An orphan biosynthetic gene cluster found in select gut bacterial isolates has been directly linked to long-term persistence in the human gut, host chromosome instability, and colorectal cancer formation. Without knowing the encoded molecule's identity or how it is delivered into host cells, further experiments to evaluate its specific role(s), mode(s) of action, therapeutic potential, and potential treatment areas are severely limited. We propose to identify and structurally characterize the specific bacterial encoded molecules that control host phenotypic responses. We will employ three complementary approaches: 1) bioassay-guided fractionation to enrich bacterial molecule(s) regulating the physiologically-relevant phenotypes, including antibacterial, biofilm disassembly, DNA damage, and immunomodulatory assays~ 2) differential liquid chromatography/mass spectrometric analysis of locus positive versus locus negative cells to identify gene cluster-specific molecules~ and 3) protein mediated small molecule capture of unknown toxin ligands. Biological activity of the toxin is imparted through a highly unusual bacteria-human cell contact-dependent manner, which lacks genetic or phenotypic similarities to previously described toxin delivery strategies. We also propose to manipulate the bacterial biosynthetic pathway to engineer toxin derivatives with reactive bioorthogonal functionalities. Our probe-driven strategy could not only illuminate the unusual cell contact-dependent delivery mechanism directing molecules into mammalian cells, but also reveal potentially new cancer targets involved in regulating chromosome instability. A fundamental understanding of the toxin's unusual delivery mechanism could lead to a Nature-inspired approach for directing engineered molecules directly into mammalian cells in a spatially controlled manner with potential applications in probiotic therapies.

National Institute of Health (NIH)
National Cancer Institute (NCI)
NIH Director’s New Innovator Awards (DP2)
Project #
Application #
Study Section
Special Emphasis Panel (ZRG1-MOSS-C (56))
Program Officer
Daschner, Phillip J
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
Yale University
Schools of Arts and Sciences
New Haven
United States
Zip Code
Trautman, Eric P; Healy, Alan R; Shine, Emilee E et al. (2017) Domain-Targeted Metabolomics Delineates the Heterocycle Assembly Steps of Colibactin Biosynthesis. J Am Chem Soc 139:4195-4201
Tørring, Thomas; Shames, Stephanie R; Cho, Wooyoung et al. (2017) Acyl Histidines: New N-Acyl Amides from Legionella pneumophila. Chembiochem 18:638-646
Park, Hyun Bong; Perez, Corey E; Barber, Karl W et al. (2017) Genome mining unearths a hybrid nonribosomal peptide synthetase-like-pteridine synthase biosynthetic gene cluster. Elife 6:
Guntaka, Naga Sandhya; Healy, Alan R; Crawford, Jason M et al. (2017) Structure and Functional Analysis of ClbQ, an Unusual Intermediate-Releasing Thioesterase from the Colibactin Biosynthetic Pathway. ACS Chem Biol 12:2598-2608
Park, Hyun Bong; Sampathkumar, Parthasarathy; Perez, Corey E et al. (2017) Stilbene epoxidation and detoxification in a Photorhabdus luminescens-nematode symbiosis. J Biol Chem 292:6680-6694
Trautman, Eric P; Crawford, Jason M (2017) A New Nucleoside Antibiotic Chokes Bacterial RNA Polymerase. Biochemistry 56:4923-4924
Tripathi, Prabhanshu; Shine, Emilee E; Healy, Alan R et al. (2017) ClbS Is a Cyclopropane Hydrolase That Confers Colibactin Resistance. J Am Chem Soc 139:17719-17722
Trautman, Eric P; Crawford, Jason M (2016) Linking Biosynthetic Gene Clusters to their Metabolites via Pathway- Targeted Molecular Networking. Curr Top Med Chem 16:1705-16
Healy, Alan R; Vizcaino, Maria I; Crawford, Jason M et al. (2016) Convergent and Modular Synthesis of Candidate Precolibactins. Structural Revision of Precolibactin A. J Am Chem Soc 138:5426-32
Healy, Alan R; Nikolayevskiy, Herman; Patel, Jaymin R et al. (2016) A Mechanistic Model for Colibactin-Induced Genotoxicity. J Am Chem Soc 138:15563-15570

Showing the most recent 10 out of 18 publications