ABSTRACTPseudomonas aeruginosais internalized into multiple types of epithelial cellin vitroandin vivoand yet is often regarded as an exclusively extracellular pathogen. Paradoxically, ExoS, a type three secretion system (T3SS) effector, has antiphagocytic activities but is required for intracellular survival ofP. aeruginosaand its occupation of bleb niches in epithelial cells. Here, we addressed mechanisms for this dichotomy using invasive (ExoS-expressing)P. aeruginosaand corresponding effector-null isogenic T3SS mutants, effector-null mutants of cytotoxicP. aeruginosawith and without ExoS transformation, antibiotic exclusion assays, and imaging using a T3SS-GFP reporter. Except for effector-null PA103, all strains were internalized while encoding ExoS. Intracellular bacteria showed T3SS activation that continued in replicating daughter cells. Correcting thefleQmutation in effector-null PA103 promoted internalization by >10-fold with or without ExoS. Conversely, mutatingfleQin PAO1 reduced internalization by >10-fold, also with or without ExoS. Effector-null PA103 remained less well internalized than PAO1 matched forfleQstatus, but only with ExoS expression, suggesting additional differences between these strains. Quantifying T3SS activation using GFP fluorescence and quantitative reverse transcription-PCR (qRT-PCR) showed that T3SS expression was hyperinducible for strain PA103ΔexoUTversus other isolates and was unrelated tofleQstatus. These findings support the principle thatP. aeruginosais not exclusively an extracellular pathogen, with internalization influenced by the relative proportions of T3SS-positive and T3SS-negative bacteria in the population during host cell interaction. These data also challenge current thinking about T3SS effector delivery into host cells and suggest that T3SS bistability is an important consideration in studyingP. aeruginosapathogenesis.IMPORTANCEP. aeruginosais often referred to as an extracellular pathogen, despite its demonstrated capacity to invade and survive within host cells. Fueling the confusion,P. aeruginosaencodes T3SS effectors with anti-internalization activity that, paradoxically, play critical roles in intracellular survival. Here, we sought to address why ExoS does not prevent internalization of theP. aeruginosastrains that natively encode it. Results showed that ExoS exerted unusually strong anti-internalization activity under conditions of expression in the effector-null background of strain PA103, often used to study T3SS effector activity. Inhibition of internalization was associated with T3SS hyperinducibility and ExoS delivery. PA103fleQmutation, preventing flagellar assembly, further reduced internalization but did so independently of ExoS. The results revealed intracellular T3SS expression by all strains and suggested that T3SS bistability influencesP. aeruginosainternalization. These findings reconcile controversies in the literature surroundingP. aeruginosainternalization and support the principle thatP. aeruginosais not exclusively an extracellular pathogen.
Laboratory Adaptation of Bordetella pertussis Is Associated with the Loss of Type Three Secretion System Functionality
