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

Claude Petit,Eric Oswald,Julien Vignard,Nadège Bossuet-Greif,Jean-Philippe Nougayrède,Damien Dubois,Frédéric Taieb,Gladys Mirey,Philippe J Sansonetti,Jorge E Galan

doi:10.1128/mbio.02393-17

Abstract

ABSTRACTColibactins are hybrid polyketide-nonribosomal peptides produced by Escherichia coli, Klebsiella pneumoniae, and other Enterobacteriaceae harboring the pks genomic island. These genotoxic metabolites are produced by pks-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 E. coli. 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 clbP 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 in cellulo, 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.

Highlights

Colibactins are hybrid polyketide-nonribosomal peptides produced by Escherichia coli, Klebsiella pneumoniae, and other Enterobacteriaceae harboring the pks genomic island
Increasing the amount of extracellular DNA further decreased the phosphorylation of histone H2AX on S139 (p-H2AX) response (Fig. 1b and c). These results suggest that the extracellular double-strand DNA captured the genotoxin, resulting in reduced DNA damage in HeLa cells exposed to colibactin-producing E. coli
In this study, we addressed the issue of the mode of action on DNA and resulting genotoxicity in the host cell of the natural colibactins produced by pksϩ enterobacteria

Summary

Introduction

Colibactins are hybrid polyketide-nonribosomal peptides produced by Escherichia coli, Klebsiella pneumoniae, and other Enterobacteriaceae harboring the pks genomic island. A similar DNA-damaging mechanism is observed in cellulo, where interstrand cross-links are detected in the genomic DNA of cultured human cells exposed to colibactin-producing bacteria. 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. Precolibactins are cleaved by the periplasmic peptidase ClbP to remove an N-terminal precursor scaffold (myristoyl-asparagine) and generate active colibactins [23,24,25] This prodrug export and activation pathway likely represents a self-protection mechanism preventing self-toxicity to the producing bacteria. This protection is observed in cultured human cells transfected with the clbS gene [26]

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Results

Conclusion