Journal:

Article Title: Inhibition of Staphylococcal Biofilm Formation by Nitrite ▿ †

doi: 10.1128/JB.00598-07

Figure Lengend Snippet: Biofilm formation of S. aureus and S. epidermidis is impaired by sodium nitrate and sodium nitrite. Biofilm formation of S. aureus SA113 (upper panel) and S. epidermidis RP62A (lower panel) was assayed using the microtiter plate adherence assay. The cells were grown in the absence or initial presence of 1 to 5 mM sodium nitrate (NO3−) or sodium nitrite (NO2−). After 24 h, the residual nitrite concentrations in the culture supernatants were determined by colorimetric detection (24 h NO2−). Adherent cells were stained with safranin.

Article Snippet: Staphylococcus aureus strain SA113 (ATCC 35556) ( 30 ) and Staphylococcus epidermidis strain RP62A (ATCC 35984) ( 11 ) were used in this study.

Techniques: Staining

Journal:

Article Title: Inhibition of Staphylococcal Biofilm Formation by Nitrite ▿ †

doi: 10.1128/JB.00598-07

Figure Lengend Snippet: Effect of nitrate and nitrite on icaA transcription and PIA synthesis. S. aureus wild type (WT) and its isogenic narG deletion (ΔnarG) mutant were grown under biofilm conditions in 6-well cell culture plate wells in the absence or presence of 20 mM sodium nitrate (NO3−) (A) or 5 and 10 mM sodium nitrite (NO2−) (B). Cells were harvested after 6 h, and total RNA was isolated. The relative abundance levels of icaA mRNAs normalized to gyrB signals were assessed by semiquantitative RT-PCR and densitometric analysis. The levels of icaA expression in the presence of nitrate or nitrite were compared with normalized levels obtained from their matched controls set to 100% (bars). (C) PIA was extracted from cells grown for 24 h under biofilm conditions in 6-well cell culture plates in the absence (−) or presence (+) of 5 mM sodium nitrite (NO2−). PIA was detected by dot blot analysis using wheat germ agglutinin coupled to horseradish peroxidase. The PIA-deficient S. aureus SA113 ica (Δica) mutant was used as a negative control. PIA quantification using rabbit antiserum raised against S. epidermidis PIA yielded comparable results (not shown).

Article Snippet: Staphylococcus aureus strain SA113 (ATCC 35556) ( 30 ) and Staphylococcus epidermidis strain RP62A (ATCC 35984) ( 11 ) were used in this study.

Techniques: Mutagenesis, Cell Culture, Isolation, Reverse Transcription Polymerase Chain Reaction, Expressing, Dot Blot, Negative Control

Journal:

Article Title: Inhibition of Staphylococcal Biofilm Formation by Nitrite ▿ †

doi: 10.1128/JB.00598-07

Figure Lengend Snippet: Nitrite toxicity toward S. aureus requires acidification. (A) S. aureus wild type (WT) and its isogenic narG deletion (ΔnarG) mutant were seeded onto TSB agar plates buffered at the indicated pH values. Five millimolar sodium nitrate (NO3−) was included (+) or omitted (−) from the plates. After the placement of a filter disc soaked with 20 μl of a 2 M NaNO2 solution, the plates were incubated under conditions of oxygen limitation for 48 h and monitored for growth inhibition zones (indicated by dotted circles). (B) Nitrite- and SNAP-mediated inhibition of biofilm formation are abrogated in the presence of NO scavengers. Biofilm formation of S. aureus SA113 in the absence (C, control) or presence of 5 mM nitrite (NO2−) or the NO donor SNAP (0.1 mM) was quantified after 24 h by spectrophotometric measurement of the absorbance of adherent cells after safranin staining at 450 nm. The NO scavengers carboxy-PTIO (PTIO; 2.5 mM) and hemoglobin (Hb; 0.5 mM) were initially added as indicated or omitted (−). Data are means ± standard error of the means of two to four experiments performed at least in triplicate.

Article Snippet: Staphylococcus aureus strain SA113 (ATCC 35556) ( 30 ) and Staphylococcus epidermidis strain RP62A (ATCC 35984) ( 11 ) were used in this study.

Techniques: Mutagenesis, Incubation, Inhibition, Control, Staining

Journal: Journal of Innate Immunity

Article Title: Prostaglandin E 2 Regulates Activation of Mouse Peritoneal Macrophages by Staphylococcus aureus through Toll-Like Receptor 2, Toll-Like Receptor 4, and NLRP3 Inflammasome Signaling

doi: 10.1159/000499604

Figure Lengend Snippet: Lipoproteins are essential for S. aureus-induced COX-2 and mPGES-1 expression in macrophages. Mouse peritoneal macrophages were infected with S. aureus SA113 (WT), isogenic mutant lgt::ermB (Δlgt), or its complemented strain, lgt::ermB + pRBlgt (+ pRB), at MOI 3:1 or 10:1 or not infected. Cox-2 and mPges-1 mRNA expression levels were detected by qRT-PCR and normalized to those of the housekeeping gene Gapdh at the indicated timepoints (a, b). Levels of specific proteins in total macrophage extracts after S. aureus infection at MOI 10:1 were determined by western blotting. GAPDH was used as a loading control (c). Grayscale values were measured using ImageJ software (d). Results are expressed as the mean ± SD of 3 independent experiments and were analyzed by 2-way ANOVA with Bonferroni's post-test. * p < 0.05; ** p < 0.01; *** p < 0.001. COX-2, cyclooxygenases 2; mPGES-1; microsomal prostaglandin E synthases 1; MOI, multiplicity of infection.

Article Snippet: S. aureus SA113 wild-type strain (WT; ATCC 35558), an S. aureus SA113 isogenic mutant lgt ::ermB (Δ lgt ) deficient in lipoprotein maturation, and its complemented strain SA113 lgt:: ermB + pRB lgt (+ pRB) were kindly provided by Prof. Friedrich Götz of Mikrobielle Genetik, Universität Tübingen, Germany [ 29 , 30 ].

Techniques: Expressing, Infection, Mutagenesis, Quantitative RT-PCR, Western Blot, Control, Software

Journal: Journal of Innate Immunity

Article Title: Prostaglandin E 2 Regulates Activation of Mouse Peritoneal Macrophages by Staphylococcus aureus through Toll-Like Receptor 2, Toll-Like Receptor 4, and NLRP3 Inflammasome Signaling

doi: 10.1159/000499604

Figure Lengend Snippet: TLR2, TLR4, and the NLRP3 inflammasome are involved in S. aureus-induced COX-2 and mPGES-1 expression in macrophages. Macrophages from WT, NLRP3−/–, TLR2−/–, and TLR4−/– mice were infected with WT S. aureus SA113 (SA113) at MOI 3:1 or 10:1. Cox-2 and mPges-1 mRNA expression levels were determined by qRT-PCR and standardized to those of Gapdh at the indicated timepoints (a, b). Levels of the indicated proteins in total macrophage extracts after S. aureus infection at MOI 10:1 were determined by western blotting. GAPDH served as a loading control (c). Grayscale values were measured using ImageJ software (d). Results are expressed as the mean ± SD of 3 independent experiments and were analyzed by 2-way ANOVA with Bonferroni's post-test. * p < 0.05; ** p < 0.01; *** p < 0.001. ND, not detected; TLR, toll-like receptors; WT, wild-type; COX-2, cyclooxygenases-2; mPGES-1; microsomal prostaglandin E synthases-1; MOI, multiplicity of infection.

Article Snippet: S. aureus SA113 wild-type strain (WT; ATCC 35558), an S. aureus SA113 isogenic mutant lgt ::ermB (Δ lgt ) deficient in lipoprotein maturation, and its complemented strain SA113 lgt:: ermB + pRB lgt (+ pRB) were kindly provided by Prof. Friedrich Götz of Mikrobielle Genetik, Universität Tübingen, Germany [ 29 , 30 ].

Techniques: Expressing, Infection, Quantitative RT-PCR, Western Blot, Control, Software

Journal: Journal of Innate Immunity

Article Title: Prostaglandin E 2 Regulates Activation of Mouse Peritoneal Macrophages by Staphylococcus aureus through Toll-Like Receptor 2, Toll-Like Receptor 4, and NLRP3 Inflammasome Signaling

doi: 10.1159/000499604

Figure Lengend Snippet: S. aureus lipoproteins and host TLR2, TLR4, and NLRP3 are involved in secretion of PGE2 and proinflammatory cytokines and chemokines by macrophages after S. aureus infection. WT and corresponding gene-deficient macrophages were infected with multiple S. aureus strains (SA113, Δlgt, and + pRB) at MOI 10:1 or not infected. PGE2, TNF-α, IL-1β, and RANTES release into the supernatant of macrophage cultures was analyzed by ELISA 9 h after infection (a–h). Activation of the cAMP-PKA (P-PKA) and MAPK (P-ERK and P-p38) pathways was evaluated by western blotting. GAPDH served as a loading control (i, j). WT macrophages (untreated or pre-treated with 10 µM H89 for 2 h) were infected with WT S. aureus strains at MOI 10:1 or not infected. TNF-α, IL-1β, and RANTES production in the supernatants of macrophages was analyzed by ELISA 9 h after infection (k). Results are expressed as the mean ± SD of 3 independent experiments and were analyzed by one-way ANOVA followed by Tukey's multiple comparisons test or 2-way ANOVA with Bonferroni's post-test. * p < 0.05; ** p < 0.01; *** p < 0.001. PKA, protein kinase A; NLRP3, NOD-like receptor P3; TLR, toll-like receptors; WT, wild-type; COX-2, cyclooxygenases-2; mPGES-1; microsomal prostaglandin E synthases-1; MOI, multiplicity of infection.

Article Snippet: S. aureus SA113 wild-type strain (WT; ATCC 35558), an S. aureus SA113 isogenic mutant lgt ::ermB (Δ lgt ) deficient in lipoprotein maturation, and its complemented strain SA113 lgt:: ermB + pRB lgt (+ pRB) were kindly provided by Prof. Friedrich Götz of Mikrobielle Genetik, Universität Tübingen, Germany [ 29 , 30 ].

Techniques: Infection, Enzyme-linked Immunosorbent Assay, Activation Assay, Western Blot, Control

Journal: Journal of Innate Immunity

Article Title: Prostaglandin E 2 Regulates Activation of Mouse Peritoneal Macrophages by Staphylococcus aureus through Toll-Like Receptor 2, Toll-Like Receptor 4, and NLRP3 Inflammasome Signaling

doi: 10.1159/000499604

Figure Lengend Snippet: Phagocytosis of S. aureus and survival within macrophages are dependent on S. aureus lipoproteins, and these processes are inhibited by PGE2. Macrophages (untreated or pre-treated with 1 µM PGE2 for 24 h) were infected using multiple S. aureus strains (SA113, Δlgt, and + pRB) at MOI 30:1 or not infected. To analyze S. aureus invasion into macrophages 3 h after infection, the colony counting technique was used to determine CFU recovered from macrophage lysates (a, b). ND, not detected. TNF-α, IL-1β, and RANTES production in the supernatants of PGE2-pre-treated or untreated macrophages was analyzed by ELISA 9 h after infection (c–e). SR-A expression in macrophages infected with WT or Δlgt S. aureus was measured by flow cytometry and quantified as median fluorescence intensity using Flowjo 10.0 software (f). Phagocytosis of Hoechst 33,258 (blue)-labeled WT or Δlgt S. aureus within DiI-labeled macrophages (Orange) was analyzed by microscopy assay (×400, g, h). Bacterial intracellular killing was measured by tetrazolium dye reduction assay (i). Results are expressed as the mean ± SD of 3 independent experiments and analyzed by one-way ANOVA followed by Tukey's multiple comparisons test or 2-way ANOVA. * p < 0.05; ** p < 0.01; *** p < 0.001. CFU, colony forming units; PGE2, prostaglandin E2; SR-A, scavenger receptor A.

Article Snippet: S. aureus SA113 wild-type strain (WT; ATCC 35558), an S. aureus SA113 isogenic mutant lgt ::ermB (Δ lgt ) deficient in lipoprotein maturation, and its complemented strain SA113 lgt:: ermB + pRB lgt (+ pRB) were kindly provided by Prof. Friedrich Götz of Mikrobielle Genetik, Universität Tübingen, Germany [ 29 , 30 ].

Techniques: Infection, Enzyme-linked Immunosorbent Assay, Expressing, Flow Cytometry, Fluorescence, Software, Labeling, Microscopy

Journal: Journal of Innate Immunity

Article Title: Prostaglandin E 2 Regulates Activation of Mouse Peritoneal Macrophages by Staphylococcus aureus through Toll-Like Receptor 2, Toll-Like Receptor 4, and NLRP3 Inflammasome Signaling

doi: 10.1159/000499604

Figure Lengend Snippet: TLR2-, TLR4-, and NLRP3-mediated signaling activation in macrophages after S. aureus infection is regulated by PGE2 treatment. WT and corresponding gene-deficient macrophages were infected using WT S. aureus SA113 at MOI 30:1 for 3 h, and the CFU recovered from lysates was determined (a). SR-A expression in WT and corresponding gene-deficient macrophages after WT S. aureus infection was measured by flow cytometry and quantified as median fluorescence intensity using Flowjo 10.0 software. Bacterial intracellular killing was measured by tetrazolium dye reduction assay to analyze S. aureus invasion into macrophage (b). Macrophages (untreated or pre-treated with 1 µM PGE2 for 24 h) were infected with WT S. aureus SA113 at MOI 30:1 or not infected. NLRP3, TLR2, and TLR4 expression in macrophages was measured by flow cytometry and quantified as median fluorescence intensity using Flowjo 10.0 software (c–e). Activation of MAPK (P-ERK, P-JNK, and P-p38), NF-κB (P-p65), and caspase-1 (p10, pro-IL-1β, and mature IL-1β) pathways were evaluated by western blotting; GAPDH was used as a loading control (f). Grayscale values were measured using ImageJ software (g, h). Results are expressed as the mean ± SD of 3 independent experiments and were analyzed by one-way ANOVA followed by Tukey's multiple comparisons test or 2-way ANOVA with Bonferroni's post-test. * p < 0.05; ** p < 0.01; *** p < 0.001. CFU, colony forming units; SR-A, scavenger receptor A; NLRP3, NOD-like receptor P3; TLR, toll-like receptors; PGE2, prostaglandin E2.

Article Snippet: S. aureus SA113 wild-type strain (WT; ATCC 35558), an S. aureus SA113 isogenic mutant lgt ::ermB (Δ lgt ) deficient in lipoprotein maturation, and its complemented strain SA113 lgt:: ermB + pRB lgt (+ pRB) were kindly provided by Prof. Friedrich Götz of Mikrobielle Genetik, Universität Tübingen, Germany [ 29 , 30 ].

Techniques: Activation Assay, Infection, Expressing, Flow Cytometry, Fluorescence, Software, Western Blot, Control

Journal: Journal of Innate Immunity

Article Title: Prostaglandin E 2 Regulates Activation of Mouse Peritoneal Macrophages by Staphylococcus aureus through Toll-Like Receptor 2, Toll-Like Receptor 4, and NLRP3 Inflammasome Signaling

doi: 10.1159/000499604

Figure Lengend Snippet: S. aureus-induced activation of ­caspase-1 was decreased in NLRP3−/–, TLR2−/–, and TLR4−/– macrophages. WT and corresponding gene-deficient macrophages were infected with WT S. aureus SA113 (MOI 10:1). Activation of caspase-1 was examined by western blotting; GAPDH was used as a loading control. WT, wild-type; NLRP3, NOD-like receptor P3; TLR, toll-like receptors.

Article Snippet: S. aureus SA113 wild-type strain (WT; ATCC 35558), an S. aureus SA113 isogenic mutant lgt ::ermB (Δ lgt ) deficient in lipoprotein maturation, and its complemented strain SA113 lgt:: ermB + pRB lgt (+ pRB) were kindly provided by Prof. Friedrich Götz of Mikrobielle Genetik, Universität Tübingen, Germany [ 29 , 30 ].

Techniques: Activation Assay, Infection, Western Blot, Control