The Extracellular Toll-like Receptor 2 Domain Directly Binds Peptidoglycan Derived from Staphylococcus aureus

Abstract

Toll-like receptor 2 (TLR2) has been recognized to mediate cell signaling in response to peptidoglycan (PGN), a major cell wall component of Gram-positive bacteria. The mechanism by which TLR2 recognizes PGN is unknown. It is not even clear whether TLR2 directly binds to PGN. In this study, we generated a soluble form of recombinant TLR2 (sTLR2) possessing only its putative extracellular domain by using the baculovirus expression system to examine the direct interaction between sTLR2 and PGN. sTLR2 bound avidly to insoluble PGN (iPGN) from Staphylococcus aureus coated onto microtiter wells in a concentration-dependent manner. In contrast, sTLR2 exhibited a very weak binding to lipopolysaccharide. iPGN cosedimented sTLR2 after the mixture of iPGN and sTLR2 had been incubated and centrifuged. sTLR2 partially attenuated the iPGN-induced NF-B activation in TLR2-transfected HEK 293 cells and the iPGN-induced IL-8 secretion in U937 cells. One of anti-human TLR2 monoclonal antibodies, which blocked iPGN-induced NF-B activation in TLR2-transfected cells, inhibited the binding of sTLR2 to iPGN. In addition, we found that sCD14 interacted with sTLR2 and increased the binding of sTLR2 to iPGN. From these results, we conclude that the extracellular TLR2 domain directly binds to PGN.

Selective Roles for Toll-Like Receptor (TLR)2 and TLR4 in the Regulation of Neutrophil Activation and Life Span

“…In contrast, the TLR2 stimulator Pam 3 CSK 4 , at 1000 ng/ml had a greater effect than 0.1 ng/ml pLPS in assays of neutrophil L-selectin and CD11b expression, yet had much more modest effects on neutrophil life span. Similarly, S. aureus peptidoglycan, which acts via TLR2 (39,49,50), had minimal effects on neutrophil survival, whereas peptidoglycan from B. subtilis exerted antiapoptotic actions, but only at high concentrations. In monocyte cell lines, TLR2 activation resulted in activation of both proapoptotic (dependent upon Fas-associated death domain protein and caspase 8) and proinflammatory (dependent upon NF-B) pathways (5,51), and TLR2-mediated apoptosis was enhanced by NF-B inhibition (5,51).…”

Recognition of Staphylococcus aureus-derived peptidoglycan (PGN) but not intact bacteria is mediated by CD14 in microglia

“…In addition, upon exposure to S. aureus and PGN, microglia respond with the robust synthesis and release of numerous proinflammatory mediators as well as internalizing and killing S. aureus in vitro (Kielian et al, 2002(Kielian et al, , 2005. Although previous studies have demonstrated that PGN binds to CD14 and enhances TLR2-mediated signaling in macrophages (Iwaki et al, 2002;Koedel et al, 2003;Schwandner et al, 1999), the fundamental role of CD14 in S. aureus-and PGNdependent microglial activation is not known. In the current study, we evaluated the relative importance of CD14 on S. aureus and PGN recognition using primary microglia isolated from CD14 KO and WT mice.…”

Toll-Like Receptors in Health and Disease: Complex Questions Remain

“…There is evidence for a direct interaction of the receptor with agonists such as peptidoglycan (27), but it is a reasonable speculation that TLR2 signaling may also be at least in part dependent upon lipid raft or membrane complex formation, and that it may not in all circumstances truly bind the putative "ligand". Recent work has highlighted the role of the coreceptor, Dectin-1, in TLR2 signaling (28,29).…”