Journal: Nature Communications

Article Title: Human GBP1 binds LPS to initiate assembly of a caspase-4 activating platform on cytosolic bacteria

doi: 10.1038/s41467-020-16889-z

Figure Lengend Snippet: a – c Intracellular bacterial fold-replication ( a ) and release of LDH ( b , c ) in naive or IFNγ-primed wild-type, CASP4 –/– or GSDMD –/– HeLa cells, at 1 or 6-h post-infection (p.i.) with Salmonella . Cells in 96-well plates were infected for 30 min, washed and gentamicin was added to kill extracellular bacteria. At the indicated time points supernatant was collected to determine the release of LDH, and then cells were lysed and the number of viable intracellular bacteria was determined by counting colony forming units (CFUs). The bacterial fold-replication was calculated relative to 1 h p.i. d Percentage of CHQ-resistant cytosolic Salmonella in naive or IFNγ-primed HeLa at 1.5 h p.i. Cells were infected for 30 min as in ( a ) and then treated with gentamicin ± CHQ for an additional 1 h before cells were lysed and bacteria counted by CFUs. The percentage of cytosolic bacteria was calculated as the ratio of (CHQ + gentamicin resistant / gentamicin resistant ). e – g Release of LDH from naive or IFNγ-primed cells, 5 h after transfection with LPS (2.5 µg/50,000 cells) or 3–4 h after electroporation with LPS (300 ng/50,0000 cells). h Western blot analysis of full length (p43) and cleaved (p32) caspase-4 in the supernatants and cell lysates from naive or IFNγ-primed HaCaT cells, upon transfection with E. coli LPS LPS (2.5 µg/50,000 cells). i Streptavidin pull-down assay of the binding of biotin-conjugated LPS to endogenous caspase-4 from the lysates of naive or IFNγ-primed HBEC3-KT. Cells in 6-well plates were transfected with LPS-biotin (10 µg) or left untransfected, and biotinylated substrate was pulled down using equal amounts of streptavidin magnetic beads, which were then eluted in equal volumes of SDS-PAGE reducing sample buffer. Streptavidin-bound and -unbound fractions were analyzed by immunoblotting for caspase-4. Graphs show the mean ± SD, and data are pooled from two to six independent experiments performed in triplicate ( a – g ) or representative of two ( h , i ) independent experiments. *** P < 0.001; ns, not significant; two-tailed t -test.

Article Snippet: Freshly purified GBP1 (1 µM) was incubated on ice alone or with a two-fold molar excess of LPS or LPS-derivatives for 5 h. Ultrapure O11:B4 E.coli LPS (Invivogen), Salmonella Typhimurium Smooth LPS (Enzo Life Science), Rhodobacter sphaeroides ultrapure LPS (Invivogen), E. coli F585 diphosphoryl Lipid A (Sigma-Aldrich), Salmonella minnesota 595 Lipid A (Invivogen), synthetic monophosphorylated Lipid A (Invivogen), E.coli EH100 LPS Ra mutant (Sigma-Aldrich), E.coli J5 LPS Rc mutant (Sigma-Aldrich), E.coli F583 LPS Rd mutant (Sigma-Aldrich), E. coli R515 LPS Re mutant (Enzo Life Science) was used.

Techniques: Infection, Transfection, Electroporation, Western Blot, Pull Down Assay, Binding Assay, Magnetic Beads, SDS Page, Two Tailed Test

Journal: Nature Communications

Article Title: Human GBP1 binds LPS to initiate assembly of a caspase-4 activating platform on cytosolic bacteria

doi: 10.1038/s41467-020-16889-z

Figure Lengend Snippet: a Immunoblots for GBP1, caspase-4 and GAPDH (loading control) in cell lysates from IFNγ-primed wild-type or GBP1 –/– HeLa. b , c Release of LDH from naive or IFNγ-primed wild-type or GBP1 –/– HeLa after Salmonella infection ( b ) or after 5 h transfection with E. coli LPS (2.5 µg / 50,000 cells) ( c ). d , e Immunoblots for full length (p43) and cleaved (p32) caspase-4 in combined supernatants and cell lysates from naive or IFNγ-primed wild-type and GBP1 –/– HeLa, upon transfection with E. coli LPS for 5 h ( d ) or Salmonella infection ( e ). f Release of LDH from IFNγ-primed wild-type or GBP1 –/– HeLa, 3 h after electroporation with LPS (300 ng/50,000 cells). g Release of LDH in IFNγ-primed HBEC3-KT or HaCaT cells treated with non-targeting control siRNA (NT) or with siRNAs targeting CASP4 , GSDMD or GBP1 , after E. coli LPS transfection. Cells were treated with siRNAs for 24 h and transfected with LPS (2.5 µg / 50,000 cells) for 5 h. Graphs show the mean ± SD, and data are pooled from two ( c , f ), three ( g ) or four ( b ) independent experiments performed in triplicate, or representative of three independent experiments ( d , e ). *** P < 0.001; ns, not significant; two-tailed t -test.

Article Snippet: Freshly purified GBP1 (1 µM) was incubated on ice alone or with a two-fold molar excess of LPS or LPS-derivatives for 5 h. Ultrapure O11:B4 E.coli LPS (Invivogen), Salmonella Typhimurium Smooth LPS (Enzo Life Science), Rhodobacter sphaeroides ultrapure LPS (Invivogen), E. coli F585 diphosphoryl Lipid A (Sigma-Aldrich), Salmonella minnesota 595 Lipid A (Invivogen), synthetic monophosphorylated Lipid A (Invivogen), E.coli EH100 LPS Ra mutant (Sigma-Aldrich), E.coli J5 LPS Rc mutant (Sigma-Aldrich), E.coli F583 LPS Rd mutant (Sigma-Aldrich), E. coli R515 LPS Re mutant (Enzo Life Science) was used.

Techniques: Western Blot, Infection, Transfection, Electroporation, Two Tailed Test

Journal: Nature Communications

Article Title: Human GBP1 binds LPS to initiate assembly of a caspase-4 activating platform on cytosolic bacteria

doi: 10.1038/s41467-020-16889-z

Figure Lengend Snippet: a Fluorescence confocal microscopy of naive and IFNγ-primed HeLa co-expressing caspase-4-eGFP (green) and mCherry-GBP1 (red) and infected with Salmonella for 1 h. DNA was stained with Hoechst (blue). Representative confocal images are shown and scale bar corresponds to 5 µm. b Fluorescence confocal microscopy of IFNγ-primed or naive HeLa cells co-expressing mCherry-GBP1 (red), Dox-inducible eGFP-GBP1, −2, −3 or −4 (green) and caspase-4-V5 (gray), and infected with Salmonella for 1 h. DNA was stained with Hoechst (blue). eGFP-GBPs were expressed by inducing cells with 1 µg/mL Dox for 16 h. Caspase-4-V5 was visualized by immunostaining with an anti-V5 antibody. Representative confocal images are shown and scale bar corresponds to 10 µm. c Percentage of caspase-4-V5 positive Salmonella at 1 h p.i., quantified out of the mCherry-GBP1-positive bacteria. At least 50 GBP1-positive bacteria were counted per coverslip. d Percentage of cell death in HeLa cells co-expressing constitutive mCherry-GBP1 and Dox-inducible eGFP or eGFP-GBP1, −2, −3 or −4. FLAG-GBP3 and HA-GBP4 were constitutively expressed together using a bicistronic plasmid. Cells were transfected with the indicated plasmids for 24 h. eGFP-GBPs were induced for 16 h with 1 µg/mL Dox, whereas eGFP was induced for 3 h. Cells were then transfected with E. coli -derived LPS (2.5 µg/50,000 cells) for 6 h and cell death values were normalized considering IFNγ-primed HeLa as 100% and naive cells co-expressing mCherry-GBP1 and eGFP as 0%. Graphs show the mean ± SD, and data are pooled from two independent experiments performed in duplicate ( c ), pooled from three independent experiments performed in triplicate ( d ) or are representative from two ( b ) or three ( a ) independent experiments. ** P < 0.01; *** P < 0.001; one-way ANOVA.

Article Snippet: Freshly purified GBP1 (1 µM) was incubated on ice alone or with a two-fold molar excess of LPS or LPS-derivatives for 5 h. Ultrapure O11:B4 E.coli LPS (Invivogen), Salmonella Typhimurium Smooth LPS (Enzo Life Science), Rhodobacter sphaeroides ultrapure LPS (Invivogen), E. coli F585 diphosphoryl Lipid A (Sigma-Aldrich), Salmonella minnesota 595 Lipid A (Invivogen), synthetic monophosphorylated Lipid A (Invivogen), E.coli EH100 LPS Ra mutant (Sigma-Aldrich), E.coli J5 LPS Rc mutant (Sigma-Aldrich), E.coli F583 LPS Rd mutant (Sigma-Aldrich), E. coli R515 LPS Re mutant (Enzo Life Science) was used.

Techniques: Fluorescence, Confocal Microscopy, Expressing, Infection, Staining, Immunostaining, Plasmid Preparation, Transfection, Derivative Assay

Journal: Nature Communications

Article Title: Human GBP1 binds LPS to initiate assembly of a caspase-4 activating platform on cytosolic bacteria

doi: 10.1038/s41467-020-16889-z

Figure Lengend Snippet: a Streptavidin pull-down assay for eGFP-GBP1-4 using biotin or biotin-conjugated LPS. HeLa cells stably expressing Dox-inducible eGFP-GBP1, −2, −3, or −4 were primed with IFNγ and 1 µg/mL Dox was added for 16 h. 1 million cells were lysed and incubated with 2 µg LPS-biotin or biotin, and the biotinylated substrates were pulled down using equal amounts of streptavidin magnetic beads, which were then eluted in equal volumes of SDS-PAGE reducing sample buffer. Streptavidin-bound and -unbound fractions were analyzed by western blot using an antibody against GFP. b SPR sensorgram of E. coli LPS (O111:B4) binding to human GBP1 immobilized on a CM5 chip surface. Sensorgram was obtained by using different LPS concentrations (47, 94, 188, 375, 750, and 1500 nM). Gray lines correspond to SPR data and orange lines to model fits using a two-state-reaction model. c Saturation curve of the titration of LPS on GBP1 immobilized on a CM5 chip. d Calculated dissociation constants ( K D ) for LPS binding to immobilized GBP1 (GBP1 im ) or GBP1 binding to immobilized E. coli LPS (LPS im ). Dissociation constants for LPS-caspase-4 and LPS-caspase-11 were previously published by Shi et al. . e , f Size exclusion chromatograms of recombinant, LPS-free His-tagged GBP1 incubated with various LPS derivatives. Following purification, GBP1 (1 µM) was incubated on ice with LPS (2 µM) for 5 h before being subjected to size-exclusion analysis on a Superdex 200 10/30 GL column. Protein size was estimated using molecular weight standards. Curves were corrected by subtracting LPS-specific absorbance at 280 nm. Individual fractions were run on a 12% acrylamide gel and immunoblotted against His 6 to confirm the presence of GBP1 in elution peaks ( f ). g GTPase activity analysis of recombinant GBP1. GBP1 (500 nM) was incubated with GTP (5 µM) with or without ultrapure LPS (5 µM) for 30 min before the reaction was stopped. Luminescence was normalized to a buffer-only control. Graphs show the mean ± SD, and data are representative from three ( a – d , g ) or five ( e , f ) independent experiments performed with at least three independently expressed and purified batches of recombinant His-GBP1.

Article Snippet: Freshly purified GBP1 (1 µM) was incubated on ice alone or with a two-fold molar excess of LPS or LPS-derivatives for 5 h. Ultrapure O11:B4 E.coli LPS (Invivogen), Salmonella Typhimurium Smooth LPS (Enzo Life Science), Rhodobacter sphaeroides ultrapure LPS (Invivogen), E. coli F585 diphosphoryl Lipid A (Sigma-Aldrich), Salmonella minnesota 595 Lipid A (Invivogen), synthetic monophosphorylated Lipid A (Invivogen), E.coli EH100 LPS Ra mutant (Sigma-Aldrich), E.coli J5 LPS Rc mutant (Sigma-Aldrich), E.coli F583 LPS Rd mutant (Sigma-Aldrich), E. coli R515 LPS Re mutant (Enzo Life Science) was used.

Techniques: Pull Down Assay, Stable Transfection, Expressing, Incubation, Magnetic Beads, SDS Page, Western Blot, Binding Assay, Titration, Recombinant, Purification, Molecular Weight, Acrylamide Gel Assay, Activity Assay

Journal: Nature Communications

Article Title: Human GBP1 binds LPS to initiate assembly of a caspase-4 activating platform on cytosolic bacteria

doi: 10.1038/s41467-020-16889-z

Figure Lengend Snippet: Observed molecular weights of GBP1 peaks after incubation with various LPS.

Article Snippet: Freshly purified GBP1 (1 µM) was incubated on ice alone or with a two-fold molar excess of LPS or LPS-derivatives for 5 h. Ultrapure O11:B4 E.coli LPS (Invivogen), Salmonella Typhimurium Smooth LPS (Enzo Life Science), Rhodobacter sphaeroides ultrapure LPS (Invivogen), E. coli F585 diphosphoryl Lipid A (Sigma-Aldrich), Salmonella minnesota 595 Lipid A (Invivogen), synthetic monophosphorylated Lipid A (Invivogen), E.coli EH100 LPS Ra mutant (Sigma-Aldrich), E.coli J5 LPS Rc mutant (Sigma-Aldrich), E.coli F583 LPS Rd mutant (Sigma-Aldrich), E. coli R515 LPS Re mutant (Enzo Life Science) was used.

Techniques: Incubation

Journal: Nature Communications

Article Title: Human GBP1 binds LPS to initiate assembly of a caspase-4 activating platform on cytosolic bacteria

doi: 10.1038/s41467-020-16889-z

Figure Lengend Snippet: a Size exclusion chromatograms of recombinant His-tagged GBP1 incubated with E. coli LPS, or with E. coli LPS pre-treated with CaCl 2 (5 mM), Polymyxin B (10 µg/mL) or with alkaline phosphatase. Curves were corrected by subtracting the respective LPS-specific absorbance at 280 nm. Black curves representing control condition were overlayed. b Release of LDH from IFNγ-primed HeLa 5 h after transfection with E. coli LPS, or after transfection with LPS previously treated with alkaline phosphatase. c 3D structure of human GBP1 (PDB 1f5n), highlighting five different negatively charged patches (A to E). Residues comprising patch E are only visible in PDB 6k1z. For each patch, the indicated residues were all mutated to alanines and analyzed for GBP1-LPS interaction by size exclusion chromatography. Purple indicates GTPase domain, green indicates helical domain. d Size exclusion chromatograms of different His-tagged GBP1 mutants incubated with E. coli LPS. Curves were corrected by subtracting LPS-specific absorbance at 280 nm. e Fluorescence confocal microscopy of naive HeLa expressing eGFP-GBP1 wt , eGFP-GBP1 KKK61-63AAA or eGFP-GBP1 KK87-88AA and infected with Salmonella -dsRed for 1 h. DNA was stained with Hoechst (blue). Representative confocal images are shown and scale bar corresponds to 5 µm. f Percentage of eGFP-GBP1 positive Salmonella at 1 h p.i., as quantified by counting between 100–200 bacteria per coverslip. Graphs show the mean ± SD, and data are pooled from three independent experiments performed in duplicate ( f ), four independent experiments performed in triplicate ( b ), or are representative from three ( a , d , e ) independent experiments. *** P < 0.001; ns, not significant, two-tailed t -test.

Article Snippet: Freshly purified GBP1 (1 µM) was incubated on ice alone or with a two-fold molar excess of LPS or LPS-derivatives for 5 h. Ultrapure O11:B4 E.coli LPS (Invivogen), Salmonella Typhimurium Smooth LPS (Enzo Life Science), Rhodobacter sphaeroides ultrapure LPS (Invivogen), E. coli F585 diphosphoryl Lipid A (Sigma-Aldrich), Salmonella minnesota 595 Lipid A (Invivogen), synthetic monophosphorylated Lipid A (Invivogen), E.coli EH100 LPS Ra mutant (Sigma-Aldrich), E.coli J5 LPS Rc mutant (Sigma-Aldrich), E.coli F583 LPS Rd mutant (Sigma-Aldrich), E. coli R515 LPS Re mutant (Enzo Life Science) was used.

Techniques: Recombinant, Incubation, Transfection, Size-exclusion Chromatography, Fluorescence, Confocal Microscopy, Expressing, Infection, Staining, Two Tailed Test