Genotoxic agents alter the genetic information by damaging DNA inside living cells, leading to genetic instability and carcinogenesis. Several bacteria from the human microbiota produce genotoxic virulence factors, called genotoxin. Thus, cells from the gastro-intestinal tract firstly, and cells from other tissues after systemic passage, are susceptible to suffer DNA damage from those compounds. Among these genotoxins, the Cytolethal Distending Toxin (CDT) is produced by several pathogenic bacteria, some of them being associated with an increased colorectal cancer risk. Some CDT-producing bacteria have been found around tumours, however the carcinogenic potential of CDT in the gastro-intestinal tract has not been directly proven to date. On the other hand, CDT of Helicobacter hepaticus has been shown to induce several cancerous markers in mice (chronic inflammation, hepatic infections and dysplastic nodules). In cell culture models, CDT induces DNA breaks, disturbs the cell cycle and may lead to cell senescence or cell death. Preliminary results show that cell sensitivity to the toxin depends on cell type, the toxin-producing bacteria and cell cycle phases. This raises several questions, particularly concerning the CDT mechanisms of action that may explain its physiopathology. As cell susceptibility to CDT might be linked to CDT internalisation (i.e. host cell recognition) or ability to induce DNA damage, we are developing tools and imaging systems to answer these issues. First, we designed a cellular assay to compare CDT activities and relate its mode of action to cellular effects. Secondly, a live-cell microscopy approach is developed to explore the impact of CDT on cell cycle. Overall, the goal of this project is to develop innovative cellular approaches to better characterise CDT mechanism of action as a first step to get more insight on the effects of the toxin on human health.