Colocalization of the <i>P. aeruginosa exsA</i> mutant with acidified vacuoles in epithelial cells compared to that of wild-type bacteria or a <i>popB</i> (translocon) mutant.

<p>Confocal microscopy images of human corneal epithelial cells at 5 h post-infection with GFP-expressing <i>P. aeruginosa</i> (green). Prior to imaging, infected cultures were infused with LysoTracker (LT) DND-22 (pseudo-colored red). Panels depict (A) Uninfected control, (B) PAO1 <i>exsA</i> mutant (C) PAO1 <i>popB</i> (translocon) mutant and (D) wild-type PAO1. Uninfected cells appeared healthy. The intracellular <i>exsA</i> mutant appeared more frequently in LT (+) (acidified) vacuoles which co-localized yellow (arrows) than either the intracellular <i>popB</i> mutant or wild-type PAO1. PAO1-infected cells which displayed bleb-niche formation (1D inset) showed reduced fluorescence (< 10% fluorescence intensity of PAO1-infected non-blebbing cells, p < 0.001 Welch’s corrected t-Test). Occasional bleb-niches contained LT (+) vacuoles containing bacteria (1D inset, yellow). Representative images are shown. Magnification ~ 600 x.