Detection of DNA damage by alkaline comet assay in mouse colonic mucosa.

We recently characterized the association between DNA damage and immunoresponse in vivo in colonic mucosa of mice infected with a Salmonella Typhimurium strain expressing a genotoxin, known as typhoid toxin. In this protocol, we describe the specific steps for assessing DNA damage by the alkaline comet assay of colonic mucosal samples. The description of the comet assay protocol follows the international guidelines (Minimum Information for Reporting on the Comet Assay [Moller et al., 2020]). For complete details on the use and execution of this protocol, please refer to Martin et al. (2021).

Short-Term and Long-Term Carcinogenic Effects of Food Contaminants (4-Hydroxynonenal and Pesticides) on Colorectal Human Cells: Involvement of Genotoxic and Non-Genomic Mechanisms.

To investigate environmental impacts upon colorectal carcinogenesis (CRC) by diet, we assessed two western diet food contaminants: 4-hydroxynonenal (HNE), a major lipid peroxidation product neoformed during digestion, and a mixture of pesticides. We used human colonic cell lines ectopically eliciting varied genetic susceptibilities to CRC: the non-transformed human epithelial colonic cells (HCECs) and their five isogenic cell lines with the loss of APC (Adenomatous polyposis coli) and TP53 (Tumor protein 53) and/or ectopic expression of mutated KRAS (Kristen-ras). These cell lines have been exposed for either for a short time (2-24 h) or for a long period (3 weeks) to 1 µM HNE and/or 10 µM pesticides. After acute exposure, we did not observe any cytotoxicity or major DNA damage. However, long-term exposure to pesticides alone and in mixture with HNE induced clonogenic transformation in normal HCECs, as well as in cells representing later stages of carcinogenesis. It was associated with genotoxic and non-genomic mechanisms (cell growth, metabolic reprogramming, cell mobility and epithelial-mesenchymal transition) depending on genetic susceptibility. This study demonstrated a potential initiating and promoting effect of food contaminants on CRC after long-term exposure. It supports that these contaminants can accelerate carcinogenesis when mutations in oncogenes or tumor suppressor genes occur.

Chronic exposure to Cytolethal Distending Toxin (CDT) promotes a cGAS-dependent type I interferon response.

The Cytolethal Distending Toxin (CDT) is a bacterial genotoxin produced by pathogenic bacteria causing major foodborne diseases worldwide. CDT activates the DNA Damage Response and modulates the host immune response, but the precise relationship between these outcomes has not been addressed so far. Here, we show that chronic exposure to CDT in HeLa cells or mouse embryonic fibroblasts promotes a strong type I interferon (IFN) response that depends on the cytoplasmic DNA sensor cyclic guanosine monophosphate (GMP)-adenosine monophosphate (AMP) synthase (cGAS) through the recognition of micronuclei. Indeed, despite active cell cycle checkpoints and in contrast to other DNA damaging agents, cells exposed to CDT reach mitosis where they accumulate massive DNA damage, resulting in chromosome fragmentation and micronucleus formation in daughter cells. These mitotic phenotypes are observed with CDT from various origins and in cancer or normal cell lines. Finally, we show that CDT exposure in immortalized normal colonic epithelial cells is associated to cGAS protein loss and low type I IFN response, implying that CDT immunomodulatory function may vary depending on tissue and cell type. Thus, our results establish a direct link between CDT-induced DNA damage, genetic instability and the cellular immune response that may be relevant in the context of natural infection associated to chronic inflammation or carcinogenesis.

Cytolethal Distending Toxin Promotes Replicative Stress Leading to Genetic Instability Transmitted to Daughter Cells.

The cytolethal distending toxin (CDT) is produced by several Gram-negative pathogenic bacteria. In addition to inflammation, experimental evidences are in favor of a protumoral role of CDT-harboring bacteria such as Escherichia coli, Campylobacter jejuni, or Helicobacter hepaticus. CDT may contribute to cell transformation in vitro and carcinogenesis in mice models, through the genotoxic action of CdtB catalytic subunit. Here, we investigate the mechanism of action by which CDT leads to genetic instability in human cell lines and colorectal organoids from healthy patients' biopsies. We demonstrate that CDT holotoxin induces a replicative stress dependent on CdtB. The slowing down of DNA replication occurs mainly in late S phase, resulting in the expression of fragile sites and important chromosomic aberrations. These DNA abnormalities induced after CDT treatment are responsible for anaphase bridge formation in mitosis and interphase DNA bridge between daughter cells in G1 phase. Moreover, CDT-genotoxic potential preferentially affects human cycling cells compared to quiescent cells. Finally, the toxin induces nuclear distension associated to DNA damage in proliferating cells of human colorectal organoids, resulting in decreased growth. Our findings thus identify CDT as a bacterial virulence factor targeting proliferating cells, such as human colorectal progenitors or stem cells, inducing replicative stress and genetic instability transmitted to daughter cells that may therefore contribute to carcinogenesis. As some CDT-carrying bacterial strains were detected in patients with colorectal cancer, targeting these bacteria could be a promising therapeutic strategy.

Functional Study of Haemophilus ducreyi Cytolethal Distending Toxin Subunit B.

The Cytolethal Distending Toxin (CDT) is produced by many Gram-negative pathogenic bacteria responsible for major foodborne diseases worldwide. CDT induces DNA damage and cell cycle arrest in host-cells, eventually leading to senescence or apoptosis. According to structural and sequence comparison, the catalytic subunit CdtB is suggested to possess both nuclease and phosphatase activities, carried by a single catalytic site. However, the impact of each activity on cell-host toxicity is yet to be characterized. Here, we analyze the consequences of cell exposure to different CDT mutated on key CdtB residues, focusing on cell viability, cell cycle defects, and DNA damage induction. A first class of mutant, devoid of any activity, targets putative catalytic (H160A), metal binding (D273R), and DNA binding residues (R117A-R144A-N201A). The second class of mutants (A163R, F156-T158, and the newly identified G114T), which gathers mutations on residues potentially involved in lipid substrate binding, has only partially lost its toxic effects. However, their defects are alleviated when CdtB is artificially introduced inside cells, except for the F156-T158 double mutant that is defective in nuclear addressing. Therefore, our data reveal that CDT toxicity is mainly correlated to CdtB nuclease activity, whereas phosphatase activity may probably be involved in CdtB intracellular trafficking.