Journal: bioRxiv

Article Title: Klebsiella pneumoniae reduces SUMOylation to limit host defence responses

doi: 10.1101/2020.06.29.179275

Figure Lengend Snippet: A. ELISA of IL-8 secreted by A549 cells transfected with either empty control plasmid (pcDNA3) or SUMO1-HA tagged 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 hours. Values are presented as the mean ± SD of three independent experiments measured in duplicate. ***P ≤ 0.001; *P ≤ 0.05; versus pcDNA3 determined using two way-ANOVA with Holm-Sidak’s multiple comparisons 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 hours. Values are presented as the mean ± SD of three independent experiments measured in duplicate. ***P ≤ 0.001; *P ≤ 0.05; versus AS determined using two way-ANOVA with Holm-Sidak’s multiple comparisons test. C.ELISA of TNFα 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 hours. Values are presented as the mean ± SD of three independent experiments measured in duplicate. ***P ≤ 0.001; *P ≤ 0.05; versus pcDNA3 determined using two way-ANOVA with Holm-Sidak’s multiple comparisons test. D.ELISA of TNFα 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 hours. Values are presented as the mean ± SD of three independent experiments measured in duplicate. ***P ≤ 0.001; *P ≤ 0.05; versus Negative Kp52145 determined using two way-ANOVA with Holm-Sidak’s multiple comparisons test. 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 JNK (P-JNK), ERK (P-ERK) and p38 (P-p38) phosphorylation 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. Tubulin immunoblotting was used as the loading control. n.i. – non-infected control G.ELISA of IL-8 or TNFα 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 hours. Values are presented as the mean ± SD of three independent experiments measured in duplicate. ***P ≤ 0.001; *P ≤ 0.05; determined using two way-ANOVA with Holm-Sidak’s multiple comparisons test. H.Percentage of intracellular survival in MH-S cells transfected with either empty control plasmid (pcDNA3) or SUMO1-HA tagged plasmid (pSUMO1). Cells were infected with Kp52145 for 30 min (MOI 100:1), 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. Percentage of intracellular survival was determined by dividing the number of colony forming units (CFU) obtained at 3 h of infection over the number obtained at 1 h and considering pcDNA3 as 100 %. Values are presented as the mean ± SD of three independent experiments measured in duplicate. ***P ≤ 0.001; *P ≤ 0.05; versus pcDNA3 determined using unpaired t-test with correction for Holm-Sidak’s multiple comparisons test. I.Percentage of intracellular survival in antagomir transfected MH-S cells. Cells were infected with Kp52145 for 30 min (MOI 100:1), 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. Percentage of intracellular survival was determined by dividing the number of colony forming units (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 mean ± SD of three independent experiments measured in duplicate. **P ≤ 0.01; *P ≤ 0.05; versus negative control determined using one way-ANOVA with Holm-Sidak’s multiple comparisons test. In panels E and F, data are representative of at least three independent experiments.

Article Snippet: The following plasmids were used: pcDNA3 (Invitrogen), pSUMO1 (pcDNA3-HA-SUMO1 was a gift from Junying Yuan, Addgene plasmid # 21154), pSENP2-GFP (pEGFP-C2 SENP2 was a gift from Mary Dasso, Addgene plasmid # 13382), pSENP2-FLAG (FLAG-SENP2 was a gift from Edward Yeh, Addgene plasmid # 18047), GSK3β-WT (HA GSK3 beta wt pcDNA3 was a gift from Jim Woodgett, Addgene plasmid # 14753), GSK3β-S9A (HA GSK3 beta S9A pcDNA3 was a gift from Jim Woodgett, Addgene plasmid # 14754).

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