The human gut bacterial genotoxin colibactin alkylates DNA

Abstract

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.

Fig. 1.. pks⁺ E. coli synthesize cyclopropane-containing metabolites that may alkylate DNA.

(A) pks genomic island. Open reading frames encoding nonribosomal peptide synthetase (NRPS) (purple), polyketide synthase (PKS) (brown), hybrid NRPS/PKS (blue), peptidase (ClbP) (green), aminomalonate synthesis and transfer (grey), and other (black) enzymes are highlighted. (B) Selected cyclopropane-containing candidate precolibactins isolated and structurally characterized from pks⁺ ΔclbP E. coli, including lactam, pyridone, and macrocyclic scaffolds. (C) Illudin S and (+)-duocarmycin A are DNA alkylating metabolites that contain a cyclopropane ring.

Fig. 2.. High-resolution accurate mass (HRAM) LC-MS³ DNA adductomic analysis identifies DNA adducts in HeLa cells and mice exposed to pks⁺ E. coli.

(A) Structural features of DNA adducts and detection by neutral loss monitoring. (B) Full scan extracted ion chromatogram (EIC) of DNA adducts 1 and 2 (m/z 540.1772) in HeLa cells exposed to colibactin-producing E. coli and negative controls (HeLa cells exposed to non-colibactin producing pBeloBAC E. coli, HeLa cells alone, or when no DNA was present). (C) 1. Full scan EIC of DNA adducts 1 and 2 (m/z 540.1772). 2. Signal corresponding to the data dependent MS² events (RT = 16.87 and 17.45 min). RT = retention time. 3. Signal corresponding to MS³ events (RT = 16.88 and 17.46 min) triggered by the neutral loss of adenine. 4. MS² mass spectrum resulting from fragmentation of m/z 540.1772 which triggered the MS³ event. (D) Flowchart of experiment detecting DNA adducts 1 and 2 in mouse colonic epithelial cells. (E) Bacterial load in the feces of mice colonized with pBelo (n = 3) or pks⁺ E. coli (n = 8) for 2 weeks. (F) EIC counts of DNA adducts 1 and 2 per μg of DNA in colonic epithelial cells isolated from mice colonized with pBelo (n = 3) or pks⁺ E. coli (n = 8) for 2 weeks. EIC counts were determined by area under the curve integrations of the most abundant MS² fragmentation ion (m/z 387.1118 ± 0.0008) of the adducts 1 and 2 precursor ion (m/z 540.1772). (G) Representative MS/MS EIC of DNA adducts 1 and 2 (m/z 387.1118 [M+H-Ade-H₂O]⁺), the most abundant fragment ion of m/z 540.1772. Each symbol in (E) and (G) represents an individual mouse; error bars represent mean +/− the standard error of the mean (SEM); **** P < 0.0001 (unpaired t test).

Fig. 3.. Comparing DNA adducts generated in vivo with a synthetic standard confirms their chemical structures.

(A) In vitro DNA alkylation reaction used to generate a synthetic standard of adducts 1 and 2. ctDNA = calf-thymus DNA; PLE = pig liver esterase. (B) Chemical structures of diastereomeric DNA adducts 6 and 7 showing key 2D-NMR correlations that support the hemiaminal and N3-adenine assignments. (C) EICs of the L-[1-¹³C]Cys-labeled in vivo adducts 1 and 2 (m/z 541.1805), co-injection of synthetic standard (m/z 540.1772) with in vivo adducts 1 and 2 (m/z 541.1805 and residual unlabeled m/z 540.1772), and synthetic standard (m/z 540.1772).

Fig. 4.. The characterized DNA adducts may derive from a colibactin-DNA interstrand cross-link.

(A) Proposed model for colibactin DNA alkylation and formation of DNA adducts 1 and 2 (DNA = deoxyribonucleic acid; Ade = adenine). (B) Arrayed microwell comets from untreated HeLa cells and HeLa cells treated with pBelo or pks⁺ E. coli for 1 h at 37 °C [multiplicity of infection (MOI) = 1,000]. Scale bar indicates 100 μm. (C) Quantification of % DNA in tail from untreated HeLa cells and HeLa cells treated with pBelo or pks⁺ E. coli for 1 h at 37 °C (MOI = 1,000). Data points and error bars represent mean +/− SEM, respectively, of three independent experiments. Student’s t-test was performed to compare each treated dose to the corresponding negative controls (* P < 0.05).