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

• Staphylococcus aureus, but not the lipoprotein-deficient mutant, induces nitric oxide (NO) production in murine macrophages.
• Loss of NO induction by the lipoprotein-deficient mutant is mediated through the decreased expression of inducible NO synthase (iNOS).
• The lipoprotein-deficient mutant hardly stimulates Toll-like receptor 2 and its signaling pathways required for iNOS induction.

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 (ΔltaS), lipoproteins (Δlgt), or d-alanine (ΔdltA) to stimulate NO production in a murine macrophage cell line, RAW 264.7, and the primary macrophages derived from C57BL/6 mice. Wild-type, ΔltaS, and ΔdltA strains induced NO production in a dose-dependent manner but this response was not observed when the cells were stimulated with the Δlgt strain. Moreover, purified lipoproteins triggered NO production in macrophages. Coincident with NO induction, the wild-type, ΔltaS, and ΔdltA strains induced expression of inducible NO synthase (iNOS) at both mRNA and protein levels whereas Δlgt failed to induce iNOS protein or mRNA. Transient transfection followed by a reporter gene assay and Western blotting experiments demonstrated that wild-type, ΔltaS, and ΔdltA strains, but not the Δlgt strain, induced substantial activation of NF-κB and STAT1 phosphorylation, both of which are known to be crucial for iNOS expression. Moreover, wild-type, ΔltaS, and ΔdltA strains increased Toll-like receptor 2 (TLR2) activation, which is known to mediate S. aureus-induced innate immunity, whereas the Δlgt 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.