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Invasive   Pseudomonas aeruginosa   (PA) can enter epithelial cells wherein they mediate formation of plasma membrane bleb-niches for intracellular compartmentalization. This phenotype, and capacity for intracellular replication, requires the ADP-ribosyltransferase (ADPr) activity of ExoS, a PA type III secretion system (T3SS) effector protein. Thus, PA T3SS mutants lack these capacities and instead traffic to perinuclear vacuoles. Here, we tested the hypothesis that the T3SS, via the ADPr activity of ExoS, allows PA to evade acidic vacuoles that otherwise suppress its intracellular viability. The acidification state of bacteria-occupied vacuoles within infected corneal epithelial cells was studied using LysoTracker to visualize acidic, lysosomal vacuoles. Steady state analysis showed that within cells wild-type PAO1 localized to both membrane bleb-niches and vacuoles, while both  exsA  (transcriptional activator) and  popB  (effector translocation) T3SS mutants were only found in vacuoles. The acidification state of occupied vacuoles suggested a relationship with ExoS expression, i.e. vacuoles occupied by the  exsA  mutant (unable to express ExoS) were more often acidified than either  popB  mutant or wild-type PAO1 occupied vacuoles (p < 0.001). An  exoS-gfp  reporter construct pJNE05 confirmed that high  exoS  transcriptional output coincided with low occupation of acidified vacuoles, and  vice versa , for both  popB  mutants and wild-type bacteria. Complementation of a triple effector null mutant of PAO1 with  exoS  (pUCP exoS ) reduced the number of acidified bacteria-occupied vacuoles per cell; pUCP exoS E381D which lacks ADPr activity did not. The H + -ATPase inhibitor bafilomycin rescued intracellular replication to wild-type levels for  exsA  mutants, showing its viability is suppressed by vacuolar acidification. Taken together, the data show that the mechanism by which ExoS ADPr activity allows intracellular replication by PA involves suppression of vacuolar acidification. They also show that variability in ExoS expression by wild-type PA inside cells can differentially influence the fate of individual intracellular bacteria, even within the same cell.

Pseudomonas aeruginosa Utilizes the Type III Secreted Toxin ExoS to Avoid Acidified Compartments within Epithelial Cells