for total IPs immediately and following a 60-min period. The reactions were terminated by removal of the medium and one wash with ice-cold PBS. Cells in each dish were harvested into 1.0 ml of ice-cold PBS and collected into screw-capped polypropylene tubes. Four milliliters methanol-chloroform (2:1) solution was added to each tube, mixed, and allowed to react at ambient temperature for at least 30 min. Then a mixture of 0.125 ml of 0.5 M EDTA (pH 8.0), 1.2 ml of chloroform, and 1.0 ml of deionized H₂O was added. The mixtures were vortexed and then centrifuged at 500 × g for 8 min. The aqueous phase, which contained the water-soluble IPs, was collected and passed through a Dowex AG 1-X8 anion-exchange resin column (formate form resin and Poly-Prep chromatography columns; Bio-Rad) followed by a wash of 5.0 ml of deionized H₂O. The IPs were eluted with a 5.0-ml solution containing 0.1 M formic acid and 1.0 M ammonium formate. A 300-μl aliquot of each sample was added to 5.0 ml of scintillation fluid (Ecolume; ICN, Costa Mesa, Calif.), and the radioactivity (counts/minute) was measured in the tritium channel (RackBeta scintillation counter; LKB-Wallac).

Procedural controls included (i) omission of the LiCl, which would be expected to reduce the amount of accumulated IPs, and (ii) testing the effects of 0.05% ethanol, the solvent for the myo-inositol, in the IP assay. HGF incubated as usual with either 1.0 or 15% FBS and with 10.0 mM LiCl led to an increase in radiolabeled IPs of 125 and 240%, respectively, over 60 min. In contrast, parallel samples for which LiCl was deleted yielded negligible radiolabeled IPs at both the beginning of the assay (10 and 14% of the 1.0% FBS–LiCl and 15% FBS–LiCl controls, respectively) and at 60 min (6 and 7% of the controls). These results verified that incubation of HGF with LiCl indeed allowed the accumulation of radiolabeled inositols. Ethanol at its assay concentration led to an insignificant change in accumulated IPs over 60 min.

IP assays were also run with T. denticola cells pretreated with either PMSF orN-α-p-tosyl-l-lysine chloromethyl ketone (TLCK; Sigma) at 150 μg/ml, as described previously (2). At least two independent experiments were conducted for each condition tested.

For IP assays in which OM extract of T. denticola ATCC 35405 was substituted for the whole bacteria, the OM extract was serially diluted 1/4 to 1/16 in CIM containing 1.0% FBS, and 3.0 ml of the suspension was added to each dish. The effect of pretreatment of the OM extract with PMSF was also determined.