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  • 99
    ATCC s aureus staphylococcal organisms
    Comparison of the orders of the genes that surround the chromosomal replication origins of S. carnosus and S. aureus <t>N315.</t> Sections of the oriC regions of S. aureus and S. carnosus illustrating the gene inversion observed in the staphylococcal genomes
    S Aureus Staphylococcal Organisms, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Millipore staphylococcus aureus
    Comparison of the orders of the genes that surround the chromosomal replication origins of S. carnosus and S. aureus <t>N315.</t> Sections of the oriC regions of S. aureus and S. carnosus illustrating the gene inversion observed in the staphylococcal genomes
    Staphylococcus Aureus, supplied by Millipore, used in various techniques. Bioz Stars score: 94/100, based on 1321 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    ATCC staphylococcus aureus
    Comparison of the orders of the genes that surround the chromosomal replication origins of S. carnosus and S. aureus <t>N315.</t> Sections of the oriC regions of S. aureus and S. carnosus illustrating the gene inversion observed in the staphylococcal genomes
    Staphylococcus Aureus, supplied by ATCC, used in various techniques. Bioz Stars score: 94/100, based on 6882 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ATCC s aureus pcx19ωhemb s aureus sa113
    Comparison of the orders of the genes that surround the chromosomal replication origins of S. carnosus and S. aureus <t>N315.</t> Sections of the oriC regions of S. aureus and S. carnosus illustrating the gene inversion observed in the staphylococcal genomes
    S Aureus Pcx19ωhemb S Aureus Sa113, supplied by ATCC, used in various techniques. Bioz Stars score: 95/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore s aureus rn4220
    Biofilm formation in vitro . A: Attached biomass of reference strain <t>RN4220</t> and S54F9 in the wells of microtiter trays based on the binding of crystal violet. Mean OD 594 with SEM. B: 3D projection of the bottom of a microtiter well stained for bacterial cells with green Syto9. 630 x magnification.
    S Aureus Rn4220, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 50 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Comparison of the orders of the genes that surround the chromosomal replication origins of S. carnosus and S. aureus N315. Sections of the oriC regions of S. aureus and S. carnosus illustrating the gene inversion observed in the staphylococcal genomes

    Journal: Applied and Environmental Microbiology

    Article Title: Genome Analysis of the Meat Starter Culture Bacterium Staphylococcus carnosus TM300 ▿ TM300 ▿ †

    doi: 10.1128/AEM.01982-08

    Figure Lengend Snippet: Comparison of the orders of the genes that surround the chromosomal replication origins of S. carnosus and S. aureus N315. Sections of the oriC regions of S. aureus and S. carnosus illustrating the gene inversion observed in the staphylococcal genomes

    Article Snippet: We compared the S. carnosus genome with those of the following representatives of sequenced staphylococcal species: S. aureus N315, Mu50, MW2, COL, MSSA476, MRSA252, and RF122; S. epidermidis RP62A and ATCC 12228; S. haemolyticus JCSC 1435; and S. saprophyticus ATCC 15305.

    Techniques:

    Biofilm formation in vitro . A: Attached biomass of reference strain RN4220 and S54F9 in the wells of microtiter trays based on the binding of crystal violet. Mean OD 594 with SEM. B: 3D projection of the bottom of a microtiter well stained for bacterial cells with green Syto9. 630 x magnification.

    Journal: Journal of Bone and Joint Infection

    Article Title: Combined Staining Techniques for Demonstration of Staphylococcus aureus Biofilm in Routine Histopathology

    doi: 10.7150/jbji.22799

    Figure Lengend Snippet: Biofilm formation in vitro . A: Attached biomass of reference strain RN4220 and S54F9 in the wells of microtiter trays based on the binding of crystal violet. Mean OD 594 with SEM. B: 3D projection of the bottom of a microtiter well stained for bacterial cells with green Syto9. 630 x magnification.

    Article Snippet: Both S. aureus S54F9 and S. aureus RN4220 (a reference strain) were diluted to a final cell density of OD 0.005 in TSB (Sigma, USA) with 1% glucose.

    Techniques: In Vitro, Binding Assay, Staining

    Binding affinity of KAMPs to P. aeruginosa LPS or S. aureus LTA measured by the competitive binding assay . LPS- or LTA-bound BODIPY TR cadaverine in 10 mM NaCl or 5 mM HEPES (pH 7.5) was competitively displaced by agents having affinity of LPS or LTA, resulting in increased fluorescence signal. (A) KAMP-19 and KAMP-10 showed relatively low binding affinities to P. aeruginosa LPS in NaCl ( left ) and no binding in HEPES ( right ). (B) Using the same media, both KAMPs were highly effective against P. aeruginosa . (C) KAMP-18C and KAMP-36 displayed lower binding affinities for LTA in HEPES compared with NaCl. However, (D) these peptides showed similar (KAMP-18C) or stronger (KAMP-36) bactericidal activities in HEPES against S. aureus . Mean ± SD is shown. ** p

    Journal: Frontiers in Microbiology

    Article Title: Membrane-Active Epithelial Keratin 6A Fragments (KAMPs) Are Unique Human Antimicrobial Peptides with a Non-αβ Structure

    doi: 10.3389/fmicb.2016.01799

    Figure Lengend Snippet: Binding affinity of KAMPs to P. aeruginosa LPS or S. aureus LTA measured by the competitive binding assay . LPS- or LTA-bound BODIPY TR cadaverine in 10 mM NaCl or 5 mM HEPES (pH 7.5) was competitively displaced by agents having affinity of LPS or LTA, resulting in increased fluorescence signal. (A) KAMP-19 and KAMP-10 showed relatively low binding affinities to P. aeruginosa LPS in NaCl ( left ) and no binding in HEPES ( right ). (B) Using the same media, both KAMPs were highly effective against P. aeruginosa . (C) KAMP-18C and KAMP-36 displayed lower binding affinities for LTA in HEPES compared with NaCl. However, (D) these peptides showed similar (KAMP-18C) or stronger (KAMP-36) bactericidal activities in HEPES against S. aureus . Mean ± SD is shown. ** p

    Article Snippet: Purified P. aeruginosa LPS and S. aureus LTA (Sigma-Aldrich) were prepared in distilled water.

    Techniques: Binding Assay, Competitive Binding Assay, Fluorescence

    Staphylococcus aureus MVs promote bacterial survival in human whole blood and in the presence of neutrophils ex vivo and in vivo . (A) Survival of S. aureus MSSA476 in blood is increased in the presence of 20 μg MV (0.1 μg/μl) isolated from MSSA476 grown in LB and BHI [marked as LB (MVs) or BHI (MVs) in the figure] compared to absence of MVs (marked as control in the figure). The number of inoculated bacteria at time point 0 was set to 100% and the number of surviving bacteria after 3 h is represented as the percentage of inoculation. (B) S. aureus MSSA476 survival in blood is increased in the presence of MVs, in a dose-dependent manner (5–20 μg of total MVs, i.e., 0.025–0.1 μg/μl). The number of surviving bacteria after 3 h in the absence of MVs was arbitrary set as 1, and the number of surviving bacteria in the presence of MVs is represented as the fold change compared to bacteria in the absence of MV. (C) Survival of USA300 MRSA in human blood is increased in the presence of MVs isolated from USA300 (MV-Hla) and USA300ΔHla (MV-ΔHla) grown in BHI. The percentage of survival was calculated as described in (A) . (D) Sonication of purified MVs followed by proteinase K (PK) treatment abolished the effect of MVs on bacterial survival in human whole blood. The fold change of survival was calculated as described in (B) . (E) Survival of opsonized S. aureus MSSA476 in the presence of neutrophils is enhanced by supplementation of MVs isolated from bacteria growing in LB and BHI. Percentage of survival was calculated as described in (A) . (F) S. aureus MSSA476 were labeled with FITC and incubated with human whole blood in the absence or presence of MVs isolated from MSSA476 grown in LB and BHI. Data represents geometric mean of the fluorescence intensity (GMFI). (G) Bacterial loads in the blood (CFU/ml) of 8-week-old C57BL/6 mice were counted 24 h after the mice were intravenously infected with S. aureus MSSA476 supplemented with PBS or an exogenous source of MVs isolated from MSSA476 grown in BHI. (H) HaCaT (100 μg of total MVs, i.e., 0.1 μg/μl) and freshly purified neutrophils were treated with MVs (5–20 μg of total MVs, i.e., 0.025–0.1 μg/μl) isolated from S. aureus MSSA476 grown in LB or BHI at the time points indicated. Percentage of cytotoxicity was calculated by measuring the amount of LDH released from the cytosol of damaged cells into the supernatant after exposure to MVs. (I) Viability staining of neutrophils in the presence (20 μg of total MVs, i.e., 0.1 μg/μl) or absence of MVs were performed using propidium iodide (PI). Live imaging was performed after 0 and 0.45 or 1.5 h using fluorescence microscopy. Scale bar is shown. The data represent as the mean ± SEM of at least three independent experiments except for (D) , which the data are expressed as the mean ± SEM of two independent experiments performed in triplicate. Mice study corresponds to one experiment performed with 10 mice/group. The significance is indicated by asterisks ( ∗ ): ∗ P

    Journal: Frontiers in Microbiology

    Article Title: Staphylococcus aureus Membrane-Derived Vesicles Promote Bacterial Virulence and Confer Protective Immunity in Murine Infection Models

    doi: 10.3389/fmicb.2018.00262

    Figure Lengend Snippet: Staphylococcus aureus MVs promote bacterial survival in human whole blood and in the presence of neutrophils ex vivo and in vivo . (A) Survival of S. aureus MSSA476 in blood is increased in the presence of 20 μg MV (0.1 μg/μl) isolated from MSSA476 grown in LB and BHI [marked as LB (MVs) or BHI (MVs) in the figure] compared to absence of MVs (marked as control in the figure). The number of inoculated bacteria at time point 0 was set to 100% and the number of surviving bacteria after 3 h is represented as the percentage of inoculation. (B) S. aureus MSSA476 survival in blood is increased in the presence of MVs, in a dose-dependent manner (5–20 μg of total MVs, i.e., 0.025–0.1 μg/μl). The number of surviving bacteria after 3 h in the absence of MVs was arbitrary set as 1, and the number of surviving bacteria in the presence of MVs is represented as the fold change compared to bacteria in the absence of MV. (C) Survival of USA300 MRSA in human blood is increased in the presence of MVs isolated from USA300 (MV-Hla) and USA300ΔHla (MV-ΔHla) grown in BHI. The percentage of survival was calculated as described in (A) . (D) Sonication of purified MVs followed by proteinase K (PK) treatment abolished the effect of MVs on bacterial survival in human whole blood. The fold change of survival was calculated as described in (B) . (E) Survival of opsonized S. aureus MSSA476 in the presence of neutrophils is enhanced by supplementation of MVs isolated from bacteria growing in LB and BHI. Percentage of survival was calculated as described in (A) . (F) S. aureus MSSA476 were labeled with FITC and incubated with human whole blood in the absence or presence of MVs isolated from MSSA476 grown in LB and BHI. Data represents geometric mean of the fluorescence intensity (GMFI). (G) Bacterial loads in the blood (CFU/ml) of 8-week-old C57BL/6 mice were counted 24 h after the mice were intravenously infected with S. aureus MSSA476 supplemented with PBS or an exogenous source of MVs isolated from MSSA476 grown in BHI. (H) HaCaT (100 μg of total MVs, i.e., 0.1 μg/μl) and freshly purified neutrophils were treated with MVs (5–20 μg of total MVs, i.e., 0.025–0.1 μg/μl) isolated from S. aureus MSSA476 grown in LB or BHI at the time points indicated. Percentage of cytotoxicity was calculated by measuring the amount of LDH released from the cytosol of damaged cells into the supernatant after exposure to MVs. (I) Viability staining of neutrophils in the presence (20 μg of total MVs, i.e., 0.1 μg/μl) or absence of MVs were performed using propidium iodide (PI). Live imaging was performed after 0 and 0.45 or 1.5 h using fluorescence microscopy. Scale bar is shown. The data represent as the mean ± SEM of at least three independent experiments except for (D) , which the data are expressed as the mean ± SEM of two independent experiments performed in triplicate. Mice study corresponds to one experiment performed with 10 mice/group. The significance is indicated by asterisks ( ∗ ): ∗ P

    Article Snippet: In order to perform proteomic analysis, fractionation of MVs from S. aureus MSSA476 grown in LB and BHI broth was carried out by density gradient centrifugation using Optiprep (Sigma–Aldrich, Germany) as previously described , with minor modifications.

    Techniques: Ex Vivo, In Vivo, Isolation, Sonication, Purification, Labeling, Incubation, Fluorescence, Mouse Assay, Infection, Staining, Imaging, Microscopy

    The size and protein cargo of S. aureus -derived MVs were markedly different depending on growth conditions. (A) TEM-immunogold analysis of MV-generation on S. aureus MSSA476 (black arrow) grown on LA and blood agar plate. Gold particles (white arrows) indicate the presence of peptidoglycan/peptidoglycan-precursors. The scale bar is shown. (B) Atomic force micrographs of S. aureus MSSA476 cultivated on LA, BHI- and blood agar plates. Arrows indicate MVs on the bacterial surface and the released MVs. The scale bar is shown. (C) The average size distribution of MVs was determined using dynamic light scattering. The data are expressed as mean ± SEM (standard error of the mean) of four independent measurements performed on independent samples. (D) TEM-immunogold labeling of peptidoglycan/peptidoglycan-precursors in purified vesicles (black arrows) obtained from S. aureus MSSA476 grown in BHI and LB purified by flotation through an OptiPrep density gradient. Gold particles (white arrows) indicate the presence of peptidoglycan/peptidoglycan-precursors. Scale bars are shown. (E) S. aureus -derived vesicles from MSSA476 grown in LB and BHI were purified by flotation through an OptiPrep density gradient. Venn diagram illustrates common and unique proteins associated with MV identified using LPI/in-solution approaches. (F) Localization of the MV-associated proteins shown as the percentage in a pie chart. (G) Histograms representing the distribution of identified vesicular proteins according to their molecular functions, role in biological processes and cellular components. (H) Supplementation of MVs (20 μg of total MV, i.e., 0.1 μg/μl) in the culture media did not influence bacterial growth in LB media (left panel) or BHI (right panel). Data represent as the mean ± SEM of three independent experiments. The significance is indicated by asterisks ( ∗ ): ∗∗ P ≤ 0.01.

    Journal: Frontiers in Microbiology

    Article Title: Staphylococcus aureus Membrane-Derived Vesicles Promote Bacterial Virulence and Confer Protective Immunity in Murine Infection Models

    doi: 10.3389/fmicb.2018.00262

    Figure Lengend Snippet: The size and protein cargo of S. aureus -derived MVs were markedly different depending on growth conditions. (A) TEM-immunogold analysis of MV-generation on S. aureus MSSA476 (black arrow) grown on LA and blood agar plate. Gold particles (white arrows) indicate the presence of peptidoglycan/peptidoglycan-precursors. The scale bar is shown. (B) Atomic force micrographs of S. aureus MSSA476 cultivated on LA, BHI- and blood agar plates. Arrows indicate MVs on the bacterial surface and the released MVs. The scale bar is shown. (C) The average size distribution of MVs was determined using dynamic light scattering. The data are expressed as mean ± SEM (standard error of the mean) of four independent measurements performed on independent samples. (D) TEM-immunogold labeling of peptidoglycan/peptidoglycan-precursors in purified vesicles (black arrows) obtained from S. aureus MSSA476 grown in BHI and LB purified by flotation through an OptiPrep density gradient. Gold particles (white arrows) indicate the presence of peptidoglycan/peptidoglycan-precursors. Scale bars are shown. (E) S. aureus -derived vesicles from MSSA476 grown in LB and BHI were purified by flotation through an OptiPrep density gradient. Venn diagram illustrates common and unique proteins associated with MV identified using LPI/in-solution approaches. (F) Localization of the MV-associated proteins shown as the percentage in a pie chart. (G) Histograms representing the distribution of identified vesicular proteins according to their molecular functions, role in biological processes and cellular components. (H) Supplementation of MVs (20 μg of total MV, i.e., 0.1 μg/μl) in the culture media did not influence bacterial growth in LB media (left panel) or BHI (right panel). Data represent as the mean ± SEM of three independent experiments. The significance is indicated by asterisks ( ∗ ): ∗∗ P ≤ 0.01.

    Article Snippet: In order to perform proteomic analysis, fractionation of MVs from S. aureus MSSA476 grown in LB and BHI broth was carried out by density gradient centrifugation using Optiprep (Sigma–Aldrich, Germany) as previously described , with minor modifications.

    Techniques: Derivative Assay, Transmission Electron Microscopy, Labeling, Purification

    Staphylococcus aureus MVs promote extracellular trap formation in human neutrophils in vitro independent of ROS generation. Neutrophils were incubated with PBS (control) or MVs isolated from bacteria grown in LB or BHI. NET induction was evaluated using various approaches: (A) Immunostaining using a primary antibody against myeloperoxidase (green). The nucleus is stained with Hoechst (blue). Scale bar is shown. (B) Quantification of extracellular DNA and (C) measuring neutrophils degranulation through determining the elastase release. The absorbance at 405 nm in the absence of MVs (control) was normalized to 1, and the absorbance in the presence of MVs (LB and BHI) and the positive control (Triton) is represented as the fold change of elastase release. (D) DCF-based ROS assays were performed to evaluate the effect of S. aureus MSSA476 MVs on ROS production by neutrophils. (E) Neutrophils were pre-treated with the ROS scavenger BHA for 30 min before addition of either PMA or MSSA476 MVs to determine whether MVs induced NET production. Data represent as the mean ± SEM of at least three independent experiments. The significance is indicated by asterisks ( ∗ ): ∗∗ P ≤ 0.01; ∗∗∗ P ≤ 0.001; ∗∗∗∗ P ≤ 0.0001.

    Journal: Frontiers in Microbiology

    Article Title: Staphylococcus aureus Membrane-Derived Vesicles Promote Bacterial Virulence and Confer Protective Immunity in Murine Infection Models

    doi: 10.3389/fmicb.2018.00262

    Figure Lengend Snippet: Staphylococcus aureus MVs promote extracellular trap formation in human neutrophils in vitro independent of ROS generation. Neutrophils were incubated with PBS (control) or MVs isolated from bacteria grown in LB or BHI. NET induction was evaluated using various approaches: (A) Immunostaining using a primary antibody against myeloperoxidase (green). The nucleus is stained with Hoechst (blue). Scale bar is shown. (B) Quantification of extracellular DNA and (C) measuring neutrophils degranulation through determining the elastase release. The absorbance at 405 nm in the absence of MVs (control) was normalized to 1, and the absorbance in the presence of MVs (LB and BHI) and the positive control (Triton) is represented as the fold change of elastase release. (D) DCF-based ROS assays were performed to evaluate the effect of S. aureus MSSA476 MVs on ROS production by neutrophils. (E) Neutrophils were pre-treated with the ROS scavenger BHA for 30 min before addition of either PMA or MSSA476 MVs to determine whether MVs induced NET production. Data represent as the mean ± SEM of at least three independent experiments. The significance is indicated by asterisks ( ∗ ): ∗∗ P ≤ 0.01; ∗∗∗ P ≤ 0.001; ∗∗∗∗ P ≤ 0.0001.

    Article Snippet: In order to perform proteomic analysis, fractionation of MVs from S. aureus MSSA476 grown in LB and BHI broth was carried out by density gradient centrifugation using Optiprep (Sigma–Aldrich, Germany) as previously described , with minor modifications.

    Techniques: In Vitro, Incubation, Isolation, Immunostaining, Staining, Positive Control

    Biochemical characterization of the SACOL1827 protein. (A) Pulldown Assay showing association of SACOL1827 with core-RNAP. Lane order: L1, LMW Markers; L2, Monoclonal Anti-poly Histidine–Agarose antibody (with beads); L3, SACOL1827; L4, SACOL1827 (with beads); L5, core-RNAP; L6, SACOL1827 + core-RNAP (with beads); L7, core-RNAP (with beads); L8, HMW Markers. (B) Transcription run-off assay. Lane order: L1, DNA size markers; L2, transcription run-off conducted with core-RNAP + purified SACOL1827; L3, transcription run-off conducted with core-RNAP only; L4, transcription run-off conducted with purified SACOL1827 only.

    Journal: PLoS ONE

    Article Title: Identification and Characterization of ?S, a Novel Component of the Staphylococcus aureus Stress and Virulence Responses

    doi: 10.1371/journal.pone.0003844

    Figure Lengend Snippet: Biochemical characterization of the SACOL1827 protein. (A) Pulldown Assay showing association of SACOL1827 with core-RNAP. Lane order: L1, LMW Markers; L2, Monoclonal Anti-poly Histidine–Agarose antibody (with beads); L3, SACOL1827; L4, SACOL1827 (with beads); L5, core-RNAP; L6, SACOL1827 + core-RNAP (with beads); L7, core-RNAP (with beads); L8, HMW Markers. (B) Transcription run-off assay. Lane order: L1, DNA size markers; L2, transcription run-off conducted with core-RNAP + purified SACOL1827; L3, transcription run-off conducted with core-RNAP only; L4, transcription run-off conducted with purified SACOL1827 only.

    Article Snippet: To test the ability of S. aureus SACOL1827 to bind to core-RNAP we generated recombinant protein using standard E. coli overexpression techniques, and the 6HIS-tagging vector pET24d (Novagen), as described previously .

    Techniques: Purification

    MerA and HypR are both required for NaOCl stress survival in S. aureus . S. aureus COL wild-type (WT), Δ hypR and Δ merA mutants (A) , and their complemented strains and Cys mutants (Δ merA merA , Δ merA merAC43S , Δ hypR hypR , Δ hypR hypRC33A ) (B, C) were grown in RPMI until an OD 500 of 0.5 and treated with 3.5 m M NaOCl. Survival assays were performed by spotting 10 μL of serial dilutions after 3 and 4 h of NaOCl exposure onto LB agar plates. The active site Cys43 of MerA and the redox-sensing Cys33 of HypR are important for NaOCl stress survival. LB, Luria Bertani.

    Journal: Antioxidants & Redox Signaling

    Article Title: Redox-Sensing Under Hypochlorite Stress and Infection Conditions by the Rrf2-Family Repressor HypR in Staphylococcus aureus

    doi: 10.1089/ars.2017.7354

    Figure Lengend Snippet: MerA and HypR are both required for NaOCl stress survival in S. aureus . S. aureus COL wild-type (WT), Δ hypR and Δ merA mutants (A) , and their complemented strains and Cys mutants (Δ merA merA , Δ merA merAC43S , Δ hypR hypR , Δ hypR hypRC33A ) (B, C) were grown in RPMI until an OD 500 of 0.5 and treated with 3.5 m M NaOCl. Survival assays were performed by spotting 10 μL of serial dilutions after 3 and 4 h of NaOCl exposure onto LB agar plates. The active site Cys43 of MerA and the redox-sensing Cys33 of HypR are important for NaOCl stress survival. LB, Luria Bertani.

    Article Snippet: The hypR gene ( SACOL0641 ) was amplified from chromosomal DNA of S. aureus COL by PCR using primers 0641-pET-for-NheI and 0641-pET-rev-BamHI , digested with Nhe I and Bam HI and inserted into plasmid pET11b (Novagen) that was digested using the same restriction enzymes to generate plasmid pET11b- hypR .

    Techniques:

    MerA and HypR are required for survival of S. aureus COL in murine macrophages. The survival of S. aureus strains was analyzed 2, 4, and 24 h postinfection (p.i.) of the murine macrophage cell line J-774A.1 and the CFUs were determined. (A, B) The percentages in survival of the Δ hypR and Δ merA mutants and complemented strains were calculated in 5–6 biological replicate experiments and the survival at the 2-h time point was set to 100%. (C) The average percentage in survival was calculated for each mutant and complemented strain in relation to the wild type (WT), which was set to 100%. Results of 5–6 biological replicates are presented as scatter dots in (A, B) and mean values of percentage survival in comparison to the wild type (C) . Error bars represent the SEM and the statistics was calculated using one-way ANOVA and Tukey's multiple comparisons post hoc test using the GraphPad Prism software. The p -values were determined as follows for the scatter dots (A, B) : p = 0.0078 for WT/Δ hypR ; p = 0.0303 for WT/Δ merA ; p = 0.0461 for WTpRB473/Δ hypRhypR ; and p = 0.1234 for WTpRB473/Δ merAmerA . For the percentage survival (C) , the p -values were determined as p

    Journal: Antioxidants & Redox Signaling

    Article Title: Redox-Sensing Under Hypochlorite Stress and Infection Conditions by the Rrf2-Family Repressor HypR in Staphylococcus aureus

    doi: 10.1089/ars.2017.7354

    Figure Lengend Snippet: MerA and HypR are required for survival of S. aureus COL in murine macrophages. The survival of S. aureus strains was analyzed 2, 4, and 24 h postinfection (p.i.) of the murine macrophage cell line J-774A.1 and the CFUs were determined. (A, B) The percentages in survival of the Δ hypR and Δ merA mutants and complemented strains were calculated in 5–6 biological replicate experiments and the survival at the 2-h time point was set to 100%. (C) The average percentage in survival was calculated for each mutant and complemented strain in relation to the wild type (WT), which was set to 100%. Results of 5–6 biological replicates are presented as scatter dots in (A, B) and mean values of percentage survival in comparison to the wild type (C) . Error bars represent the SEM and the statistics was calculated using one-way ANOVA and Tukey's multiple comparisons post hoc test using the GraphPad Prism software. The p -values were determined as follows for the scatter dots (A, B) : p = 0.0078 for WT/Δ hypR ; p = 0.0303 for WT/Δ merA ; p = 0.0461 for WTpRB473/Δ hypRhypR ; and p = 0.1234 for WTpRB473/Δ merAmerA . For the percentage survival (C) , the p -values were determined as p

    Article Snippet: The hypR gene ( SACOL0641 ) was amplified from chromosomal DNA of S. aureus COL by PCR using primers 0641-pET-for-NheI and 0641-pET-rev-BamHI , digested with Nhe I and Bam HI and inserted into plasmid pET11b (Novagen) that was digested using the same restriction enzymes to generate plasmid pET11b- hypR .

    Techniques: Mutagenesis, Software

    Northern blot analysis of hypR-merA transcription in S. aureus COL under NaOCl, diamide, H 2 O 2 , and aldehyde stress and in hypR Cys-Ala mutants. (A) Northern blot analysis was performed using RNA isolated from S. aureus COL wild type before (co) and 15 and 30 min after exposure to 1 m M NaOCl, 2 m M diamide, 10 m M H 2 O 2 , 0.75 m M formaldehyde, and 0.5 m M methylglyoxal stress. (B) Transcription of the hypR-merA operon was analyzed in the COL wild-type and in the Δ hypR mutant under 1 m M NaOCl stress indicating strong derepression of hypR-merA transcription under control conditions in the absence of HypR. (C, D) Northern blot analysis of hypR-merA operon transcription in the Δ hypR deletion mutant and in Δ hypR mutants complemented with hypR , hypRC33A , hypRC99A , and hypRC142A before and 15 and 30 min after exposure to 1 m M NaOCl (C) or 2 m M diamide stress (D) . The results indicate that Cys33 is required for redox sensing of HypR in vivo . For stress experiments, S. aureus cells were grown in RPMI medium and treated with thiol-reactive compounds at an OD 500 of 0.5. The arrows point toward the hypR-merA bicistronic mRNA (1.8 kb) in the wild type or the truncated hypR-merA transcript ( TR-merA ) (1.5 kb) in the hypR mutant. The methylene blue stain is the RNA loading control indicating the 16S and 23S rRNAs. OD 500 , optical density at 500 nms. H 2 O 2 , hydrogen peroxide.

    Journal: Antioxidants & Redox Signaling

    Article Title: Redox-Sensing Under Hypochlorite Stress and Infection Conditions by the Rrf2-Family Repressor HypR in Staphylococcus aureus

    doi: 10.1089/ars.2017.7354

    Figure Lengend Snippet: Northern blot analysis of hypR-merA transcription in S. aureus COL under NaOCl, diamide, H 2 O 2 , and aldehyde stress and in hypR Cys-Ala mutants. (A) Northern blot analysis was performed using RNA isolated from S. aureus COL wild type before (co) and 15 and 30 min after exposure to 1 m M NaOCl, 2 m M diamide, 10 m M H 2 O 2 , 0.75 m M formaldehyde, and 0.5 m M methylglyoxal stress. (B) Transcription of the hypR-merA operon was analyzed in the COL wild-type and in the Δ hypR mutant under 1 m M NaOCl stress indicating strong derepression of hypR-merA transcription under control conditions in the absence of HypR. (C, D) Northern blot analysis of hypR-merA operon transcription in the Δ hypR deletion mutant and in Δ hypR mutants complemented with hypR , hypRC33A , hypRC99A , and hypRC142A before and 15 and 30 min after exposure to 1 m M NaOCl (C) or 2 m M diamide stress (D) . The results indicate that Cys33 is required for redox sensing of HypR in vivo . For stress experiments, S. aureus cells were grown in RPMI medium and treated with thiol-reactive compounds at an OD 500 of 0.5. The arrows point toward the hypR-merA bicistronic mRNA (1.8 kb) in the wild type or the truncated hypR-merA transcript ( TR-merA ) (1.5 kb) in the hypR mutant. The methylene blue stain is the RNA loading control indicating the 16S and 23S rRNAs. OD 500 , optical density at 500 nms. H 2 O 2 , hydrogen peroxide.

    Article Snippet: The hypR gene ( SACOL0641 ) was amplified from chromosomal DNA of S. aureus COL by PCR using primers 0641-pET-for-NheI and 0641-pET-rev-BamHI , digested with Nhe I and Bam HI and inserted into plasmid pET11b (Novagen) that was digested using the same restriction enzymes to generate plasmid pET11b- hypR .

    Techniques: Northern Blot, Isolation, Mutagenesis, In Vivo, Staining

    MC extracellular trap (MCET) induction and phagocytosis of Staphylococcus aureus bioparticles . (A) MCETs were visualized without fixation using the live/dead viability/cytotoxicity kit (Invitrogen) for mammalian cells. Significantly more MCETs are found after 24 h hypoxia versus normoxia, but not after 3 h. Results are shown from the analysis of n = 3 independent experiments, each with four individual images. (B) Representative fluorescence micrograph of MCET induction (red: dead cells/MCETs in a fiber like structure; green living cells). (C) Mast cells (MCs) (2 × 10 6 cells/ml) were preincubated 3 or 24 h under hypoxia (37°C, 1% O 2 , 5% CO 2 ) or normoxia (37°C, 21% O 2 , 5% CO 2 ). Then phycoerythrin (PE)-labeled S. aureus (Wood strain, bioparticles; Sigma) at an MOI of 60 was incubated with MCs for 30 min under the respective oxygen condition. CTR represents uninfected control. The cells were washed with PBS and centrifuged to remove non-phagocytosed bacteria. PE-fluorescence was measured using a Beckman Coulter EPICS XL flow cytometer. The red fluorescence intensity per MC (% gated) was recorded and represents the mean relative phagocytosis of PE-labeled S. aureus per MC of n = 3 independent experiments.

    Journal: Frontiers in Immunology

    Article Title: Hypoxia Modulates the Response of Mast Cells to Staphylococcus aureus Infection

    doi: 10.3389/fimmu.2017.00541

    Figure Lengend Snippet: MC extracellular trap (MCET) induction and phagocytosis of Staphylococcus aureus bioparticles . (A) MCETs were visualized without fixation using the live/dead viability/cytotoxicity kit (Invitrogen) for mammalian cells. Significantly more MCETs are found after 24 h hypoxia versus normoxia, but not after 3 h. Results are shown from the analysis of n = 3 independent experiments, each with four individual images. (B) Representative fluorescence micrograph of MCET induction (red: dead cells/MCETs in a fiber like structure; green living cells). (C) Mast cells (MCs) (2 × 10 6 cells/ml) were preincubated 3 or 24 h under hypoxia (37°C, 1% O 2 , 5% CO 2 ) or normoxia (37°C, 21% O 2 , 5% CO 2 ). Then phycoerythrin (PE)-labeled S. aureus (Wood strain, bioparticles; Sigma) at an MOI of 60 was incubated with MCs for 30 min under the respective oxygen condition. CTR represents uninfected control. The cells were washed with PBS and centrifuged to remove non-phagocytosed bacteria. PE-fluorescence was measured using a Beckman Coulter EPICS XL flow cytometer. The red fluorescence intensity per MC (% gated) was recorded and represents the mean relative phagocytosis of PE-labeled S. aureus per MC of n = 3 independent experiments.

    Article Snippet: To analyze bacterial uptake, MCs were incubated with fluorescent S. aureus bioparticles (Sigma), and the percentage of fluorescent MCs was determined after removal of free, non-associated bacteria to demonstrate the engulfment of S. aureus under hypoxia.

    Techniques: Fluorescence, Labeling, Incubation, Flow Cytometry, Cytometry

    Transmission electron micrographs of S. aureus HG001 cells grown in RPMI medium. Staphylococcal cells were grown without any stress for 120 min after an OD of 0.5 was reached (A), or staphylococcal cells were exposed to vancomycin (B) or ampicillin (C)

    Journal: Antimicrobial Agents and Chemotherapy

    Article Title: Impact of Antibiotics with Various Target Sites on the Metabolome of Staphylococcus aureus

    doi: 10.1128/AAC.03104-14

    Figure Lengend Snippet: Transmission electron micrographs of S. aureus HG001 cells grown in RPMI medium. Staphylococcal cells were grown without any stress for 120 min after an OD of 0.5 was reached (A), or staphylococcal cells were exposed to vancomycin (B) or ampicillin (C)

    Article Snippet: S. aureus HG001 ( ) was grown in RPMI 1640 R7509 medium (Sigma-Aldrich) with vigorous agitation at 37°C as previously described ( ).

    Techniques: Transmission Assay

    Auranofin inhibits bacterial TrxR. ( A ) Purified recombinant M. tuberculosis TrxB2 was preincubated with NADPH and DTNB with or without auranofin for 15 min. Reactions were initiated by the addition of TrxC. Shown is a dose–response plot for the initial rate of TrxB2 activity in the presence or absence of auranofin. ( Inset ) Representative progress plot of TrxB2 activity in the presence or absence of auranofin used for generating the dose–response plot. The colors of the lines on the progress plot correspond to the same color points on the dose–response plot. The purple line on the progress plot corresponds to the untreated control, which is not graphed on the dose-response plot. Auranofin concentrations tested were 800, 400, 200, 100, 50, and 25 nM. ( B ) Dose–response plots for S. aureus TrxB activity with and without a 15-min preincubation with auranofin with corresponding progress plots showing that auranofin inhibits TrxB activity. Assays were carried out as with TrxB2. Dose–response plots represent at least three determinations ± SE.

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Auranofin exerts broad-spectrum bactericidal activities by targeting thiol-redox homeostasis

    doi: 10.1073/pnas.1504022112

    Figure Lengend Snippet: Auranofin inhibits bacterial TrxR. ( A ) Purified recombinant M. tuberculosis TrxB2 was preincubated with NADPH and DTNB with or without auranofin for 15 min. Reactions were initiated by the addition of TrxC. Shown is a dose–response plot for the initial rate of TrxB2 activity in the presence or absence of auranofin. ( Inset ) Representative progress plot of TrxB2 activity in the presence or absence of auranofin used for generating the dose–response plot. The colors of the lines on the progress plot correspond to the same color points on the dose–response plot. The purple line on the progress plot corresponds to the untreated control, which is not graphed on the dose-response plot. Auranofin concentrations tested were 800, 400, 200, 100, 50, and 25 nM. ( B ) Dose–response plots for S. aureus TrxB activity with and without a 15-min preincubation with auranofin with corresponding progress plots showing that auranofin inhibits TrxB activity. Assays were carried out as with TrxB2. Dose–response plots represent at least three determinations ± SE.

    Article Snippet: M. tuberculosis trxb2 (Rv3913), trxC (Rv3914), and S. aureus trxB were cloned into the pET28a or pET28b expression vectors (EMD Millipore) and purified by Ni-NTA chromatography.

    Techniques: Purification, Recombinant, Activity Assay