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Structural and Functional Characterization of the Type Three Secretion System (T3SS) Needle of Pseudomonas aeruginosa

The type three secretion system (T3SS) is a macromolecular protein nano-syringe used by different bacterial pathogens to inject effectors into host cells. The extracellular part of the syringe is a needle-like filament formed by the polymerization of a 9-kDa protein whose structure and proper locali...

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Autores principales: Lombardi, Charlotte, Tolchard, James, Bouillot, Stephanie, Signor, Luca, Gebus, Caroline, Liebl, David, Fenel, Daphna, Teulon, Jean-Marie, Brock, Juliane, Habenstein, Birgit, Pellequer, Jean-Luc, Faudry, Eric, Loquet, Antoine, Attrée, Ina, Dessen, Andréa, Job, Viviana
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2019
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6455054/
https://www.ncbi.nlm.nih.gov/pubmed/31001211
http://dx.doi.org/10.3389/fmicb.2019.00573
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author Lombardi, Charlotte
Tolchard, James
Bouillot, Stephanie
Signor, Luca
Gebus, Caroline
Liebl, David
Fenel, Daphna
Teulon, Jean-Marie
Brock, Juliane
Habenstein, Birgit
Pellequer, Jean-Luc
Faudry, Eric
Loquet, Antoine
Attrée, Ina
Dessen, Andréa
Job, Viviana
author_facet Lombardi, Charlotte
Tolchard, James
Bouillot, Stephanie
Signor, Luca
Gebus, Caroline
Liebl, David
Fenel, Daphna
Teulon, Jean-Marie
Brock, Juliane
Habenstein, Birgit
Pellequer, Jean-Luc
Faudry, Eric
Loquet, Antoine
Attrée, Ina
Dessen, Andréa
Job, Viviana
author_sort Lombardi, Charlotte
collection PubMed
description The type three secretion system (T3SS) is a macromolecular protein nano-syringe used by different bacterial pathogens to inject effectors into host cells. The extracellular part of the syringe is a needle-like filament formed by the polymerization of a 9-kDa protein whose structure and proper localization on the bacterial surface are key determinants for efficient toxin injection. Here, we combined in vivo, in vitro, and in silico approaches to characterize the Pseudomonas aeruginosa T3SS needle and its major component PscF. Using a combination of mutagenesis, phenotypic analyses, immunofluorescence, proteolysis, mass spectrometry, atomic force microscopy, electron microscopy, and molecular modeling, we propose a model of the P. aeruginosa needle that exposes the N-terminal region of each PscF monomer toward the outside of the filament, while the core of the fiber is formed by the C-terminal helix. Among mutations introduced into the needle protein PscF, D76A, and P47A/Q54A caused a defect in the assembly of the needle on the bacterial surface, although the double mutant was still cytotoxic on macrophages in a T3SS-dependent manner and formed filamentous structures in vitro. These results suggest that the T3SS needle of P. aeruginosa displays an architecture that is similar to that of other bacterial needles studied to date and highlight the fact that small, targeted perturbations in needle assembly can inhibit T3SS function. Therefore, the T3SS needle represents an excellent drug target for small molecules acting as virulence blockers that could disrupt pathogenesis of a broad range of bacteria.
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spelling pubmed-64550542019-04-18 Structural and Functional Characterization of the Type Three Secretion System (T3SS) Needle of Pseudomonas aeruginosa Lombardi, Charlotte Tolchard, James Bouillot, Stephanie Signor, Luca Gebus, Caroline Liebl, David Fenel, Daphna Teulon, Jean-Marie Brock, Juliane Habenstein, Birgit Pellequer, Jean-Luc Faudry, Eric Loquet, Antoine Attrée, Ina Dessen, Andréa Job, Viviana Front Microbiol Microbiology The type three secretion system (T3SS) is a macromolecular protein nano-syringe used by different bacterial pathogens to inject effectors into host cells. The extracellular part of the syringe is a needle-like filament formed by the polymerization of a 9-kDa protein whose structure and proper localization on the bacterial surface are key determinants for efficient toxin injection. Here, we combined in vivo, in vitro, and in silico approaches to characterize the Pseudomonas aeruginosa T3SS needle and its major component PscF. Using a combination of mutagenesis, phenotypic analyses, immunofluorescence, proteolysis, mass spectrometry, atomic force microscopy, electron microscopy, and molecular modeling, we propose a model of the P. aeruginosa needle that exposes the N-terminal region of each PscF monomer toward the outside of the filament, while the core of the fiber is formed by the C-terminal helix. Among mutations introduced into the needle protein PscF, D76A, and P47A/Q54A caused a defect in the assembly of the needle on the bacterial surface, although the double mutant was still cytotoxic on macrophages in a T3SS-dependent manner and formed filamentous structures in vitro. These results suggest that the T3SS needle of P. aeruginosa displays an architecture that is similar to that of other bacterial needles studied to date and highlight the fact that small, targeted perturbations in needle assembly can inhibit T3SS function. Therefore, the T3SS needle represents an excellent drug target for small molecules acting as virulence blockers that could disrupt pathogenesis of a broad range of bacteria. Frontiers Media S.A. 2019-03-29 /pmc/articles/PMC6455054/ /pubmed/31001211 http://dx.doi.org/10.3389/fmicb.2019.00573 Text en Copyright © 2019 Lombardi, Tolchard, Bouillot, Signor, Gebus, Liebl, Fenel, Teulon, Brock, Habenstein, Pellequer, Faudry, Loquet, Attrée, Dessen and Job. http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
spellingShingle Microbiology
Lombardi, Charlotte
Tolchard, James
Bouillot, Stephanie
Signor, Luca
Gebus, Caroline
Liebl, David
Fenel, Daphna
Teulon, Jean-Marie
Brock, Juliane
Habenstein, Birgit
Pellequer, Jean-Luc
Faudry, Eric
Loquet, Antoine
Attrée, Ina
Dessen, Andréa
Job, Viviana
Structural and Functional Characterization of the Type Three Secretion System (T3SS) Needle of Pseudomonas aeruginosa
title Structural and Functional Characterization of the Type Three Secretion System (T3SS) Needle of Pseudomonas aeruginosa
title_full Structural and Functional Characterization of the Type Three Secretion System (T3SS) Needle of Pseudomonas aeruginosa
title_fullStr Structural and Functional Characterization of the Type Three Secretion System (T3SS) Needle of Pseudomonas aeruginosa
title_full_unstemmed Structural and Functional Characterization of the Type Three Secretion System (T3SS) Needle of Pseudomonas aeruginosa
title_short Structural and Functional Characterization of the Type Three Secretion System (T3SS) Needle of Pseudomonas aeruginosa
title_sort structural and functional characterization of the type three secretion system (t3ss) needle of pseudomonas aeruginosa
topic Microbiology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6455054/
https://www.ncbi.nlm.nih.gov/pubmed/31001211
http://dx.doi.org/10.3389/fmicb.2019.00573
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