The human gut bacterial genotoxin colibactin alkylates DNA

Bacterial warhead targets DNA The bacterial toxin colibactin causes double-stranded DNA breaks and is associated with the occurrence of bacterially induced colorectal cancer in humans. However, isolation of colibactin is difficult, and its mode of action is poorly understood. Wilson et al. studied Escherichia coli that contain the biosynthetic gene island called pks, which is associated with colibactin production (see the Perspective by Bleich and Arthur). They identified the DNA adducts that resulted from incubating pks+E. coli in human cells. To overcome the lack of colibactin for direct analysis, mimics of the pks product were synthesized. From the resulting synthetic adenine-colibactin adducts, it became evident that alkylation via a cyclopropane “warhead” breaks the DNA strands. Similar DNA adducts were then identified in the gut epithelia of mice infected with pks+E. coli. Science, this issue p. eaar7785; see also p. 689 DNA adducts in cells and animals exposed to colibactin-producing gut microbes shed light on the mode of action of a cancer-linked genotoxin. INTRODUCTION Members of the human gut microbiota have been implicated in the development and progression of colorectal cancer (CRC). These CRC-associated microorganisms may influence carcinogenesis through a variety of mechanisms, including the production of genotoxins. Colibactin is a genotoxic secondary metabolite made by organisms harboring the pks genomic island, including certain gut commensal Escherichia coli strains (pks+ E. coli). Transient infection of mammalian cells with pks+ E. coli causes cell cycle arrest, DNA double-strand breaks, and senescence. Moreover, colibactin-producing E. coli accelerate tumor progression in multiple mouse models of colitis-associated CRC and are overrepresented in patients with familial adenomatous polyposis and CRC. Despite colibactin’s strong links to cancer, the active genotoxic metabolite has eluded all isolation attempts, limiting our mechanistic understanding of this association. RATIONALE Over the past decade, multiple complementary approaches have provided indirect information about colibactin’s chemical structure. Interestingly, the isolation and structural characterization of metabolites from mutant strains of pks+ E. coli revealed that colibactin likely contains a cyclopropane ring, a reactive structural motif found in DNA alkylating natural products. This led us and others to hypothesize that colibactin may covalently modify DNA. To obtain information about the active genotoxin’s chemical structure and its mode of action, we sought to identify and structurally characterize colibactin-DNA adducts from human cells infected with pks+ E. coli. RESULTS Using untargeted liquid chromatography–mass spectrometry–based DNA adductomics, we compared the DNA adducts present in mammalian cell lines transiently infected with either pks+ E. coli or a mutant strain missing the pks genes. We discovered two adenine adducts that were specific to the cells exposed to pks+ E. coli. These adducts were confirmed to be pks-associated by feeding isotopic labeled versions of known colibactin biosynthetic precursors to the E. coli–mammalian cell system. The pks-dependent adducts were also found in human cells exposed to clinical colibactin-producing E. coli isolates and in the colonic epithelial cells of mice monocolonized with pks+ E. coli. Chemical synthesis and in vitro DNA alkylation reactions enabled the preparation of an authentic standard of the adducts. Structural characterization revealed a mixture of two diastereomeric adducts that both contain a 5-hydroxypyrrolidin-2-one ring system with an attached N3-substituted adenine ring. These DNA adducts are generated from ring opening of a reactive, cyclopropane-containing electrophilic warhead, confirming the importance of this structural feature for colibactin’s in vivo activity. Because these adducts are too small to derive from the final colibactin structure, we hypothesize that they arise from decomposition of a larger, unstable colibactin-DNA interstrand cross-link. Using a CometChip assay, we detected interstrand cross-link formation in cells infected with pks+ E. coli at the same time point at which we identified the characterized DNA adducts, supporting this proposal. CONCLUSION Our results provide direct evidence that the gut bacterial genotoxin colibactin alkylates DNA in vivo, providing mechanistic insights into how colibactin may contribute to CRC. The ability of pks+ E. coli to generate DNA adducts in mammalian cells and in mice strengthens support for the involvement of colibactin in cancer development or progression. Bulky DNA adducts, especially interstrand cross-links, are often cytotoxic and can lead to mutations if not accurately repaired. Colibactin-mediated DNA damage and the ensuing genomic instability could thus potentially be an underlying mediator of colorectal carcinogenesis. The colibactin-derived DNA adducts we identified could serve as a biomarker of pks+ E. coli exposure and will ultimately help to address the question of whether DNA damage inflicted by colibactin-producing gut bacteria contributes to CRC development and progression in humans. Gut commensal E. coli strains associated with CRC produce a DNA-alkylating genotoxin. (Top) The cyclopropane ring found in pks-dependent metabolites led us to hypothesize that colibactin alkylates DNA. Me, methyl. (Bottom) Untargeted DNA adductomics revealed colibactin-derived DNA adducts in human cells exposed to colibactin-producing E. coli. These adducts also form in mice colonized with pks+ E. coli, confirming that colibactin alkylates DNA in vivo and strengthening its link to cancer. Certain Escherichia coli strains residing in the human gut produce colibactin, a small-molecule genotoxin implicated in colorectal cancer pathogenesis. However, colibactin’s chemical structure and the molecular mechanism underlying its genotoxic effects have remained unknown for more than a decade. Here we combine an untargeted DNA adductomics approach with chemical synthesis to identify and characterize a covalent DNA modification from human cell lines treated with colibactin-producing E. coli. Our data establish that colibactin alkylates DNA with an unusual electrophilic cyclopropane. We show that this metabolite is formed in mice colonized by colibactin-producing E. coli and is likely derived from an initially formed, unstable colibactin-DNA adduct. Our findings reveal a potential biomarker for colibactin exposure and provide mechanistic insights into how a gut microbe may contribute to colorectal carcinogenesis.