Strains of Escherichia coli present in the human gut produce colibactin, a small molecule genotoxin implicated in colorectal cancer pathogenesis. In spite of significant efforts over the past decade, the chemical structure of colibactin and molecular mechanism by which it exerts its genotoxic effects remain unknown. Here, we report on the use of our untargeted DNA adductomic methodology to discover covalent DNA modifications present in human cell lines treated with colibactin-producing E. coli. Two putative DNA adducts, with the same [M+H]+ m/z of 540.1765, were found only in the cells treated with pks+ E. coli. Both peaks triggered MS3 fragmentation events upon observation of the neutral loss of adenine (135.0545 amu) in the MS2 fragmentation spectra, and confirmed to be associated with pks through cell infection assays using individual stable isotope labeled L-amino acids known to be used by the pks NRPS-PKS assembly line and integrated into pks-associated metabolites. The accurate mass measurement of m/z 540.1765 [M+H]+ (calculated, 540.1772) yielded a molecular formula of C23H25N9O5S with 16 degrees of unsaturation. MS2 fragmentation of both adducts identified the same major fragment ions indicating these compounds were likely stereoisomeric. Using this information and knowledge regarding the chemical nature of colibactin, potential structures were postulated and confirmed through synthesis of adduct standards and confirmation of identical mass spectral and chromatographic behaviour, including co-injection of the synthetic standard and colibactin-adduct containing E.coli +pks sample. Finally, the adducts were detected using targeted high resolution LC-MS2 analysis in DNA extracted from the colonic epithelial cells of Germ-free wild-type C57BL/6J mice which had been incolated with pks+ E. coli for 2 weeks, and were not found in the same mice inculated with pks- E. coli. Additional experiments are underway to identify of crosslinking adducts, thought to be central to the genotoxicity of colibactin.

E.coli-produced genotoxin colibactin: DNA adduct identification using untargeted adductomics and in vivo detection using high resolution mass spectrometry

2019 Fall National Meeting

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