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

Area of Science:

• Microbiology
• Biochemistry
• Drug Discovery

Background:

• Multidrug-resistant Gram-negative infections, especially Pseudomonas aeruginosa, are a global health threat due to their impermeable outer membrane (OM).
• Targeting essential outer membrane proteins like BamA is a strategy to overcome this resistance.

Purpose of the Study:

• To provide direct experimental evidence that DNA aptamers targeting BamA can destabilize the P. aeruginosa OM.
• To elucidate the mechanism of aptamer-induced OM destabilization and its impact on antibiotic efficacy.

Main Methods:

• DNA aptamers (Apt 60, Apt 173) targeting BamA were selected and characterized.
• Permeability assays (N-phenyl-1-naphthylamine uptake) and scanning electron microscopy (SEM) were used to assess OM integrity.
• Molecular docking and molecular dynamics (MD) simulations elucidated aptamer-protein interactions.
• Synergistic effects with Azithromycin were evaluated in a Galleria mellonella infection model.

Main Results:

• Apt 60 and Apt 173 directly destabilized the P. aeruginosa OM, causing permeabilization and morphological damage (membrane blebbing).
• Aptamers were shown to interact with BamA's extracellular loop 6.
• The aptamer-antibiotic combination significantly enhanced Azithromycin efficacy at sub-inhibitory concentrations.
• Improved host survival was observed in the Galleria mellonella model with no detectable aptamer toxicity.

Conclusions:

• DNA aptamers targeting BamA offer a novel strategy to disrupt the P. aeruginosa OM, overcoming low-permeability resistance.
• This aptamer-driven approach represents a new mode of action for nucleic acid antimicrobials.
• The synergistic combination of aptamers and antibiotics holds potential for treating challenging Gram-negative infections.