pu 6  (Addgene inc)

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Porcine extraintestinal pathogenic Escherichia coli delivers two serine protease autotransporters coordinately optimizing the bloodstream infection

doi: 10.3389/fcimb.2023.1138801

Figure Lengend Snippet: ExPEC strain PU-1 caused a heavy bacterial load in infected mouse blood. (A) The curves showed the bacterial loads in bloods from the mice challenged with different E. coli strains until 24 h post infection. The ExPEC strains DCE7 and DCE1 were used as controls here, respectively. Statistical significance of PU-1 infection group was determined by a one-way ANOVA test based on comparisons with the DCE7 infection group (**P < 0.01, *P < 0.05). (B, C) Blood routine testing detected the white blood cell (WBC) and neutrophil cell (NEU) counts of peripheral blood from the mice infected with indicated ExPEC strains at the 6 h post-infection. Statistical significance was determined by a one-way ANOVA test based on comparisons with the wild-type group (**P < 0.01, *P < 0.05). NS, no significance.

Article Snippet: The recombinant His 6 -Vat PU-1 and His 6 -Tsh PU-1 proteins were purified by Ni-NTA Spin Columns (QIAGEN) from BL21 (DE3) carrying the recombinant pET-21a plasmid after IPTG induction.

Techniques: Infection

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Porcine extraintestinal pathogenic Escherichia coli delivers two serine protease autotransporters coordinately optimizing the bloodstream infection

doi: 10.3389/fcimb.2023.1138801

Figure Lengend Snippet: Identification of two serine protease autotransporters as the potential facilitators for optimal blood infection in strain PU-1. (A) The transcriptional changes of indicated genes in ExPEC strain PU-1 response to host blood and serum. The data were normalized to the housekeeping gene tus transcript. Mean values and SDs of triplicate samples are indicated. Statistical significance was determined by a two-way ANOVA test based on comparisons with the bacterial cells cultured in LB medium (**P < 0.01). (B) Phylogenetic analysis of SPATEs from E coli . A neighbor-joining tree (bootstrap n = 1000; Poisson correction) was constructed based on a ClustalW alignment of the amino acid sequences of SPATEs using the MEGA software version 5.0. (C) Incubations of indicated bacterial strains within fresh blood and serum. The survival rates were calculated by measuring the bacterial counts. The porcine ExPEC strain DCE1 from phylogenetic group A were used as a control here. Statistical significance was determined by a one-way ANOVA test based on comparisons with the wild-type group (**P < 0.01).

Article Snippet: The recombinant His 6 -Vat PU-1 and His 6 -Tsh PU-1 proteins were purified by Ni-NTA Spin Columns (QIAGEN) from BL21 (DE3) carrying the recombinant pET-21a plasmid after IPTG induction.

Techniques: Infection, Cell Culture, Construct, Software

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Porcine extraintestinal pathogenic Escherichia coli delivers two serine protease autotransporters coordinately optimizing the bloodstream infection

doi: 10.3389/fcimb.2023.1138801

Figure Lengend Snippet: Mouse infection assay identified the pathogenic roles of VatPU-1 and TshPU-1. (A) Effect of vat PU-1 or tsh PU-1 deletion on strain PU-1 pathogenicity. Survival curve of mice infected with 1 × 10 6 CFU/mouse bacteria (ten mice per group). (B) Systemic infection experiment was conducted to assess bacterial load in mouse blood. Bacterial reisolation from the blood at 16 h post-inoculation was quantified by plate count. (C, D) Porcine and human bloods were then used to assess the bacterial survival of indicated ExPEC strains. The survival rates were calculated by measuring the bacterial counts. Statistical significance was determined by a one-way ANOVA test (**P < 0.01, *P < 0.05).

Article Snippet: The recombinant His 6 -Vat PU-1 and His 6 -Tsh PU-1 proteins were purified by Ni-NTA Spin Columns (QIAGEN) from BL21 (DE3) carrying the recombinant pET-21a plasmid after IPTG induction.

Techniques: Infection

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Porcine extraintestinal pathogenic Escherichia coli delivers two serine protease autotransporters coordinately optimizing the bloodstream infection

doi: 10.3389/fcimb.2023.1138801

Figure Lengend Snippet: Vat PU-1 and Tsh PU-1 bound to and cleaved diverse O-glycosylated mucin-like proteins. (A) SPATEs bind to the hemoglobin. Human hemoglobin was coated onto a 96-well microplate and incubated with the purified proteins. Binding was detected by an indirect ELISA. (B) The assessment of mucin degradation by using a column penetration assay. Quantification of indicated ExPEC strains in fractions eluted from columns filled with gel-forming mucus (1 to 5 fractions: top to bottom of the column). (C) Mucin extracted from porcine respiratory tract was incubated overnight with filtered culture supernatants of indicated ExPEC strains. Mucin degradation was observed by Western blot using an anti-MUC2 antibody. (D) The degradation mediated by Vat PU-1 and Tsh PU-1 to the extracellular domain of O-glycosylated mucin-like protein CD43 on human leukocytes. The PMNs were isolated from human blood and incubated with the purified Vat PU-1 , Tsh PU-1 , or denatured proteins at 37°C for 30 min. Flow cytometry was employed to analyze these samples using monoclonal antibodies against the extracellular domains of CD43. Flow cytometry data are representative of at least three independent experiments. ** P value < 0.05, which is considered statistically significant.

Article Snippet: The recombinant His 6 -Vat PU-1 and His 6 -Tsh PU-1 proteins were purified by Ni-NTA Spin Columns (QIAGEN) from BL21 (DE3) carrying the recombinant pET-21a plasmid after IPTG induction.

Techniques: Incubation, Purification, Binding Assay, Indirect ELISA, Western Blot, Isolation, Flow Cytometry

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Porcine extraintestinal pathogenic Escherichia coli delivers two serine protease autotransporters coordinately optimizing the bloodstream infection

doi: 10.3389/fcimb.2023.1138801

Figure Lengend Snippet: The impairment mediated by Vat PU-1 and Tsh PU-1 to the PMNs’ chemotaxis and transmigration through endothelial cell monolayers. (A, B) Assessment of PMNs’ chemotaxis. The calcein- labeled PMNs were treated with indicated proteins, or PBS vehicle control in the upper chamber of Transwell; IL-8 or fMLP were added to the lower chamber. Penetration of cells through the membrane was measured after 4 h fluorometrically. (C, D) Assessment of PMNs’ transmigration. The preincubated PMNs were applied to the upper chamber of Transwell supporting HBMEC, and transmigrated PMNs were enumerated as mentioned before. Statistical significance was determined by a one-way ANOVA test based on comparisons with the control group (** P <0.01). Error bars represent the SDs for three independent experiments.

Article Snippet: The recombinant His 6 -Vat PU-1 and His 6 -Tsh PU-1 proteins were purified by Ni-NTA Spin Columns (QIAGEN) from BL21 (DE3) carrying the recombinant pET-21a plasmid after IPTG induction.

Techniques: Chemotaxis Assay, Transmigration Assay, Labeling

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Porcine extraintestinal pathogenic Escherichia coli delivers two serine protease autotransporters coordinately optimizing the bloodstream infection

doi: 10.3389/fcimb.2023.1138801

Figure Lengend Snippet: The assessment Vat PU-1 and Tsh PU-1 in modulation of host immune responses during blood infection. (A, B) Blood routine testing detected the white blood cell (WBC) and neutrophil cell (NEU) counts of peripheral blood from the mice infected with indicated ExPEC strains at the 6 h post-infection. (C, D) Detection of IL-6 and IL-8 levels in bloods from the mice infected with indicated ExPEC strains at the 6 h post-infection. Statistical significance was determined by a one-way ANOVA test based on comparisons with the wild-type group (**P < 0.01).

Article Snippet: The recombinant His 6 -Vat PU-1 and His 6 -Tsh PU-1 proteins were purified by Ni-NTA Spin Columns (QIAGEN) from BL21 (DE3) carrying the recombinant pET-21a plasmid after IPTG induction.

Techniques: Infection

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Porcine extraintestinal pathogenic Escherichia coli delivers two serine protease autotransporters coordinately optimizing the bloodstream infection

doi: 10.3389/fcimb.2023.1138801

Figure Lengend Snippet: ExPEC strain PU-1 caused a heavy bacterial load in infected mouse blood. (A) The curves showed the bacterial loads in bloods from the mice challenged with different E. coli strains until 24 h post infection. The ExPEC strains DCE7 and DCE1 were used as controls here, respectively. Statistical significance of PU-1 infection group was determined by a one-way ANOVA test based on comparisons with the DCE7 infection group (**P < 0.01, *P < 0.05). (B, C) Blood routine testing detected the white blood cell (WBC) and neutrophil cell (NEU) counts of peripheral blood from the mice infected with indicated ExPEC strains at the 6 h post-infection. Statistical significance was determined by a one-way ANOVA test based on comparisons with the wild-type group (**P < 0.01, *P < 0.05). NS, no significance.

Article Snippet: The recombinant His 6 -Vat PU-1 and His 6 -Tsh PU-1 proteins were purified by Ni-NTA Spin Columns (QIAGEN) from BL21 (DE3) carrying the recombinant pET-21a plasmid after IPTG induction.

Techniques: Infection

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Porcine extraintestinal pathogenic Escherichia coli delivers two serine protease autotransporters coordinately optimizing the bloodstream infection

doi: 10.3389/fcimb.2023.1138801

Figure Lengend Snippet: Identification of two serine protease autotransporters as the potential facilitators for optimal blood infection in strain PU-1. (A) The transcriptional changes of indicated genes in ExPEC strain PU-1 response to host blood and serum. The data were normalized to the housekeeping gene tus transcript. Mean values and SDs of triplicate samples are indicated. Statistical significance was determined by a two-way ANOVA test based on comparisons with the bacterial cells cultured in LB medium (**P < 0.01). (B) Phylogenetic analysis of SPATEs from E coli . A neighbor-joining tree (bootstrap n = 1000; Poisson correction) was constructed based on a ClustalW alignment of the amino acid sequences of SPATEs using the MEGA software version 5.0. (C) Incubations of indicated bacterial strains within fresh blood and serum. The survival rates were calculated by measuring the bacterial counts. The porcine ExPEC strain DCE1 from phylogenetic group A were used as a control here. Statistical significance was determined by a one-way ANOVA test based on comparisons with the wild-type group (**P < 0.01).

Article Snippet: The recombinant His 6 -Vat PU-1 and His 6 -Tsh PU-1 proteins were purified by Ni-NTA Spin Columns (QIAGEN) from BL21 (DE3) carrying the recombinant pET-21a plasmid after IPTG induction.

Techniques: Infection, Cell Culture, Construct, Software

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Porcine extraintestinal pathogenic Escherichia coli delivers two serine protease autotransporters coordinately optimizing the bloodstream infection

doi: 10.3389/fcimb.2023.1138801

Figure Lengend Snippet: Mouse infection assay identified the pathogenic roles of VatPU-1 and TshPU-1. (A) Effect of vat PU-1 or tsh PU-1 deletion on strain PU-1 pathogenicity. Survival curve of mice infected with 1 × 10 6 CFU/mouse bacteria (ten mice per group). (B) Systemic infection experiment was conducted to assess bacterial load in mouse blood. Bacterial reisolation from the blood at 16 h post-inoculation was quantified by plate count. (C, D) Porcine and human bloods were then used to assess the bacterial survival of indicated ExPEC strains. The survival rates were calculated by measuring the bacterial counts. Statistical significance was determined by a one-way ANOVA test (**P < 0.01, *P < 0.05).

Article Snippet: The recombinant His 6 -Vat PU-1 and His 6 -Tsh PU-1 proteins were purified by Ni-NTA Spin Columns (QIAGEN) from BL21 (DE3) carrying the recombinant pET-21a plasmid after IPTG induction.

Techniques: Infection

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Porcine extraintestinal pathogenic Escherichia coli delivers two serine protease autotransporters coordinately optimizing the bloodstream infection

doi: 10.3389/fcimb.2023.1138801

Figure Lengend Snippet: Vat PU-1 and Tsh PU-1 bound to and cleaved diverse O-glycosylated mucin-like proteins. (A) SPATEs bind to the hemoglobin. Human hemoglobin was coated onto a 96-well microplate and incubated with the purified proteins. Binding was detected by an indirect ELISA. (B) The assessment of mucin degradation by using a column penetration assay. Quantification of indicated ExPEC strains in fractions eluted from columns filled with gel-forming mucus (1 to 5 fractions: top to bottom of the column). (C) Mucin extracted from porcine respiratory tract was incubated overnight with filtered culture supernatants of indicated ExPEC strains. Mucin degradation was observed by Western blot using an anti-MUC2 antibody. (D) The degradation mediated by Vat PU-1 and Tsh PU-1 to the extracellular domain of O-glycosylated mucin-like protein CD43 on human leukocytes. The PMNs were isolated from human blood and incubated with the purified Vat PU-1 , Tsh PU-1 , or denatured proteins at 37°C for 30 min. Flow cytometry was employed to analyze these samples using monoclonal antibodies against the extracellular domains of CD43. Flow cytometry data are representative of at least three independent experiments. ** P value < 0.05, which is considered statistically significant.

Article Snippet: The recombinant His 6 -Vat PU-1 and His 6 -Tsh PU-1 proteins were purified by Ni-NTA Spin Columns (QIAGEN) from BL21 (DE3) carrying the recombinant pET-21a plasmid after IPTG induction.

Techniques: Incubation, Purification, Binding Assay, Indirect ELISA, Western Blot, Isolation, Flow Cytometry

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Porcine extraintestinal pathogenic Escherichia coli delivers two serine protease autotransporters coordinately optimizing the bloodstream infection

doi: 10.3389/fcimb.2023.1138801

Figure Lengend Snippet: The impairment mediated by Vat PU-1 and Tsh PU-1 to the PMNs’ chemotaxis and transmigration through endothelial cell monolayers. (A, B) Assessment of PMNs’ chemotaxis. The calcein- labeled PMNs were treated with indicated proteins, or PBS vehicle control in the upper chamber of Transwell; IL-8 or fMLP were added to the lower chamber. Penetration of cells through the membrane was measured after 4 h fluorometrically. (C, D) Assessment of PMNs’ transmigration. The preincubated PMNs were applied to the upper chamber of Transwell supporting HBMEC, and transmigrated PMNs were enumerated as mentioned before. Statistical significance was determined by a one-way ANOVA test based on comparisons with the control group (** P <0.01). Error bars represent the SDs for three independent experiments.

Article Snippet: The recombinant His 6 -Vat PU-1 and His 6 -Tsh PU-1 proteins were purified by Ni-NTA Spin Columns (QIAGEN) from BL21 (DE3) carrying the recombinant pET-21a plasmid after IPTG induction.

Techniques: Chemotaxis Assay, Transmigration Assay, Labeling

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Porcine extraintestinal pathogenic Escherichia coli delivers two serine protease autotransporters coordinately optimizing the bloodstream infection

doi: 10.3389/fcimb.2023.1138801

Figure Lengend Snippet: The assessment Vat PU-1 and Tsh PU-1 in modulation of host immune responses during blood infection. (A, B) Blood routine testing detected the white blood cell (WBC) and neutrophil cell (NEU) counts of peripheral blood from the mice infected with indicated ExPEC strains at the 6 h post-infection. (C, D) Detection of IL-6 and IL-8 levels in bloods from the mice infected with indicated ExPEC strains at the 6 h post-infection. Statistical significance was determined by a one-way ANOVA test based on comparisons with the wild-type group (**P < 0.01).

Article Snippet: The recombinant His 6 -Vat PU-1 and His 6 -Tsh PU-1 proteins were purified by Ni-NTA Spin Columns (QIAGEN) from BL21 (DE3) carrying the recombinant pET-21a plasmid after IPTG induction.

Techniques: Infection

Journal: The Journal of Biological Chemistry

Article Title: DNA polymerase η promotes nonhomologous end joining upon etoposide exposure dependent on the scaffolding protein Kap1

doi: 10.1016/j.jbc.2022.101861

Figure Lengend Snippet: Pol η deficiency leads to NHEJ repair defect and cellular hypersensitivity to ETO. A , Pol η shRNA–treated cells complemented with Flag-Pol η or not were transfected with NHEJ reporter (I-SceI endonuclease encoding plasmid and pDsRed2-N1). NHEJ repair efficiency (GFP + /DsRed + ) were analyzed by FACS after 96 h. The lower panels show the Pol η levels through immunoblotting. Tubulin: loading control. B , the recruitment of Pol η to ETO-induced DSBs is independent of its catalytic activity. The proportions of GFP-Pol η WT- or CI (catalytic inactive) mutant-expressing cells with more than 30 foci were determined. Data represent means ± SEM from three independent experiments. C , the role of Pol η in NHEJ is independent of its catalytic activity. EJ5 cells overexpressing Flag-vector, or Flag-Pol η WT, or CI were transfected with I-SceI endonuclease. NHEJ repair efficiency (GFP + ) was analyzed by FACS after 48 h. The lower panels show immunoblots indicating the Pol η levels in different conditions. D , MRC5 (NC, siPol η, and siKu80), XPV, and GFP-Pol η–complemented XPV (XPV-GFP-Pol η) cells were treated with indicated ETO and further incubated for 7 to 10 days. The number of clones was determined. Surviving fraction was expressed as a percentage of mock-treated cells. Experiment was repeated three times, giving similar results. The representative curve is shown. Error bar: s.d., n = 3. E , immunoblots indicating the Pol η and Ku80 levels in ( D ). Tubulin: loading control. DSB, double-strand break; ETO, etoposide; FACS, fluorescence activated cell sorting; NHEJ, nonhomologous end joining.

Article Snippet: Antibodies sources were as follows: mouse anti-Flag (F1804, 1:1000) from Sigma; anti-Pol η (ab17725, 1:1000), anti-γH2AX (ab11174, 1:1000), anti-Rad18 (ab57447, 1:1000), anti-DNA-PKcs (ab18356, 1:1000), and anti-Rad51 (ab133534, 1:200) from Abcam; anti-53BP1 (4937S, 1:1000) from Cell Signaling Technology; anti-Myc (MMS-150R-500, 1:1000) from Covance; anti-His (HT501–02, 1:1000) from Beijing TransGen Biotech Co, Ltd; anti-β-actin (M20010, 1:2000) from Abmart; anti-β-Tubulin (AbM59005–37-PU, 1:4000), anti-Ku80 (AbM51083-6-PU, 1:1000), and anti-Ku70 (AbM51079-4-PU, 1:1000) from Beijing Protein Innovation; anti-GFP (sc-8334, 1:500), anti-Kap1 (sc-515790, 1:1000), anti-PCNA (sc-56, 1:1000), and anti-PARP1 (sc-8007, 1:1000) from Santa Cruz Biotechnology.

Techniques: shRNA, Transfection, Plasmid Preparation, Western Blot, Activity Assay, Mutagenesis, Expressing, Incubation, Clone Assay, Fluorescence, FACS