The genotoxin colibactin is a secondary metabolite produced by several Enterobacteriaceae that triggers interstrand crosslinking of DNA and thereby induces DNA damage in eukaryotic cells. The presence of colibactin-producing bacteria has been correlated to the promotion of colorectal cancer and urinary tract tumors. Further, a specific mutational signature is associated with the genotoxic activity of colibactin. Colibactin is encoded by a 54-kb gene cluster, referred to as pks (polyketide synthase) island, which consists of 19 clb genes (clbA-clbS).
Here, we present a new strategy to repress the production of colibactin via targeted gene knockdown using antisense oligomers. Specifically, various peptide nucleic acid (PNAs) 10mers were designed to inhibit translation of clbA, clbP, clbQ, and clbR. The efficiency of PNAs to dampen colibactin production was evaluated at different levels. First, PNA-mediated decrease of target protein level was validated in vitro as well as in living bacterial cells. Second, we provide evidence that PNAs can decrease the in vitro crosslinking capacity of colibactin-producing bacteria. Third, cell culture infection experiments revealed a reduction of the colibactin-induced mammalian DNA damage response when bacteria were pretreated with specific PNAs.
In summary, we identified three promising PNAs, two of them targeting clbA, involved in the activation of colibactin synthesis, and one targeting the key transcriptional activator ClbR. Further research is needed to evaluate off-target effects of the PNAs and to optimize their delivery to target infected mammalian cells and organoids.