Aptamer‐mediated outer membrane destabilization overcomes low permeability resistance in Pseudomonas aeruginosa

Abstract

The rise of multidrug‐resistant infections, particularly those caused by Gram‐negative pathogens like Pseudomonas aeruginosa with its highly impermeable outer membrane (OM), poses a critical threat to global health. Here, we provide the first direct experimental evidence, through permeability assays (N‐phenyl‐1‐naphthylamine uptake) and scanning electron microscopy (SEM), that DNA aptamers (Apt 60 and Apt 173) targeting the essential OM protein, BamA, can physically destabilize the P. aeruginosa OM. These aptamers were selected using a hybrid non‐SELEX approach and determined by molecular docking and molecular dynamics (MD) based structural refinement to interact with BamA's lateral gate, specifically extracellular loop 6. Ultimately, they were able to induce significant OM permeabilization and morphological damage, including membrane blebbing. While previous research with other BamA‐targeting aptamers suggested an impact on membrane integrity, the direct mechanism of destabilization and its visual confirmation were not previously elucidated. Consequently, Apt 60 and Apt 173 sensitize P. aeruginosa to Azithromycin, enhancing its efficacy at sub‐inhibitory concentrations. This synergistic combination significantly improves host survival in a Galleria mellonella infection model without detectable aptamer‐induced toxicity. These findings unveil a novel aptamer‐driven OM disruption strategy to overcome low‐permeability resistance in P. aeruginosa, marking a new mode of action for nucleic acid antimicrobials.