Mycobacterium tuberculosis Type VII Secretion System Effectors Differentially Impact the ESCRT Endomembrane Damage Response

EsxH antagonizes ESCRT-III recruitment. (A, B, and C) IF images of CHMP1A (A), CHMP1B (B), and CHMP4B (C) with DsRed-expressing H37Rv or ΔesxH and Δpe5-ppe4 (Δppe) mutants 3 hpi in BMDMs. Images are maximum-intensity projections. Scale bar, 10 μm. Boxed areas in the merged image are shown in higher magnification in the rightmost panel. Mtb, M. tuberculosis. (D to H) Automated image analysis was used to quantify the MFI of CHMP1A (D and E), CHMP1B (F), and CHMP4B (G and H) colocalized with individual bacilli in 5 fields from a 12-mm coverslip. In panels E and H, BMDMs were infected with the PKH-labeled ΔesxH or ΔesxH::esxH mutant for 3 h. Data are means ± SEM from one representative experiment from three (for the WT and ΔesxH mutant in panels A to D, F, and G and the Δpe5-ppe4 mutant in panels A, C, D, and G) or two (for panels E and H and the Δpe5-ppe4 mutant in panels B and F) independent experiments. *, P ≤ 0.05, **, P ≤ 0.01, ***, P ≤ 0.001, and ****, P ≤ 0.0001, Student’s t test. ns, not significant.

EsxG-EsxH alters phagosomal GAL3, ubiquitin, and LAMP1. (A, C, and E) IF images of GAL3 (A), ubiquitin (FK2 antibody) (C), and LAMP1 (E) in BMDMs that were infected with DsRed-expressing H37Rv (WT) or the ΔesxH mutant for 3 h. Images are maximum-intensity projections. Scale bar, 10 μm. Boxed areas in the merged image are shown in higher magnification in the rightmost panel. Mtb, M. tuberculosis. (B, D, and F) Automated image analysis was used to quantify the MFI of GAL3 (B), ubiquitin (D), and LAMP1 (F) colocalized with individual bacilli from 5 fields of a 12-mm coverslip. Data are means ± SEM from one representative experiment from three (A, B, E, and F) or two (C and D) independent experiments. ****, P ≤ 0.0001, Student's t test.

EsxG-EsxH impairs ESCRT-III recruitment to damaged lysosomes. (A and C) HeLa cells transfected with M. tuberculosis (Mtb) EsxG-EsxH (GH) or the vector control were treated with LLOME or the solvent control and stained for CHMP1B (A) or CHMP4A (C). ESCRT-III and EsxG-EsxH are shown in green and red, respectively. EsxG-EsxH was visualized with an anti-EsxG-EsxH monoclonal antibody. (B and D) Automated image analysis was used to quantify the number of CHMP1B (A) or CHMP4A (B) punctae on 30 macrophages per sample. Data are means ± SEM from one representative experiment from at least three independent experiments. ***, P ≤ 0.001, and ****, P ≤ 0.0001, Student's t test. (E) HeLa cells were transfected with Mtb EsxG-EsxH and loaded with SRB. Live-cell imaging was used to visualize SRB before and after addition of LLOME, after which cells were fixed and stained to visualize EsxG-EsxH (green) and CHMP4A (magenta). Image panels of representative cells are shown at the times indicated from each recording. Individual cells are outlined by white dashed lines. Images are maximum-intensity projections. Scale bars, 10 µm. (F and G) Automated image analysis was used to quantify the number of CHMP4A punctae, the reduction in SRB signal, and the MFI of EsxG-EsxH on a per cell basis. The number of CHMP4A punctae and the reduction is SRB signal were compared in cells with an EsxG-EsxH MFI greater than and less than 500. Data are means ± SEM from one representative experiment from at least three independent experiments. ***, P ≤ 0.001, Student’s t test. ns, not significant. (H) HeLa cells transfected with Mtb or M. smegmatis (Msmeg) EsxG-EsxH were treated with LLOME, and CHMP4A and EsxG-EsxH were visualized. Automated image analysis was used to quantify the EsxG-EsxH MFI and the number of CHMP4A punctae in individual cells. The correlation between EsxG-EsxH expression and number of CHMP4A punctae is shown (R value). Data are means ± SEM from four independent experiments in which at least 100 cells were evaluated.

EsxG-EsxH relocalizes in response to membrane damage. HeLa cells transfected with M. tuberculosis (Mtb) EsxG-EsxH were treated with LLOME, and CHMP1B (A) or CHMP4A (B) (red) and EsxG-EsxH (green) was visualized by IF. (C) HeLa cells expressing Mtb EsxG-EsxH were treated with LLOME or the solvent control for 2.5 min. EsxG-EsxH is shown in red. (D) HeLa cells expressing Mtb EsxG-EsxH were treated with LLOME for 1.0 min and then incubated in excess LLOME-free medium for 5 to 30 min as indicated and stained for CHMP4A (green) and EsxG-EsxH (red). (E) Automated image analysis was used to quantify the number of CHMP4A punctae on 30 macrophages per sample from panel D. Data are means ± SEM from one representative experiment from at least two independent experiments. ***, P ≤ 0.001, Student's t test. (F and G) HeLa cells expressing Ms EsxG-EsxH (green) (F) or LacZ (vector control [red]) (G) were treated with LLOME or the solvent control. (H) U2OS cells transfected with Mtb EsxG-EsxH were treated with silica (SiO₂) nanoparticles for 15 min and stained for CHMP4A (red) and EsxG-EsxH (green). White arrows indicate the silica nanoparticles. (A to H) Both EsxG-EsxH and LacZ were detected with anti-V5 antibody. Nuclei were stained with DAPI. Images are maximum-intensity projections. Scale bar 10 μm.

EsxG-EsxH alters HRS localization during infection. (A) BMDMs were uninfected (UI) or infected with H37Rv (WT), the ΔesxH or ΔesxG mutant, or the ΔesxG complemented strain for 3 h, and HRS was examined by IF. Mtb, M. tuberculosis. (B) The number of HRS punctae was quantified. Data are means ± SEM from three independent experiments. ****, P < 0.0001, and ***, P ≤ 0.0005, one-way ANOVA with Tukey’s multiple-comparison test. ns, not significant. (C) BMDMs were uninfected or infected with the WT or ΔesxH mutant for 1 to 4 h at a multiplicity of infection (MOI) of 10 to 50 as indicated. HRS and β-actin were examined by Western blotting. (D) Immunoelectron microscopy of BMDMs infected for 3 h with the WT and ΔesxH mutant. Red arrows indicate anti-HRS gold particles on M. tuberculosis phagosomes, while vesicular and cytosolic gold particles are indicated with orange and black arrows, respectively. Bacteria are labeled “B.” (E) The subcellular localization of anti-HRS gold particles was quantified from two independent experiments by an investigator blind to sample identity. At least 25 images with at least 174 bacilli per sample were analyzed. The number of anti-HRS gold particles in each sample is indicated (n). ****, P <0.0001, Fisher’s exact test.

HRS is not required for ESCRT-III endomembrane damage response. (A and B) HRS does not assemble with ESCRT machinery on LLOME-disrupted endolysosomes. U20S cells were treated with LLOME or the solvent control for 10 min and then stained for HRS and EEA1 (A) or for HRS and CHMP4A (B). Boxed areas are magnified at right. The middle two columns show each indicated stain in grayscale; overlap of both stains in leftmost and rightmost columns appears white. Individual cells are outlined by white dashed lines; scale bars equal 10 μm (2 μm in magnified views). (C) RAW cells were treated with siRNA to deplete HRS or TSG101 for 2 days. Macrophages were then treated with LLOME for 15 min, and ubiquitin (FK2 antibody [red]) and CHMP4B (green) were examined. FK2-positive cells are outlined by white dashed lines. (D and E) Automated image analysis was used to quantify the mean fluorescent intensity (MFI) of FK2 (D) or the number of CHMP4B (E) punctae on 50 macrophages per sample. The number of CHMP4B punctae was compared in cells with FK2 MFI greater than and less than 700. Data are means ± SEM from one representative experiment from at least two independent experiments. ****, P ≤ 0.0001, Student’s t test. ns, not significant. (F) HeLa cells were transfected with M. tuberculosis (Mtb) EsxG-EsxH or vector control, preincubated for 1 h with BAPTA-AM, and then treated with LLOME for 15 min and stained for CHMP4A (green) and EsxG-EsxH (red). EsxG-EsxH was detected with anti-V5 antibody. Nuclei were stained with DAPI. Images are maximum-intensity projections. Scale bar, 10 μm.