A Mechanistic Model for Colibactin-Induced Genotoxicity

J Am Chem Soc. 2016 Dec 7;138(48):15563-15570. doi: 10.1021/jacs.6b10354. Epub 2016 Nov 28.


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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Biological Products / chemical synthesis
  • Biological Products / chemistry
  • Biological Products / toxicity*
  • DNA Cleavage / drug effects
  • Escherichia coli / chemistry*
  • Escherichia coli / genetics
  • Escherichia coli Proteins / genetics
  • Escherichia coli Proteins / metabolism
  • Molecular Structure
  • Multigene Family / genetics
  • Mutagens / chemical synthesis
  • Mutagens / chemistry
  • Mutagens / toxicity*
  • Peptide Hydrolases / genetics
  • Peptide Hydrolases / metabolism
  • Peptides / chemical synthesis
  • Peptides / chemistry
  • Peptides / genetics
  • Peptides / toxicity*
  • Phenotype
  • Polyketides / chemical synthesis
  • Polyketides / chemistry
  • Polyketides / toxicity*


  • Biological Products
  • Escherichia coli Proteins
  • Mutagens
  • Peptides
  • Polyketides
  • colibactin
  • ClbP protein, E coli
  • Peptide Hydrolases