Deciphering the interplay between the genotoxic and probiotic activities of Escherichia coli Nissle 1917

Author summary Escherichia coli Nissle 1917 (EcN) has been used as a probiotic for over a century. However, it produces the genotoxin colibactin, which has been linked to the virulence of certain E. coli strains and could promote colorectal cancer. Administering a potentially pro-carcinogenic strain as a probiotic raises public health concerns. Therefore, our aim was to separate EcN genotoxic activity from its antagonistic effect, one of the most distinctive features of the probiotic property of EcN. We demonstrated that the microcin activity of EcN requires ClbP, an enzyme essential for colibactin production, and another enzyme involved in the synthesis of the siderophore salmochelin. This interplay was found in other E. coli strains, mainly isolated from urine. Therefore, E. coli strains utilize determinants from three distinct metabolic pathways to synthesize siderophore-microcins. Interestingly, the ClbP peptidase activity mandatory for colibactin production, has no role in siderophore-microcin activity, which provides a way to construct a non-genotoxic strain that retains its antibacterial activity. This intricate interplay between probiotic and pathogenic determinants confirms that biosynthetic pathways should be studied at the level of protein domains rather than that of whole proteins. Although Escherichia coli Nissle 1917 (EcN) has been used therapeutically for over a century, the determinants of its probiotic properties remain elusive. EcN produces two siderophore-microcins (Mcc) responsible for an antagonistic activity against other Enterobacteriaceae. EcN also synthesizes the genotoxin colibactin encoded by the pks island. Colibactin is a virulence factor and a putative pro-carcinogenic compound. Therefore, we aimed to decouple the antagonistic activity of EcN from its genotoxic activity. We demonstrated that the pks-encoded ClbP, the peptidase that activates colibactin, is required for the antagonistic activity of EcN. The analysis of a series of ClbP mutants revealed that this activity is linked to the transmembrane helices of ClbP and not the periplasmic peptidase domain, indicating the transmembrane domain is involved in some aspect of Mcc biosynthesis or secretion. A single amino acid substitution in ClbP inactivates the genotoxic activity but maintains the antagonistic activity. In an in vivo salmonellosis model, this point mutant reduced the clinical signs and the fecal shedding of Salmonella similarly to the wild type strain, whereas the clbP deletion mutant could neither protect nor outcompete the pathogen. The ClbP-dependent antibacterial effect was also observed in vitro with other E. coli strains that carry both a truncated form of the Mcc gene cluster and the pks island. In such strains, siderophore-Mcc synthesis also required the glucosyltransferase IroB involved in salmochelin production. This interplay between colibactin, salmochelin, and siderophore-Mcc biosynthetic pathways suggests that these genomic islands were co-selected and played a role in the evolution of E. coli from phylogroup B2. This co-evolution observed in EcN illustrates the fine margin between pathogenicity and probiotic activity, and the need to address both the effectiveness and safety of probiotics. Decoupling the antagonistic from the genotoxic activity by specifically inactivating ClbP peptidase domain opens the way to the safe use of EcN.