Differential effect of the cytolethal distending toxin of Actinobacillus actinomycetemcomitans on co-cultures of human oral cells

Abstract

The periodontal pathogen Actinobacillus actinomycetemcomitans expresses a cytolethal distending toxin (CDT) that typically arrests the growth of eukaryotic cells at either the G 0 /G 1 or G 2 /M phase of the cell cycle. It was previously found that CDT failed to arrest the growth of human periodontal ligament fibroblasts (HPLFs) when grown in pure culture. In contrast, proliferation of an oral epithelial cell line was rapidly inhibited by the toxin. In this study, the feasibility of using mixed-cell cultures and cell-specific markers to evaluate the response of oral cells, when in heterogeneous populations, to CDT was established. Proliferation of epithelial cells was rapidly inhibited and the cells were selectively eliminated in co-culture with HPLFs or cementoblasts by 24-48 h postintoxication. Epithelial cells and HPLFs were detected and counted in co-cultures following cellspecific immunolabelling with antibodies against simian virus 40 large T antigen and the Ab-1 surface antigen, respectively. These results demonstrated that the activities of potential virulence factors, such as CDT, from periodontal pathogens can be successfully examined in mixed-cell cultures. This approach is especially relevant to infectious diseases that affect tissues with a diverse cellular composition, such as the periodontium.

Cytolethal Distending Toxin Damages the Oral Epithelium of Gingival Explants

“…4B and inset, respectively). These results were consistent with those of previous studies in which primary periodontal ligament fibroblasts were found to be resistant to the cell-cycle-inhibitory activity of the Cdt (Kang et al, 2005;Kanno et al, 2005).…”

“…Cultured HGF exhibited a morphology typical of fibroblasts and bound the CD90/Thy-1 (Ab-1) antibody. We previously showed that the Ab-1 marker is expressed by oral fibroblasts but not by immortalized labial epithelial cells (Kang et al, 2005).…”

“…The Cdt is a secreted heterotrimer that damages the DNA of susceptible cells, leading to cell-cycle-mediated growth arrest or, in some cases, apoptosis (Sugai et al, 1998;Mayer et al, 1999). The rapid growth arrest of Cdt-treated immortalized oral epithelial cell lines, co-cultured with periodontal ligament fibroblasts (Kang et al, 2005;Kanno et al, 2005), prompted us to advance the hypothesis that this putative virulence factor affects the integrity of human gingival epithelial cells (HGEC) in situ.…”

Aggregatibacter actinomycetemcomitansCytolethal Distending Toxin Induces Apoptosis in Nonproliferating Macrophages by a Phosphatase-Independent Mechanism

“…Cdt holotoxin induces arrest in the G 2 phase of the cell cycle in a variety of proliferating cells, including epithelial cells, fibroblasts, human periodontal ligament cells, and lymphocytes (2,3,5,8,19,20,24,25,27,28,38,41). Interestingly, Shenker et al reported that the specific activity of Cdt against stimulated primary T lymphocytes was five-to 10-fold greater than that against HeLa cells (39) and subsequently showed that the Jurkat T-cell line is hypersensitive to Cdt intoxication (36).…”

Bacterial genotoxins

“…AactCDT has been shown to induce cell cycle arrest in the oral epithelial cell line GMSM-K [187,188], and in some studies also in human periodontal ligament fibroblasts (HPLFs) and human gingival fibroblasts (HGFs) [183,189], although the effects on this cell type have not been consistently reproducible [187,190]. The discrepancy may be due to the amounts of active toxin used in the studies, which may trigger a different set of responses in the target cells, depending on the efficiency of uptake and intracellular trafficking route.…”