<italic toggle="yes">Pseudomonas aeruginosa</italic> Pore-Forming Exolysin and Type IV Pili Cooperate To Induce Host Cell Lysis

ABSTRACT Clinical strains of Pseudomonas aeruginosa lacking the type III secretion system genes employ a toxin, exolysin (ExlA), for host cell membrane disruption. Here, we demonstrated that ExlA export requires a predicted outer membrane protein, ExlB, showing that ExlA and ExlB define a new active...

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Autores principales:	Pauline Basso, Michel Ragno, Sylvie Elsen, Emeline Reboud, Guillaume Golovkine, Stephanie Bouillot, Philippe Huber, Stephen Lory, Eric Faudry, Ina Attrée
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2017
Materias:	Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/f6d0809c3fa644599ba0f26864033712
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spelling	oai:doaj.org-article:f6d0809c3fa644599ba0f268640337122021-11-15T15:51:06Z<italic toggle="yes">Pseudomonas aeruginosa</italic> Pore-Forming Exolysin and Type IV Pili Cooperate To Induce Host Cell Lysis10.1128/mBio.02250-162150-7511https://doaj.org/article/f6d0809c3fa644599ba0f268640337122017-03-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mBio.02250-16https://doaj.org/toc/2150-7511ABSTRACT Clinical strains of Pseudomonas aeruginosa lacking the type III secretion system genes employ a toxin, exolysin (ExlA), for host cell membrane disruption. Here, we demonstrated that ExlA export requires a predicted outer membrane protein, ExlB, showing that ExlA and ExlB define a new active two-partner secretion (TPS) system of P. aeruginosa. In addition to the TPS signals, ExlA harbors several distinct domains, which include one hemagglutinin domain, five arginine-glycine-aspartic acid (RGD) motifs, and a C-terminal region lacking any identifiable sequence motifs. However, this C-terminal region is important for the toxic activity, since its deletion abolishes host cell lysis. Using lipid vesicles and eukaryotic cells, including red blood cells, we demonstrated that ExlA has a pore-forming activity which precedes cell membrane disruption of nucleated cells. Finally, we developed a high-throughput cell-based live-dead assay and used it to screen a transposon mutant library of an ExlA-producing P. aeruginosa clinical strain for bacterial factors required for ExlA-mediated toxicity. The screen resulted in the identification of proteins involved in the formation of type IV pili as being required for ExlA to exert its cytotoxic activity by promoting close contact between bacteria and the host cell. These findings represent the first example of cooperation between a pore-forming toxin of the TPS family and surface appendages in host cell intoxication. IMPORTANCE The course and outcome of acute, toxigenic infections by Pseudomonas aeruginosa clinical isolates rely on the deployment of one of two virulence strategies: delivery of effectors by the well-known type III secretion system or the cytolytic activity of the recently identified two-partner secreted toxin, exolysin. Here, we characterize several features of the mammalian cell intoxication process mediated by exolysin. We found that exolysin requires the outer membrane protein ExlB for export into extracellular medium. Using in vitro recombinant protein and ex vivo assays, we demonstrated a pore-forming activity of exolysin. A cellular cytotoxicity screen of a transposon mutant library, made in an exolysin-producing clinical strain, identified type IV pili as bacterial appendages required for exolysin toxic function. This work deciphers molecular mechanisms underlying the activity of novel virulence factors used by P. aeruginosa clinical strains lacking the type III secretion system, including a requirement for the toxin-producing bacteria to be attached to the targeted cell to induce cytolysis, and defines new targets for developing antivirulence strategies.Pauline BassoMichel RagnoSylvie ElsenEmeline ReboudGuillaume GolovkineStephanie BouillotPhilippe HuberStephen LoryEric FaudryIna AttréeAmerican Society for MicrobiologyarticleMicrobiologyQR1-502ENmBio, Vol 8, Iss 1 (2017)
institution	DOAJ
collection	DOAJ
language	EN
topic	Microbiology QR1-502
spellingShingle	Microbiology QR1-502 Pauline Basso Michel Ragno Sylvie Elsen Emeline Reboud Guillaume Golovkine Stephanie Bouillot Philippe Huber Stephen Lory Eric Faudry Ina Attrée <italic toggle="yes">Pseudomonas aeruginosa</italic> Pore-Forming Exolysin and Type IV Pili Cooperate To Induce Host Cell Lysis
description	ABSTRACT Clinical strains of Pseudomonas aeruginosa lacking the type III secretion system genes employ a toxin, exolysin (ExlA), for host cell membrane disruption. Here, we demonstrated that ExlA export requires a predicted outer membrane protein, ExlB, showing that ExlA and ExlB define a new active two-partner secretion (TPS) system of P. aeruginosa. In addition to the TPS signals, ExlA harbors several distinct domains, which include one hemagglutinin domain, five arginine-glycine-aspartic acid (RGD) motifs, and a C-terminal region lacking any identifiable sequence motifs. However, this C-terminal region is important for the toxic activity, since its deletion abolishes host cell lysis. Using lipid vesicles and eukaryotic cells, including red blood cells, we demonstrated that ExlA has a pore-forming activity which precedes cell membrane disruption of nucleated cells. Finally, we developed a high-throughput cell-based live-dead assay and used it to screen a transposon mutant library of an ExlA-producing P. aeruginosa clinical strain for bacterial factors required for ExlA-mediated toxicity. The screen resulted in the identification of proteins involved in the formation of type IV pili as being required for ExlA to exert its cytotoxic activity by promoting close contact between bacteria and the host cell. These findings represent the first example of cooperation between a pore-forming toxin of the TPS family and surface appendages in host cell intoxication. IMPORTANCE The course and outcome of acute, toxigenic infections by Pseudomonas aeruginosa clinical isolates rely on the deployment of one of two virulence strategies: delivery of effectors by the well-known type III secretion system or the cytolytic activity of the recently identified two-partner secreted toxin, exolysin. Here, we characterize several features of the mammalian cell intoxication process mediated by exolysin. We found that exolysin requires the outer membrane protein ExlB for export into extracellular medium. Using in vitro recombinant protein and ex vivo assays, we demonstrated a pore-forming activity of exolysin. A cellular cytotoxicity screen of a transposon mutant library, made in an exolysin-producing clinical strain, identified type IV pili as bacterial appendages required for exolysin toxic function. This work deciphers molecular mechanisms underlying the activity of novel virulence factors used by P. aeruginosa clinical strains lacking the type III secretion system, including a requirement for the toxin-producing bacteria to be attached to the targeted cell to induce cytolysis, and defines new targets for developing antivirulence strategies.
format	article
author	Pauline Basso Michel Ragno Sylvie Elsen Emeline Reboud Guillaume Golovkine Stephanie Bouillot Philippe Huber Stephen Lory Eric Faudry Ina Attrée
author_facet	Pauline Basso Michel Ragno Sylvie Elsen Emeline Reboud Guillaume Golovkine Stephanie Bouillot Philippe Huber Stephen Lory Eric Faudry Ina Attrée
author_sort	Pauline Basso
title	<italic toggle="yes">Pseudomonas aeruginosa</italic> Pore-Forming Exolysin and Type IV Pili Cooperate To Induce Host Cell Lysis
title_short	<italic toggle="yes">Pseudomonas aeruginosa</italic> Pore-Forming Exolysin and Type IV Pili Cooperate To Induce Host Cell Lysis
title_full	<italic toggle="yes">Pseudomonas aeruginosa</italic> Pore-Forming Exolysin and Type IV Pili Cooperate To Induce Host Cell Lysis
title_fullStr	<italic toggle="yes">Pseudomonas aeruginosa</italic> Pore-Forming Exolysin and Type IV Pili Cooperate To Induce Host Cell Lysis
title_full_unstemmed	<italic toggle="yes">Pseudomonas aeruginosa</italic> Pore-Forming Exolysin and Type IV Pili Cooperate To Induce Host Cell Lysis
title_sort	<italic toggle="yes">pseudomonas aeruginosa</italic> pore-forming exolysin and type iv pili cooperate to induce host cell lysis
publisher	American Society for Microbiology
publishDate	2017
url	https://doaj.org/article/f6d0809c3fa644599ba0f26864033712
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<italic toggle="yes">Pseudomonas aeruginosa</italic> Pore-Forming Exolysin and Type IV Pili Cooperate To Induce Host Cell Lysis

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