SCV formation can be associated with reduced TLR2-activity.

<p><b>A,C and D:</b> HEK293 cells were transfected with or without TLR2 cDNA (100 ng/well in <b>A+D</b> or 200 ng/well in <b>C</b>) and stimulated with <i>S. aureus</i> strains for 18–20 hours. IL-8 secretion was quantified as indicator of TLR2 activation. All diagrams show the results obtained in three independent experiments given as mean values ± SD (standard deviation). <b>A:</b> Stimulation of HEK293 cells w/o pTLR2 with <i>S. aureus</i> SH1000 and its isogenic thymidine-auxotrophic mutant SCV SH1000 <i>ΔthyA</i>; *p(SH1000:<i>ΔthyA) = </i>0.049. <b>B:</b> Comparison of SpA expression in SH1000 (TLR2^high) and its isogenic SCV SH1000 <i>ΔthyA</i> (TLR2^low). Left: Western blot analysis of SpA expression in bacterial lysates using anti-SpA mAb and anti-murine IgG-HRP as secondary antibody. Right: Control blot incubated with human serum and biotinylated anti-human IgG+streptavidin-HRP. One representative experiment of n = 3 experiment is shown. <b>C:</b> HEK293 cells w/o TLR2 were stimulated with SA113 or its mutants <i>ΔhemB</i> and Δ<i>sdh</i> (grown on Mueller Hinton (MH) or Columbia agar (CA)) or with 1 µg/ml of the TLR2 ligand Pam₃CSK₄ (P3) or left unstimulated (<b>−</b>). <b>D:</b> Stimulation with five different clinical <i>S. aureus</i> SCV (isolate source: CF = cystic fibrosis, INV = invasive) and SA113. (<b>−</b>) = unstimulated.</p

Capsular polysaccharides mask TLR2-activity in <i>S. aureus</i>.

<p><b>A:</b> HEK293 cells were transfected with pTLR2 (200 ng/well) and stimulated with 1×10⁶ CFU/well of <i>S. aureus</i> Newman-132 mutant strain after culture with or without IPTG. Stimulation with bacteria was performed for 2.5 hours in the absence of antibiotics. Thereafter cells were washed and medium exchanged by RPMI medium containing ciprofloxacin to kill any residual bacteria. Cellular supernatants were harvested after 24 hours and analyzed for IL-8 concentrations. (<b>−</b>) refers to unstimulated cells, P3: Pam₃CSK₄ was used as positive control (200 ng/ml). Results were normalized to Newman-132+IPTG ( = 160.4 pg/ml ±121.6) = 100%; Newman-132 -IPTG = 292.4 pg/ml ±268.5. The diagram shows the results obtained in four experiments given as mean values ± SD.</p

Protein A expression correlates with TLR2-activity.

<p><b>A:</b> Comparison of TLR2-induced IL-8 secretion levels after stimulation of HEK293 cells transfected with 200 ng/well of pTLR2 with SA113, clinical <i>S. aureus</i> isolates (CF36, INV66, INV02), 1 µg/ml Pam₃CSK₄ (P3) or when left unstimulated (<b>−</b>). <b>B:</b> Comparison of SpA expression in SA113 and clinical <i>S. aureus</i> isolates (CF36, INV66, INV02). Left: Western blot analysis of SpA expression in bacterial lysates using anti-SpA mAb and anti-murine IgG-HRP. Right: Control blot incubated with human serum and biotinylated anti-human IgG-+ streptavidin-HRP to visualize protein loading. One representative experiment of n ≥ 3 experiments is shown. <b>C+D:</b> Stimulation of TLR2-transfected HEK293 cells with 5 µg/ml cell wall (CW; <b>C</b>) or lipoprotein preparations (SALP; <b>D</b>) prepared from <i>S. aureus</i> strains Cowan I (SAC; SpA^high, grey bars) or Wood46 (SpA^low, black bars). As indicated crude CW (left) were treated with hydrofluoric acid (HF) and RNAse A and DNAse I (middle) followed by digestion with proteinase K (PK, right). SALP were treated with proteinase K (PK, left) or lysostaphin (LS, right). (<b>−</b>) refers to unstimulated cells. The experiments shown were performed in triplicates and show mean values ± SEM of IL-8 concentrations determined in the supernatants. They are representative of n = 2 independent experiments. <b>E+F: </b><b>Analysis of </b><b><i>agr</i></b><b> activity. E: Hemolysin production.</b> Strains to be tested for hemolysin production were cross-streaked to RN4220, which produces β-hemolysin. After incubation for 36 hours strains were analyzed following the description by Taber et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096416#pone.0096416-Traber1" target="_blank">[15]</a>: Wood46 and CF36 displayed the typical pattern for α- and δ-hemolysin expression, INV02 produced α-, β- and δ-hemolysins, SAC (Cowan I) and INV66 expressed only low amounts of δ-hemolysin and SA113 was negative for α-, β- and δ-hemolysins. In conclusion, SA113, SAC and INV66 were categorized as <i>agr</i>-, Wood46, CF36 and INV02 were typed as <i>agr</i>+. <b>F: Analysis of δ-toxin expression by MALDI-TOF.</b> δ-toxin expression is dependent on <i>agr</i> activity as reported in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096416#pone.0096416-Josten1" target="_blank">[17]</a>. Delta toxin (MW 3007) and delta toxin G10S (MW 3037) peaks were detectable in Wood46, INV002 and CF36 and absent in SAC, SA113 and INV66.</p

Disruption of cell wall integrity facilitates recognition of TLR2 ligands.

<p><b>A+B:</b> HEK293 cells transfected with 100 ng/well of TLR2 plasmid were stimulated with clinical <i>S. aureus</i> isolates (<b>A</b>) or with heat-inactivated, boiled or live SA113 (<b>B</b>) and compared to Pam₃CSK₄ (P3; 0.2 µg/ml (<b>A</b>); 1 µg/ml (<b>B</b>)) and to unstimulated cells (<b>−</b>). Stimulation was carried out in the presence of different antibiotics: penicillin (Pen), vancomycin (Vanco), fosfomycin (Fosfo), ciprofloxacin (Cipro) or linezolid (Lz). <i>S. aureus</i> isolates were susceptible to all antibiotics tested. The diagrams shown provide the mean values ± SD of IL-8 values summarized from n = 3 independent experiments. The most relevant statistical results are highlighted as (*) or (**): Penicillin: *p (live:100°C) = 0.03, *p (live:60°C) = 0.01, **p (100°C: 60°C) = 0.001 and **p (live+Pen: live+Lz) = 0.002; not indicated: Vancomycin: **p (live:100°C) = 0.001; Linezolid: *p(live:100°C) = 0.006. <b>C:</b> HEK293 cells transfected with (white or grey bars) or without (black bars) 100 ng/well of TLR2 plasmid were stimulated with clinical SCV strains, 0.2 µg/ml of Pam₃CSK₄ (P3) or left unstimulated<b>.</b> Live bacteria (white bars) were compared to crude preps (boiled bacterial suspensions; grey bars). IL-8 levels in the supernatants were determined to quantify TLR2-activity. The diagram shows the mean values ± SD obtained in three independent experiments.</p

TLR2 ligands enhance <i>S. aureus</i>-induced cytokine secretion.

<p><b>A:</b> Adherent human monocytes were stimulated with <i>S. aureus</i> strains: e.g. SA113 and its isogenic mutant Δ<i>lgt</i>, which is deficient in lipoprotein maturation. Secreted IL-6 (left) and TNF (right) concentrations were quantified in the supernatants. The diagrams show the mean values ± SD from cells from six healthy donors. <b>B:</b> HEK293 cells transfected with or without TLR2 plasmid (100 ng/well) were stimulated with <i>S. aureus</i> Lpp SitC (left; 5 ng/ml) or <i>S. aureus</i> strain SA113 (right). (<b>−</b>) refers to unstimulated cells. IL-8 was detected in the supernatants after 24h of stimulation. All diagrams show the mean values ± SD obtained in three experiments.</p

The TLR2-stimulatory capacity varies among <i>S. aureus</i> isolates.

<p>HEK293 cells transfected with or without TLR2 plasmid (100 ng/well) were stimulated with either cystic fibrosis (CF) or invasive (INV) <i>S. aureus</i> isolates. IL-8 production was quantified 24 hours after stimulation. <b>A:</b> TLR2-activity was tested in 53 CF (left) and 53 INV (right) isolates. The dots provide the values obtained for single strains, the bars indicate the means. No significant difference was observed when comparing CF to INV isolates. <b>B:</b> The diagram shows one representative experiment with 16 CF (left, grey) and 16 INV (right, black) isolates. The origins of the isolates with absent to low TLR2-activity (arrows) are indicated in the graph. (<b>−</b>) refers to unstimulated cells.</p

<i>S. aureus</i> strains used in this study.

<p>n.a. = not applicable.</p