The Impact of ExoS on <italic toggle="yes">Pseudomonas aeruginosa</italic> Internalization by Epithelial Cells Is Independent of <italic toggle="yes">fleQ</italic> and Correlates with Bistability of Type Three Secretion System Gene Expression

ABSTRACT Pseudomonas 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 intrac...

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Autores principales:	Abby R. Kroken, Camille K. Chen, David J. Evans, Timothy L. Yahr, Suzanne M. J. Fleiszig
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2018
Materias:	bistability ExoS FleQ Pseudomonas aeruginosa epithelial cells host cell invasion Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/fed2cbe1a18146dbb4eaa4deb742abf0
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spelling	oai:doaj.org-article:fed2cbe1a18146dbb4eaa4deb742abf02021-11-15T16:00:26ZThe Impact of ExoS on <italic toggle="yes">Pseudomonas aeruginosa</italic> Internalization by Epithelial Cells Is Independent of <italic toggle="yes">fleQ</italic> and Correlates with Bistability of Type Three Secretion System Gene Expression10.1128/mBio.00668-182150-7511https://doaj.org/article/fed2cbe1a18146dbb4eaa4deb742abf02018-07-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mBio.00668-18https://doaj.org/toc/2150-7511ABSTRACT Pseudomonas 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. IMPORTANCE P. aeruginosa is often referred to as an extracellular pathogen, despite its demonstrated capacity to invade and survive within host cells. Fueling the confusion, P. aeruginosa encodes 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 the P. aeruginosa strains 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. PA103 fleQ mutation, 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 influences P. aeruginosa internalization. These findings reconcile controversies in the literature surrounding P. aeruginosa internalization and support the principle that P. aeruginosa is not exclusively an extracellular pathogen.Abby R. KrokenCamille K. ChenDavid J. EvansTimothy L. YahrSuzanne M. J. FleiszigAmerican Society for MicrobiologyarticlebistabilityExoSFleQPseudomonas aeruginosaepithelial cellshost cell invasionMicrobiologyQR1-502ENmBio, Vol 9, Iss 3 (2018)
institution	DOAJ
collection	DOAJ
language	EN
topic	bistability ExoS FleQ Pseudomonas aeruginosa epithelial cells host cell invasion Microbiology QR1-502
spellingShingle	bistability ExoS FleQ Pseudomonas aeruginosa epithelial cells host cell invasion Microbiology QR1-502 Abby R. Kroken Camille K. Chen David J. Evans Timothy L. Yahr Suzanne M. J. Fleiszig The Impact of ExoS on <italic toggle="yes">Pseudomonas aeruginosa</italic> Internalization by Epithelial Cells Is Independent of <italic toggle="yes">fleQ</italic> and Correlates with Bistability of Type Three Secretion System Gene Expression
description	ABSTRACT Pseudomonas 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. IMPORTANCE P. aeruginosa is often referred to as an extracellular pathogen, despite its demonstrated capacity to invade and survive within host cells. Fueling the confusion, P. aeruginosa encodes 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 the P. aeruginosa strains 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. PA103 fleQ mutation, 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 influences P. aeruginosa internalization. These findings reconcile controversies in the literature surrounding P. aeruginosa internalization and support the principle that P. aeruginosa is not exclusively an extracellular pathogen.
format	article
author	Abby R. Kroken Camille K. Chen David J. Evans Timothy L. Yahr Suzanne M. J. Fleiszig
author_facet	Abby R. Kroken Camille K. Chen David J. Evans Timothy L. Yahr Suzanne M. J. Fleiszig
author_sort	Abby R. Kroken
title	The Impact of ExoS on <italic toggle="yes">Pseudomonas aeruginosa</italic> Internalization by Epithelial Cells Is Independent of <italic toggle="yes">fleQ</italic> and Correlates with Bistability of Type Three Secretion System Gene Expression
title_short	The Impact of ExoS on <italic toggle="yes">Pseudomonas aeruginosa</italic> Internalization by Epithelial Cells Is Independent of <italic toggle="yes">fleQ</italic> and Correlates with Bistability of Type Three Secretion System Gene Expression
title_full	The Impact of ExoS on <italic toggle="yes">Pseudomonas aeruginosa</italic> Internalization by Epithelial Cells Is Independent of <italic toggle="yes">fleQ</italic> and Correlates with Bistability of Type Three Secretion System Gene Expression
title_fullStr	The Impact of ExoS on <italic toggle="yes">Pseudomonas aeruginosa</italic> Internalization by Epithelial Cells Is Independent of <italic toggle="yes">fleQ</italic> and Correlates with Bistability of Type Three Secretion System Gene Expression
title_full_unstemmed	The Impact of ExoS on <italic toggle="yes">Pseudomonas aeruginosa</italic> Internalization by Epithelial Cells Is Independent of <italic toggle="yes">fleQ</italic> and Correlates with Bistability of Type Three Secretion System Gene Expression
title_sort	impact of exos on <italic toggle="yes">pseudomonas aeruginosa</italic> internalization by epithelial cells is independent of <italic toggle="yes">fleq</italic> and correlates with bistability of type three secretion system gene expression
publisher	American Society for Microbiology
publishDate	2018
url	https://doaj.org/article/fed2cbe1a18146dbb4eaa4deb742abf0
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The Impact of ExoS on <italic toggle="yes">Pseudomonas aeruginosa</italic> Internalization by Epithelial Cells Is Independent of <italic toggle="yes">fleQ</italic> and Correlates with Bistability of Type Three Secretion System Gene Expression

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