Colonization Persistence of the Live Bacterial Therapeutics Chassis Escherichiacoli Nissle 1917 Deficient of the Colibactin Peptidase (ClbP)

Escherichia coli Nissle 1917 (EcN) has a rich history spanning more than a century of use as a probiotic. In recent years, there has been an increasing application of this strain as a platform for the advancement of engineered live therapeutic agents. However, this renewed interest has prompted a reevaluation of the safety aspects surrounding its utility as a live biotherapeutic chassis due to the production of colibactin from the Polyketide Synthase (pks) gene cluster. A multitude of investigations have demonstrated colibactin's genotoxic attributes, showing its ability to alkylate DNA, leading to double-strand breaks. Despite other reports concluding that the mucus layer of the gut can act as a protective barrier against DNA damage, these findings have raised concerns regarding EcN potential association with the development of colorectal cancer. Here, we explore the impact of inactivating the colibactin peptidase (ClbP), which cleaves pre-colibactin into its active form, colibactin, that is associated with its probiotic properties and colonization. Our results reveal that this inactivation has no significant effect on the strain's in-vitro characteristics such as growth or antibiotic sensitivity. Furthermore, we demonstrate that the deletion of ΔclbP does not adversely affect its ability to colonize mice and does not affect toxicity. Thus, we conclude that this ΔclbP deficient strain can be used as a live biotherapeutic.