Characterisation of a Novel Neutrophil-derived Antimicrobial Agent

Background: The rapid emergence of antimicrobial resistance (AMR) particularly within the ESKAPE pathogen group, currently poses a significant threat to many lives. Characterisation of new antimicrobial proteins in the innate immune system offers novel antimicrobial agents for antibiotic development. The aim of this study was to determine the involvement of peptidyl-arginine deiminases (PADs), isotypes 2 and 4, in the bacterial killing processes of neutrophils.

Methods: The localisation of PAD2 and PAD4 within isolated neutrophil primary granules, secondary granules, and the phagosome was determined by western-blot analysis. Involvement of PADs within the bacterial killing process of neutrophils was investigated in extracellular bacterial killing assays, in the presence of a PAD-inhibitor. In-vitro bactericidal activity of PADs was assessed against Pseudomonas aeruginosa (P. aeruginosa) relative to bactericidal permeability-increasing protein (BPI). The effect of exogenous Ca2+ and the relevant neutrophil phagosome pH range on bacterial activity was assessed. Bacterial killing was determined by difference in optical density at 600 nm of the bacterial solution or by colony forming units enumeration relative to the control.

Results: PAD2 and PAD4 reside in primary granules and cytosol of neutrophils, but are absent from the secondary granules. During the phagocytosis process, PAD2 and PAD4 are both deregulated into the neutrophil phagosome. Addition of a PAD-inhibitor to the degranulated proteins from TNF-α (p < 0.0001) and IL-8 (p = 0.0064) stimulated neutrophils, significantly reduced bacterial killing. At 20 nM, PAD2 and PAD4 show similar killing of P. aeruginosa as BPI, killing 65.5 ± 5.9% and 65.3 ± 6.5% of the inoculum, respectively. PAD2 and PAD4 exhibit bacterial activity at pH 6.5-8.5 and 5.5-8.5, respectively, with optimum activity occurring at pH 6.5-7.5.

Conclusion: These findings indicate that PAD2 and PAD4 function as direct anti-bacterial proteins in circulating neutrophils. This supports further research into the antimicrobial mechanisms of PADs.