The Chemical Ecology of Colibactin
by Roberto
Microbial genotoxic compounds – molecules that damage DNA – are found aplenty in nature. Doxorubicin and bleomycin come to mind; powerful DNA damaging agents developed as anti-cancer therapeutics but whose extreme toxicity is part of what makes some cancer chemotherapies arduous and risky. The production of such compounds is generally associated with soil bacteria, particularly members of the genus Streptomyces. That's why in 2006, the report that "Escherichia coli Induces DNA Double-Strand Breaks in Eukaryotic Cells" was big news. Less striking, but more accurate, would have been to qualify that title by stating that only some strains of E. coli do this, but that's neither here nor there. Fact is, strains of E. coli that carry a gene cluster known alternatively as pks or clb produce the genotoxic hybrid polyketide-nonribosomal peptide known as colibactin. This was indeed an unexpected result a couple of decades ago.
Fig. 1. The structure of colibactin. In orange are the two electrophilic cyclopropane "warheads" that react with adenine yielding the interstrand crosslink characteristic of colibactin-damaged DNA. Source
You can imagine the concerns that arose from the knowledge that many of us harbor genotoxic E. coli in our guts. What ensued was a flurry of research on the properties of colibactin as a potential mutagen and carcinogen. Though named as if it were a siderophore (think enterobactin, aerobactin, yersiniabactin – I suspect the initial inspection of the pks gene cluster led to the incorrect suspicion that it encoded an iron chelator), colibactin proved to be a potent yet extremely unstable DNA damaging agent. This instability meant that it was impossible to purify and elucidating its structure proved to be extremely challenging; more than ten years went by without a structure. Then, in 2019, as often happens in science, two papers reported the structure and molecular mechanism of action pretty much at the same time (with Emily Balskus and Jason Crawford as senior authors). Just glancing at the molecule (Fig. 1) you can already venture a guess at its reactivity. Those two orange-colored cyclopropane moieties look menacing and unstable. Their separation is perfect for an attack by adenine residues on DNA, a damaging event. No wonder that, in chemists' parlance, these three-carbon rings are referred to as warheads.
Research on colibactin yielded much evidence associating the presence of colibactin-producing E. coli in the gut with colon cancer. Yet, little evidence pointed clearly at causality, except in the very artificial setting of infecting germ-free mice with colibactin-producing strains. Importantly, the E. coli strain Nissle 1917, which for over a century has been widely used as a probiotic without any indication of long-term adverse effects, produces colibactin. This prompted investigators to look into other possible effects of colibactin production. Indeed, when tested for its effects on the mouse gut microbiome, colibactin proved critical in shaping this microbial community. The most obvious explanation for this, that colibactin is an antibiotic, turned out to not be the case. Then came a simple, yet brilliant insight.
Fig. 2. Plaque assay obtained from 24‑h co-cultures between pks+ or pks− E. coli with E. coli harboring prophage lambda. Ten-fold serial dilutions of co-culture supernatants were spotted onto wild-type (WT) E. coli (top) and the lambda-resistant ∆lamB mutant (bottom). Source
What else might a DNA-damaging molecule do to bacteria if it does not kill them? Think, for example, of what happens when you treat E. coli K‑12 with the genotoxin mitomycin C. Recall, K‑12 is a lambda lysogen. And yes, treatment with mitomycin C leads to prophage induction (as does any treatment that damages DNA). The SOS response is turned on, leading to cleavage of the lambda repressor, the prophage excises and enters the lytic cycle. That is what Justin Silpe, Emily Balskus and colleagues hypothesized would happen if a lambda lysogen were exposed to colibactin. As their paper attests, they were right! They co-cultured an E. coli producing colibactin with an E. coli lambda lysogen and observed prophage induction using a plaque assay (Fig. 2). As a control, they detected no induction in the co-culture with the strain that did not produce colibactin. The phage produced were indeed lambda; when they plated the supernatants on a lambda-resistant strain they saw no plaques, as expected. They then went on to show that this prophage induction was not limited to lambda or even E. coli lysogens. Colibactin exposure resulted in the induction of many different prophages present in a diversity of bacterial species. This induction upon co-cultivation even happened when the authors mixed the colibactin-producing E. coli with complex microbial communities derived from mouse feces.
Fig. 3. Plaque assays (as in Fig. 2) obtained from pks+ E. coli co-cultured with pks− E. coli harboring lambda and a vector encoding the clbS-like genes of the indicated organism (or the ΔclbS plasmid as control). The heat map and clustering of the ClbS-like proteins are based on amino acid identity to pks+ E. coli ClbS. Source
The authors then turned to the perennial ecological/evolutionary questions: If a microbe releases a compound toxic, how come not all neighbors die? How widespread is colibactin resistance? Colibactin-producing strains carry a resistance gene, clbS. ClbS is a hydrolase that targets the cyclopropane ring and cleaves it: how clever! (Pun intended.) Through a bioinformatic approach, the authors searched for genes similar to clbS in bacterial genomes and found several in a wide diversity of bacteria. When they cloned and expressed the clbS homologs in a lambda lysogen, these strains no longer induced the prophage when co-cultured with a colibactin producer (Fig. 3). Further tests showed that in their native context, these genes protected the strains from lysis when they were grown in the presence of a colibactin producer. A key insight from all these findings is that the presence of a colibactin producer can have dramatic effects on the composition of a microbial community. Which raises the question, did colibactin evolve to target the mammalian host and cause cancer? The authors do not think so. In their words: "The knowledge that colibactin induces prophages in diverse bacteria, combined with the finding that non-colibactin-producing bacteria from distinct environmental origins have functional clbS-like genes, leads us to speculate that colibactin production is more widespread than currently recognized, and that this genotoxin is likely to have evolved to target bacteria rather than a mammalian host." In retrospect, by seeing colibactin as a carcinogen, investigators appear to have been barking up the wrong ecological/evolutionary tree!
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