A Mechanistic Model for Colibactin-Induced Genotoxicity

Abstract

Precolibactins and colibactins represent a family of natural products that are encoded by the clb gene cluster and are produced by certain commensal, extraintestinal, and probiotic E. coli. clb+ E. coli induce megalocytosis and DNA double-strand breaks in eukaryotic cells, but paradoxically, this gene cluster is found in the probiotic Nissle 1917. Evidence suggests precolibactins are converted to genotoxic colibactins by colibactin peptidase (ClbP)-mediated cleavage of an N-acyl- D-Asn side chain, and all isolation efforts have employed ΔclbP strains to facilitate accumulation of precolibactins. It was hypothesized that colibactins form unsaturated imines that alkylate DNA by cyclopropane ring opening (2 → 3). However, as no colibactins have been isolated, this hypothesis has not been tested experimentally. Additionally, precolibactins A–C (7–9) contain a pyridone that cannot generate the unsaturated imines that form the basis of this hypothesis. To resolve this, we prepared 13 synthetic colibactin derivatives and evaluated their DNA binding and alkylation activity. We show that unsaturated imines, but not the corresponding pyridone derivatives, potently alkylate DNA. The imine, unsaturated lactam, and cyclopropane are essential for efficient DNA alkylation. A cationic residue enhances activity. These studies suggest that precolibactins containing a pyridone are not responsible for the genotoxicity of the clb cluster. Instead, we propose that these are off-pathway fermentation products produced by a facile double cyclodehydration route that manifests in the absence of viable ClbP. The results presented herein provide a foundation to begin to connect metabolite structure with the disparate phenotypes associated with clb+ E. coli.

Domain-Targeted Metabolomics Delineates the Heterocycle Assembly Steps of Colibactin Biosynthesis

“…12 ClbH is hypothesized to incorporate this functional group using a 1-aminocyclopropane-1-carboxylate (ACC) extender unit, although its biosynthesis remains unknown. In plants, ACC is an intermediate in ethylene biosynthesis and is derived from methionine via a pyridoxal phosphate (PLP)-dependent mechanism.…”

“…The lactams and pyridones can currently be accessed synthetically through common linear intermediates via selective cyclization reactions. 11g,12 When the linear products are synthesized as their deacylated (ClbP-cleaved) forms, they readily cyclize to lactams, which exhibit potent DNA alkylation activities in vitro. 12 N -acylated linear precursors, however, readily form pyridones through a double cyclodehydration sequence under mildly acidic or basic conditions.…”

“…11g,12 When the linear products are synthesized as their deacylated (ClbP-cleaved) forms, they readily cyclize to lactams, which exhibit potent DNA alkylation activities in vitro. 12 N -acylated linear precursors, however, readily form pyridones through a double cyclodehydration sequence under mildly acidic or basic conditions. 11g Control experiments established that pyridones do not alkylate DNA.…”

ClbS Is a Cyclopropane Hydrolase That Confers Colibactin Resistance

“…This imine has been synthesized, fully characterized, and shown to extensively damage DNA in vitro . 9 This substrate is the N -methylamide analog of metabolite 1 , which was detected in clbP -encoding bacterial cultures in vivo and which has also been characterized by synthesis. 10 The structures of fully-elaborated (pre)-colibactins, accounting for all of the genes in the clb gene cluster, are unknown.…”

“…C4 was previously proposed to be electrophilic, 13 although 2 did not lead to observable DNA interstrand cross-links in vitro with plasmid DNA. 9 It is also interesting that decomposition likely proceeds through the reactive organic peroxide. A role for this in vitro sequence in DNA alkylation and genotoxicity remains unknown, but many natural products damage DNA via the generation of reactive oxygen species (ROS).…”