FIG 2.

ExoU elicits oxidative stress and induces autophagy at the mitochondria during P. aeruginosa infection. (A) MitoSOX-labeled WT BMDMs were treated with sterile saline (control) or inoculated with P. aeruginosa strain PA103, PA103 exoT::Tc, PA103 ΔexoU exoT::Tc, or PA103 ΔpcrV at an MOI of 20:1. At 180 min postinoculation, images were acquired by fluorescence microscopy. Images are representative of 3 to 7 biological replicates. The scale bar is equal to 10 μm. (B) Average fluorescence intensity of images from panel A, including additional strains as indicated. Data for several of the groups were determined to be non-normally distributed (D’Agostino and Pearson normality test); thus, all groups were compared by one-way ANOVA with Kruskal-Wallis post hoc test. All statistical comparison data are reported in Table S1. As a control, WT BMDMs were treated with either cDMEM (Ctrl) or H₂O₂ (400 μM) for 180 min. Images were acquired by fluorescence microscopy and intensities determined (2 biological replicates). (C) WT BMDMs treated with sterile saline (control) or inoculated with P. aeruginosa strain PA103 or PA103 ΔexoU exoT::Tc for 60 min or 180 min. Enriched mitochondrial (M) and cytosol (C) fractions (2 μg of total protein) were assayed by Western blotting to measure prohibitin as a marker of mitochondrial enrichment (29 kDa). Blots were run on separate gels and images compiled. (D) WT or Nlrc4^−/− BMDMs were treated with sterile saline (control) or inoculated with P. aeruginosa strain PA103, PA103 exoT::Tc, PA103 ΔexoU exoT::Tc, or PA103 ΔpcrV. At 180 min postinoculation, enriched mito-MAM fractions were isolated, and LC3-I, LC3-II, and prohibitin were measured in lysates by Western blotting. (E) Densitometry analysis of blots from panel D showing LC3-II signal normalized to prohibitin. ***, P value ≤ 0.0007; **, P value ≤ 0.008; *, P value ≤ 0.04. All blots and densitometry analyses are representative of 3 independent experiments. (F) Colocalization of LC3 with mitochondria. WT BMDMs were treated with sterile saline (control) or inoculated with P. aeruginosa strain PA103 or PA103 ΔexoU exoT::Tc for 180 min at an MOI of 20:1. Images of red (MitoTracker) and green (LC3) fluorescence were acquired by confocal microscopy. Colocalization of LC3 with mitochondria appears as yellow to orange spots. (G) Colocalization analysis using Pearson’s correlation coefficient. **, P value = 0.003; *, P value = 0.01. Images are representative of 4 independent experiments.