3a), confirming the requirement of dltA for the effective inhibition of superoxide production in macrophages by S. aureus. The viability of engulfed S. aureus was then assessed based on their colony-forming ability. The number of colony-forming units obtained with the dltA mutant was much smaller than that obtained with the parental strain or with the same mutant strain that had acquired the corresponding

wild-type operon (Fig. 3b), indicating that S. aureus lacking the expression of dltA was more efficiently killed in macrophages. Furthermore, the dltA mutant survived killing in macrophages when the cultures were supplemented with N-acetyl-l-cysteine, a superoxide scavenger (Fig. 3c). We next examined whether the recognition selleck chemical and engulfment of S. aureus alter the activity of macrophages other than

superoxide production. Acalabrutinib in vitro For this purpose, macrophages were incubated with various S. aureus stains, and their whole-cell lysates were assayed for α-N-acetylglucosaminidase, a major lysosomal enzyme. However, its activity did not change after incubation with any of the bacterial strains tested (Fig. 3d), suggesting that the lysosomal activity is not influenced by S. aureus. These results indicated that a lack of expression of dltA or tagO in S. aureus causes augmented production of superoxide and accelerated killing of engulfed bacteria in macrophages, and thus suggested a role for the d-alanylation of WTA in the survival of S. aureus in macrophages. We next determined the level of NF-κB-dependent gene expression in TLR2-expressing HEK293 cells, to investigate the role of dltA and tagO in the activation of TLR2. The expression of an NF-KB-induced gene coding for luciferase depended on the presence of TLR2 in HEK293 cells as well as the addition of S. aureus to them (Fig. 4a), indicating that the level of active NF-κB reflects the Carnitine palmitoyltransferase II activation of TLR2-initiated signalling by bacteria. HEK293 cells incubated with the dltA

mutant produced much less luciferase than those treated with the parental strain, and luciferase levels recovered when the dltABCD operon was introduced into the mutant (Fig. 4b). Similarly, a decrease in the level of active NF-κB was observed when the mutants for tagO, SA0614 and SA0615, which all gave reduced levels of phosphorylated JNK in macrophages (see Fig. 1b), were tested (Fig. 4c). In contrast, the other mutant strains with no effect on the phosphorylation of JNK activated NF-κB as effectively as the parental strain (Fig. 4c). These results suggested that d-alanylated WTA is required for S. aureus to effectively induce the TLR2-mediated activation of NF-κB. Taken together, the effects of dltA and tagO on JNK phosphorylation, superoxide production, the survival of engulfed bacteria, and the activation of TLR2-mediated signalling are consistent with the concept that a component of S. aureus, i.e.