Multiplexing bacteriocin synthesis to kill and prevent antimicrobial resistance

Antibiotic resistance represents an emergency for global public health. This calls for using alternative drugs and developing innovative therapies based on a clear understanding of their mechanisms of action and resistance in bacteria. Bacteriocins represent a unique class of natural molecules selectively eliminating bacteria. These secreted proteins exhibit a narrower spectrum of activity compared to conventional broad–spectrum antimicrobials by interacting with specific protein and lipid receptors on bacterial cell envelopes. Despite their diverse molecular structures, the commonality of being genetically encoded makes bacteriocins amenable to synthetic biology design. In using cell–free gene expression (CFE) and continuous-exchange CFE (CECFE), we produced controlled combinations (cocktails) of bacteriocins in single synthesis reactions for the first time. A first set of bacteriocin cocktails comprising both linear and circular proteins allowed the targeting of different bacterial species. Other cocktails were designed to target one bacterial species and considering bacteriocins pathways to cross the cell–envelope. Such combinations demonstrated efficient bacterial eradication and prevention of resistance. We illustrate the effectiveness of these bacteriocin mixtures in eradicating various human pathogenic–multiresistant–isolates. Finally, we highlight their potential as targeted and versatile tools in antimicrobial therapy by testing a combination of bacteriocins for treatment in vivo in the animal model Galleria mellonella.