Ko K S, Glogauer M, McCulloch C A, Ellen R P
Department of Periodontics, Faculty of Dentistry, University of Toronto, Ontario, Canada.
Infect Immun. 1998 Feb;66(2):703-9. doi: 10.1128/IAI.66.2.703-709.1998.
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.
齿垢密螺旋体是一种可培养的口腔螺旋体，它会扰乱口腔来源培养细胞中的细胞骨架，但它影响肌动蛋白组装的细胞内信号通路在很大程度上尚不清楚。由于齿垢密螺旋体的外膜会破坏通常需要精确控制细胞内钙的肌动蛋白依赖性过程，我们研究了外膜提取物对人牙龈成纤维细胞（HGF）中细胞内钙释放、配体门控和钙释放激活钙通道以及相关机械敏感阳离子通量的影响。单细胞比率荧光法表明，在用Fura-2加载的静息细胞中，基线细胞内Ca2+浓度（[Ca2+]i）不受外膜提取物处理的影响，但正常的自发[Ca2+]i振荡频率在20至30分钟内显著增加，随后完全被阻断。外膜提取物分别抑制ATP诱导和毒胡萝卜素诱导的细胞内钙库中钙的释放40%和30%。在用毒胡萝卜素耗尽细胞内钙并通过乙二醇双（2-氨基乙基醚）四乙酸（EGTA）耗尽细胞外钙后，向细胞外池中添加Ca2+，与对照HGF相比，外膜提取物处理的细胞中[Ca2+]i的补充减少了59%。在加载胶原包被的氧化铁珠以刺激整合素依赖性钙释放的细胞中，基线[Ca2+]i几乎增加了一倍，但在对照和外膜提取物处理的细胞中没有显著差异。磁体对珠子产生的拉力导致[Ca2+]i比基线增加>300%。预先用外膜提取物孵育的细胞在拉伸诱导的[Ca2+]i瞬变中表现出剂量依赖性和时间依赖性的降低，这既不是由于细胞中珠子的丢失也不是由于细胞死亡。将外膜提取物加热到60摄氏度并煮沸可消除齿垢密螺旋体外膜的抑制活性，但苯甲基磺酰氟处理则不能。因此，齿垢密螺旋体外膜中的非脂多糖、非胰凝乳蛋白酶、热敏感蛋白显然可以抑制细胞内钙库中钙的释放以及通过质膜摄取钙，可能是通过干扰钙释放激活通道来实现的。
