Supplementary Material for: Inhibition of Pseudomonas aeruginosa biofilm formation by peptidyl-arginine deiminases 2 and 4

Pseudomonas aeruginosa (P. aeruginosa) is a significant pathogen associated with chronic infections, particularly in immunocompromised individuals. Its ability to form biofilms, 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. The aim of this study was to investigate the impact of PAD enzymes on biofilm development and virulence factor production by P. aeruginosa, with focus on the multidrug resistant strain, PGO2330. Crystal-violet biofilm assays and confocal scanning laser microscopy were employed to assess the effects of PADs on biofilm formation. Cell motility assays, qPCR for quorum sensing (QS) gene expression and QS-related virulence factor assays were performed. This study reports that 20 nM of PAD2 and PAD4 reduced PGO2330 biofilm formation to 67.9±5.6% (p=0.0002) and 68.2±4.2% (p=0.0002), respectively. This effect was likely due to PAD2 and PAD4 reducing PGO2330 surface attachment (p<0.0001) and twitching motility (p<0.0001 and p=0.0190, respectively). PGO2330 exposed to PAD2 and PAD4 showed reduced lasR, lasI, rhlR, rhll and mvrf gene expression and reduced levels of extracellular DNA, rhamnolipid, pyocyanin and protease activity. Overall, the results revealed that in addition to inhibiting P. aeruginosa biofilm formation, PADs decrease the production of QS-related virulence factors, advancing knowledge of the antimicrobial properties of PAD enzymes and supporting further research into the development of PAD-based therapeutics.