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

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-α) 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.

Fig 1

Isolation of SSL3-binding proteins from porcine spleen. (A) Electrophoretic analysis of recombinant His-tagged SSL3, SSL5, and SSL7. These purified proteins were separated by SDS-PAGE with a 12.5% polyacrylamide gel and stained with CBB. (B and C) Isolation of SSL-binding proteins from the lysate of porcine spleen. The proteins bound to His-tagged SSLs conjugated to Sepharose (SSL1 to SSL14 conjugated to Sepharose and unconjugated Sepharose [B] and SSL3 and SSL7 conjugated to Sepharose and unconjugated Sepharose [C]) were separated by SDS-PAGE with a 10% polyacrylamide gel and stained with CBB. The ∼100-kDa band bound to the SSL3-Sepharose conjugate was subjected to peptide mass fingerprinting analysis. Arrowheads, the protein recovered by the SSL3-Sepharose conjugate.

Fig 2

Binding of human and mouse TLR2 to the SSL3-Sepharose conjugate. (A and B) His-tagged SSL3, SSL5, and SSL7 conjugated to Sepharose were incubated with the lysate of human neutrophils (A) or mouse macrophage-like cell line RAW 264.7 (B) at 4°C for 1 h. The bound proteins were analyzed by immunoblotting using anti-human TLR-2 (A) or anti-mouse TLR2 (B) antibody. Figures are representative of seven experiments. (C) The C-terminal sequences of SSL3 and SSL5 and of SSL3M and SSL5M (mutants with a point mutation in the conserved sialic acid-binding sequence of SSL3 and SSL5) and of SSL7. The amino acid residues conserved in sialic acid-binding SSLs and replaced in the mutants are highlighted in bold. (D) Human peripheral mononuclear cells were incubated with 10 μg/ml of FITC-labeled SSL3, SSL5, SSL7, SSL3M, or SSL5M at 4°C for 30 min. The cells were subjected to flow cytometric analysis. Monocytes were gated to determine FSC versus SSC. The fluorescence intensities associated with monocytes were measured. Histograms are representative of three experiments. (E and F) His-tagged SSL3 or SSL3M-Sepharose conjugate was incubated with the lysate of human neutrophil lysate (E) or RAW 264.7 cells (F) at 4°C for 1 h. The binding protein was analyzed by immunoblotting using anti-human TLR2 (E) or anti-mouse TLR2 (F) antibody. Results are representative of five experiments. (G) His-tagged SSL3 and SSL7 conjugated to Sepharose and control (unconjugated) Sepharose were incubated with the mixture of the recombinant fusion protein consisting of the ECD of mouse TLR2 with the Fc portion of human IgG (1 μg) at 4°C for 1 h. Protein binding was analyzed by immunoblotting using anti-mouse TLR2 antibody.

Fig 3

Effects of SSL3 on cytokine production from mouse macrophages. (A and B) Mouse bone marrow macrophages (generated from 2 × 10⁵ bone marrow cells/well [A]) and peritoneal macrophages (1 × 10⁵ cells/well [B]) were treated with heat-killed S. aureus (S.a.; 4 × 10⁷ CFU/ml) in the presence or absence of SSLs (1 μg/ml) at 37°C for 24 h. After recovering culture supernatant, the IL-12 concentration in the culture supernatant was measured by ELISA. (C) RAW 264.7 cells were stimulated with 1 μg/ml of PGN in the presence or absence of SSL3 or SSL7 (1 μg/ml) at 37°C for 24 h, and then the TNF-α concentration in the culture supernatant was measured by ELISA. (D) RAW 264.7 cells were stimulated with 1 μg/ml PGN in the presence of SSL3 (0, 0.01, 0.033, and 0.1 μg/ml) or boiled SSL3 (0.1 μg/ml) at 37°C for 24 h. The TNF-α concentration in the culture supernatant was measured as described above. (E) RAW 264.7 cells (1 × 10⁵) were stimulated with 1 μg/ml of PGN in the presence or absence of SSL3 or SSL3M (1 μg/ml) for 24 h, and the TNF-α concentration in the culture supernatant was measured as described above. (F) RAW 264.7 cells (1 × 10⁴ cells/well) were cultivated in the presence or absence of SSL3 (0, 0.1, 1, and 10 μg/ml) at 37°C for 24 h. Cell viability was measured by staining with crystal violet. (G) RAW 264.7 cells (1 × 10⁵) were cultivated in the presence or absence of PGN (1 μg/ml), LPS (100 ng/ml), SSL3 (1 μg/ml), or SSL7 (1 μg/ml) at 37°C for 24 h. The TNF-α concentration in the culture supernatant was measured by ELISA. Data shown as means ± standard deviations of triplicate wells. The statistical difference was assessed by using Student's t test: n.s., not significant; ***, P < 0.001; **, P < 0.01; *, P < 0.05. Results are representative of more than three experiments.

Fig 4

Effects of SSL3 on TNF-α production from RAW 264.7 cells by means of stimulation with Pam₃CSK₄, MALP-2, or ultrapure LPS. (A and B) RAW 264.7cells (1 × 10⁵) were stimulated with Pam₃CSK₄ (10 ng/ml [A]) or MALP-2 (1 ng/ml [B]) in the presence or absence of SSL3 (1 μg/ml) for 24 h. TNF-α in the culture supernatant was measured by ELISA. (C and D) RAW 264.7 cells were stimulated with 1 μg/ml of PGN or 100 ng/ml of LPS in the presence or absence of SSL3 (0.1 μg/ml [C] or 1 μg/ml [D]) at 37°C for 24 h, and then the TNF-α concentration in the culture supernatant was measured as described above. Data are shown as means ± standard deviations of triplicate wells. The statistical difference was assessed by Student's t test: n.s., not significant; ***, P < 0.001; **, P < 0.01; *, P < 0.05. Results are from more than three experiments.