The colibactin warhead crosslinks DNA

Abstract

Members of the human microbiota are increasingly being correlated to human health and disease states, but the majority of the underlying microbial metabolites that regulate host-microbe interactions remain largely unexplored. Select strains of Escherichia coli present in the human colon have been linked to the initiation of inflammation-induced colorectal cancer through an unknown small-molecule-mediated process. The responsible non-ribosomal peptide-polyketide hybrid pathway encodes 'colibactin', which belongs to a largely uncharacterized family of small molecules. Genotoxic small molecules from this pathway that are capable of initiating cancer formation have remained elusive due to their high instability. Guided by metabolomic analyses, here we employ a combination of NMR spectroscopy and bioinformatics-guided isotopic labelling studies to characterize the colibactin warhead, an unprecedented substituted spirobicyclic structure. The warhead crosslinks duplex DNA in vitro, providing direct experimental evidence for colibactin's DNA-damaging activity. The data support unexpected models for both colibactin biosynthesis and its mode of action.

Figure 1. Key colibactin pathway (clb)-dependent shunt metabolites

Analogous to the decarboxylation of clb-assembly line derailment product 14 to 15, characterized shunt precolibactin 27 most likely arises from decarboxylation of transient derailment product 26. ClbP-mediated cleavage appears to be promiscuous in our analysis, leading to N-terminal N-acyl-D-asparagines, such as 1, and detectable C-terminal products, such as 21 and 30. Structures are numbered in accordance with increasing biosynthetic complexity as illustrated in Figure 3.

Figure 2. Colibactin pathway (clb)-dependent molecular network

(a) clb-dependent metabolites detected in IHE3034-derived wildtype (clb+ or wt) and clbP (ΔclbP) mutant cultures. A heat map of ionization intensities for the ΔclbP metabolites is shown. (b) System-wide ¹³C-isotopic incorporations determined from HRMS analysis of L-[U-¹³C]-amino acid substrates, Asn, Ala, Met, Gly, Cys, and Ser. If a specific amino acid incorporation was detected, the metabolite node was color coded as follows: Asn (red), Ala (blue), Met (green), Gly (orange), and Cys (purple). For L-[¹³C₅]-Met, we only observed ¹³C₄ products, indicating amino-butyryl incorporation (green), which were not labeled by [2,2,3,3-D]–ACC. We also observed 1, 2, and 3 Cys incorporations as denoted by the colored map. L-[U-¹³C]-Ser (¹³C₃ and ¹³C₂) labeled metabolites were not detected. Grey nodes were not detected in the labeling experiments. Connectivity strength is represented by the thickness of the lines linking individual nodes.

Figure 3. Proposed assembly line biosynthetic model for precolibactin A

(a) Proposed biosynthesis for the clb assembly line derailment product 15 and its structurally related shunt metabolites (1, 10-21; Supplementary Table S5). Experimentally supported metabolites are indicated with bold numbers, which can result from thioester hydrolysis. Arrows represent NRPS and PKS enzymes with each acronym representing a distinct catalytic domain. Malonyl-CoA and amino acid substrate incorporations, supported by universally ¹³C-labeled amino acid feeding experiments, are indicated at their proposed cognate carrier proteins (T domains). (b) Predicted and experimentally supported assembly line biosynthesis of advanced derailment products (24-31) and precolibactin A (32). Our studies indicate that the proposed ClbH-dependent ACC formation is ultimately derived from the aminobutyryl moiety of L-Met. (c) Proposed structure of precolibactin A (32) and detected N-acyl-D-Asn ClbP cleavage products (1-9; Supplementary Table S5). NRPS and PKS domains: C, condensation; A, adenylation; T, thiolation sequence of acyl- and peptidyl-carrier proteins; E, epimerization; KS, ketosynthase; AT, acyl-transferase; KR, ketoreductase; DH, dehydratase; ER, enoyl-reductase; Cy, condensation/cyclization; Ox, oxidase; TE, thioesterase. *, Denotes evolutionarily deteriorated cis-AT domain in a trans-AT PKS. The bioinformatics predicted thiazoline and thiazole-containing tail was supported by HRMS, MS/MS, and isotopic labeling studies, and its predicted heterocycle order and stereochemistry (#) need further validation.

Figure 4. The colibactin warhead crosslinks DNA

(a) DNA alkylation by the warhead is hypothesized to occur through a homo-Michael addition reaction, followed by a (b) pseudo-intramolecular Michael addition reaction, generating a DNA interstrand crosslink. (c) DNA crosslinking was observed using an EcoRI-linearized pBR322 plasmid in the presence of 27 (0.5-1.0 mM) with or without reducing agents. DMSO (-) and 15 controls did not lead to detectable activity. Reactions were performed at 37 °C for 20 h (Gel 1). Under these conditions, positive control mitomycin C + DTT caused substantial DNA degradation. Consequently, experiment was repeated with a shorter incubation time and reduced temperature for mitomycin C + DTT (2 h, 20 °C), while the DMSO (-) control and 27 were incubated at 37 °C for 20 h with and without β-ME (Gel 2). mit C, mitomycin C; DTT, dithiothreitol; β-ME, β-mercaptoethanol; I, single-stranded DNA; II, cross-linked DNA.