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ACS Chem Biol. 2018 Dec 21;13(12):3286-3293. doi: 10.1021/acschembio.8b00714. Epub 2018 Nov 20.

Model Colibactins Exhibit Human Cell Genotoxicity in the Absence of Host Bacteria.

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Department of Microbial Pathogenesis , Yale School of Medicine , New Haven , Connecticut 06536 , United States.
Chemical Biology Institute , Yale University , West Haven , Connecticut 06516 , United States.
Department of Chemistry , Yale University , New Haven , Connecticut 06520 , United States.
Department of Molecular, Cellular, and Developmental Biology , Yale University , New Haven , Connecticut 06520 , United States.
Yale Center for Molecular Discovery , West Haven , Connecticut 06516 , United States.
Department of Pharmacology , Yale School of Medicine , New Haven , Connecticut 06520 , United States.


Colibactins are genotoxic secondary metabolites produced in select Enterobacteriaceae, which induce downstream DNA double-strand breaks (DSBs) in human cell lines and are thought to promote the formation of colorectal tumors. Although key structural and functional features of colibactins have been elucidated, the full molecular mechanisms regulating these phenotypes remain unknown. Here, we demonstrate that free model colibactins induce DSBs in human cell cultures and do not require delivery by host bacteria. Through domain-targeted editing, we demonstrate that a subset of native colibactins generated from observed module skipping in the nonribosomal peptide synthetase-polyketide synthase (NRPS-PKS) biosynthetic assembly line share DNA alkylation phenotypes with the model colibactins in vitro. However, module skipping eliminates the strong DNA interstrand cross-links formed by the wild-type pathway in cell culture. This product diversification during the modular NRPS-PKS biosynthesis produces a family of metabolites with varying observed mechanisms of action (DNA alkylation versus cross-linking) in cell culture. The presence of membranes separating human cells from model colibactins attenuated genotoxicity, suggesting that membrane diffusion limits colibactin activity and could account for the reported bacterium-human cell-to-cell contact phenotype. Additionally, extracellular supplementation of the colibactin resistance protein ClbS was able to intercept colibactins in an Escherichia coli-human cell transient infection model. Our studies demonstrate that free model colibactins recapitulate cellular phenotypes associated with module-skipped products in the native colibactin pathway and define specific protein domains that are required for efficient DNA interstrand cross-linking in the native pathway.

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