Journal: Acta Crystallographica. Section D, Structural Biology

Article Title: The role of conserved surface hydrophobic residues in the carbapenemase activity of the class D β-lactamases

doi: 10.1107/S2059798317008671

Figure Lengend Snippet: Antimicrobial susceptibility profile produced by the β-lactamase OXA-143

Article Snippet: The resulting plasmids were transformed into Escherichia coli JM83 and A. baumannii ATCC 17978, respectively.

Techniques: Produced, Control

Journal: PLoS ONE

Article Title: Isolation of Mycobacterium avium Subspecies paratuberculosis Reactive CD4 T Cells from Intestinal Biopsies of Crohn's Disease Patients

doi: 10.1371/journal.pone.0005641

Figure Lengend Snippet: A) Comparison of cytokine response in MAP reactive (n = 17) and E. coli reactive (n = 8) T-cell clones from the same CD patient (CD-46). T-cell clones were stimulated with MAP or E. coli antigens (10 µg/ml) for 48 hours using HLA class II matched irradiated adherent cells as APC. Supernatants from duplicate wells were sampled and tested for cytokine production. Cytokine production in control wells was subtracted. One symbol represents one clone. Error bars indicate mean±SEM. B) IL-17 and IFN-γ production in MAP reactive T-cell clones (n = 26) from four different CD patients in response to MAP antigen. C) Intracellular staining of IFN-γ and IL-17 after stimulation with PMA/ionomycin in a Th1 (TCC906.A.8.4.15) clone and a Th1/Th17 clone (TCC946.A.8.2b.17). Filled histogram represent PMA/ionomycin samples and open histograms represent unstimulated samples. D) CCR6 expression in a Th1 clone and (top) and a Th1/Th17 clone (bottom). Filled histograms represent CCR6 expression and open histograms represent isotype control.

Article Snippet: The following strains were used to prepare the antigens: Bacterioides thetaiotaomicron CCUG 12297, Lactobacillus gasseri CCUG 39972, Bifidobacterium bifidum CCUG 45217, Escherichia coli ATCC 43893 (enteroinvasive), M. avium subsp. paratuberculosis 2E, Mycobacterium avium subspecies avium D4, Mycobacterium intracellulare MNC72, Mycobacterium gordonae MNC 64, Mycobacterium tuberculosis clinical isolate.

Techniques: Comparison, Clone Assay, Irradiation, Control, Staining, Expressing

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: Scientific Reports

Article Title: Hypoionic shock treatment enables aminoglycosides antibiotics to eradicate bacterial persisters

doi: 10.1038/srep14247

Figure Lengend Snippet: ( a ) Survival ratio of stationary phase MC4100 (streptomycin-resistant) or BW25113 (streptomycin-sensitive) E. coli cells after being treated with tobramycin- (500 μg/ml) or streptomycin-containing (2 mg/ml) distilled water. Asterisks indicate no detection of survival. ( b ) Time-dependent fluorescence-activated cell sorting analysis (left) and direct fluorescent microscopy imaging (right; at the 30 minutes point) of stationary phase cells that were treated with tobramycin-containing (500 μg/ml) distilled water before incubated with propidium iodide (50 μg/ml) in PBS or LB for the indicated length of time. A.U., arbitrary units. ( c ) Regular micrographs of the cells taken at the indicated time point. Scale bar, 5 μm.

Article Snippet: Stationary phase Gram-negative Escherichia coli bacteria cells of strains BW25113 and MC4100 or Gram-positive Staphylococcus aureus bacteria cells of the strain ATCC 25923 were prepared by overnight culturing frozen stock bacteria cells at 37 °C, 200 r.p.m. in 20 ml of Luria-Bertani (LB) growth medium.

Techniques: Fluorescence, FACS, Microscopy, Imaging, Incubation