Figure 5
OMVs containing OmpAAb trigger mitochondrial fragmentation. (A) Illustration showing the transwell assay set-up (created with BioRender.com). (B) A549 cells seeded in the bottom chamber of the transwell plate were incubated with the indicated bacteria (corresponding to MOI 50) in the top chamber for 6 h. The bacteria were not in contact with the cells. Immunofluorescence was performed using anti-TOM20 antibody to stain mitochondria (red) and DAPI to stain the nucleus (blue). Scale bar represents 10 µm. (C) The scatter plots represent the quantification of mitochondrial area (red) and perimeter (orange). Error bars represent standard deviation, n = 32–46 cells. One-way ANOVA with Tukey’s multiple comparisons test ****p \(\le \) 0.0001. (D) Western blot analysis of indicated bacterial lysates and OMVs using anti-Flag and anti-GroEL antibodies. (E) A549 cells were treated with 400 µg/ml of the indicated OMVs for 6 h. Immunofluorescence was performed using anti-TOM20 antibody to stain mitochondria (red) and DAPI to stain the nucleus (blue). Scale bar represents 10 µm. (F) The scatter plots represent the quantification of mitochondrial area (red) and perimeter (orange). Error bars represent standard deviation, n = 35–40 cells. One-way ANOVA with Tukey’s multiple comparisons test *p \(\le \) 0.05, **p \(\le \) 0.01. (G) Western blot analysis of indicated OMVs using anti-Flag and anti-BamA antibodies. (H) A549 cells were treated with 400 µg/ml of the indicated OMVs for 6 h. Immunofluorescence was performed using anti-TOM20 antibody to stain mitochondria (red) and DAPI to stain the nucleus (blue). Scale bar represents 10 µm. (I) The scatter plots represent the quantification of mitochondrial area (red) and perimeter (orange). Error bars represent standard deviation, n = 37–50 cells. One-way ANOVA with Tukey’s multiple comparisons test *p \(\le \) 0.05, **p \(\le \) 0.01. (J) Cytotoxicity was assessed by LDH release assay after 6 h of OMV treatment in A549 cells. The experiments were done in triplicates. Error bars represent standard deviation. Two-tailed p value using unpaired t-test **p \(\le \) 0.01. % cytotoxicity was calculated by normalizing the LDH release in the treated groups with untreated cells (representing no cytotoxicity) and TritonX-100 treated cells (representing the highest cytotoxicity). (K) A549 cells were treated with 400 µg/ml of the indicated OMVs (fluorescently labelled with Vybrant Dio) for 6 h. Immunofluorescence was performed using anti-TOM20 antibody to stain mitochondria (red) and OMVs were labelled in green. Z-stack confocal imaging was performed on the cells, orthogonal views presented here. Arrows indicate OMVs (green) colocalizing with mitochondria (red). Scale bar represents 10 µm. (L) The bar graph represents the % of indicated OMVs colocalizing with mitochondria. The cells were stained with phalloidin and intracellular OMVs were scored for their colocalization with mitochondria or not. Error bars represent standard deviation. Mitochondrial area and perimeter quantifications were performed using an unbiased automated CellProfiler pipeline (see “Materials and methods” section for details). All the experiments shown here were performed three times independently.
