Figure 2.
Bacterial T3SS effector CipB family triggers mitophagy. (A)C. violaceum induces autophagy depending on Cpi-1/-1a T3SS. 293T cells stably expressing GFP-LC3B (cyan) were infected with the indicated C. violaceum strains (MOI = 5, 3 h). C. violaceum (magenta) was stained with anti–C. violaceum antibody. ∆Cpi-1/-1a, C. violaceum mutant strain with gene deletion of the ATPase CivC of the Cpi-1/-1a T3SS; ∆Cpi-2, C. violaceum mutant strain with gene deletion of the ATPase CsaN of the Cpi-2 T3SS. These abbreviations have the same meaning throughout the article. Scale bar, 10 µm. (B) Quantification of GFP-LC3B puncta as shown in A. Mean ± SEM, n = 25 cells in each group. P values were determined by one-way ANOVA with Tukey’s test. (C) Immunoblot analysis of LC3B-II and p62 in 293T cells after infection with the indicated C. violaceum strains as in A. (D) Immunoblot analyzing Tom20 in 293T cells infected with the indicated C. violaceum strains (MOI = 5) for 5 h. (E) T3SS architectures of C. violaceum. The basal body of the T3SS spans the bacterial inner membrane (IM), peptidoglycan (PG), and outer membrane (OM). The basal body associates with an extracellular needle, which elongates across host cell membrane and forms a channel-like translocon that inserts into the host plasma membrane. (F) Western blot analysis of LC3B-II and p62 in 293T cells transfected with RFP/RFP-CipB or treated with CCCP (10 µM) plus Baf A1 (0.5 µM) for 21 h. (G and H) Confocal microscopy analysis of LC3B puncta in HeLa cells. Cells were transfected with RFP/RFP-CipB (magenta) for 14 h and then were stained with LC3B antibody (yellow). Nuclei (gray) were stained with DAPI. Scale bar, 10 µm. Quantification of LC3B puncta (mean ± SEM, n = 30 cells in each group) in H. (I and J) Immunofluorescence of p62 in HeLa cells transfected with GFP/GFP-CipB (magenta) for 21 h (I). p62 (yellow) was stained with anti-p62 antibody. Nuclei (gray) were stained with DAPI. Scale bar, 10 µm. Quantification of the number of p62 puncta (J). Data are shown as mean ± SEM (n = 30 cells in each group). (K and L) Confocal microscopy analysis of LC3B puncta in HeLa cells. Cells were transfected with RFP, RFP-CipB, RFP-SipB, or RFP-IpaB (magenta), respectively, for 14 h. LC3B puncta (yellow) were detected by immunofluorescence (K). Nuclei (gray) were stained with DAPI. Scale bar, 10 µm. Quantification of the number of LC3B puncta per cell (L). Data are shown as mean ± SEM (n = 30 cells in each group). P values were determined by one-way ANOVA with Dunnett’s test. (M and N) Confocal microscopy analysis of mitochondrial morphology in HeLa cells; cells were treated with CCCP (10 µM) for 6 h or transfected RFP, RFP-CipB (magenta) for 14 h (M). Mitochondria (yellow) were stained with anti-Tom20 antibody. Scale bar, 10 µm. The percentage of cells displaying mitochondrial fragmentation was quantified by analyzing 100 randomly selected transfection-positive cells (N). Mean ± SEM, n = 3 independent experiments. (O and P) 293T cells expressing GFP-LC3B (magenta) were transfected with RFP, FUNDC1-RFP, or RFP-CipB (yellow) for 14 h. Cells were stained with anti-Tom20 antibody (cyan) and analyzed by confocal microscopy. The white arrows were indicating the LC3B puncta colocalizing or contacting with Tom20 (O). Scale bar, 10 μm. Quantification of the number of the GFP-LC3B puncta colocalizing or contacting with Tom20 in a cell (P). n = 30 cells; data are presented as mean ± SEM (n = 30), and ****P < 0.001. P values were determined by one-way ANOVA with Dunnett’s test. (Q and R) CipB induces mitophagy in 293T cells. Cells were transfected with RFP or RFP-CipB for 21 h. The mitochondrial proteins (Tom20, Tim23, and HSP60) and p62 were detected by western blotting (Q). Quantification of normalized Tom20, Tim23, HSP60, and p62 (R). Data are represented as mean ± SEM (n = 3, three independent experiments). (S)ATG5 deficiency blocks CipB-induced mitophagy in 293T cells. WT 293T cells and ATG5-knockout 293T cells were transfected with Flag-BFP or Flag-CipB for 21 h, respectively. The mitochondrial proteins (Tom20 and Tim23) were detected by western blotting. (T and U) Immunoblot analysis of Tom20 in 293T cells transfected with RFP, RFP-CipB, RFP-SipB, and RFP-IpaB for 21 h (T). Quantification of normalized Tom20 (U). Data are represented as mean ± SEM (n = 3, three independent experiments). P values were determined by one-way ANOVA with Dunnett’s test. All data are determined for triplicates of three independent experiments. Unpaired two-sided Student’s t tests were used to measure significance (H, J, N, and R). ns, not significant; **P < 0.01; ***P < 0.001; ****P < 0.0001. Source data are available for this figure: SourceData F2.

Bacterial T3SS effector CipB family triggers mitophagy. (A) C. violaceum induces autophagy depending on Cpi-1/-1a T3SS. 293T cells stably expressing GFP-LC3B (cyan) were infected with the indicated C. violaceum strains (MOI = 5, 3 h). C. violaceum (magenta) was stained with anti–C. violaceum antibody. ∆Cpi-1/-1a, C. violaceum mutant strain with gene deletion of the ATPase CivC of the Cpi-1/-1a T3SS; ∆Cpi-2, C. violaceum mutant strain with gene deletion of the ATPase CsaN of the Cpi-2 T3SS. These abbreviations have the same meaning throughout the article. Scale bar, 10 µm. (B) Quantification of GFP-LC3B puncta as shown in A. Mean ± SEM, n = 25 cells in each group. P values were determined by one-way ANOVA with Tukey’s test. (C) Immunoblot analysis of LC3B-II and p62 in 293T cells after infection with the indicated C. violaceum strains as in A. (D) Immunoblot analyzing Tom20 in 293T cells infected with the indicated C. violaceum strains (MOI = 5) for 5 h. (E) T3SS architectures of C. violaceum. The basal body of the T3SS spans the bacterial inner membrane (IM), peptidoglycan (PG), and outer membrane (OM). The basal body associates with an extracellular needle, which elongates across host cell membrane and forms a channel-like translocon that inserts into the host plasma membrane. (F) Western blot analysis of LC3B-II and p62 in 293T cells transfected with RFP/RFP-CipB or treated with CCCP (10 µM) plus Baf A1 (0.5 µM) for 21 h. (G and H) Confocal microscopy analysis of LC3B puncta in HeLa cells. Cells were transfected with RFP/RFP-CipB (magenta) for 14 h and then were stained with LC3B antibody (yellow). Nuclei (gray) were stained with DAPI. Scale bar, 10 µm. Quantification of LC3B puncta (mean ± SEM, n = 30 cells in each group) in H. (I and J) Immunofluorescence of p62 in HeLa cells transfected with GFP/GFP-CipB (magenta) for 21 h (I). p62 (yellow) was stained with anti-p62 antibody. Nuclei (gray) were stained with DAPI. Scale bar, 10 µm. Quantification of the number of p62 puncta (J). Data are shown as mean ± SEM (n = 30 cells in each group). (K and L) Confocal microscopy analysis of LC3B puncta in HeLa cells. Cells were transfected with RFP, RFP-CipB, RFP-SipB, or RFP-IpaB (magenta), respectively, for 14 h. LC3B puncta (yellow) were detected by immunofluorescence (K). Nuclei (gray) were stained with DAPI. Scale bar, 10 µm. Quantification of the number of LC3B puncta per cell (L). Data are shown as mean ± SEM (n = 30 cells in each group). P values were determined by one-way ANOVA with Dunnett’s test. (M and N) Confocal microscopy analysis of mitochondrial morphology in HeLa cells; cells were treated with CCCP (10 µM) for 6 h or transfected RFP, RFP-CipB (magenta) for 14 h (M). Mitochondria (yellow) were stained with anti-Tom20 antibody. Scale bar, 10 µm. The percentage of cells displaying mitochondrial fragmentation was quantified by analyzing 100 randomly selected transfection-positive cells (N). Mean ± SEM, n = 3 independent experiments. (O and P) 293T cells expressing GFP-LC3B (magenta) were transfected with RFP, FUNDC1-RFP, or RFP-CipB (yellow) for 14 h. Cells were stained with anti-Tom20 antibody (cyan) and analyzed by confocal microscopy. The white arrows were indicating the LC3B puncta colocalizing or contacting with Tom20 (O). Scale bar, 10 μm. Quantification of the number of the GFP-LC3B puncta colocalizing or contacting with Tom20 in a cell (P). n = 30 cells; data are presented as mean ± SEM (n = 30), and ****P < 0.001. P values were determined by one-way ANOVA with Dunnett’s test. (Q and R) CipB induces mitophagy in 293T cells. Cells were transfected with RFP or RFP-CipB for 21 h. The mitochondrial proteins (Tom20, Tim23, and HSP60) and p62 were detected by western blotting (Q). Quantification of normalized Tom20, Tim23, HSP60, and p62 (R). Data are represented as mean ± SEM (n = 3, three independent experiments). (S)ATG5 deficiency blocks CipB-induced mitophagy in 293T cells. WT 293T cells and ATG5-knockout 293T cells were transfected with Flag-BFP or Flag-CipB for 21 h, respectively. The mitochondrial proteins (Tom20 and Tim23) were detected by western blotting. (T and U) Immunoblot analysis of Tom20 in 293T cells transfected with RFP, RFP-CipB, RFP-SipB, and RFP-IpaB for 21 h (T). Quantification of normalized Tom20 (U). Data are represented as mean ± SEM (n = 3, three independent experiments). P values were determined by one-way ANOVA with Dunnett’s test. All data are determined for triplicates of three independent experiments. Unpaired two-sided Student’s t tests were used to measure significance (H, J, N, and R). ns, not significant; **P < 0.01; ***P < 0.001; ****P < 0.0001. Source data are available for this figure: SourceData F2.

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