Journal: International Journal of Biochemistry and Molecular Biology

Article Title: Comparison of the functions of glutathionylspermidine synthetase/amidase from E. coli and its predicted homologues YgiC and YjfC

doi:

Figure Lengend Snippet: Analysis of thiol content in E. coli cells. Analysis of the thiol content of wild type and gsp, yjfC, and ygiC gene knockout E. coli strains grown in LB media to stationary phase under anaerobic conditions was performed using HPLC. Labeled peaks represent DTNB derivatives of G-Sp and GSH. Peak immediately followed GSH (14 min) corresponds to DTNB derivative of γ-glutamylcysteine. Peaks at 18 min and 21 min are 2-nitro-5-thiobenzoate and the excess of DTNB correspondently.

Article Snippet: Materials E. coli K-12 genomic DNA was from ATCC (Manassas).

Techniques: Gene Knockout, Labeling

Journal: Cell reports

Article Title: CD89 Is a Potent Innate Receptor for Bacteria and Mediates Host Protection from Sepsis.

doi: 10.1016/j.celrep.2019.03.062

Figure Lengend Snippet: Figure 1. The Recombinant Soluble CD89 Receptor Interacts Directly with Bacteria (A and B) Dose-dependent binding of soluble recombinant CD89 (sCD89) to fixed S.p (A) and E. coli (B). Binding to albumin (Alb) was used as a control. (C) Comparison of sCD89 binding to various types of fixed bacteria. (D) Interaction of sCD89 with live (green bar) versus fixed (black bars) 106 E. coli or 106 S. p. (E and F) S.p (E) and E. coli (F) binding to BMMs grown from CD89 transgenic mice (CD89Tg) or from littermates, visualized by confocal laser-scanning mi- croscopy. Right: quantification of binding (n = 4). All data are presented as mean ± SEM. **p < 0.01, t test. (G and H) S.p (G) and E. coli (H) binding to BMMs isolated from CD89Tg mice or from littermates in the presence or absence of the anti-CD89 blocking antibody MIP8a F(ab’)2 (10 mg/mL) or of sCD89 (500 mg/mL), analyzed by flow cytometry. MFI, mean fluorescence intensity. Data are presented as mean ± SEM; n = 5. *p < 0.05, **p < 0.01; t test. See also Figure S1.

Article Snippet: The Escherichia coli (E. coli-K12, Strain SMG 123 (PTA-7555)), Staphylococcus aureus subsp. aureus Rosenbach (S. aureus, ATCC 25923), Streptococcus pyogenes Rosenbach (S. pyogenes, ATCC 19615) and Escherichia coli-K12 WzxE (Coli genetic stock center, Yale university) were used for sCD89-bacteria interaction assays shown in Figure 1C.

Techniques: Recombinant, Bacteria, Binding Assay, Control, Comparison, Transgenic Assay, Isolation, Blocking Assay, Cytometry

Journal: Cell reports

Article Title: CD89 Is a Potent Innate Receptor for Bacteria and Mediates Host Protection from Sepsis.

doi: 10.1016/j.celrep.2019.03.062

Figure Lengend Snippet: Figure 2. Bacterium-CD89 Interaction on Mouse Cells Induces Activating ITAM Signaling, Leading to Inflammatory Cytokine Production, Bacterial Phagocytosis, and Killing (A and B) IL-6, TNF-a, and IL-1 production in the supernatant of BMMs obtained from CD89Tg and CD89R209L transgenic mice and littermate controls. Cells were incubated for 16 h in the presence of S.p (A) and E. coli (B) and cytokines in the supernatants were measured by ELISA. All data are presented as mean ± SEM; n = 3. *p < 0.05, **p < 0.01; t test. (C) Confocal analysis of E. coli-pHrodo phagocytosis by BMMs obtained from CD89Tg mice compared with littermates in the presence or absence of MIP8a F(ab)’2 or sCD89 in a dose-dependent manner (100–800 mg/mL). Left: representative images. Right: quantification. Data are presented as mean ± SEM; n = 3. ***p < 0.001, t test. (D) ROS production over 30 min by littermate, CD89Tg, and CD89R209L transgenic BMMs stimulated by live S.p (left) or E. coli (right), measured by confocal microscopy. All data are presented as mean ± SEM; n = 15. **p < 0.01, t test. (E) Quantification of bacterial survival after 2 h of incubation with BMMs from CD89Tg, CD89R209L Tg, and littermate mice. Data are presented as mean ± SEM; n = 3. ***p < 0.001, t test.

Article Snippet: The Escherichia coli (E. coli-K12, Strain SMG 123 (PTA-7555)), Staphylococcus aureus subsp. aureus Rosenbach (S. aureus, ATCC 25923), Streptococcus pyogenes Rosenbach (S. pyogenes, ATCC 19615) and Escherichia coli-K12 WzxE (Coli genetic stock center, Yale university) were used for sCD89-bacteria interaction assays shown in Figure 1C.

Techniques: Transgenic Assay, Incubation, Enzyme-linked Immunosorbent Assay, Confocal Microscopy

Journal: Cell reports

Article Title: CD89 Is a Potent Innate Receptor for Bacteria and Mediates Host Protection from Sepsis.

doi: 10.1016/j.celrep.2019.03.062

Figure Lengend Snippet: Figure 3. IgA-Deficient CVID Phagocytes Mediate Phagocytosis, ROS Production, and Bacterial Killing through CD89 Interaction (A) Representative plots of CD89 expression on blood monocytes isolated from healthy donors (HDs) (left) and CVID patients (right) using a phycoerythrin (PE)-conjugated anti-CD89 antibody and its isotype control. (B) Binding of S.p or E. coli to blood monocytes from HDs (purple symbols) or from CVID patients (red symbols) in the presence of monomeric IgA (500 mg/mL) or of MIP8a F(ab’)2 (10 mg/mL). All data are presented as mean ± SEM; n = 4. ***p < 0.001, t test. (C) Phagocytosis of E. coli-pHrodo by human blood monocytes and/or macrophages isolated from HDs or from CVID patients. Left: representative images. Scale bars, 200 mm. Right: quantification (n = 3). All data are presented as mean ± SEM. ns, not significant. (D) IL-6, TNF-a, and IL-1 production in the supernatant of monocytes obtained from CVID patients. Cells were incubated for 16 h in the presence of E. coli or S.p and in the presence or absence of MIP8a F(ab)’2 (500 mg/mL), and cytokines in the supernatants were measured by ELISA. All data are presented as mean ± SEM; n = 3. *p < 0.05, **p < 0.01, ****p < 0.0001; t test.

Article Snippet: The Escherichia coli (E. coli-K12, Strain SMG 123 (PTA-7555)), Staphylococcus aureus subsp. aureus Rosenbach (S. aureus, ATCC 25923), Streptococcus pyogenes Rosenbach (S. pyogenes, ATCC 19615) and Escherichia coli-K12 WzxE (Coli genetic stock center, Yale university) were used for sCD89-bacteria interaction assays shown in Figure 1C.

Techniques: Expressing, Isolation, Control, Binding Assay, Incubation, Enzyme-linked Immunosorbent Assay

Journal: Cell reports

Article Title: CD89 Is a Potent Innate Receptor for Bacteria and Mediates Host Protection from Sepsis.

doi: 10.1016/j.celrep.2019.03.062

Figure Lengend Snippet: Figure 4. Role of CD89-Bacterium Interaction under Physiological Conditions (A) Competitive ELISA assays between sCD89 and S.p (blue line) or E. coli (red line) and ns-IgA. (B) Competitive ELISA assays between sCD89 and S.p (blue line) or E. coli (red line) and pd-IgA. (C) Bacterial phagocytosis by BMMs obtained from CD89Tg mice (left) compared with littermates (right). Bacteria were allowed to be phagocytosed by BMMs from the indicated mice in the presence or absence of ns-IgA at physiological concentration (2 mg/mL) or MIP8a F(ab)’2 (500 mg/mL). Cells were washed and analyzed by flow cytometry. Data are presented as mean ± SEM; n = 3. *p < 0.05, ***p < 0.001; t test. (D) S.p (left) or E. coli (right) phagocytosis by BMDCs obtained from CD89Tg mice compared with littermates. Bacteria were allowed to be phagocytosed by BMDCs from the indicated mice in the presence or absence of ns-IgA at physiological concentration (2 mg/mL). Cells were washed and analyzed by flow cytometry. Data are presented as mean ± SEM; n = 3. *p < 0.05, ***p < 0.0001; t test. (E) Representative images of E. coli (blue) and CD11c (red) staining by BMDCs derived from CD89Tg or wild-type (WT) mice captured by imaging flow cytometry (scale bars, 5 mm) and the percentages of the bacterial phagocytosis score. See also Figures S1C and S5C.

Article Snippet: The Escherichia coli (E. coli-K12, Strain SMG 123 (PTA-7555)), Staphylococcus aureus subsp. aureus Rosenbach (S. aureus, ATCC 25923), Streptococcus pyogenes Rosenbach (S. pyogenes, ATCC 19615) and Escherichia coli-K12 WzxE (Coli genetic stock center, Yale university) were used for sCD89-bacteria interaction assays shown in Figure 1C.

Techniques: Competitive ELISA, Bacteria, Concentration Assay, Cytometry, Staining, Derivative Assay, Imaging

Journal: Cell reports

Article Title: CD89 Is a Potent Innate Receptor for Bacteria and Mediates Host Protection from Sepsis.

doi: 10.1016/j.celrep.2019.03.062

Figure Lengend Snippet: Figure 5. CD89-Bacterium Interaction Protects against Infection-Related Mortality in Mice (A) Survival of CD89Tg mice (red line) and littermates (black line) after intranasal inoculation (at time 0) with S. pneumonia (n = 25). Kaplan-Meier curves and log rank test were used to compare mortality rates. All data are presented as mean ± SEM. *p < 0.05. (B) Decreased lung contents of S.p in CD89 transgenic compared with littermate mice. All data are presented as mean ± SEM; n = 8. ***p < 0.001, t test. (C) H&E staining of lung sections from representative CD89Tg and littermate animals after intranasal infection. Scale bars, 200 mm. (D) Alveolitis invasion score of monomorphic inflammatory cells. All data are presented as mean ± SEM; n = 6. ***p < 0.001, t test. (E) mRNA expression of cytokines (IL-1, TNF-a, and IL-6) was assessed by qRT-PCR of 5 independent lung tissue RNA samples collected 6 and 48 h after intranasal infection. mRNA levels were normalized to b-actin mRNA levels. All data are presented as mean ± SEM; n = 6. *p < 0.05, t test. (F) Increased survival of CD89Tg mice (red line, n = 26) compared with littermates (black line, n = 22) after CLP. Kaplan-Meier curves and log rank test were used to compare mortality rates. All data are presented as mean ± SEM. **p < 0.01. (G–I) 48 h after CLP, peritoneal fluid was evaluated for total bacteria (G), E. coli (H), and Enterococcus (I) in CD89Tg mice and littermates. All data are presented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001; t test. (J) IL-1, TNF-a, and IL-6 levels in peritoneal lavage, assessed by ELISA 6 and 48 h after CLP. All data are presented as mean ± SEM; n = 3. *p < 0.05, t test. See also Figures S6A–S6C.

Article Snippet: The Escherichia coli (E. coli-K12, Strain SMG 123 (PTA-7555)), Staphylococcus aureus subsp. aureus Rosenbach (S. aureus, ATCC 25923), Streptococcus pyogenes Rosenbach (S. pyogenes, ATCC 19615) and Escherichia coli-K12 WzxE (Coli genetic stock center, Yale university) were used for sCD89-bacteria interaction assays shown in Figure 1C.

Techniques: Infection, Transgenic Assay, Staining, Expressing, Quantitative RT-PCR, Bacteria, Enzyme-linked Immunosorbent Assay

Journal: Cell reports

Article Title: CD89 Is a Potent Innate Receptor for Bacteria and Mediates Host Protection from Sepsis.

doi: 10.1016/j.celrep.2019.03.062

Figure Lengend Snippet: Figure 7. CD89 Protection against Sepsis Is Independent of CRP and IgA Antibodies during the Early Phase of Infection (A) Increased survival of CD89TgCRP-KO animals after intranasal infection with S.p compared with CRP-KO mice (n = 12 per group). CD89Tg mice and their littermates were used as controls. Kaplan-Meier curves and log rank test were used to compare mortality rates. All data are presented as mean ± SEM. (B) Decreased lung counts of S.p in CD89TgCRP-KO mice at 48 h compared with CRP-KO mice (n = 4). All data are presented as mean ± SEM. **p < 0.01, t test. (C) Expression of cytokine mRNA (IL-1, TNF-a, and IL-6) was assessed by qPCR of independent lung tissue RNA samples collected 6 and 24 h after intranasal infection. Cytokine mRNA levels were normalized to b-actin mRNA levels, as indicated in Figure 5E (n = 4). All data are presented as mean ± SEM. *p < 0.05, **p < 0.01; t test. (D) Increased survival of CD89TgCRP-KO (n = 10) compared with CRP-KO mice (n = 10) after CLP. Kaplan-Meier curves and log rank test were used to compare mortality rates. CD89Tg mice and their littermates were used as controls. All data are presented as mean ± SEM. (E) Peritoneal fluid counts of bacteria 48 h after CLP (n = 4). All data are presented as mean ± SEM. **p < 0.01, t test. (F) IL-1, TNF-a, and IL-6 levels in peritoneal lavage, assessed by ELISA 6 and 48 h after CLP (n = 4). All data are presented as mean ± SEM. *p < 0.05, **p < 0.01; t test. (G) Phagocytosis of S.p (left) and E. coli (right) after incubation with BMMs isolated from CD89TgCRP-KO or CRP-KO mice. (H) Phagocytosis of S.p (left) and E. coli (right) after incubation with BMMs isolated from CD89TgCRP-KO or CRP-KO mice in the presence of MIP8a anti-CD89 F(ab’)2. All data are presented as mean ± SEM. *p < 0.05, **p < 0.01; t test. (I) Measurement of mouse IgA antibodies against the indicated bacteria 48 or 168 h after S.p infection (left) or CLP (right) in CD89Tg or CD89TgCRP-KO mice. Data are presented as mean ± SEM. See also Figure S7.

Article Snippet: The Escherichia coli (E. coli-K12, Strain SMG 123 (PTA-7555)), Staphylococcus aureus subsp. aureus Rosenbach (S. aureus, ATCC 25923), Streptococcus pyogenes Rosenbach (S. pyogenes, ATCC 19615) and Escherichia coli-K12 WzxE (Coli genetic stock center, Yale university) were used for sCD89-bacteria interaction assays shown in Figure 1C.

Techniques: Infection, Expressing, Bacteria, Enzyme-linked Immunosorbent Assay, Incubation, Isolation

Journal: Infection and Immunity

Article Title: Roles of OmpX, an Outer Membrane Protein, on Virulence and Flagellar Expression in Uropathogenic Escherichia coli

doi: 10.1128/IAI.00721-20

Figure Lengend Snippet: Colonization by the parent strain (CFT073) and ompX mutant in the bladders and kidneys of mice with UTIs. The female mice (n = 3 for each group) were infected with the parent strain or ompX mutant. At 48 h postinfection, cell numbers of bacteria isolated from the bladder and kidneys were determined as CFU. We repeated experiments independently three times, but one mouse infected with the parent strain died within 48 h of infection. Each data point represents a sample from an individual mouse (n = 8 for the parent strain and n = 9 for the ompX mutant). Horizontal bars show median values. *, P < 0.05 relative to the value for the parent strain.

Article Snippet: CFT073 , Parent strain (ATCC 700928) , ATCC 700928.

Techniques: Mutagenesis, Infection, Bacteria, Isolation

Journal: Infection and Immunity

Article Title: Roles of OmpX, an Outer Membrane Protein, on Virulence and Flagellar Expression in Uropathogenic Escherichia coli

doi: 10.1128/IAI.00721-20

Figure Lengend Snippet: Adhesion to and internalization in kidney epithelial cells (HTB-44) of the parent strain (CFT073) and the ompX mutant (A and B) or the parent strain and the ompX mutant carrying pTrc99K (empty vector) or pTrc99KompX (ompX expression plasmid) (C). Values are percent CFU of adhered/internalized (A) and internalized (B and C) bacteria relative to total bacterial cell numbers. Data are means from three independent experiments; error bars indicate standard deviations. *, P < 0.05 relative to values for CFT073 (A and B) or CFT073/pTrc99K (C).

Article Snippet: CFT073 , Parent strain (ATCC 700928) , ATCC 700928.

Techniques: Mutagenesis, Plasmid Preparation, Expressing, Bacteria

Journal: Infection and Immunity

Article Title: Roles of OmpX, an Outer Membrane Protein, on Virulence and Flagellar Expression in Uropathogenic Escherichia coli

doi: 10.1128/IAI.00721-20

Figure Lengend Snippet: Aggregation within kidney epithelial cells (HTB-44) for the parent strain and the ompX mutant or the parent strain and the ompX mutant carrying pTH18kr (empty vector) or pTH18krompX (ompX expression plasmid). Bacteria carrying a green fluorescence protein (GFP) expression plasmid, pTurboGFP-B, and HTB-44 cells stained with rhodamine-phalloidin were imaged with green and red fluorescence, respectively, using a 100× objective. Images were taken from above (A), and cross-sectional images (B) correspond to the white boxes in panel A. The experiment was repeated twice, and similar results were obtained. (C) Aggregated bacteria within HTB-44 cells were quantified by representing levels of colonized bacteria as areas (in square pixels) of GFP. Microscopy data are means from three fields of view, and error bars indicate standard deviations. **, P < 0.01 relative to the value for the parent strain CFT073.

Article Snippet: CFT073 , Parent strain (ATCC 700928) , ATCC 700928.

Techniques: Mutagenesis, Plasmid Preparation, Expressing, Bacteria, Fluorescence, Staining, Microscopy

Journal: Infection and Immunity

Article Title: Roles of OmpX, an Outer Membrane Protein, on Virulence and Flagellar Expression in Uropathogenic Escherichia coli

doi: 10.1128/IAI.00721-20

Figure Lengend Snippet: Motilities and flagellar production for the parent strain (CFT073) and the ompX mutant or the parent strain and the ompX mutant carrying pTrc99K (empty vector) or pTrc99KompX (ompX expression plasmid). (A) Bacterial migration on LB medium containing 0.3% agar. (B) Diameters reflecting bacterial migration on the agar. Data are means from three independent experiments; error bars indicate standard deviations. (C) Flagella and bacterial cells were stained with Victoria blue/tannic acid were pictured using a 100× objective. (D) Ratios of bacteria observed with flagella to ∼120 to 150 randomly selected bacteria on microscopy, presented as percentages. Data are means, and error bars indicate standard deviations. **, P < 0.01 relative to the value for CFT073.

Article Snippet: CFT073 , Parent strain (ATCC 700928) , ATCC 700928.

Techniques: Mutagenesis, Plasmid Preparation, Expressing, Migration, Staining, Bacteria, Microscopy

Journal: Infection and Immunity

Article Title: Roles of OmpX, an Outer Membrane Protein, on Virulence and Flagellar Expression in Uropathogenic Escherichia coli

doi: 10.1128/IAI.00721-20

Figure Lengend Snippet: FliC expression in the ompX mutant and contribution of fliC to bacterial adhesion to and internalization within the kidney epithelial cells. (A) Western blots of cell lysates and secreted proteins from the parent strain (CFT073) and the ompX mutant containing a VSVG-tagged FliC expression plasmid (pTH18krfliC-VSVG) and pTrc99A (empty vector) or pTrc99AompX (ompX expression plasmid). Locations of molecular mass standards (in kilodaltons) are shown on the left. VSVG-tagged FliC was visualized by probing with a VSVG antibody. Adhesion to and internalization in kidney epithelial cells (HTB-44) of the parent strain, fliC mutant, and fliC/ompX double mutant (B and C) or the parent strain and the fliC mutant carrying pTrc99A (empty vector) or pTrc99AfliC (fliC expression plasmid) (D). Values are percent CFU of adhered/internalized (B) and internalized (C and D) bacteria relative to total bacterial cell numbers. Data are means from three independent experiments; error bars indicate standard deviations. *, P < 0.05 relative to CFT073 (B and C) or CFT073/pTrc99A (D).

Article Snippet: CFT073 , Parent strain (ATCC 700928) , ATCC 700928.

Techniques: Expressing, Mutagenesis, Western Blot, Plasmid Preparation, Bacteria

Journal: Infection and Immunity

Article Title: Roles of OmpX, an Outer Membrane Protein, on Virulence and Flagellar Expression in Uropathogenic Escherichia coli

doi: 10.1128/IAI.00721-20

Figure Lengend Snippet: Aggregation within kidney epithelial cells (HTB-44) for the parent strain and the fliC mutant or the parent strain and the fliC mutant carrying pTH18kr (empty vector) or pTH18krfliC-VSVG (fliC expression plasmid). Bacteria carrying a GFP expression plasmid, pTurboGFP-B, and HTB-44 cells stained with rhodamine-phalloidin were imaged with green and red fluorescence, respectively, using a 100× objective. Images were taken from above (A), and cross-sectional images (B) correspond to the white boxes in panel A. The experiment was repeated twice, and similar results were obtained. (C) Aggregated bacteria within HTB-44 cells were quantified by determining levels of colonized bacteria as areas (in square pixels) of GFP. Microscopy data are means from three fields of view, and error bars indicate standard deviations. *, P < 0.05, and **, P < 0.01, relative to CFT073.

Article Snippet: CFT073 , Parent strain (ATCC 700928) , ATCC 700928.

Techniques: Mutagenesis, Plasmid Preparation, Expressing, Bacteria, Staining, Fluorescence, Microscopy

Journal: Infection and Immunity

Article Title: Roles of OmpX, an Outer Membrane Protein, on Virulence and Flagellar Expression in Uropathogenic Escherichia coli

doi: 10.1128/IAI.00721-20

Figure Lengend Snippet: Transcript levels and promoter activities of flagellum-related and fimbrial genes in the parent strain (CFT073) and the ompX mutant. (A) Transcript levels were determined relative to that of rpoD. Data are means for two biological replicates; error bars indicate the ranges. (B) β-Galactosidase activities corresponding to flhD, fliA, and fliC promoter activities in the parent strain and the ompX mutant containing pNNflhD-P, pNNfliA-P, or pNNfliC-P, the lacZ reporter plasmid. Data are means from three independent experiments; error bars indicate standard deviations. **, P < 0.01 relative to CFT073.

Article Snippet: CFT073 , Parent strain (ATCC 700928) , ATCC 700928.

Techniques: Mutagenesis, Plasmid Preparation

Journal: Infection and Immunity

Article Title: Roles of OmpX, an Outer Membrane Protein, on Virulence and Flagellar Expression in Uropathogenic Escherichia coli

doi: 10.1128/IAI.00721-20

Figure Lengend Snippet: Strains and plasmids used in this study a

Article Snippet: CFT073 , Parent strain (ATCC 700928) , ATCC 700928.

Techniques: Plasmid Preparation, Mutagenesis, Expressing