Introduction: Pseudomonas aeruginosa is the major respiratory pathogen in patients with cystic fibrosis (CF). Chronic infections result in accelerated pulmonary function decline. New effective therapeutic options are needed. Proteomic studies have identified peptidyl-arginine deaminases (PADs) within neutrophil granules, possibly indicating their involvement in microbial killing.

Aims: We hypothesised that PADs participate in the vacuolar bacterial killing process of neutrophils. To assess this, PAD's in-vitro bactericidal effect against P. aeruginosa and localisation in neutrophils was evaluated.

Methods: In bacterial killing assays, P. aeruginosa (PA01) was exposed to increasing concentrations of PAD2, PAD4 or bactericidal permeability-increasing protein (BPI; positive control) and colony-forming units or optical density (600 nm) were recorded. Subcellular fractionation of neutrophils was performed by sucrose gradient ultracentrifugation following N2 cavitation. Presence of PADs in neutrophil compartments was assessed by western blot analysis.

Results: In combination, 20 nM PAD2 and PAD4 killed 74±19% of P. aeruginosa in 1hr (p < 0.001). Post-32 min exposure, PAD2, PAD4 and BPI exhibited MIC50 values of 18.9, 10.4, and 7.0 nM respectively. PAD2 and PAD4 were localised to neutrophil primary granules. Together these results support the antimicrobial participation of PADs following degranulation into neutrophil phagosomes.

Conclusion: PAD2 and PAD4 possess potent anti-pseudomonal activity, warranting further research to validate the use of PAD-based antimicrobial agents for CF patients.

This study was supported by Pfizer Healthcare Ireland.