Colocalization of <i>P. aeruginosa</i> with acidified versus non-acidified vacuoles in relation to <i>exoS</i> transcriptional output.

<p>Confocal and Differential Interference Contrast (DIC) microscopy of human corneal epithelial cells at 5 h post-infection with <i>P. aeruginosa</i> PAO1 complemented with a reporter construct pJNE05 encoding the <i>exoS</i> promoter fused to <i>gfp</i> (green), and p67T1 which constitutively expresses dTomato (red). Bacteria were classified as having a high <i>exoS</i> transcriptional output using a threshold value of 1000 units of GFP fluorescent intensity (green) based on expression levels observed under T3SS-inducing conditions (see Results). Prior to imaging, epithelial cells were infused with LysoTracker DND-22 (blue). ExoS-expressing bacteria (high output, green) [solid arrows] were located primarily outside of acidified (blue) intracellular compartments, which often contained bacteria with low <i>exoS</i> output [dashed arrows]. Blebs are indicated with open arrows. Representative images are shown from two independent experiments. Magnification ~ 600 x.