The microbiota in the human gastrointestinal system is predicted to produce hundreds of unique small molecules and secondary metabolites that may influence host health and disease (1). Many such molecules are produced by sophisticated multienzymatic assembly lines that are encoded by bacterial biosynthetic gene clusters. One class of molecules, colibactins, are produced from the gene cluster called the polyketide synthase (pks) island. The pks island occurs in certain strains of Escherichia coli and is prevalent in the microbiota of colorectal cancer (CRC) patients (2–5). However, despite more than a decade of research into the potential carcinogenic role of colibactin, little is known about its structure or mechanism of action. On page 709 of this issue, Wilson et al.(6) show that colibactin alkylates DNA in cultured cells and in vivo, forming covalent modifications known as DNA adducts. These colibactin-DNA adducts are chemical evidence of DNA damage and represent a detectable signature of exposure to colibactin. Misrepaired DNA adducts may generate mutations that contribute to colorectal tumorigenesis.
Colibactin was first described as an unknown product of a 54-kilobase genomic island that encodes a hybrid nonribosomal peptide synthetase–polyketide synthase gene cluster, the pksisland, in some commensal and extraintestinal pathogenic E. coli strains (2). Exposure to pks+ E. coli induces DNA double-strand breaks and an increased gene mutation frequency in mammalian cells in culture (3). This raised speculation that products of pks were microbial-derived genotoxins that could promote cancer. The tumorigenic potential of pks products was demonstrated in a study showing that pks+ E. coli was abundant in colon tissue from CRC patients and promoted CRC in mouse models (4). This tumorigenic effect was later demonstrated in several other mouse models of CRC (5, 7, 8).
Because of its instability, the structure of colibactin has been elusive (2). Most previous work to determine the structure of colibactin has focused on identifying stable precursors using mutant strains of E. coli missing the colibactin-producing peptidase ClbP, which activates colibactin precursors by removing an N-myristoyl-D-asparagine “prodrug group” (9). However, the precursors do not necessarily represent a final colibactin structure. Previous research suggested that colibactin alkylates DNA and forms a DNA adduct via a cyclopropane functional group, called the “warhead,” which is structurally similar to other natural products that alkylate DNA (9, 10). The importance of the cyclopropane ring was confirmed by identification of the colibactin resistance protein ClbS, which inactivates the cyclopropane ring to provide self-protection against DNA damage in the pks+ bacteria (11). Recently, colibactin-DNA adducts with similar structures were detected in vitro using purified DNA and colibactin-producing bacteria (10). However, there was no direct evidence or structural characterization of these colibactin-DNA adducts in a biological setting.
Wilson et al. used an untargeted mass spectrometry DNA adductomics approach to structurally and mechanistically define a DNA alkylation end product of colibactin exposure. They identified two stereoisomeric colibactin adducts to the DNA nucleotide adenine in cultured mammalian cells and in colonic epithelial cells of formerly germfree (sterile) mice colonized with a single pks+ E. colistrain, providing direct evidence that these DNA adducts occur in vivo. As the authors note, the structure they uncovered does not necessarily represent the immediate colibactin-DNA adduct but is likely a degradation product of a larger colibactin adduct. This study provides important information about the structure and mechanism of action of colibactin. Furthermore, it describes a mass spectrometry method that could be used to identify other intractable compounds.
Precolibactins are synthesized from the pks island in E. coli before being activated by ClbP. When E. coli has direct contact with a mammalian cell, data suggest that the unstable, active colibactin reaches the nucleus where it alkylates DNA. A stable colibactin-DNA adduct was identified by Wilson et al., revealing the structural identity of a biomarker for colibactin exposure and potentially for CRC risk.
GRAPHIC: JOSHUA BIRD/SCIENCE
The adenine-colibactin adducts elucidated by Wilson et al. provide insight into how the cyclopropane functional group could react to alkylate DNA so effectively. These structures support a reaction mechanism whereby the cyclopropane ring is conjugated to an α,β-unsaturated imine formed from an intramolecular cyclodehydration that occurs once ClbP deacetylates the prodrug group. The presence of this proposed imine increases the reactivity of the cyclopropane ring to alkylate DNA (12) (see the figure). This alkylation generates DNA adducts that could lead to mutations in oncogenes or tumor suppressors that drive CRC tumorigenesis.
The identity of colibactin has been a long-standing question in the field of microbiota-influenced CRC. An important question to be resolved by further studies is how to distinguish the precise type of DNA damage responsible for the carcinogenic effects of colibactin. For example, what are the kinetics and relative levels of monoadducts versus interstrand DNA cross-links that can also result from alkylation and have been shown to occur after exposure to pks+ E. coli (6, 13)? Many other questions remain. For example, many bacterial biosynthetic gene clusters produce several bioactive molecules; is more than one colibactin variant produced from the pks island? Also, are there other roles for colibactin in mediating the interaction between the bacteria and human host? Undoubtedly, E. coli did not acquire pks to destroy its ecosystem by inducing DNA damage that may lead to cancer; instead, it is likely that pks imparts an important microbiological function, such as colonization and persistence in the gut (14).
From a clinical perspective, is there a way to predict which resident E. coli strains will colonize the gut mucosa and permit colibactin delivery? Colibactin requires direct cell-to-cell contact to exert its genotoxicity (2); thus, how does colibactin get from the bacteria into the nucleus of gastrointestinal epithelial cells, where it can cause DNA damage? Finally, how can we further apply our knowledge to improving clinical outcomes and treatment? This work has revealed a potential metabolite biomarker for colibactin exposure: adenine-colibactin adducts. However, it remains unknown whether adenine-colibactin adducts can distinguish precancerous tissue from healthy epithelium. We also do not yet know whether misrepaired adenine-colibactin adducts lead to gene mutations associated with known CRC subtypes and/or response to therapy. Future studies and the structural insight provided by Wilson et al. are expected to provide the next step toward applying microbiota signatures to improve prognosis and treatment for CRC.
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