Comparison of the biofilm-forming capacity of <i>S</i>. <i>aureus</i> isolates from prosthetic joint infection (PJI) and uninfected prosthetic joint (PJU) groups.

<p>Values were calculated as percentage capacity to form biofilms relative to <i>S</i>. <i>aureus</i> control strain UAMS-1. Box ends represent the 25^th and 75^th percentiles, and whisker ends represent the minimum and maximum. There was no difference in biofilm-forming capacity between isolates in the PJI and PJU groups.</p

Single Nucleotide Polymorphisms (SNPs) in fibronectin binding protein B (<i>fnbB)</i> in <i>fnbB-</i>containing isolates.

<p>*When false discovery rate control is applied, this raw p-value no longer maintains statistical significance (p = 1.00).</p><p>No SNP was associated with the prosthetic joint infected (PJI) or uninfected (PJU) isolates in the derivation cohort, external validation cohort, or late <i>S</i>. <i>aureus</i> bacteremia (SAB) group. Late SAB was defined as SAB occurring >1 year after placement or manipulation of prostheses.</p

Demographic and clinical characteristics of patients in the derivation cohort with <i>S</i>. <i>aureus</i> bacteremia and infected or uninfected prostheses.

<p>*Late infection is defined as bloodstream infection occurring >1 year after the prostheses was implanted or surgically manipulated.</p><p>Demographic and clinical characteristics of patients in the derivation cohort with <i>S</i>. <i>aureus</i> bacteremia and infected or uninfected prostheses.</p

Down-regulation of <i>Dusp3</i> and <i>Psme3</i> in A/J are responsible for increased NF-κB signaling activity.

<p>(<b>A</b>) The expression of <i>Dcaf7</i>, <i>Dusp3</i>, and <i>Psme3</i> in A/J was significantly lower than C57BL/6J under both non-infected and <i>S. aureus</i> infected conditions. Eight-week-old male A/J and C57BL/6J mice (n = 6) were challenged (i.p.) by <i>S. aureus</i> Sanger 476 at 10⁷ CFU/g or DPBS. At two hours post-infection whole blood RNAs was extracted by RNeasy followed by RT-PCR and qPCR. <i>Dcaf7</i> (0.81 fold; p<0.05), <i>Dusp3</i> (0.27 fold; p<0.01), and <i>Psme3</i> (0.83 fold; p<0.05) were down-regulated in A/J mice at baseline (0 hr) as compared with resistant C57BL/6J. Two genes exhibited elevated expression in susceptible A/J mice (baseline): <i>Fam134c</i> (1.82 fold; p<0.01) and <i>Slc4a1</i> (1.31fold; p<0.05). The baseline difference in expression between susceptible (A/J) and resistant (C57BL/6J) mice of all the five genes remained unchanged at 2 hr post <i>S. aureus</i>-infection. (<b>B</b>) <i>Dusp3</i> and <i>Psme3</i> inhibit NF-κB signaling activity in RAW264.7 macrophages. RAW264.7 cells co-transfected with NF-κB-luciferase and pRL-TK plasmids were then transfected with siRNA of each individual candidate genes (<i>Dcaf7</i>, <i>Dusp3</i>, <i>Psme3</i>) or scrambled siRNA. Then transfected RAW cells were stimulated by either medium alone, medium containing LTA (10 µg/ml) or medium containing <i>S. aureus</i> particles (10 µg/ml) for 7 hours. Cells were directly lysed by 1× passive lysis buffer and luciferase activity was assayed and normalized to renilla activity as previously described <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004149#ppat.1004149-Yan1" target="_blank">[15]</a>. As shown, knockdown of both <i>Dusp3</i> and <i>Psme3</i> significantly up-regulates NF-κB luciferase activity (p<0.01). (<b>C</b>) Knockdown of <i>Dusp3</i> or <i>Psme3</i> enhanced the activation of NF-κB signaling upon <i>S. aureus</i> stimulation. BMDMs from C57BL/6J were transfected with either scrambled, <i>Dusp3</i> or <i>Psme3</i> siRNA, then stimulated with <i>S. aureus</i> for 15 minutes. Whole cell lysate was loaded for western-blot. Knockdown of <i>Dusp3</i> or <i>Psme3</i> dramatically increased degradation of IκBα and phosphorylation of p65 (Ser536) as compared with scrambled siRNA control. (<b>D</b>) Enhanced NF-κB signaling upon <i>S. aureus</i> stimulation in CSS11 BMDMs. BMDMs from either C57BL/6J or CSS11 were stimulated with <i>S. aureus</i> for 15 minutes. Whole cell lysate was loaded for Western blot. BMDMs from CSS11 exhibited increased degradation of IκBα and phosphorylation of p65 (Ser536) as compared with BMDMs from C57BL/6J. (<b>E</b>) Bay inhibition of NF-κB dramatically suppressed cytokine production. RAW264.7 macrophages were pre-treated with 4 µM Bay 11-7085 for one hour, then stimulated with 10 µg/ml <i>S. aureus</i> particles in 2 µM Bay for 3 hours. RNA was extracted and subjected to reverse-transcription PCR and qPCR. Inhibition of NF-κB by Bay inhibitor dramatically suppressed cytokine production upon <i>S. aureus</i> stimulation, including IL-1β (p<0.01), IL-6 (p<0.05) and TNF-α (p<0.05). (<b>F</b>) Inhibition of NF-κB enhanced <i>Dusp3</i> and <i>Psme3</i> expression. The inhibition of NF-κB activity by Bay inhibitor significantly enhanced the expression of both <i>Dusp3</i> (p<0.05) and <i>Psme3</i> (p<0.05), which indicated a reciprocal relationship between NF-κB signaling activity and <i>Dusp3</i> or <i>Psme3</i> expression.</p

Overall strategy for identifying genes associated with <i>S. aureus</i> susceptibility on chromosome 11 of A/J mice.

<p>Flow chart of the strategy for identifying <i>S. aureus</i> susceptible genes on chromosome 11 of A/J mice.</p

Down-regulation of <i>Dusp3</i> and <i>Psme3</i> in A/J is associated with over-production of pro-inflammatory cytokines.

<p>Both <i>Dusp3</i> and <i>Psme3</i> inhibit NF-κB activity, which is responsible for the production of inflammatory cytokines. The reduced expression of <i>Dusp3</i> and <i>Psme3</i> in A/J mice is associated with elevated NF-κB activity, which leads to increased inflammatory cytokines. <i>Dusp3</i> and <i>Psme3</i>, together with other factors from murine chromosome 8 and 18, contribute to <i>S. aureus</i> susceptibility of A/J strain.</p

Comparison of the fibronectin binding capacity of <i>S</i>. <i>aureus</i> isolates from prosthetic joint infection (PJI) and uninfected prosthetic joint (PJU) groups.

<p>Values were calculated as percentage capacity to bind fibronectin relative to <i>S</i>. <i>aureus</i> control strain 8325–4. Box ends represent the 25^th and 75^th percentiles, and whisker ends represent the minimum and maximum. There was no difference in fibronectin binding capacity between isolates in the PJI and PJU groups.</p

Quantitative-PCR confirmed elevation of cytokine production in macrophages transfected by <i>Dusp3</i> and <i>Psme3</i> siRNA or BMDMs from CSS11(GM-CSF, IL-1β, IL-6, and TNF-α).

<p>(<b>A</b>) Down-regulation of <i>Dusp3</i> and <i>Psme3</i> by siRNA led to increased cytokine RNA expression upon <i>S. aureus</i> challenge in RAW264.7 macrophages. At three hours post-infection total RNA was extracted followed by reverse-transcription PCR and SYBR-Green qPCR. The expression of all genes were normalized to 18s rRNA. The expression level of GM-CSF, IL-1β, IL-6, and TNF-α was higher in <i>Dusp3</i> knockdown RAW cells, and the level of GM-CSF and IL-6 was higher in <i>Psme3</i> knockdown RAW cells. p-value smaller than 0.05 was considered significant. (<b>B</b>) BMDMs cytokine RNA production in CSS11 mice was significantly higher than in C57BL/6J upon <i>S. aureus</i> infection. 2×10⁶ BMDMs were seeded to single wells in a 6-well plate the day before infection. At three hours post-infection, RNA was extracted using RNeasy followed by RT-PCR and qPCR. The expression levels of GM-CSF, IL-1β, IL-6, and TNF-α were significantly higher in BMDMs from CSS11 mice. The expression of all genes were normalized to 18s rRNA. p-value smaller than 0.05 was considered significant. (<b>C</b>) Down-regulation of <i>Dusp3</i> and <i>Psme3</i> by siRNA led to increased cytokine RNA expression upon <i>S. aureus</i> challenge in BMDMs of C57BL/6J. The expression level of IL-6 was higher in <i>Dusp3</i> siRNA transfected BMDMs, and the expression of TNF-α was higher in both <i>Dusp3</i> and <i>Psme3</i> siRNA transfected BMDMs.</p

siRNA knockdown of <i>Dusp3</i> and <i>Psme3</i> result in significant elevation of cytokine production, consistent with the pattern of bone marrow derived macrophages from CSS11 as compared with C57BL/6J (GM-CSF, IL-1β, IL-6 and TNF-α).

<p>(<b>A</b>) Bone-marrow derived macrophages (BMDMs) from A/J and C57BL/6J have similar <i>S. aureus</i> phagocytosis ability. 2×10⁶ BMDMs were seeded to single wells in a 6-well plate the day before phagocytosis and incubated with hexidium iodide stained <i>S. aureus</i>. Phagocytic efficiency as determined by the mean fluorescence intensity (MFI) is not significantly different in BMDMs from C57BL/6J and CSS11 mice. Representative histogram of 3 separate experiments. (<b>B</b>) BMDMs from CSS11 mice produced significantly higher cytokine levels as compared to C57BL/6J. 4×10⁵ BMDMs from both C57BL/6J and CSS11 mice were seeded to single-wells of a 24-well plate the day before infection. Infection was simulated by adding <i>S. aureus</i> particles at 10 µg/ml. At 24 hours post-infection the supernatants were harvested and subjected to Luminex cytokine assaying. BMDMs from CSS11 mice significantly enhanced cytokine production, including GM-CSF, IL-1β, IL-6, and TNF-α. (<b>C</b>) Down-regulation of <i>Dusp3</i> and <i>Psme3</i> by siRNA led to up-regulation of cytokine production upon <i>S. aureus</i> challenge in RAW264.7 macrophages. RAW264.7 cells were transfected by either scramble or <i>Dusp3</i> or <i>Psme3</i> siRNA, and then infected with <i>S. aureus</i> particles at 10 µg/ml as before <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004149#ppat.1004149-Ahn1" target="_blank">[11]</a>. At 24 hours post-infection, the supernatants were harvested and subjected to Luminex-multiplex cytokine assaying. The down-regulation of <i>Dusp3</i> significantly enhanced cytokine production, including GM-CSF, IL-1β, IL-6, and TNF-α, as compared to scramble siRNA control. The down-regulation of <i>Psme3</i> also significantly elevated GM-CSF and IL-6 production. (<b>D</b>) Down-regulation of <i>Dusp3</i> or <i>Psme3</i> by siRNA led to up-regulation of cytokine production upon <i>S. aureus</i> challenge in BMDMs. BMDMs from C57BL/6J were transfected by either scrambled, <i>Dusp3</i> or <i>Psme3</i> siRNA, and then infected with <i>S. aureus</i> particles at 10 µg/ml. At 24 hours post-infection, the supernatants were harvested and subjected to cytokine analysis. The down-regulation of <i>Dusp3</i> significantly enhanced cytokine production, including IL-6 and TNF-α, as compared to scrambled siRNA control. The down-regulation of <i>Psme3</i> also significantly elevated TNF-α production.</p

Single Nucleotide Polymorphisms (SNPs) in the binding regions (Repeats 1–11) of fibronectin binding protein B (FnBPB) in the derivation cohort (A) and the external validation cohort (B).

<p>Red boxes indicate SNPs that were only present in isolates from the prosthetic joint infection group (PJI), blue boxes indicate SNPs that were only present in the uninfected (PJU) group, and purple boxes indicate SNPs that were present in both PJI and PJU isolates.</p