ABSTRACT: The clb gene cluster encodes the biosynthesis of metabolites known as precolibactins and colibactins. The clb pathway is found in gut commensal Escherichia coli, and clb metabolites are thought to initiate colorectal cancer via DNA crosslinking. Here we report confirmation of the structural assignment of the complex clb product precolibactin 886 via a biomimetic synthetic pathway. We show that an ?-ketoimine linear precursor undergoes spontaneous cyclization to precolibactin 886 on HPLC purification. Studies of this ?-ketoimine and the related ?-dicarbonyl revealed that these compounds are unexpectedly susceptible to nucleophilic cleavage under mildly basic conditions. This cleavage pathway forms other known clb metabolites or biosynthetic intermediates and explains the difficulties in isolating fully mature biosynthetic products. This cleavage also accounts for a recently identified colibactin-adenine adduct. The colibactin peptidase ClbP deacylates synthetic precolibactin 886 to form a non-genotoxic pyridone, which suggests precolibactin 886 lies off the path of the major biosynthetic route.
Project description:The colibactins are hybrid polyketide-nonribosomal peptide natural products produced by certain strains of commensal and extraintestinal pathogenic Escherichia coli. The metabolites are encoded by the clb gene cluster as prodrugs termed precolibactins. clb(+) E. coli induce DNA double-strand breaks in mammalian cells in vitro and in vivo and are found in 55-67% of colorectal cancer patients, suggesting that mature colibactins could initiate tumorigenesis. However, elucidation of their structures has been an arduous task as the metabolites are obtained in vanishingly small quantities (?g/L) from bacterial cultures and are believed to be unstable. Herein we describe a flexible and convergent synthetic route to prepare advanced precolibactins and derivatives. The synthesis proceeds by late-stage union of two complex precursors (e.g., 28 + 17 ? 29a, 90%) followed by a base-induced double dehydrative cascade reaction to form two rings of the targets (e.g., 29a ? 30a, 79%). The sequence has provided quantities of advanced candidate precolibactins that exceed those obtained by fermentation, and is envisioned to be readily scaled. These studies have guided a structural revision of the predicted metabolite precolibactin A (from 5a or 5b to 7) and have confirmed the structures of the isolated metabolites precolibactins B (3) and C (6). Synthetic precolibactin C (6) was converted to N-myristoyl-d-asparagine and its corresponding colibactin by colibactin peptidase ClbP. The synthetic strategy outlined herein will facilitate mechanism of action and structure-function studies of these fascinating metabolites, and is envisioned to accommodate the synthesis of additional (pre)colibactins as they are isolated.
Project description:Colibactin is an as-yet-uncharacterized genotoxic secondary metabolite produced by human gut bacteria. Here we report the biosynthetic discovery of two new precolibactin molecules from Escherichia coli, including precolibactin-886, which uniquely incorporates the highly sought genotoxicity-associated aminomalonate building block into its unprecedented macrocyclic structure. This work provides new insights into the biosynthetic logic and mode of action of this colorectal-cancer-linked microbial chemical.
Project description: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 ?clbP 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.
Project description:Certain commensal Escherichia coli contain the clb biosynthetic gene cluster that codes for small molecule prodrugs known as precolibactins. Precolibactins are converted to colibactins by N-deacylation; the latter are postulated to be genotoxic and to contribute to colorectal cancer formation. Though advances toward elucidating (pre)colibactin biosynthesis have been made, the functions and mechanisms of several clb gene products remain poorly understood. Here we report the 2.1 Å X-ray structure and molecular function of ClbS, a gene product that confers resistance to colibactin toxicity in host bacteria and which has been shown to be important for bacterial viability. The structure harbors a potential colibactin binding site and shares similarity to known hydrolases. In vitro studies using a synthetic colibactin analog and ClbS or an active site residue mutant reveal cyclopropane hydrolase activity that converts the electrophilic cyclopropane of the colibactins into an innocuous hydrolysis product. As the cyclopropane has been shown to be essential for genotoxic effects in vitro, this ClbS-catalyzed ring-opening provides a means for the bacteria to circumvent self-induced genotoxicity. Our study provides a molecular-level view of the first reported cyclopropane hydrolase and support for a specific mechanistic role of this enzyme in colibactin resistance.
Project description:Colibactin represents a structurally undefined class of bacterial genotoxin inducing DNA damage and genomic instability in mammalian cells, thus promoting tumour development and exacerbating lymphopenia in animal models. The colibactin biosynthetic gene cluster (clb) has been known for ten years and it encodes a hybrid nonribosomal peptide synthetase (NRPS)/polyketide synthase (PKS) assembly line. Nevertheless, the final chemical product(s) remain unknown. Previously, we and others reported several colibactin pathway-related metabolites including N-myristoyl-d-asparagine (1) as part of a prodrug precursor that is cleaved from the putative precolibactin to form active colibactin by the peptidase ClbP. Herein, we report two new colibactin pathway-related metabolites (2 and 3) isolated from a clbP mutant of the probiotic E. coli Nissle 1917 strain. Their structures were established by HRMS and NMR. Compound 2 shows an additional 4-aminopenatanoic acid moiety with respect to 1, while 3 is characterized by the presence of an unusual 7-methyl-4-azaspiro[2.4]hept-6-en-5-one residue. Moreover, we propose the biosynthetic pathway towards both intermediates on the basis of extensive gene inactivation and feeding experiments. The identification of 2 and 3 provides further insight into colibactin biosynthesis including the involvement and formation of a rare 1-aminocyclopropanecarboxylic acid unit. Thus, our work establishes additional steps of the pathway forming the bacterial genotoxin colibactin.
Project description:Colibactins are genotoxic secondary metabolites whose biosynthesis is encoded in the clb gene cluster harbored by certain strains of gut commensal Escherichia coli. Using synthetic colibactin analogues, we previously provided evidence that colibactins alkylate DNA by addition of a nucleotide to an electrophilic cyclopropane intermediate. However, natural colibactin-nucleobase adducts have not been identified, to the best of our knowledge. Here we present the first identification of such adducts, derived from treatment of pUC19 DNA with clb + E. coli. Previous biosynthetic studies established cysteine and methionine as building blocks in colibactin biosynthesis; accordingly, we used cysteine (? cysE) and methionine (? metA) auxotrophic strains cultured in media supplemented with l-[U-13C]Cys or l-[U-13C]Met to facilitate the identification of nucleobases bound to colibactins. Using MS2 and MS3 analysis, in conjunction with the known oxidative instability of colibactin cyclopropane-opened products, we were able to characterize adenine adducts derived from cyclopropane ring opening. This study provides the first reported detection of nucleobase adducts derived from clb + E. coli and lends support to our earlier model suggesting DNA alkylation by addition of a nucleotide to an electrophilic cyclopropane.
Project description:Colibactin is a genotoxic hybrid nonribosomal peptide/polyketide secondary metabolite produced by various pathogenic and probiotic bacteria residing in the human gut. The presence of colibactin metabolites has been correlated to colorectal cancer formation in several studies. The specific function of many gene products in the colibactin gene cluster can be predicted. However, the role of ClbQ, a type II editing thioesterase, has not been established. The importance of ClbQ has been demonstrated by genetic deletions that abolish colibactin cytotoxic activity, and recent studies suggest an atypical role in releasing pathway intermediates from the assembly line. Here we report the 2.0 Å crystal structure and biochemical characterization of ClbQ. Our data reveal that ClbQ exhibits greater catalytic efficiency toward acyl-thioester substrates as compared to precolibactin intermediates and does not discriminate among carrier proteins. Cyclized pyridone-containing colibactins, which are off-pathway derivatives, are not viable substrates for ClbQ, while linear precursors are, supporting a role of ClbQ in facilitating the promiscuous off-loading of premature precolibactin metabolites and novel insights into colibactin biosynthesis.
Project description:Modular polyketide synthases (PKSs) and nonribosomal peptide synthetases (NRPSs) comprise giant multidomain enzymes responsible for the "assembly line" biosynthesis of many genetically encoded small molecules. Site-directed mutagenesis, protein biochemical, and structural studies have focused on elucidating the catalytic mechanisms of individual multidomain proteins and protein domains within these megasynthases. However, probing their functions at the cellular level typically has invoked the complete deletion (or overexpression) of multidomain-encoding genes or combinations of genes and comparing those mutants with a control pathway. Here we describe a "domain-targeted" metabolomic strategy that combines genome editing with pathway analysis to probe the functions of individual PKS and NRPS catalytic domains at the cellular metabolic level. We apply the approach to the bacterial colibactin pathway, a genotoxic NRPS-PKS hybrid pathway found in certain Escherichia coli. The pathway produces precolibactins, which are converted to colibactins by a dedicated peptidase, ClbP. Domain-targeted metabolomics enabled the characterization of "multidomain signatures", or functional readouts of NRPS-PKS domain contributions to the pathway-dependent metabolome. These multidomain signatures provided experimental support for individual domain contributions to colibactin biosynthesis and delineated the assembly line timing events of colibactin heterocycle formation. The analysis also led to the structural characterization of two reactive precolibactin metabolites. We demonstrate the fate of these reactive intermediates in the presence and absence of ClbP, which dictates the formation of distinct product groups resulting from alternative cyclization cascades. In the presence of the peptidase, the reactive intermediates are converted to a known genotoxic scaffold, providing metabolic support of our mechanistic model for colibactin-induced genotoxicity. Domain-targeted metabolomics could be more widely used to characterize NRPS-PKS pathways with unprecedented genetic and metabolic precision.
Project description:The gut bacterial genotoxin colibactin is linked to the development of colorectal cancer. In the final stages of colibactin's biosynthesis, an inactive precursor (precolibactin) undergoes proteolytic cleavage by ClbP, an unusual inner-membrane-bound periplasmic peptidase, to generate the active genotoxin. This enzyme presents an opportunity to monitor and modulate colibactin biosynthesis, but its active form has not been studied in vitro and limited tools exist to measure its activity. Here, we describe the in vitro biochemical characterization of catalytically active, full-length ClbP. We elucidate its substrate preferences and use this information to develop a fluorogenic activity probe. This tool will enable the discovery of ClbP inhibitors and streamline identification of colibactin-producing bacteria.
Project description:Colibactins are hybrid polyketide-nonribosomal peptides produced by Escherichia coli, Klebsiella pneumoniae, and other Enterobacteriaceae harboring the pks genomic island. These genotoxic metabolites are produced by pks-encoded peptide-polyketide synthases as inactive prodrugs called precolibactins, which are then converted to colibactins by deacylation for DNA-damaging effects. Colibactins are bona fide virulence factors and are suspected of promoting colorectal carcinogenesis when produced by intestinal E. coli Natural active colibactins have not been isolated, and how they induce DNA damage in the eukaryotic host cell is poorly characterized. Here, we show that DNA strands are cross-linked covalently when exposed to enterobacteria producing colibactins. DNA cross-linking is abrogated in a clbP mutant unable to deacetylate precolibactins or by adding the colibactin self-resistance protein ClbS, confirming the involvement of the mature forms of colibactins. A similar DNA-damaging mechanism is observed in cellulo, where interstrand cross-links are detected in the genomic DNA of cultured human cells exposed to colibactin-producing bacteria. The intoxicated cells exhibit replication stress, activation of ataxia-telangiectasia and Rad3-related kinase (ATR), and recruitment of the DNA cross-link repair Fanconi anemia protein D2 (FANCD2) protein. In contrast, inhibition of ATR or knockdown of FANCD2 reduces the survival of cells exposed to colibactin-producing bacteria. These findings demonstrate that DNA interstrand cross-linking is the critical mechanism of colibactin-induced DNA damage in infected cells.IMPORTANCE Colorectal cancer is the third-most-common cause of cancer death. In addition to known risk factors such as high-fat diets and alcohol consumption, genotoxic intestinal Escherichia coli bacteria producing colibactin are proposed to play a role in colon cancer development. Here, by using transient infections with genotoxic E. coli, we showed that colibactins directly generate DNA cross-links in cellulo Such lesions are converted into double-strand breaks during the repair response. DNA cross-links, akin to those induced by metabolites of alcohol and high-fat diets and by widely used anticancer drugs, are both severely mutagenic and profoundly cytotoxic lesions. This finding of a direct induction of DNA cross-links by a bacterium should facilitate delineating the role of E. coli in colon cancer and engineering new anticancer agents.