Bacterial outer membrane vesicles as mediators of colibactin toxicity

Abstract

Colibactin is the product of a hybrid non-ribosomal peptide/polyketide synthase complex (<em>pks island</em>) found in some strains of <em>Escherichia coli.</em> Bacterial strains harboring the pks island show peculiar toxicity toward mammalian cells in culture with a distinctive phenotype that includes DNA damage, cell cycle arrest, and megalocytosis of the infected cells. It has been shown by our group that the pks island can be found in the normal gut microflora and its presence is positively correlated with colorectal cancer (CRC). Despite the notable interest in elucidating the mode of action of colibactin, its structure, the detailed mechanism of action, and the mechanism by which colibactin is transported to host cells remains unknown. The broad objective of this investigation was to develop molecular strategies towards the isolation of colibactin in pursuance of its structure and eventually, its mode of action. To do so, we first made a variant strain of the <em>pks+ E. coli </em>IHE3034 deficient of clbP gene, a key enzyme involved in the activation of colibactin. In our strain, removal of clbP did not cause a complete decrease in the megalocytosis phenotype (toxicity) on infected cells as expected but caused the accumulation of an unknown product of 994 Da. In addition, since the production of colibactin takes place in the space between the inner and outer membranes, we explored the involvement of bacterial outer membrane vesicles (OMVs) in colibactin toxicity. In all cases, we compared a natural producer of colibactin, strain IHE3034 with the mutant &Delta;<em>clbP</em>. We further found that (1) colibactin production does not have any detectable effects on the chemical composition, size, and amount of bacterial OMVs, (2) OMVs were sufficient to elicit the colibactin hallmarks of genotoxicity, including megalocytosis and DNA Damages on treated cells. However, we also found that (3) OMVs from both the pks+ strain and the &Delta;<em>clbP</em> mutant did not cause interstrand crosslinks, contrary to what was expected based on the proposed genotoxic mode of action of colibactin. Interestingly and an unforeseen outcome, we found that OMVs from the strain incapable to produce the active colibactin &Delta;clbP mutant, caused a substantial amount of toxicity towards cells. From these efforts, we conclude that OMVs vesicles are involved in the genotoxicity of colibactin although we have yet to find the compound in these vesicles.