Journal: mBio

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

doi: 10.1128/mBio.01765-18

Figure Lengend Snippet: 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.

Article Snippet: To generate the M. smegmatis EsxG-T2A-EsxH-V5 plasmid (pEM01), Genewiz synthesized DNA that was identical to the insert of pET018, except that the M. tuberculosis EsxG and EsxH sequences were replaced with M. smegmatis EsxG and EsxH, and then the insert was cloned into the HindIII and XbaI restriction sites of pET018.

Techniques: Transfection, Plasmid Preparation, Control, Solvent, Staining, Live Cell Imaging, Expressing