Type IV Pili Can Mediate Bacterial Motility within Epithelial Cells

ABSTRACT Pseudomonas aeruginosa is among bacterial pathogens capable of twitching motility, a form of surface-associated movement dependent on type IV pili (T4P). Previously, we showed that T4P and twitching were required for P. aeruginosa to cause disease in a murine model of corneal infection, to...

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Autores principales:	Vincent Nieto, Abby R. Kroken, Melinda R. Grosser, Benjamin E. Smith, Matteo M. E. Metruccio, Patrick Hagan, Mary E. Hallsten, David J. Evans, Suzanne M. J. Fleiszig
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2019
Materias:	bacterial exit bacterial motility epithelial cells intracellular bacteria Pseudomonas aeruginosa twitching motility Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/01f26b1439aa46768f337c40ca045667
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spelling	oai:doaj.org-article:01f26b1439aa46768f337c40ca0456672021-11-15T16:22:11ZType IV Pili Can Mediate Bacterial Motility within Epithelial Cells10.1128/mBio.02880-182150-7511https://doaj.org/article/01f26b1439aa46768f337c40ca0456672019-08-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mBio.02880-18https://doaj.org/toc/2150-7511ABSTRACT Pseudomonas aeruginosa is among bacterial pathogens capable of twitching motility, a form of surface-associated movement dependent on type IV pili (T4P). Previously, we showed that T4P and twitching were required for P. aeruginosa to cause disease in a murine model of corneal infection, to traverse human corneal epithelial multilayers, and to efficiently exit invaded epithelial cells. Here, we used live wide-field fluorescent imaging combined with quantitative image analysis to explore how twitching contributes to epithelial cell egress. Results using time-lapse imaging of cells infected with wild-type PAO1 showed that cytoplasmic bacteria slowly disseminated throughout the cytosol at a median speed of >0.05 μm s−1 while dividing intracellularly. Similar results were obtained with flagellin (fliC) and flagellum assembly (flhA) mutants, thereby excluding swimming, swarming, and sliding as mechanisms. In contrast, pilA mutants (lacking T4P) and pilT mutants (twitching motility defective) appeared stationary and accumulated in expanding aggregates during intracellular division. Transmission electron microscopy confirmed that these mutants were not trapped within membrane-bound cytosolic compartments. For the wild type, dissemination in the cytosol was not prevented by the depolymerization of actin filaments using latrunculin A and/or the disruption of microtubules using nocodazole. Together, these findings illustrate a novel form of intracellular bacterial motility differing from previously described mechanisms in being directly driven by bacterial motility appendages (T4P) and not depending on polymerized host actin or microtubules. IMPORTANCE Host cell invasion can contribute to disease pathogenesis by the opportunistic pathogen Pseudomonas aeruginosa. Previously, we showed that the type III secretion system (T3SS) of invasive P. aeruginosa strains modulates cell entry and subsequent escape from vacuolar trafficking to host lysosomes. However, we also showed that mutants lacking either type IV pili (T4P) or T4P-dependent twitching motility (i) were defective in traversing cell multilayers, (ii) caused less pathology in vivo, and (iii) had a reduced capacity to exit invaded cells. Here, we report that after vacuolar escape, intracellular P. aeruginosa can use T4P-dependent twitching motility to disseminate throughout the host cell cytoplasm. We further show that this strategy for intracellular dissemination does not depend on flagellin and resists both host actin and host microtubule disruption. This differs from mechanisms used by previously studied pathogens that utilize either host actin or microtubules for intracellular dissemination independently of microbe motility appendages.Vincent NietoAbby R. KrokenMelinda R. GrosserBenjamin E. SmithMatteo M. E. MetruccioPatrick HaganMary E. HallstenDavid J. EvansSuzanne M. J. FleiszigAmerican Society for Microbiologyarticlebacterial exitbacterial motilityepithelial cellsintracellular bacteriaPseudomonas aeruginosatwitching motilityMicrobiologyQR1-502ENmBio, Vol 10, Iss 4 (2019)
institution	DOAJ
collection	DOAJ
language	EN
topic	bacterial exit bacterial motility epithelial cells intracellular bacteria Pseudomonas aeruginosa twitching motility Microbiology QR1-502
spellingShingle	bacterial exit bacterial motility epithelial cells intracellular bacteria Pseudomonas aeruginosa twitching motility Microbiology QR1-502 Vincent Nieto Abby R. Kroken Melinda R. Grosser Benjamin E. Smith Matteo M. E. Metruccio Patrick Hagan Mary E. Hallsten David J. Evans Suzanne M. J. Fleiszig Type IV Pili Can Mediate Bacterial Motility within Epithelial Cells
description	ABSTRACT Pseudomonas aeruginosa is among bacterial pathogens capable of twitching motility, a form of surface-associated movement dependent on type IV pili (T4P). Previously, we showed that T4P and twitching were required for P. aeruginosa to cause disease in a murine model of corneal infection, to traverse human corneal epithelial multilayers, and to efficiently exit invaded epithelial cells. Here, we used live wide-field fluorescent imaging combined with quantitative image analysis to explore how twitching contributes to epithelial cell egress. Results using time-lapse imaging of cells infected with wild-type PAO1 showed that cytoplasmic bacteria slowly disseminated throughout the cytosol at a median speed of >0.05 μm s−1 while dividing intracellularly. Similar results were obtained with flagellin (fliC) and flagellum assembly (flhA) mutants, thereby excluding swimming, swarming, and sliding as mechanisms. In contrast, pilA mutants (lacking T4P) and pilT mutants (twitching motility defective) appeared stationary and accumulated in expanding aggregates during intracellular division. Transmission electron microscopy confirmed that these mutants were not trapped within membrane-bound cytosolic compartments. For the wild type, dissemination in the cytosol was not prevented by the depolymerization of actin filaments using latrunculin A and/or the disruption of microtubules using nocodazole. Together, these findings illustrate a novel form of intracellular bacterial motility differing from previously described mechanisms in being directly driven by bacterial motility appendages (T4P) and not depending on polymerized host actin or microtubules. IMPORTANCE Host cell invasion can contribute to disease pathogenesis by the opportunistic pathogen Pseudomonas aeruginosa. Previously, we showed that the type III secretion system (T3SS) of invasive P. aeruginosa strains modulates cell entry and subsequent escape from vacuolar trafficking to host lysosomes. However, we also showed that mutants lacking either type IV pili (T4P) or T4P-dependent twitching motility (i) were defective in traversing cell multilayers, (ii) caused less pathology in vivo, and (iii) had a reduced capacity to exit invaded cells. Here, we report that after vacuolar escape, intracellular P. aeruginosa can use T4P-dependent twitching motility to disseminate throughout the host cell cytoplasm. We further show that this strategy for intracellular dissemination does not depend on flagellin and resists both host actin and host microtubule disruption. This differs from mechanisms used by previously studied pathogens that utilize either host actin or microtubules for intracellular dissemination independently of microbe motility appendages.
format	article
author	Vincent Nieto Abby R. Kroken Melinda R. Grosser Benjamin E. Smith Matteo M. E. Metruccio Patrick Hagan Mary E. Hallsten David J. Evans Suzanne M. J. Fleiszig
author_facet	Vincent Nieto Abby R. Kroken Melinda R. Grosser Benjamin E. Smith Matteo M. E. Metruccio Patrick Hagan Mary E. Hallsten David J. Evans Suzanne M. J. Fleiszig
author_sort	Vincent Nieto
title	Type IV Pili Can Mediate Bacterial Motility within Epithelial Cells
title_short	Type IV Pili Can Mediate Bacterial Motility within Epithelial Cells
title_full	Type IV Pili Can Mediate Bacterial Motility within Epithelial Cells
title_fullStr	Type IV Pili Can Mediate Bacterial Motility within Epithelial Cells
title_full_unstemmed	Type IV Pili Can Mediate Bacterial Motility within Epithelial Cells
title_sort	type iv pili can mediate bacterial motility within epithelial cells
publisher	American Society for Microbiology
publishDate	2019
url	https://doaj.org/article/01f26b1439aa46768f337c40ca045667
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Type IV Pili Can Mediate Bacterial Motility within Epithelial Cells

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