Journal: Microorganisms

Article Title: Reduced Susceptibility and Increased Resistance of Bacteria against Disinfectants: A Systematic Review

doi: 10.3390/microorganisms9122550

Figure Lengend Snippet: Study characteristics of identified research studies about disinfectant resistance.

Article Snippet: Cottell, et al. (2009), Great Britain and Germany, Journal of Hospital Infection , Determining the minimum inhibitory concentrations of triclosan with broth- and agar-dilution methods. Using the British Society for Antimicrobial Chemotherapy guidelines, the antibiotic susceptibilities were determined. Exploring further linkages between triclosan exposure and the emergence or lack of bacterial antibiotic resistance. , Triclosan MICs were significantly higher for the mutant strains S. aureus T3 and E. coli TM3 compared with the parent strains E. coli ATCC 8739 and S. aureus NCIMB 9518. Significantly higher MIC was also observed in the triclosan-tolerant strain A. johnsonii RC compared to the sensitive counterpart..

Techniques: Infection, Mutagenesis, Derivative Assay, Functional Assay, Activity Assay, Plasmid Preparation, Produced, Bacteria, Inhibition, Control, Reverse Transcription, Gene Expression, Concentration Assay, Isolation

Journal: Microorganisms

Article Title: Reduced Susceptibility and Increased Resistance of Bacteria against Disinfectants: A Systematic Review

doi: 10.3390/microorganisms9122550

Figure Lengend Snippet: MIC of biocides and the expressed susceptibility/resistance of bacteria to disinfectants.

Article Snippet: Cottell, et al. (2009), Great Britain and Germany, Journal of Hospital Infection , Determining the minimum inhibitory concentrations of triclosan with broth- and agar-dilution methods. Using the British Society for Antimicrobial Chemotherapy guidelines, the antibiotic susceptibilities were determined. Exploring further linkages between triclosan exposure and the emergence or lack of bacterial antibiotic resistance. , Triclosan MICs were significantly higher for the mutant strains S. aureus T3 and E. coli TM3 compared with the parent strains E. coli ATCC 8739 and S. aureus NCIMB 9518. Significantly higher MIC was also observed in the triclosan-tolerant strain A. johnsonii RC compared to the sensitive counterpart..

Techniques: Bacteria, Concentration Assay

Journal: Microorganisms

Article Title: Reduced Susceptibility and Increased Resistance of Bacteria against Disinfectants: A Systematic Review

doi: 10.3390/microorganisms9122550

Figure Lengend Snippet: Disinfectant and bacterial mechanisms for the most commonly used disinfectants.

Article Snippet: Cottell, et al. (2009), Great Britain and Germany, Journal of Hospital Infection , Determining the minimum inhibitory concentrations of triclosan with broth- and agar-dilution methods. Using the British Society for Antimicrobial Chemotherapy guidelines, the antibiotic susceptibilities were determined. Exploring further linkages between triclosan exposure and the emergence or lack of bacterial antibiotic resistance. , Triclosan MICs were significantly higher for the mutant strains S. aureus T3 and E. coli TM3 compared with the parent strains E. coli ATCC 8739 and S. aureus NCIMB 9518. Significantly higher MIC was also observed in the triclosan-tolerant strain A. johnsonii RC compared to the sensitive counterpart..

Techniques: Membrane, Inhibition, Transformation Assay, Transduction, Plasmid Preparation, Gene Expression, Expressing, Concentration Assay, Activity Assay, Disruption, Lysis, Molecular Weight, Over Expression, Modification

Journal: Frontiers in Immunology

Article Title: Metallothionein 3-Zinc Axis Suppresses Caspase-11 Inflammasome Activation and Impairs Antibacterial Immunity

doi: 10.3389/fimmu.2021.755961

Figure Lengend Snippet: The MT3-Zn 2+ axis suppresses TRIF signaling resulting in decreased IRF3 phosphorylation. When MT3 is absent, TRIF-IRF3-STAT1 signaling and non-canonical inflammasome activation are exaggerated. A lack of MT3 augments immunity to gram-negative bacteria, an effect, that is further enhanced by the combined absence of MT3 and caspase-11 in vivo . Thus, while MT3 curtails caspase-11 activation, the two molecules act together in compromising antibacterial immunity.

Article Snippet: MT3 , Applied Biosystems , Hs00359394_g1.

Techniques: Phospho-proteomics, Activation Assay, Bacteria, In Vivo

Journal: Frontiers in Immunology

Article Title: Metallothionein 3-Zinc Axis Suppresses Caspase-11 Inflammasome Activation and Impairs Antibacterial Immunity

doi: 10.3389/fimmu.2021.755961

Figure Lengend Snippet: See also Supplementary Table S1 and Files S1 , S2 |Protein interaction network of Mus musculus MT3 to determine functionally enriched GO BP categories using the STRING database.

Article Snippet: MT3 , Applied Biosystems , Hs00359394_g1.

Techniques: Transduction, Cell Differentiation

Journal: Frontiers in Immunology

Article Title: Metallothionein 3-Zinc Axis Suppresses Caspase-11 Inflammasome Activation and Impairs Antibacterial Immunity

doi: 10.3389/fimmu.2021.755961

Figure Lengend Snippet: See also Supplementary Figure S1 MT3 suppresses caspase-11 inflammasome activation in BMDMϕ. qRT-PCR analysis of Mt3 expression in WT BMDMϕ stimulated with (A) iLPS (2 μg/ml) or vehicle control, 3-5 independent experiments and (B) exLPS (10 μg/ml) for 48h, 3 independent experiments, two-tailed t-test. (C) Western Blots of pro- and active-caspase-11, pro-caspase-1, pro-IL1β and β-actin in cell lysates and active-caspase-1 and active-IL-1β in supernatants of WT and Mt3 -/- BMDMϕ stimulated with iLPS (10 μg/ml) or vehicle for 48h. Bar graphs are densitometric analysis of targets normalized to β-actin, 3-4 independent experiments, one-way ANOVA, data are mean ± SEM. (D) Western Blots of pro- and active-caspase-11 and β-actin in lysate + supernatant samples from WT and Mt3 -/- BMDMϕ stimulated with iLPS (2 μg/ml) or vehicle for 48h. Bar graphs are densitometric analysis of targets normalized to β-actin. *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: MT3 , Applied Biosystems , Hs00359394_g1.

Techniques: Activation Assay, Quantitative RT-PCR, Expressing, Control, Two Tailed Test, Western Blot

Journal: Frontiers in Immunology

Article Title: Metallothionein 3-Zinc Axis Suppresses Caspase-11 Inflammasome Activation and Impairs Antibacterial Immunity

doi: 10.3389/fimmu.2021.755961

Figure Lengend Snippet: See also Supplementary Figure S2 . MT3 curtails CASPASE-4 and caspase-11 signaling and antibacterial immunity in hMϕ and in vivo . (A) MT3 and MT2A expression analyzed by qRT-PCR in hMϕ transfected with scramble siRNA or MT3 siRNA for 24h, 3 independent experiments, two-tailed t-test. (B) Scramble siRNA or MT3 siRNA treated hMϕ stimulated with iLPS (10 μg/ml) or vehicle for 48h. Immunoblots of pro-CASPASE-4 and active-CASPASE-4 in cell extracts, 3 independent experiments, one-way ANOVA. (C) Active-IL-1β measured by ELISA in supernatants of hMϕ treated as above, 3 independent experiments, one-way ANOVA. (D) E . coli growth inhibition in hMϕ transfected with MT3 siRNA and infected with 25 E . coli (K12): 1 hMϕ for 24h compared to scramble siRNA treated hMϕ, 3 independent experiments, two-tailed t-test. (E) E . coli growth inhibition in WT and Mt3 -/- BMDMϕ infected with 25 E . coli (K12):1 hMϕ for 24h, 4 independent experiments, two-tailed t-test. (F) WT and Mt3 -/- mice infected i.p. with 1X10 9 E . coli for 6h, log CFUs of E . coli in blood, kidney and peritoneal lavage samples, n = 12-15 per group, two-tailed t-test. (G) Western blots of inflammasome mediators in kidney homogenates of WT and Mt3 -/- mice infected as above, n = 6 per group, two-tailed t-test. (H) WT and Mt3 -/- mice infected i.p. with 1 X10 9 E . coli for 1h and IL-1β measured in peritoneal lavage and serum by ELISA. n = 3 per group, two-tailed t-test. (I) WT and Mt3 -/- mice primed i.p. with poly(I:C) (10 mg/kg) for 6h and challenged with LPS (2 mg/kg) i.p. After 18h, IL-1β was measured in peritoneal lavage and serum by ELISA, n = 3/group, two-tailed t-test. (J) Bacterial growth in spleen, lung and kidney of WT and Mt3 -/- mice infected i.n. with K. pneumoniae (4 X10 4 CFUs/mouse) for 48h, n = 8-12 per group, two-tailed t-test, data are mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: MT3 , Applied Biosystems , Hs00359394_g1.

Techniques: In Vivo, Expressing, Quantitative RT-PCR, Transfection, Two Tailed Test, Western Blot, Enzyme-linked Immunosorbent Assay, Inhibition, Infection

Journal: Frontiers in Immunology

Article Title: Metallothionein 3-Zinc Axis Suppresses Caspase-11 Inflammasome Activation and Impairs Antibacterial Immunity

doi: 10.3389/fimmu.2021.755961

Figure Lengend Snippet: See also Supplementary Figure S3 Caspase-11 synergizes with MT3 in impairing bacterial clearance. WT, C asp-11 -/- , Mt3 -/- and Casp-11 -/- Mt3 -/- mice were infected i.p. with E . coli (1 X10 9 CFUs/mouse) for 6h. (A) Bacterial CFUs measured in kidney, blood and peritoneal lavage, n = 3-6 per group, one-way ANOVA. (B) Western blots of pro-GSDMD, active-GSDMD (p31), pro-caspase-1, active-caspase-1, pro-IL1β and active-IL-1β in kidney homogenates, n = 3-6 per group, one-way ANOVA, data are mean ± SEM. (C) WT and Mt3 -/- mice treated i.p. with MCC950 (1 mg/mouse) or PBS and infected i.p. with E . coli (1 X10 9 CFUs/mouse) for 6h. IL1β was measured by ELISA in peritoneal lavage, n = 6 per group, one-way ANOVA, data are mean ± SEM. Bacterial CFUs in whole blood and kidney, n = 4 per group, one-way ANOVA, data are mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: MT3 , Applied Biosystems , Hs00359394_g1.

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

Journal: Frontiers in Immunology

Article Title: Metallothionein 3-Zinc Axis Suppresses Caspase-11 Inflammasome Activation and Impairs Antibacterial Immunity

doi: 10.3389/fimmu.2021.755961

Figure Lengend Snippet: See also Supplementary Figure S4 Myeloid-MT3 suppresses non-canonical inflammasome activation and blunts gram-negative bacterial clearance in vivo . (A) Generation of Mt3 fl/fl mice by inserting loxp sites flanking exon 3 of the Mt3 gene using the CRISPR-Cas9 gene targeting approach. Mt3 fl/fl mice crossed with Lys2Cre mice to obtain Lys2Cre Mt3 fl/fl mice. (B) Efficacy of myeloid Mt3 deletion assessed by genotyping peritoneal Mϕ (PMϕ) and BMDMϕ from Lys2Cre , Mt3 fl/fl and Lys2Cre Mt3 fl/fl mice. Gel electrophoresis analysis demonstrating efficient deletion of the Mt3 gene from BMDMϕ and PMϕ of Lys2Cre Mt3 fl/fl mice. (C) Western blots of pro-caspase-11, active-caspase-11, pro-GSDMD, active-GSDMD (p31), pro-caspase-1, active-caspase-1, pro-IL1β and active-IL-1β in whole kidney homogenates of mice infected as above, n = 3-5 per group, two-tailed t-test. (D) Bacterial CFUs in kidney and whole blood of Lys2Cre and Lys2Cre Mt3 fl/fl mice infected i.p. with E . coli (1 X10 9 CFUs/mouse) for 6h, n = 3-5 per group, two-tailed t-test, data are mean ± SEM. **p < 0.01, ***p < 0.001.

Article Snippet: MT3 , Applied Biosystems , Hs00359394_g1.

Techniques: Activation Assay, In Vivo, CRISPR, Nucleic Acid Electrophoresis, Western Blot, Infection, Two Tailed Test

Journal: Frontiers in Immunology

Article Title: Metallothionein 3-Zinc Axis Suppresses Caspase-11 Inflammasome Activation and Impairs Antibacterial Immunity

doi: 10.3389/fimmu.2021.755961

Figure Lengend Snippet: See also Supplementary Figure S5 MT3 thwarts TRIF-IRF3-STAT1 signaling to suppress non-canonical inflammasome activation. (A) Functional enrichment analysis of differentially expressed genes using RNA-seq data from resting WT and Mt3 -/- BMDMϕ (NCBI SRA: PRJNA533616) FDR, false detection rates. (B, C) Heat map (left) and table (right) show differentially expressed IFN-related genes in resting Mt3 -/- BMDMϕ compared to resting WT BMDMϕ obtained from RNA-seq analysis. (D) Western blots of pIRF3, pSTAT1, STAT1, GBP2 and GBP5 in vehicle or iLPS (10 μg/ml)-treated WT and Mt3 -/- BMDMϕ lysates, 3-4 independent experiments, one-way ANOVA. (E) Western blots of TRIF in lysates from WT and Mt3 -/- BMDMϕ stimulated as above, 3 independent experiments, one-way ANOVA. (F) Scramble and Ticam1 siRNA treated WT and Mt3 -/- BMDMϕ treated with iLPS (10 μg/ml) or vehicle for 48h. Immunoblots of TRIF (2 independent experiments), pro-caspase-11, and active-caspase-11 in lysates and active-IL-1β in supernatants, 3 independent experiments, one-way ANOVA, data are mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001; NS, not significant.

Article Snippet: MT3 , Applied Biosystems , Hs00359394_g1.

Techniques: Activation Assay, Functional Assay, RNA Sequencing, Western Blot

Journal: Frontiers in Immunology

Article Title: Metallothionein 3-Zinc Axis Suppresses Caspase-11 Inflammasome Activation and Impairs Antibacterial Immunity

doi: 10.3389/fimmu.2021.755961

Figure Lengend Snippet: See also Supplementary Figures S6 , 7 MT3-Zn 2+ axis drives negative regulation of the non-canonical inflammasome. (A) SEC-ICP-MS of WT and Mt3 -/- BMDMϕ exposed to vehicle or iLPS (10 ug/ml) for the indicated time points, chromatograms depict Zn 2+ distribution in cell lysates across various molecular masses, arrow indicates Zn 2+ associated with the MT-peak (18-21 min.) on the chromatogram, Y axis is off-set to allow easy comparison under the same scale. (B) Bar graphs of total Zn 2+ and MT-Zn 2+ in WT and Mt3 -/- BMDMϕ post iLPS (10 μg/ml) or vehicle exposure. Two-way t-test against respective BMDMϕ controls at each time point, 3 independent experiments, data are mean ± SD. (C) WT BMDMϕ treated with iLPS (10 μg/ml) or vehicle for 24h in Zn 2+ sufficient or Zn 2+ deficient Opti-MEM media, immunoblots of pIRF3, pro-caspase-11, active-caspase-11 and pro-IL-1β in lysates and active-IL-1β in media supernatants, one-way ANOVA, data are mean ± SEM. (D, E) Mt3 -/- BMDMϕ transfected with Pro-Ject™ or Pro-Ject™ complexed with apo-MT3, 4Zn 2+ MT3 or 6Zn 2+ MT3 and treated with iLPS (10 μg/ml) or vehicle for 24h in Zn 2+ deficient Opti-MEM media. (D) Chromatograms depict Zn 2+ distribution in cell lysates across various molecular masses, arrow indicates Zn 2+ signal associated with the MT-peak (18-21 min.) on the chromatogram, Y axis is off-set to allow easy comparison under the same scale. (E) Western blots of pIRF3, pro-caspase-11, active-caspase-11 and pro-IL1β in lysates and active-IL-1β in supernatants, 3 independent experiments, one-way ANOVA, data are mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001; NS, not significant.

Article Snippet: MT3 , Applied Biosystems , Hs00359394_g1.

Techniques: Comparison, Western Blot, Transfection

Journal: Frontiers in Immunology

Article Title: Metallothionein 3-Zinc Axis Suppresses Caspase-11 Inflammasome Activation and Impairs Antibacterial Immunity

doi: 10.3389/fimmu.2021.755961

Figure Lengend Snippet: Reagents and resources.

Article Snippet: MT3 , Applied Biosystems , Hs00359394_g1.

Techniques: Expressing, Plasmid Preparation, Control, Extraction, Blocking Assay, Injection, Cell Culture, Enzyme-linked Immunosorbent Assay, Transgenic Assay, Filtration, Transfection, Reverse Transcription, Cytotoxicity Assay, Isolation, Software, Imaging, Real-time Polymerase Chain Reaction

Journal: Microorganisms

Article Title: Reduced Susceptibility and Increased Resistance of Bacteria against Disinfectants: A Systematic Review

doi: 10.3390/microorganisms9122550

Figure Lengend Snippet: Study characteristics of identified research studies about disinfectant resistance.

Article Snippet: Cottell, et al. (2009), Great Britain and Germany, Journal of Hospital Infection , Determining the minimum inhibitory concentrations of triclosan with broth- and agar-dilution methods. Using the British Society for Antimicrobial Chemotherapy guidelines, the antibiotic susceptibilities were determined. Exploring further linkages between triclosan exposure and the emergence or lack of bacterial antibiotic resistance. , Triclosan MICs were significantly higher for the mutant strains S. aureus T3 and E. coli TM3 compared with the parent strains E. coli ATCC 8739 and S. aureus NCIMB 9518. Significantly higher MIC was also observed in the triclosan-tolerant strain A. johnsonii RC compared to the sensitive counterpart..

Techniques: Infection, Mutagenesis, Derivative Assay, Functional Assay, Activity Assay, Plasmid Preparation, Produced, Bacteria, Inhibition, Control, Reverse Transcription, Gene Expression, Concentration Assay, Isolation

Journal: Microorganisms

Article Title: Reduced Susceptibility and Increased Resistance of Bacteria against Disinfectants: A Systematic Review

doi: 10.3390/microorganisms9122550

Figure Lengend Snippet: MIC of biocides and the expressed susceptibility/resistance of bacteria to disinfectants.

Article Snippet: Cottell, et al. (2009), Great Britain and Germany, Journal of Hospital Infection , Determining the minimum inhibitory concentrations of triclosan with broth- and agar-dilution methods. Using the British Society for Antimicrobial Chemotherapy guidelines, the antibiotic susceptibilities were determined. Exploring further linkages between triclosan exposure and the emergence or lack of bacterial antibiotic resistance. , Triclosan MICs were significantly higher for the mutant strains S. aureus T3 and E. coli TM3 compared with the parent strains E. coli ATCC 8739 and S. aureus NCIMB 9518. Significantly higher MIC was also observed in the triclosan-tolerant strain A. johnsonii RC compared to the sensitive counterpart..

Techniques: Bacteria, Concentration Assay

Journal: Microorganisms

Article Title: Reduced Susceptibility and Increased Resistance of Bacteria against Disinfectants: A Systematic Review

doi: 10.3390/microorganisms9122550

Figure Lengend Snippet: Disinfectant and bacterial mechanisms for the most commonly used disinfectants.

Article Snippet: Cottell, et al. (2009), Great Britain and Germany, Journal of Hospital Infection , Determining the minimum inhibitory concentrations of triclosan with broth- and agar-dilution methods. Using the British Society for Antimicrobial Chemotherapy guidelines, the antibiotic susceptibilities were determined. Exploring further linkages between triclosan exposure and the emergence or lack of bacterial antibiotic resistance. , Triclosan MICs were significantly higher for the mutant strains S. aureus T3 and E. coli TM3 compared with the parent strains E. coli ATCC 8739 and S. aureus NCIMB 9518. Significantly higher MIC was also observed in the triclosan-tolerant strain A. johnsonii RC compared to the sensitive counterpart..

Techniques: Membrane, Inhibition, Transformation Assay, Transduction, Plasmid Preparation, Gene Expression, Expressing, Concentration Assay, Activity Assay, Disruption, Lysis, Molecular Weight, Over Expression, Modification

Journal: Frontiers in Microbiology

Article Title: The W-Acidic Motif of Histidine Kinase WalK Is Required for Signaling and Transcriptional Regulation in Streptococcus mutans

doi: 10.3389/fmicb.2022.820089

Figure Lengend Snippet: The extended CTT of WalK is unique in S. mutans and required for its interaction with WalR. (A) Phylogenetic analysis of S. mutans HKs. Evolutionary relationship of all 14 HKs is shown in a circular tree, which are grouped into two based on six key residues following their catalytic histidine with conservation for each group below. HKs with a conserved HisKA motif are colored in green. HKs with HisKA_3 motif are grouped in orange. HKs are named with four digits in their protein ID: NP_72****.1. (B) Alignment of streptococcus WalK C-terminal sequences. Completely conserved residues are shown in white with a red background and boxed in blue. Highly conserved residues are in red with a white background and boxed in blue. Marked on top are protein secondary structures and residue numbers in S. mutans . (C) Mutations in the CTT disrupt the WalRK interaction. A GST fusion protein with full-length S. mutans WalR was used to pull-down S. mutans WalK (196–450) WT and mutant proteins (top gel). As a negative control, GST alone was used to pull down WalK WT and mutant proteins (middle gel). Shown in the bottom gel are 10% of the WalK protein levels used above. CK shows GST-WalR or GST used in the pull-down. (D–F) Quantification of the WalRK interaction by ITC experiments. WalK (196–450) WT and mutant proteins were titrated against the full-length WalR, resulting in raw titration curves at the top and their global fittings at the bottom. The derived thermodynamic parameters are shown within.

Article Snippet: The acquired wiff files were searched with Maxquant V1.5 against S. mutans serotype C (strain UA159, ATCC 700610) in UniProt.

Techniques: Residue, Mutagenesis, Negative Control, Titration, Derivative Assay

Journal: Frontiers in Microbiology

Article Title: The W-Acidic Motif of Histidine Kinase WalK Is Required for Signaling and Transcriptional Regulation in Streptococcus mutans

doi: 10.3389/fmicb.2022.820089

Figure Lengend Snippet: The CTT of S. mutans WalK is indispensable for its enzymatic activities. (A) Autokinase activity of WalK (31–450) and its tail mutants. The WalK loadings were shown in the lower gel stained by CBB, while the phosphorylation of WalK was detected by ATPγS and anti-thiophosphate antibodies shown in the upper gel. H217A is an autokinase inactive mutant. (B) Phosphotransferase of WalK. The phosphotransferase activity was examined by the reduced phosphorylation of WalK incubated with WalR and detected using ATPγS and anti-thiophosphate antibody over time. Quantitative analysis of phosphotransferase activity normalized to 0 min is shown below the gel. (C) Phosphatase of WalK. Phosphorylated WalR was incubated with WalK and its derivatives at 1:5 (WalK:WalR), separated from the dephosphorylated WalR in a Phos-tag gel and stained with CBB. Quantitative analysis of phosphatase activity is below the gel. The phosphorylated/dephosphorylated WalR was estimated, normalized to its initial amount at 0 s of the gel. Data presented are means ± standard deviations (error bars) for three independent experiments. Student’s t -tests were used to compare mutants to WT at each time point (*** p < 0.001 and **** p < 0.0001).

Article Snippet: The acquired wiff files were searched with Maxquant V1.5 against S. mutans serotype C (strain UA159, ATCC 700610) in UniProt.

Techniques: Activity Assay, Staining, Phospho-proteomics, Mutagenesis, Incubation

Journal: Frontiers in Microbiology

Article Title: The W-Acidic Motif of Histidine Kinase WalK Is Required for Signaling and Transcriptional Regulation in Streptococcus mutans

doi: 10.3389/fmicb.2022.820089

Figure Lengend Snippet: The CTT of S. mutans WalK contributes to the interaction with the WalR DBD. (A) The domain architectures of WalK and WalR. (B) WalK (196–450) interacts with WalR in GST pull-down experiments. The 10% loading controls for WalK, GST, GST-WalR full-length (FL), GST-RD, and GST-DBD are shown in lanes 1, 2, 4, 6, and 8, respectively. (C) Mutations in the WalK CTT disrupt the interaction with the WalR DBD. As a negative control, GST alone was used to pull down WalK (middle gel). Shown in the bottom gel are 10% of the WalK protein used above. CK shows GST-DBD or GST used in the pull-down. (D) Phosphotransferase activity of WalK is diminished toward the RD of WalR alone. Phosphorylated WalK detected by anti-thiophosphate antibody was incubated with WalR full-length, RD, DBD, and D52A, shown from top to bottom, respectively.

Article Snippet: The acquired wiff files were searched with Maxquant V1.5 against S. mutans serotype C (strain UA159, ATCC 700610) in UniProt.

Techniques: Negative Control, Activity Assay, Incubation

Journal: Frontiers in Microbiology

Article Title: The W-Acidic Motif of Histidine Kinase WalK Is Required for Signaling and Transcriptional Regulation in Streptococcus mutans

doi: 10.3389/fmicb.2022.820089

Figure Lengend Snippet: The CTT of S. mutans WalK is important for WalK in competition with promoter DNA. (A,B) WalK (196–450) competes off fluorescein labeled 25-mer promoter DNA from binding to WalR or DBD in a dose-dependent manner. (C) Comparison of the relative ability of CTT mutants (W443A, Δtail) of WalK to compete with DBD in promoter binding. All samples were mixed and incubated for 15 min at RT before loading onto gels. Final concentrations of proteins and DNA used in the reactions were marked above the panels. All EMSA gels were imaged under UV to show DNA in the upper panel and stained with CBB to visualize protein loading in the lower panel.

Article Snippet: The acquired wiff files were searched with Maxquant V1.5 against S. mutans serotype C (strain UA159, ATCC 700610) in UniProt.

Techniques: Labeling, Binding Assay, Comparison, Incubation, Staining

Journal: Frontiers in Microbiology

Article Title: The W-Acidic Motif of Histidine Kinase WalK Is Required for Signaling and Transcriptional Regulation in Streptococcus mutans

doi: 10.3389/fmicb.2022.820089

Figure Lengend Snippet: Effect of WalK mutations on biofilm development of S. mutans . (A–C) SEM analyses of mature biofilms grown for 72 h. (D–F) Quantification of biofilms by fluorescent staining. All biofilms were quantified for their thickness and horizontal growth shown below each 3D image. (G) Phosphorylation state of WalR in vivo . Phosphorylated WalR was separated from its non-phosphorylated state in a Phos-tag gel and detected using an anti-WalR antibody. The non-phosphorylated (CK) and phosphorylated WalR (His-tagged, treated with AcP) were loaded in the first two lanes. The cytoplasmic phosphorylation ratio of WalR shown in the bar chart was determined by band intensities and averaged from three independent experiments with error bars showing standard deviation.

Article Snippet: The acquired wiff files were searched with Maxquant V1.5 against S. mutans serotype C (strain UA159, ATCC 700610) in UniProt.

Techniques: Staining, Phospho-proteomics, In Vivo, Standard Deviation

Journal: Frontiers in Microbiology

Article Title: The W-Acidic Motif of Histidine Kinase WalK Is Required for Signaling and Transcriptional Regulation in Streptococcus mutans

doi: 10.3389/fmicb.2022.820089

Figure Lengend Snippet: Protein profiling in S. mutans biofilms. (A) Protein profiling of S mutans WT and Δtail strains from a quantitative mass spectroscopy experiment. The x -axis indicates the fold change of LFQ in the Δtail strain. The y -axis of log ( P ) indicates a significance level of the t- test. The black curves separate those proteins at a level of false discovery rate (FDR) = 0.01 and minimal fold changes (S0) = 0.1. Below the curve in gray are those unchanged proteins. Those proteins that were upregulated are colored in red, and those that were downregulated are colored in blue. Total and altered proteins are listed in MS Dataset. (B) Proteins that were most altered in the Δtail strain. Proteins were selected at a difference cutoff of > 1.5 except GbpA and GbpB. Five short, functionally unknown, peptides were excluded. The functional annotations of the proteins are shown in . (C,D) qRT-PCR analyses of key genes known to be regulated by WalRK. Transcriptional profiles of the genes gtfBCD and gbpABC were normalized to 16S RNA. Data presented are means ± standard deviations (error bars) for three independent experiments. Student’s t -tests were used to compare Δ tail strain to WT strain (** p < 0.005 and *** p < 0.001).

Article Snippet: The acquired wiff files were searched with Maxquant V1.5 against S. mutans serotype C (strain UA159, ATCC 700610) in UniProt.

Techniques: Mass Spectrometry, Functional Assay, Quantitative RT-PCR

Journal: Frontiers in Microbiology

Article Title: The W-Acidic Motif of Histidine Kinase WalK Is Required for Signaling and Transcriptional Regulation in Streptococcus mutans

doi: 10.3389/fmicb.2022.820089

Figure Lengend Snippet: The CTT of WalK is required for S. aureus WalRK interaction and enzymatic activity. (A) Conservation of the WalK CTT across Gram-positive bacteria. Representative sequences of WalK C-termini were aligned and boxed. The residues are numbered according to S. mutans WalK. Completely conserved residues are colored in white in a red background. The less conserved residues are highlighted in yellow. (B) Alignment of staphylococcus WalK tail sequences. Completely conserved residues are shown in white in a red background and boxed in blue. Highly conserved residues are in red in a white background and boxed in blue. Marked on top are protein secondary structures and residue numbers in S. aureus . (C) Mutations in the CTT disrupt WalRK interaction. A GST fusion protein with full-length S. aureus WalR was used to pull-down S. aureus WalK (364–608) WT and mutant derivatives shown in the top panel. As a negative control, GST alone was used to pull down WalK WT and its mutants shown on the center panel. Shown in the bottom panel are 10% levels of WalK proteins used in each lane. CK shows GST-WalR or GST used in the pull-down. (D) Phosphotransferase of WalK (364–608) was disrupted by mutations in the WalK CTT. The phosphotransferase activity was represented by the reduction in WalK phosphorylation relative to T0, the initial phos-WalK. After the addition of WalR, the mixtures were incubated for the indicated time and stopped by the addition of SDS-loading buffer. Loading controls are shown in the bottom gel. The enzymatic activity was quantified shown below. (E) Phosphatase of WalK (364–608) was disrupted by mutations in the WalK CTT. Phos-tag gel is shown in the top, and a regular SDS-PAGE below shows the total protein used. The phosphatase activity was quantified by the reduction in the phos-WalR relative to the amount of T0, the initial phos-WalR. The reactions were incubated for the indicated time and stopped by the addition of SDS-loading buffer. Data presented are means ± standard deviations (error bars) for three independent experiments. Student’s t -tests were used to compare mutants to WT at each time point (** p < 0.005 and * p < 0.05).

Article Snippet: The acquired wiff files were searched with Maxquant V1.5 against S. mutans serotype C (strain UA159, ATCC 700610) in UniProt.

Techniques: Activity Assay, Bacteria, Residue, Mutagenesis, Negative Control, Phospho-proteomics, Incubation, SDS Page

Journal: Cells

Article Title: HSP90 Modulates T2R Bitter Taste Receptor Nitric Oxide Production and Innate Immune Responses in Human Airway Epithelial Cells and Macrophages

doi: 10.3390/cells11091478

Figure Lengend Snippet: HSP90 inhibition reduces T2R-stimulated intracellular NO production in H441 cells grown at the air–liquid interface (ALI). ( A ) : Representative image of DAF-FM-loaded H441 ALIs stimulated for 10 min with 1 mM sodium benzoate (NaBenz.) or denatonium benzoate (denat benz.); fluorescence increased with denatonium benzoate but not sodium benzoate. ( B ) : Average trace and bar graph (mean ± SEM) of four experiments as in ( A ). Significance determined by Student’s t -test; ** p < 0.01. ( C ) : Average trace and bar graph (mean ± SEM of three experiments) showing response in cultures pre-loaded with BAPTA-AM and stimulated in the absence of extracellular Ca 2+ (0-Ca 2+ o ) vs. control cultures pre-incubated with 0.1% DMSO only and stimulated in the presence of extracellular Ca 2+ . ( D ) : Denatonium-induced DAF-FM fluorescence increases in H441 ALIs were inhibited by pretreatment with geldanamycin or L-NAME but not HSP70 inhibitor VER-15508. Average trace and bar graph of results from four independent experiments are shown. Significance determined by one-way ANOVA with Dunnett’s post-test comparing all values to control (denatonium only); ** p < 0.01.

Article Snippet: H441 cells were transfected with Wt or D88N HSP90 (Kindly provided by W. Sessa, Addgene plasmids #22487 or #22480, respectively) using lipofectamine 3000 and the specific H441 protocol provided on the Thermo Fisher Scientific website.

Techniques: Inhibition, Fluorescence, Control, Incubation

Journal: Cells

Article Title: HSP90 Modulates T2R Bitter Taste Receptor Nitric Oxide Production and Innate Immune Responses in Human Airway Epithelial Cells and Macrophages

doi: 10.3390/cells11091478

Figure Lengend Snippet: HSP90 inhibition reduces T2R-stimulated NO diffusion into the airway surface liquid (ASL) in H441 cells grown at the air–liquid interface (ALI) ( A ): Representative images and bar graph of 4 independent experiments of fluorescence at the apical plane of ALI when 100 µL of solution containing cell impermeable DAF-2 was placed on top (1.1 cm 2 Transwell) either containing sodium benzoate (top) or denatonium benzoate (bottom). Cultures were either pretreated with 0.1% DMSO (vehicle control), 10 µM PLC inhibitor U73122, or 10 µM inactive analogue U73343 prior to the experiment. Significance determined by one-way ANOVA with Dunnett’s post-test comparing all values to HBSS only control. ( B ): Bar graph of experiments performed as in ( A ) but testing inhibition of denatonium-induced or quinine-induced ASL DAF-2 fluorescence ± NOS inhibitor L-NAME or inactive D-NAME (10 µM). Bar graph shows the mean ± SEM of 3–5 independent experiments imaged at identical conditions. Significance by one-way ANOVA with Bonferroni post-test comparing all values to respective HBSS control; ** p < 0.01. ( C ) : Denatonium-stimulated H441 DAF-2 ASL fluorescence increases were reduced in the presence of GPCR signaling inhibitor YM254890 or HSP90 inhibitors geldanamycin, 17-AAG, or BIIB 021. HSP70 inhibitor VER-15508 had no effect. Bar graph shows the mean ± SEM of four independent experiments. Significance by one-way ANOVA with Dunnett’s post-test comparing all values to control (0.1% DMSO only); * p < 0.05 and ** p < 0.01. ( D ): H441s were treated with siRNA as described in the methods. ASL DAF-2 responses during denatonium stimulation were reduced by eNOS siRNA but not with scramble, nNOS, or PAR-2 siRNA. Bar graph shows the mean ± SEM of four independent experiments (separate siRNA transfections). Significance by one-way ANOVA with Dunnett’s post-test comparing all values to control; ** p < 0.01.

Article Snippet: H441 cells were transfected with Wt or D88N HSP90 (Kindly provided by W. Sessa, Addgene plasmids #22487 or #22480, respectively) using lipofectamine 3000 and the specific H441 protocol provided on the Thermo Fisher Scientific website.

Techniques: Inhibition, Diffusion-based Assay, Fluorescence, Control, Transfection

Journal: Cells

Article Title: HSP90 Modulates T2R Bitter Taste Receptor Nitric Oxide Production and Innate Immune Responses in Human Airway Epithelial Cells and Macrophages

doi: 10.3390/cells11091478

Figure Lengend Snippet: HSP90 inhibition reduces T2R-stimulated intracellular NO production in primary sinonasal epithelial cells grown at the air–liquid interface (ALI). ( A ): Intracellular DAF-FM increases were measured in response to T2R38-agonist PTC (1 mM) followed by NO donor SNAP (25 µM) as positive control. PTC stimulated NO production in ALIs from PAV/PAV (homozygous functional T2R38) but not AVI/AVI (homozygous non-functional T2R38) ALIs (nonfunctional T2R38) patients. Geldanamycin pretreatment inhibited the NO production in PAV/PAV ALIs. Trace and bar graph show the mean ± SEM of 8–10 experiments per condition using ALIs from 4–5 patients. Significance determined by one-way ANOVA with Tukey–Kramer post-test comparing all values; ** p < 0.01. ( B ): Traces of DAF-FM fluorescence in PAV/AVI (heterozygous T2R38) cultures stimulated with T2R14/39 agonist apigenin (100 µM) shown with 0.1% DMSO vehicle control. Pretreatment with T2R14/39 antagonist 4′-fluoro-6-methoxyflavanone (4′-F-6-MF) or HSP90 inhibitor geldanamycin but not 0.1% DMSO (inhibitor vehicle control) reduced apigenin-induced but not SNAP-induced DAF-FM fluorescence increases. ( C ): Traces of DAF-FM fluorescence in PAV/AVI (heterozygous T2R38) cultures stimulated with T2R14 agonist quercetin (50 µM). Pretreatment with T2R14/39 antagonist 4′-fluoro-6-methoxyflavanone (4′-F-6-MF) or HSP90 inhibitor geldanamycin but not 0.1% DMSO (inhibitor vehicle control) reduced quercetin-induced but not SNAP-induced DAF-FM fluorescence increases. ( D ): Bar graph of intracellular DAF-FM fluorescence increases after 2 min stimulation from experiments as in ( C , D ). Stimulation (DMSO vehicle control, apigenin, quercetin, or SNAP) listed on top and pretreatment (DMSO vehicle control, 4′-F-6-MF, or geldanamycin) listed on the bottom. Each data point is an independent experiment ( n = 4–8 per condition). Significance by Bonferroni post-test; * p < 0.05 vs. bracketed bars; # p < 0.05 vs. DMSO alone.

Article Snippet: H441 cells were transfected with Wt or D88N HSP90 (Kindly provided by W. Sessa, Addgene plasmids #22487 or #22480, respectively) using lipofectamine 3000 and the specific H441 protocol provided on the Thermo Fisher Scientific website.

Techniques: Inhibition, Positive Control, Functional Assay, Fluorescence, Control

Journal: Cells

Article Title: HSP90 Modulates T2R Bitter Taste Receptor Nitric Oxide Production and Innate Immune Responses in Human Airway Epithelial Cells and Macrophages

doi: 10.3390/cells11091478

Figure Lengend Snippet: HSP90 inhibition reduces T2R-stimulated NO diffusion into the airway surface liquid (ASL) in primary sinonasal epithelial cells grown at the air–liquid interface (ALI) Experiments were performed as in to measure NO diffusion into the ASL but with primary nasal ALIs. ( A ): PTC (500 µM) or 3oxoC12HSL (100 µM) stimulated extracellular DAF-2 fluorescence in PAV/PAV and AVI/AVI cultures, as indicated. PAV/PAV cultures were also pretreated with HSP90 inhibitors geldanamycin, 17-AAG, or BIIB 021 or HSP70 inhibitor VER-155008. ( B ): shows experiments with apigenin ± 4′-F-6-MF, geldanamycin, 17-AAG, or PLC inhibitor U73122 and inactive analogue U73343. Control Transwells containing no cells were similarly incubated with vehicle only or apigenin to test for any cell-independent reaction of apigenin with DAF-2. Significance by one way ANOVA with Bonferroni post-test; * p < 0.05 vs. bracketed bars; ** p < 0.01 vs. bracketed bars; ## p < 0.05 for the same condition in PAV/PAV vs AVI/AVI cultures.

Article Snippet: H441 cells were transfected with Wt or D88N HSP90 (Kindly provided by W. Sessa, Addgene plasmids #22487 or #22480, respectively) using lipofectamine 3000 and the specific H441 protocol provided on the Thermo Fisher Scientific website.

Techniques: Inhibition, Diffusion-based Assay, Fluorescence, Control, Incubation

Journal: Cells

Article Title: HSP90 Modulates T2R Bitter Taste Receptor Nitric Oxide Production and Innate Immune Responses in Human Airway Epithelial Cells and Macrophages

doi: 10.3390/cells11091478

Figure Lengend Snippet: HSP90 inhibition reduces T2R-stimulated ciliary beating in primary sinonasal epithelial cells. ( A ): Left shows representative normalized CBF responses (representative experiments shown) to T2R14/39 agonist apigenin in human primary sinonasal ALIs ± T2R14/39 inhibitor 4′-fluoro-6-methoxyflavanone. Right shows normalized CBF responses (representative experiments shown) to apigenin ± geldanamycin (10 µM; 5 min pretreatment). Mean baseline CBF was not with vehicle or 4′-fluoro-6-methoxyflavanone pretreatment (7.5 ± 1.1 Hz or 8.2 ± 0.9 Hz, respectively; not significant by Students’ t -test). Mean baseline CBF was also not different before or after vehicle or geldanamycin pretreatment (6.9 ± 1.7 Hz or 7.9 ± 1.2 Hz, respectively; not significant by Students’ t -test). ( B ): Bar graph of the mean ± SEM of CBF responses from five independent experiments as shown in ( A ) using ALIs from four different patients. Significance determined by one-way ANOVA with Bonferroni post-test; * p < 0.05. ( C ): Left shows representative normalized CBF responses (representative experiments shown) to T2R14/39 agonist quercetin in human ALIs ± T2R14/39 inhibitor 4′-fluoro-6-methoxyflavanone. Mean baseline CBF was not with vehicle or 4′-fluoro-6-methoxyflavanone pretreatment (7.3 ± 1.2 Hz or 7.9 ± 0.6 Hz, respectively; not significant by Students’ t -test). Right shows normalized CBF responses (representative experiments shown) to quercetin ± geldanamycin (10 µM; 5 min pretreatment). Mean baseline CBF was not different before or after vehicle or geldanamycin pretreatment (7.4 ± 1.3 Hz or 7.0 ± 0.9 Hz, respectively; not significant by Students’ t -test). ( D ): Bar graph of the mean ± SEM of CBF responses from five independent experiments as shown in C using ALIs from five different patients. Significance determined by one-way ANOVA with Bonferroni post-test; ** p < 0.01.

Article Snippet: H441 cells were transfected with Wt or D88N HSP90 (Kindly provided by W. Sessa, Addgene plasmids #22487 or #22480, respectively) using lipofectamine 3000 and the specific H441 protocol provided on the Thermo Fisher Scientific website.

Techniques: Inhibition

Journal: Cells

Article Title: HSP90 Modulates T2R Bitter Taste Receptor Nitric Oxide Production and Innate Immune Responses in Human Airway Epithelial Cells and Macrophages

doi: 10.3390/cells11091478

Figure Lengend Snippet: HSP90 inhibition reduces epithelial ciliary response to P. aeruginosa conditioned medium. ( A ): Graph shows real-time measurement of CBF (mean ± SEM of six independent experiments using ALIs from three patients) during prolonged geldanamycin treatment, followed by stimulation with purinergic agonist ATP. ( B ): Primary nasal ALIs genotyped for functional T2R38 (TAS2R38 PAV/PAV) or non-functional T2R38 (TAS2R38 AVI/AVI) were stimulated with diluted HBSS in which P. aeruginosa PAO-1 had been incubated overnight (conditioned HBSS; cHBSS, diluted with unconditioned HBSS). Peak CBF responses to PAO-1 cHBSS were greater in PAV/PAV cells vs. AVI/AVI cells. Representative trace shown from five experiments using cultures from separate individual patients. ( C ): PAV/PAV cells were stimulated with cHBSS from PAO-1 or PAO-JP2, which lacks the ability to produce AHLs. PAO-1 cHBSS stimulated CBF increases that were greater than CBF increases observed with PAO-JP2 cHBSS. Representative trace shown from five experiments using cultures from separate individual patients. ( D ): PAV/PAV cells were stimulated with PAO-1 cHBSS ± geldanamycin pretreatment. Representative trace shown from five experiments using cultures from separate individual patients. ( E ): Bar graph showing peak CBF (mean ± SEM with individual data points showing individual experiments) observed from experiments as in F-H . Asterisks represent significance compared with PAV/PAV + PAO-1 cHBSS at each individual concentration, determined by Sidak’s multiple comparison test; * p < 0.05 and ** p < 0.01.

Article Snippet: H441 cells were transfected with Wt or D88N HSP90 (Kindly provided by W. Sessa, Addgene plasmids #22487 or #22480, respectively) using lipofectamine 3000 and the specific H441 protocol provided on the Thermo Fisher Scientific website.

Techniques: Inhibition, Functional Assay, Incubation, Concentration Assay, Comparison

Journal: Cells

Article Title: HSP90 Modulates T2R Bitter Taste Receptor Nitric Oxide Production and Innate Immune Responses in Human Airway Epithelial Cells and Macrophages

doi: 10.3390/cells11091478

Figure Lengend Snippet: HSP90 inhibition reduces nasal epithelial bacterial killing mediated by T2Rs and NO. P. aeruginosa PAO-1 bacteria were incubated with nasal ALI cultures as described in the methods. ( A ): Bar graph showing live (Syto9)/dead (propidium iodide [PI]) staining quantified by fluorescence plate reader. First two bars represent bacteria incubated in the absence of nasal cells treated with saline only or saline + colistin. This illustrates max (saline) and min (colistin) live/dead ratios. Significance by one way ANOVA with Bonferroni post-test; ** p < 0.01 between bracketed groups; # p < 0.05 and ## p < 0.01 vs. PAV/PAV cultures with no inhibitor. ( B ): Representative image (left) and bar graph (right) showing CFU counts from experiments as shown in ( A ). HSP90 inhibitor geldanamycin reduced bacterial killing (increased CFUs) while HSP70 inhibitor VER 155008 did not. Significance by one-way ANOVA with Dunnett’s post test comparing all values to PAV/PAV control (no inhibitor); ## p < 0.01 vs. PAV/PAV control.

Article Snippet: H441 cells were transfected with Wt or D88N HSP90 (Kindly provided by W. Sessa, Addgene plasmids #22487 or #22480, respectively) using lipofectamine 3000 and the specific H441 protocol provided on the Thermo Fisher Scientific website.

Techniques: Inhibition, Bacteria, Incubation, Staining, Fluorescence, Saline, Control

Journal: Cells

Article Title: HSP90 Modulates T2R Bitter Taste Receptor Nitric Oxide Production and Innate Immune Responses in Human Airway Epithelial Cells and Macrophages

doi: 10.3390/cells11091478

Figure Lengend Snippet: HSP90 inhibition reduces T2R-stimulated NO production in primary human M0 MΦs. ( A ): DAF-FM-loaded MΦs exhibited increases in fluorescence in response to 1 mM denatonium benzoate that were strongly inhibited by geldanamycin. Left shows average traces and right shows bar graphs (mean ± SEM) from eight independent experiments using MΦs from two donors. DAF-FM fluorescence increase was also inhibited by BIIB 021. Control = denatonium benzoate after pretreatment with 0.1% DMSO. Significance by one way ANOVA with Bonferroni posttest; * p < 0.05. ( B ): Low-level Ca 2+ responses to denatonium benzoate were not affected by geldanamycin. Top shows representative traces in the absence or presence of 1 µM geldanamycin. Bottom shows bar graph of six independent experiments using MΦs from three different donors. Response to purinergic agonist ATP shown as control. ( C ): NO production in MΦs treated with HSP90 or control non-targeting siRNAs. Left shows representative traces and right shows bar graph of data from four independent experiments per condition. Significance by Student’s t -test; ** p < 0.01.

Article Snippet: H441 cells were transfected with Wt or D88N HSP90 (Kindly provided by W. Sessa, Addgene plasmids #22487 or #22480, respectively) using lipofectamine 3000 and the specific H441 protocol provided on the Thermo Fisher Scientific website.

Techniques: Inhibition, Fluorescence, Control

Journal: Cells

Article Title: HSP90 Modulates T2R Bitter Taste Receptor Nitric Oxide Production and Innate Immune Responses in Human Airway Epithelial Cells and Macrophages

doi: 10.3390/cells11091478

Figure Lengend Snippet: HSP90 inhibition reduces T2R-stimulated FITC- E. coli phagocytosis in primary human M0 MΦs. ( A ): Representative image of MΦs with phagocytosed FITC-labeled E. coli . ( B ): Left shows time course of phagocytosis responses during 30 min incubation in HBSS as described in the methods after pretreatment with geldanamycin or HBSS for times indicated on the y-axis. Each data point is the mean ± SEM of three independent experiments using MΦs from three different donors. Right shows separate experiments of baseline phagocytosis over 30 min (HBSS only) of FITC- E. coli after 2 h pretreatment with HBSS only (containing 0.1% DMSO as vehicle control), 1 µM VER-15508, 1 µM geldanamycin, or geldanamycin plus VER-15508. Significance determined by one-way ANOVA with Dunnett’s post-test comparing values to HBSS pretreatment; * p < 0.05, ** p < 0.01. Bar graph shows the mean ± SEM of six experiments using MΦs from three donors. ( C ): Stimulated 30 min phagocytosis of FITC- E. coli (HBSS only control or 1 mM denat. benz. ± pertussis toxin [PTX]) was measured after pre-incubation with HBSS + 0.1% DMSO or 1 µM geldanamycin. PTX and geldanamycin both inhibited denatonium-induced phagocytosis. Significance determined by one-way ANOVA with Bonferroni post-test; ** p < 0.01 vs. HBSS control and ## p < 0.01 vs. bracketed groups. ( D ): Geldanamycin reduced phagocytosis increases observed with both denatonium and quinine. Bar graph shows the mean ± SEM of six independent experiments using cells from six different individual patients. Significance by one way ANOVA with Tukey–Kramer post-test comparing all bars; ** p < 0.01 vs. HBSS alone; ## p < 0.01 vs. bracketed bar. ( E ): Assays were carried out in MΦs previously treated with siRNAs directed against eNOS, iNOS, HSP90, or non-targeting control sequences. Bar graph shows increase in phagocytosis relative to HBSS in the same macrophage background over four independent experiments. Significance compared with no siRNA control using one-way ANOVA with Bonferroni post-test and pairwise comparisons; * p < 0.05 and ** p < 0.01.

Article Snippet: H441 cells were transfected with Wt or D88N HSP90 (Kindly provided by W. Sessa, Addgene plasmids #22487 or #22480, respectively) using lipofectamine 3000 and the specific H441 protocol provided on the Thermo Fisher Scientific website.

Techniques: Inhibition, Labeling, Incubation, Control

Journal: Cells

Article Title: HSP90 Modulates T2R Bitter Taste Receptor Nitric Oxide Production and Innate Immune Responses in Human Airway Epithelial Cells and Macrophages

doi: 10.3390/cells11091478

Figure Lengend Snippet: HSP90 inhibition reduces T2R-stimulated pHrodo- S. aureus phagocytic responses in primary human M0 MΦs. ( A ): Representative images of pHrodo-labeled S. aureus phagocytosis in primary human MΦs ± denatonium benzoate (1 mM) stimulation after D-NAME or L-NAME pretreatment (10 µM; 45 min). ( B ) : Bar graph of pHrodo- S. aureus fluorescence after experiments as in A . Significance by Bonferroni post-test with paired comparisons; ** p < 0.01. ( C ): Representative images of pHrodo-labeled S. aureus phagocytosis in primary human MΦs ± denatonium benzoate (1 mM) or 3oxoC12HSL (100 µM) after no-pretreatment (0.1% DMSO only as vehicle control)) or pretreatment with HSP90 inhibitors geldanamycin or BIIB 021 (pretreatment as in ). ( D ): Bar graph of pHrodo- S. aureus phagocytosis during stimulation with HBSS only (unstimulated control), 1 mM denatonium benzoate, or 100 µM 3oxoC12HSL ± geldanamycin or BIIB 021 (pretreatment as in ). Significance by one-way ANOVA with Bonferroni post-test; * p < 0.05 or ** p < 0.01. ( E ): Bar graph of pHrodo- S. aureus phagocytosis during stimulation with HBSS only (unstimulated control) or 1 mM denatonium benzoate ± pertussis toxin (PTX), geldanamycin, BIIB 021, 17-AAG, or VER 15508. PTX (500 ng/mL) pretreatment was 18 h. MΦs were pretreated with other inhibitors as in . Significance by one-way ANOVA with Bonferroni post-test; ** p < 0.01.

Article Snippet: H441 cells were transfected with Wt or D88N HSP90 (Kindly provided by W. Sessa, Addgene plasmids #22487 or #22480, respectively) using lipofectamine 3000 and the specific H441 protocol provided on the Thermo Fisher Scientific website.

Techniques: Inhibition, Labeling, Fluorescence, Control

Journal: Journal of Functional Biomaterials

Article Title: Antibacterial Designs for Implantable Medical Devices: Evolutions and Challenges

doi: 10.3390/jfb13030086

Figure Lengend Snippet: A photothermal antibacterial surface: ( a ) schematic illustration of the coordinated assembly of tannic acid (TA) and Fe 3+ ions (iron chloride hexahydrate) on gold (can be other materials), yielding the Au-TA/Fe; ( b ) near-infrared (NIR) irradiation (808 nm, 2.2 W·cm −2 ) induced temperature changes on the material surface immersed in phosphate-buffered saline (PBS), with corresponding thermal images inserted; ( c ) SEM images of adherent bacteria ( E. coli or MRSA) on materials surface with/without NIR irradiation (5 min), together with the typical photographs of bacterial colonies re-cultured from materials surface of different processing histories. Adapted from reference with permission from the American Chemical Society.

Article Snippet: 3 , Long-term efficacy: salt-responsive polyzwitterionic brushes on a nanopatterned surface , P. aeruginosa (BNCC 337005); Escherichia coli (ATCC 25922) , Rabbit red blood cells (2 h- incubation); L929 fibroblasts (cultured for 24 h) , Subcutaneous implant model in mice; Placed in for 5 days , Not specific , [ ] .

Techniques: Irradiation, Saline, Bacteria, Cell Culture

Journal: Journal of Functional Biomaterials

Article Title: Antibacterial Designs for Implantable Medical Devices: Evolutions and Challenges

doi: 10.3390/jfb13030086

Figure Lengend Snippet: A cell-selective titanium surface: ( a ) SEM surface morphology of the microbes ( E. coli ) cultured for 24 h on titanium doped with both calcium and silver (Ti-Ag/Ca), with a high magnification image, inserted; ( b ) typical morphology of rat bone marrow stem cells (BMSCs) cultured for 1 h on Ti-Ag/Ca, with a high magnification image inserted; ( c , d ) potential mechanism underlying the actions of Ti-Ag/Ca on microbes and mammalian cells, respectively . Reused with permission from the Royal Society of Chemistry.

Article Snippet: 3 , Long-term efficacy: salt-responsive polyzwitterionic brushes on a nanopatterned surface , P. aeruginosa (BNCC 337005); Escherichia coli (ATCC 25922) , Rabbit red blood cells (2 h- incubation); L929 fibroblasts (cultured for 24 h) , Subcutaneous implant model in mice; Placed in for 5 days , Not specific , [ ] .

Techniques: Cell Culture

Journal: Journal of Functional Biomaterials

Article Title: Antibacterial Designs for Implantable Medical Devices: Evolutions and Challenges

doi: 10.3390/jfb13030086

Figure Lengend Snippet: Typical flaws in our reports on antibacterial surfaces.

Article Snippet: 3 , Long-term efficacy: salt-responsive polyzwitterionic brushes on a nanopatterned surface , P. aeruginosa (BNCC 337005); Escherichia coli (ATCC 25922) , Rabbit red blood cells (2 h- incubation); L929 fibroblasts (cultured for 24 h) , Subcutaneous implant model in mice; Placed in for 5 days , Not specific , [ ] .

Techniques: In Vitro, In Vivo, Cell Culture, Bacteria, Incubation, Irradiation, Cell Function Assay, Titanium Dioxide, Infection, Plasmid Preparation, Injection

Journal: Infection and Immunity

Article Title: Role of the Carboxy Terminus of SecA in Iron Acquisition, Protein Translocation, and Virulence of the Bacterial Pathogen Acinetobacter baumannii

doi: 10.1128/IAI.02925-14

Figure Lengend Snippet: Strains and plasmids used in this study

Article Snippet: Detection by Southern blotting of a single 2.4-kb KpnI-PvuII restriction fragment, the size of which was increased by 2 kb because of the transposon insertion in the 2010 derivative ( and ), indicates that this is the only ATCC 19606 T coding region disrupted by the transposition insertion.

Techniques: Plasmid Preparation, Recombinant, PCR Cloning, Expressing, Mutagenesis, Amplification, Clone Assay

Journal: Infection and Immunity

Article Title: Role of the Carboxy Terminus of SecA in Iron Acquisition, Protein Translocation, and Virulence of the Bacterial Pathogen Acinetobacter baumannii

doi: 10.1128/IAI.02925-14

Figure Lengend Snippet: Analysis of the genetic locus affected in the A. baumannii ATCC 19606T 2010 insertion derivative. (A) Genetic map of the locus harboring the secA gene (ORF 2), which was truncated due to a transposon insertion near its 3′ end (vertical black arrow), and the ORF1 and ORF 3 flanking coding regions. The horizontal arrows represent the location and direction of transcription of predicted coding regions. The locations of the KpnI (K) and PvuII (P) restriction sites used to confirm the EZ::TN insertion site are indicated. The three long black horizontal bars represent cloned DNA regions used as probes in Southern blotting (pMU734), to complement the 2010 insertion derivative (pMU472), or to overproduce a His-tagged SecA derivative (pMU481). The short black horizontal bar flanked by primer numbers indicate the 5′-end secA region used to test transcriptional expression by qRT-PCR. (B and C) Southern blotting of KpnI-PvuII-digested total DNA isolated from the ATCC 19606T parental strain (lanes 2) and the 2010 derivative (lanes 3) probed with pMU734 (B) or the aph gene (C). (D) Western blotting of cytoplasmic proteins isolated from the ATCC 19606T parental strain (lane 1) and the 2010 derivative (lane 2) probed with anti-SecA antibodies. The white arrow indicates the truncated SecA protein produced in the 2010 insertion derivative.

Article Snippet: Detection by Southern blotting of a single 2.4-kb KpnI-PvuII restriction fragment, the size of which was increased by 2 kb because of the transposon insertion in the 2010 derivative ( and ), indicates that this is the only ATCC 19606 T coding region disrupted by the transposition insertion.

Techniques: Clone Assay, Southern Blot, Expressing, Quantitative RT-PCR, Isolation, Western Blot, Produced

Journal: Infection and Immunity

Article Title: Role of the Carboxy Terminus of SecA in Iron Acquisition, Protein Translocation, and Virulence of the Bacterial Pathogen Acinetobacter baumannii

doi: 10.1128/IAI.02925-14

Figure Lengend Snippet: Transcriptional analysis of the secA gene in ATCC 19606T and 2010 cells. qRT-PCR was used to detect secA transcripts produced in the ATCC 19606T parental strain and the isogenic 2010 secA insertion mutant using the primers 3976 and 3977, which anneal to the 5′ end of the this gene, using recA expression for normalization. The error bars represent the standard error (SE) of the mean of data collected from three independent biological samples.

Article Snippet: Detection by Southern blotting of a single 2.4-kb KpnI-PvuII restriction fragment, the size of which was increased by 2 kb because of the transposon insertion in the 2010 derivative ( and ), indicates that this is the only ATCC 19606 T coding region disrupted by the transposition insertion.

Techniques: Quantitative RT-PCR, Produced, Mutagenesis, Expressing

Journal: Infection and Immunity

Article Title: Role of the Carboxy Terminus of SecA in Iron Acquisition, Protein Translocation, and Virulence of the Bacterial Pathogen Acinetobacter baumannii

doi: 10.1128/IAI.02925-14

Figure Lengend Snippet: Growth of A. baumannii ATCC 19606T and 2010 under different culture conditions. (A) Growth curves of the ATCC 19606T parental strain and the 2010 derivative incubated in LB broth. The OD600 of 1-ml samples taken from a 50-ml LB broth culture was determined hourly for 8 h. (B) Growth of the ATCC 19606T parental strain, the 2010 mutant, its complemented derivative 2010.C, and the t6 BauA mutant incubated in LB broth in the absence of DIP or the presence of 100 μM or 200 μM DIP. Bacterial growth (OD600) was determined after overnight incubation (12 to 14 h) at 37°C in a shaking incubator set at 200 rpm. The error bars show standard errors (SEs) of the means from an assay done in triplicate. Inset, detection of BauA in outer membrane fractions isolated from 2010 (lane 1) and 2010.C (lane 2) cells. SDS-PAGE size-fractionated proteins were probed with anti-BauA polyclonal antibodies.

Article Snippet: Detection by Southern blotting of a single 2.4-kb KpnI-PvuII restriction fragment, the size of which was increased by 2 kb because of the transposon insertion in the 2010 derivative ( and ), indicates that this is the only ATCC 19606 T coding region disrupted by the transposition insertion.

Techniques: Incubation, Mutagenesis, Membrane, Isolation, SDS Page

Journal: Infection and Immunity

Article Title: Role of the Carboxy Terminus of SecA in Iron Acquisition, Protein Translocation, and Virulence of the Bacterial Pathogen Acinetobacter baumannii

doi: 10.1128/IAI.02925-14

Figure Lengend Snippet: Cross-feeding bioassays and HPLC analysis of iron-restricted culture supernatants. (A to C) Cross-feeding bioassays were done with LB agar plates containing 200 μM DIP and seeded with 2010 (A), t6 acinetobactin uptake-deficient mutant (B), or s1 acinetobactin synthesis-deficient mutant (C) overnight-cultured bacteria. Sterile filter discs impregnated with 10 μl of 10 mM FeCl3, sterile distilled water, purified acinetobactin (Ab), or cell-free supernatants from 2010 overnight cultures, which contained a subinhibitory concentration of DIP, were deposited on the surface of each plate. Plates were incubated overnight at 37°C before results were recorded. The error bars show the standard errors (SEs) of the means from an assay performed in triplicate. (D) HPLC analysis of M9 minimal medium supernatants from ATCC 19606T (19606), 2010, and t6 overnight cultures. The elution peaks corresponding to acinetobactin (Ab) and the precursor DHBA are indicated. HPLC of sterile M9 minimal medium was used as a negative control.

Article Snippet: Detection by Southern blotting of a single 2.4-kb KpnI-PvuII restriction fragment, the size of which was increased by 2 kb because of the transposon insertion in the 2010 derivative ( and ), indicates that this is the only ATCC 19606 T coding region disrupted by the transposition insertion.

Techniques: Mutagenesis, Cell Culture, Bacteria, Sterility, Purification, Concentration Assay, Incubation, Negative Control

Journal: Infection and Immunity

Article Title: Role of the Carboxy Terminus of SecA in Iron Acquisition, Protein Translocation, and Virulence of the Bacterial Pathogen Acinetobacter baumannii

doi: 10.1128/IAI.02925-14

Figure Lengend Snippet: Detection of acinetobactin transport proteins and transcripts. (A) Genetic organization of the bauDCEBA operon coding for acinetobactin transport functions. (B to D) SDS-PAGE of size-fractionated outer membrane (B) and cytoplasmic membrane (C and D) proteins, which were isolated from ATCC 19606T and 2010 cells cultured in LB broth or LB broth containing 100 μM DIP, were blotted onto nitrocellulose filters and probed with anti-BauA (B), anti-BauB (C), or anti-BauE (D) antibodies. The positions of molecular mass markers are shown on the left-hand side of each blot. (E to G) Agarose gel electrophoresis of bauA (E), bauB (F), and bauD (G) RT-PCR amplicons using as the template total RNA isolated from ATCC 19606T and 2010 cells cultured in LB broth or LB broth containing 100 μM DIP. Lane M, molecular size marker.

Article Snippet: Detection by Southern blotting of a single 2.4-kb KpnI-PvuII restriction fragment, the size of which was increased by 2 kb because of the transposon insertion in the 2010 derivative ( and ), indicates that this is the only ATCC 19606 T coding region disrupted by the transposition insertion.

Techniques: SDS Page, Membrane, Isolation, Cell Culture, Agarose Gel Electrophoresis, Reverse Transcription Polymerase Chain Reaction, Marker

Journal: Infection and Immunity

Article Title: Role of the Carboxy Terminus of SecA in Iron Acquisition, Protein Translocation, and Virulence of the Bacterial Pathogen Acinetobacter baumannii

doi: 10.1128/IAI.02925-14

Figure Lengend Snippet: Proteins differentially produced by the ATCC 19606T parental strain and the 2010 secA isogenic mutant. Proteins were grouped based on their predicted cellular functions. Numbers between parentheses indicate the number of proteins included in each functional category.

Article Snippet: Detection by Southern blotting of a single 2.4-kb KpnI-PvuII restriction fragment, the size of which was increased by 2 kb because of the transposon insertion in the 2010 derivative ( and ), indicates that this is the only ATCC 19606 T coding region disrupted by the transposition insertion.

Techniques: Produced, Mutagenesis, Functional Assay

Journal: Infection and Immunity

Article Title: Role of the Carboxy Terminus of SecA in Iron Acquisition, Protein Translocation, and Virulence of the Bacterial Pathogen Acinetobacter baumannii

doi: 10.1128/IAI.02925-14

Figure Lengend Snippet: Growth of the ATCC 19606T parental strain and the 2010 SecA isogenic mutant with different salt concentrations. Experiments were done using 96-well plates containing 200 μl LB broth that were inoculated with 20 μl of overnight cultures and then incubated at 37°C in a plate-shaking incubator. The OD600 was determined hourly for 11 h. The error bars show the standard errors (SEs) of the means from an assay done in triplicate.

Article Snippet: Detection by Southern blotting of a single 2.4-kb KpnI-PvuII restriction fragment, the size of which was increased by 2 kb because of the transposon insertion in the 2010 derivative ( and ), indicates that this is the only ATCC 19606 T coding region disrupted by the transposition insertion.

Techniques: Mutagenesis, Incubation

Journal: Infection and Immunity

Article Title: Role of the Carboxy Terminus of SecA in Iron Acquisition, Protein Translocation, and Virulence of the Bacterial Pathogen Acinetobacter baumannii

doi: 10.1128/IAI.02925-14

Figure Lengend Snippet: Role of SecA in virulence. G. mellonella caterpillars (n = 30) were injected with 1 × 105 bacteria of the ATCC 19606T parental strain (19606), the t6 bauA insertion derivative, or the 2010 secA insertion derivative in the absence (A) or the presence (B) of 50 μM FeCl3. Negative controls included noninjected caterpillars or caterpillars injected with comparable volumes of PBS or PBS plus 50 μM FeCl3. Caterpillar death was determined daily for 5 days during incubation at 37°C in darkness.

Article Snippet: Detection by Southern blotting of a single 2.4-kb KpnI-PvuII restriction fragment, the size of which was increased by 2 kb because of the transposon insertion in the 2010 derivative ( and ), indicates that this is the only ATCC 19606 T coding region disrupted by the transposition insertion.

Techniques: Injection, Bacteria, Incubation

Journal: Nature Communications

Article Title: A general strategy for expanding polymerase function by droplet microfluidics

doi: 10.1038/ncomms11235

Figure Lengend Snippet: ( a ) We have developed a fluorescent reporter system that produces an optical signal when a primer–template complex is extended to full-length product. The reporter consists of a primer–template complex (pink and green) containing a downstream fluorophore that is quenched when a DNA-quencher (black) anneals to the unextended region. ( b ) The assay was designed with a metastable probe to allow dissociation at elevated temperatures, where thermophilic polymerases function with optimal activity. Red arrow marks the maximium fluorescence observed in the absence of the quencher probe. ( c ) Flourophore (F)/quencher (Q) pairs were screened to identify a dye pair with the maximum signal-to-noise ratio. ( d ) Primer-extension analysis by denaturing PAGE (top) and fluorescence (bottom) for 9n and 9n-GLK polymerases using dNTP and NTP substrates. Negative control: no NTPs. Positive control: dNTPs or no DNA-quencher probe. ( e ) Single-emulsion droplets containing a functional 9n-GLK polymerase that extends a primer–template complex with RNA (top) and non-functional (bottom) wild-type 9n polymerase. The panel shows a cartoon depiction of the droplet, a bright-field micrograph of encapsulated E. coli (arrow), a fluorescence micrograph of the same field of view and an overlay of the two images. Scale bars, 10 μm. ( f ) Flow cytometry analysis of 9n and 9n-GLK polymerases following NTP extension in water-in-oil-in-water (w/o/w) droplets.

Article Snippet: Accuprime DNA Polymerase was obtained from Invitrogen (Grand Island, NY).

Techniques: Activity Assay, Fluorescence, Negative Control, Positive Control, Emulsion, Functional Assay, Flow Cytometry

Journal: Nature Communications

Article Title: A general strategy for expanding polymerase function by droplet microfluidics

doi: 10.1038/ncomms11235

Figure Lengend Snippet: ( a ) Overview of the microfluidic polymerase enrichment strategy. A pool of polymerase genes containing functional (green) and non-functional (blue) members are expressed in E. coli and encapsulated in w/o droplets generated in a microfluidics device. Polymerases are liberated from their bacteria by heat lysis and incubated at 55 °C to allow for primer extension. Using a second microfluidics device, droplets are emulsified into a bulk aqueous phase to generate water-in-oil-in-water compartments (w/o/w). Fluorescent w/o/ws are FACS sorted and the vectors encoding functional polymerases are recovered. ( b ) Vector design. The 9n-GLK vector was engineered to contain a unique NotI restriction site. Control digestion showing that NotI only cuts PCR-amplified DNA from the 9n-GLK vector. ( c ) Following a complete cycle of selection and amplification (see ) PCR-amplified DNA was digested with NotI to measure the enrichment of 9n-GLK from libraries that were doped at levels of 1:100, 1:1,000 and 1:10,000 (9n-GLK to 9n). NotI digestion of the PCR-amplified DNA reveals an enrichment of ∼1,200-fold per round of microfluidics selection.

Article Snippet: Accuprime DNA Polymerase was obtained from Invitrogen (Grand Island, NY).

Techniques: Functional Assay, Generated, Bacteria, Lysis, Incubation, Plasmid Preparation, Control, Amplification, Selection

Journal: Nature Communications

Article Title: A general strategy for expanding polymerase function by droplet microfluidics

doi: 10.1038/ncomms11235

Figure Lengend Snippet: ( a ) Constitutional structure for the linearized backbone of threose nucleic acid (TNA). ( b ) Positions 409, 485 and 664 mapped onto the structure of 9n DNA polymerase (PDB: 4K8X). Polymerases isolated after one round of selection were analysed for TNA synthesis activity in the absence of Mn 2+ . Activity is defined as the amount of full-length product generated in 18 h. Basal activity of wild-type 9n polymerase (dashed grey line). ( c ) Time course of TNA synthesis for 9n-YRI and 9n-NVA polymerases compared with wild-type 9n. ( d ) Fidelity analysis of 9n-YRI polymerase in the presence and absence of manganese ions yields a mutational profile of 8 errors per 100 bases and 2 errors per 1,000 bases, respectively.

Article Snippet: Accuprime DNA Polymerase was obtained from Invitrogen (Grand Island, NY).

Techniques: Isolation, Selection, Activity Assay, Generated

Journal: eLife

Article Title: IL-21/type I interferon interplay regulates neutrophil-dependent innate immune responses to Staphylococcus aureus

doi: 10.7554/eLife.45501

Figure Lengend Snippet: ( A, B ) Intra-tracheal infection with the USA 300 strain of MRSA induced a significant increase in pulmonary Il21 mRNA ( A ) and IL-21 protein ( B ) 24 hr after infection. ( C ) PBS or 2 μg of IL-21 was administered i.t. to WT mice one day prior to MRSA infection, and lung MRSA CFU were quantitated 7 and 24 hr later. ( D, E ) Lung immunopathology was assessed in H and E-stained sections of lung tissue from naïve uninfected mice and mice pre-treated with PBS or IL-21 and then infected for 7 or 24 hr with MRSA (in upper left panel, the bar = 500 μm and inset bar = 10 μm) ( D ), and pathology ( E ) scores were assessed. ( F–K ) animals were infected with MRSA as above. ( F ) Total lung neutrophil cellularity was quantitated by flow cytometry after staining with Ly6G and CD11b. ( G ) RNA-Seq analysis was performed on total lung tissue mRNA (pools of 5 animals) isolated 7 or 24 hr after treatment of WT mice with PBS or IL-21. Boxed regions include genes mentioned in the text. ( H–K ) RT-PCR was used to assess expression of Gzma ( H ) and Gzmb ( I ) mRNA in lungs treated with IL-21 for 7 hr, and ELISA was used to assess the induction of granzyme B ( J ) and IFNγ protein ( K ) in corresponding bronchoalveolar lavage fluid at 7 hr. Data are representative of either three ( A, B, C, F, H–K ) or two ( D, E, G ) independent experiments and validation of RNA-Seq was performed by RT-PCR of mRNA from additional mice.

Article Snippet: Neutrophils were then purified (80–90%) with a mouse neutrophil isolation kit (Miltenyi Biotec) and were used directly in either MRSA killing assays or were in vitro stimulated with IL-21 and then RNA isolated for RT-PCR analysis.

Techniques: Infection, Staining, Flow Cytometry, RNA Sequencing, Isolation, Reverse Transcription Polymerase Chain Reaction, Expressing, Enzyme-linked Immunosorbent Assay, Biomarker Discovery

Journal: eLife

Article Title: IL-21/type I interferon interplay regulates neutrophil-dependent innate immune responses to Staphylococcus aureus

doi: 10.7554/eLife.45501

Figure Lengend Snippet: ( A–B ) Total cellularity of lung was assessed in BAL fluid ( A ) and lung ( B ) 24 hr after IL-21 intratracheal instillation. ( C ) Percentage of neutrophils was assessed by flow cytometry at 24 hr after IL-21 treatment. ( D–E ) Levels of chemokines were measured in BAL fluid at 24 hr after IL-21 treatment.

Article Snippet: Neutrophils were then purified (80–90%) with a mouse neutrophil isolation kit (Miltenyi Biotec) and were used directly in either MRSA killing assays or were in vitro stimulated with IL-21 and then RNA isolated for RT-PCR analysis.

Techniques: Flow Cytometry

Journal: eLife

Article Title: IL-21/type I interferon interplay regulates neutrophil-dependent innate immune responses to Staphylococcus aureus

doi: 10.7554/eLife.45501

Figure Lengend Snippet: ( A ) Bone-marrow neutrophils were purified by negative selection using a neutrophil isolation kit (Miltenyi) and either not stimulated or stimulated in vitro with IL-21 (100 ng/ml) for 24 hr in the absence (control) or presence of peptidoglycan (PG) (2 or 5 μg/ml); IL-21R expression on gated Ly6G + CD11b + neutrophils was detected by flow cytometry (blue = isotype control; red = anti-IL-21R). ( B, C ) IL-21 lowers the MRSA CFU in a neutrophil-dependent fashion. Mice were treated i.p. with an isotype control mAb or neutrophil-depleted with anti-Ly6G (1A8 mAb), and the efficacy of neutrophil depletion is shown in BAL and lung ( B ). Mice were then treated with PBS or IL-21 i.t., infected i.t. with MRSA, and CFU quantitated at 24 hr ( C ). CFU quantitation was performed on the left single lobe, whereas immune populations and RNA were determined using the right lobes. ( D, E ) Levels of Gzmb and Gzma mRNAs in lung tissue of untreated or neutrophil-depleted mice (treated as in panel C ) were quantitated by RT-PCR. ( F ) Levels of Gzma and Gzmb mRNAs in purified cell-sorted lung neutrophils (>98% pure Ly6G + CD11b + ) from either untreated or IL-21-treated WT or Il21r KO mice were measured by RT-PCR and normalized to Rpl7 expression. ( G, H ) Lung neutrophils were elicited by i.t treatment with heat-killed S. aureus 24 hr prior to isolation, purified, stimulated in vitro for 4 hr with either PBS or IL-21 (100 ng/ml), and Gzma ( G ) and Gzmb ( H ) mRNAs assayed by RT-PCR and normalized to Rpl7 expression. ( I ) Purified HKSA-elicited lung neutrophils were incubated in vitro with MRSA for 3 hr in the presence of PBS or IL-21 either without or with the granzyme B inhibitor, Z-AAD-CMK. MRSA CFU was quantitated by plating serial dilutions on blood agar plates. Representative experiments are shown; each experiment was performed three times with similar results.

Article Snippet: Neutrophils were then purified (80–90%) with a mouse neutrophil isolation kit (Miltenyi Biotec) and were used directly in either MRSA killing assays or were in vitro stimulated with IL-21 and then RNA isolated for RT-PCR analysis.

Techniques: Purification, Selection, Isolation, In Vitro, Control, Expressing, Flow Cytometry, Infection, Quantitation Assay, Reverse Transcription Polymerase Chain Reaction, Incubation

Journal: eLife

Article Title: IL-21/type I interferon interplay regulates neutrophil-dependent innate immune responses to Staphylococcus aureus

doi: 10.7554/eLife.45501

Figure Lengend Snippet: ( A ) Quantitation of IL-21R MFI in neutrophils treated with or without IL-21 in the presence or absence of peptidoglycan (PG). ( B ) Lung neutrophils from HKSA-stimulated mice were purified via a magnetic neutrophil purification kit (Miltenyi). Shown is CD11b versus Ly6G staining.

Article Snippet: Neutrophils were then purified (80–90%) with a mouse neutrophil isolation kit (Miltenyi Biotec) and were used directly in either MRSA killing assays or were in vitro stimulated with IL-21 and then RNA isolated for RT-PCR analysis.

Techniques: Quantitation Assay, Purification, Staining

Journal: eLife

Article Title: IL-21/type I interferon interplay regulates neutrophil-dependent innate immune responses to Staphylococcus aureus

doi: 10.7554/eLife.45501

Figure Lengend Snippet: ( A ) Human peripheral blood neutrophils were purified by a neutrophil negative selection kit (StemCell Technologies). Shown is CD56 versus CD66b and CD8 versus CD66b staining. ( B ) Western blotting of granzyme B from neutrophils, PBMC, or A549 cells containing 0, 1, or 5% PBMC extracts, confirming that the low level of contamination of purified neutrophils cannot account for the granzyme B expression observed in this population. Also shown is western blotting for actin as a loading control.

Article Snippet: Neutrophils were then purified (80–90%) with a mouse neutrophil isolation kit (Miltenyi Biotec) and were used directly in either MRSA killing assays or were in vitro stimulated with IL-21 and then RNA isolated for RT-PCR analysis.

Techniques: Purification, Selection, Staining, Western Blot, Expressing, Control

Journal: eLife

Article Title: IL-21/type I interferon interplay regulates neutrophil-dependent innate immune responses to Staphylococcus aureus

doi: 10.7554/eLife.45501

Figure Lengend Snippet: ( A, B ) Purified peripheral blood neutrophils were stimulated with PBS (control) or IL-21 (100 ng/ml) for 4 hr, and IL21R mRNA was measured by real-time PCR and normalized to RPL7 expression ( A ), and IL-21R protein levels were measured by flow cytometry ( B ); left panel shows isotype control shaded and anti-IL21R black line, with a summary in the right panel). ( C ) RNA-Seq was performed on neutrophils after 4 or 24 hr incubation with PBS or IL-21 in the absence or presence of heat-killed S. aureus (10 6 /ml). We used HKSA rather than live bacteria in order to allow analysis at 24 hr, as live bacteria would have overgrown the system by then. Genes differentially expressed (fold-change >2.0) are shown. Shown is a representative RNA-Seq analysis. ( D – G ) Human peripheral blood neutrophils were stimulated for 4 hr in vitro in the presence or absence of IL-21 and GZMA ( D ), GZMB ( E ), GNLY ( F ), and IFNG ( G ) mRNA levels were quantitated by RT-PCR and normalized to RPL7 expression. ( H ) Purified human neutrophils were stimulated with IL-21 for 4 hr, fixed, permealized, and stained for intracellular granzyme B protein (gated on CD66b + cells); MFIs are summarized in right panel. ( I ) Granzyme B protein was measured by ELISA in supernatants from human peripheral blood neutrophils cultured for 24 hr in either the absence or presence of IL-21 (100 ng/ml). ( J, K ) Human neutrophils were incubated in vitro with either MRSA ( J ) or S. pyogenes ( K ) for 3 hr with PBS or IL-21 (100 ng/ml). MRSA and S. pyogenes CFU were quantitated by plating serial dilutions on blood agar plates. Results shown are representative of 3 independent experiments, except panel C shows one of two similar independent RNA-Seq experiments, each from a different donor.

Article Snippet: Neutrophils were then purified (80–90%) with a mouse neutrophil isolation kit (Miltenyi Biotec) and were used directly in either MRSA killing assays or were in vitro stimulated with IL-21 and then RNA isolated for RT-PCR analysis.

Techniques: Purification, Control, Real-time Polymerase Chain Reaction, Expressing, Flow Cytometry, RNA Sequencing, Incubation, Bacteria, In Vitro, Reverse Transcription Polymerase Chain Reaction, Staining, Enzyme-linked Immunosorbent Assay, Cell Culture

Journal: eLife

Article Title: IL-21/type I interferon interplay regulates neutrophil-dependent innate immune responses to Staphylococcus aureus

doi: 10.7554/eLife.45501

Figure Lengend Snippet: Reactive oxygen species were measured by flow cytometry in CellRox Red loaded peripheral blood neutrophils that were stimulated at 37°C for 30 min with PBS, IL-21, HKSA, or HKSA +IL-21.

Article Snippet: Neutrophils were then purified (80–90%) with a mouse neutrophil isolation kit (Miltenyi Biotec) and were used directly in either MRSA killing assays or were in vitro stimulated with IL-21 and then RNA isolated for RT-PCR analysis.

Techniques: Flow Cytometry

Journal: eLife

Article Title: IL-21/type I interferon interplay regulates neutrophil-dependent innate immune responses to Staphylococcus aureus

doi: 10.7554/eLife.45501

Figure Lengend Snippet: ( A ) WT and Il21r KO mice were infected intratracheally with MRSA (2 × 10 7 ) and CFU in the lung were quantitated at 4 and 24 hr post-infection. ( B ) WT and Il21r KO mice were infected in parallel with WT mice that had been treated with PBS or 2 μg IL-21, and CFU in the lung were quantitated at 7 and 24 hr post infection. ( C–E ) Lung immunopathology was assessed in H and E-stained lung sections (bar in upper left panel = 500 μm; bar in inset = 10 μm) ( C ); lung neutrophil cellularity as assessed by pathology score ( D ) and flow cytometry ( E ) were assessed at 4 and 24 hr post-infection. ( F ) WT or Il21r KO mice were pre-treated with an isotype control antibody or anti-Ly6G to deplete neutrophils, infected with MRSA, and CFU quantitated at 24 hr. Representative experiments are shown; each experiment was performed three times with similar results.

Article Snippet: Neutrophils were then purified (80–90%) with a mouse neutrophil isolation kit (Miltenyi Biotec) and were used directly in either MRSA killing assays or were in vitro stimulated with IL-21 and then RNA isolated for RT-PCR analysis.

Techniques: Infection, Staining, Flow Cytometry, Control

Journal: eLife

Article Title: IL-21/type I interferon interplay regulates neutrophil-dependent innate immune responses to Staphylococcus aureus

doi: 10.7554/eLife.45501

Figure Lengend Snippet: ( A ) WT mice were treated intratracheally with 50 μg of control Fc (from IgG1) or IL-21R-Fc protein for 2 days prior to infection with MRSA and lung CFU were quantitated at 4 and 24 hr post-infection with MRSA. ( B ) Histology of lungs in control Fc or IL-21R-Fc pre-treated mice at 4 and 24 hr post-infection (bar in upper left panel = 500 μm; bar in inset = 10 μm). ( C ) IFNα levels in BAL fluid were measured by ELISA 4 hr after infection in mice pretreated with control Fc or IL-21R-Fc. ( D ) Gzmb mRNA was measured in lungs of WT or Il21r KO mice pre-treated with isotype control or anti-IFNAR1 antibodies prior to MRSA infection, and normalized to Rpl7 mRNA expression. ( E ) Gzmb mRNA was measured in lungs of mice treated with either control Fc or IL-21R-Fc prior to MRSA infection and normalized to Rpl7 expression. ( F ) Like IL-21, IFNβ also induced GZMB mRNA in human peripheral blood neutrophils and normalized to RPL7 expression. ( G ) IFNβ induces increased in vitro killing of MRSA by human peripheral blood neutrophils and this was prevented by a granzyme B inhibitor, Z-AAD-CMK. ( H–J ) Co-cultures of CD4 + T cells and dendritic cells were stimulated with SEB either in the presence of control Fc or IL-21R-Fc, and mRNA was quantitated by RT-PCR after 24 hr and normalized to Rpl7 expression. Representative experiments are shown; each experiment was performed three times with similar results.

Article Snippet: Neutrophils were then purified (80–90%) with a mouse neutrophil isolation kit (Miltenyi Biotec) and were used directly in either MRSA killing assays or were in vitro stimulated with IL-21 and then RNA isolated for RT-PCR analysis.

Techniques: Control, Infection, Enzyme-linked Immunosorbent Assay, Expressing, In Vitro, Reverse Transcription Polymerase Chain Reaction

Journal: eLife

Article Title: IL-21/type I interferon interplay regulates neutrophil-dependent innate immune responses to Staphylococcus aureus

doi: 10.7554/eLife.45501

Figure Lengend Snippet: MRSA infection, as detailed in the legend to . ( A, B ) Pathology and neutrophil infiltration scores were assessed from H and E-stained lung sections shown in . ( C ) RNA-Seq was performed on total lung mRNA either before infection (Ctrl) or at 4 hr after MRSA infection of mice pretreated with either Fc protein or an IL-21R-Fc fusion protein. Representative of 2 independent RNA-Seq experiments.

Article Snippet: Neutrophils were then purified (80–90%) with a mouse neutrophil isolation kit (Miltenyi Biotec) and were used directly in either MRSA killing assays or were in vitro stimulated with IL-21 and then RNA isolated for RT-PCR analysis.

Techniques: Infection, Staining, RNA Sequencing

Journal: eLife

Article Title: IL-21/type I interferon interplay regulates neutrophil-dependent innate immune responses to Staphylococcus aureus

doi: 10.7554/eLife.45501

Figure Lengend Snippet: ( A ) Peripheral blood neutrophils from normal donors (NDs) or AD-HIES patients (PTs) were assayed using an in vitro MRSA killing assay in the absence or presence of IL-21 (100 ng/ml). ( B ) RT-PCR was used to quantitate GZMB mRNA in neutrophils 4 hr after in vitro stimulation without or with IL-21. Expression was normalized to RPL7 expression ( C ) RNA-Seq was performed on normal donor and AD-HIES patient neutrophils stimulated for 4 hr with PBS, IL-21, IFNβ, or IL-21 +IFNβ. Genes differentially expressed (fold-change >1.5) in two independent RNA-Seq analyses are shown. ( D ) RT-PCR normalized to RPL7 expression was used to validate the expression pattern of GBP1 and GBP2 in neutrophils from additional normal donors and AD-HIES patients. ( E–G ) Mutant Stat3 transgenic mice were treated i.t. with PBS or 2 μg IL-21, infected i.t. 24 hr later with MRSA, and at 7 hr post-infection lung MRSA CFU quantitated ( E ), and IFNα levels were measured in the serum ( F ) and BAL fluid ( G ).

Article Snippet: Neutrophils were then purified (80–90%) with a mouse neutrophil isolation kit (Miltenyi Biotec) and were used directly in either MRSA killing assays or were in vitro stimulated with IL-21 and then RNA isolated for RT-PCR analysis.

Techniques: In Vitro, Reverse Transcription Polymerase Chain Reaction, Expressing, RNA Sequencing, Mutagenesis, Transgenic Assay, Infection

Journal: eLife

Article Title: IL-21/type I interferon interplay regulates neutrophil-dependent innate immune responses to Staphylococcus aureus

doi: 10.7554/eLife.45501

Figure Lengend Snippet: ( A ) In wild-type (WT) mice, MRSA produces SEB, which bridges between T cells and dendritic cells by interacting with TCR and MHCII, leading to the production of IL-21 by CD4 + T cells. IL-21 stimulates neutrophils to release granzymes, with enhanced killing of MRSA. Type I IFN (e.g., produced by dendritic cells, as shown in the cartoon) can also induce granzyme production to promote killing of MRSA, but IL-21 inhibits dendritic cell production of type I IFN. ( B ) In the absence of either IL-21R or a functional STAT3 signaling response, the IL-21-induced neutrophil granzyme production does not occur. However, IL-21-mediated repression of type I IFN production by dendritic cells also no longer occurs, which leads to enhanced production of type I IFN and hence increased killing of MRSA through this pathway. The functional cross-talk of and relative potency of the IL-21- and type 1 IFN-mediated pathways and levels of each cytokine influence the outcome.

Article Snippet: Neutrophils were then purified (80–90%) with a mouse neutrophil isolation kit (Miltenyi Biotec) and were used directly in either MRSA killing assays or were in vitro stimulated with IL-21 and then RNA isolated for RT-PCR analysis.

Techniques: Produced, Functional Assay

Journal: eLife

Article Title: IL-21/type I interferon interplay regulates neutrophil-dependent innate immune responses to Staphylococcus aureus

doi: 10.7554/eLife.45501

Figure Lengend Snippet:

Article Snippet: Neutrophils were then purified (80–90%) with a mouse neutrophil isolation kit (Miltenyi Biotec) and were used directly in either MRSA killing assays or were in vitro stimulated with IL-21 and then RNA isolated for RT-PCR analysis.

Techniques: In Vivo, Flow Cytometry, Blocking Assay, Recombinant, Enzyme-linked Immunosorbent Assay, Staining, Isolation

Journal:

Article Title: Biodegradation of Aromatic Compounds by Escherichia coli

doi: 10.1128/MMBR.65.4.523-569.2001

Figure Lengend Snippet: Historical perspective on the mineralization of aromatic compounds by E. coli

Article Snippet: A vitamin B 12 auxotroph derivative of the W wild-type strain, E. coli ATCC 11105 (hereafter referred as E. coli W) ( 63 ), is a well-known penicillin G acylase producer.

Techniques: Isolation, Molecular Cloning, Sequencing, Expressing, Construct, Bacteria

Journal:

Article Title: Biodegradation of Aromatic Compounds by Escherichia coli

doi: 10.1128/MMBR.65.4.523-569.2001

Figure Lengend Snippet: Aromatic acids degraded by different E. coli strains a

Article Snippet: A vitamin B 12 auxotroph derivative of the W wild-type strain, E. coli ATCC 11105 (hereafter referred as E. coli W) ( 63 ), is a well-known penicillin G acylase producer.

Techniques:

Journal:

Article Title: Biodegradation of Aromatic Compounds by Escherichia coli

doi: 10.1128/MMBR.65.4.523-569.2001

Figure Lengend Snippet: Pathway for the catabolism of HPA (4HPA and 3HPA) in E. coli. (A) Genetic map of the chromosomal hpa (in E. coli W) and hpc (in E. coli C) regions. Relevant genes are indicated by blocks: genes with similar shading participate in the same enzymatic step or in the same functional unit (route) of the pathway. The hpc genes are indicated in brackets. Regulatory and transport genes are shown by solid and vertically striped blocks, respectively. The genes flanking the hpa cluster (tsr, orf12, orf13, and orf14) are contiguous in the genome of E. coli K-12 and are represented by thick lines. orf12 and orf13 correspond to the yjiY gene from E. coli K-12. orf14 corresponds to the yjiA gene from E. coli K-12. The arrows show the directions of gene transcription. Bent arrows represent the Pr, Pg, Px, Pa1, Pa2 and PBC promoters. REP sequences are shown. The HpaR repressor and HpaA activator are represented by a square and hexagon, respectively; empty and solid symbols indicate inactive and active regulators, respectively; − and + indicate transcriptional repression and activation, respectively. The inducer molecule (HPA and HPC) is represented by a solid circle. (B) Biochemistry of the HPA catabolic pathway. The metabolites are 4HPA and 3HPA, HPC (homoprotocatechuate), CHMS (5-carboxymethyl-2-hydroxy-muconic semialdehyde), CHM (5-carboxymethyl-2-hydroxy-muconic acid), OPET (5-oxo-pent-3-ene-1,2,5-tricarboxylic acid), HHDD (2-hydroxy-hept-2,4-diene-1,7-dioic acid), OHED (2-oxo-hept-3-ene-1,7-dioic acid), and HHED (2,4-dihydroxy-hept-2-ene-1,7-dioic acid). The enzymes are HpaBC (HPA monooxygenase), HpaD (HPC 2,3-dioxygenase), HpaE (CHMS dehydrogenase), HpaF (CHM isomerase), HpaG (OPET decarboxylase), HpaH (hydratase), HpaI (HHED aldolase), and Sad (succinic semialdehyde dehydrogenase). The HPA transport protein (HpaX) is represented by a thick arrow. Out and In indicate outside and inside the cell, respectively.

Article Snippet: A vitamin B 12 auxotroph derivative of the W wild-type strain, E. coli ATCC 11105 (hereafter referred as E. coli W) ( 63 ), is a well-known penicillin G acylase producer.

Techniques: Functional Assay, Activation Assay

Journal:

Article Title: Biodegradation of Aromatic Compounds by Escherichia coli

doi: 10.1128/MMBR.65.4.523-569.2001

Figure Lengend Snippet: Pathway for the catabolism of 3HPP in E. coli. (A) Genetic map of the chromosomal mhp cluster. Relevant genes are indicated by blocks: genes with similar shading participate in the same enzymatic step or in the same functional unit (route) of the pathway. Regulatory and transport genes are shown by solid and vertically striped blocks, respectively. Genes flanking the mhp cluster (lacI and yaiL) are represented by thick lines. The arrows show the directions of gene transcription. Bent arrows represent the Pr and Pa promoters. The location of the BIME is shown. The inactive and active forms of the MhpR activator are represented by empty and solid hexagons, respectively. + indicates transcriptional activation. The inducer molecule (3HPP and 3HCI) is represented by a solid circle. (B) Biochemistry of the 3HPP catabolic pathway. The metabolites are 3HPP, DHPP (2,3-dihydroxyphenlypropionic acid), HKNDA (2-hydroxy-6-keto-nona-2,4-diene 1,9-dioic acid), HPDA (2-hydroxy-penta-2,4-dienoic acid), and HKP (4-hydroxy-2-ketopentanoic acid). The enzymes are MhpA (3HPP monooxygenase), MhpB, (DHPP 1,2 dioxygenase), MhpC (HKNDA hydrolase), MhpD (HPDA hydratase), MhpE (HKP aldolase), and MhpF (acetaldehyde dehydrogenase [acylating]). The 3HPP transport protein (MhpT) is represented by a thick arrow. Out and In indicate outside and inside the cell, respectively.

Article Snippet: A vitamin B 12 auxotroph derivative of the W wild-type strain, E. coli ATCC 11105 (hereafter referred as E. coli W) ( 63 ), is a well-known penicillin G acylase producer.

Techniques: Functional Assay, Activation Assay

Journal:

Article Title: Biodegradation of Aromatic Compounds by Escherichia coli

doi: 10.1128/MMBR.65.4.523-569.2001

Figure Lengend Snippet: Pathway for the catabolism of PP in E. coli. (A) Genetic map of the chromosomal hca cluster. Relevant genes are indicated by blocks: genes with similar shading encode the subunits of the PP dioxygenase. Regulatory (solid block) and putative transport (vertically striped block) genes are also shown. The horizontally striped block indicates the gene encoding the PP dihydrodiol dehydrogenase. The empty block represents a gene of unknown function. The csiE gene flanking the hca cluster is represented by a thick line. The arrows show the directions of gene transcription. Bent arrows represent the Pr and Pe promoters. The inactive and active forms of the HcaR activator are represented by empty and solid hexagons, respectively. + indicates transcriptional activation. The inducer molecule (PP and CI) is represented by a solid circle. (B) Biochemistry of the PP catabolic pathway. The metabolites are PP, CI, PP dihydrodiol, CI-dihydrodiol, DHPP, and DHCI (see the legends to Fig. ​Fig.22 and ​and3).3). The enzymes are HcaEFCD (PP dioxygenase), HcaB (PP-dihydrodiol dehydrogenase), and MhpBCDEF (see the legend to Fig. ​Fig.2).2). The putative PP/CI transport protein (HcaT) is represented by a thick arrow. Out and In indicate outside and inside the cell, respectively.

Article Snippet: A vitamin B 12 auxotroph derivative of the W wild-type strain, E. coli ATCC 11105 (hereafter referred as E. coli W) ( 63 ), is a well-known penicillin G acylase producer.

Techniques: Blocking Assay, Activation Assay

Journal:

Article Title: Biodegradation of Aromatic Compounds by Escherichia coli

doi: 10.1128/MMBR.65.4.523-569.2001

Figure Lengend Snippet: Pathway for the catabolism of PA in E. coli. (A) Genetic map of the chromosomal paa cluster. Relevant genes are indicated by blocks: genes with similar shading participate in the same enzymatic step or in the same functional unit of the pathway. Genes flanking the paa cluster (maoA and ydbC) are represented by thick lines. The arrows show the directions of gene transcription. Bent arrows represent the Pz, Pa, and Px promoters. The locations of the IS2 and IS30 insertion sequences within ydbA in E. coli K-12 are shown. The regulatory gene (paaX) is represented by a black block. The inactive and active forms of the PaaX repressor are indicated by empty and solid squares, respectively. − and + indicate transcriptional repression and activation, respectively. The inducer molecule (PA-CoA) is represented by a solid circle. (B) Biochemistry of the PA catabolic pathway. The first intermediate of the pathway is PA-CoA (phenylacetyl CoA). The enzymes are PaaK (PA-CoA ligase), PaaABCDE (putative multicomponent oxygenase), PaaZ (putative ring-cleavage enzyme), and PaaFGHIJ (putative β-oxidation-like enzymatic system).

Article Snippet: A vitamin B 12 auxotroph derivative of the W wild-type strain, E. coli ATCC 11105 (hereafter referred as E. coli W) ( 63 ), is a well-known penicillin G acylase producer.

Techniques: Functional Assay, Blocking Assay, Activation Assay

Journal:

Article Title: Biodegradation of Aromatic Compounds by Escherichia coli

doi: 10.1128/MMBR.65.4.523-569.2001

Figure Lengend Snippet: Upper pathway for the catabolism of aromatic amines (PEA, tyramine, and dopamine) in E. coli. (A) Genetic map of the chromosomal cluster for the initial catabolism of aromatic amines. Relevant genes are indicated by blocks. Alternative gene names are in parentheses. The paaZ gene from the paa cluster (see Fig. ​Fig.5)5) is indicated by a thick line. The arrows show the directions of gene transcription. Bent arrows represent the PmaoB PmaoA and Ppad promoters. The regulatory gene maoB (feaR) is shown by a solid block. The inactive and active forms of the MaoB activator are represented by empty and solid hexagons, respectively. + indicates transcriptional activation. The inducer molecule (PEA, tyramine) is represented by a solid circle. (B) Biochemistry of the initial catabolism of aromatic amines. The metabolites are PAL (phenylacetaldehyde), 4HPAL (4-hydroxyphenylacetaldehyde), DHPAL (3,4-dihydroxyphenylacetaldehyde), PA, 4HPA, and HPC (homoprotocatechuate). The enzymes are MaoA (monoamine oxidase) and PadA (phenylacetaldehyde dehydrogenase).

Article Snippet: A vitamin B 12 auxotroph derivative of the W wild-type strain, E. coli ATCC 11105 (hereafter referred as E. coli W) ( 63 ), is a well-known penicillin G acylase producer.

Techniques: Blocking Assay, Activation Assay

Journal:

Article Title: Biodegradation of Aromatic Compounds by Escherichia coli

doi: 10.1128/MMBR.65.4.523-569.2001

Figure Lengend Snippet: Relevant enzymatic activities of E. coli for the metabolism of chorismate-derived compounds. The enzymes catalyzing the different reactions are those described in Table ​Table4.4. SAM, S-adenosylmethionine; SAH, S-adenosylhomocysteine; R, octaprenyl side chain.

Article Snippet: A vitamin B 12 auxotroph derivative of the W wild-type strain, E. coli ATCC 11105 (hereafter referred as E. coli W) ( 63 ), is a well-known penicillin G acylase producer.

Techniques: Derivative Assay

Journal:

Article Title: Biodegradation of Aromatic Compounds by Escherichia coli

doi: 10.1128/MMBR.65.4.523-569.2001

Figure Lengend Snippet: Biotransformation activities of E. coli on some aromatic compounds. The known or putative proteins catalyzing the different enzymatic reactions are indicated in boldface type and are described in Table ​Table44.

Article Snippet: A vitamin B 12 auxotroph derivative of the W wild-type strain, E. coli ATCC 11105 (hereafter referred as E. coli W) ( 63 ), is a well-known penicillin G acylase producer.

Techniques:

Journal:

Article Title: Biodegradation of Aromatic Compounds by Escherichia coli

doi: 10.1128/MMBR.65.4.523-569.2001

Figure Lengend Snippet: Comparison of hpa clusters involved in HPA catabolism in different proteobacteria from the γ subgroup. Arrows show the directions of gene transcription. Genes are indicated by blocks: red (regulatory genes), blue (transport genes), green (genes encoding the initial HPA monooxygenase), yellow (ring cleavage dioxygenase gene), and purple (genes encoding the meta-cleavage dehydrogenative route). A broken line indicates an unknown distance. The hpaR gene from K. pneumoniae corresponds to the previously characterized moaI gene (in parentheses). References of the sequences are as follows: E. coli (strain W) (accession no. Z37980), S. enterica serovar Dublin (accession no. AF144422), S. enterica serovar Typhimurium and Y. pestis (obtained from the ERGO database website) K. pneumoniae (accession no. L41068 and AJ000054 and ERGO database), and P. aeruginosa PAO1 (Pseudomonas Genome Project, at http://www.pseudomonas.com/).

Article Snippet: A vitamin B 12 auxotroph derivative of the W wild-type strain, E. coli ATCC 11105 (hereafter referred as E. coli W) ( 63 ), is a well-known penicillin G acylase producer.

Techniques: Comparison

Journal:

Article Title: Biodegradation of Aromatic Compounds by Escherichia coli

doi: 10.1128/MMBR.65.4.523-569.2001

Figure Lengend Snippet: Comparison of gene clusters involved in HPP catabolism in different bacteria. Arrows show the directions of gene transcription. Genes are indicated by blocks: red (regulatory genes), blue (transport genes), green (genes encoding the HPP monooxygenase), yellow (ring cleavage dioxygenase gene), orange (genes encoding the meta-cleavage hydrolytic route), and black (genes of unknown function). The ohpR gene from Rhodococcus sp. strain V49 encodes a regulator that, in contrast to the regulators of the other four gene clusters, does not belong to the IclR protein family. β and γ indicate the β and γ subgroups of proteobacteria. The references of the sequences are as follows: E. coli strain K-12 (EcoGene database), K. pneumoniae (ERGO database), C. testosteroni TA441 (accession no. AB024335), R. globerulus PWD1 (accession no. U89712), and Rhodococcus sp. strain V49 (accession no. AF274045).

Article Snippet: A vitamin B 12 auxotroph derivative of the W wild-type strain, E. coli ATCC 11105 (hereafter referred as E. coli W) ( 63 ), is a well-known penicillin G acylase producer.

Techniques: Comparison, Bacteria

Journal:

Article Title: Biodegradation of Aromatic Compounds by Escherichia coli

doi: 10.1128/MMBR.65.4.523-569.2001

Figure Lengend Snippet: Comparison of gene clusters involved in PA catabolism in different bacteria. Arrows show the directions of gene transcription. Genes are indicated by blocks: red (regulatory genes), dark blue (transport genes), light blue (genes encoding the PA-CoA ligase), green (genes encoding a putative multicomponent oxygenase), brown (genes encoding a putative ring cleavage enzyme), purple (genes encoding a putative β-oxidation-like pathway), and dotted (genes of unknown function). The paaI ortholog in P. putida U has not been described previously (182, 217), and it was named phaP here. orf3 is an incomplete orf gene that encodes a putative transcriptional regulator of the TetR family. An asterisk indicates a truncated gene. A discontinuous line means that the sequence has not been yet reported. The broken line shows that the genes are not adjacent. γ and β represent the γ and β subgroups of proteobacteria. The references of the sequences are as follows: E. coli (strain W) (accession no. X97452), K. pneumoniae (ERGO database), P. putida U and KT2440 (accession no. AF029714 and database referenced at website http: //www.ncbi.nlm.nih.gov/Microb_blast/unfinishedgenome.html, respectively), A. evansii KB740 (paa or pac genes under accession no. AF176259 or AJ278756, respectively), D. radiodurans R1 (accession no. AE002069), and B. halodurans C-125 (accession no. AB011837).

Article Snippet: A vitamin B 12 auxotroph derivative of the W wild-type strain, E. coli ATCC 11105 (hereafter referred as E. coli W) ( 63 ), is a well-known penicillin G acylase producer.

Techniques: Comparison, Bacteria, Sequencing

Journal:

Article Title: Biodegradation of Aromatic Compounds by Escherichia coli

doi: 10.1128/MMBR.65.4.523-569.2001

Figure Lengend Snippet: Schematic representation of the gene clusters and the encoded catabolic pathways for the aerobic degradation of aromatic compounds in E. coli. The gene clusters mhp, mao, paa, hca, hpau (upper route), and hpam (meta-cleavage route) are indicated by blocks and correspond to those described in Fig. ​Fig.11 to ​to6.6. The locations of the clusters refer to the E. coli K-12 linkage map (the hpa cluster is absent in E. coli K-12). The regulatory proteins are indicated by different symbols that reflect the different regulatory protein families. + and − indicate transcriptional activation and repression, respectively. The transporters are represented by thick arrows spanning the cellular envelope. Abbreviations of the metabolites and enzymes are the same than those used in Fig. ​Fig.11 to ​to6.6. The different colors correspond to the different catabolic pathways (or to different routes within the same pathway). A discontinuous arrow indicates more than one enzymatic step. TCA, tricarboxylic acids.

Article Snippet: A vitamin B 12 auxotroph derivative of the W wild-type strain, E. coli ATCC 11105 (hereafter referred as E. coli W) ( 63 ), is a well-known penicillin G acylase producer.

Techniques: Activation Assay

Journal:

Article Title: Biodegradation of Aromatic Compounds by Escherichia coli

doi: 10.1128/MMBR.65.4.523-569.2001

Figure Lengend Snippet: Selected biotransformations of aromatic compounds in recombinant E. coli strains. The enzymes catalyzing the different reactions are indicated in boldface type. (A) Production of 3,4-dihydroxyphenylalanine (l-Dopa) from tyrosine through the 4HPA monooxygenase (HpaBC) from E. coli W. (B) Biotransformation of styrene into (S)-styrene oxide (epoxystyrene), phenylacetaldehyde (PAL), and PA by the StyAB, StyC, and StyD enzymes from different Pseudomonas strains. Formation of (S)-styrene oxide from styrene was also reported using the xylene monooxygenase (XylAM) from the TOL plasmid of P. putida. (C) Conversion of benzene into l-Dopa using the toluene dioxygenase and toluene cis-dihydrodiol dehydrogenase (DH) from P. putida F1 and the tyrosine phenol-lyase from C. freundii. (D) Biotransformation of glucose into the dye indigo in a recombinant E. coli strain that converts glucose into indole and then oxidizes the latter through naphthalene dioxygenase (DOx) or xylene monooxygenase (MOx) from P. putida. (E) Conversion of l-phenylalanine to cinnamic acid (CI) and ammonia through the phenylalanine ammonia-lyase of R. toruloides.

Article Snippet: A vitamin B 12 auxotroph derivative of the W wild-type strain, E. coli ATCC 11105 (hereafter referred as E. coli W) ( 63 ), is a well-known penicillin G acylase producer.

Techniques: Recombinant, Plasmid Preparation