A Secreted Glutamate-Specific Serine Protease from Staphylococcus aureus Activates NF-κB Signaling and Enhances Vascular Leakage

Abstract

Staphylococcus aureus secretes multiple proteases that contribute to tissue damage and modulation of host immune responses. The glutamate-specific serine endopeptidase (VSPase) is secreted by S. aureus strain C-66; however, its effects on inflammatory signaling and vascular permeability remain poorly understood. In this study, we investigated the pro-inflammatory and vascular permeability-enhancing activities of VSPase and compared them with those of its catalytically inactive S237L mutant. In RAW 264.7 macrophages, VSPase induced the mRNA expression of TNF-α, IL-1β, IL-12β, MIP-2, cyclooxygenase-2, and prostaglandin E synthase. VSPase also increased TNF-α secretion in a concentration-dependent manner, whereas S237L elicited little or no comparable response. VSPase caused a rapid and transient reduction in cytosolic IκBα levels and increased NF-κB DNA-binding activity in nuclear extracts. Competition and supershift assays confirmed the specificity of the DNA–protein complex and demonstrated the presence of the p65 subunit. In a guinea pig Evans blue extravasation assay, intradermal administration of VSPase increased vascular leakage in a dose-related manner, whereas S237L produced minimal effects. Collectively, these findings demonstrate that VSPase induces pro-inflammatory mediator expression in association with activation of a p65-containing NF-κB complex and enhances vascular permeability in vivo. The markedly reduced activity of S237L further suggests that the catalytic activity of VSPase is closely associated with its inflammatory and vascular effects. VSPase may therefore contribute to host inflammatory responses and vascular dysfunction during staphylococcal infection.