Limits...
The colibactin warhead crosslinks DNA.

Vizcaino MI, Crawford JM - Nat Chem (2015)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Chemistry, Yale University, New Haven, Connecticut 06510, USA [2] Chemical Biology Institute, Yale University, West Haven, Connecticut 06516, USA.

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.

Show MeSH

Related in: MedlinePlus

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.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC4499846&req=5

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.

Mentions: Colibactin belongs to a subset of hybrid polyketide-nonribosomal peptides that undergo a prodrug activation mechanism18-21. During colibactin maturation, the inner membrane peptidase ClbP cleaves “precolibactins” in the periplasm22,23, liberating N-terminal N-acyl-D-asparagines and the unknown C-terminal “colibactins”17,20,24. To focus our colibactin structural characterization efforts, we initially assessed secondary metabolic flux of clb pathway-dependent molecular features in wildtype (clb+) and clbP mutant (ΔclbP) strains (Supplementary Table S1) by comparative metabolomics (Supplementary Table S2) and network analyses (Supplementary Fig. S2). By comparing MS ionization intensities of closely related features in a given experiment, a qualitative assessment of metabolite abundances can be determined relative to one another in a molecular network cluster. We analyzed the organic-extractable metabolomes from clb+ and ΔclbP heterologous systems, containing the clb pathways from the meningitis isolate E. coli IHE3034 expressed in E. coli DH10B, and homologous systems of the probiotic E. coli Nissle 191717. ClbP was previously determined to cleave 1517, and our metabolomic analyses indicate that ClbP is promiscuous. Consequently, we focused on the structural characterization of the most abundant advanced precolibactins from the clbP mutants (Fig. 2, Supplementary Table S2) to correlate the colibactin biosynthetic pathway to precolibactin structure(s).


The colibactin warhead crosslinks DNA.

Vizcaino MI, Crawford JM - Nat Chem (2015)

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.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4499846&req=5

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.
Mentions: Colibactin belongs to a subset of hybrid polyketide-nonribosomal peptides that undergo a prodrug activation mechanism18-21. During colibactin maturation, the inner membrane peptidase ClbP cleaves “precolibactins” in the periplasm22,23, liberating N-terminal N-acyl-D-asparagines and the unknown C-terminal “colibactins”17,20,24. To focus our colibactin structural characterization efforts, we initially assessed secondary metabolic flux of clb pathway-dependent molecular features in wildtype (clb+) and clbP mutant (ΔclbP) strains (Supplementary Table S1) by comparative metabolomics (Supplementary Table S2) and network analyses (Supplementary Fig. S2). By comparing MS ionization intensities of closely related features in a given experiment, a qualitative assessment of metabolite abundances can be determined relative to one another in a molecular network cluster. We analyzed the organic-extractable metabolomes from clb+ and ΔclbP heterologous systems, containing the clb pathways from the meningitis isolate E. coli IHE3034 expressed in E. coli DH10B, and homologous systems of the probiotic E. coli Nissle 191717. ClbP was previously determined to cleave 1517, and our metabolomic analyses indicate that ClbP is promiscuous. Consequently, we focused on the structural characterization of the most abundant advanced precolibactins from the clbP mutants (Fig. 2, Supplementary Table S2) to correlate the colibactin biosynthetic pathway to precolibactin structure(s).

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Chemistry, Yale University, New Haven, Connecticut 06510, USA [2] Chemical Biology Institute, Yale University, West Haven, Connecticut 06516, USA.

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.

Show MeSH
Related in: MedlinePlus