Staphylococcal Superantigen-Like Protein 3 Binds to the Toll-Like Receptor 2 Extracellular Domain and Inhibits Cytokine Production Induced by Staphylococcus aureus, Cell Wall Component, or Lipopeptides in Murine Macrophages

Abstract

c Staphylococcal superantigen-like proteins (SSLs) are a family of exoproteins sharing structural similarity with superantigens, but no superantigenic activity. Corresponding host target proteins or receptors against a portion of SSLs in the family have been identified. In this study, we show that SSL3 specifically binds to Toll-like receptor 2 (TLR2) and inhibits the stimulation of macrophages by TLR2 ligands. An approximately 100-kDa protein was recovered by using recombinant His-tagged SSL3-conjugated Sepharose from the lysate of porcine spleen, and the protein was identified as porcine TLR2 by peptide mass fingerprinting analysis. The SSL3-conjugated Sepharose recovered human and mouse TLR2 but not TLR4 from human neutrophils and mouse macrophage RAW 264.7 cells, as well as a recombinant TLR2 extracellular domain chimera protein. The production levels of interleukin 12 (IL-12) from mouse macrophages treated with heat-killed Staphylococcus aureus and of tumor necrosis factor alpha (TNF-␣) from RAW 264.7 cells induced by peptidoglycan or lipopeptide TLR2 ligands were strongly suppressed in the presence of SSL3. The mutation of consensus sialic acid-containing glycan-binding residues in SSL3 did not abrogate the binding ability to TLR2 or inhibitory activity on TLR2, indicating that the interaction of SSL3 with TLR2 was independent of the sialic acid-containing glycan-binding residues. These findings demonstrate that SSL3 is able to bind the extracellular domain of TLR2 and interfere with TLR2 function. The present study provides a novel mechanism of SSL3 in immune evasion of S. aureus via interfering with its recognition by innate immune cells.

Structural basis for inhibition of TLR2 by staphylococcal superantigen-like protein 3 (SSL3)

“…SSL3 residue Arg308, previously described to be crucial for sialic acid binding, was found to be involved in, yet not crucial for, binding and activity of SSL3 (5). Yokoyama et al (6) reported that mutation of Phe297-Glu298, residues also involved in Lewis X binding, results in decreased binding to cells, but has no effect on binding to TLR2 itself. Our crystallographic data show that the distance from the Lewis X binding site of SSL3 to the nearest N-linked glycosylation site in both mouse and human TLR2 is too large for interaction to occur (Fig.…”

“…Among these secreted virulence factors are the staphylococcal superantigen-like proteins (SSLs), a family of 14 proteins located on two genomic clusters (2)(3)(4). Recently, we and others identified SSL3 as a potent inhibitor of Tolllike receptor 2 (TLR2) (5,6), an innate immunity receptor that is a dominant factor in immune recognition of S. aureus (7)(8)(9)(10).…”

IgG4 Subclass-Specific Responses to Staphylococcus aureus Antigens Shed New Light on Host-Pathogen Interaction

“…These immune modulators interact with both the human innate and acquired immune systems on many levels. Innate responses affected are chemotaxis, which is modulated by CHIPS (56), extravasation, modulated by SSL3 and SSL5 (57), complement activity, which is modulated by SCIN (58), and Toll-like receptor 2 (TLR2) signaling, which is affected by SSL3 (59). SEC and TSST-1 modulate adaptive responses by non-antigen-directed binding of major histocompatibility complex (MHC) class II with T cell receptors, resulting in polyclonal T cell activation (60).…”

Staphylococcus pseudintermedius, both commensal and pathogen

“…Furthermore, staphylococcal superantigens, such as (SSL) proteins, are also capable of disrupting the host's innate immune response. Among these, SSL3 blocks TLR2 activation through direct extracellular interaction with this receptor, inhibiting TNF-α production (6,51,98,112). Recently, a new virulence factor of S. aureus has been discovered.…”