P. aeruginosa infection suppresses NLRP3 inflammasome.

(A–E) Wild-type BMDM cells were primed overnight with 1 μg/ml Pam3CSK4 and incubated with E. coli or P. aeruginosa (ΔRetS PAO1) at an MOI of 30 and 20 μg/ml OMVs for 2 h. Cells were then supplemented with fresh medium containing 100 μg/ml Gentamycin. Cells were lysed and immunoblotted as indicated 16 h post infection (A). Band intensities of cleaved caspase-11 (top) and GSDMD (bottom) were quantified and compared to that of β-actin (B). Cell culture supernatants were collected for an ELISA assay to determine the secreted IL-1β protein levels 16 h post infection (C). Cytotoxicity was determined by LDH release assay in cell culture supernatants 16 h post infection (D). Cell viability was determined by an ATP quantification assay in cell pellets 16 h post infection (E). (F) Wild-type BMDM cells were primed overnight with 1 μg/ml Pam3CSK4 and incubated with E. coli or ΔRetS PAO1 at an MOI of 30 and 20 μg/ml OMVs for 2 h. Cells were then supplemented with fresh medium containing 100 μg/ml Gentamycin. Cells were subjected to cellular component fractionation and immunoblotted as indicated 16 h post infection. (G–J) Aim2–/– BMDM cells were primed overnight with 1 μg/ml Pam3CSK4, followed by transfection of 2 μg/ml LPS and 2 μg/ml extracted BMDM mitochondrial DNA using DOTAP with or without the incubation of ΔRetS PAO1 at an MOI of 30 for 2 h. Cells were then supplemented with fresh medium containing 100 μg/ml Gentamycin. Cells were lysed and immunoblotted 16 h post infection (G). Cell culture supernatants were collected for an ELISA assay to determine the secreted IL-1β protein levels 16 h post infection (H). Cytotoxicity was determined by LDH release assay in cell culture supernatants 16 h post infection (I). Cell viability was determined by an ATP quantification assay in cell pellets 16 h post infection (J). (K–N) Casp11–/–;Aim2–/– BMDM cells were primed overnight with 1 μg/ml Pam3CSK4, followed by transfection of 2 μg/ml LPS and 2 μg/ml extracted BMDM mitochondrial DNA using DOTAP with or without the incubation of ΔRetS PAO1 at an MOI of 30 for 2 h. Cells were then supplemented with fresh medium containing 100 μg/ml Gentamycin. Cells were lysed and immunoblotted 16 h post infection (K). Cell culture supernatants were collected for an ELISA assay to determine the secreted IL-1β protein levels 16 h post infection (L). Cytotoxicity was determined by LDH release assay in cell culture supernatants 16 h post infection (M). Cell viability was determined by an ATP quantification assay in cell pellets 16 h post infection (N). Data were shown as means±SD. Experiments were repeated three times with similar results.

P. aeruginosa VgrG2b is cleaved by caspase-11.

(A) Screening strategy for identifying caspase-11 substrates in P. aeruginosa. Briefly, P. aeruginosa cDNAs were cloned into GFP vectors containing a mitochondrial localization signal. RFP vectors with a mitochondrial localization signal were used as transfection controls. HEK293T cells were co-transfected with these vectors and caspase-11. Cells losing GFP signals on mitochondria were sequenced. (B, C) Plasmids encoding VgrG2b and mouse caspase-11 p22/p10 (B) or human caspase-4 p22/p10 (C) were co-transfected into HEK293T cells for 24 h, followed by immunoblotting with antibodies against the indicated proteins. (D) Wild-type BMDM cells were primed overnight with 1 μg/ml Pam3CSK4 and incubated with ΔRetS PAO1 or VgrG2b-Myc knockin ΔRetS PAO1 at an MOI of 30 and 20 μg/ml OMVs for 2 h. Cells were then supplemented with fresh medium containing 100 μg/ml Gentamycin. Cells were lysed and immunoblotted as indicated 16 h post infection. (E) Wild-type BMDM cells were primed overnight with 1 μg/ml Pam3CSK4 and incubated with VgrG2b-Myc knockin ΔRetS PAO1 at an MOI of 30 and 20 μg/ml OMVs for 2 h. Cells were then supplemented with fresh medium containing 100 μg/ml Gentamycin. Cells were fixed and stained with antibodies against Myc or caspase-11 16 h post infection. Nucleus was stained with DAPI. Scale bar, 5 μm. (F) Casp11+/+ and Casp11–/– BMDM cells were primed overnight with 1 μg/ml Pam3CSK4, followed by transfection of 2 μg/ml LPS using DOTAP with or without the incubation of VgrG2b-Myc knockin ΔRetS PAO1 at an MOI of 30 for 2 h. Cells were then supplemented with fresh medium containing 100 μg/ml Gentamycin. Cells were lysed and immunoblotted 16 h post infection. (G) Casp11–/– BMDM cells were infected with lentiviruses encoding a control vector of caspase-11. Cells were primed overnight with 1 μg/ml Pam3CSK4, followed by transfection of 2 μg/ml LPS using DOTAP with or without the incubation of VgrG2b-Myc knockin ΔRetS PAO1 at an MOI of 30 for 2 h. Cells were then supplemented with fresh medium containing 100 μg/ml Gentamycin. Cells were lysed and immunoblotted as indicated 16 h post infection. (H) Casp11–/– BMDM cells were infected with lentiviruses encoding wild-type or mutant caspase-11. Cells were primed overnight with 1 μg/ml Pam3CSK4, followed by transfection of 2 μg/ml LPS using DOTAP with or without the incubation of VgrG2b-Myc knockin ΔRetS PAO1 at an MOI of 30 for 2 h. Cells were then supplemented with fresh medium containing 100 μg/ml Gentamycin. Cells were lysed and immunoblotted as indicated 16 h post infection. (I) Human macrophages were primed overnight with 1 μg/ml Pam3CSK4, followed by transfection of 2 μg/ml LPS using DOTAP with or without the incubation of VgrG2b-Myc knockin ΔRetS PAO1 at an MOI of 30 for 2 h. Cells were then supplemented with fresh medium containing 100 μg/ml Gentamycin. Cells were lysed and immunoblotted as indicated 16 h post infection. (J–M) Wild-type BMDM cells were primed overnight with 1 μg/ml Pam3CSK4, followed by incubation of VgrG2b-Myc knockin (KI) or knockout (KO) ΔRetS PAO1 at an MOI of 30 and 20 μg/ml OMVs for 2 h. Cells were then supplemented with fresh medium containing 100 μg/ml Gentamycin. Cells were lysed and immunoblotted as indicated 16 h post infection (J). Cell culture supernatants were collected for an ELISA assay to determine the secreted IL-1β protein levels 16 h post infection (K). Cytotoxicity was determined by LDH release assay in cell culture supernatants 16 h post infection (L). Cell viability was determined by an ATP quantification assay in cell pellets 16 h post infection (M). Data were shown as means±SD. Experiments were repeated three times with similar results.

Caspase-11 cleaves VgrG2b at D883.

(A) Plasmids encoding VgrG2b and mutant caspase-11 p22/p10 were co-transfected into HEK293T cells for 24 h, followed by immunoprecipitation with a control IgG or antibody against Myc. Precipitates were immunoblotted as indicated. (B) Plasmids encoding caspase-11 p22/p10 and VgrG2b mutants were co-transfected into HEK293T cells for 24 h, followed by immunoblotting with antibodies against the indicated proteins. (C) Plasmids encoding caspase-4 p22/p10 and VgrG2b variants were co-transfected into HEK293T cells for 24 h, followed by immunoblotting with antibodies against the indicated proteins. (D) Plasmids encoding VgrG2a-N-VgrG2b-C chimera and caspase-11 p22/p10 were co-transfected into HEK293T cells for 24 h, followed by immunoblotting with antibodies against the indicated proteins. (E) Plasmids encoding VgrG2b C-terminus and mutant caspase-11 p22/p10 were co-transfected into HEK293T cells for 24 h, followed by immunoprecipitation with a control IgG or antibody against Myc. Precipitates were immunoblotted as indicated. (F) Recombinant VgrG2b were incubated with caspase-11 p22/p10 for 4 h, followed by immunoblotting with antibodies against the indicated proteins. (G) Wild-type BMDM cells were primed overnight with 1 μg/ml Pam3CSK4, followed by transfection of 2 μg/ml LPS using DOTAP with or without the incubation of VgrG2b wild-type (KI) or mutant (D883A) knockin ΔRetS PAO1 at an MOI of 30 for 2 h. Cells were then supplemented with fresh medium containing 100 μg/ml Gentamycin. Cells were lysed and immunoblotted 16 h post infection. (H–L) Wild-type BMDM cells were primed overnight with 1 μg/ml Pam3CSK4, followed by transfection of 2 μg/ml LPS using DOTAP with or without the incubation of VgrG2b wild-type (KI) or mutant (D883A) knockin ΔRetS PAO1 at an MOI of 30 for 2 h. Cells were then supplemented with fresh medium containing 100 μg/ml Gentamycin. Cells were lysed and immunoblotted as indicated 16 h post infection (H). Band intensities of cleaved caspase-11 (top) and GSDMD (bottom) were quantified and compared to that of β-actin (I). Cell culture supernatants were collected for an ELISA assay to determine the secreted IL-1β protein levels 16 h post infection (J). Cytotoxicity was determined by LDH release assay in cell culture supernatants 16 h post infection (K). Cell viability was determined by an ATP quantification assay in cell pellets 16 h post infection (L). Data were shown as means±SD. Experiments were repeated three times with similar results.

VgrG2b C-terminus interacts with NLRP3.

(A) Yeast two-hybrid screening was performed using VgrG2b C-terminus as bait and a mouse bone marrow cDNA library was screened. The interaction strength between bait and preys were visualized by X-α-gal assays. (B, C) Plasmids encoding VgrG2b C-terminus (B) or full-length (C) and NLRP3 were co-transfected into HEK293T cells for 24 h, followed by immunoprecipitation with a control IgG or antibody against Myc. Precipitates were immunoblotted as indicated. (D) VgrG2b C-terminus proteins were introduced into BMDM cells with the help of cell-penetrating peptides (CPP) for 6 h. Cells were lysed and immunoprecipitated with a control IgG or antibody against Myc. Precipitates were immunoblotted as indicated. (E) BMDM cells were primed overnight with 1 μg/ml Pam3CSK4, followed by transfection of 2 μg/ml LPS using DOTAP with the incubation of VgrG2b knockin (KI) ΔRetS PAO1 at an MOI of 30 for 2 h. Cells were then supplemented with fresh medium containing 100 μg/ml Gentamycin. Cells were lysed and immunoprecipitated with a control IgG or antibody against NLRP3 16 h post infection. Precipitates were immunoblotted as indicated. (F) BMDM cells were primed overnight with 1 μg/ml Pam3CSK4, followed by transfection of 2 μg/ml LPS using DOTAP with the incubation of VgrG2b knockin (KI) ΔRetS PAO1 at an MOI of 30 for 2 h. Cells were then supplemented with fresh medium containing 100 μg/ml Gentamycin. Cells were fixed and stained with antibodies against Myc and NLRP3 16 h post infection. Nucleus was stained with DAPI. Scale bar, 5 μm. (G) Scheme for NLRP3 truncations. (H, I) Plasmids encoding VgrG2b C-terminus and NLRP3 truncations were co-transfected into HEK293T cells for 24 h, followed by immunoprecipitation with a control IgG or antibody against FLAG. Precipitates were immunoblotted as indicated. (J) Casp11+/+ and Casp11–/– BMDM cells were primed overnight with 1 μg/ml Pam3CSK4, followed by transfection of 2 μg/ml LPS using DOTAP with the incubation of VgrG2b-Myc knockin (KI) ΔRetS PAO1 at an MOI of 30 for 2 h. Cells were then supplemented with fresh medium containing 100 μg/ml Gentamycin. Cells were lysed and immunoprecipitated with a control IgG or antibody against NLRP3 16 h post infection. Precipitates were immunoblotted as indicated. Data were shown as means±SD. Experiments were repeated three times with similar results.

VgrG2b C-terminus suppresses the NLRP3 inflammasome.

(A) Plasmids encoding VgrG2b C-terminus and the indicated murine proteins were co-transfected into HEK293T cells for 24 h, followed by immunoblotting with antibodies against the indicated proteins. (B) Wild-type BMDM cells were infected with lentiviruses encoding a control vector or VgrG2b C-terminus and primed with 1 μg/ml LPS for 3 h, followed by stimulation of 10 μM nigericin for 30 min, 0.5 μM gramicidin for 1 h and 2 mM ATP for 30 min. Cells were lysed and immunoblotted as indicated. (C–G) Wild-type BMDM cells were primed overnight with 1 μg/ml Pam3CSK4, followed by transfection of 2 μg/ml LPS using DOTAP with or without the transfection of VgrG2b C-terminus proteins using cell-penetrating peptides (CPP) for 2 h. Cells were then supplemented with fresh medium. Cells were lysed and immunoblotted 16 h post transfection (C). Band intensities of cleaved caspase-11 (top) and GSDMD (bottom) were quantified and compared to that of β-actin (D). Cell culture supernatants were collected for an ELISA assay to determine the secreted IL-1β protein levels 16 h post infection (E). Cytotoxicity was determined by LDH release assay in cell culture supernatants 16 h post infection (F). Cell viability was determined by an ATP quantification assay in cell pellets 16 h post infection (G). (H, I) Wild-type BMDM cells were primed overnight with 1 μg/ml Pam3CSK4, followed by transfection of 2 μg/ml LPS using DOTAP with or without the transfection of VgrG2b C-terminus proteins using cell-penetrating peptides (CPP) for 2 h. Cells were then supplemented with fresh medium. Cells were lysed and immunoprecipitated with a control IgG or antibody against NLRP3 16 h post transfection. Precipitates were immunoblotted with antibody against Nur77 (H) or NEK7 (I). (J) Plasmids encoding VgrG2b C-terminus, NEK7 and NLRP3 were co-transfected into HEK293T cells for 24 h, followed by immunoprecipitation with a control IgG or antibody against NLRP3. Precipitates were immunoblotted as indicated. Data were shown as means±SD. Experiments were repeated three times with similar results.

P. aeruginosa suppresses NLRP3 inflammasome in vivo.

(A–E) Wild-type mice were intraperitoneally challenged with poly(I:C) at a dose of 2 mg/kg body weight for 6 h and then intranasally infected with 1x109 cfu of VgrG2b (KI) or mutant (D883A) knockin ΔRetS PAO1, followed by survival calculation at the indicated days (A). Serum protein levels of IL-1β (B), TNF-α (C) and IL-6 (D) were examined through ELISA assays 24 h post infection. Bacterial load was examined in mouse lung tissues 4 days post infection (E). (F–J) Wild-type mice were intraperitoneally challenged with poly(I:C) at a dose of 2 mg/kg body weight for 6 h and then intranasally infected with 1x109 cfu of VgrG2b (KI) or mutant (D883A) knockin ΔRetS PAO1. Bronchoalveolar lavage fluids (BALF) were collected 16 h post infection. Cells were immunoblotted as indicated (F). Cell viability was also determined by an ATP quantification assay in cell pellets (G). Protein levels of IL-1β (H), TNF-α (I) and IL-6 (J) in BALF supernatants were examined through ELISA assays. (K–O) Wild-type mice were intraperitoneally challenged with poly(I:C) at a dose of 2 mg/kg body weight for 6 h and then intranasally infected with 1x109 cfu of ΔRetS PAO1 and administrated with 20 μg TAT sequence containing VgrG2b C-terminus peptide, followed by survival calculation at the indicated days (K). Serum protein levels of IL-1β (L), TNF-α (M) and IL-6 (N) were examined through ELISA assays 24 h post infection. Bacterial load was examined in mouse lung tissues 4 days post infection (O). Data were shown as means±SD. Experiments were repeated three times with similar results.