Exotoxin S secreted by internalized Pseudomonas aeruginosa delays lytic host cell death.

The Pseudomonas aeruginosa toxin ExoS, secreted by the type III secretion system (T3SS), supports intracellular persistence via its ADP-ribosyltransferase (ADPr) activity. For epithelial cells, this involves inhibiting vacuole acidification, promoting vacuolar escape, countering autophagy, and niche construction in the cytoplasm and within plasma membrane blebs. Paradoxically, ExoS and other P. aeruginosa T3SS effectors can also have antiphagocytic and cytotoxic activities. Here, we sought to reconcile these apparently contradictory activities of ExoS by studying the relationships between intracellular persistence and host epithelial cell death. Methods involved quantitative imaging and the use of antibiotics that vary in host cell membrane permeability to selectively kill intracellular and extracellular populations after invasion. Results showed that intracellular P. aeruginosa mutants lacking T3SS effector toxins could kill (permeabilize) cells when extracellular bacteria were eliminated. Surprisingly, wild-type strain PAO1 (encoding ExoS, ExoT and ExoY) caused cell death more slowly, the time extended from 5.2 to 9.5 h for corneal epithelial cells and from 10.2 to 13.0 h for HeLa cells. Use of specific mutants/complementation and controls for initial invasion showed that ExoS ADPr activity delayed cell death. Triggering T3SS expression only after bacteria invaded cells using rhamnose-induction in T3SS mutants rescued the ExoS-dependent intracellular phenotype, showing that injected effectors from extracellular bacteria were not required. The ADPr activity of ExoS was further found to support internalization by countering the antiphagocytic activity of both the ExoS and ExoT RhoGAP domains. Together, these results show two additional roles for ExoS ADPr activity in supporting the intracellular lifestyle of P. aeruginosa; suppression of host cell death to preserve a replicative niche and inhibition of T3SS effector antiphagocytic activities to allow invasion. These findings add to the growing body of evidence that ExoS-encoding (invasive) P. aeruginosa strains can be facultative intracellular pathogens, and that intracellularly secreted T3SS effectors contribute to pathogenesis. Author summary: While the ADPr domain of the T3SS effector ExoS plays multiple roles in the intracellular lifestyle of P. aeruginosa, ExoS can also be cytotoxic and/or antiphagocytic. Here, we show that when P. aeruginosa enters the cytosol of epithelial cells, cell death is triggered independently of T3SS effector toxins, but ExoS ADPr activity delays this to enable continued intracellular survival and replication. Using rhamnose induction to express the T3SS only after invasion restored this ExoS-dependent phenotype, showing that intracellularly secreted effectors can enable intracellular pathogenesis. ExoS ADPr activity also countered antiphagocytic activity of ExoS and ExoT RhoGAP domains. These results show two additional roles for ExoS ADPr activity in promoting internalization of P. aeruginosa and protecting the intracellular niche, continuing to challenge the notions that P. aeruginosa is exclusively an extracellular pathogen, that it needs to inject T3SS effectors across plasma membranes, and that ExoS is necessarily cytotoxic to host cells. [ABSTRACT FROM AUTHOR]