The Colibactin Genotoxin Generates DNA Interstrand Cross-Links in Infected Cells

Abstract

Colibactins are hybrid polyketide-nonribosomal peptides produced by <i>Escherichia coli</i>, <i>Klebsiella pneumoniae</i>, and other <i>Enterobacteriaceae</i> harboring the <i>pks</i> genomic island. These genotoxic metabolites are produced by <i>pks</i>-encoded peptide-polyketide synthases as inactive prodrugs called precolibactins, which are then converted to colibactins by deacylation for DNA-damaging effects. Colibactins are bona fide virulence factors and are suspected of promoting colorectal carcinogenesis when produced by intestinal <i>E. coli</i> Natural active colibactins have not been isolated, and how they induce DNA damage in the eukaryotic host cell is poorly characterized. Here, we show that DNA strands are cross-linked covalently when exposed to enterobacteria producing colibactins. DNA cross-linking is abrogated in a <i>clbP</i> mutant unable to deacetylate precolibactins or by adding the colibactin self-resistance protein ClbS, confirming the involvement of the mature forms of colibactins. A similar DNA-damaging mechanism is observed <i>in cellulo</i>, where interstrand cross-links are detected in the genomic DNA of cultured human cells exposed to colibactin-producing bacteria. The intoxicated cells exhibit replication stress, activation of ataxia-telangiectasia and Rad3-related kinase (ATR), and recruitment of the DNA cross-link repair Fanconi anemia protein D2 (FANCD2) protein. In contrast, inhibition of ATR or knockdown of FANCD2 reduces the survival of cells exposed to colibactin-producing bacteria. These findings demonstrate that DNA interstrand cross-linking is the critical mechanism of colibactin-induced DNA damage in infected cells.<b>IMPORTANCE</b> Colorectal cancer is the third-most-common cause of cancer death. In addition to known risk factors such as high-fat diets and alcohol consumption, genotoxic intestinal <i>Escherichia coli</i> bacteria producing colibactin are proposed to play a role in colon cancer development. Here, by using transient infections with genotoxic <i>E. coli</i>, we showed that colibactins directly generate DNA cross-links <i>in cellulo</i> Such lesions are converted into double-strand breaks during the repair response. DNA cross-links, akin to those induced by metabolites of alcohol and high-fat diets and by widely used anticancer drugs, are both severely mutagenic and profoundly cytotoxic lesions. This finding of a direct induction of DNA cross-links by a bacterium should facilitate delineating the role of <i>E. coli</i> in colon cancer and engineering new anticancer agents.