Resistance of human periodontal ligament fibroblasts to the cytolethal distending toxin of Actinobacillus actinomycetemcomitans.

BACKGROUND

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).

METHODS

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.

RESULTS

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.

CONCLUSIONS

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.