A synthetic Biology framework to deal with Antimicrobial Resistance

Antimicrobial resistance (AMR) represents a significant challenge to human health. The rate of novel drug development is currently outpaced by the emergence of new resistance variants. Synthetic biology offers potential solutions to address this issue by enhancing bacterial susceptibility to antimicrobials. In this project, we propose and present several proofs of concept for implementing a synergistic action that operates on two complementary fronts: an in-cell approach focused on delivering genetic circuits directly into target bacteria to repress gene expression, and an out-cell approach designed to inhibit bacterial communication, specifically quorum sensing (QS), which is known to trigger AMR-associated gene expression. For the in-cell approach, we evaluate engineered M13 bacteriophages targeting Escherichia coli as a delivery platform for transferring CRISPR interference (CRISPRi) based genetic circuits that can provide robust transcriptional repression directly within the target bacterium. Additionally, we demonstrate the functionality of this optimized CRISPRi technology on pathogenic Acinetobacter baumannii strains, indicating the potential applicability of the system to other bacteria for which phage engineering is feasible. Lastly, in our out-cell approach, we characterize a novel enzyme capable of degrading a signalling molecule produced by the pathogen Pseudomonas aeruginosa. The efficacy of this degradation is confirmed through testing on an engineered P. aeruginosa biosensor that replicates the genetic context of the pathogen. This indicates that engineering probiotics to express such enzymes may be a promising strategy in the fight against AMR.