Kanno Fumio, Korostoff Jonathan, Volgina Alla, DiRienzo Joseph M
Department of Periodontics, School of Dental Medicine, University of Pennsylvania, 240 South 40th Street, Philadelphia, PA 19104-6030, USA.
J Periodontol. 2005 Jul;76(7):1189-201. doi: 10.1902/jop.2005.76.7.1189.
The cytolethal distending toxin (CDT) of Actinobacillus actinomycetemcomitans is a typical member of this Gram-negative bacterium holotoxin family that targets a wide spectrum of eukarytotic cells, typically causing cell cycle arrest at either the G(1) or G(2)/M phase of the cell cycle. In view of the possible role of the CDT as a prominent A. actinomycetemcomitans virulence factor in periodontal diseases, we have examined the effects of the toxin on primary cultures of human periodontal ligament fibroblasts (HPLF).
HPLF and an immortalized human gingival epithelial cell line, GMSM-K, were exposed to recombinant A. actinomycetemcomitans CDT. Effects of the toxin on cell proliferation and cell cycle were assessed by a cell viability assay and flow cytometry, respectively. Double-strand DNA damage was detected by pulsed field gel electrophoresis. Binding of the toxin and its individual subunits to HPLF was examined by immunofluorescence microscopy.
Viability of HPLF was not reduced following prolonged exposure to the CDT. There was no indication of cell cycle arrest or double-strand DNA damage. GMSM-K cells exhibited morphological alterations and a rapid decrease in cell viability within 6 and 12 hours, respectively, following exposure to the toxin for 5 minutes. These effects were dependent on toxin dose and age of the cultures and occurred more rapidly compared to CDT-treated HeLa cells. CDT-treated GMSM-K cells displayed cell cycle arrest at the S phase of growth and double-strand DNA damage was observed by 6 hours post-intoxication. Holotoxin and the CdtA subunit were detected on the surface of both HPLF and epithelial cells.
These results demonstrate that HPLF are resistant to the cytotoxic effects of the A. actinomycetemcomitans CDT. The mechanism of resistance is not known but may be related to the inability of the toxin to cause DNA damage. The difference in sensitivities of HPLF and oral epithelial cells to the CDT has important implications for the role of this putative microbial virulence factor in periodontal pathogenesis.
伴放线放线杆菌的细胞致死性膨胀毒素（CDT）是这种革兰氏阴性细菌全毒素家族的典型成员，可作用于多种真核细胞，通常导致细胞周期在G(1)期或G(2)/M期停滞。鉴于CDT作为伴放线放线杆菌在牙周疾病中一种重要毒力因子的潜在作用，我们研究了该毒素对人牙周膜成纤维细胞（HPLF）原代培养物的影响。
将HPLF和永生化人牙龈上皮细胞系GMSM-K暴露于重组伴放线放线杆菌CDT。分别通过细胞活力测定和流式细胞术评估毒素对细胞增殖和细胞周期的影响。通过脉冲场凝胶电泳检测双链DNA损伤。通过免疫荧光显微镜检查毒素及其单个亚基与HPLF的结合。
长时间暴露于CDT后，HPLF的活力并未降低。没有细胞周期停滞或双链DNA损伤的迹象。GMSM-K细胞在暴露于毒素5分钟后，分别在6小时和12小时内出现形态改变和细胞活力迅速下降。这些效应取决于毒素剂量和培养物的传代次数，与经CDT处理的HeLa细胞相比，发生得更快。经CDT处理的GMSM-K细胞在生长的S期出现细胞周期停滞，中毒后6小时观察到双链DNA损伤。在HPLF和上皮细胞表面均检测到全毒素和CdtA亚基。
这些结果表明，HPLF对伴放线放线杆菌CDT的细胞毒性作用具有抗性。抗性机制尚不清楚，但可能与毒素无法引起DNA损伤有关。HPLF和口腔上皮细胞对CDT敏感性的差异对这种假定的微生物毒力因子在牙周发病机制中的作用具有重要意义。
