Bacteria on and within us – the human microbiota – play an integral role in regulating human physiology, disease states, and therapeutic responses.1 Cyclomodulins are microbiota toxins and effectors that modulate eukaryotic cell cycle progression, proliferation, differentiation, or apoptosis.2 The majority of cyclomodulins are peptidic in nature, but a limited number of non-proteogenic cyclomodulins have been discovered. Certain strains of gut commensal and extraintestinal pathogenic E. coli produce cyclomodulins known as precolibactins.3-5 Precolibactins are encoded by a 52-kb NRPS–PKS genomic island referred to as the clb or pks gene cluster.

Several lines of investigation suggest clb+ E. coli are genotoxic and contribute to colorectal cancer (CRC) progression. Clb+ E. coli induce DNA double-strand breaks (DSBs) in eukaryotic cells in vitro3 and in vivo6 and cells infected with low levels of clb+ E. coli accumulate DNA damage and continue to proliferate with increased mutation frequency and transformed phenotype.6 Additionally, intestinal mucosa colonization by clb+ E. coli is more prevalent in patients with stage III or IV CRC than those with stage I CRC and these bacteria accelerate tumor progression in multiple intestinal neoplasia (MIN) mice.7

However, the structural elucidation of the precolibactins has been an arduous task, and natural sources do not provide sufficient quantities of material for mechanism of action studies. Biosynthetically, precolibactins are assembled by the NRPS–PKS machinery, transported to the periplasm by the 12-transmembrane MATE transporter ClbM,8 and deacylated by colibactin peptidase (ClbP), a dedicated protease anchored within the inner bacterial periplasmic membrane.9 Deacylation converts precolibactins to cytotoxic colibactins and likely constitutes a prodrug resistance mechanism.10-12 Owing to the instability of colibactins, all isolation efforts to date have focused on obtaining precolibactins by employing clbp deletion strains, which eliminates the final deacylation thereby allowing precolibactins to accumulate.

We have been studying the synthesis, biosynthesis, and mechanism of action of the (pre)colibactins in collaboration with the Crawford laboratory at Yale. We developed a synthetic route to many of the most complex clb isolates, corrected the structure of the predicted metabolite precolibactin A (as 1, Fig. 1), and confirmed the structures of precolibactins B and C (2, 3, respectively).

Fig. 1.  Structures of precolibactins A–C (1–3).

Fig. 1.  Structures of precolibactins A–C (13).

Our synthesis of 3 is representative (Fig. 2). N-Tert-butoxycarbonyl-D-asparagine (4) and (S)-hex-5-en-2-amine (5) were elaborated in five steps and 57% yield to the β-ketothioester 6. The commercial reagent tert-butyl (2-amino-2-thioxoethyl)carbamate (7) was converted to the aminobithiazole 8 in seven steps and 31% yield. The fragments 6 and 8 were efficiently coupling using silver trifluoroacetate to provide the linear precursor 9. Treatment of 9 with potassium carbonate in methyl sulfoxide induces double dehydrative cyclization to form precolibactin C (3) via the intermediacy of the unsaturated lactam 10 (82% overall). Our biomimetic approach of preparing linear precursors such as 9 has provided insights into colibactin structure and biosynthesis that would otherwise have been unattainable.

Fig. 2. Synthesis of precolibactin C (3).

Fig. 2. Synthesis of precolibactin C (3).

Our unique combination of synthetic and biosynthetic studies have allowed us to construct a model for precolibactin biosynthesis, elucidate their mechanism of action, and show that the metabolites obtained from clbp deletion strains fundamentally differ from those produced by wild-type strains (Fig. 3). First, we used our synthetic material to establish that precolibactins derive from linear precursors such as 11, and that in clbp deletion strains 11 undergoes a facile double dehydrative cyclization to generate pyridones (e.g., 1–3) via the unsaturated lactams 12, as we observed in the laboratory. Next, we determined that the pyridones precolibactins A–C (1–3) are inactive in an in vitro DNA alkylation assay, suggesting they do not account for the genotoxicy of the clb cluster. We then established that upon deacylation of 11 (by ClbP in the natural system, by deprotection of the corresponding N-tert-butoxycarbonyl derivative in the laboratory), cyclization to the vinylogous urea 13, followed by the unsaturated imine 14, occurs. We demonstrated that the unsaturated imines 14 potently alkylate DNA in vitro by nucleotide addition to the cyclopropane. Moreover, we have recently detected the linear precursor 9 corresponding to the precolibactin C (3) series in cellular extracts and shown that this converts to 14 and 3 in the presence or absence of ClbP, respectively. These data show that in clbp deletion strains cyclization to non-genotoxic pyridones occurs, while in the wild-type strains the unsaturated imines 14 are generated. We are currently working to determine if the imines 14 phenocopy the activity of the clb cluter in vivo, further elucidating key steps in colibactin biosynthesis, and synthesizing and studying additional complex clb metabolites.

Fig. 3.  Biosynthetic model for (pre)colbactin production.  Our data suggest that the structures of metabolites obtained from clbp deletion strains fundamentally differ from those produced by wild-type strains.

Fig. 3.  Biosynthetic model for (pre)colbactin production.  Our data suggest that the structures of metabolites obtained from clbp deletion strains fundamentally differ from those produced by wild-type strains.

  1. Alan R. Healy, Herman Nikolayevskiy, Jaymin R. Patel, Jason M. Crawford and Seth B. Herzon J. Am. Chem. Soc. 2016, 138, 15563.
  2. A. R. Healy, M. I. Vizcaino, J. M. Crawford and S. B. Herzon J. Am. Chem. Soc. 2016, 138, 5426.
  3. E. P. Trautman, A. R. Healy, E. E. Shine, S. B. Herzon and J. M. Crawford J. Am. Chem. Soc. 2017, Article ASAP, DOI: 10.1021/jacs.7b00659.

References:

  1. Donia, M. S.; Fischbach, M. A. "Small molecules from the human microbiota." Science 2015, 349, 395.

  2. Nougayrede, J. P.; Taieb, F.; De Rycke, J.; Oswald, E. "Cyclomodulins: Bacterial effectors that modulate the eukaryotic cell cycle." Trends Microbiol. 2005, 13, 103.

  3. Nougayrède, J.-P.; Homburg, S.; Taieb, F.; Boury, M.; Brzuszkiewicz, E.; Gottschalk, G.; Buchrieser, C.; Hacker, J.; Dobrindt, U.; Oswald, E. "Escherichia coli induces DNA double-strand breaks in eukaryotic cells." Science 2006, 313, 848.

  4. Balskus, E. P. "Colibactin: Understanding an elusive gut bacterial genotoxin." Nat. Prod. Rep. 2015, 32, 1534.

  5. Trautman, E. P.; Crawford, J. M. "Linking biosynthetic gene clusters to their metabolites via pathway-targeted molecular networking." Curr. Top. Med. Chem. 2015, 16, 1.

  6. Cuevas-Ramos, G.; Petit, C. R.; Marcq, I.; Boury, M.; Oswald, E.; Nougayrède, J.-P. "Escherichia coli induces DNA damage in vivo and triggers genomic instability in mammalian cells." Proc. Natl. Acad. Sci. U. S. A. 2010, 107, 11537.

  7. Bonnet, M.; Buc, E.; Sauvanet, P.; Darcha, C.; Dubois, D.; Pereira, B.; Déchelotte, P.; Bonnet, R.; Pezet, D.; Darfeuille-Michaud, A. "Colonization of the human gut by E. coli and colorectal cancer risk." Clin. Cancer Res. 2014, 20, 859.

  8. Mousa, J. J.; Yang, Y.; Tomkovich, S.; Shima, A.; Newsome, R. C.; Tripathi, P.; Oswald, E.; Bruner, S. D.; Jobin, C. "Mate transport of the E. coli-derived genotoxin colibactin." Nat. Microbiol. 2016, 1, 15009.

  9. Dubois, D.; Baron, O.; Cougnoux, A.; Delmas, J.; Pradel, N.; Boury, M.; Bouchon, B.; Bringer, M. A.; Nougayrede, J. P.; Oswald, E.; Bonnet, R. "Clbp is a prototype of a peptidase subgroup involved in biosynthesis of nonribosomal peptides." J. Biol. Chem. 2011, 286, 35562.

  10. Brotherton, C. A.; Balskus, E. P. "A prodrug resistance mechanism is involved in colibactin biosynthesis and cytotoxicity." J. Am. Chem. Soc. 2013, 135, 3359.

  11. Bian, X.; Fu, J.; Plaza, A.; Herrmann, J.; Pistorius, D.; Stewart, A. F.; Zhang, Y.; Muller, R. "In vivo evidence for a prodrug activation mechanism during colibactin maturation." Chembiochem 2013, 14, 1194.

  12. Vizcaino, M. I.; Engel, P.; Trautman, E.; Crawford, J. M. "Comparative metabolomics and structural characterizations illuminate colibactin pathway-dependent small molecules." J. Am. Chem. Soc. 2014, 136, 9244.