Structure elucidation of colibactin and its DNA cross-links

Abstract

INTRODUCTION: Research on the human microbiome has revealed extensive correlations between bacterial populations and host physiology and disease states. However, moving past correlations to understanding causal relationships between the bacteria in our bodies and our health remains a challenge. A well-studied human-bacteria relationship is that of certain gut Escherichia coli strains whose presence correlates with colorectal cancer in humans. These E. coli damage host DNA and cause tumor formation in animal models, and this genotoxic phenotype is thought to derive from a secondary metabolite—known as colibactin—that is synthesized by the bacteria. Because colibactin’s biosynthetic pathway is only partially resolved, the complete structure of colibactin has remained unknown for more than a decade. Similarly, because colibactin is unstable and is produced in vanishingly small quantities, it has yet to be isolated and characterized by means of standard spectroscopic methods. RATIONALE: Determining colibactin’s chemical structure and related biological activity will allow researchers to determine whether the metabolite is the causal agent underlying many colorectal cancers. To that end, we used an interdisciplinary approach to overcome the challenges that have impeded determination of colibactin’s structure. Inspired by an earlier study that showed that colibactin-producing bacteria cross-link DNA, we used DNA as a probe to isolate colibactin from bacterial cultures. Using a combination of isotope labeling and tandem mass spectrometry analysis, we deduced the structure of the colibactin residue when bound to two nucleobases. This information allowed us to then identify and characterize colibactin in bacterial extracts and to identify plausible biosynthetic precolibactin precursors. Last, we developed a method to recreate colibactin in the laboratory and thereby confirm these structure-function relationships. RESULTS: Colibactin is formed through the union of two complex biosynthetic intermediates. This coupling generates a nearly symmetrical structure that contains two electrophilic cyclopropane warheads. We found that each of these residues undergoes ring-opening through nucleotide addition, a determination that is consistent with earlier studies of truncated colibactin derivatives and the observation that colibactin-producing bacteria cross-link DNA. Using genome editing techniques, we were able to show that the production of colibactin’s precursor, precolibactin 1489, requires every biosynthetic gene in the colibactin gene cluster, implicating it as being derived from the long-elusive and now completed biosynthetic pathway. Because natural colibactin remains non-isolable, the chemical synthetic route to colibactin we developed will allow researchers to probe for causal relationships between the metabolite and inflammation-associated colorectal cancer. CONCLUSION: These studies reveal the structure of colibactin, which accounts for the entire gene cluster encoding its biosynthesis, a goal that has remained b...

UropathogenicE. coliinduces DNA damage in the bladder

“…1a) 15–17 . The genotoxin alkylates adenine residues on both strands of DNA, producing DNA interstrand cross-links 18–20 . These highly toxic DNA lesions initiate a DNA damage response, by phosphorylating replication protein A (pRPA) and phosphorylating the H2AX histone variant (pH2AX) (Fig.…”

The accumulation of colibactin intermediates does not affect the growth and morphology ofpks+Escherichia coli

“…In many of these reports, there is a common observation of accumulation of colibactin intermediates in E. coli cells, enough to characterize chemically. These intermediates have been the basis of efforts to elucidate the structure of active colibactin (15).…”

“…However, once in the nucleus, colibactin can form DNA cross-links and double-strand breaks that activate much of the DNA damage responses while also inducing cell cycle arrest (12–14). Recently, the groups of Nougayréde and Crawford, independently showed how specific covalent adducts between DNA and colibactin can be formed (13, 15). Furthermore, at around the same time, Balskus group found evidence of adduct formation in a germ-free mice infected with pks + E. coli, showing that the consensus mechanism of colibactin toxicity also operates in vivo (14).…”

Colibactin DNA damage signature indicates causative role in colorectal cancer

“…Raw sequencing data were pre-processed as previously described 7 . In brief, only reads which contained the expected prefix of UMI and sample barcode were kept using SAMtools 40 .…”

“…The recent discovery of a cyclopropane ring, characteristic of DNA alkylating agents, led to the isolation of colibactin-dependent N-3 adenine adducts from host DNA 6 . This observation was followed by the resolution of colibactin’s mature structure as a highly symmetrical molecule, containing identical cyclopropane warheads at each end, which can give rise to DNA cross-links 7 . Yet, it is unclear if colibactin’s mode of action generates a specific signature that is retrievable in cancers from tissues potentially exposed to respective E.coli infections.…”