Targeting metabolism and membrane: Neferine synergizes with gentamicin against <i>Escherichia coli</i> by disrupting FNR/ArcA-Mediated homeostasis

The escalating prevalence of antimicrobial-resistant bacteria, particularly in food and environmental settings, poses a severe global health threat. E. coli with rising resistance to gentamicin exemplifies this crisis, necessitating novel strategies to restore antibiotic efficacy. This study identified Neferine, a bisbenzylisoquinoline alkaloid from Nelumbo nucifera, as a potent synergistic adjuvant for gentamicin against E. coli. Integrating high-throughput screening with bioinformatic and molecular biological approaches, we systematically investigated its synergistic efficacy and underlying mechanism.Our results demonstrated that Neferine, while lacking intrinsic bactericidal activity, dramatically enhanced gentamicin’s potency, reducing its MIC by ≥16-fold, accelerating bactericidal kinetics, and delaying resistance development in vitro. Mechanistically, Neferine specifically targeted the global transcriptional regulators FNR and ArcA with high affinity (K_D = 1.3 μM and 658.6 nM, respectively), inducing conformational changes and disrupting their regulatory networks. This interaction hyper-activated the TCA cycle, leading to NAD+ /NADH imbalance, severe ATP depletion, and ROS burst, ultimately causing metabolic catastrophe. Concurrently, Neferine synergized with gentamicin to severely disrupt bacterial membrane integrity, increasing fluidity and provoking rapid depolarization. In vivo validation demonstrated that the combination significantly enhanced host survival, reduced bacterial loads in organs, and attenuated excessive inflammatory responses.In conclusion, Neferine synergizes with gentamicin through a dual mechanism: targeting FNR/ArcA to induce metabolic collapse and co-disrupting membrane integrity, offering a promising therapeutic strategy to combat multidrug-resistant E. coli infections.