Figure S5.
CipB is essential for C. violaceum infection. (A) Genomic organization of the cipB locus in C. violaceum (12472; ATCC). Red arrow represents cipB site. (B–E) CipB is essential for C. violaceum infection. CipB is required for the invasion ability of C. violaceum (B). HeLa GSDMD−/− cells were infected with the indicated C. violaceum strains (MOI = 10), followed by gentamicin protection assays (B). CipB is required for C. violaceum-induced mitophagy (C–E). 293T cells were infected with the indicated C. violaceum strains for 4 h (C), or 293T cells stably expressing GFP-LC3B (cyan) were assayed (D), followed by immunoblotting (C) or fluorescence imaging (D). C. violaceum (magenta) was labeled with anti–C. violaceum antibody. Scale bar, 10 μm. Quantification of the number of GFP-LC3B puncta (E). About 30 cells were counted and analyzed for each biological replicate. Data are shown as mean ± SEM. C.v. ΔcipB, C. violaceum mutant strain with gene deletion of cipB; C.v. ΔcipB+pCipB, C.v. ΔcipB complemented with a CipB expression vector. (F) Confirmation of the invasion ability of different C. violaceum strains. HeLa GSDMD−/− cells were treated as in B. All data are shown from three independent replicates. (G–I) CipB is required for C. violaceum–induced mitophagy in livers. Mice were infected with C.v. WT (n = 4) or C.v. ΔcipB (n = 4) strains for 10 h. Immunoblotting of LC3B-II and Tom20 in the livers of C57BL/6N mice (G). Confocal microscopy analysis of LC3B puncta (green) in livers (H). Nuclei (blue) were counterstained with DAPI. Scale bar, 20 μm; inserts, 5 µm. Quantification of the number of LC3B puncta per liver cell (I). Mean ± SEM, n = 30 cells in each group. (J and K) CipB is essential for bacterial loads in the liver (J)/spleen (K) of infected mice. Mice were infected with C.v. WT (n = 5) or C.v. ΔcipB (n = 5) strains. (L) CipB is essential for C. violaceum virulence. Survival rate of mice after C.v. WT (n = 8) or C.v. ΔcipB (n = 8) strains infection. (M) The complementation of pCipB, pCipB mLIR1&3&4, or CipB-mLIR3&4 in C.v. ΔcipB strain resulted in similar weight loss in infected mice in the acute infection period. Mice were infected intraperitoneally with indicated C. violaceum strains for 24 h. Quantification of mice weight reduction. ns, not significant; **P < 0.01. (N) The interaction of CipB and LC3C is essential for C. violaceum–induced autophagy in the spleen of infected mice. Spleen tissue was subjected to immunofluorescence by anti-LC3B (green). Nuclei (blue) were counterstained with DAPI. N = 4. Scale bar, 10 μm; inserts, 2.5 µm. Data are representative of three independent experiments and are shown as the mean ± SEM. Unpaired two-sided Student’s t tests (J and K) or Mantel–Cox tests (L) were used to measure significance. P values were determined by one-way ANOVA with Tukey’s test (E, I, and M). **P < 0.01, ***P < 0.001, ****P < 0.0001 Source data are available for this figure: SourceData FS5.

CipB is essential for C. violaceum infection. (A) Genomic organization of the cipB locus in C. violaceum (12472; ATCC). Red arrow represents cipB site. (B–E) CipB is essential for C. violaceum infection. CipB is required for the invasion ability of C. violaceum (B). HeLa GSDMD−/− cells were infected with the indicated C. violaceum strains (MOI = 10), followed by gentamicin protection assays (B). CipB is required for C. violaceum-induced mitophagy (C–E). 293T cells were infected with the indicated C. violaceum strains for 4 h (C), or 293T cells stably expressing GFP-LC3B (cyan) were assayed (D), followed by immunoblotting (C) or fluorescence imaging (D). C. violaceum (magenta) was labeled with anti–C. violaceum antibody. Scale bar, 10 μm. Quantification of the number of GFP-LC3B puncta (E). About 30 cells were counted and analyzed for each biological replicate. Data are shown as mean ± SEM. C.v. ΔcipB, C. violaceum mutant strain with gene deletion of cipB; C.v. ΔcipB+pCipB, C.v. ΔcipB complemented with a CipB expression vector. (F) Confirmation of the invasion ability of different C. violaceum strains. HeLa GSDMD−/− cells were treated as in B. All data are shown from three independent replicates. (G–I) CipB is required for C. violaceum–induced mitophagy in livers. Mice were infected with C.v. WT (n = 4) or C.v. ΔcipB (n = 4) strains for 10 h. Immunoblotting of LC3B-II and Tom20 in the livers of C57BL/6N mice (G). Confocal microscopy analysis of LC3B puncta (green) in livers (H). Nuclei (blue) were counterstained with DAPI. Scale bar, 20 μm; inserts, 5 µm. Quantification of the number of LC3B puncta per liver cell (I). Mean ± SEM, n = 30 cells in each group. (J and K) CipB is essential for bacterial loads in the liver (J)/spleen (K) of infected mice. Mice were infected with C.v. WT (n = 5) or C.v. ΔcipB (n = 5) strains. (L) CipB is essential for C. violaceum virulence. Survival rate of mice after C.v. WT (n = 8) or C.v. ΔcipB (n = 8) strains infection. (M) The complementation of pCipB, pCipB mLIR1&3&4, or CipB-mLIR3&4 in C.v. ΔcipB strain resulted in similar weight loss in infected mice in the acute infection period. Mice were infected intraperitoneally with indicated C. violaceum strains for 24 h. Quantification of mice weight reduction. ns, not significant; **P < 0.01. (N) The interaction of CipB and LC3C is essential for C. violaceum–induced autophagy in the spleen of infected mice. Spleen tissue was subjected to immunofluorescence by anti-LC3B (green). Nuclei (blue) were counterstained with DAPI. N = 4. Scale bar, 10 μm; inserts, 2.5 µm. Data are representative of three independent experiments and are shown as the mean ± SEM. Unpaired two-sided Student’s t tests (J and K) or Mantel–Cox tests (L) were used to measure significance. P values were determined by one-way ANOVA with Tukey’s test (E, I, and M). **P < 0.01, ***P < 0.001, ****P < 0.0001 Source data are available for this figure: SourceData FS5.

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal