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.
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