A genome-wide CRISPRi screen identifies homologous recombination pathway as potential target for broad-spectrum antibiotic adjuvants.

INTRODUCTION

The widespread misuse and overuse of antibiotics have driven the emergence of multidrug-resistant and pan drug-resistant bacteria, constituting a formidable global health threat. Antibiotic adjuvants that potentiate the efficacy of existing antibiotics represent a particularly promising avenue to address this challenge.

METHODS

We performed a genome-wide CRISPR interference (CRISPRi) screening to identify potential targets for broad-spectrum antibiotic adjuvants, which highlighted the homologous recombination pathway as a promising candidate. To functionally validate this pathway, we employed three strategies to suppress the expression and function of recA, a key component of homologous recombination, including a CRISPRi system delivered via transconjugation, a RecX-derived peptide (RecX-20) fused to a cell-penetrating motif, and a small-molecule inhibitor cisplatin validated by surface plasmon resonance.

RESULTS

Disruption of the homologous recombination pathway not only significantly increased bacterial susceptibility to multiple classes of antibiotics, including quinolones, β-lactams, aminoglycosides, and nitrofurantoin, but also reduced horizontal gene transfer of antibiotic resistance. In addition, recA deficiency resulted in a cascade of physiological disruptions, including membrane damage, efflux pump dysfunction, oxidative stress imbalance and metabolic disruption. All three recA-targeting strategies enhanced the antibacterial activity, with cisplatin exhibiting the most pronounced potentiating effect both in vitro and in vivo.

CONCLUSIONS

This study reveals that the homologous recombination pathway, particularly RecA, is a viable target for the development of broad-spectrum antibiotic adjuvant. Our findings provide mechanistic insights and practical strategies to restore the effectiveness of existing antibiotics and address the growing threat of antimicrobial resistance.