Presumably, TLR2 is activated by a component(s) of S. aureus located at the cell wall, such as lipoproteins and lipopeptides11–17 with some controversies as to their role as a ligand for human TLR2,18 to transmit a signal

leading to the phosphorylation IAP inhibitor of JNK and the subsequent inhibition of superoxide production in macrophages. In the present study, we took a genetic approach to search for additional bacterial components required for the exploitation of TLR2 by S. aureus and obtained evidence that genes responsible for the synthesis of d-alanylated wall teichoic acid (WTA) play a crucial role in this exploitation. An antibody (#9251) specifically recognizing the phosphorylated form of JNK and another (#9252) recognizing both the phosphorylated and unphosphorylated forms were purchased from Cell Signaling Technology (Beverly, MA). Using these antibodies, two isoforms of JNK with relative molecular mass (Mr) values of 46 000 and 54 000 MW and their

phosphorylated forms were detectable. pHY300PLK, an Escherichia coli–S. aureus shuttle vector containing a tetracycline-resistant gene, was obtained from Takara-Bio (Ohtsu, Japan). Fluorescein isothiocyanate was purchased from Molecular Probes (Eugene, OR); the synthetic lipopeptide tripalmitoyl-S-glycerylcysteine (Pam3Cys), lipopolysaccharide (LPS) from Salmonella enteritidis, and N-acetyl-l-cysteine were from Sigma-Aldrich (St Louis, MO); mannitol salt agar medium was from Nissui (Tokyo, Japan); Diogenes was from National BMN 673 clinical trial Diagnostics (Atlanta, GA); and the Dual Luciferase Assay kit was from Promega Corp. (Madison, WI). Cell surface mutants of S. aureus are derivatives of the parental wild-type S. aureus strain RN4220 (a derivative of NCTC8325-4, a restriction and agr mutant)19 (Table 1). To construct the mutant see more strains M0614 and M0615, sequences corresponding to portions of the SA0614 and SA0615 genes (nucleotide positions 50–400 and 32–507, respectively, with

the first nucleotide of the translation start codon numbered 1) were amplified by polymerase chain reaction (PCR) and inserted into the S. aureus integration vector pSF151.20 RN4220 was then transformed with the resulting plasmids pSFSA0614 and pSFSA0615, and M0614 and M0615 where the cognate genes had been disrupted by homologous recombination were selected. RN4220 and all the mutant strains were grown in Luria–Bertani medium at 37° (except for M0702 which was grown at 30°) to full growth, washed once with phosphate-buffered saline (PBS), and used in the subsequent experiments. Macrophages from the peritoneal cavity of thioglycollate-injected C57BL/6 mice were prepared and maintained in RPMI-1640 medium supplemented with 10% [volume/volume (v/v)] heat-inactivated fetal bovine serum at 37° with 5% (v/v) CO2 in air.21 Mice carrying disrupted tlr2 in a C57BL/6 background22 were provided by Dr Shizuo Akira of Osaka University.