title: 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.
authors.affiliation: Department of Chemistry, Yale University , New Haven, Connecticut 06520, United States.|Chemical Biology Institute, Yale University , West Haven, Connecticut 06516, United States.
authors.forename: Alan R
authors.initials: AR
authors.lastname: Healy
authors.affiliation: Department of Chemistry, Yale University , New Haven, Connecticut 06520, United States.
authors.forename: Herman
authors.initials: H
authors.lastname: Nikolayevskiy
authors.affiliation: Chemical Biology Institute, Yale University , West Haven, Connecticut 06516, United States.|Department of Molecular, Cellular, and Developmental Biology, Yale University , New Haven, Connecticut 06520, United States.
authors.forename: Jaymin R
authors.initials: JR
authors.lastname: Patel
authors.affiliation: Department of Chemistry, Yale University , New Haven, Connecticut 06520, United States.|Chemical Biology Institute, Yale University , West Haven, Connecticut 06516, United States.|Department of Microbial Pathogenesis, Yale School of Medicine , New Haven, Connecticut 06536, United States.
authors.forename: Jason M
authors.initials: JM
authors.lastname: Crawford
authors.affiliation: Department of Chemistry, Yale University , New Haven, Connecticut 06520, United States.|Department of Pharmacology, Yale School of Medicine , New Haven, Connecticut 06520, United States.
authors.forename: Seth B
authors.initials: SB
authors.lastname: Herzon
mesh_terms: D001688:Biological Products / Q000138:chemical synthesis / Q000737:chemistry / Q000633:toxicity*; D053837:DNA Cleavage / Q000187:drug effects; D004926:Escherichia coli / Q000737:chemistry* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D015394:Molecular Structure; D005810:Multigene Family / Q000235:genetics; D009153:Mutagens / Q000138:chemical synthesis / Q000737:chemistry / Q000633:toxicity*; D010447:Peptide Hydrolases / Q000235:genetics / Q000378:metabolism; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry / Q000235:genetics / Q000633:toxicity*; D010641:Phenotype; D061065:Polyketides / Q000138:chemical synthesis / Q000737:chemistry / Q000633:toxicity*
chemical_list: D001688:Biological Products; D029968:Escherichia coli Proteins; D009153:Mutagens; D010455:Peptides; D061065:Polyketides; C569566:colibactin; C577770:ClbP protein, E coli; D010447:Peptide Hydrolases