Probing Microbiome Genotoxicity: A Stable Colibactin Provides Insight into Structure-Activity Relationships and Facilitates Mechanism of Action Studies.

Colibactin is a genotoxic metabolite produced by commensal-pathogenic members of the human microbiome that possess the clb (aka pks) biosynthetic gene cluster. clb+ bacteria induce tumorigenesis in models of intestinal inflammation and have been causally linked to oncogenesis in humans. While colibactin is believed underlie these effects, it has not been possible to study the molecule directly due to its instability. Herein, we report the synthesis and biological studies of colibactin 742 (4), a stable colibactin derivative. We show that colibactin 742 (4) induces DNA interstrand-cross-links, activation of the Fanconi Anemia DNA repair pathway, and G2/M arrest in a manner similar to clb+E. coli. The linear precursor 9, which mimics the biosynthetic precursor to colibactin, also recapitulates the bacterial phenotype. In the course of this work, we discovered a novel cyclization pathway that was previously undetected in MS-based studies of colibactin, suggesting a refinement to the natural product structure and its mode of DNA binding. Colibactin 742 (4) and its precursor 9 will allow researchers to study colibactin's genotoxic effects independent of the producing organism for the first time.

Depurination of Colibactin-Derived Interstrand Cross-Links.

Colibactin is a genotoxic gut microbiome metabolite long suspected of playing an etiological role in colorectal cancer. Evidence suggests that colibactin forms DNA interstrand cross-links (ICLs) in eukaryotic cells and activates ICL repair pathways, leading to the production of ICL-dependent DNA double-strand breaks (DSBs). Here we show that colibactin ICLs can evolve directly to DNA DSBs. Using the topology of supercoiled plasmid DNA as a proxy for alkylation adduct stability, we find that colibactin-derived ICLs are unstable toward depurination and elimination of the 3' phosphate. This ICL degradation pathway leads progressively to single strand breaks (SSBs) and subsequently DSBs. The spontaneous conversion of ICLs to DSBs is consistent with the finding that nonhomologous end joining repair-deficient cells are sensitized to colibactin-producing bacteria. The results herein refine our understanding of colibactin-derived DNA damage and underscore the complexities underlying the DSB phenotype.

Employing chemical synthesis to study the structure and function of colibactin, a "dark matter" metabolite.

Covering: 2015 to 2020 The field of natural products is dominated by a discovery paradigm that follows the sequence: isolation, structure elucidation, chemical synthesis, and then elucidation of mechanism of action and structure-activity relationships. Although this discovery paradigm has proven successful in the past, researchers have amassed enough evidence to conclude that the vast majority of nature's secondary metabolites - biosynthetic "dark matter" - cannot be identified and studied by this approach. Many biosynthetic gene clusters (BGCs) are expressed at low levels, or not at all, and in some instances a molecule's instability to fermentation or isolation prevents detection entirely. Here, we discuss an alternative approach to natural product identification that addresses these challenges by enlisting synthetic chemistry to prepare putative natural product fragments and structures as guided by biosynthetic insight. We demonstrate the utility of this approach through our structure elucidation of colibactin, an unisolable genotoxin produced by pathogenic bacteria in the human gut.

Structure and bioactivity of colibactin.

Colibactin is a secondary metabolite produced by certain strains of bacteria found in the human gut. The presence of colibactin-producing bacteria has been correlated to colorectal cancer in humans. Colibactin was first discovered in 2006, but because it is produced in small quantities and is unstable, it has yet to be isolated from bacterial cultures. Here we summarize advances in the field since ~2017 that have led to the identification of the structure of colibactin as a heterodimer containing two DNA-reactive electrophilic cyclopropane residues. Colibactin has been shown to form interstrand cross-links by alkylation of adenine residues on opposing strands of DNA. The structure of colibactin contains two thiazole rings separated by a two-carbon linker that is thought to exist as an α-aminoketone following completion of the biosynthetic pathway. However, synthetic studies have now established that this α-aminoketone is unstable toward aerobic oxidation; the resulting oxidation products are in turn unstable toward nucleophilic cleavage under mild conditions. These data provide a simple molecular-level explanation for colibactin's instability and potentially also explain the observation that cell-to-cell contact is required for genotoxic effects.

Theaflavins from black tea affect growth, development, and motility in Dictyostelium discoideum.

Theaflavins, flavonoids found in black tea, exhibit a variety of health-promoting activities, but the mechanisms by which they act are not clear. Here, we assess the effects of black tea extract and isolated theaflavins on Dictyostelium discoideum, a model organism exhibiting an unusual life cycle relying on conserved pathways involved in human disease. Dictyostelium has been used to characterize the activities of numerous bioactive small molecules, including catechins, from which theaflavins are produced during the preparation of black tea. We show that theaflavins block growth, development, and motility in Dictyostelium, results that suggest catechins and theaflavins exert similar activities in this organism.

The Fanconi anemia pathway repairs colibactin-induced DNA interstrand cross-links.

Colibactin is a secondary metabolite produced by bacteria present in the human gut and is implicated in the progression of colorectal cancer and inflammatory bowel disease. This genotoxin alkylates deoxyadenosines on opposite strands of host cell DNA to produce DNA interstrand cross-links (ICLs) that block DNA replication. While cells have evolved multiple mechanisms to resolve ("unhook") ICLs encountered by the replication machinery, little is known about which of these pathways promote resistance to colibactin-induced ICLs. Here, we use Xenopus egg extracts to investigate replication-coupled repair of plasmids engineered to contain site-specific colibactin-ICLs. We show that replication fork stalling at a colibactin-ICL leads to replisome disassembly and activation of the Fanconi anemia ICL repair pathway, which unhooks the colibactin-ICL through nucleolytic incisions. These incisions generate a DNA double-strand break intermediate in one sister chromatid, which can be repaired by homologous recombination, and a monoadduct ("ICL remnant") in the other. Our data indicate that translesion synthesis past the colibactin-ICL remnant depends on Polη and a Polκ-REV1-Polζ polymerase complex. Although translesion synthesis past colibactin-induced DNA damage is frequently error-free, it can introduce T>N point mutations that partially recapitulate the mutation signature associated with colibactin exposure in vivo. Taken together, our work provides a biochemical framework for understanding how cells tolerate a naturally-occurring and clinically-relevant ICL.

The microbial genotoxin colibactin exacerbates mismatch repair mutations in colorectal tumors.

Certain Enterobacteriaceae strains contain a 54-kb biosynthetic gene cluster referred to as "pks" encoding the biosynthesis of a secondary metabolite, colibactin. Colibactin-producing E. coli promote colorectal cancer (CRC) in preclinical models, and in vitro induce a specific mutational signature that is also detected in human CRC genomes. Yet, how colibactin exposure affects the mutational landscape of CRC in vivo remains unclear. Here we show that colibactin-producing E. coli-driven colonic tumors in mice have a significantly higher SBS burden and a larger percentage of these mutations can be attributed to a signature associated with mismatch repair deficiency (MMRd; SBS15), compared to tumors developed in the presence of colibactin-deficient E. coli. We found that the synthetic colibactin 742 but not an inactive analog 746 causes DNA damage and induces transcriptional activation of p53 and senescence signaling pathways in non-transformed human colonic epithelial cells. In MMRd colon cancer cells (HCT 116), chronic exposure to 742 resulted in the upregulation of BRCA1, Fanconi anemia, and MMR signaling pathways as revealed by global transcriptomic analysis. This was accompanied by increased T>N single-base substitutions (SBS) attributed to the proposed pks+E. coli signature (SBS88), reactive oxygen species (SBS17), and mismatch-repair deficiency (SBS44). A significant co-occurrence between MMRd SBS44 and pks-associated SBS88 signature was observed in a large cohort of human CRC patients (n=2,945), and significantly more SBS44 mutations were found when SBS88 was also detected. Collectively, these findings reveal the host response mechanisms underlying colibactin genotoxic activity and suggest that colibactin may exacerbate MMRd-associated mutations.

On the Stability and Spectroscopic Properties of 5-Hydroxyoxazole-4-carboxylic Acid Derivatives.

5-Hydroxyoxazole-4-carboxylic acid residues were advanced as substructures within the secondary bacterial metabolites precolibactins 969 and 795a. However, oxazoles containing both 5-hydroxy and 4-carboxy substituents are unprecedented. We have found these oxazoles are unstable with respect to hydrolytic ring opening and decarboxylation. Comparison of reported and theoretical 13C NMR chemical shifts between synthetic intermediates and the isolates revealed discrepancies in the oxazole region. These results suggest that precolibactins 969 and 795a may not contain 5-hydroxyoxazole-4-carboxylic acid residues.

Addendum: Synthesis and reactivity of precolibactin 886.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Synthesis and reactivity of precolibactin 886.

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 α-ketoimine linear precursor undergoes spontaneous cyclization to precolibactin 886 on HPLC purification. Studies of this α-ketoimine and the related α-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.

Structure elucidation of colibactin and its DNA cross-links.

Colibactin is a complex secondary metabolite produced by some genotoxic gut Escherichia coli strains. The presence of colibactin-producing bacteria correlates with the frequency and severity of colorectal cancer in humans. However, because colibactin has not been isolated or structurally characterized, studying the physiological effects of colibactin-producing bacteria in the human gut has been difficult. We used a combination of genetics, isotope labeling, tandem mass spectrometry, and chemical synthesis to deduce the structure of colibactin. Our structural assignment accounts for all known biosynthetic and cell biology data and suggests roles for the final unaccounted enzymes in the colibactin gene cluster.