TREPONEMA-DENTICOLA OUTER-MEMBRANE INHIBITS CALCIUM FLUX IN GINGIVAL FIBROBLASTS

Treponema denticola is a cultivable oral spirochete which perturbs the cytoskeleton in cultured cells of oral origin, but intracellular sign alling pathways by which it affects actin assembly are largely unknown . As the outer membrane (OM) of Treponema denticola disrupts actin-dep endent processes that normally require precise control of intracellula r 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 fibrobla sts (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 sp ontaneous [Ca2+](i) oscillations were dramatically increased in freque ncy for 20 to 30 min followed by complete blockade, OM extract inhibit ed ATP-induced and thapsigargin-induced release of calcium from intrac ellular stores by 40 and 30%, respectively. Addition of Ca2+ to the ex tracellular pool following depletion of intracellular Ca2+ by thapsiga rgin and extracellular Ca2+ by EGTA yielded 59% less replenishment of [Ca2+](i) in OM extract-treated than in control HGF. In cells loaded w ith collagen-coated ferric oxide beads to stimulate integrin-dependent calcium release, baseline [Ca2+](i) was nearly doubled but was not si gnificantly different in control and OM extract-treated cells, Magneti cally generated tensile forces on the beads induced >300% increases of [Ca2+](i) above baseline. Cells preincubated with OM extract exhibite d dose-dependent and time dependent reductions in stretch-induced [Ca2 +](i) transients, which were due to neither loss of beads from the cel ls nor cell death, The T. denticola OM inhibitory activity was elimina ted by heating the OM extract to 60 degrees C and by boiling but not b y phenylmethylsulfonyl fluoride treatment, Thus nonlipopolysaccharide, nonchymotrypsin, heat-sensitive protein(s) in T. denticola OM can evi dently inhibit both release of calcium from internal stores and uptake of calcium through the plasma membrane, possibly by interference with calcium release-activated channels.