Individual contributions of exotoxins S and T on internalized P. aeruginosa.

Pseudomonas aeruginosa is a high-priority pathogen and significant burden to health care worldwide. Although often regarded as an extracellular pathogen, P. aeruginosa is also capable of existing intracellularly in multiple cell types, including epithelial cells, goblet cells, and macrophages. This designation is attributed to two of its type three secretion system (T3SS)-dependent exotoxins, ExoS and ExoT, which inactivate host proteins that facilitate phagocytosis. However, studies investigating intracellular bacteria show that ExoS can paradoxically facilitate survival and replication, seemingly overriding the anti-internalization properties of itself and ExoT through its ADP-ribosylation activity. Here, we set out to define the individual and combined contributions of ExoS and ExoT by examining how each of their two functional domain activities affects epithelial cell invasion. Through in vitro biochemical assays, we found that ExoS is capable of ADP-ribosylating ExoT and their shared human cofactor, 14-3-3. We also found that ExoT ADP-ribosylates itself, unexpectedly enhancing its GTPase-activating protein (GAP) activity. Using fluorescence microscopy, we found that GAP activity of either exotoxin does not block internalization events, but is instead associated with fewer internalized bacteria entering a phase of rapid cytoplasmic replication. We demonstrate that ExoS ADP-ribosyltransferase activity is positively associated with vacuolar exit and cytoplasmic replication, and delivery of ExoS from extracellular bacteria triggers the intracellular T3SS⁻ subpopulation to become T3SS+ and exit the vacuole. Overall, this study highlights the underappreciated ability of P. aeruginosa to become intracellular and delineates how the enzymatic domains of ExoS and ExoT dictate the intracellular localization of the pathogen.