Figure 7.
The mitophagy receptor function of CipB is essential for C. violaceum–induced mitophagy and pathogenesis during animal infection. (A and B) Expression of CipB, CipB-mLIR1&3&4 or CipB-mLIR3&4 restored the invasive ability of C. violaceum ΔcipB strain. (A) Invasion rates and intracellular growth were determined by CFU assays. (B) Quantification of the percentage of invasion rate. CipB mLIR 1&3&4: CipB simultaneously mutated LIR1, LIR3, and LIR4; CipB mLIR3&4: CipB simultaneously mutated LIR3 and LIR4. ΔcipB+pVector, ΔcipB complemented with an empty vector; ΔcipB+pCipB, ΔcipB complemented with a CipB expression vector; ΔcipB+pCipB mLIR1&3&4, ΔcipB complemented with CipB mLIR 1&3&4; ΔcipB+pCipB mLIR3&4, ΔcipB complemented with CipB mLIR3&4. The abbreviations and symbols used in this article have consistent meanings throughout. (C) Immunoblot analysis of Flag-LC3C-II in 293T cells infected indicated strains (MOI = 5) for 4 h. WT, C. violaceum WT; ΔcipB, C. violaceum mutant strain with gene deletion of CipB. (D) Co-IP analysis of Flag-CipB with RFP-tagged mouse ATG8s protein (mATG8s). Cells were co-transfected with Flag-CipB and RFP-mATG8s for 14 h. Cell lysates were immunoprecipitated with anti-FLAG M2 affinity gel. The immunoprecipitate was eluted with SDS loading buffer and analyzed by immunoblotting. (E–G) Levels of LC3B activation in the liver/spleen of mice. Mice were intraperitoneally injected with 12 × 106 CFU indicated strains for 24 h. Liver/spleen tissues were analyzed by immunoblot (E) or immunofluorescence (F). n = 3 (liver), n = 4 (spleen). LC3B (green) were stained with anti-LC3B antibody. Nuclei (blue) were stained with DAPI. The mean number of LC3B puncta in liver/spleen cells was determined based on the analysis of ∼30 cells per biological replicate (G). Data are shown as mean ± SEM. (H and I) Level of Tom20 in liver tissue in mice (n = 4) as treated in E. (I) shows quantification of levels of Tom20. (J–L) Effects of CipB on C. violaceum infection in mice. C57BL/6N mice were infected intraperitoneally with 12 × 106 CFU indicated C. violaceum strains. Bacterial load in the liver (J) and spleen (K) was counted, and daily survival rate was calculated (L). The data are representative of three independent experiments. P values were determined by one-way ANOVA with Tukey’s test. (B, G, and I–K). P values were determined by Mantel–Cox tests (L). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Source data are available for this figure: SourceData F7.

The mitophagy receptor function of CipB is essential for C. violaceum–induced mitophagy and pathogenesis during animal infection. (A and B) Expression of CipB, CipB-mLIR1&3&4 or CipB-mLIR3&4 restored the invasive ability of C. violaceum ΔcipB strain. (A) Invasion rates and intracellular growth were determined by CFU assays. (B) Quantification of the percentage of invasion rate. CipB mLIR 1&3&4: CipB simultaneously mutated LIR1, LIR3, and LIR4; CipB mLIR3&4: CipB simultaneously mutated LIR3 and LIR4. ΔcipB+pVector, ΔcipB complemented with an empty vector; ΔcipB+pCipB, ΔcipB complemented with a CipB expression vector; ΔcipB+pCipB mLIR1&3&4, ΔcipB complemented with CipB mLIR 1&3&4; ΔcipB+pCipB mLIR3&4, ΔcipB complemented with CipB mLIR3&4. The abbreviations and symbols used in this article have consistent meanings throughout. (C) Immunoblot analysis of Flag-LC3C-II in 293T cells infected indicated strains (MOI = 5) for 4 h. WT, C. violaceum WT; ΔcipB, C. violaceum mutant strain with gene deletion of CipB. (D) Co-IP analysis of Flag-CipB with RFP-tagged mouse ATG8s protein (mATG8s). Cells were co-transfected with Flag-CipB and RFP-mATG8s for 14 h. Cell lysates were immunoprecipitated with anti-FLAG M2 affinity gel. The immunoprecipitate was eluted with SDS loading buffer and analyzed by immunoblotting. (E–G) Levels of LC3B activation in the liver/spleen of mice. Mice were intraperitoneally injected with 12 × 106 CFU indicated strains for 24 h. Liver/spleen tissues were analyzed by immunoblot (E) or immunofluorescence (F). n = 3 (liver), n = 4 (spleen). LC3B (green) were stained with anti-LC3B antibody. Nuclei (blue) were stained with DAPI. The mean number of LC3B puncta in liver/spleen cells was determined based on the analysis of ∼30 cells per biological replicate (G). Data are shown as mean ± SEM. (H and I) Level of Tom20 in liver tissue in mice (n = 4) as treated in E. (I) shows quantification of levels of Tom20. (J–L) Effects of CipB on C. violaceum infection in mice. C57BL/6N mice were infected intraperitoneally with 12 × 106 CFU indicated C. violaceum strains. Bacterial load in the liver (J) and spleen (K) was counted, and daily survival rate was calculated (L). The data are representative of three independent experiments. P values were determined by one-way ANOVA with Tukey’s test. (B, G, and I–K). P values were determined by Mantel–Cox tests (L). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Source data are available for this figure: SourceData F7.

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