Dual-action antimicrobial peptide therapy: disrupting biofilm formation and targeting bacterial DNA for enhanced treatment of Pseudomonas aeruginosa endophthalmitis

ABSTRACT Intraocular infections caused by Pseudomonas aeruginosa are difficult to treat due to bacterial biofilm-mediated antibiotic resistance. We used cationic polypeptide (XXYY) n to evaluate its effect and mechanism against Pseudomonas aeruginosa biofilm and its efficacy against Pseudomonas aeruginosa intraocular infection. Microbroth dilution showed that the MIC 90 of (LLKK) 3 C against Pseudomonas aeruginosa was 32 μg/mL. After continuous passage for 10 generations, the stability of antibacterial activity of (LLKK) 3 C against Pseudomonas aeruginosa was better than that of amikacin. TEM, SEM and live/dead staining demonstrated that (LLKK) 3 C was able to destroy the integrity of bacterial and biofilm and exert antibacterial activity by interacting with phosphatidyl glycerol on the membrane to destroy the bacterial membrane. Electrophoretic gel and Alphafold3 demonstrated that (LLKK) 3 C binds to bacterial DNA through charge interactions. In the rabbit endophthalmitis model, the intraocular bacterial load decreased after intravitreal injection (LLKK) 3 C treatment (vs untreated, P < 0.05). Studies have shown that (LLKK) 3 C has synergistic antibacterial effects through the dual mechanism of targeting membrane destruction and DNA binding, which provides a new treatment strategy for biomembrane-associated intraocular infections. IMPORTANCE Ocular infections caused by Pseudomonas aeruginosa often lead to severe vision loss due to antibiotic-resistant bacterial biofilms, which shield bacteria from conventional treatments. This study introduces (LLKK)3C, that uniquely attacks biofilms through a dual-action mechanism: physically disrupting bacterial membranes and binding to bacterial DNA. Unlike traditional antibiotics like amikacin, (LLKK)3C maintains robust antibacterial activity even after prolonged exposure, reducing the risk of resistance development. In a rabbit endophthalmitis model, (LLKK)3C significantly reduced intraocular bacterial loads, demonstrating its potential as a targeted therapy for sight-threatening infections. By addressing the dual challenges of biofilm resistance and drug stability, (LLKK)3C offers a promising strategy to improve clinical outcomes for patients with difficult-to-treat eye infections.