Yang P F, Song M, Grove D A, Ellen R P
Faculty of Dentistry, University of Toronto, Ontario, Canada.
Infect Immun. 1998 Feb;66(2):696-702. doi: 10.1128/IAI.66.2.696-702.1998.
Previous reports have shown that Treponema denticola causes rearrangement of filamentous actin (F-actin) in human gingival fibroblasts (HGF). The purpose of this investigation was to determine the effect of T. denticola on the generation of inositol phosphates (IPs) in relation to a time course for F-actin disruption in HGF. Cultured HGF were exposed to washed cells of T. denticola ATCC 35405 for 140 min. Changes in the fluorescence intensity of rhodamine-phalloidin-labeled F-actin in serial optical sections of single HGF were quantified by confocal microscopy image analysis. The percentage of cells with stress fiber disruption was also determined by fluorescence microscopy. Challenge with T. denticola caused a significant reduction in F-actin within the first hour, especially at the expense of F-actin in the ventral third of the cells, and a significant increase in the percentage of HGF with altered stress fiber patterns. Significant concentration-dependent disruption of stress fibers was also caused by HGF exposure to a Triton X-100 extract of T. denticola outer membrane (OM). IPs were measured by a radiotracer assay based on the incorporation of myo-[3H]inositol into IPs in HGF incubated with LiCl to inhibit endogenous phosphatases. HGF challenge with several strains of T. denticola and the OM extract of T. denticola ATCC 35405 resulted in a diminished accumulation of radiolabeled IPs relative to both 15 and 1% fetal bovine serum, which served as strongly positive and background control agonists, respectively. The significantly diminished IP response to T. denticola ATCC 35405 occurred within 60 min, concomitant with significant reduction of total F-actin and disruption of stress fibers. Pretreatment with the proteinase inhibitor phenylmethylsulfonyl fluoride, which had previously been found to block T. denticola's degradation of endogenous fibronectin and detachment of HGF from the extracellular matrix, had little effect on F-actin stress fiber disruption and the IP response. Therefore, in addition to its major surface chymotrypsin-like properties, T. denticola expresses cytopathogenic activities that diminish the generation of IPs during the time course associated with significant cytoskeletal disruption in fibroblasts.
先前的报告显示，具核梭杆菌可导致人牙龈成纤维细胞（HGF）中丝状肌动蛋白（F-肌动蛋白）重排。本研究的目的是确定具核梭杆菌对肌醇磷酸（IPs）生成的影响，并研究其与HGF中F-肌动蛋白破坏的时间进程之间的关系。将培养的HGF暴露于具核梭杆菌ATCC 35405的洗涤细胞中140分钟。通过共聚焦显微镜图像分析对单个HGF连续光学切片中罗丹明-鬼笔环肽标记的F-肌动蛋白的荧光强度变化进行定量。还通过荧光显微镜确定了应力纤维破坏细胞的百分比。用具核梭杆菌攻击在第一小时内导致F-肌动蛋白显著减少，尤其是细胞腹侧三分之一处的F-肌动蛋白减少，并且具有改变的应力纤维模式的HGF百分比显著增加。HGF暴露于具核梭杆菌外膜（OM）的Triton X-100提取物也导致应力纤维的浓度依赖性显著破坏。基于将肌醇-[3H]肌醇掺入与LiCl孵育的HGF中的IPs来抑制内源性磷酸酶，通过放射性示踪剂测定法测量IPs。用几株具核梭杆菌和具核梭杆菌ATCC 35405的OM提取物攻击HGF导致相对于15%和1%胎牛血清，放射性标记的IPs积累减少，15%胎牛血清和1%胎牛血清分别作为强阳性和背景对照激动剂。对具核梭杆菌ATCC 35405的IP反应显著降低在60分钟内发生，同时总F-肌动蛋白显著减少和应力纤维破坏。先前发现蛋白酶抑制剂苯甲基磺酰氟可阻断具核梭杆菌对内源性纤连蛋白的降解以及HGF从细胞外基质的脱离，用其预处理对F-肌动蛋白应力纤维破坏和IP反应几乎没有影响。因此，除了其主要的表面类胰凝乳蛋白酶特性外，具核梭杆菌还表达细胞致病活性，在与成纤维细胞中显著细胞骨架破坏相关的时间进程中减少IPs的生成。
