Inhibition of <italic>Pseudomonas aeruginosa</italic> Biofilm Formation by Peptidyl-Arginine Deiminases 2 and 4.

INTRODUCTION: Pseudomonas aeruginosa is a significant pathogen associated with acute and chronic infections, particularly in immunocompromised individuals. Its capacity for biofilm formation, combined with antibiotic resistance, plays a critical role in the persistence of these infections. Peptidyl-arginine deiminases (PADs), including PAD2 and PAD4 isoforms, are involved in neutrophil phagocytic killing of P. aeruginosa. This study aimed to investigate the impact of PAD enzymes on biofilm development and virulence factor production in P. aeruginosa, with focus on the multidrug resistant strain, PGO2330. METHODS: Biofilm formation was assessed using crystal violet assays and confocal scanning laser microscopy. Quorum sensing (QS) gene expression and QS-related virulence factor production were quantified using qPCR and virulence factor assays. RESULTS: Exposure to 20 nm of PAD2 or PAD4 reduced PGO2330 surface attachment (p < 0.0001) and biofilm formation to 67.9 ± 5.6% (p < 0.0001) and 68.2 ± 4.2% (p = 0.0004), respectively. Moreover, rPAD2 and rPAD4 citrullinated multiple protein substrates of P. aeruginosa, yet citrullination activity was not required by rPADs to reduce P. aeruginosa biofilm formation. PGO2330 exposed to PAD2 and PAD4 showed reduced lasR, lasI, rhlR, rhlI, and mvfR gene expression and reduced levels of extracellular DNA, rhamnolipids, pyocyanin, and protease activity. CONCLUSION: These results demonstrate that PADs inhibit P. aeruginosa biofilm formation and decrease the production of QS-related virulence factors, highlighting their potential as novel antimicrobials and supporting further research into the development of PAD-based therapeutics.