Herzon, S. | bioRxiv Science

bioRxiv2019

Depurination of colibactin-derived interstrand cross-links.

Colibactin is a genotoxic gut microbiome metabolite long suspected of playing an etiological role in colorectal cancer progression. Evidence suggests colibactin forms DNA interstrand cross-links (ICLs) in eukaryotic cells and activates ICL repair pathways, leading to the production of ICL-dependent DNA double-strand breaks (DSBs). Here we show that colibactin ICLs can evolve directly to DNA DSBs. Using the topology of supercoiled plasmid DNA as a proxy for alkylation adduct stability, we show that colibactin-derived ICLs are unstable toward depurination and elimination of the 3' phosphate. This pathway leads progressively to the formation of nicks SSBs and cleavage DSBs and is consistent with the earlier determination that non-homologous end joining repair-deficient cells are sensitized to colibactin-producing bacteria. The results herein further our understanding of colibactin-derived DNA damage and underscore the complexities underlying the DSB phenotype.

microbiology↗

bioRxiv2018

Model colibactins exhibit human cell genotoxicity in the absence of host bacteria

Colibactins are genotoxic secondary metabolites produced in select Enterobacteriaceae, which induce downstream DNA double-strand breaks (DSBs) in human cell lines and are thought to promote the formation of colorectal tumors. Although key structural and functional features of colibactins have been elucidated, the full molecular mechanisms regulating these phenotypes remain unknown. Here, we demonstrate that free model colibactins induce DSBs in human cell cultures and do not require delivery by host bacteria. Through domain-targeted editing, we demonstrate that a subset of native colibactins generated from observed module skipping in the nonribosomal peptide synthetase-polyketide synthase (NRPS-PKS) biosynthetic assembly line share DNA alkylation phenotypes with the model colibactins in vitro. However, module skipping eliminates the strong DNA interstrand cross-links formed by the wildtype pathway in cell culture. This product diversification during the modular NRPS-PKS biosynthesis produces a family of metabolites with varying observed mechanisms of action - DNA alkylation versus crosslinking - in cell culture. The presence of membranes separating human cells from model colibactins attenuated genotoxicity, suggesting that membrane diffusion limits colibactin activity and could account for the observed bacteria-human cell-to-cell contact phenotype. Additionally, extracellular supplementation of the colibactin resistance protein ClbS was able to intercept colibactins in an E. coli-human cell transient infection model. Our studies demonstrate that free model colibactins recapitulate cellular phenotypes associated with moduleskipped products in the native colibactin pathway and define specific protein domains that are required for efficient DNA interstrand crosslinking in the native pathway.

cancer biology↗