Enhancing the probiotic properties of escherichia coli nissle: when less is more

Abstract

There is a pressing need for the development of novel antibacterial therapies in light of the current antimicrobial resistance (AMR) crisis. Probiotics often possess inherent antibacterial activity, which can be underpinned by various mechanisms including competition for resources, stimulation of the host immune response and production of bacteriocins. Bacteriocins are antimicrobial peptides which are produced by almost all known bacterial genera. They display immense diversity with regards to structure, function and target species. Equipping probiotic bacteria with the ability to synthesise heterologous bacteriocins against a pathogen of interest is emerging as a popular strategy to enhance probiotic antibacterial effectiveness. Although native bacteriocin production is tightly regulated and induced only in response to specific signals, production from engineered strains is typically manipulated to be high and dysregulated. The impact of this on probiotic fitness and antibacterial efficacy is often not considered. We engineered the popular probiotic strain E. coli Nissle (EcN) to produce different amounts of the bacteriocin microcin C (McC) in response to native regulatory signals. This was achieved through strategic modification of the wild-type promoter (Pmcc). We found that synthesis of high levels of McC reduces producer fitness and causes substantial transcriptomic disruption, whilst low level McC production has minimal impact on physiology. Moreover, in a Galleria mellonella model, the low level McC producing strain conferred greatest protection against a target pathogen. Our results highlight that, when engineering probiotics for production of heterologous cargo, it is possible to have “too much of a good thing” – an important consideration for future researchers.