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5 results on '"Laureen Logger"'

1. In situ and high-resolution Cryo-EM structure of the Type VI secretion membrane complex

Author

Riccardo Pellarin, Victoria Schmidt, Yassine Cherrak, Laureen Logger, Rémi Fronzes, Romain Kooger, Chiara Rapisarda, Eric Durand, Eric Cascales, Martin Pilhofer, Institut Européen de Chimie et Biologie (IECB), Université de Bordeaux (UB)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Laboratoire d'ingénierie des systèmes macromoléculaires (LISM), Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Aix Marseille Université (AMU), Institute of Biochemistry [ETH Zürich], Department of Biology [ETH Zürich] (D-BIOL), Eidgenössische Technische Hochschule - Swiss Federal Institute of Technology [Zürich] (ETH Zürich)- Eidgenössische Technische Hochschule - Swiss Federal Institute of Technology [Zürich] (ETH Zürich), Bioinformatique structurale - Structural Bioinformatics, Centre National de la Recherche Scientifique (CNRS)-Institut Pasteur [Paris], Paris-Centre de Recherche Cardiovasculaire (PARCC - UMR-S U970), Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Hôpital Européen Georges Pompidou [APHP] (HEGP), Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Hôpitaux Universitaires Paris Ouest - Hôpitaux Universitaires Île de France Ouest (HUPO)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Hôpitaux Universitaires Paris Ouest - Hôpitaux Universitaires Île de France Ouest (HUPO), European Institute of Chemistry and Biology, Université Sciences et Technologies - Bordeaux 1, This work was funded by the Centre National de la Recherche Scientifique, the Aix‐Marseille Université, and grants from the Agence Nationale de la Recherche (ANR‐14‐CE14‐0006‐02, ANR‐17‐CE11‐0039‐01) and the Fondation pour la Recherche Médicale (DEQ20180339165) to EC. ED was supported by the INSERM and an EMBO short‐term fellowship (ASTF 417 – 2015). YC is supported by a Doctoral school PhD fellowship from the FRM (ECO20160736014). VS is supported by a post‐doctoral fellowship from the association Espoir contre la Mucoviscidose. LL was supported by a fourth year PhD fellowship from the FRM (FDT20160435498). MP is funded by the European Research Council, the Swiss National Science Foundation and the Helmut Horten Foundation. RF and CR were supported by IDEX Bordeaux through a 'chaire d'excellence' to RF., ANR-17-CE11-0039,T6-PLATFORM,Une approche multidisciplinaire et intégrative pour comprendre l'assemblage, la structure et la dynamique d'une plateforme de queue contractile(2017), ANR-14-CE14-0006,B-War,Guerre bactérienne: Architecture et Fonction du Système de Sécrétion de Type VI(2014), Aix Marseille Université (AMU)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Hôpital Européen Georges Pompidou [APHP] (HEGP), Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Hôpitaux Universitaires Paris Ouest - Hôpitaux Universitaires Île de France Ouest (HUPO)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Hôpitaux Universitaires Paris Ouest - Hôpitaux Universitaires Île de France Ouest (HUPO)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM), Université Sciences et Technologies - Bordeaux 1 (UB), Eidgenössische Technische Hochschule - Swiss Federal Institute of Technology in Zürich [Zürich] (ETH Zürich)-Eidgenössische Technische Hochschule - Swiss Federal Institute of Technology in Zürich [Zürich] (ETH Zürich), Bioinformatique structurale, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Université Paris Descartes - Paris 5 (UPD5)-Hôpital Européen Georges Pompidou [APHP] (HEGP), Hôpitaux Universitaires Paris Ouest - Hôpitaux Universitaires Île de France Ouest (HUPO)-Assistance publique - Hôpitaux de Paris (AP-HP) (APHP)-Hôpitaux Universitaires Paris Ouest - Hôpitaux Universitaires Île de France Ouest (HUPO)-Assistance publique - Hôpitaux de Paris (AP-HP) (APHP)-Institut National de la Santé et de la Recherche Médicale (INSERM), and Centre National de la Recherche Scientifique (CNRS)-Université de Bordeaux (UB)-Institut National de la Santé et de la Recherche Médicale (INSERM)

Subjects
In situ, MESH: Protein Structure, Quaternary, Cryo-electron microscopy, [SDV]Life Sciences [q-bio], MESH: Type VI Secretion Systems, Single particle analysis, MESH: Escherichia coli Proteins, law.invention, 03 medical and health sciences, law, MESH: Protein Binding, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Secretion, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], MESH: Bacterial Secretion Systems, 030304 developmental biology, Type VI secretion system, 0303 health sciences, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], MESH: Escherichia coli, 030306 microbiology, Chemistry, Negative stain, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM], [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM], [SDV.MP]Life Sciences [q-bio]/Microbiology and Parasitology, Membrane, Biophysics, MESH: Cryoelectron Microscopy, MESH: Membrane Proteins, Electron microscope, MESH: Models, Molecular, MESH: Cell Membrane
Abstract

Bacteria have evolved macromolecular machineries that secrete effectors and toxins to survive and thrive in diverse environments. The type VI secretion system (T6SS) is a contractile machine that is related to Myoviridae phages. The T6SS is composed of a baseplate that contains a spike onto which an inner tube is built, surrounded by a contractile sheath. Unlike phages that are released to and act in the extracellular medium, the T6SS is an intracellular machine inserted in the bacterial membranes by a trans-envelope complex. This membrane complex (MC) comprises three proteins: TssJ, TssL and TssM. We previously reported the low-resolution negative stain electron microscopy structure of the enteroaggregative Escherichia coli MC and proposed a rotational 5-fold symmetry with a TssJ:TssL:TssM stoichiometry of 2:2:2. Here, cryo-electron tomography analysis of the T6SS MC confirmed the 5-fold symmetry in situ and identified the regions of the structure that insert into the bacterial membranes. A high resolution model obtained by single particle cryo-electron microscopy reveals its global architecture and highlights new features: five additional copies of TssJ, yielding a TssJ:TssL:TssM stoichiometry of 3:2:2, a 11-residue loop in TssM, protruding inside the lumen of the MC and constituting a functionally important periplasmic gate, and hinge regions. Based on these data, we revisit the model on the mechanism of action of the MC during T6SS assembly and function.

Published
2018
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2. Structure–Function Analysis of the C-Terminal Domain of the Type VI Secretion TssB Tail Sheath Subunit

Author

Laureen Logger, Stéphanie Blangy, Badreddine Douzi, Silvia Spinelli, Christian Cambillau, Eric Cascales, Laboratoire d'ingénierie des systèmes macromoléculaires (LISM), Aix Marseille Université (AMU)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Architecture et fonction des macromolécules biologiques (AFMB), Institut National de la Recherche Agronomique (INRA)-Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS), Environnement, Bioénergie, Microalgues et Plantes (EBMP), Institut de Biosciences et Biotechnologies d'Aix-Marseille (ex-IBEB) (BIAM), Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Aix Marseille Université (AMU), Centre National de la Recherche Scientifique (CNRS)-Aix Marseille Université (AMU)-Institut National de la Recherche Agronomique (INRA), Bioénergie et Microalgues (EBM), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS), Centre National de la Recherche Scientifique (CNRS)-Aix Marseille Université (AMU)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)-Aix Marseille Université (AMU)-Direction de Recherche Fondamentale (CEA) (DRF (CEA))

Subjects
0301 basic medicine, Models, Molecular, Protein Conformation, Protein subunit, [SDV]Life Sciences [q-bio], 030106 microbiology, Protein domain, Crystallography, X-Ray, 03 medical and health sciences, chemistry.chemical_compound, Protein structure, Protein Domains, Structural Biology, Type VI Secretion Systems/*chemistry, Escherichia coli, sheath, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], Molecular Biology, ComputingMilieux_MISCELLANEOUS, Type VI secretion system, Escherichia coli Proteins/*chemistry, contractile injection system, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], Effector, C-terminus, Escherichia coli Proteins, X-ray, protein secretion, [SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, Escherichia coli/*chemistry, Type VI Secretion Systems, [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM], [SDV.BBM.BP]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biophysics, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM], 030104 developmental biology, chemistry, Cytoplasm, Biophysics, structure, Protein Multimerization, DNA
Abstract

International audience; The type VI secretion system (T6SS) is a specialized macromolecular complex dedicated to the delivery of protein effectors into both eukaryotic and bacterial cells. The general mechanism of action of the T6SS is similar to the injection of DNA by contractile bacteriophages. The cytoplasmic portion of the T6SS is evolutionarily, structurally and functionally related to the phage tail complex. It is composed of an inner tube made of stacked Hcp hexameric rings, engulfed within a sheath and built on a baseplate. This sheath undergoes cycles of extension and contraction, and the current model proposes that the sheath contraction propels the inner tube toward the target cell for effector delivery. The sheath comprises two subunits: TssB and TssC that polymerize under an extended conformation. Here, we show that isolated TssB forms trimers, and we report the crystal structure of a C-terminal fragment of TssB. This fragment comprises a long helix followed by a helical hairpin that presents surface-exposed charged residues. Site-directed mutagenesis coupled to functional assay further showed that these charges are required for proper assembly of the sheath. Positioning of these residues in the extended T6SS sheath structure suggests that they may mediate contacts with the baseplate.

Published
2018
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3. Molecular Dissection of the Interface between the Type VI Secretion TssM Cytoplasmic Domain and the TssG Baseplate Component

Author

Marie Guérin, Eric Durand, Laureen Logger, Eric Cascales, Marie-Stéphanie Aschtgen, Laboratoire d'ingénierie des systèmes macromoléculaires (LISM), Aix Marseille Université (AMU)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), and Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Aix Marseille Université (AMU)

Subjects
0301 basic medicine, Multiprotein complex, [SDV]Life Sciences [q-bio], DNA Mutational Analysis, 030106 microbiology, [SDV.BC.BC]Life Sciences [q-bio]/Cellular Biology/Subcellular Processes [q-bio.SC], Biology, Bacterial cell structure, 03 medical and health sciences, Structural Biology, Protein Interaction Mapping, [SDV.BC.IC]Life Sciences [q-bio]/Cellular Biology/Cell Behavior [q-bio.CB], Escherichia coli, Secretion, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], Molecular Biology, ComputingMilieux_MISCELLANEOUS, Type VI secretion system, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], Escherichia coli Proteins, Membrane Proteins, [SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, [SDV.BBM.MN]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular Networks [q-bio.MN], Type VI Secretion Systems, [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, Transmembrane protein, Transport protein, 030104 developmental biology, Secretory protein, Biochemistry, Cytoplasm, Mutagenesis, Site-Directed, Biophysics, Protein Multimerization, Protein Binding
Abstract

The type VI secretion system (T6SS) is a multiprotein complex that catalyses toxin secretion through the bacterial cell envelope of various Gram-negative bacteria including important human pathogens. This machine uses a bacteriophage-like contractile tail to puncture the prey cell and inject harmful toxins. The T6SS tail comprises an inner tube capped by the cell-puncturing spike and wrapped by the contractile sheath. This structure is built on an assembly platform, the baseplate, which is anchored to the bacterial cell envelope by the TssJLM membrane complex (MC). This MC serves as both a tail docking station and a channel for the passage of the inner tube. The TssM transmembrane protein is a key component of the MC as it connects the inner and outer membranes. In this study, we define the TssM topology, highlighting a large but poorly studied 35-kDa cytoplasmic domain, TssMCyto, located between two transmembrane segments. Protein–protein interaction assays further show that TssMCyto oligomerises and makes contacts with several baseplate components. Using computer predictions, we delineate two subdomains in TssMCyto, including a nucleotide triphosphatase (NTPase) domain, followed by a 110-aa extension. Finally, site-directed mutagenesis coupled to functional assays reveals the contribution of these subdomains and conserved motifs to the interaction with T6SS partners and to the function of the secretion apparatus.

Published
2016
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4. Biogenesis and structure of a Type VI secretion membrane core complex

Author

Abdelrahim Zoued, Annick Dujeancourt, Laureen Logger, Rémi Fronzes, Benjamin Bardiaux, Eric Cascales, Van Son Nguyen, Silvia Spinelli, Aline Desmyter, Marie-Stéphanie Aschtgen, Eric Durand, Christian Cambillau, Gérard Pehau-Arnaudet, Alain Roussel, Laboratoire d'ingénierie des systèmes macromoléculaires (LISM), Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Aix Marseille Université (AMU), Architecture et fonction des macromolécules biologiques (AFMB), Centre National de la Recherche Scientifique (CNRS)-Aix Marseille Université (AMU)-Institut National de la Recherche Agronomique (INRA), Biologie Structurale de la Sécrétion Bactérienne, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Bioinformatique structurale - Structural Bioinformatics, Centre National de la Recherche Scientifique (CNRS)-Institut Pasteur [Paris], This work was funded by Agence Nationale de la Recherche (ANR) grants ANR-10-JCJC-1303-03 to E.C., Bip:Bip to R.F., ANR-14-CE14-0006-02 to C.C. and E.C., and supported by the French Infrastructure for Integrated Structural Biology (FRISBI) ANR-10-INSB-05-01. E.D. was supported by a post-doctoral fellowship from the Fondation pour la Recherche Médicale (SPF20101221116) and ANR grants Bip:Bip and ANR-10-JCJC-1303-03. V.S.N. was supported by a PhD grant from the French Embassy in Vietnam (792803C). A.Z., L.L. and M.S.A. were recipients of doctoral fellowships from the French Ministère de la Recherche. A.Z. received a Fondation pour la Recherche Médicale fellowship (FDT20140931060)., ANR-10-JCJC-1303,A Fun T6SS,Assemblage et Fonction des Systèmes de Sécrétion de Type VI bactériens(2010), ANR-10-INBS-0005,FRISBI,Infrastructure Française pour la Biologie Structurale Intégrée(2010), ANR-14-CE14-0006,B-War,Guerre bactérienne: Architecture et Fonction du Système de Sécrétion de Type VI(2014), Institut Pasteur [Paris] - Centre National de la Recherche Scientifique (CNRS), Aix Marseille Université (AMU) - Institut National de la Santé et de la Recherche Médicale (INSERM) - Centre National de la Recherche Scientifique (CNRS), Centre National de la Recherche Scientifique (CNRS) - Aix Marseille Université (AMU) - Institut National de la Recherche Agronomique (INRA), Microscopie ultrastructurale - Ultrapole (CITECH), Institut Pasteur [Paris], Bioinformatique structurale, This work was funded by Agence Nationale de la Recherche (ANR) grants ANR-10-JCJC-1303-03 to E.C., Bip:Bip to R.F., ANR-14-CE14-0006-02 to C.C. and E.C., and supported by the French Infrastructure for Integrated Structural Biology (FRISBI) ANR-10-INSB-05-01. E.D. was supported by a post-doctoral fellowship from the Fondation pour la Recherche Médicale (SPF20101221116) and ANR grants Bip:Bip and ANR-10-JCJC-1303-03. V.S.N. was supported by a PhD grant from the French Embassy in Vietnam (792803C). A.Z., L.L. and M.S.A. were recipients of doctoral fellowships from the French Ministère de la Recherche. A.Z. received a Fondation pour la Recherche Médicale fellowship (FDT20140931060), We thank O. Francetic for providing anti-DglA and anti-OmpF antibodies. We thank E. Marza, P. Violinova Krasteva and H. Remaut for comments on the manuscript, and T. Mignot, M. Guzzo and L. Espinosa for advice about the fluorescence microscopy experiments and the statistical analyses. We also thank the members of the R.F. and E.C. research groups for discussions and suggestions, and R. Lloubès, J. Sturgis and A. Galinier for encouragement. We thank the ERSF and Soleil Synchrotron radiation facilities for beamline allocation, ANR-10-JCJC-1303, A Fun T6SS, Assemblage et Fonction des Systèmes de Sécrétion de Type VI bactériens(2010), ANR-14-CE14-0006, B-War, Guerre bactérienne: Architecture et Fonction du Système de Sécrétion de Type VI(2014), ANR-10-INBS-05-01/10-INBS-0005, FRISBI, Infrastructure Française pour la Biologie Structurale Intégrée(2010), CASCALES, ERIC, Jeunes Chercheuses et Jeunes Chercheurs - Assemblage et Fonction des Systèmes de Sécrétion de Type VI bactériens - - A Fun T6SS2010 - ANR-10-JCJC-1303 - JCJC - VALID, Infrastructure Française pour la Biologie Structurale Intégrée - - FRISBI2010 - ANR-10-INBS-0005 - INBS - VALID, Appel à projets générique - Guerre bactérienne: Architecture et Fonction du Système de Sécrétion de Type VI - - B-War2014 - ANR-14-CE14-0006 - Appel à projets générique - VALID, Aix Marseille Université (AMU)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Institut National de la Recherche Agronomique (INRA)-Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), and ANR-10-INBS-05-01/10-INBS-0005,FRISBI,Infrastructure Française pour la Biologie Structurale Intégrée(2010)

Subjects
Models, Molecular, Cytoplasm, MESH : Bacterial Secretion Systems, MESH : Escherichia coli/chemistry, MESH: Escherichia coli Proteins, MESH : Cytoplasm/chemistry, [SDV.BC.BC]Life Sciences [q-bio]/Cellular Biology/Subcellular Processes [q-bio.SC], Crystallography, X-Ray, [SDV.BBM.BM] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, MESH : Multiprotein Complexes/biosynthesis, MESH: Protein Structure, Tertiary, Bacterial secretion, MESH : Membrane Proteins/biosynthesis, MESH: Lipopeptides, MESH : Escherichia coli Proteins/chemistry, MESH: Bacterial Secretion Systems, Bacterial Secretion Systems, [SDV.BBM.BC] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], MESH : Cell Membrane/chemistry, "structure of a type VI", MESH : Membrane Proteins/chemistry, MESH : Escherichia coli Proteins/biosynthesis, 0303 health sciences, [SDV.BIBS] Life Sciences [q-bio]/Quantitative Methods [q-bio.QM], Multidisciplinary, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], MESH: Escherichia coli, Escherichia coli Proteins, MESH: Periplasm, MESH : Escherichia coli/metabolism, MESH: Protein Subunits, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM], [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM], Biochemistry, Periplasm, MESH : Cytoplasm/metabolism, MESH: Membrane Proteins, Cell envelope, Bacterial outer membrane, Porosity, MESH : Lipopeptides/biosynthesis, MESH : Protein Structure, Tertiary, MESH: Models, Molecular, [SDV.BBM.BS] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], MESH : Models, Molecular, MESH : Lipopeptides/chemistry, MESH: Microscopy, Electron, Biology, MESH : Protein Subunits/biosynthesis, Lipopeptides, 03 medical and health sciences, MESH: Porosity, Escherichia coli, [SDV.BC.BC] Life Sciences [q-bio]/Cellular Biology/Subcellular Processes [q-bio.SC], Electron microscopy, Secretion, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], "Biogenesis", MESH : Cell Membrane/metabolism, "secretion membrane", MESH : Protein Subunits/chemistry, 030304 developmental biology, Type VI secretion system, X-ray crystallography, MESH : Periplasm/metabolism, MESH : Periplasm/chemistry, 030306 microbiology, MESH: Cytoplasm, Cell Membrane, Membrane Proteins, [SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, Periplasmic space, MESH: Multiprotein Complexes, MESH: Crystallography, X-Ray, [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, MESH : Microscopy, Electron, MESH : Multiprotein Complexes/chemistry, Protein Structure, Tertiary, Microscopy, Electron, Protein Subunits, Membrane protein, Multiprotein Complexes, Biophysics, [SDV.MP.BAC] Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, MESH : Porosity, MESH : Crystallography, X-Ray, Biogenesis, MESH: Cell Membrane
Abstract

International audience; Bacteria share their ecological niches with other microbes. The bacterial type VI secretion system is one of the key players in microbial competition, as well as being an important virulence determinant during bacterial infections. It assembles a nano-crossbow-like structure in the cytoplasm of the attacker cell that propels an arrow made of a haemolysin co-regulated protein (Hcp) tube and a valine–glycine repeat protein G (VgrG) spike and punctures the prey’s cell wall. The nano-crossbow is stably anchored to the cell envelope of the attacker by a membrane core complex. Here we show that this complex is assembled by the sequential addition of three type VI subunits (Tss)— TssJ, TssM and TssL—and present a structure of the fully assembled complex at 11.6 A ̊ resolution, determined by negative-stain electron microscopy. With overall C5 symmetry, this 1.7-megadalton complex comprises a large base in the cytoplasm. It extends in the periplasm via ten arches to form a double-ring structure containing the carboxy-terminal domain of TssM (TssMct) and TssJ that is anchored in the outer membrane. The crystal structure of the TssMct–TssJ complex coupled to whole-cell accessibility studies suggest that large conformational changes induce transient pore formation in the outer membrane, allowing passage of the attacking Hcp tube/VgrG spike.

Published
2015
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5. Dissection of the TssB-TssC Interface during Type VI Secretion Sheath Complex Formation

Author

Laure Journet, Yannick R. Brunet, Laureen Logger, Eric Cascales, Christian Cambillau, Badreddine Douzi, Xiang Y. Zhang, State Key Laboratory of Chemical Engineering (SKLCE), East China University of Science and Technology, Laboratoire d'ingénierie des systèmes macromoléculaires (LISM), Aix Marseille Université (AMU)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Architecture et fonction des macromolécules biologiques (AFMB), Institut National de la Recherche Agronomique (INRA)-Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS), Architecture et fonction des Macromolécules Biologiques - UMR 6098 (AFMB), Université de Provence - Aix-Marseille 1-Centre National de la Recherche Scientifique (CNRS), Institut Gilbert-Laustriat : Biomolécules, Biotechnologie, Innovation Thérapeutique, Université Louis Pasteur - Strasbourg I-Centre National de la Recherche Scientifique (CNRS), ANR-10-JCJC-1303,A Fun T6SS,Assemblage et Fonction des Systèmes de Sécrétion de Type VI bactériens(2010), Douzi, Badreddine, Jeunes Chercheuses et Jeunes Chercheurs - Assemblage et Fonction des Systèmes de Sécrétion de Type VI bactériens - - A Fun T6SS2010 - ANR-10-JCJC-1303 - JCJC - VALID, Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Aix Marseille Université (AMU), and Centre National de la Recherche Scientifique (CNRS)-Aix Marseille Université (AMU)-Institut National de la Recherche Agronomique (INRA)

Subjects
[SDV.BBM.BS] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], Substitution mutation, [SDV.BBM.BP] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biophysics, lcsh:Medicine, [SDV.BBM.BM] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, Protein–protein interaction, Bacteriophage, Viral Proteins, 03 medical and health sciences, Protein structure, Secretion systems, Fluorescence microscope, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Secretion, Bacteriophages, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], lcsh:Science, [SDV.BBM.BC] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], Vibrio cholerae, 030304 developmental biology, Type VI secretion system, Fluorescence microscopy, 0303 health sciences, Multidisciplinary, biology, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], 030306 microbiology, lcsh:R, Protein interactions, [SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, biology.organism_classification, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM], [SDV.BBM.BP]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biophysics, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM], Microscopy, Fluorescence, Biochemistry, Cytoplasm, Pseudomonas aeruginosa, Mutagenesis, Site-Directed, Biophysics, lcsh:Q, Cell envelope, Research Article
Abstract

International audience; The Type VI secretion system (T6SS) is a versatile machine that delivers toxins into either eukaryotic or bacterial cells. At a molecular level, the T6SS is composed of a membrane complex that anchors a long cytoplasmic tubular structure to the cell envelope. This structure is thought to resemble the tail of contractile bacteriophages. It is composed of the Hcp protein that assembles into hexameric rings stacked onto each other to form a tube similar to the phage tail tube. This tube is proposed to be wrapped by a structure called the sheath, composed of two proteins, TssB and TssC. It has been shown using fluorescence microscopy that the TssB and TssC proteins assemble into a tubular structure that cycles between long and short conformations suggesting that, similarly to the bacteriophage sheath, the T6SS sheath undergoes elongation and contraction events. The TssB and TssC proteins have been shown to interact and a specific α-helix of TssB is required for this interaction. Here, we confirm that the TssB and TssC proteins interact in enteroaggregative E. coli. We further show that this interaction requires the N-terminal region of TssC and the conserved α-helix of TssB. Using site-directed mutagenesis coupled to phenotypic analyses, we demonstrate that an hydrophobic motif located in the N-terminal region of this helix is required for interaction with TssC, sheath assembly and T6SS function.

Published
2013
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