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

Abstract

Colibactins 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.

The reactivity of an unusual amidase may explain colibactin’s DNA cross-linking activity

“…Detection of 6 in the absence of ClbJ and ClbP indicated that ClbJ was not required for its biosynthesis. To confirm this result, we fed [1,[2][3][4][5][6][7][8][9][10][11][12][13] C2]Gly to a glycine auxotrophic BACpksΔclbP strain. We observed a +2 mass shift in the known glycine-derived precolibactin 3, but not in 6 (Figure S13), confirming that pathway A is not the source of 6.…”

“…[5][6][7] Recent studies have indicated colibactin's genotoxicity likely arises from a direct interaction with DNA, as we and others have reported the accumulation of interstrand cross-links in human cell lines incubated with pks + E. coli. [8][9] However, the underlying chemical and enzymatic basis for this activity remains unclear.…”

Colibactin‐positive Escherichia coli induce a procarcinogenic immune environment leading to immunotherapy resistance in colorectal cancer

“…These CoPEC strains were more prevalent in most aggressive CRC tumors (TNM stage) . Colibactin is a genotoxin that induces oxidative stress and DNA double‐strand breaks in epithelial cells, leading to genomic instability . In addition to this genotoxic effect, CoPEC strains induce cellular senescence associated with the production of growth factors, such as hepatocyte growth factor, leading to an increase of tumor growth in chemically induced colitis‐associated CRC (CAC) models .…”

“…27,29 Colibactin is a genotoxin that induces oxidative stress and DNA double-strand breaks in epithelial cells, leading to genomic instability. 24,[30][31][32] In addition to this genotoxic effect, CoPEC strains induce cellular senescence associated with the production of growth factors, such as hepatocyte growth factor, leading to an increase of tumor growth in chemically induced colitis-associated CRC (CAC) models. 33 In addition, experiments using other CAC mouse models (IL-10 −/− /AOM and APC Min/+ IL10 −/− models) showed that CoPEC were able to strongly persist in the gut and to induce epithelial damage and cell proliferation.…”

Photoactivated Colibactin Probes Induce Cellular DNA Damage

“…[11a,b] This is supported by arecent study showing that infection of cultured human cells with pks + bacteria results in cross-linking of genomic DNAa nd, furthermore,t hat the addition of extracellular DNAreduced this effect. [23] Healy et al recently reported that colibactin analogues featuring the unsaturated imine found in 6 only alkylated purified DNA( whilst dimethyl analogues and those representing the pyridone class of colibactins were inactive). [15a] Subsequent work using optimized assay conditions revealed that these compounds are also capable of DNAc rosslinking,h owever the adducts are more labile than those induced by pks + E. coli.…”