Treponema denticola outer membrane inhibits calcium flux in gingival fibroblasts

Abstract

Treponema denticola is a cultivable oral spirochete which perturbs the cytoskeleton in cultured cells of oral origin, but intracellular signalling pathways by which it affects actin assembly are largely unknown. As the outer membrane (OM) of Treponema denticola disrupts actin-dependent processes that normally require precise control of intracellular calcium, we studied the effects of an OM extract on internal calcium release, ligand-gated and calcium release-activated calcium channels, and related mechanosensitive cation fluxes in human gingival fibroblasts (HGF). Single-cell ratio fluorimetry demonstrated that in resting cells loaded with Fura-2, baseline intracellular Ca2+ concentration ([Ca2+]i) was not affected by treatment with OM extract, but normal spontaneous [Ca2+]i oscillations were dramatically increased in frequency for 20 to 30 min followed by complete blockade. OM extract inhibited ATP-induced and thapsigargin-induced release of calcium from intracellular stores by 40 and 30%, respectively. Addition of Ca2+ to the extracellular pool following depletion of intracellular Ca2+ by thapsigargin and extracellular Ca2+ by EGTA yielded 59% less replenishment of [Ca2+]i in OM extract-treated than in control HGF. In cells loaded with collagen-coated ferric oxide beads to stimulate integrin-dependent calcium release, baseline [Ca2+]i was nearly doubled but was not significantly different in control and OM extract-treated cells. Magnetically generated tensile forces on the beads induced >300% increases of [Ca2+]i above baseline. Cells preincubated with OM extract exhibited dose-dependent and time-dependent reductions in stretch-induced [Ca2+]i transients, which were due to neither loss of beads from the cells nor cell death. The T. denticola OM inhibitory activity was eliminated by heating the OM extract to 60 degrees C and by boiling but not by phenylmethylsulfonyl fluoride treatment. Thus nonlipopolysaccharide, nonchymotrypsin, heat-sensitive protein(s) in T. denticola OM can evidently inhibit both release of calcium from internal stores and uptake of calcium through the plasma membrane, possibly by interference with calcium release-activated channels.

FIG. 1

T. denticola OM extract induces an early increase of spiking frequency that is followed by significant (P < 0.05) diminution at 70 min. Cells were preincubated with OM extract from T. denticola (▴) or untreated (control; ▪). Spikes were defined as calcium transients that were >10 nM above baseline values and that returned to baseline levels. Frequency was measured as the number of spikes for 5 cells per 20 min. Data are means ± SE, with each data point representing an average of 40 cells in a 20-min time interval in eight independent experiments.

FIG. 2

Increase of [Ca²⁺]_i in single cells that were untreated (control) or treated with OM extract for 30 min and then incubated with ionomycin (3 μM; arrow). The baselines of the two traces were at the same level (100 nM), but the OM-treated trace has been offset vertically by 50 nM merely to facilitate visualization of the two responses. Note that both control and OM-treated cells respond robustly to ionomycin and show a sharp reduction of intracellular calcium within 50 s.

FIG. 3

T. denticola OM extract reduces the amplitude of stretch-induced calcium transients. Cells were incubated with collagen-coated ferric oxide beads and were either untreated (control) or treated with OM extract for 1 min. [Ca²⁺]_i was measured in cells that were mechanically stretched by application of magnetic fields.

FIG. 4

T. denticola OM extract inhibits stretch-induced calcium responses within 20 min after incubation with OM extract, and the inhibition persists for up to 70 min (P < 0.05). Cells were treated as described for Fig. 3. Measurements of peak calcium increases induced by mechanical stretching were made at the indicated times, and the percent change compared to the increase obtained at time zero was computed. The responses of control cells did not change significantly over 70 min. Data are shown as mean ± SE percent of control values (n = 10 cells per data point).

FIG. 5

T. denticola OM extract causes a dose-dependent reduction of the amplitude of stretch-induced calcium transients in fibroblasts. Cells were loaded with ferric oxide beads and incubated with OM extract at the indicated dilutions for 30 min. Data are means ± SE percent change of peak calcium transient compared with untreated controls (n = 10 cells per data point).

FIG. 6

Histogram of stretch-induced calcium responses in cells that were treated with OM extract for 30 min (OM), 5 mM EGTA before stretching (EGTA), EGTA plus OM extract for 30 min, 1 mM gadolinium chloride (GdCl₃), gadolinium chloride plus OM extract for 30 min, 1 μM thapsigargin (Tg), or thapsigargin plus OM extract for 30 min. OM extract reduces the amplitude of stretch-induced calcium transients >3-fold. Data are means ± SE percent of control values (n = 20 per experimental condition).

FIG. 7

Histogram of repeated stretch-induced calcium responses of fibroblasts to OM extract treated by heating, boiling, or chymotrypsin inhibitor. Heat (60°C for 30 min) or boiling (10 min) abrogated the inhibitory effect of OM extract on stretch-induced calcium transients. The chymotrypsin inhibitor PMSF did not affect inhibition. OM extract was treated by heating, boiling, or PMSF before incubation with cells and stretching as described. Cells were measured before (control) and after incubation with OM extract for 1, 18, 35, 52, 69 min. Data are means ± SE percent of control values (N = 10 cells per bar).

FIG. 8

Putative mechanisms for T. denticola OM perturbation of intracellular calcium ion fluxes in HGF. Channels and sources marked by X’s and thin arrows, representing Ca²⁺ release from internal stores and Ca²⁺ flow through calcium release-activated calcium (CRAC) channels, are blocked by nonproteolytic OM protein(s). Inhibition of mobilization of intracellular Ca²⁺ may be, in part, secondary to diminished IP responses (40) and calcium-activated Ca²⁺ release. In their simplest interpretation, the findings suggest a hypothesis that OM proteins interfere rapidly and directly with CRAC channels and perhaps with receptors for some agonists of the IP pathway at the external interface of the plasma membrane prior to and concomitant with disruption of F-actin at the ventral interface with the substratum (40) and cytoskeletal proteins which complex with integrins upon mechanical stretching by magnetic forces acting on the bound, collagen-coated ferric oxide beads (Bead) (17). T. denticola OM has no effect on integrin-gated (Ligand-gated) Ca²⁺-permeable channels, and the data suggest little direct effect on stretch-activated ion-permeable channels (thick arrows). Proteolytic enzymes in the OM have little if any immediate effect on intracellular Ca²⁺ and IPs but may indirectly affect actin subsequently by degrading extracellular matrix proteins like endogenous fibronectin (Fn) and components of the substratum (ECM [extracellular matrix]).