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Several commensal and pathogenic Gram-negative bacteria produce DNA-damaging toxins that are considered bona fide carcinogenic agents. The microbiota of colorectal cancer (CRC) patients is enriched in genotoxin-producing bacteria, but their role in the pathogenesis of CRC is poorly understood. The adenomatous polyposis coli (APC) gene is mutated in familial adenomatous polyposis and in the majority of sporadic CRCs. We investigated whether the loss of APC alters the response of colonic epithelial cells to infection by Salmonella enterica, the only genotoxin-producing bacterium associated with cancer in humans. Using 2D and organotypic 3D cultures, we found that APC deficiency was associated with sustained activation of the DNA damage response, reduced capacity to repair different types of damage, including DNA breaks and oxidative damage, and failure to induce cell cycle arrest. The reduced DNA repair capacity and inability to activate adequate checkpoint responses was associated with increased genomic instability in APC-deficient cells exposed to the genotoxic bacterium. Inhibition of the checkpoint response was dependent on activation of the phosphatidylinositol 3-kinase pathway. These findings highlight the synergistic effect of the loss of APC and infection with genotoxin-producing bacteria in promoting a microenvironment conducive to malignant transformation.

Bacterial genotoxins cause DNA damage in eukaryotic cells, resulting in activation of the DNA damage response (DDR) in vitro. These toxins are produced by Gram-negative bacteria, enriched in the microbiota of inflammatory bowel disease (IBD) and colorectal cancer (CRC) patients. However, their role in infection remains poorly characterized. We address the role of typhoid toxin in modulation of the host-microbial interaction in health and disease. Infection with a genotoxigenic Salmonella protects mice from intestinal inflammation. We show that the presence of an active genotoxin promotes DNA fragmentation and senescence in vivo, which is uncoupled from an inflammatory response and unexpectedly associated with induction of an anti-inflammatory environment. The anti-inflammatory response is lost when infection occurs in mice with acute colitis. These data highlight a complex context-dependent crosstalk between bacterial-genotoxin-induced DDR and the host immune response, underlining an unexpected role for bacterial genotoxins.

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 how to assess the extent and features of infiltrating macrophages by double immunofluorescence. Total macrophage population was determined using an F4/80 antibody, whereas the specific M2-like population was assessed using a CD206 antibody. For complete details on the use and execution of this protocol, please refer to Martin et al. (2021).

Expression of typhoid toxin in Salmonella Typhimurium causes DNA damage, activating the DNA damage response (DDR), in absence of an inflammatory response in the colonic mucosa of infected mice. The anti-inflammatory effect is tissue specific and is not observed in the liver, suggesting that the local immune microenvironment modulates the DDR outcome.

To assess the role of the immune cells in the DDR outcome induced by the genotoxigenic Salmonella, we have initiated the development of an immunocompetent 3D colonic mucosal model based on a collagen matrix containing colonic fibroblasts and different subtypes of immune cells, overlayed with colonic epithelial cells.

Embedding of peripheral blood mononuclear cells in the collagen matrix did not influenced either the tissue integrity or the activation of the DDR, observed exclusively upon infection with the genotoxigenic strain. However, embedding of T cells, monocytes, or non-polarized macrophages altered the pattern of the DDR and the toxin specific effect was lost. Presence of macrophages was further associated with alteration of the epithelial layer integrity. This effect was infection-dependent, but not toxin specific.

Our data demonstrated that addition of immune cells to a 3D mucosal model altered the DDR induced by a genotoxigenic bacterium, highlighting the need to develop and optimize immunocompetent in vitro models.