Abstract
ABSTRACTPseudomonas aeruginosa is internalized into multiple types of epithelial cell in vitro and in vivo and 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 of P. aeruginosa and its occupation of bleb niches in epithelial cells. Here, we addressed mechanisms for this dichotomy using invasive (ExoS-expressing) P. aeruginosa and corresponding effector-null isogenic T3SS mutants, effector-null mutants of cytotoxic P. aeruginosa with 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 the fleQ mutation in effector-null PA103 promoted internalization by >10-fold with or without ExoS. Conversely, mutating fleQ in PAO1 reduced internalization by >10-fold, also with or without ExoS. Effector-null PA103 remained less well internalized than PAO1 matched for fleQ status, 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 PA103ΔexoUT versus other isolates and was unrelated to fleQ status. These findings support the principle that P. aeruginosa is 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 studying P. aeruginosa pathogenesis.
Highlights
IMPORTANCE P. aeruginosa is often referred to as an extracellular pathogen, despite its demonstrated capacity to invade and survive within host cells
Differentiated human telomerase-immortalized corneal epithelial cells, bronchial epithelial cells (NuLi-1), and HeLa cells were compared for infection with green fluorescent protein (GFP)-expressing P. aeruginosa strain PAO1 and a T3SS mutant, both expressing constitutive GFP from plasmid pSMC2
While HeLa cells infected with PAO1 were rounded as previously reported [34, 35], the presence or absence of rounding differed for corneal and bronchial epithelial cells, suggesting reduced susceptibility to T3SS effector delivery or cytoskeleton-disruptive effects (Fig. 1)
Summary
IMPORTANCE P. aeruginosa is often referred to as an extracellular pathogen, despite its demonstrated capacity to invade and survive within host cells. While often regarded as an extracellular pathogen, P. aeruginosa can invade multiple types of epithelial cell in vitro and in vivo. In vivo, this was first illustrated by our group using transmission electron microscopy to show bacteria within the cytoplasm of epithelial cells during corneal infections in mice [16]. Our published data have shown variability in internalization capacity among clinical isolates [23] Those less able to invade tended to have acute cytotoxic activity, leading us to propose classification of P. aeruginosa isolates as either cytotoxic or invasive strains based on their interactions with epithelial cells. Further investigation showed that these alternate phenotypes relate directly to the different T3SS effectors encoded, with cytotoxic strains encoding ExoU and not ExoS and the reverse for invasive strains [10]
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