Limited proteolysis of neutrophil granule proteins by the bacterial protease RgpB 2 depletes neutrophil antimicrobial capacity

Neutrophils are highly abundant in the gingival tissues where they play an essential role in immune homeostasis by preventing microbial invasion. Here, we show that the oral periodontal pathogen Porphyromonas gingivalis utilizes its cysteine proteases (gingipains) to disengage phagosomal antimicrobial capacity without inducing neutrophil apoptosis. Arginine gingipains are a sub-family of trypsin-like proteases produced by P. gingivalis that cleave several host proteins at arginine residues. We found that RgpB action is not limited to the extracellular or plasma membrane-associated host proteins but can proteolytically modify several intracellular proteins in neutrophils. We used 2D-DIGE coupled with mass spectrometry to identify neutrophil proteins susceptible to RgpB-mediated cleavage and determined the impact of these modifications on neutrophil antimicrobial capacity and inflammatory responses. RgpB cleaved several cytoskeletal and cytoplasmic proteins, including metabolic enzymes and antimicrobial proteins such as neutrophil elastase, myeloperoxidase, and proteinase 3 within neutrophil granules. RgpB also directly primed neutrophils, increasing integrin expression and inducing broad transcriptional changes, consistent with proinflammatory reprogramming. However, despite their primed status and augmented inflammatory capacity, RgpB-treated neutrophils were conducive to intracellular bacterial survival due to the reduced activity of granule proteins and oxidative burst. Thus, our data show a previously unknown role for P. gingivalis proteases in the systematic inactivation of neutrophil microbicidal capacity via proteolysis of intracellular proteins. Peripheral neutrophils were obtained from whole blood from healthy human donors using Percoll density gradient. Neutrophils were treated with a vehicle control or purified bacterial protease (RgpB) for 1 h. Following treatment, neutrophils were washed twice with PBS to remove residual RgpB and RNA was exracted. We profiled differenital gene expression to determine how RgpB exposure impacts the inflammatory priming of neutrophils.