Engineered antimicrobial-derived peptides to manipulate mixed microbial systems

Due to the complexity of microbial communities and coarseness of currently available manipulation techniques (e.g., transplantation and antibiotic treatment), it is often difficult to fully elucidate the interactions between members that define community structure and function from the top down. Thus, it is imperative to be able to observe and manipulate subpopulations within microbial communities to enable a fine-detail understanding of the full spectrum and mechanism of community functions. However, there is a technological gap that prevents targeted manipulation of subpopulations within intact microbial mixtures and communities. In this work, we develop molecular probes to manipulate specific subpopulations within multispecies microbial populations and validate these methods using model synthetic populations in vitro. We leverage the narrow spectrum of class II peptide bacteriocin (a bacterially synthesized antimicrobial peptide), pediocin PA-1, as a model to develop molecular probes. We first demonstrate the narrow-spectrum activity of pediocin and quantify its potency against a panel of bacteria. Next, we conjugate chemical handles on the bacteriocin and show that the binding spectrum is largely unchanged. Then, using truncated variants, also conjugated to chemical handles, we show functional non-bactericidal binders that largely maintain their specificity. Finally, with the unmodified and modified bacteriocins, we show that specific bacteria can be depleted through killing or cell sorting within a mixture of highly similar bacteria. While we developed the system with a single bacteriocin, we expect that the elucidated design rules may be applicable to a variety of natural bacteriocins to develop a generalized approach for manipulating specific bacterial members within a community. Development of such molecular probes would be transformative to advancing the mechanistic underpinnings of microbial community and microbiota structure-function relationships.

We demonstrate a novel approach for top-down manipulation of microbial co-cultures by leveraging the narrow-spectrum activity of a class IIa bacteriocin (bacterial-derived antimicrobial peptide). We expect this approach to be applicable to other microbial communities/microbiota when expanded to other bacteriocins and may prove to be an invaluable tool in studying structure-function relationships in microbial communities and engineering them for a variety of applications.