Lipoprotein in the cell wall of Staphylococcus aureus is a major inducer of nitric oxide production in murine macrophages

Staphylococcus aureus is a Gram-positive bacterium that causes inflammation at infection sites by inducing various inflammatory mediators such as nitric oxide (NO). To identify the staphylococcal virulence factors contributing to NO production, we compared the ability of ethanol-killed wild-type S. aureus and mutant strains lacking lipoteichoic acid (Delta ltaS), lipoproteins (Delta lgt), or D-alanine (Delta dltA) to stimulate NO production in a murine macrophage cell line, RAW 264.7, and the primary macrophages derived from C57BL/6 mice. Wild-type, Delta ltaS, and Delta dltA strains induced NO production in a dose-dependent manner but this response was not observed when the cells were stimulated with the Delta lgt strain. Moreover, purified lipoproteins triggered NO production in macrophages. Coincident with NO induction, the wild-type, Delta ltaS, and Delta dltA strains induced expression of inducible NO synthase (iNOS) at both mRNA and protein levels whereas Delta lgt failed to induce iNOS protein or mRNA. Transient transfection followed by a reporter gene assay and Western blotting experiments demonstrated that wild-type, Delta ltaS, and Delta dltA strains, but not the Delta lgt strain, induced substantial activation of NF-kappa B and STAT1 phosphorylation, both of which are known to be crucial for iNOS expression. Moreover, wild-type, Delta ltaS, and Delta dltA strains increased Toll-like receptor 2 (TLR2) activation, which is known to mediate S. aureus-induced innate immunity, whereas the Delta lgt strain did not. Collectively, these results suggest that lipoproteins in the cell wall of S. aureus play a major role in the induction of NO production in murine macrophages through activation of the TLR2 receptor. (C) 2015 Elsevier Ltd. All rights reserved.