Figure 1.
SDS-PAGE analysis of a macroamphiphile glycopolymer from S. hygroscopicus.
Lane 1, Mycobactrium tuberculosis LAM and LM (top and bottom bands, respectively); lane 2, S. hygroscopicus macroamphiphile glycopolymer; lane 3, S. aureus LTA. The gel was revealed by periodic acid-silver nitrate staining. LAM, lipoarabinomannan; LM, lipomannan; LTA, lipoteichoic acid.
Figure 2.
An anti-LTA antibody recognizes S. hygroscopicus macroamphiphile glycopolymer.
100 ng of S. hygroscopicus or S. coelicolor macroamphiphile glycopolymers (ShLTA and ScLTA, respectively), S. aureus LTA (SaLTA), Pam3CSK4 synthetic lipopeptide or M. tuberculosis lipoglycans mixture were coated in microtiter plate wells and probed with an antibody directed against S. epidermis LTA (anti-LTA). The results are mean ± SD and are representative of three separate experiments.
Table 1.
Relative abundance of phosphorus, glycerol and glucosamines in S. hygroscopicus LTA as determined by biochemical and NMR analyses.
Table 2.
Positive ion MALDI-TOF/MS and MS/MS analyses of structural motifs generated by HF hydrolysis (48%, 48 h at 4°C) of ShLTA.
Figure 3.
1D 1H (A, D), 2D 1H-13C HMQC (F, G), 1H-1H HOHOHA τm 110 ms (E) and 1H-1H NOESY τm 500 ms (B, C, H) spectra of ShLTA.
1H (D), 1H-13C HMQC, 1H-1H HOHOHA and 1H-1H NOESY (H) were recorded in D2O, 1H (A) and 1H-1H NOESY (B, C) in H2O/D2O (9∶1) at 298K. Expanded regions (δ1H: 8.10–8.40) (A), (δ1H: 8.10–8.40, δ1H: 2.00–2.30) (B), (δ1H: 8.10–8.40, δ1H: 3.20–5.70) (C), (δ1H: 5.05–5.50) (D), (δ1H: 5.05–5.50, δ1H: 3.20–5.70) (E), (δ1H: 5.05–5.50, δ13C: 95–100) (F), (δ1H: 3.90–4.40, δ13C: 65–80) (G) and (δ1H: 3.90–4.40, δ1H: 3.20–5.70) (H) are shown. GI, α-GlcN, GII, α-GlcNAc, gI to gIV, Gro units (see Table 3 and Figure 4). GIII corresponds to a weakly abundant GlcNAc that could not be further characterized.
Table 3.
Proton and carbon chemical shifts of S. hygroscopicus LTA.
Figure 4.
The PGP backbone is shown, with n, m, p and q estimated to be ca. 10, 4, 1 and 4 respectively. The relative position of the different motifs along the backbone is unknown. The LTA lipid anchor, presumptively a glycosyl-containing diacylglyceride glycolipid, is not shown. GI, GII and gI to gIV refer to spin systems as defined in Figure 3 and Table 3.
Figure 5.
ShLTA activity is inhibited by an anti-LTA antibody and is dependent on TLR1, TLR6 and CD14.
A. Stimuli were pre-incubated for 30 min at 37°C with 5 µg.ml−1 of anti-LTA or an IgG1 isotype control before HEK-TLR2 cells addition. ShLTA, Pam3CSK4 and M. tuberculosis lipoglycans were tested at a concentration of 10 ng.ml−1. B, C. HEK-TLR2 cells were pre-incubated for 30 min at 37°C, before stimuli addition, with 5 µg.ml−1 of various monoclonal antibodies: anti-TLR1 and anti-TLR6 (B), anti-CD14 (C) or IgG1 isotype control. ShLTA was tested at a concentration of 10 ng.ml−1. Pam3CSK4 (5 ng.ml−1) and FSL-1 (0.5 ng.ml−1) were used as positive controls of TLR2/TLR1 and TLR2/TLR6 agonists, respectively. NF-κB activation was determined by reading OD at 630 nm. The results are mean ± SD and are representative of three separate experiments. *, P<0.05; **, P<0.01; ***, P<0.001; ns, not significant.
Figure 6.
H2O2 (A) or lipoprotein lipase (B) treatments alter ShLTA capacity to stimulate TLR2.
HEK-TLR2 cells were stimulated with 10 ng.ml−1 ShLTA or M. smegmatis lipoproteins (MsLP) previously treated or not with 1% H2O2 for 24 h at 37°C (A) or with Pseudomonas sp. lipoprotein lipase (B). NF-κB activation was determined by reading OD at 630 nm. The results are mean ± SD and are representative of three separate experiments. *, P<0.05; **, P<0.01; ***, P<0.001.
Figure 7.
ShLTA stimulates NF-κB activation (A) and IL-6 (B) and TNF-α (C) production by human THP-1 monocyte/macrophage cell line.
A. THP-1 cells were stimulated with 1 or 10 µg.ml−1 of ShLTA, SaLTA or Pam3CSK4. NF-κB activation was determined by reading OD at 630 nm. B, C. THP-1 cells were differentiated with 20 ng.ml−1 of PMA for 24 h and then stimulated with 1 or 10 µg.ml−1 of ShLTA, SaLTA or Pam3CSK4. IL-6 and TNF-α were assayed in the supernatant by sandwich ELISA. The results are mean ± SD and are representative of three separate experiments.