Anti-virulence activity of peptide C7-3 and its derivatives against Pseudomonas aeruginosa.

BACKGROUND: Pseudomonas aeruginosa is a highly resistant pathogenic bacterium known for causing infections,particularly in immunocompromised patients. Its ability to form biofilms contributes to itsvirulence and antibiotic resistance. In other bacteria, C7-3 and its derivatives act asnitrite reductase inhibitors and inhibit bacterial biofilm formation.Given the similarity in the nitritereductase gene with P. aeruginosa, this study aims to characterize and evaluate the potential anti-virulence effect of C7-3 and its derivatives on the P. aeruginosastrain PAO1. METHODS: BLAST analysis was performed to investigate sequence similarity and evolutionary relationships of the Neisseria gonorrhoeae AniA protein. Predicted allosteric binding sites were identified using PASSER machine learning models. Flexible peptide docking of C7-3, C7-3m1, and C7-3m2 against N. gonorrhoeae (5tb7) and P. aeruginosa (1nir) was conducted using the CABS-dock platform. Antimicrobial activity against P. aeruginosa was evaluated using agar diffusion and broth microdilution assays. Antibiofilm activity was assessed by crystal violet staining, scanning electron microscopy (SEM), and SYTO9 fluorescence labelling. In addition, real-time PCR was performed to examine the expression of biofilm-associated genes in peptide-treated and untreated P. aeruginosa. RESULTS: The P. aeruginosa target protein shared 67.6% sequence similarity with N. gonorrhoeae AniA. Predicted binding pockets in P. aeruginosa exhibited greater hydrophobicity, whereas the gonococcal target displayed predominantly electrostatic features. Docking analysis demonstrated stronger peptide affinity toward the P. aeruginosa enzyme, with C7-3 showing the most favourable binding energy. Structural analysis revealed that the P. aeruginosa enzyme exists as a homodimer, while the N. gonorrhoeae enzyme forms a homotrimer. The tested peptides exhibited antimicrobial activity against P. aeruginosa PAO1, with an MIC value of 3.3 mM/mL. In addition, the peptides reduced biofilm formation in a concentration-dependent manner. SEM and fluorescence analyses demonstrated disruption of biofilm architecture and reduced biofilm biomass following peptide treatment. Gene expression analysis further showed modulation of quorum-sensing- and biofilm-associated genes, including downregulation of lasI, lasR, and rhlR, indicating interference with biofilm regulatory pathways in P. aeruginosa PAO1. CONCLUSION: Study results highlight the potential anti-biofilm activity of C7-3 peptide and its derivatives againstP. aeruginosa strain PAO1.