Discovering and deciphering the pathogenic and probiotic activities from the bacterial colibactin pathway

Select strains of E. coli in our gut encode the “colibactin” pathway, a nonribosomal peptide synthetase-polyketide synthase hybrid pathway phenotypically linked to inflammatory bowel disease and colorectal cancer patients. A variety of cell biology and animal model studies have previously been reported for the pathway, but the responsible small molecules driving the phenotypes have remained enigmatic. Employing molecular networking tools, we developed a “pathway-targeted” molecular networking approach to map the colibactin pathway from both the meningitis pathogen E. coli IHE3034 and the probiotic E. coli Nissle 1917. A combination of bacterial genetics, metabolomic network analyses, system-wide isotopic labeling studies, 1- and 2D-NMR, bioinformatics, and/or synthesis supported the structures and biosynthesis of at least 32 molecules from the colibactin pathway. In vitro activity studies – 5-hydroxytryptamine-7 receptor antagonist, bacterial growth-inhibitory, and DNA interstrand crosslinking assays – for some of these molecules provide functional insights and pave the way for connecting bacterial small molecule chemistry to host physiology in animal model systems.