title: Internalization of Pseudomonas aeruginosa Strain PAO1 into Epithelial Cells Is Promoted by Interaction of a T6SS Effector with the Microtubule Network.
abstract: Invasion of nonphagocytic cells through rearrangement of the actin cytoskeleton is a common immune evasion mechanism used by most intracellular bacteria. However, some pathogens modulate host microtubules as well by a still poorly understood mechanism. In this study, we aim at deciphering the mechanisms by which the opportunistic bacterial pathogen Pseudomonas aeruginosa invades nonphagocytic cells, although it is considered mainly an extracellular bacterium. Using confocal microscopy and immunofluorescence, we show that the evolved VgrG2b effector of P. aeruginosa strain PAO1 is delivered into epithelial cells by a type VI secretion system, called H2-T6SS, involving the VgrG2a component. An in vivo interactome of VgrG2b in host cells allows the identification of microtubule components, including the γ-tubulin ring complex (γTuRC), a multiprotein complex catalyzing microtubule nucleation, as the major host target of VgrG2b. This interaction promotes a microtubule-dependent internalization of the bacterium since colchicine and nocodazole, two microtubule-destabilizing drugs, prevent VgrG2b-mediated P. aeruginosa entry even if the invasion still requires actin. We further validate our findings by demonstrating that the type VI injection step can be bypassed by ectopic production of VgrG2b inside target cells prior to infection. Moreover, such uncoupling between VgrG2b injection and bacterial internalization also reveals that they constitute two independent steps. With VgrG2b, we provide the first example of a bacterial protein interacting with the γTuRC. Our study offers key insight into the mechanism of self-promoting invasion of P. aeruginosa into human cells via a directed and specific effector-host protein interaction.
  
  
  OBJECTIVE
  Innate immunity and specifically professional phagocytic cells are key determinants in the ability of the host to control P. aeruginosa infection. However, among various virulence strategies, including attack, this opportunistic bacterial pathogen is able to avoid host clearance by triggering its own internalization in nonphagocytic cells. We previously showed that a protein secretion/injection machinery, called the H2 type VI secretion system (H2-T6SS), promotes P. aeruginosa uptake by epithelial cells. Here we investigate which H2-T6SS effector enables P. aeruginosa to enter nonphagocytic cells. We show that VgrG2b is delivered by the H2-T6SS machinery into epithelial cells, where it interacts with microtubules and, more particularly, with the γ-tubulin ring complex (γTuRC) known as the microtubule-nucleating center. This interaction precedes a microtubule- and actin-dependent internalization of P. aeruginosa. We thus discovered an unprecedented target for a bacterial virulence factor since VgrG2b constitutes, to our knowledge, the first example of a bacterial protein interacting with the γTuRC.
authors.affiliation: CNRS & Aix-Marseille Université, Laboratoire d'Ingénierie des Systèmes Macromoléculaires (UMR7255), Institut de Microbiologie de la Méditerranée, Marseille, France.
authors.forename: Thibault G
authors.initials: TG
authors.lastname: Sana
authors.affiliation: Research Center for Molecular Medicine of the Austrian Academy of Sciences, Vienna, Austria.
authors.forename: Christoph
authors.initials: C
authors.lastname: Baumann
authors.affiliation: Centre de Biologie du Développement, Université Paul Sabatier, Toulouse, France.
authors.forename: Andreas
authors.initials: A
authors.lastname: Merdes
authors.affiliation: CNRS & Aix-Marseille Université, Laboratoire d'Ingénierie des Systèmes Macromoléculaires (UMR7255), Institut de Microbiologie de la Méditerranée, Marseille, France.
authors.forename: Chantal
authors.initials: C
authors.lastname: Soscia
authors.affiliation: Department of Microbiology and Ecosystem Science, University of Vienna, Vienna, Austria.
authors.forename: Thomas
authors.initials: T
authors.lastname: Rattei
authors.forename: Abderrahman
authors.initials: A
authors.lastname: Hachani
authors.affiliation: Department of Life Sciences, MRC Centre for Molecular Bacteriology and Infection, Imperial College London, London, United Kingdom.
authors.forename: Cerith
authors.initials: C
authors.lastname: Jones
authors.affiliation: Research Center for Molecular Medicine of the Austrian Academy of Sciences, Vienna, Austria.
authors.forename: Keiryn L
authors.initials: KL
authors.lastname: Bennett
authors.affiliation: Department of Life Sciences, MRC Centre for Molecular Bacteriology and Infection, Imperial College London, London, United Kingdom.
authors.forename: Alain
authors.initials: A
authors.lastname: Filloux
authors.affiliation: Research Center for Molecular Medicine of the Austrian Academy of Sciences, Vienna, Austria.
authors.forename: Giulio
authors.initials: G
authors.lastname: Superti-Furga
authors.affiliation: CNRS & Aix-Marseille Université, Laboratoire d'Ingénierie des Systèmes Macromoléculaires (UMR7255), Institut de Microbiologie de la Méditerranée, Marseille, France.
authors.forename: Romé
authors.initials: R
authors.lastname: Voulhoux
authors.affiliation: CNRS & Aix-Marseille Université, Laboratoire d'Ingénierie des Systèmes Macromoléculaires (UMR7255), Institut de Microbiologie de la Méditerranée, Marseille, France bleves@imm.cnrs.fr.
authors.forename: Sophie
authors.initials: S
authors.lastname: Bleves
mesh_terms: D001426:Bacterial Proteins / Q000378:metabolism*; D004705:Endocytosis; D004847:Epithelial Cells / Q000382:microbiology* / Q000502:physiology*; D006367:HeLa Cells; D054884:Host-Pathogen Interactions; D006801:Humans; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D008870:Microtubules / Q000378:metabolism*; D011485:Protein Binding; D025941:Protein Interaction Mapping; D021381:Protein Transport; D011550:Pseudomonas aeruginosa / Q000502:physiology*; D000069376:Type VI Secretion Systems; D037521:Virulence Factors / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D000069376:Type VI Secretion Systems; D037521:Virulence Factors