A new mode of action for the cytolethal distending toxin
Résumé
The Cytolethal Distending Toxin (CDT) is a virulence factor produced by many pathogenic bacteria like E. coli, H. hepaticus, H. ducreyi etc. The CDT Production allows bacteria to persistently colonize the body, evade the immune system, induce inflammation and trigger genetic instability. In fact, CDT induces DNA double-stranded breaks (DSB), leading to cell cycle arrest and to cytotoxicity, associated with a characteristic cellular distension. Our objectives are to characterize the type of DNA damage induced by CDT and study the cell sensitivity to different CDT doses, with the cell cycle as read-out. Thanks to comet assay and immunofluorescence, we have shown that at doses 1000 times lower than those used in the literature, CDT induces multiple single-strand breaks. When cells are going through S phase, a replicative stress is induced and DNA damage degenerate into DSB. Indeed, with a PCNA marker, we observed a S phase slowing down and an increase of the number of cells in late S-phase. Moreover, these CDT-induced DNA damage cause the activation of the pathway involving the RPA, ATR and CHK1 proteins, characteristics of a replicative stress. Finally, the activation of the ATM pathway, due to DSB induction, happens later after the CDT treatment. Therefore, the importance of the S-phase passage for the CDT cytotoxicity suggests that proliferating cells are more sensitive to CDT than quiescent cells. Another part of our project is to better characterize the CDT catalytic activity. As CdtB, the catalitic sub-unit of CDT, display DNase and phosphatase activities, we are developing specific mutants to dissociate these activities. The experiments are progressing and the results will be presented. Our work underlines the complex mechanism of CDT action. It will help in the characterization of CDT-expressing bacteria and may open new therapeutic approaches, for example by targeting the mechanisms of associated genotoxicity.