plasmids encoding pegfp c2 senp2  (Addgene inc)

Journal: mBio

Article Title: Klebsiella pneumoniae Reduces SUMOylation To Limit Host Defense Responses

doi: 10.1128/mBio.01733-20

Figure Lengend Snippet: The deSUMOylase SENP2 mediates K. pneumoniae -induced decrease in SUMO-conjugated proteins. (A) Immunoblot analysis of Ubc9 and tubulin levels in lysates of A549 cells infected with Kp52145 for the indicated times. Ubc9 bands were quantified from three independent experiments using Image Studio Lite (LI-COR) and normalized to α-tubulin. The graph represents fold change compared to noninfected control cells. n.s., not significant versus n.i. determined using one-way ANOVA with Tukey’s multiple-comparison test. (B) Immunoblot analysis of SAE1, SAE2, and tubulin levels in lysates of A549 cells infected with Kp52145 for the indicated times. SAE1 and SAE2 bands were quantified from three independent experiments using Image Studio Lite (LI-COR) and normalized to α-tubulin. The graph represents fold change compared to noninfected control cells. n.s., not significant versus n.i. determined using one-way ANOVA with Tukey’s multiple-comparison test. (C) Immunoblot analysis of SUMO1 and tubulin levels in lysates of control (AS), SENP1, or SENP2 siRNA-transfected A549 cells infected with Kp52145 for 5 h. AS, AllStars control, nonsilencing siRNA. SUMO1 smears were quantified from three independent experiments using Image Studio Lite (LI-COR) and normalized to α-tubulin. The graph represents fold change compared to AS-transfected noninfected control cells. n.s., not significant; ***, P ≤ 0.001; *, P ≤ 0.05 by one-way ANOVA with Bonferroni’s multiple-comparison test. (D) Fluorescence microscopy of pSENP2-GFP-transfected A549 cells grown on glass coverslips. Cells were infected with Kp52145 for 5 h or left uninfected (n.i.). Coverslips were stained with Hoechst (4′,6-diamidino-2-phenylindole [DAPI]) for nucleus identification. The percentage of SENP2 localized in either nucleus or cytoplasm is represented on the graph and is the result of independent counting of 100 cells from each of 3 independent experiments. (E) Immunoblot analysis of SENP2 and tubulin levels in cytosolic extracts of A549 cells infected with Kp52145 for 5 h or left uninfected. (F) Fluorescence microscopy of pSENP2-GFP transfected A549 cells grown on glass coverslips. Cells were treated with the proteasomal inhibitor MG262 (5 μM, 2 h before infection), the nuclear export inhibitor leptomycin B (LMB; 10 nM, 2 h before infection), or DMSO (vehicle solution) and infected with Kp52145 for 5 h or left uninfected (n.i.). Coverslips were stained with Hoechst (DAPI) for nucleus identification. The percentage of SENP2 localized in either nucleus or cytoplasm is represented on the graphs and is the result of independent counting of 100 cells from each of 3 independent experiments. (G) Immunoblot analysis of SUMO1 (antibody sc-9060) and tubulin levels in lysates of the nuclear export inhibitor leptomycin B- (5 μM, 2 h before infection) or DMSO (vehicle solution)-treated A549 infected with Kp52145 for 5 h or left uninfected. SUMO1 smears were quantified using Image Studio Lite (LI-COR) and normalized to α-tubulin. The graph represents fold change compared to DMSO noninfected control cells. ***, P ≤ 0.001; **, P ≤ 0.01; *, P ≤ 0.05 by one-way ANOVA with Bonferroni’s multiple-comparison test. (H) Immunoblot analysis of K48 linkage-specific polyubiquitin and GFP levels in immunoprecipitates of A549 infected with Kp52145 for 5 h. Cells were immunoprecipitated using anti-GFP antibody. Preimmune mouse IgG served as a negative control. (I) Immunoblot analysis of SENP2 and tubulin levels in cytosolic extracts of control (AS), RBX1, Skp1, Cul-1, βTrCP or Skp2 siRNA-transfected A549 cells left uninfected. (J) Immunoblot analysis of cullin-1 and tubulin levels in lysates of A549 cells infected with Kp52145 for 5 h or left uninfected. Lysates of A549 treated with 5 mM H 2 O 2 for 10 min were used as a positive control for cullin-1 deNEDDylation. (K) Immunoblot analysis of Cul-1 and tubulin levels in lysates of control (AS) or CSN5 siRNA-transfected A549 cells infected with Kp52145 for 5 h or left uninfected. (L) Immunoblot analysis of K48 linkage-specific polyubiquitin and GFP levels in immunoprecipitates of control (AS) or CSN5 siRNA-transfected A549. Cells were transfected with a SENP2-GFP plasmid 24 h after the siRNA transfection and infected the following day with Kp52145 for 5 h or left uninfected. Cells were immunoprecipitated using anti-GFP antibody. Preimmune mouse IgG served as negative control. (M) Immunoblot analysis of SENP2 and tubulin levels in cytosolic extracts of control (AS) or CSN5 siRNA-transfected A549 cells infected with Kp52145 for 5 h or left uninfected. (N) Immunoblot analysis of SUMO1 and tubulin levels in lysates of control (AS) or CSN5 siRNA-transfected A549 cells infected with Kp52145 for 5 h or left uninfected. SUMO1 smears were quantified from three independent experiments using Image Studio Lite (LI-COR) and normalized to α-tubulin. The graph represents fold change compared to AS-transfected noninfected control cells. ***, P ≤ 0.001; **, P ≤ 0.01; *, P ≤ 0.05 by one-way ANOVA with Bonferroni’s multiple-comparison test. In all panels, data are representative of at least three independent experiments.

Article Snippet: Cells were transfected using Lipofectamine 2000 with plasmids encoding pEGFP-C2 SENP2 (gift from Mary Dasso, Addgene plasmid 13382) and infected 24 h later.

Techniques: Western Blot, Infection, Control, Comparison, Transfection, Fluorescence, Microscopy, Staining, Immunoprecipitation, Negative Control, Positive Control, Plasmid Preparation

Journal: mBio

Article Title: Klebsiella pneumoniae Reduces SUMOylation To Limit Host Defense Responses

doi: 10.1128/mBio.01733-20

Figure Lengend Snippet: SUMOylation increases host defenses against K. pneumoniae . (A) ELISA of IL-8 secreted by A549 cells transfected with either empty control plasmid (pcDNA3) or SUMO1-hemagglutinin (HA) tag plasmid (pSUMO1), which were left untreated (n.i.) or infected with Kp52145 for 3 h of contact, after which, the medium was replaced with medium containing gentamicin (100 μg ml −1 ) to kill extracellular bacteria and incubated for a further 4 h. Values are presented as the means ± SDs from three independent experiments measured in duplicates. **, P ≤ 0.01 by two-way ANOVA with Holm-Sidak’s multiple-comparison test. (B) ELISA of IL-8 secreted by control (AS) or SENP2 siRNA-transfected A549, which were left untreated (n.i.) or infected with Kp52145 for 3 h of contact, after which, the medium was replaced with medium containing gentamicin (100 μg ml −1 ) to kill extracellular bacteria and incubated for a further 4 h. Values are presented as the means ± SDs from three independent experiments measured in duplicates. **, P ≤ 0.01 by two-way ANOVA with Holm-Sidak’s multiple-comparison test. (C) ELISA of TNF-α secreted by empty control (pcDNA3) or SUMO1-HA plasmid-transfected MH-S, which were left untreated (n.i.) or infected with Kp52145 for 1 h of contact, after which, the medium was replaced with medium containing gentamicin (100 μg ml −1 ) to kill extracellular bacteria and incubated for a further 4 h. Values are presented as the means ± SDs from three independent experiments measured in duplicates. ***, P ≤ 0.001 by two-way ANOVA with Holm-Sidak’s multiple-comparison test. (D) ELISA of TNF-α secreted by antagomir-transfected MH-S, which were left untreated (n.i.) or infected with Kp52145 for 1 h of contact, after which, the medium was replaced with medium containing gentamicin (100 μg ml −1 ) to kill extracellular bacteria and incubated for a further 4 h. Values are presented as the means ± SDs from three independent experiments measured in duplicates. ***, P ≤ 0.001 by two-way-ANOVA with Holm-Sidak’s multiple-comparison tes; Neg, negative control. (E) Immunoblot analysis of IκBα and tubulin levels in lysates of empty (pcDNA3) or SUMO1-HA (pSUMO1) plasmid transfected A549 or MH-S cells, infected with Kp52145 for the indicated time points. (F) Immunoblot analysis of phosphorylated Jun N-terminal protein kinase (P-JNK), P-ERK, and P-p38 levels in lysates of pcDNA3- or pSUMO1-transfected A549 or MH-S cells infected with Kp52145 for the indicated time points. Tubulin immunoblotting was used as the loading control. n.i., noninfected control. (G) ELISA of IL-8 or TNF-α secreted by empty control (pcDNA3) or SUMO1-HA plasmid-transfected A549 or MH-S, respectively, which were treated with the NF-κB inhibitor BAY 11-7082 (5 μM, 2 h before infection) or DMSO (vehicle solution). Cells were left untreated (n.i.) or infected with Kp52145 for 1 h (MH-S) or 3 h (A549) of contact, after which, the medium was replaced with medium containing gentamicin (100 μg ml −1 ) to kill extracellular bacteria and incubated for a further 4 h. Values are presented as the means ± SDs from three independent experiments measured in duplicates. ***, P ≤ 0.001; **, P ≤ 0.01; *, P ≤ 0.05 by two-way ANOVA with Holm-Sidak’s multiple-comparison test. (H) Percent intracellular survival in MH-S cells transfected with either empty control plasmid (pcDNA3) or SUMO1-HA tag plasmid (pSUMO1). Cells were infected with Kp52145 for 30 min (MOI, 100:1), and wells were washed and incubated with medium containing gentamicin (100 μg ml −1 ) for 2.5 h. Intracellular bacteria were quantified by lysis, serial dilution, and viable counting on LB agar plates. Percent intracellular survival was determined by dividing the number of CFU obtained at 3 h of infection over the number obtained at 1 h and considering pcDNA3 as 100%. Values are presented as the means ± SDs from three independent experiments measured in duplicates. *, P ≤ 0.05 by unpaired t test with correction for Holm-Sidak’s multiple-comparison test. (I) Percent intracellular survival in antagomir-transfected MH-S cells. Cells were infected with Kp52145 for 30 min (MOI, 100:1), and wells were washed and incubated with medium containing gentamicin (100 μg ml −1 ) for 2.5 h. Intracellular bacteria were quantified by lysis, serial dilution, and viable counting on LB agar plates. Percent intracellular survival was determined by dividing the number of CFU obtained at 3 h of infection over the number obtained at 1 h and considering the negative control as 100%. Values are presented as the means ± SDs from three independent experiments measured in duplicates. **, P ≤ 0.01; *, P ≤ 0.05 by one-way ANOVA with Holm-Sidak’s multiple-comparison test. In panels E and F, data are representative of at least three independent experiments.

Article Snippet: Cells were transfected using Lipofectamine 2000 with plasmids encoding pEGFP-C2 SENP2 (gift from Mary Dasso, Addgene plasmid 13382) and infected 24 h later.

Techniques: Enzyme-linked Immunosorbent Assay, Transfection, Control, Plasmid Preparation, Infection, Bacteria, Incubation, Comparison, Negative Control, Western Blot, Lysis, Serial Dilution

Journal: mBio

Article Title: Klebsiella pneumoniae Reduces SUMOylation To Limit Host Defense Responses

doi: 10.1128/mBio.01733-20

Figure Lengend Snippet: Klebsiella pneumoniae ablates SUMOylation via distinct pathways in epithelial cells or macrophages. Working model of K. pneumoniae strategies to decrease SUMOylation in epithelial cells and macrophages. In epithelial cells, Kp52145 activates the signaling pathway EGFR-PI3K-AKT-ERK-GSK3β to increase the COP9 signalosome component CSN5, which inhibits NEDDylation of Cullin-1 and prevents proteasomal degradation of SENP2. SENP2 then accumulates in the cytosol, preventing SUMOylation. In macrophages, Kp52145 via TLR4-TRAM-TRIF induces the production of type I interferon, which signals through IFNAR1. Type I interferon stimulates transcription of the miRNA let-7 , which prevents SUMOylation. Both strategies lead to increased intracellular survival and subversion of host responses.

Article Snippet: Cells were transfected using Lipofectamine 2000 with plasmids encoding pEGFP-C2 SENP2 (gift from Mary Dasso, Addgene plasmid 13382) and infected 24 h later.

Techniques: