Ellen R P, Ko K S, Lo C M, Grove D A, Ishihara K
Dental Research Institute and Medical Research Council Group in Periodontal Physiology, University of Toronto, Faculty of Dentistry, Ontario, Canada.
J Mol Microbiol Biotechnol. 2000 Oct;2(4):581-6.
The purified chymotrypsin-like protease of Treponema denticola, designated dentilisin or PrtP (DDBJ accession no. D83264), can disrupt cell-cell junctions and impair the barrier function of epithelial monolayers in vitro. Serine protease inhibitors block these effects. Yet, the protease is apparently less significant in perturbing intracellular signaling pathways and cytoskeletal rearrangement in fibroblasts. The purpose of this study was to use a PrtP-deficient mutant of T. denticola to confirm that the cytopathic effects of whole bacteria and its outer membrane on epithelial cell junctions were primarily accounted for by the activity of this protease. The prtP gene of ATCC 35405 was inactivated by insertion of an erythromycin-resistance cassette, yielding mutant K1. In contrast to wildtype ATCC 35405, mutant K1 grew in tight cell aggregates; the cells had a disrupted outer sheath, as determined by electron microscopy. When compared by silver stained SDS-PAGE of sonicated extracts of whole cells, the extract of mutant K1 was missing a band at approximately 90 kDa that was present in the wildtype ATCC 35405 strain. Whole cells and Triton X-100 outer membrane (OM) extracts of K1 and the wildtype strains were compared 1) for SAAPNA degrading activity by a colorimetric assay, 2) for stress fiber disruption in human gingival fibroblasts (HGF) by fluorescence microscopy of TRITC-phalloidin stained cells, and 3) the OM extracts only for perturbation of HEp-2 epithelial monolayers by electrical cell-substrate impedance sensing (ECIS). Mutant K-1 cells and OM had no SAPPNA degrading activity that is characteristic of dentilisin. K1 cells had HGF stress fiber disrupting activity (86 +/- 4.5% of HGFs affected) equivalent to both 35405 wildtype strains (84 +/- 3.9% and 71 +/- 14.1% of HGF, respectively). Yet, mutant K1 OM had diminished stress fiber disrupting activity (12.9 +/- 4.6% of HGF) compared with its parent 35405's OM (94.6 +/- 2.9%). The major cytopathogenic difference between the K1 mutant and wildtype strains was in their OM's effect on epithelial cell junctions. ATCC 35405 OM completely disrupted epithelial resistance in a concentration - dependent manner; mutant K1 OM had negligible effects. These data confirm that inactivation of the prtP gene completely reverses T. denticola's disruption of epithelial junctions, but there are pleiotropic effects of the mutation that may account for its apparently diminished effects on the cytoskeleton of HGF when the cells were challenged with OM extracts.
齿垢密螺旋体纯化的类胰凝乳蛋白酶，命名为齿垢蛋白酶或PrtP（DDBJ登录号：D83264），在体外可破坏细胞间连接并损害上皮单层的屏障功能。丝氨酸蛋白酶抑制剂可阻断这些效应。然而，该蛋白酶在干扰成纤维细胞的细胞内信号通路和细胞骨架重排方面似乎作用较小。本研究的目的是使用齿垢密螺旋体的PrtP缺陷型突变体来证实全菌及其外膜对上皮细胞连接的细胞病变效应主要是由该蛋白酶的活性引起的。通过插入红霉素抗性盒使ATCC 35405的prtP基因失活，产生突变体K1。与野生型ATCC 35405相比，突变体K1以紧密的细胞聚集体形式生长；通过电子显微镜观察，细胞的外鞘被破坏。当通过全细胞超声提取物的银染SDS-PAGE进行比较时，突变体K1的提取物在约90 kDa处缺少一条野生型ATCC 35405菌株中存在的条带。比较了K1和野生型菌株的全细胞及Triton X-100外膜（OM）提取物：1）通过比色法检测SAAPNA降解活性；2）通过TRITC-鬼笔环肽染色细胞的荧光显微镜观察人牙龈成纤维细胞（HGF）中应力纤维的破坏情况；3）仅通过细胞-基质阻抗传感（ECIS）检测OM提取物对HEp-2上皮单层的扰动。突变体K-1细胞和OM没有齿垢蛋白酶特有的SAPPNA降解活性。K1细胞具有与两种35405野生型菌株相当的HGF应力纤维破坏活性（分别影响84±3.9%和71±14.1%的HGF）。然而，与亲本35405的OM（94.6±2.9%）相比，突变体K1的OM应力纤维破坏活性降低（12.9±4.6%的HGF）。K1突变体和野生型菌株之间主要的细胞致病差异在于它们的OM对上皮细胞连接产生的影响。ATCC 35405的OM以浓度依赖性方式完全破坏上皮电阻；突变体K1的OM影响可忽略不计。这些数据证实prtP基因的失活完全逆转了齿垢密螺旋体对上皮连接的破坏，但该突变存在多效性效应，这可能解释了在用OM提取物刺激细胞时，其对HGF细胞骨架的影响明显减弱的原因。
