Article Figures & Data
Figures
- Figure 1. Irgb6 significantly contributes to IFN-γ–induced cell-autonomous T. gondii killing.
(A) Schematic representation of the gene-targeting strategy for mouse Irgb6* and Irgb6 locus by Cas9-mediated genome editing. (B) Western blot analysis of the indicated protein expressions in WT and Irgb6 KO MEFs after IFN-γ stimulation or not. (C, D) Survival rate of T. gondii in the presence of IFN-γ stimulation relative to that in the non–IFN-γ–treated control by luciferase analysis at 24 h postinfection. The graphs show the mean ± SEM in four independent experiments. Two-tailed t tests were used: ***P < 0.001 versus WT, Irgb10 KO MEFs or Irgm1/m3 DKO MEFs. (E, F) Electron microscope images of T. gondii–infected WT (E) and Irgb6 KO (F) MEFs at 4 h postinfection in the presence of IFN-γ stimulation. The images are representative of three independent experiments. Red arrowheads indicate membrane blebbing. Scale bars, 1 μm.
- Figure S1. Sequence analysis of both Irgb6* and Irgb6 genomic DNA from Irgb6-deficient MEFs.
(A, B) An 891-bp coding region deletion (position 172-1062) in Irgb6* (A) and Irgb6 (B) gene is shown in red. Yellow highlights indicate the coding region of Irgb6 genes.
- Figure S2. Conserved expression of Irgb5-b4 tandem (decoy) gene in Irgb6 KO MEFs.
(A) PCR products of Irgb5-b4 and Irgb5*-b3 tandem (decoy) genes were amplified from cDNA of IFN-γ–treated WT and Irgb6 KO MEFs. (B) Chromatograms of DNA sequence analysis of the 3′ coding region of Irgb5-b4 and Irgb5*-b3 tandem (decoy) genes. Arrows indicate Irgb4 DNA sequence at position 2208T, 2286A, and 2307T. (C) Sequence data of the complete coding region (1–2,535 bp) of Irgb5-b4 tandem (decoy) gene from IFN-γ–treated Irgb6 KO MEFs. (D) PCR products of the 3′ untranslated region of Irgb4 gene in WT and Irgb6 KO MEFs.
- Figure 2. Irgb6 recruits other IFN-γ–inducible GTPases and ubiquitin on the T. gondii PVM.
(A, B, C, D, E, F, G) Confocal microscope images (left) and the graphs (right) represent the localization of Irga6 (A), Gbp1 (B), Gbp2 (C), Gbp1-5 (D), Irgb10 (E), p62 (F), and ubiquitin (G) (red) to T. gondii vacuoles (green), and DAPI (blue) at 4 h postinfection in IFN-γ–treated WT and Irgb6 KO MEFs. (H) Confocal microscope images represent the colocalization of Irgb6 (red) and ubiquitin (cyan blue) to T. gondii vacuoles (green), and DAPI (blue) at 4 h postinfection in IFN-γ–treated WT MEFs. (I) The pie chart represents the mean ± SEM of Irgb6 and ubiquitin double- or Irgb6 single- or ubiquitin single-positive vacuoles in IFN-γ–treated WT MEFs. All graphs show the mean ± SEM in three or four independent experiments. All images are representative of three or four independent experiments. White arrowheads indicate colocalization. Scale bars, 5 μm. Two-tailed t tests were used: *P < 0.05, **P < 0.01, and ***P < 0.001.
- Figure S3. Irgb6 involves in the cell-autonomous immunity of T. gondii in BMDM.
(A) Quantification analysis for Irga6-, Gbp1-, Gbp2-, Gbp1-5-, ubiquitin-, or p62-positive T. gondii vacuoles by confocal microscopy at 4 h postinfection in IFN-γ–treated WT, Irgb6 KO, or Irgb6/b10 DKO MEFs. (B) The parasite survival rate in WT and Irgb6 KO BMDMs by luciferase analysis at 24 h postinfection in the presence or absence of IFN-γ stimulation. (C) Quantification analysis for the infection rate of parasites in WT and Irgb6 KO BMDMs at 4 and 24 h postinfection in the presence of IFN-γ stimulation. (D) Quantification analysis for Irgb10-, Irga6-, Gbp1-, Gbp2-, Gbp1-5-, ubiquitin-, or p62-positive T. gondii vacuoles by confocal microscopy at 4 h postinfection in IFN-γ–treated WT and Irgb6 KO BMDMs. (A, B, C, D) All graphs are means ± SEM from three independent experiments. *P < 0.05, **P < 0.01, and ***P < 0.001 from the two-tailed t test. NS, not significant.
- Figure 3. Regulator IRG proteins, but not other effector IRG proteins, are required for loading of Irgb6 and ubiquitin on T. gondii PVM.
(A, B) Confocal microscope images (left) and the graphs (right) represent the localization of Irgb6 (A) and ubiquitin (B) (red) to T. gondii vacuoles (green), and DAPI (blue) at 4 h postinfection in IFN-γ–treated WT and Irga6 KO MEFs. (C, D) Confocal microscope images (left) and the graphs (right) represent the localization of Irgb6 (C) and ubiquitin (D) (red) to T. gondii vacuoles (green), and DAPI (blue) at 4 h postinfection in IFN-γ–treated WT and Irgb10 KO MEFs. (E, F) Confocal microscope images (left) and the graphs (right) represent the localization of Irgb6 (E) and ubiquitin (F) (red) to T. gondii vacuoles (green), and DAPI (blue) at 4 h postinfection in IFN-γ–treated WT and Irgm1/m3 DKO MEFs. All graphs show the mean ± SEM in three independent experiments. All images are representative of three independent experiments. White arrowheads indicate colocalization. Scale bars, 5 μm. ND, not detected; NS, not significant.
- Figure S4. The GTP-binding motif of Irgb6 is essential for localization on the PVM and IFN-γ–induced parasite killing activity.
(A, B) The representative images (A) and the ratio (B) of Irgb6-positive parasites by confocal microscopy analysis at 4 h postinfection in IFN-γ–treated WT MEFs reconstituted with EM, K69A-Irgb6-FLAG, S70N-Irgb6-FLAG, or WT-Irgb6-FLAG. Scale bars, 5 μm. (C, D) The representative images (C) and the ratio (D) of Irgb6-positive parasites by confocal microscopy analysis at 4 h postinfection in IFN-γ–treated Irgb6 KO MEFs reconstituted with EM, K69A-Irgb6-FLAG, S70N-Irgb6-FLAG, or WT-Irgb6-FLAG. Scale bars, 5 μm. (E) The parasite survival rate in the presence of IFN-γ stimulation relative to that in the non–IFN-γ–treated control by luciferase analysis at 24 h postinfection in Irgb6 KO MEFs reconstituted with EM, K69A-Irgb6-FLAG, S70N-Irgb6-FLAG, or WT-Irgb6-FLAG. (B, D, E) All graphs show the mean ± SEM in three or four independent experiments. (A, C) All images are representative of three or four independent experiments. (A, C) White arrowheads indicate colocalization. **P < 0.01 and ***P < 0.001 from the two-tailed t test. ND, not detected.
- Figure 4. Irgb6 recognizes PI5P and PS at T. gondii PVM.
(A) The representative image from three independent experiments of PIP strips showing the binding of His-tagged recombinant Irgb6 protein to PI3P, PI4P, PI5P, and PS. (B) Confocal microscope images from two independent experiments of the localization of PS (green) with T. gondii (red), and DAPI (blue) in WT MEFs. Scale bars, 5 μm. (C) Confocal microscope images from three independent experiments of the localization of PIs (green) with T. gondii (red), and DAPI (blue) in WT MEFs. Scale bars, 5 μm. (D, E) Confocal microscope images (D) and the graphs (E) from three independent experiments represent the localization of the indicated HA-tagged probes recognizing each specific PIs (green) with T. gondii (red), and DAPI (blue) in WT MEFs. Scale bars, 5 μm. White arrowheads indicate colocalization.
- Figure S5. K275 and R371 in the α-helices of Irgb6 are important for Irgb6 accumulation on the T. gondii PVM.
(A) The representative image from two independent experiments of PIP strip showing that anti-PIP2 antibody recognizes PI3P, PI4P, PI5P, PI(3,5)P2, PI(3,4,5)P3, and PA. (B) The ratio of FLAG-tagged WT Irgb6-positive parasites or FLAG-tagged the point mutants Irgb6-positive parasites by confocal microscopy analysis at 4 h postinfection in Irgb6 KO MEFs reconstituted with the indicated proteins in the presence of IFN-γ stimulation. The graph shows the mean ± SEM in three independent experiments. ND, not detected.
- Figure 5. Basic amino acids in the C-terminal α-helices of Irgb6 are required for recognition of phospholipids and immune responses against T. gondii.
(A) Schematic representation of WT Irgb6 and C-terminal α-helices–deleted mutants of Irgb6. (B, C) The ratio (B) and representative images (C) of colocalization of FLAG-tagged WT Irgb6 or FLAG-tagged C-terminal deletion mutants Irgb6 (red) to T. gondii vacuoles (green) by confocal microscopy analysis at 4 h postinfection in Irgb6 KO MEFs reconstituted with the indicated proteins in the presence of IFN-γ stimulation. Scale bars, 5 μm. (D) The helical wheel projection of the two α-helical regions of Irgb6 and alignment according to its amphiphilic properties. Yellow indicates nonpolar amino acids, purple indicates acidic amino acids, blue indicates polar amino acids, and green indicates basic amino acids. (E, F) The ratio (E) and representative images (F) of colocalization of FLAG-tagged WT Irgb6 or FLAG-tagged the point mutants Irgb6 (red) to T. gondii vacuoles (green) by confocal microscopy analysis at 4 h postinfection in Irgb6 KO MEFs reconstituted with the indicated proteins in the presence of IFN-γ stimulation. Scale bars, 5 μm. (G) The representative images from two independent experiments of PIP strip membranes incubated with His-tagged recombinant WT-Irgb6 or K275A/R371A-Irgb6 protein. (H) Coomassie blue staining of the purified His-tagged recombinant WT-Irgb6 and K275A/R371A-Irgb6 protein. (I) T. gondii survival rate in the presence of IFN-γ stimulation relative to that in the non–IFN-γ–treated control by luciferase analysis at 24 h postinfection in Irgb6 KO MEFs reconstituted with empty (EM), WT-Irgb6, or K275A/R371A-Irgb6. (J) 3D models of the distribution of K275 (red) and R371 (blue) in Irgb6. (B, E, I) All graph shows the mean ± SEM in three independent experiments. (C, F, G, H) All images are representative of two or three independent experiments. (C, F) White arrowheads indicate colocalization. **P < 0.01 and ***P < 0.001 from the two-tailed t test. ND, not detected.
- Figure 6. Irgb6 provides host defense against T. gondii infection in vivo.
(A, B) In vivo bioluminescence imaging (A) and quantification of T. gondii numbers (B) in WT and Irgb6 KO mice on day 3, 5, and 6 postinfection. (C) Parasite numbers in the indicated tissues of the mice by luciferase analysis on day 5 postinfection. (D) Release of proinflammatory cytokines in the peritoneal fluids of T. gondii–infected WT and Irgb6 KO mice on day 5 postinfection. (E) Survival rates of T. gondii–infected WT and Irgb6 KO mice. All images are representative of 5–10 mice per group, and all graphs are means ± SEM from two independent experiments. Two-tailed t tests were used. NS, not significant; *P < 0.05, **P < 0.01, and ***P < 0.001 versus WT mice.
Supplementary Materials
Table S1 Primer list used in this study.
