Targeting MCR-3 and membrane biosynthesis: mechanistic insights into pterostilbene-colistin synergy

Colistin is a crucial antibiotic for treating infections caused by multidrug-resistant Enterobacterales. However, the emergence of plasmid-mediated <i>mcr</i> genes has significantly compromised their clinical efficacy. Natural adjuvants, such as pterostilbene, offer a promising strategy to restore antibiotic efficacy against resistant bacteria. This study aims to elucidate the molecular mechanisms underlying the synergistic action between pterostilbene and colistin (polymyxin E) against <i>mcr-3</i>-positive <i>Escherichia coli</i>. The <i>in vitro</i> synergistic activity between pterostilbene and colistin was confirmed by checkerboard assays. This effect was further supported by bacterial viability and membrane potential analysis. In the <i>Galleria mellonella</i> infection model, combination therapy also demonstrated therapeutic efficacy. Molecular docking revealed that pterostilbene directly targets key catalytic residues (H380 and E111) of the MCR-3 enzyme, inhibiting its phosphoethanolamine transferase activity, a finding validated by site-directed mutagenesis. Transcriptomic analysis further revealed that pterostilbene downregulated genes involved in fatty acid biosynthesis and central carbon metabolism while upregulating fatty acid degradation pathways. These metabolic disruptions were associated with compromised membrane integrity, as confirmed by electron microscopy and membrane permeability assays. Supplementation with exogenous phospholipids attenuated the synergistic effect, supporting a lipid-mediated mechanism. This study demonstrates that pterostilbene enhances colistin efficacy through dual mechanisms: direct inhibition of MCR-3 enzymatic function and disruption of bacterial membrane stability. These findings provide a strong theoretical foundation for developing pterostilbene-based adjuvants to combat <i>mcr</i>-mediated colistin resistance.IMPORTANCEColistin remains one of the last therapeutic options for infections caused by multidrug-resistant Enterobacteriaceae, but plasmid-borne <i>mcr</i> genes threaten their utility. We identified that the natural compound pterostilbene restores polymyxin activity against <i>mcr-3</i>-positive <i>Escherichia coli</i> through dual mechanisms: direct inhibition of MCR-3 catalytic residues and disruption of membrane stability via metabolic reprogramming. These dual actions compromise resistance and enhance bacterial killing both <i>in vitro</i> and <i>in vivo</i>. Our findings provide mechanistic insight into a plant-derived molecule that could be developed to counteract colistin resistance, highlighting a promising approach for extending the lifespan of critical last-line antibiotics.