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Macrocyclic colibactin induces DNA double-strand breaks via copper-mediated oxidative cleavage

Matters Arising to this article was published on 21 September 2020

Abstract

Colibactin is an assumed human gut bacterial genotoxin, whose biosynthesis is linked to the clb genomic island that has a widespread distribution in pathogenic and commensal human enterobacteria. Colibactin-producing gut microbes promote colon tumour formation and enhance the progression of colorectal cancer via cellular senescence and death induced by DNA double-strand breaks (DSBs); however, the chemical basis that contributes to the pathogenesis at the molecular level has not been fully characterized. Here, we report the discovery of colibactin-645, a macrocyclic colibactin metabolite that recapitulates the previously assumed genotoxicity and cytotoxicity. Colibactin-645 shows strong DNA DSB activity in vitro and in human cell cultures via a unique copper-mediated oxidative mechanism. We also delineate a complete biosynthetic model for colibactin-645, which highlights a unique fate of the aminomalonate-building monomer in forming the C-terminal 5-hydroxy-4-oxazolecarboxylic acid moiety through the activities of both the polyketide synthase ClbO and the amidase ClbL. This work thus provides a molecular basis for colibactin’s DNA DSB activity and facilitates further mechanistic study of colibactin-related colorectal cancer incidence and prevention.

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Fig. 1: Structures and proposed biosynthesis of precolibactins.
Fig. 2: Genes and proposed mechanisms of aminomalonate-utilizing PKSs in the biosynthesis of precolibactins.
Fig. 3: Maturation of colibactin.
Fig. 4: Analysis of DNA damage by colibactin cleavage in vitro.
Fig. 5: Colibactin-induced DNA damage in cell cultures.

Data availability

The authors declare that all the data supporting the findings of this study are available within the paper and the Supplementary Information, and/or from the corresponding authors upon reasonable request.

References

  1. 1.

    Nicholson, J. K. et al. Host–gut microbiota metabolic interactions. Science 336, 1262–1267 (2012).

    CAS  Article  Google Scholar 

  2. 2.

    Cho, I. & Blaser, M. J. The human microbiome: at the interface of health and disease. Nat. Rev. Genet. 13, 260–270 (2012).

    CAS  Article  Google Scholar 

  3. 3.

    Sharon, G. et al. Specialized metabolites from the microbiome in health and disease. Cell Metab. 20, 719–730 (2014).

    CAS  Article  Google Scholar 

  4. 4.

    Donia, M. S. et al. A systematic analysis of biosynthetic gene clusters in the human microbiome reveals a common family of antibiotics. Cell 158, 1402–1414 (2014).

    CAS  Article  Google Scholar 

  5. 5.

    Donia, M. S. & Fischbach, M. A. Small molecules from the human microbiota. Science 349, 1254766 (2015).

    Article  Google Scholar 

  6. 6.

    Bode, H. B. The microbes inside us and the race for colibactin. Angew. Chem. Int. Ed. 54, 10408–10411 (2015).

    CAS  Article  Google Scholar 

  7. 7.

    Balskus, E. P. Colibactin: understanding an elusive gut bacterial genotoxin. Nat. Prod. Rep. 32, 1534–1540 (2015).

    CAS  Article  Google Scholar 

  8. 8.

    Faïs, T., Delmas, J., Barnich, N., Bonnet, R. & Dalmasso, G. Colibactin: more than a new bacterial toxin. Toxins 10, e151 (2018).

    Article  Google Scholar 

  9. 9.

    Nougayrède, J. P. et al. Escherichia coli induces DNA double-strand breaks in eukaryotic cells. Science 313, 848–851 (2006).

    Article  Google Scholar 

  10. 10.

    Cuevas-Ramos, G. et al. Escherichia coli induces DNA damage in vivo and triggers genomic instability in mammalian cells. Proc. Natl Acad. Sci. USA 107, 11537–11542 (2010).

    CAS  Article  Google Scholar 

  11. 11.

    Secher, T., Samba-Louaka, A., Oswald, E. & Nougayrède, J. P. Escherichia coli producing colibactin triggers premature and transmissible senescence in mammalian cells. PLoS ONE 8, e77157 (2013).

    CAS  Article  Google Scholar 

  12. 12.

    Cougnoux, A. et al. Bacterial genotoxin colibactin promotes colon tumour growth by inducing a senescence-associated secretory phenotype. Gut 63, 1932–1942 (2014).

    CAS  Article  Google Scholar 

  13. 13.

    Payros, D. et al. Maternally acquired genotoxic Escherichia coli alters offspring’s intestinal homeostasis. Gut Microbes 5, 313–325 (2014).

    Article  Google Scholar 

  14. 14.

    Arthur, J. C. et al. Intestinal inflammation targets cancer-inducing activity of the microbiota. Science 338, 120–123 (2012).

    CAS  Article  Google Scholar 

  15. 15.

    Tomkovich, S. et al. Locoregional effects of microbiota in a preclinical model of colon carcinogenesis. Cancer Res. 77, 2620–2632 (2017).

    CAS  Article  Google Scholar 

  16. 16.

    Buc, E. et al. High prevalence of mucosa-associated E. coli producing cyclomodulin and genotoxin in colon cancer. PLoS ONE 8, e56964 (2013).

    CAS  Article  Google Scholar 

  17. 17.

    Bondarev, V. et al. The genus Pseudovibrio contains metabolically versatile bacteria adapted for symbiosis. Environ. Microbiol. 15, 2095–2113 (2013).

    CAS  Article  Google Scholar 

  18. 18.

    Engel, P., Vizcaino, M. I. & Crawford, J. M. Gut symbionts from distinct hosts exhibit genotoxic activity via divergent colibactin biosynthesis pathways. Appl. Environ. Microbiol. 81, 1502–1512 (2015).

    Article  Google Scholar 

  19. 19.

    Brotherton, C. A. & Balskus, E. P. A prodrug resistance mechanism is involved in colibactin biosynthesis and cytotoxicity. J. Am. Chem. Soc. 135, 3359–3362 (2013).

    CAS  Article  Google Scholar 

  20. 20.

    Bian, X. In vivo evidence for a prodrug activation mechanism during colibactin maturation. ChemBioChem 14, 1194–1197 (2013).

    CAS  Article  Google Scholar 

  21. 21.

    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. 136, 9244–9247 (2014).

    CAS  Article  Google Scholar 

  22. 22.

    Brotherton, C. A., Wilson, M., Byrd, G. & Balskus, E. P. Isolation of a metabolite from the pks island provides insights into colibactin biosynthesis and activity. Org. Lett. 17, 1545–1548 (2015).

    CAS  Article  Google Scholar 

  23. 23.

    Bian, X., Plaza, A., Zhang, Y. & Müller, R. Two more pieces of the colibactin genotoxin puzzle from Escherichia coli show incorporation of an unusual 1-aminocyclopropanecarboxylic acid moiety. Chem. Sci. 6, 3154–3160 (2015).

    CAS  Article  Google Scholar 

  24. 24.

    Vizcaino, M. I. & Crawford, J. M. The colibactin warhead crosslinks DNA. Nat. Chem. 7, 411–417 (2015).

    CAS  Article  Google Scholar 

  25. 25.

    Li, Z.-R. et al. Critical intermediates reveal new biosynthetic events in the enigmatic colibactin pathway. ChemBioChem 16, 1715–1719 (2015).

    CAS  Article  Google Scholar 

  26. 26.

    Brachmann, A. O. et al. Colibactin biosynthesis and biological activity depends on the rare aminomalonyl polyketide precursor. Chem. Commun. 51, 13138–13141 (2015).

    CAS  Article  Google Scholar 

  27. 27.

    Zha, L., Wilson, M. R., Brotherton, C. A. & Balskus, E. P. Characterization of polyketide synthase machinery from the pks island facilitates isolation of a candidate precolibactin. ACS Chem. Biol. 11, 1287–1295 (2016).

    CAS  Article  Google Scholar 

  28. 28.

    Li, Z.-R. et al. Divergent biosynthesis yields a cytotoxic aminomalonate-containing precolibactin. Nat. Chem. Biol. 12, 773–775 (2016).

    CAS  Article  Google Scholar 

  29. 29.

    Zha, L. et al. Colibactin assembly line enzymes use S-adenosylmethionine to build a cyclopropane ring. Nat. Chem. Biol. 13, 1063–1065 (2017).

    CAS  Article  Google Scholar 

  30. 30.

    Khanna, K. K. & Jackson, S. P. DNA double-strand breaks: signaling, repair and the cancer connection. Nat. Genet. 27, 247–254 (2001).

    CAS  Article  Google Scholar 

  31. 31.

    Guntaka, N. S., Healy, A. R., Crawford, J. M., Herzon, S. B. & Bruner, S. D. Structure and functional analysis of ClbQ, an unusual intermediate-releasing thioesterase from the colibactin biosynthetic pathway. ACS Chem. Biol. 12, 2598–2608 (2017).

    CAS  Article  Google Scholar 

  32. 32.

    Bossuet-Greif, N. et al. Escherichia coli ClbS is a colibactin resistance protein. Mol. Microbiol. 99, 897–908 (2016).

    CAS  Article  Google Scholar 

  33. 33.

    Tripathi, P. et al. ClbS is a cyclopropane hydrolase that confers colibactin resistance. J. Am. Chem. Soc. 139, 17719–17722 (2017).

    CAS  Article  Google Scholar 

  34. 34.

    Colis, L. C. et al. The cytotoxicity of (–)-lomaiviticin A arises from induction of double-strand breaks in DNA. Nat. Chem. 6, 504–510 (2014).

    CAS  Article  Google Scholar 

  35. 35.

    Melvin, M. S. et al. Double-strand DNA cleavage by copper·prodigiosin. J. Am. Chem. Soc. 122, 6333–6334 (2000).

    CAS  Article  Google Scholar 

  36. 36.

    Povirk, L. F., Wübker, W., Köhnlein, W. & Hutchinson, F. DNA double-strand breaks and alkali-labile bonds produced by bleomycin. Nucleic Acids Res. 4, 3573–3580 (1977).

    CAS  Article  Google Scholar 

  37. 37.

    Stubbe, J. A. & Kozarich, J. W. Mechanisms of bleomycin-induced DNA degradation. Chem. Rev. 87, 1107–1136 (1987).

    CAS  Article  Google Scholar 

  38. 38.

    Chen, J. & Stubbe, J. Bleomycins: towards better therapeutics. Nat. Rev. Cancer 5, 102–112 (2005).

    CAS  Article  Google Scholar 

  39. 39.

    Pitie, M. & Pratviel, G. Activation of DNA carbon–hydrogen bonds by metal complexes. Chem. Rev. 110, 1018–1059 (2010).

    CAS  Article  Google Scholar 

  40. 40.

    Chaturvedi, K. S., Hung, C. S., Crowley, J. R., Stapleton, A. E. & Henderson, J. P. The siderophore yersiniabactin binds copper to protect pathogens during infection. Nat. Chem. Biol. 8, 731–736 (2012).

    CAS  Article  Google Scholar 

  41. 41.

    Humphreys, K. J., Johnson, A. E., Karlin, K. D. & Rokita, S. E. Oxidative strand scission of nucleic acids by a multinuclear copper(ii) complex. J. Biol. Inorg. Chem. 7, 835–842 (2002).

    CAS  Article  Google Scholar 

  42. 42.

    Rogakou, E. P., Pilch, D. R., Orr, A. H., Ivanova, V. S. & Bonner, W. M. DNA double-stranded breaks induce histone H2AX phosphorylation on serine 139. J. Biol. Chem. 273, 5858–5868 (1998).

    CAS  Article  Google Scholar 

  43. 43.

    Schultz, L. B., Chehab, N. H., Malikzay, A. & Halazonetis, T. D. p53 binding protein 1 (53bp1) is an early participant in the cellular response to DNA double-strand breaks. J. Cell Biol. 151, 1381–1390 (2000).

    CAS  Article  Google Scholar 

  44. 44.

    Collins, A. The comet assay for DNA damage and repair. Mol. Biotechnol. 26, 249–261 (2004).

    CAS  Article  Google Scholar 

  45. 45.

    Burger, R. M., Peisach, J. & Horwitz, S. B. Activated bleomycin: a transient complex of drug, iron, and oxygen that degrades DNA. J. Biol. Chem. 256, 11636–11644 (1981).

    CAS  PubMed  Google Scholar 

  46. 46.

    Chan, Y. A. et al. Hydroxymalonyl-acyl carrier protein (ACP) and aminomalonyl-ACP are two additional type I polyketide synthase extender units. Proc. Natl. Acad. Sci. USA 103, 14349–14354 (2006).

    CAS  Article  Google Scholar 

  47. 47.

    Holmes, T. C. et al. Molecular insights into the biosynthesis of guadinomine: a type III secretion system inhibitor. J. Am. Chem. Soc. 134, 17797–17806 (2012).

    CAS  Article  Google Scholar 

  48. 48.

    Jiang, Y. et al. The reactivity of an unusual amidase may explain colibactin’s DNA cross-linking activity. Preprint at bioRxiv https://doi.org/10.1101/567248 (2019).

  49. 49.

    Xue, M. et al. Structure elucidation of colibactin. Preprint at bioRxiv https://doi.org/10.1101/574053 (2019).

  50. 50.

    Stern, B. R. et al. Copper and human health: biochemistry, genetics, and strategies for modeling dose–response relationships. J. Toxicol. Env. Heal. B 10, 157–222 (2007).

    CAS  Article  Google Scholar 

  51. 51.

    Borah, S., Melvin, M. S., Lindquist, N. & Manderville, R. A. Copper-mediated nuclease activity of a tambjamine alkaloid. J. Am. Chem. Soc. 120, 4557–4562 (1998).

    CAS  Article  Google Scholar 

  52. 52.

    Pommier, Y. Drugging topoisomerases: lessons and challenges. ACS Chem. Biol. 8, 82–95 (2013).

    CAS  Article  Google Scholar 

  53. 53.

    Woo, C. M., Li, Z., Paulson, E. K. & Herzon, S. B. Structural basis for DNA cleavage by the potent antiproliferative agent (–)-lomaiviticin A. Proc. Natl Acad. Sci. USA 113, 2851–2856 (2016).

    CAS  Article  Google Scholar 

  54. 54.

    Wilson, M. R. et al. The human gut bacterial genotoxin colibactin alkylates DNA. Science 363, eaar7785 (2019).

    Article  Google Scholar 

  55. 55.

    Bossuet-Greif, N. et al. The colibactin genotoxin generates DNA interstrand cross-links in infected cells. mBio 9, e02393-17 (2018).

    Article  Google Scholar 

  56. 56.

    Olier, M. et al. Genotoxicity of Escherichia coli Nissle 1917 strain cannot be dissociated from its probiotic activity. Gut Microbes 3, 501–509 (2012).

    Article  Google Scholar 

  57. 57.

    Pérez-Berezo, T. et al. Identification of an analgesic lipopeptide produced by the probiotic Escherichia coli strain Nissle 1917. Nat. Commun. 8, 1314 (2017).

    Article  Google Scholar 

  58. 58.

    Bleich, R. M. & Arthur, J. C. Revealing a microbial carcinogen. Science 363, 689–690 (2019).

    CAS  Article  Google Scholar 

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Acknowledgements

This work was supported by an NSFC grant (no. 41576140), a China Ocean Mineral Resources Research and Development Association grant (COMRRDA17SC01) a grant from National Key R&D Programmes of China and a grant from the Hong Kong Branch of Southern Marine Science and Engineering Guangdong Laboratory (Guangzhou) to P.-Y.Q., grants from the National Institutes of Health (DP2AT009148), the Alfred P. Sloan Foundation and the Chan Zuckerberg Biohub Investigator Program to W.Z. and a grant from the National Institutes of Health (R01-GM85770) to B.S.M. We thank D. Lin and L. Feng for NMR measurements, Y. K. Tam and N. Harris for assistance with mass spectrometry experiments, Y. Huang and S. Jia for helpful discussions and A. Cheung for manuscript proofreading. Z.-R.L. acknowledges support from the International House at UC Berkeley, Chevron Corporation and UC Berkeley as the fellow of Chevron-Xenel PhD Gateway Fellowship. S.M.K.M. acknowledges the NSERC-PDF fellowship.

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Z.-R.L. performed the experiments, J.Y.H.L. conducted some of the fermentation work, Z.-R.L., J.L., W.C. and S.M.K.M. analysed NMR data, Z.-R.L. and W.-P.Z. performed the gene analysis, Z.-R.L., W.Z. and P.-Y.Q. designed the study and wrote the manuscript, with input from J.L. and B.S.M.

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Correspondence to Wenjun Zhang or Pei-Yuan Qian.

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Materials and Methods, Supplementary text, Supplementary Figs. 1–27, Supplementary Tables 1–7 and Supplementary references 1–28.

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Li, ZR., Li, J., Cai, W. et al. Macrocyclic colibactin induces DNA double-strand breaks via copper-mediated oxidative cleavage. Nat. Chem. 11, 880–889 (2019). https://doi.org/10.1038/s41557-019-0317-7

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