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MATE transport of the E. coli-derived genotoxin colibactin

Abstract

Various forms of cancer have been linked to the carcinogenic activities of microorganisms13. The virulent gene island polyketide synthase (pks) produces the secondary metabolite colibactin, a genotoxic molecule(s) causing double-stranded DNA breaks4 and enhanced colorectal cancer development5,6. Colibactin biosynthesis involves a prodrug resistance strategy where an N-terminal prodrug scaffold (precolibactin) is assembled, transported into the periplasm and cleaved to release the mature product710. Here, we show that ClbM, a multidrug and toxic compound extrusion (MATE) transporter, is a key component involved in colibactin activity and transport. Disruption of clbM attenuated pks+ E. coli-induced DNA damage in vitro and significantly decreased the DNA damage response in gnotobiotic Il10−/− mice. Colonization experiments performed in mice or zebrafish animal models indicate that clbM is not implicated in E. coli niche establishment. The X-ray structure of ClbM shows a structural motif common to the recently described MATE family. The 12-transmembrane ClbM is characterized as a cation-coupled antiporter, and residues important to the cation-binding site are identified. Our data identify ClbM as a precolibactin transporter and provide the first structure of a MATE transporter with a defined and specific biological function.

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Figure 1: clbM is important for in vitro colibactin activity and production.
Figure 2: clbM is essential for NC101-induced genotoxic response in vivo.
Figure 3: Structural and functional characterization of ClbM.

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Acknowledgements

This work was supported by funding from R01 DK73338 (C.J.), R01DK47700 (C.J.), R01 GM0865700 (S.D.B.) and by the University of Florida (S.D.B.) and the UF DoM Gatorade Fund (C.J.). The authors thank V. de Crecy-Lagard for discussions.

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Contributions

C.J., S.D.B. and E.O. conceived the project and designed the experiments. J.J.M., Y.Y., S.D.B. and C.J. wrote the manuscript. J.J.M. purified, crystallized, collected data and solved the structure of ClbM. J.J.M. and R.C.N. performed the ethidium fluorescence assay and LC-MS analysis of compound 1 accumulation. J.J.M., P.T. and Y.Y. performed the ClbM cellular localization experiment. Y.Y. made ΔclbM and ΔacrA in NC101 and performed the in vitro infection and mouse colonization studies. S.T. carried out the zebrafish colonization study. A.S. constructed MGpks+ and MGpks+ mutants and examined minimal inhibitory concentrations of various antibiotics and toxic compounds. Y.Y. performed statistical analyses. J.J.M. and Y.Y. contributed equally to this work.

Corresponding authors

Correspondence to Steven D. Bruner or Christian Jobin.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Information

Supplementary Figures 1–9 and Tables 1,2. (PDF 1329 kb)

Supplementary Video 1

NC101-colonized, fixed 6-d.p.f. zebrafish. Reconstruction from z-stack images; DAPI stained whole mount zebrafish with tdTomato-labelled bacteria. (MOV 1444 kb)

Supplementary Video 2

ΔclbM-colonized, fixed 6-d.p.f. zebrafish. Reconstruction from z-stack images; DAPI stained whole mount zebrafish with tdTomato-labelled bacteria. (MOV 1559 kb)

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Mousa, J., Yang, Y., Tomkovich, S. et al. MATE transport of the E. coli-derived genotoxin colibactin. Nat Microbiol 1, 15009 (2016). https://doi.org/10.1038/nmicrobiol.2015.9

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