Structure elucidation of colibactin and its DNA cross-links

Abstract

Colibactin is a complex secondary metabolite produced by some genotoxic gut Escherichia coli strains. The presence of colibactin-producing bacteria correlates with the frequency and severity of colorectal cancer in humans. However, because colibactin has not been isolated or structurally characterized, studying the physiological effects of colibactin-producing bacteria in the human gut has been difficult. We used a combination of genetics, isotope labeling, tandem mass spectrometry, and chemical synthesis to deduce the structure of colibactin. Our structural assignment accounts for all known biosynthetic and cell biology data and suggests roles for the final unaccounted enzymes in the colibactin gene cluster.

Fig. 1.. Structures and reaction pathways of selected clb biosynthetic products.

(A) Established mechanism of DNA mono-alkylation by clb metabolites formed in wild-type cultures. (B) Structures of clb metabolites formed in ΔclbP clb⁺ E. coli cultures. The green spheres in structure 5 denote the carbon atoms derived from glycine.

Fig. 2.. Selected HRMS signals deriving from treatment of linearized pUC19 DNA with clb⁺ E. coli, followed by digestion.

(A) Natural abundance and stable isotope derivatives of 9. The highest-intensity labeled peaks (green) were selected for analysis, except for Ser, which was extensively metabolized. All selected ions were confirmed by means of tandem MS. [M+2H]²⁺ ions are marked. (B) Structure of the colibactin-bis (adenine) adduct 9. (C) Structures of the daughter ions 10 to 12. (D) The DNA adducts 13 and 14.

Fig. 3.. Proposed biosynthesis of (pre)colibactin.

The early stages in the biosynthetic pathway are grayed for clarity. The heterodimerization is highlighted in the red box (top right). Intermediates B to E are also possible substrates for thioesterase ClbQ, although promiscuous ClbQ has a known preference for hydrolyzing intermediates toward the middle of the assembly line. Amino acids are depicted at their sites of pathway entry. Domain abbreviations are C, condensation; A, adenylation; E, epimerization; KS, ketosynthase; KR, ketoreductase; DH, dehydratase; ER, enoylreductase; AT*, inactivated acyltransferase (AT); Cy, dual condensation/cyclization; and Ox, oxidase.

Fig. 4.. Stimulation, genetic dependence, and isotopic labeling of natural colibactin (17).

(A) Genetic dependence of colibactin (17) production in clb⁺ DH10B and Nissle 1917. n = 3 biological replicates; error represents standard deviation. n.d., not detected. (B) Isotopic labeling pattern of colibactin (17). (C) Results of isotopic labeling studies of colibactin (17) in Nissle 1917 (ΔclbS). [U-¹³C]-Gly labeling was conducted in both Nissle 1917 (ΔclbS), which also led to glycine-derived serine labeling, and clb⁺ DH10B.The highest-intensity labeled peaks (green) were selected for analysis. [M+H]⁺ ions are marked unless otherwise noted. (D) Ions observed in the tandem MS of colibactin (17). The two structures of ion 20 are equally plausible based on the MS data.

Fig. 5.. Genetic, tandem MS, and isotopic labeling support for precolibactin 1489 (18).

(A) Precolibactin 1489 (18) biosynthesis requires all biosynthetic enzymes in the clb gene cluster. Precolibactin 886 (8a) is still produced in clbL, clbO, or clbQ mutants. n = 5 biological replicates; error represents standard deviation. (B) Position of isotopic labels in precolibactin 1489 (18), as established by means of tandem MS analysis. (C) Isotopic labeling studies of precolibactin 1489 (18) in a clb⁺ DH10B strain deficient in ClbP catalytic activity. (D) Proposed structures of ions 21 to 23 (fig. S125C) derived from the tandem MS of precolibactin 1489 (18).

Fig. 6.. Synthesis of the colibactin precursor 38.

(A) Retrosynthetic analysis of colibactin (17). (B) Synthesis of the β-ketoester 25. (C) Synthesis of the α-silyloxyketone 26 and the linear precursors 24 and 38.

Fig. 7.. Confirmation of the predicted structure of colibactin (17).

(A) Cyclization of intermediate 38 to colibactin (17). (B) LC-MS coinjection analysis of colibactin (17): natural (top), synthetic (middle), and coinjection (bottom). (C) Tandem MS data of natural colibactin (17, top) and synthetic colibactin (17, bottom). Collision energy = 30 eV. Additional data is available in fig. S127. (D) DNA cross-linking assay by using linearized pUC19 DNA and synthetic intermediate 38. (E) Tandem MS data of the bis(adenine) adduct 9 derived from natural and synthetic colibactin (17).