Descriptor: "MESH: Bacterial Secretion Systems" - Searchworks@Jio Institute Digital Library Search Results

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27 results on '"MESH: Bacterial Secretion Systems"'

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

2. RD5-mediated lack of PE_PGRS and PPE-MPTR export in BCG vaccine strains results in strong reduction of antigenic repertoire but little impact on protection

Author: Louis S. Ates, Merel P.M. Damen, Roland Brosch, Daria Bottai, Wafa Frigui, Fadel Sayes, Laleh Majlessi, Wilbert Bitter, Marcel A. Behr, Roy Ummels, Jeroen W. J. van Heijst, Pathogénomique mycobactérienne intégrée - Integrated Mycobacterial Pathogenomics, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), University of Amsterdam [Amsterdam] (UvA), Vrije Universiteit Amsterdam [Amsterdam] (VU), VU University Medical Center [Amsterdam], University of Pisa - Università di Pisa, McGill University Health Center [Montreal] (MUHC), LSA and RB acknowledge the support in part by grants of the Agence national de la Recherche ANR-14-JAMR-001-02, ANR-10-LABX-62-IBEID, and ANR-16-CE35-0009, the Fondation pour la Recherche Médicale FRM (DEQ20130326471) and the European Union's Horizon 2020 Research and Innovation Program grant TBVAC2020 643381. LSA and JWJvH are supported by the Netherlands Organisation for Scientific Research (Vidi grant 91717305 to JWJvH)., VU University medical center, Medical Microbiology and Infection Prevention, AII - Infectious diseases, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), ANR-16-CE35-0009,TBemerg,Naissance d'un tueur: facteurs génétiques et adaptations métaboliques impliquées dans l'émergence des bacilles tuberculeux épidémiques(2016), Experimental Immunology, AIMMS, and Molecular Microbiology
Subjects: MESH: Mycobacterium tuberculosis, Pathology and Laboratory Medicine, MESH: Genome, Bacterial, Biochemistry, Mice, Animal Cells, [SDV.MHEP.MI]Life Sciences [q-bio]/Human health and pathology/Infectious diseases, Microbial Physiology, MESH: Animals, Bacterial Physiology, Biology (General), Immune Response, MESH: Bacterial Proteins, 3. Good health, Actinobacteria, Multigene Family, MESH: Membrane Proteins, Cellular Types, Tuberculosis, QH301-705.5, Virulence Factors, Immune Cells, Immunology, Virulence, Microbiology, Mycobacterium tuberculosis, 03 medical and health sciences, Antigen, SDG 3 - Good Health and Well-being, Bacterial Proteins, Genetics, Secretion, Molecular Biology, Blood Cells, Bacteria, Organisms, Biology and Life Sciences, Protein Secretion, Proteins, medicine.disease, [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, Mice, Inbred C57BL, 030104 developmental biology, MESH: Multigene Family, Parasitology, Immunologic diseases. Allergy, Physiological Processes, BCG vaccine, MESH: Female, 0301 basic medicine, Physiology, Secretion Systems, MESH: Virulence, [SDV.MHEP.PSR]Life Sciences [q-bio]/Human health and pathology/Pulmonology and respiratory tract, White Blood Cells, Immune Physiology, [SDV.BC.IC]Life Sciences [q-bio]/Cellular Biology/Cell Behavior [q-bio.CB], Medicine and Health Sciences, MESH: Tuberculosis, Tuberculosis Vaccines, MESH: Bacterial Secretion Systems, Bacterial Secretion Systems, Mice, Inbred BALB C, Vaccines, Immune System Proteins, biology, T Cells, Protein Transport, Infectious Diseases, Cell Processes, Female, Pathogens, Tuberculosis vaccines, Research Article, Infectious Disease Control, MESH: Mice, Inbred BALB C, Immune system, MESH: Mice, Inbred C57BL, Virology, medicine, Animals, Antigens, MESH: Mice, Antigens, Bacterial, Membrane Proteins, Bacteriology, Cell Biology, RC581-607, biology.organism_classification, MESH: Tuberculosis Vaccines, [SDV.GEN.GH]Life Sciences [q-bio]/Genetics/Human genetics, Genome, Bacterial, MESH: Antigens, Bacterial
Abstract: Tuberculosis is the deadliest infectious disease worldwide. Although the BCG vaccine is widely used, it does not efficiently protect against pulmonary tuberculosis and an improved tuberculosis vaccine is therefore urgently needed. Mycobacterium tuberculosis uses different ESX/Type VII secretion (T7S) systems to transport proteins important for virulence and host immune responses. We recently reported that secretion of T7S substrates belonging to the mycobacteria-specific Pro-Glu (PE) and Pro-Pro-Glu (PPE) proteins of the PGRS (polymorphic GC-rich sequences) and MPTR (major polymorphic tandem repeat) subfamilies required both a functional ESX-5 system and a functional PPE38/71 protein for secretion. Inactivation of ppe38/71 and the resulting loss of PE_PGRS/PPE-MPTR secretion were linked to increased virulence of M. tuberculosis strains. Here, we show that a predicted total of 89 PE_PGRS/PPE-MPTR surface proteins are not exported by certain animal-adapted strains of the M. tuberculosis complex including M. bovis. This Δppe38/71-associated secretion defect therefore also occurs in the M. bovis-derived tuberculosis vaccine BCG and could be partially restored by introduction of the M. tuberculosis ppe38-locus. Epitope mapping of the PPE-MPTR protein PPE10, further allowed us to monitor T-cell responses in splenocytes from BCG/M. tuberculosis immunized mice, confirming the dependence of PPE10-specific immune-induction on ESX-5/PPE38-mediated secretion. Restoration of PE_PGRS/PPE-MPTR secretion in recombinant BCG neither altered global antigenic presentation or activation of innate immune cells, nor protective efficacy in two different mouse vaccination-infection models. This unexpected finding stimulates a reassessment of the immunomodulatory properties of PE_PGRS/PPE-MPTR proteins, some of which are contained in vaccine formulations currently in clinical evaluation., Author summary One of the major findings of the pioneering Mycobacterium tuberculosis H37Rv genome sequencing project was the identification of the highly abundant PE and PPE proteins, named after their N-terminal motifs Pro–Glu (PE) or Pro–Pro–Glu (PPE). Within the 20 years of research since then, many claims were made that PE/PPE proteins, including the two large subgroups encoded by repetitive sequences with very high GC content (PE_PGRS and PPE-MPTR families), are exported to the bacterial surface or beyond, and show broad immunomodulatory impact on host-pathogen interaction. We thus screened strains from different branches of the M. tuberculosis complex, including the attenuated Mycobacterium bovis BCG vaccine strains, for their capacity to export PE_PGRS/PPE-MPTR proteins. Strikingly, we found that BCG strains were unable to export the plethora of PE_PGRS/PPE-MPTR proteins due to the absence of the region of difference RD5, which in M. tuberculosis encodes PPE38, required for PE_PGRS/PPE-MPTR export. Surprisingly, the restoration of PE_PGRS/PPE-MPTR export by genetic complementation in recombinant BCG did not result in immunomodulatory changes or altered protection in mouse models. Our results thus put into perspective the numerous reports on virulence-associated immunomodulatory impact of individual PE_PGRS and PPE-MPTR proteins and open novel questions on their biological function(s).
Published: 2018

3. Analysis of Bacterial Pilus Assembly by Shearing and Immunofluorescence Microscopy

Author: Olivera Francetic, Areli Luna-Rico, Jenny-Lee Thomassin, Biochimie des Interactions Macromoléculaires / Biochemistry of Macromolecular Interactions, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), The work in our group is funded by the Institut Pasteur, CNRS and ANR grant 14-CE09-0004. A.L.R. was funded by the Pasteur-Paris University PhD program. J.L.T. was funded by the ANR grant 14-CE09-0004 and by the NSERC postdoctoral fellowship., Springer, ANR-14-CE09-0004,FiberSpace,Pili de type IV et pseudopili: structure, dynamique, assemblage et fonction moléculaire(2014), and Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)
Subjects: 0301 basic medicine, Pilus assembly, Tricine SDS-PAGE, Motility, MESH: Klebsiella oxytoca, MESH: Microscopy, Fluorescence, Immunofluorescence Microscopy, Immunofluorescence microscopy, Pilus, MESH: Fimbriae, Bacterial, 03 medical and health sciences, Immuno-detection, MESH: Bacterial Adhesion, MESH: Bacterial Secretion Systems, MESH: Bacterial Proteins, Shearing (physics), Chemistry, Shearing assay, SUPERFAMILY, MESH: Macromolecular Substances, Type 2 secretion pseudopili, [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, 030104 developmental biology, Secretory protein, Bacterial Pilus, Biophysics
Abstract: International audience; Bacterial surface appendages of the type 4 pilus superfamily play diverse roles in adherence, aggregation, motility, signaling, and macromolecular transport. Here we describe two analytical approaches to study assembly of type 4 pili and of pseudopili produced by type 2 protein secretion systems: the shearing assay and immunofluorescence microscopy. These complementary antibody-based methods allow for semiquantitative analysis of fiber assembly. The shearing assay can be scaled up to yield crude extracts of pili that can be further analyzed by electron and atomic force microscopy or by mass spectrometry.
Published: 2018

4. Pathogenic Leptospira interrogans Exoproteins Are Primarily Involved in Heterotrophic Processes

Author: Benjamin Thomas, Elisa Pappalardo, Gabriela Pretre, Mathieu Picardeau, Svenja Hester, Azad Eshghi, Biologie des Spirochètes / Biology of Spirochetes, Institut Pasteur [Paris], Sir William Dunn School of Pathology [Oxford], University of Oxford [Oxford], Gabriela Pretre is a recipient of a Bernado Houssay grant. This work (including a postdoctoral grant) was funded by the FUI 14 (Fonds Unique Interministériel) 'COVALEPT' and BPIFrance. Azad Eshghi was funded by a Carnot grant from the 'Institut Carnot- Pasteur Maladies Infectieuses'., The authors would like to thank David A. Haake for rabbit antisera against FlaA2, GroEL and LigA, Kristel Lourdault for the characterization of ligA mutant, and Karim Sebastien for his help with guinea pig heart punctures., Institut Pasteur [Paris] (IP), and University of Oxford
Subjects: Male, MESH: Protein Transport, [SDV]Life Sciences [q-bio], Guinea Pigs, Immunology, Virulence, MESH: Virulence, Biology, Microbiology, MESH: Guinea Pigs, MESH: Leptospira interrogans/genetics, 03 medical and health sciences, Bacterial Proteins, Leptospira, MESH: Heterotrophic Processes, medicine, Animals, Humans, Leptospirosis, MESH: Animals, MESH: Bacterial Secretion Systems, Bacterial Secretion Systems, MESH: Bacterial Proteins, MESH: Leptospira interrogans/growth & development, 030304 developmental biology, 0303 health sciences, MESH: Humans, 030306 microbiology, Effector, Heterotrophic Processes, MESH: Leptospira interrogans/pathogenicity, biology.organism_classification, medicine.disease, Molecular Pathogenesis, MESH: Male, 3. Good health, Transport protein, Protein Transport, [SDV.MP]Life Sciences [q-bio]/Microbiology and Parasitology, Infectious Diseases, Parasitology, Leptospira interrogans, Function (biology), Bacteria
Abstract: Leptospirosis is a life-threatening and emerging zoonotic disease with a worldwide annual occurrence of more than 1 million cases. Leptospirosis is caused by spirochetes belonging to the genus Leptospira . The mechanisms of disease manifestation in the host remain elusive, and the roles of leptospiral exoproteins in these processes have yet to be determined. Our aim in this study was to assess the composition and quantity of exoproteins of pathogenic Leptospira interrogans and to construe how these proteins contribute to disease pathogenesis. Label-free quantitative mass spectrometry of proteins obtained from Leptospira spirochetes cultured in vitro under conditions mimicking infection identified 325 exoproteins. The majority of these proteins are conserved in the nonpathogenic species Leptospira biflexa , and proteins involved in metabolism and energy-generating functions were overrepresented and displayed the highest relative abundance in culture supernatants. Conversely, proteins of unknown function, which represent the majority of pathogen-specific proteins (presumably involved in virulence mechanisms), were underrepresented. Characterization of various L. interrogans exoprotein mutants in the animal infection model revealed host mortality rates similar to those of hosts infected with wild-type L. interrogans . Collectively, these results indicate that pathogenic Leptospira exoproteins primarily function in heterotrophic processes (the processes by which organisms utilize organic substances as nutrient sources) to maintain the saprophytic lifestyle rather than the virulence of the bacteria. The underrepresentation of proteins homologous to known virulence factors, such as toxins and effectors in the exoproteome, also suggests that disease manifesting from Leptospira infection is likely caused by a combination of the primary and potentially moonlight functioning of exoproteins.
Published: 2015

5. Injection of T3SS effectors not resulting in invasion is the main targeting mechanism of Shigella toward human lymphocytes

Author: Pinaud, Laurie, Samassa, Fatoumata, Porat, Ziv, Ferrari, Mariana L., Belotserkovsky, Ilia, Parsot, Claude, Sansonetti, Philippe J., Campbell-Valois, François-Xavier, Phalipon, Armelle, Pathogénie microbienne moléculaire, Institut Pasteur [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM), Weizmann Institute of Science [Rehovot, Israël], Chaire Microbiologie et Maladies infectieuses, Collège de France (CdF (institution)), This study was supported by the French Ministry of Higher Education and Research (L.P. and F.S.), French Medical Research Foundation Grants FDT20150532160 and SPF20121226366 (to L.P., M.L.F., and F.-X.C.-V.), European Research Council Grants 232798 and 339579 (to M.L.F. and I.B.), the Marie Curie Fellowship Program (to F.-X.C.-V.), the National Science and Engineering Research Council of Canada and the Faculty of Science of the University of Ottawa (F.-X.C.-V.), and the French Government’s Investissement d’Avenir Program, Laboratoire d’Excellence 'Integrative Biology of Emerging Infectious Diseases' (Grant ANR-10-LABX-62-IBEID)., ANR-10-LABX-0062,IBEID,Integrative Biology of Emerging Infectious Diseases(2010), European Project: 232798,EC:FP7:ERC,ERC-2008-AdG,HOMEOEPITH(2009), European Project: 339579,EC:FP7:ERC,ERC-2013-ADG,DECRYPT(2014), Institut Pasteur [Paris] (IP)-Institut National de la Santé et de la Recherche Médicale (INSERM), and Collège de France - Chaire Microbiologie et Maladies infectieuses
Subjects: host–pathogen cross talk, MESH: Dysentery, Bacillary, Virulence Factors, Adaptive Immunity, MESH: Virulence, MESH: Shigella, MESH: Type III Secretion Systems, Bacterial Proteins, Cell Movement, Type III Secretion Systems, Humans, Lymphocytes, MESH: Bacterial Secretion Systems, Bacterial Secretion Systems, MESH: Bacterial Proteins, MESH: Cell Movement, Dysentery, Bacillary, MESH: Virulence Factors, MESH: Humans, Bacteria, Virulence, bacterial effectors, MESH: Host-Pathogen Interactions, biochemical phenomena, metabolism, and nutrition, Biological Sciences, [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, T3SS, human lymphocytes, MESH: Bacteria, Host-Pathogen Interactions, bacteria, MESH: Lymphocytes, Shigella, MESH: Adaptive Immunity
Abstract: International audience; The enteroinvasive bacterium Shigella is a facultative intracellular bacterium known, in vitro, to invade a large diversity of cells through the delivery of virulence effectors into the cell cytoplasm via a type III secretion system (T3SS). Here, we provide evidence that the injection of T3SS effectors does not necessarily result in cell invasion. Indeed, we demonstrate through optimization of a T3SS injection reporter that effector injection without subsequent cell invasion, termed the injection-only mechanism, is the main strategy used by Shigella to target human immune cells. We show that in vitro-activated human peripheral blood B, CD4+ T, and CD8+ T lymphocytes as well as switched memory B cells are mostly targeted by the injection-only mechanism. B and T lymphocytes residing in the human colonic lamina propria, encountered by Shigella upon its crossing of the mucosal barrier, are also mainly targeted by injection-only. These findings reveal that cells refractory to invasion can still be injected, thus extending the panel of host cells manipulated to the benefit of the pathogen. Future analysis of the functional consequences of the injection-only mechanism toward immune cells will contribute to the understanding of the priming of adaptive immunity, which is known to be altered during the course of natural Shigella infection.
Published: 2017

6. Tracking Proteins Secreted by Bacteria: What's in the Toolbox?

Author: Maffei, Benoit, Francetic, Olivera, Subtil, Agathe, Biologie cellulaire de l'Infection microbienne - Cellular Biology of Microbial Infection, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Biochimie des Interactions Macromoléculaires / Biochemistry of Macromolecular Interactions, This work was supported by the European Research Council Starting Grant NUChLEAR N°282046 to AS, the Institut Pasteur and the Centre National de la Recherche Scientifique. The OF group is funded by the French ANR grant FiberSpace N°ANR-14-CE09-0004., We thank Tony Pugsley for critical reading of the manuscript., ANR-14-CE09-0004,FiberSpace,Pili de type IV et pseudopili: structure, dynamique, assemblage et fonction moléculaire(2014), European Project: 282046,ERC,ERC-2011-StG_20101109,NUCHLEAR, and Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)
Subjects: Microbiology (medical), Proteomics, MESH: Protein Transport, Immunology, Review, Protein Sorting Signals, secretion machinery, Bacterial Physiological Phenomena, exoproteome, Microbiology, Bacterial Proteins, Yeasts, [SDV.BC.IC]Life Sciences [q-bio]/Cellular Biology/Cell Behavior [q-bio.CB], reporter, Bacteriophages, MESH: Bacteriophages, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], MESH: Bacterial Secretion Systems, Bacterial Secretion Systems, MESH: Bacterial Proteins, Bacteria, MESH: Proteomics, MESH: Yeasts, MESH: Bacterial Outer Membrane Proteins, live imaging, Protein Transport, MESH: Bacteria, Infectious Diseases, MESH: Bacterial Physiological Phenomena, secretion signal, MESH: Protein Sorting Signals, Bacterial Outer Membrane Proteins
Abstract: International audience; Bacteria have acquired multiple systems to expose proteins on their surface, release them in the extracellular environment or even inject them into a neighboring cell. Protein secretion has a high adaptive value and secreted proteins are implicated in many functions, which are often essential for bacterial fitness. Several secreted proteins or secretion machineries have been extensively studied as potential drug targets. It is therefore important to identify the secretion substrates, to understand how they are specifically recognized by the secretion machineries, and how transport through these machineries occurs. The purpose of this review is to provide an overview of the biochemical, genetic and imaging tools that have been developed to evaluate protein secretion in a qualitative or quantitative manner. After a brief overview of the different tools available, we will illustrate their advantages and limitations through a discussion of some of the current open questions related to protein secretion. We will start with the question of the identification of secreted proteins, which for many bacteria remains a critical initial step toward a better understanding of their interactions with the environment. We will then illustrate our toolbox by reporting how these tools have been applied to better understand how substrates are recognized by their cognate machinery, and how secretion proceeds. Finally, we will highlight recent approaches that aim at investigating secretion in real time, and in complex environments such as a tissue or an organism.
Published: 2017

7. Camelid nanobodies used as crystallization chaperones for different constructs of PorM, a component of the type IX secretion system from Porphyromonas gingivalis

Author: Christian Cambillau, Christine Kellenberger, Alain Roussel, Aline Desmyter, Anaïs Gaubert, Jennifer Roche, Philippe Leone, Thi Trang Nhung Trinh, Yoan Duhoo, 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), Centre National de la Recherche Scientifique (CNRS)-Aix Marseille Université (AMU)-Institut National de la Recherche Agronomique (INRA), Architecture et fonction des macromolécules biologiques ( AFMB ), and Centre National de la Recherche Scientifique ( CNRS ) -Aix Marseille Université ( AMU ) -Institut National de la Recherche Agronomique ( INRA )
Subjects: MESH: Sequence Homology, Amino Acid, [SDV.NEU.NB]Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC]/Neurobiology, Plasma protein binding, MESH: Amino Acid Sequence, Biochemistry, MESH: Recombinant Proteins, [SDV.MHEP.MI]Life Sciences [q-bio]/Human health and pathology/Infectious diseases, MESH: Animals, [ SDV.BIBS ] Life Sciences [q-bio]/Quantitative Methods [q-bio.QM], Peptide sequence, MESH: Bacterial Proteins, MESH : Porphyromonas gingivalis, MESH : Protein Conformation, alpha-Helical, [SDV.MHEP.ME]Life Sciences [q-bio]/Human health and pathology/Emerging diseases, [ SDV.MHEP.ME ] Life Sciences [q-bio]/Human health and pathology/Emerging diseases, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], MESH: Escherichia coli, MESH : Amino Acid Sequence, MESH : Protein Binding, MESH: Camelus, [SDV.BIBS]Life Sciences [q-bio]/Quantitative Methods [q-bio.QM], MESH : Sequence Homology, Amino Acid, MESH : Genetic Vectors, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM], [ SDV.MHEP.MI ] Life Sciences [q-bio]/Human health and pathology/Infectious diseases, MESH : Crystallization, [ SDV.NEU.NB ] Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC]/Neurobiology, [SDV.MP.VIR]Life Sciences [q-bio]/Microbiology and Parasitology/Virology, MESH: Camelids, New World, MESH: Porphyromonas gingivalis, MESH: Models, Molecular, Camelus, MESH: Gene Expression, MESH : Cloning, Molecular, Biophysics, MESH: Sequence Alignment, [ SDV.MP.VIR ] Life Sciences [q-bio]/Microbiology and Parasitology/Virology, MESH: Single-Domain Antibodies, Microbiology, 03 medical and health sciences, Bacterial Proteins, Genetics, MESH: Protein Binding, Secretion, Molecular replacement, Protein Interaction Domains and Motifs, 5fwo, MESH: Cloning, Molecular, Amino Acid Sequence, Porphyromonas gingivalis, MESH: Protein Conformation, alpha-Helical, [ SDV.IMM.II ] Life Sciences [q-bio]/Immunology/Innate immunity, MESH: Protein Interaction Domains and Motifs, MESH : Molecular Chaperones, Periplasmic space, [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, MESH : Camelids, New World, MESH : Gene Expression, 030104 developmental biology, MESH: Binding Sites, Protein Conformation, beta-Strand, PorM, Molecular Chaperones, type IX secretion system, 0301 basic medicine, Models, Molecular, Protein Conformation, alpha-Helical, MESH : Escherichia coli, [SDV.BIO]Life Sciences [q-bio]/Biotechnology, Gene Expression, [ SDV.MP.BAC ] Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, Crystallography, X-Ray, [SDV.IMM.II]Life Sciences [q-bio]/Immunology/Innate immunity, MESH : Bacterial Secretion Systems, Research Communications, [ SDV.BBM.BC ] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM], Structural Biology, MESH: Genetic Vectors, 5lmj, MESH : Bacterial Proteins, Cloning, Molecular, MESH: Bacterial Secretion Systems, Bacterial Secretion Systems, MESH : Protein Conformation, beta-Strand, MESH: Crystallization, 5lmw, MESH: Kinetics, MESH : Sequence Alignment, MESH : Camelus, Condensed Matter Physics, Recombinant Proteins, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM], MESH : Single-Domain Antibodies, Thermodynamics, MESH: Protein Conformation, beta-Strand, nb02, MESH : Kinetics, nb01, MESH: Thermodynamics, MESH: Molecular Chaperones, Crystallization, Camelids, New World, Protein Binding, crystallization chaperones, MESH : Recombinant Proteins, MESH : Models, Molecular, Genetic Vectors, Context (language use), Biology, MESH : Peptide Library, Peptide Library, Escherichia coli, Animals, nb130, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], Peptide library, MESH : Thermodynamics, nb19, Binding Sites, Sequence Homology, Amino Acid, [ SDV.SP.PHARMA ] Life Sciences [q-bio]/Pharmaceutical sciences/Pharmacology, [ SDV.BIO ] Life Sciences [q-bio]/Biotechnology, Single-Domain Antibodies, biology.organism_classification, MESH: Crystallography, X-Ray, Kinetics, [SDV.SP.PHARMA]Life Sciences [q-bio]/Pharmaceutical sciences/Pharmacology, MESH : Animals, MESH: Peptide Library, MESH : Crystallography, X-Ray, camelid nanobodies, Sequence Alignment, 5lz0, MESH : Binding Sites, MESH : Protein Interaction Domains and Motifs, [ SDV.BBM.BS ] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM]
Abstract: PorM is a membrane protein that is involved in the assembly of the type IX secretion system (T9SS) inPorphyromonas gingivalis, a major bacterial pathogen that is responsible for periodontal disease in humans. In the context of structural studies of PorM to better understand T9SS assembly, four camelid nanobodies were selected, produced and purified, and their specific interaction with the N-terminal or C-terminal part of the periplasmic domain of PorM was investigated. Diffracting crystals were also obtained, and the structures of the four nanobodies were solved by molecular replacement. Furthermore, two nanobodies were used as crystallization chaperones and turned out to be valuable tools in the structure-determination process of the periplasmic domain of PorM.
Published: 2017

8. Biological Diversity and Evolution of Type IV Secretion Systems

Author: Peter J. Christie, Laura Gomez Valero, Carmen Buchrieser, McGovern Medical School [Houston, Texas], Biologie des Bactéries intracellulaires - Biology of Intracellular Bacteria, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), and Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS)
Subjects: 0301 basic medicine, DNA transfer, Multiprotein complex, 030106 microbiology, MESH: Biological Evolution, Computational biology, Pathogenesis, Effector translocation, Legionella Dot/Icm, Legionella pneumophila, Article, MESH: Biodiversity, Type IV Secretion Systems, 03 medical and health sciences, chemistry.chemical_compound, Type IV secretion, Type (biology), Bacterial Proteins, Phylogenomics, Secretion, MESH: Bacterial Secretion Systems, Bacterial Secretion Systems, MESH: Bacterial Proteins, MESH: Type IV Secretion Systems, biology, Conjugation, MESH: Agrobacterium tumefaciens, [SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, Agrobacterium tumefaciens, Biodiversity, biology.organism_classification, Biological Evolution, Traffic ATPases, MESH: Legionella pneumophila, [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, Coupling protein, chemistry, Bacteria, DNA
Abstract: International audience; The bacterial type IV secretion systems (T4SSs) are a highly functionally and structurally diverse superfamily of secretion systems found in many species of gram-negative and-positive bacteria. Collectively, the T4SSs can translocate DNA and monomeric and multimeric protein substrates to a variety of bacterial and eukaryotic cell types. Detailed phylogenomics analyses have established that the T4SSs evolved from ancient conjugation machines whose original functions were to disseminate mobile DNA elements within and between bacterial species. How members of the T4SS superfamily evolved to recognize and translocate specific substrate repertoires to prokaryotic or eukaryotic target cells is a fascinating question from evolutionary, biological, and structural perspectives. In this chapter, we will summarize recent findings that have shaped our current view of the biological diversity of the T4SSs. We focus mainly on two subtypes, designated as the types IVA (T4ASS) and IVB (T4BSS) systems that respectively are represented by the paradigmatic Agrobacterium tumefaciens VirB/VirD4 and Legionella pneumophila Dot/Icm T4SSs. We present current information about the composition and architectures of these representative systems. We also describe how these and a few related T4ASS and T4BSS members evolved as specialized nanomachines through acquisition of novel domains or subunits, a process that ultimately generated extensive genetic and structural mosaicism among this secretion superfamily. Finally, we present new phylogenomics information establishing that the T4BSSs are much more broadly distributed than initially envisioned.
Published: 2017

9. Structural Similarity of Secretins from Type II and Type III Secretion Systems

Author: Anthony P. Pugsley, Andréa Dessen, Ingrid Guilvout, Tommaso Tosi, Viviana Job, Guy Schoehn, Leandro F. Estrozi, Institut de biologie structurale (IBS - UMR 5075 ), Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), 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)-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), Génétique Moléculaire, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Biologie structurale des interactions entre virus et cellule hôte (UVHCI), Université Joseph Fourier - Grenoble 1 (UJF)-European Molecular Biology Laboratory [Grenoble] (EMBL)-Centre National de la Recherche Scientifique (CNRS), Brazilian National Laboratory for Biosciences (LNBio), Centro Nacional de Pesquisa em Energia e materiais, Centra Nacional de Pesquisa-Centra Nacional de Pesquisa, Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), 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), and Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS)
Subjects: Models, Molecular, MESH: Protein Structure, Quaternary, Structural similarity, [SDV]Life Sciences [q-bio], MESH: Klebsiella oxytoca, Biology, digestive system, Type three secretion system, Microbiology, Saucer, 03 medical and health sciences, fluids and secretions, Secretin, Structural Biology, Secretion, MESH: Bacterial Secretion Systems, Protein Structure, Quaternary, Bacterial Secretion Systems, Molecular Biology, MESH: Structural Homology, Protein, 030304 developmental biology, MESH: Secretin, 0303 health sciences, Type II secretion system, MESH: Bacterial Outer Membrane Proteins, Cryoelectron Microscopy, 030302 biochemistry & molecular biology, Klebsiella oxytoca, Secretory protein, Structural Homology, Protein, Bacterial Outer Membrane Proteins, MESH: Pseudomonas aeruginosa, Pseudomonas aeruginosa, Biophysics, MESH: Cryoelectron Microscopy, Bacterial outer membrane, MESH: Models, Molecular, hormones, hormone substitutes, and hormone antagonists
Abstract: International audience; Secretins, the outer membrane components of several secretion systems in Gram-negative bacteria, assemble into channels that allow exoproteins to traverse the membrane. The membrane-inserted, multimeric regions of PscC, the Pseudomonas aeruginosa type III secretion system secretin, and PulD, the Klebsiella oxytoca type II secretion system secretin, were purified after cell-free synthesis and their structures analyzed by single particle cryoelectron microscopy. Both homomultimeric, barrel-like structures display a "cup and saucer" architecture. The "saucer" region of both secretins is composed of two distinct rings, with that of PulD being less segmented than that of PscC. Both secretins have a central chamber that is occluded by a plug linked to the chamber walls through hairpin-like structures. Comparisons with published structures from other bacterial systems reveal that secretins have regions of local structural flexibility, probably reflecting their evolved functions in protein secretion and needle assembly.
Published: 2014

10. A Specific Polymorphism in Mycobacterium tuberculosis H37Rv Causes Differential ESAT-6 Expression and Identifies WhiB6 as a Novel ESX-1 Component

Author: Carlos Martin, Nacho Aguilo, Roland Brosch, Sofía Samper, Luis Solans, Jesús Gonzalo-Asensio, Wafa Frigui, Alexandre Pawlik, Departamento de Microbiología, Medicina Preventiva y Salud Publica [Zaragoza], Facultad de Medicina [Zaragoza], University of Zaragoza - Universidad de Zaragoza [Zaragoza]-University of Zaragoza - Universidad de Zaragoza [Zaragoza], Centro de Investigación Biomédica en Red Enfermedades Respiratorias (CIBERES), Hospital Universitario Miguel Servet, Pathogénomique mycobactérienne intégrée, Institut Pasteur [Paris] (IP), This work was supported by the European Community's Framework Programme 7 grants NEWTBVAC 241745 and MM4TB 260872, the Spanish Ministry of Economy and Competitiveness grants BIO2011-23555 and FIS12/1970, the Fondation pour la Recherche Médicale FRM no. DEQ20130326471, and the Pyrenees biomedical network Refbio grant SecRegulTBC. Jesús Gonzalo-Asensio was supported by the Juan de la Cierva Programme (JCI-2009-03799) from the Spanish Ministry of Science and Innovation. Luis Solans was supported by grant BES-2009-013037., We thank Marie-Agnès Dillies and Jean-Yves Coppée from the Transcriptome Platform PF2 at the Institut Pasteur for advice and Ida Rosenkrands from Statens Serum Institute for kindly providing the polyclonal anti-SigA antibodies, and we also gratefully acknowledge Alberto Cebollada for help with the sequence analysis of M. tuberculosis strains., European Project: 241745,EC:FP7:HEALTH,FP7-HEALTH-2009-single-stage,NEWTBVAC(2010), European Project: 260872,EC:FP7:HEALTH,FP7-HEALTH-2010-single-stage,MM4TB(2011), and Institut Pasteur [Paris]
Subjects: MESH: Bacterial Secretion Systems, Tuberculosis, Immunology, Virulence, Molecular Genomics, Polymorphism, Single Nucleotide, Regulon, complex mixtures, Microbiology, Virulence factor, Mycobacterium tuberculosis, 03 medical and health sciences, Bacterial Proteins, [SDV.MHEP.MI]Life Sciences [q-bio]/Human health and pathology/Infectious diseases, [SDV.BBM.GTP]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Genomics [q-bio.GN], MESH: Promoter Regions, Genetic, medicine, Secretion, Promoter Regions, Genetic, Bacterial Secretion Systems, Gene, 030304 developmental biology, Genetics, Antigens, Bacterial, 0303 health sciences, biology, 030306 microbiology, MESH: Bacterial Proteins / metabolism, MESH: Polymorphism, Single Nucleotide, Gene Expression Regulation, Bacterial, biochemical phenomena, metabolism, and nutrition, bacterial infections and mycoses, biology.organism_classification, medicine.disease, [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, MESH: Mycobacterium tuberculosis / metabolism, Infectious Diseases, MESH: Regulon, ESAT-6, bacteria, Parasitology, MESH: Antigens, Bacterial, MESH: Gene Expression Regulation, Bacterial, MESH: Mycobacterium tuberculosis / genetics
Abstract: The ESX-1 secreted virulence factor ESAT-6 is one of the major and most well-studied virulence factors of Mycobacterium tuberculosis , given that its inactivation severely attenuates virulent mycobacteria. In this work, we show that clinical isolates of M. tuberculosis produce and secrete larger amounts of ESAT-6 than the widely used M. tuberculosis H37Rv laboratory strain. A search for the genetic polymorphisms underlying this observation showed that whiB6 ( rv3862c ), a gene upstream of the ESX-1 genetic locus that has not previously been found to be implicated in the regulation of the ESX-1 secretory apparatus, presents a unique single nucleotide insertion in its promoter region in strains H37Rv and H37Ra. This polymorphism is not present in any of the other publicly available M. tuberculosis complex genomes or in any of the 76 clinical M. tuberculosis isolates analyzed in our laboratory. We demonstrate that in consequence, the virulence master regulator PhoP downregulates whiB6 expression in H37Rv, while it upregulates its expression in clinical strains. Importantly, reintroduction of the wild-type (WT) copy of whiB6 in H37Rv restored ESAT-6 production and secretion to the level of clinical strains. Hence, we provide clear evidence that in M. tuberculosis —with the exception of the H37Rv strain—ESX-1 expression is regulated by WhiB6 as part of the PhoP regulon, which adds another level of complexity to the regulation of ESAT-6 secretion with a potential role in virulence adaptation.
Published: 2014

11. The Rate of Folding Dictates Substrate Secretion by the Escherichia coli Hemolysin Type 1 Secretion System

Author: Lutz Schmitt, Sander H. J. Smits, Patrick J. Bakkes, Stefan Jenewein, I. Barry Holland, Institut de génétique et microbiologie [Orsay] (IGM), and Université Paris-Sud - Paris 11 (UP11)-Centre National de la Recherche Scientifique (CNRS)
Subjects: Protein Folding, MESH: Protein Folding, Mutant, Mutation, Missense, MESH: Carrier Proteins, MESH: Escherichia coli Proteins, ATP-binding cassette transporter, Biology, Biochemistry, MESH: Membrane Transport Proteins, Hemolysin Proteins, 03 medical and health sciences, Maltose-binding protein, Bacterial Proteins, Membrane Biology, Escherichia coli, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Secretion, MESH: Bacterial Secretion Systems, Bacterial Secretion Systems, MESH: Bacterial Proteins, Molecular Biology, MESH: Periplasmic Binding Proteins, 030304 developmental biology, MESH: Mutation, Missense, 0303 health sciences, MESH: Escherichia coli, 030306 microbiology, Escherichia coli Proteins, MESH: Bacterial Outer Membrane Proteins, Membrane Transport Proteins, Cell Biology, MESH: Multiprotein Complexes, MESH: Amino Acid Substitution, Fusion protein, [SDV.MP]Life Sciences [q-bio]/Microbiology and Parasitology, Amino Acid Substitution, MESH: Hemolysin Proteins, Membrane protein, Membrane protein complex, Multiprotein Complexes, Periplasmic Binding Proteins, biology.protein, Protein folding, Carrier Proteins, Bacterial Outer Membrane Proteins
Abstract: International audience; Secretion of the Escherichia coli toxin hemolysin A (HlyA) is catalyzed by the membrane protein complex HlyB-HlyD-TolC and requires a secretion sequence located within the last 60 amino acids of HlyA. The Hly translocator complex exports a variety of passenger proteins when fused N-terminal to this secretion sequence. However, not all fusions are secreted efficiently. Here, we demonstrate that the maltose binding protein (MalE) lacking its natural export signal and fused to the HlyA secretion signal is poorly secreted by the Hly system. We anticipated that folding kinetics might be limiting secretion, and we therefore introduced the "folding" mutation Y283D. Indeed this mutant fusion protein was secreted at a much higher level. This level was further enhanced by the introduction of a second MalE folding mutation (V8G or A276G). Secretion did not require the molecular chaperone SecB. Folding analysis revealed that all mutations reduced the refolding rate of the substrate, whereas the unfolding rate was unaffected. Thus, the efficiency of secretion by the Hly system is dictated by the folding rate of the substrate. Moreover, we demonstrate that fusion proteins defective in export can be engineered for secretion while still retaining function.
Published: 2010

12. 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

13. Cytosolic Access of Mycobacterium tuberculosis: Critical Impact of Phagosomal Acidification Control and Demonstration of Occurrence In Vivo

Author: Fadel Sayes, Ok-Ryul Song, Laleh Majlessi, Matthias I. Gröschel, Roxane Simeone, Priscille Brodin, Roland Brosch, Pathogénomique mycobactérienne intégrée, Institut Pasteur [Paris] (IP), Centre d’Infection et d’Immunité de Lille - INSERM U 1019 - UMR 9017 - UMR 8204 (CIIL), Institut Pasteur de Lille, Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Lille-Centre Hospitalier Régional Universitaire [Lille] (CHRU Lille)-Centre National de la Recherche Scientifique (CNRS), This work was supported by the European Community’s Framework Programme 7 grants NEWTBVAC 241745, MM4TB 260872 and INTRACELLTB 260901, the Institut Pasteur (PTR441), the Agence Nationale de Recherche, the Feder (12001407 (D-AL) Equipex Imaginex BioMed), the Region Nord Pas de Calais and the Fondation pour la Recherche Médicale FRM noDEQ20130326471 (to RB). MIG is a recipient of a Jan Kornelis de Cock Stichting scholarship., European Project: 241745,EC:FP7:HEALTH,FP7-HEALTH-2009-single-stage,NEWTBVAC(2010), European Project: 260872,EC:FP7:HEALTH,FP7-HEALTH-2010-single-stage,MM4TB(2011), European Project: 260901,EC:FP7:ERC,ERC-2010-StG_20091118,INTRACELLTB(2010), SAYES, FADEL, Discovery and preclinical development of new generation tuberculosis vaccines - NEWTBVAC - - EC:FP7:HEALTH2010-01-01 - 2014-02-28 - 241745 - VALID, More Medicines for Tuberculosis - MM4TB - - EC:FP7:HEALTH2011-02-01 - 2016-01-31 - 260872 - VALID, A Chemical Genomics Approach of Intracellular Mycobacterium tuberculosis Towards Defining Specific Host Pathogen Interactions - INTRACELLTB - - EC:FP7:ERC2010-12-01 - 2015-11-30 - 260901 - VALID, Institut Pasteur [Paris], Centre National de la Recherche Scientifique (CNRS)-Centre Hospitalier Régional Universitaire [Lille] (CHRU Lille)-Université de Lille-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut Pasteur de Lille, and Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP)
Subjects: MESH: Hydrogen-Ion Concentration, MESH: Mycobacterium tuberculosis, Phagocyte, MESH: Mice, Mutant Strains, [SDV]Life Sciences [q-bio], chemistry.chemical_compound, Mice, Phagosomes, MESH: Animals, MESH: Tuberculosis, Biology (General), MESH: Bacterial Secretion Systems, Bacterial Secretion Systems, Cation Transport Proteins, Phagosome, biology, Bafilomycin, Hydrogen-Ion Concentration, 3. Good health, Transport protein, [SDV] Life Sciences [q-bio], MESH: Leukocyte Common Antigens, medicine.anatomical_structure, Research Article, MESH: Cell Line, Tumor, QH301-705.5, Phagosome acidification, Immunology, Spleen, Microbiology, Mycobacterium tuberculosis, MESH: Cation Transport Proteins, MESH: Phagosomes, Virology, Cell Line, Tumor, Genetics, medicine, Animals, Humans, Tuberculosis, Secretion, MESH: Mice, Molecular Biology, MESH: Humans, Macrophages, MESH: Macrophages, RC581-607, biology.organism_classification, Mice, Mutant Strains, chemistry, Leukocyte Common Antigens, Parasitology, Immunologic diseases. Allergy
Abstract: Mycobacterium tuberculosis (Mtb) uses efficient strategies to evade the eradication by professional phagocytes, involving—as recently confirmed—escape from phagosomal confinement. While Mtb determinants, such as the ESX-1 type VII secretion system, that contribute to this phenomenon are known, the host cell factors governing this important biological process are yet unexplored. Using a newly developed flow-cytometric approach for Mtb, we show that macrophages expressing the phagosomal bivalent cation transporter Nramp-1, are much less susceptible to phagosomal rupture. Together with results from the use of the phagosome acidification inhibitor bafilomycin, we demonstrate that restriction of phagosomal acidification is a prerequisite for mycobacterial phagosomal rupture and cytosolic contact. Using different in vivo approaches including an enrichment and screen for tracking rare infected phagocytes carrying the CD45.1 hematopoietic allelic marker, we here provide first and unique evidence of M. tuberculosis-mediated phagosomal rupture in mouse spleen and lungs and in numerous phagocyte types. Our results, linking the ability of restriction of phagosome acidification to cytosolic access, provide an important conceptual advance for our knowledge on host processes targeted by Mtb evasion strategies., Author Summary The intracellular fate of the agent of the human tuberculosis agent in phagocytes is a question of great biological relevance. Among the mycobacterial survival strategies, the escape of Mycobacterium tuberculosis from phagosomes has been subject of scientific debate for a long time. However, technically improved methods recently reinforced the occurrence of this phenomenon. Here, we focused on the host factors involved in phagosomal rupture and provide first and singular evidence of M. tuberculosis-mediated phagosomal rupture in vivo in mouse lungs and inside the granuloma. We show that partial blockage of phagosomal acidification, induced by mycobacteria, is a prerequisite for efficient vacuolar breakage by M. tuberculosis and link maturation arrest, cytosolic contact and the corresponding immune responses. From our results we conclude that vacuolar breakage induced by M. tuberculosis is not an ex vivo artifact of cell cultures, but an important process that occurs inside infected phagocytes within organs during several days that strongly determines the outcome of infection with this key pathogen.
Published: 2015

14. Structure of a type IV secretion system

Author: Sarah Connery, Annick Dujeancourt, Adam Redzej, Francesca Gubellini, Angel Rivera-Calzada, Gabriel Waksman, Elena V. Orlova, Fang Lu, Harry H. Low, Rémi Fronzes, Nathalie Braun, University College of London [London] (UCL), Birkbeck College [University of London], Biologie Structurale de la Sécrétion Bactérienne, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), This work was financed by grant 098302 from the Wellcome Trust to G.W. and by equipment grant 079605 from the Wellcome Trust. R.F. and A.D. were financed by Institut Pasteur and the CNRS. F.G. was the recipient of ‘Bourse Roux’ from Institut Pasteur., and Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)
Subjects: Models, Molecular, ATPase, [PHYS.PHYS.PHYS-BIO-PH]Physics [physics]/Physics [physics]/Biological Physics [physics.bio-ph], Virulence, MESH: Escherichia coli Proteins, MESH: Microscopy, Electron, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, medicine.disease_cause, Cell membrane, 03 medical and health sciences, chemistry.chemical_compound, Escherichia coli, medicine, MESH: Adenosine Triphosphatases, [CHIM.CRIS]Chemical Sciences/Cristallography, Secretion, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, MESH: Bacterial Secretion Systems, Bacterial Secretion Systems, 030304 developmental biology, Adenosine Triphosphatases, 0303 health sciences, Inner membrane complex, Multidisciplinary, biology, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], 030306 microbiology, MESH: Escherichia coli, Escherichia coli Proteins, Cell Membrane, MESH: Multiprotein Complexes, biology.organism_classification, Cell biology, Microscopy, Electron, medicine.anatomical_structure, chemistry, Multiprotein Complexes, biology.protein, [INFO.INFO-BI]Computer Science [cs]/Bioinformatics [q-bio.QM], Bacteria, DNA, MESH: Models, Molecular, MESH: Cell Membrane
Abstract: The three-dimensional structure of the type IV secretion system encoded by the Escherichia coli R388 conjugative plasmid. This study reports the use of electron microscopy to reconstruct a large, 3-megadalton complex of the bacterial type IV secretion (T4S) system from Escherichia coli, made up of eight proteins assembled in an intricate stoichiometric relationship to form a stalk spanning the membrane to unite a core outer-membrane-associated complex with an inner membrane complex. The structure reveals a novel architecture that differs markedly from those known from other bacterial secretion systems. T4S systems are used by many bacterial pathogens to deliver virulence factors and to transfer genetic material and also show potential as a tool for the genetic modification of human cells. Bacterial type IV secretion systems translocate virulence factors into eukaryotic cells1,2, distribute genetic material between bacteria and have shown potential as a tool for the genetic modification of human cells3. Given the complex choreography of the substrate through the secretion apparatus4, the molecular mechanism of the type IV secretion system has proved difficult to dissect in the absence of structural data for the entire machinery. Here we use electron microscopy to reconstruct the type IV secretion system encoded by the Escherichia coli R388 conjugative plasmid. We show that eight proteins assemble in an intricate stoichiometric relationship to form an approximately 3 megadalton nanomachine that spans the entire cell envelope. The structure comprises an outer membrane-associated core complex1 connected by a central stalk to a substantial inner membrane complex that is dominated by a battery of 12 VirB4 ATPase subunits organized as side-by-side hexameric barrels. Our results show a secretion system with markedly different architecture, and consequently mechanism, to other known bacterial secretion systems1,4,5,6.
Published: 2014

15. Membrane and chaperone recognition by the major translocator protein PopB of the type III secretion system of Pseudomonas aeruginosa

Author: Viviana Job, Jean-Pierre Simorre, Karen F. Discola, Ina Attree, François Boulay, Andreas Förster, Andréa Dessen, Thomas, Frank, Institut de biologie structurale (IBS - UMR 5075 ), Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), 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)-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), Biologie du Cancer et de l'Infection (BCI ), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), 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)
Subjects: MESH: Protein Transport, MESH: Protein Structure, Quaternary, [SDV.BBM.BS] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], Crystallography, X-Ray, Biochemistry, Cell Line, Type three secretion system, Mice, 03 medical and health sciences, MESH: Protein Structure, Tertiary, Bacterial Proteins, Animals, Secretion, MESH: Animals, Protein Structure, Quaternary, MESH: Bacterial Secretion Systems, Bacterial Secretion Systems, Molecular Biology, Ternary complex, MESH: Bacterial Proteins, MESH: Mice, 030304 developmental biology, Antigens, Bacterial, 0303 health sciences, biology, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], Effector, 030302 biochemistry & molecular biology, Cell Biology, Translocon, MESH: Crystallography, X-Ray, Protein Structure, Tertiary, Cell biology, MESH: Cell Line, Protein Transport, Membrane protein, Cytoplasm, Chaperone (protein), Protein Structure and Folding, Pseudomonas aeruginosa, MESH: Pseudomonas aeruginosa, biology.protein, MESH: Molecular Chaperones, MESH: Antigens, Bacterial, Molecular Chaperones
Abstract: International audience; The type III secretion system is a widespread apparatus used by pathogenic bacteria to inject effectors directly into the cytoplasm of eukaryotic cells. A key component of this highly conserved system is the translocon, a pore formed in the host membrane that is essential for toxins to bypass this last physical barrier. In Pseudomonas aeruginosa the translocon is composed of PopB and PopD, both of which before secretion are stabilized within the bacterial cytoplasm by a common chaperone, PcrH. In this work we characterize PopB, the major translocator, in both membrane-associated and PcrH-bound forms. By combining sucrose gradient centrifugation experiments, limited proteolysis, one-dimensional NMR, and β-lactamase reporter assays on eukaryotic cells, we show that PopB is stably inserted into bilayers with its flexible N-terminal domain and C-terminal tail exposed to the outside. In addition, we also report the crystal structure of the complex between PcrH and an N-terminal region of PopB (residues 51-59), which reveals that PopB lies within the concave face of PcrH, employing mostly backbone residues for contact. PcrH is thus the first chaperone whose structure has been solved in complex with both type III secretion systems translocators, revealing that both molecules employ the same surface for binding and excluding the possibility of formation of a ternary complex. The characterization of the major type III secretion system translocon component in both membrane-bound and chaperone-bound forms is a key step for the eventual development of antibacterials that block translocon assembly.
Published: 2014

16. A safe bacterial microsyringe for in vivo antigen delivery and immunotherapy

Author: Bertrand Toussaint, Mitra Ahmadi, Xavier Chauchet, Sandrine Martin, David Laurin, François Cretin, Alexis Broisat, Charlotte Genestet, Julien Verove, Candice Trocme, Joel Plumas, Catherine Ghezzi, Caroline Aspord, Yan Wang, Audrey Le Gouellec, Benoît Polack, Université Grenoble Alpes, Inserm, CEA, BIG-Biologie du Cancer et de l’Infection, Grenoble, France, Laboratoire d'Ingénierie des Macromolécules (LIM), Institut de biologie structurale (IBS - UMR 5075 ), Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), 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)-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)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), TheREx, Techniques de l'Ingénierie Médicale et de la Complexité - Informatique, Mathématiques et Applications, Grenoble - UMR 5525 (TIMC-IMAG), Université Joseph Fourier - Grenoble 1 (UJF)-Institut polytechnique de Grenoble - Grenoble Institute of Technology (Grenoble INP )-VetAgro Sup - Institut national d'enseignement supérieur et de recherche en alimentation, santé animale, sciences agronomiques et de l'environnement (VAS)-Centre National de la Recherche Scientifique (CNRS)-Université Joseph Fourier - Grenoble 1 (UJF)-Institut polytechnique de Grenoble - Grenoble Institute of Technology (Grenoble INP )-VetAgro Sup - Institut national d'enseignement supérieur et de recherche en alimentation, santé animale, sciences agronomiques et de l'environnement (VAS)-Centre National de la Recherche Scientifique (CNRS), Institut d'oncologie/développement Albert Bonniot de Grenoble (INSERM U823), Université Joseph Fourier - Grenoble 1 (UJF)-CHU Grenoble-EFS-Institut National de la Santé et de la Recherche Médicale (INSERM), Biochimie et biophysique des systèmes intégrés (BBSI), Université Joseph Fourier - Grenoble 1 (UJF)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), Université Joseph Fourier - Grenoble 1 (UJF)-Institut polytechnique de Grenoble - Grenoble Institute of Technology (Grenoble INP )-VetAgro Sup - Institut national d'enseignement supérieur et de recherche en alimentation, santé animale, sciences agronomiques et de l'environnement (VAS)-Centre National de la Recherche Scientifique (CNRS), Radiopharmaceutiques biocliniques, Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Santé et de la Recherche Médicale (INSERM), Association nationale de prévention en alcoologie et addictologie, ANPAA30, Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), 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), VetAgro Sup - Institut national d'enseignement supérieur et de recherche en alimentation, santé animale, sciences agronomiques et de l'environnement (VAS)-Institut polytechnique de Grenoble - Grenoble Institute of Technology (Grenoble INP )-Centre National de la Recherche Scientifique (CNRS)-Université Joseph Fourier - Grenoble 1 (UJF)-VetAgro Sup - Institut national d'enseignement supérieur et de recherche en alimentation, santé animale, sciences agronomiques et de l'environnement (VAS)-Institut polytechnique de Grenoble - Grenoble Institute of Technology (Grenoble INP )-Centre National de la Recherche Scientifique (CNRS)-Université Joseph Fourier - Grenoble 1 (UJF), Centre National de la Recherche Scientifique (CNRS)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Joseph Fourier - Grenoble 1 (UJF), and VetAgro Sup - Institut national d'enseignement supérieur et de recherche en alimentation, santé animale, sciences agronomiques et de l'environnement (VAS)-Institut polytechnique de Grenoble - Grenoble Institute of Technology (Grenoble INP )-Centre National de la Recherche Scientifique (CNRS)-Université Joseph Fourier - Grenoble 1 (UJF)
Subjects: [SDV.BIO]Life Sciences [q-bio]/Biotechnology, MESH: Immunotherapy, medicine.medical_treatment, MESH: Photosensitizing Agents, Epitopes, T-Lymphocyte, Active immunotherapy, CD8-Positive T-Lymphocytes, [SDV.IMM.II]Life Sciences [q-bio]/Immunology/Innate immunity, Epitope, MESH: Cancer Vaccines, Mice, Furocoumarins, Neoplasms, Drug Discovery, MESH: Animals, MESH: Neoplasms, [INFO.INFO-BT]Computer Science [cs]/Biotechnology, Cytotoxicity, MESH: Bacterial Secretion Systems, Bacterial Secretion Systems, MESH: Furocoumarins, 0303 health sciences, Antigen Presentation, Immunity, Cellular, Photosensitizing Agents, MESH: Dendritic Cells, MESH: CD8-Positive T-Lymphocytes, 3. Good health, Pseudomonas aeruginosa, MESH: Pseudomonas aeruginosa, Molecular Medicine, Original Article, Female, MESH: Antigens, Immunotherapy, MESH: Xenograft Model Antitumor Assays, MESH: Mutation, MESH: Cell Line, Tumor, Lymphoid Tissue, Heterologous, Biology, Vaccines, Attenuated, Cancer Vaccines, Microbiology, MESH: Immunity, Cellular, MESH: Epitopes, T-Lymphocyte, 03 medical and health sciences, Immune system, Antigen, In vivo, Cell Line, Tumor, MESH: Vaccines, Attenuated, medicine, Genetics, Animals, Humans, Antigens, Molecular Biology, MESH: Mice, 030304 developmental biology, Pharmacology, MESH: Humans, 030306 microbiology, Dendritic Cells, Xenograft Model Antitumor Assays, [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, Disease Models, Animal, Mutation, MESH: Antigen Presentation, MESH: Lymphoid Tissue, Cancer research, MESH: Disease Models, Animal, MESH: Female
Abstract: International audience; The industrial development of active immunotherapy based on live-attenuated bacterial vectors has matured. We developed a microsyringe for antigen delivery based on the type III secretion system (T3SS) of P. aeruginosa. We applied the "killed but metabolically active" (KBMA) attenuation strategy to make this bacterial vector suitable for human use. We demonstrate that attenuated P. aeruginosa has the potential to deliver antigens to human antigen-presenting cells in vitro via T3SS with considerable attenuated cytotoxicity as compared with the wild-type vector. In a mouse model of cancer, we demonstrate that this KBMA strain, which cannot replicate in its host, efficiently disseminates into lymphoid organs and delivers its heterologous antigen. The attenuated strain effectively induces a cellular immune response to the cancerous cells while lowering the systemic inflammatory response. Hence, a KBMA P. aeruginosa microsyringe is an efficient and safe tool for in vivo antigen delivery.
Published: 2013

17. The type II secretion system – a dynamic fiber assembly nanomachine

Author: Olivera Francetic, David A. Cisneros, Mangayarkarasi Nivaskumar, Manuel Campos, Génétique Moléculaire, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Université Paris Diderot - Paris 7 (UPD7), The study of protein secretion in our team has been funded by the Institut Pasteur grant PTR339 and by the ANR grant SecPath. MC was a scholar of the French Ministry of Science and Education, MN is the recipient of the Pasteur-Paris University (PPU) International PhD program fellowship and DAC was funded by EMBO long term and Roux fellowships., We are grateful to our collaborators Michaël Nilges, Guillaume Bouvier, Peter J. Bond, Gérard Péhau-Arnaudet, Nadia Izadi-Pruneyre, Gisèle Amorim, Daniel Ladant and Gouzel Karimova for their contributions to the pseudopilus project. We are grateful to all members of the Molecular Genetics Unit for many stimulating discussions. We thank Tony Pugsley for generous support and for the critical reading of the manuscript., and Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)
Subjects: MESH: Bacterial Secretion Systems, MESH: Fimbriae, Bacterial/metabolism, Pilus assembly, MESH: Cell Membrane/genetics, MESH: Protein Transport, MESH: Bacteria/genetics, MESH: Bacteria/metabolism, Biology, Flagellum, MESH: Cell Membrane/metabolism, MESH: Bacterial Proteins/genetics, Microbiology, Pilus, MESH: Bacterial Proteins/metabolism, MESH: Fimbriae, Bacterial/genetics, 03 medical and health sciences, Bacterial Proteins, Pseudopilins, Type IV pili, Secretion, Bacterial Secretion Systems, Molecular Biology, 030304 developmental biology, 0303 health sciences, Type II protein secretion, Bacteria, Type II secretion system, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], 030306 microbiology, Cell Membrane, [SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, General Medicine, Periplasmic space, [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, Cell biology, Protein Transport, Secretory protein, Fimbriae, Bacterial, Biogenesis
Abstract: International audience; Type II secretion systems (T2SSs) share common origins and structure with archaeal flagella (archaella) and pili, bacterial competence systems and type IV pili. All of these systems use a conserved ATP-powered machinery to assemble helical fibers that are anchored in the plasma membrane. The T2SSs assemble pseudopili, periplasmic filaments that promote extracellular secretion of folded periplasmic proteins. Comparative analysis of T2SSs and related fiber assembly nanomachines might provide important clues on their functional specificities and dynamics. This review focuses on recent developments in the study of pseudopilus structure and biogenesis, and discusses mechanistic models of pseudopilus function in protein secretion.
Published: 2013

18. Structural insights into Helicobacter pylori oncoprotein CagA interaction with β1 integrin

Author: Laurent Terradot, Cyril Dian, Claudia Ertl, Burcu Kaplan-Türköz, Luisa F. Jiménez-Soto, Arthur Louche, Tommaso Tosi, Rainer Haas, Han Remaut, European Synchrotron Radiation Facility (ESRF), Institut de biologie structurale (IBS - UMR 5075 ), Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), 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)-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), Department of Haematology, Oncology and Clinical Immunology, University Hospital of Düsseldorf, Institut de biologie et chimie des protéines [Lyon] (IBCP), Centre National de la Recherche Scientifique (CNRS)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon, Structural Biology Brussels, Department of Bio-engineering Sciences, Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Université Claude Bernard Lyon 1 (UCBL), and Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS)
Subjects: Models, Molecular, MESH: Antigens, CD29, Protein Conformation, [SDV]Life Sciences [q-bio], virulence factor, 0302 clinical medicine, Protein structure, MESH: Protein Conformation, oncogene, pathogenicity, Cloning, Molecular, Internalization, MESH: Bacterial Secretion Systems, Bacterial Secretion Systems, MESH: Bacterial Proteins, media_common, MESH: Mutagenesis, Host cell membrane, 0303 health sciences, Multidisciplinary, gastric ulcer, Integrin beta1, Biological Sciences, Transport protein, Cell biology, Protein Transport, 030220 oncology & carcinogenesis, MESH: Models, Molecular, MESH: Protein Transport, media_common.quotation_subject, Biology, MESH: Two-Hybrid System Techniques, Microbiology, 03 medical and health sciences, Bacterial Proteins, Antigen, Two-Hybrid System Techniques, CagA, Secretion, MESH: Cloning, Molecular, 030304 developmental biology, X-ray crystallography, Antigens, Bacterial, Helicobacter pylori, biology.organism_classification, bacterial infections and mycoses, digestive system diseases, Mutagenesis, MESH: Helicobacter pylori, MESH: Antigens, Bacterial
Abstract: Infection with the gastric pathogen Helicobacter pylori is a risk factor for the development of gastric cancer. Pathogenic strains of H. pylori carry a type IV secretion system (T4SS) responsible for the injection of the oncoprotein CagA into host cells. H. pylori and its cag -T4SS exploit α5β1 integrin as a receptor for CagA translocation. Injected CagA localizes to the inner leaflet of the host cell membrane, where it hijacks host cell signaling and induces cytoskeleton reorganization. Here we describe the crystal structure of the N-terminal ∼100-kDa subdomain of CagA at 3.6 Å that unveils a unique combination of folds. The core domain of the protein consists of an extended single-layer β-sheet stabilized by two independent helical subdomains. The core is followed by a long helix that forms a four-helix helical bundle with the C-terminal domain. Mapping of conserved regions in a set of CagA sequences identified four conserved surface-exposed patches (CSP1–4), which represent putative hot-spots for protein–protein interactions. The proximal part of the single-layer β-sheet, covering CSP4, is involved in specific binding of CagA to the β1 integrin, as determined by yeast two-hybrid and in vivo competition assays in H. pylori cell-culture infection studies. These data provide a structural basis for the first step of CagA internalization into host cells and suggest that CagA uses a previously undescribed mechanism to bind β1 integrin to mediate its own translocation.
Published: 2012

19. Large-scale study of the interactions between proteins involved in type IV pilus biology in Neisseria meningitidis: characterization of a subcomplex involved in pilus assembly

Author: Michaella, Georgiadou, Marta, Castagnini, Gouzel, Karimova, Daniel, Ladant, Vladimir, Pelicic, Imperial College London, Biochimie des Interactions Macromoléculaires, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), This work was supported by grants from the Biotechnology and Biological Sciences Research Council (BBSRC) and European Commission (EIMID, Seventh Framework Programme). M. Georgiadou was supported by a Doctoral Training Grant from the BBSRC., European Project: 35388,EIMID, Biochimie des Interactions Macromoléculaires / Biochemistry of Macromolecular Interactions, and Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS)
Subjects: MESH: Protein Interaction Domains and Motifs, Macromolecular Substances, MESH: Protein Multimerization, MESH: Protein Interaction Mapping, Membrane Transport Proteins, MESH: Macromolecular Substances, Neisseria meningitidis, MESH: Two-Hybrid System Techniques, [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, MESH: Neisseria meningitidis, MESH: Fimbriae, Bacterial, MESH: Membrane Transport Proteins, Fimbriae, Bacterial, Two-Hybrid System Techniques, Protein Interaction Mapping, MESH: Protein Binding, Protein Interaction Domains and Motifs, Protein Multimerization, MESH: Bacterial Secretion Systems, Bacterial Secretion Systems, ComputingMilieux_MISCELLANEOUS, Protein Binding
Abstract: International audience; The functionally versatile type IV pili (Tfp) are one of the most widespread virulence factors in bacteria. However, despite generating much research interest for decades, the molecular mechanisms underpinning the various aspects of Tfp biology remain poorly understood, mainly because of the complexity of the system. In the human pathogen Neisseria meningitidis for example, 23 proteins are dedicated to Tfp biology, 15 of which are essential for pilus biogenesis. One of the important gaps in our knowledge concerns the topology of this multiprotein machinery. Here we have used a bacterial two-hybrid system to identify and quantify the interactions between 11 Pil proteins from N. meningitidis. We identified 20 different binary interactions, many of which are novel. This represents the most complex interaction network between Pil proteins reported to date and indicates, among other things, that PilE, PilM, PilN and PilO, which are involved in pilus assembly, indeed interact. We focused our efforts on this subset of proteins and used a battery of assays to determine the membrane topology of PilN and PilO, map the interaction domains between PilE, PilM, PilN and PilO, and show that a widely conserved N-terminal motif in PilN is essential for both PilM-PilN interactions and pilus assembly. Finally, we show that PilP (another protein involved in pilus assembly) forms a complex with PilM, PilN and PilO. Taken together, these findings have numerous implications for understanding Tfp biology and provide a useful blueprint for future studies.
Published: 2012

20. Unique biofilm signature, drug susceptibility and decreased virulence in Drosophila through the Pseudomonas aeruginosa two-component system PprAB

Author: Friederike Ewald, Cathy Nguyen, Marie-Odile Fauvarque, Ina Attree, Virginie Calderon, Katy Jeannot, Sophie de Bentzmann, Christophe Bordi, Christophe S. Bernard, Didier Grunwald, Patrick Plésiat, Caroline Giraud, 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), Laboratoire de microbiologie et génétique moléculaires (LMGM), Centre de Biologie Intégrative (CBI), Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS), Laboratoire de bactériologie, Université de Franche-Comté (UFC), Université Bourgogne Franche-Comté [COMUE] (UBFC)-Université Bourgogne Franche-Comté [COMUE] (UBFC), Technologies avancées pour le génôme et la clinique (TAGC), Aix Marseille Université (AMU)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), ANTE-INSERM U836, équipe 4, Muscles et pathologies, Biologie du Cancer et de l'Infection (BCI ), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), 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)-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)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Laboratoire de Biologie à Grande Échelle (BGE - UMR S1038), French cystic fibrosis foundation, French Ministry of Research and Technology, Region Rhône-Alpes, European Project, Roux-Buisson, Nathalie, ERA-NET ADHRES 27481, INCOMING, Laboratoire de microbiologie et génétique moléculaires - UMR5100 (LMGM), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS), Université Bourgogne Franche-Comté [COMUE] (UBFC), Theories and Approaches of Genomic Complexity (TAGC), Aix Marseille Université (AMU)-Institut National de la Santé et de la Recherche Médicale (INSERM), Pathogénie bactérienne et réponses cellulaires, Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), Institut de Recherches en Technologies et Sciences pour le Vivant (IRTSV), Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes (UGA), Université de Toulouse (UT)-Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS)-Université Toulouse III - Paul Sabatier (UT3), Université de Toulouse (UT)-Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS), Institut de Recherche Interdisciplinaire de Grenoble (IRIG), and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])
Subjects: MESH: Adhesins, Bacterial, Fimbria, Pathogenesis, medicine.disease_cause, MESH: Animals, Biology (General), MESH: Bacterial Secretion Systems, Bacterial Secretion Systems, ComputingMilieux_MISCELLANEOUS, 0303 health sciences, Genomics, 3. Good health, Bacterial Pathogens, Host-Pathogen Interaction, Drosophila melanogaster, [SDV.MP]Life Sciences [q-bio]/Microbiology and Parasitology, Pseudomonas aeruginosa, MESH: Pseudomonas aeruginosa, Research Article, QH301-705.5, Immunology, Virulence, MESH: Biofilms, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, Biology, Microbiology, Bacterial genetics, Cell Line, MESH: Drosophila melanogaster, MESH: Fimbriae, Bacterial, 03 medical and health sciences, Virology, medicine, Genetics, Animals, Secretion, Adhesins, Bacterial, Molecular Biology, [SDV.BC] Life Sciences [q-bio]/Cellular Biology, [SDV.MP] Life Sciences [q-bio]/Microbiology and Parasitology, 030304 developmental biology, 030306 microbiology, fungi, Biofilm, Bacteriology, RC581-607, MESH: Cell Line, Bacterial adhesin, Regulon, Biofilms, Fimbriae, Bacterial, Parasitology, Immunologic diseases. Allergy
Abstract: Bacterial biofilm is considered as a particular lifestyle helping cells to survive hostile environments triggered by a variety of signals sensed and integrated through adequate regulatory pathways. Pseudomonas aeruginosa, a Gram-negative bacterium causing severe infections in humans, forms biofilms and is a fantastic example for fine-tuning of the transition between planktonic and community lifestyles through two-component systems (TCS). Here we decipher the regulon of the P. aeruginosa response regulator PprB of the TCS PprAB. We identified genes under the control of this TCS and once this pathway is activated, analyzed and dissected at the molecular level the PprB-dependent phenotypes in various models. The TCS PprAB triggers a hyper-biofilm phenotype with a unique adhesive signature made of BapA adhesin, a Type 1 secretion system (T1SS) substrate, CupE CU fimbriae, Flp Type IVb pili and eDNA without EPS involvement. This unique signature is associated with drug hyper-susceptibility, decreased virulence in acutely infected flies and cytotoxicity toward various cell types linked to decreased Type III secretion (T3SS). Moreover, once the PprB pathway is activated, decreased virulence in orally infected flies associated with enhanced biofilm formation and dissemination defect from the intestinal lumen toward the hemolymph compartment is reported. PprB may thus represent a key bacterial adaptation checkpoint of multicellular and aggregative behavior triggering the production of a unique matrix associated with peculiar antibiotic susceptibility and attenuated virulence, a particular interesting breach for therapeutic intervention to consider in view of possible eradication of P. aeruginosa biofilm-associated infections., Author Summary We unraveled that once the two-component system PprAB regulatory pathway is activated, Pseudomonas aeruginosa displays a unique hyper-biofilm phenotype due to a molecular signature combining a T1SS high molecular weight substrate, BapA, fimbriae of the chaperone-usher pathway, Type IVb pili and eDNA. Originally, this particular hyper-biofilm that is not strictly dependent on Psl or Pel exopolysaccharide (EPS) synthesis displays increased drug susceptibility, in contrary to previously reported biofilm lifestyle associated with increased resistance to antibiotics. PprB-dependent hyper-biofilm was also observed on intestinal mucosa of orally infected Drosophila flies in which it also displays a reduced capacity to cross the epithelial barrier from the intestinal lumen toward the hemolymph that consequently resulted in a reduced capacity to kill flies. Furthermore, constitutive activation of this PprB regulatory pathway triggers a reduced secretion of T3SS effectors which may account for the decreased virulence observed in epithelial and macrophage lineages and in acute Drosophila infections induced by septic injury. We appended in this study pieces of regulatory and molecular data that highlight the possibility to combat infections due to P. aeruginosa-biofilm with particular matrix.
Published: 2012

21. ESX-1 dependent impairment of autophagic flux by Mycobacterium tuberculosis in human dendritic cells

Author: Marco Corazzari, Maria Elena Remoli, Roland Brosch, Laura Falasca, Giovanni Delogu, Roxane Simeone, Alessandra Romagnoli, Eliana M. Coccia, Delia Goletti, Valérie Gafa, Gian Maria Fimia, Mauro Piacentini, Elena Giacomini, Manuela Pardini, Marilena P. Etna, National Institute for Infectious Diseases 'Lazzaro Spallanzani', Istituto Superiore di Sanità (ISS), Pathogénomique mycobactérienne intégrée, Institut Pasteur [Paris] (IP), Università cattolica del Sacro Cuore = Catholic University of the Sacred Heart [Roma] (Unicatt), Università degli Studi di Roma Tor Vergata [Roma], The study was supported by grants from Ministry for Health of Italy: 'Ricerca Corrente' to M.P. G.M.F. and D.G., 'Ricerca Finalizzata' RF06.76.1, RF-IMI-2009-1302952 to D.G., RF07.103 to E.M.C., G.M.F. and D.G., by grants from the FP7 program of the European Community n°241745 to R.B. and E.M.C., n°40H58 to D.G. and APO-SYS Health F4-2007-200767 to M.P., European Project: 241745,EC:FP7:HEALTH,FP7-HEALTH-2009-single-stage,NEWTBVAC(2010), Istituto Superiore di Sanita [Rome], Institut Pasteur [Paris], Università cattolica del Sacro Cuore [Roma] (Unicatt), Romagnoli, Alessandra, Etna, Marilena P, Giacomini, Elena, Pardini, Manuela, Remoli, Maria Elena, Corazzari, Marco, Falasca, Laura, Goletti, Delia, Gafa, Valérie, Simeone, Roxane, Delogu, Giovanni, Piacentini, Mauro, Brosch, Roland, Fimia, Gian Maria, and Coccia, Eliana M.
Subjects: MESH: Mycobacterium bovis, MESH: Mycobacterium tuberculosis, Autophagosome maturation, [SDV]Life Sciences [q-bio], Mycobacterium tuberculosi, vaccine, Phagosomes, Sirolimu, BCG, MESH: Tuberculosis, MESH: Bacterial Secretion Systems, Bacterial Secretion Systems, Phagosome, Cells, Cultured, Mycobacterium bovis, Cultured, biology, MESH: Dendritic Cells, Effector, Cell Differentiation, respiratory system, Basic Research Paper, 3. Good health, Th1 Cell, Biological Markers, Human, MESH: Cells, Cultured, MESH: Cell Differentiation, Settore BIO/06, Cells, Dendritic Cells, Humans, Mycobacterium tuberculosis, Sirolimus, Th1 Cells, Tuberculosis, Autophagy, Tuberculosi, Dendritic Cell, complex mixtures, Settore MED/07 - MICROBIOLOGIA E MICROBIOLOGIA CLINICA, Microbiology, Immune system, MESH: Phagosomes, Mycobacterium bovi, MESH: Autophagy, Secretion, Molecular Biology, MESH: Humans, RD1 region, Cell Biology, biochemical phenomena, metabolism, and nutrition, biology.organism_classification, bacterial infections and mycoses, MESH: Th1 Cells, Biological Marker, MESH: Biomarkers, bacteria, MESH: Sirolimus, ESX1/type VII secretion system, Biomarkers
Abstract: International audience; Emerging evidence points to an important role of autophagy in the immune response mediated by dendritic cells (DC) against Mycobacterium tuberculosis (Mtb). Since current vaccination based on Bacillus Calmette-Guerin (BCG) is unable to stop the tuberculosis epidemic, a deeper comprehension of the alterations induced by Mtb in DC is essential for setting new vaccine strategies. Here, we compared the capacity of virulent (H37Rv) and avirulent (H37Ra) Mtb strains as well as BCG to modulate autophagy in human primary DC. We found that Mtb H37Rv impairs autophagy at the step of autophagosome-lysosome fusion. In contrast, neither Mtb H37Ra nor BCG strains were able to hamper autophagosome maturation. Both these attenuated strains have a functional inhibition of the 6kD early secreted antigenic target ESAT-6, an effector protein of the ESAT-6 Secretion System-1(ESX-1)/type VII secretion system. Notably, the ability to inhibit autophagy was fully restored in recombinant BCG and Mtb H37Ra strains in which ESAT-6 secretion was re-established by genetic complementation using either the ESX-1 region from Mtb (BCG::ESX-1) or the PhoP gene (Mtb H37Ra::PhoP), a regulator of ESAT-6 secretion. Importantly, the autophagic block induced by Mtb was overcome by rapamycin treatment leading to an increased interleukin-12 expression and, in turn, to an enhanced capacity to expand a Th1-oriented response. Collectively, our study demonstrated that Mtb alters the autophagic machinery through the ESX-1 system, and thereby opens new exciting perspectives to better understand the relationship between Mtb virulence and its ability to escape the DC-mediated immune response.
Published: 2012

22. Molecular interface of S100A8 with cytochrome b558 and NADPH oxidase activation

Author: Minh Vu Chuong Nguyen, Marc-André Hograindleur, Françoise Morel, Athan Baillet, Marie-Hélène Paclet, Benoît Polack, Sylvie Berthier, Techniques de l'Ingénierie Médicale et de la Complexité - Informatique, Mathématiques et Applications, Grenoble - UMR 5525 (TIMC-IMAG), VetAgro Sup - Institut national d'enseignement supérieur et de recherche en alimentation, santé animale, sciences agronomiques et de l'environnement (VAS)-Institut polytechnique de Grenoble - Grenoble Institute of Technology (Grenoble INP )-Centre National de la Recherche Scientifique (CNRS)-Université Joseph Fourier - Grenoble 1 (UJF), Thérapeutique Recombinante Expérimentale (TIMC-IMAG-TheREx), VetAgro Sup - Institut national d'enseignement supérieur et de recherche en alimentation, santé animale, sciences agronomiques et de l'environnement (VAS)-Institut polytechnique de Grenoble - Grenoble Institute of Technology (Grenoble INP )-Centre National de la Recherche Scientifique (CNRS)-Université Joseph Fourier - Grenoble 1 (UJF)-VetAgro Sup - Institut national d'enseignement supérieur et de recherche en alimentation, santé animale, sciences agronomiques et de l'environnement (VAS)-Institut polytechnique de Grenoble - Grenoble Institute of Technology (Grenoble INP )-Centre National de la Recherche Scientifique (CNRS)-Université Joseph Fourier - Grenoble 1 (UJF), 'Ministère de l'Enseignement Supérieur de la Recherche et Technologie, Paris', 'UFR de Médecine, Université Joseph Fourier, Grenoble', 'Région Rhône-Alpes, programme Emergence 2003 and ARCUS (Actions en Régions de Coopération Universitaire et Scientifique) France/Chine 2007-2008', CGD (Chronic Granulomatous Disease) research Trust 2006-2007, UK, 'Groupement des Entreprises Françaises dans la lutte contre le Cancer, délégation de Grenoble', 'Société Française de Rhumatologie', and 'Délégation à la Recherche Clinique et à l'Innovation, Centre Hospitalier Universitaire, Grenoble'
Subjects: Herpesvirus 4, Human, [SDV.BIO]Life Sciences [q-bio]/Biotechnology, Cytochrome, Neutrophils, lcsh:Medicine, NADPH Oxidase, MESH: Amino Acid Sequence, Plasma protein binding, Biochemistry, biophysics & molecular biology [F05] [Life sciences], MESH: Neutrophils, Biochemistry, MESH: Antibodies, Monoclonal, MESH: Recombinant Proteins, MESH: Protein Structure, Tertiary, Cytosol, MESH: Structure-Activity Relationship, 0302 clinical medicine, Protein structure, MESH: Cytosol, ExoS toxin, MESH: Cell-Free System, MESH: Calgranulin B, MESH: Calgranulin A, Lymphocytes, [INFO.INFO-BT]Computer Science [cs]/Biotechnology, Biochimie, biophysique & biologie moléculaire [F05] [Sciences du vivant], MESH: NADPH Oxidase, MESH: Bacterial Secretion Systems, lcsh:Science, Bacterial Secretion Systems, Peptide sequence, 0303 health sciences, Oxidase test, Multidisciplinary, NADPH oxidase, biology, Enzyme Classes, Antibodies, Monoclonal, Recombinant Proteins, Oxygen Metabolism, Enzymes, Protein Transport, Cross-Linking Reagents, 030220 oncology & carcinogenesis, Pseudomonas aeruginosa, MESH: Pseudomonas aeruginosa, Oxidoreductases, Protein Binding, Research Article, MESH: Enzyme Activation, MESH: Protein Transport, Hemeprotein, MESH: Cross-Linking Reagents, Molecular Sequence Data, Enzyme Regulation, Structure-Activity Relationship, 03 medical and health sciences, Escherichia coli, Calgranulin B, Humans, MESH: Cytochrome b Group, MESH: Protein Binding, Calgranulin A, Amino Acid Sequence, Protein Interactions, Biology, 030304 developmental biology, MESH: Humans, MESH: Molecular Sequence Data, Cell-Free System, lcsh:R, NADPH Oxidases, Proteins, MESH: Herpesvirus 4, Human, Cytochrome b Group, Fusion protein, Protein Structure, Tertiary, Regulatory Proteins, Enzyme Activation, Transmembrane Proteins, Metabolism, Enzyme Structure, biology.protein, MESH: Lymphocytes, lcsh:Q
Abstract: International audience; S100A8 and S100A9 are two calcium binding Myeloid Related Proteins, and important mediators of inflammatory diseases. They were recently introduced as partners for phagocyte NADPH oxidase regulation. However, the precise mechanism of their interaction remains elusive. We had for aim (i) to evaluate the impact of S100 proteins on NADPH oxidase activity; (ii) to characterize molecular interaction of either S100A8, S100A9, or S100A8/S100A9 heterocomplex with cytochrome b(558); and (iii) to determine the S100A8 consensus site involved in cytochrome b(558)/S100 interface. Recombinant full length or S100A9-A8 truncated chimera proteins and ExoS-S100 fusion proteins were expressed in E. coli and in P. aeruginosa respectively. Our results showed that S100A8 is the functional partner for NADPH oxidase activation contrary to S100A9, however, the loading with calcium and a combination with phosphorylated S100A9 are essential in vivo. Endogenous S100A9 and S100A8 colocalize in differentiated and PMA stimulated PLB985 cells, with Nox2/gp91(phox) and p22(phox). Recombinant S100A8, loaded with calcium and fused with the first 129 or 54 N-terminal amino acid residues of the P. aeruginosa ExoS toxin, induced a similar oxidase activation in vitro, to the one observed with S100A8 in the presence of S100A9 in vivo. This suggests that S100A8 is the essential component of the S100A9/S100A8 heterocomplex for oxidase activation. In this context, recombinant full-length rS100A9-A8 and rS100A9-A8 truncated 90 chimera proteins as opposed to rS100A9-A8 truncated 86 and rS100A9-A8 truncated 57 chimeras, activate the NADPH oxidase function of purified cytochrome b(558) suggesting that the C-terminal region of S100A8 is directly involved in the molecular interface with the hemoprotein. The data point to four strategic (87)HEES(90) amino acid residues of the S100A8 C-terminal sequence that are involved directly in the molecular interaction with cytochrome b(558) and then in the phagocyte NADPH oxidase activation.
Published: 2012

23. Pilotin-secretin recognition in the type II secretion system of Klebsiella oxytoca

Author: Tosi, Tommaso, Nickerson, Nicholas, Mollica, Luca, Jensen, Malene Ringkjøbing, Blackledge, Martin, Baron, Bruno, England, Patrick, Pugsley, Anthony, Dessen, Andréa, Institut de biologie structurale (IBS - UMR 5075 ), Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), 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)-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), Génétique Moléculaire, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Biophysique des Macromolécules et de leurs Interactions, Institut Pasteur [Paris] (IP), This work was supported by Grant ANR-09-BLAN-0291. N.N.N. was supported by a postdoctoral fellowship from the Canadian Louis Pasteur Foundation during part of this work. The co-ordinates of PulS have been deposited in the Protein Database under code 4A56., The authors wish to thank Ingrid Guilvout and Séverine Collin for their helpful discussions and members of the Molecular Genetics Unit at the Institut Pasteur for their support. We are also grateful to J. Marquez and the HTX Lab team (Partnership for Structural Biology, Grenoble, PSB) for access to and help with high throughput crystallization, Hassan Berlhali (EMBL Grenoble) for help with collection of PulS-Barium data and Carlos Contreras-Martel for his support in solving PulSV30 structure, the European Synchrotron Radiation Facility (ESRF, PSB) for access to beamlines, Bertrand Raynal (Centre of Biophysics of Macromolecules and their Interactions, Institut Pasteur) for hydrodynamic modelling calculations, and Jacques d'Alayer (Protein Microsequencing and Analysis Platform, Institut Pasteur) for N-terminal sequencing., and ANR-09-BLAN-0291,Secretin(2009)
Subjects: [SDV]Life Sciences [q-bio], MESH: Bacterial Outer Membrane Proteins, Klebsiella oxytoca, MESH: Protein Structure, Secondary, MESH: Klebsiella oxytoca, Protein Structure, Secondary, Protein Structure, Tertiary, MESH: Protein Structure, Tertiary, MESH: Protein Binding, MESH: Molecular Chaperones, MESH: Bacterial Secretion Systems, Bacterial Secretion Systems, Bacterial Outer Membrane Proteins, Molecular Chaperones, Protein Binding
Abstract: International audience; A crucial aspect of the functionality of bacterial type II secretion systems is the targeting and assembly of the outer membrane secretin. In the Klebsiella oxytoca type II secretion system, the lipoprotein PulS, a pilotin, targets secretin PulD monomers through the periplasm to the outer membrane. We present the crystal structure of PulS, an all-helical bundle that is structurally distinct from proteins with similar functions. Replacement of valine at position 42 in a charged groove of PulS abolished complex formation between a non-lipidated variant of PulS and a peptide corresponding to the unfolded region of PulD to which PulS binds (the S-domain), in vitro, as well as PulS function in vivo. Substitutions of other residues in the groove also diminished the interaction with the S-domain in vitro but exerted less marked effects in vivo. We propose that the interaction between PulS and the S-domain is maintained through a structural adaptation of the two proteins that could be influenced by cis factors such as the fatty acyl groups on PulS, as well as periplasmic transacting factors, which represents a possible paradigm for chaperonetarget protein interactions.
Published: 2011

24. Outer Membrane Targeting of Secretin PulD Protein Relies on Disordered Domain Recognition by a Dedicated Chaperone

Author: Bertrand Raynal, Bruno Baron, Andréa Dessen, Nicholas N. Nickerson, Tommasso Tosi, Patrick England, Anthony P. Pugsley, Génétique Moléculaire, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Institut de biologie structurale (IBS - UMR 5075 ), Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), 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)-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), Biophysique des Macromolécules et de leurs Interactions, This work was supported in part by French National Research Agency Grants ANR-05-0307-01 and ANR-09-BLAN-0291., ANR-05-BLAN-0307,SECRETIN,Structure and function of secretin in the bacterial type II protein secretion system(2005), and ANR-09-BLAN-0291,Secretin(2009)
Subjects: Protein Folding, MESH: Protein Structure, Quaternary, [SDV]Life Sciences [q-bio], MESH: Protein Structure, Secondary, MESH: Escherichia coli Proteins, Plasma protein binding, Biochemistry, Protein Structure, Secondary, MESH: Protein Structure, Tertiary, fluids and secretions, MESH: Nuclear Magnetic Resonance, Biomolecular, MESH: Bacterial Secretion Systems, Protein secondary structure, Bacterial Secretion Systems, MESH: Escherichia coli, Escherichia coli Proteins, Outer Membrane, MESH: Lipoproteins, Periplasm, Chaperone binding, Protein folding, MESH: Molecular Chaperones, Bacterial outer membrane, hormones, hormone substitutes, and hormone antagonists, Bacterial Outer Membrane Proteins, Protein Binding, Lipoproteins, MESH: Protein Folding, Biology, Intrinsically disordered proteins, digestive system, Microbiology, Escherichia coli, MESH: Protein Binding, Binding site, Chaperone Chaperonin, Protein Structure, Quaternary, Molecular Biology, Nuclear Magnetic Resonance, Biomolecular, Secretion, MESH: Bacterial Outer Membrane Proteins, Membrane Proteins, Cell Biology, digestive system diseases, Protein Structure, Tertiary, Intrinsically Disordered Proteins, Chaperone (protein), Biophysics, biology.protein, Molecular Chaperones
Abstract: International audience; Interaction of bacterial outer membrane secretin PulD with its dedicated lipoprotein chaperone PulS relies on a disorder-to-order transition of the chaperone binding (S) domain near the PulD C terminus. PulS interacts with purified S domain to form a 1:1 complex. Circular dichroism, one-dimensional NMR, and hydrodynamic measurements indicate that the S domain is elongated and intrinsically disordered but gains secondary structure upon binding to PulS. Limited proteolysis and mass spectrometry identified the 28 C-terminal residues of the S domain as a minimal binding site with low nanomolar affinity for PulS in vitro that is sufficient for outer membrane targeting of PulD in vivo. The region upstream of this binding site is not required for targeting or multimerization and does not interact with PulS, but it is required for secretin function in type II secretion. Although other secretin chaperones differ substantially from PulS in sequence and secondary structure, they have all adopted at least superficially similar mechanisms of interaction with their cognate secretins, suggesting that intrinsically disordered regions facilitate rapid interaction between secretins and their chaperones.
Published: 2011

25. Full virulence of Pseudomonas aeruginosa requires OprF

Author: Nathalie Connil, Alain Dufour, Laurène Fito-Boncompte, Bertrand Toussaint, Sylvie Chevalier, Hichem Chaker, Alexis Bazire, Qing Wei, Wilfried Veron, Eric Déziel, Pierre Cornelis, Laure Taupin, Marc G. J. Feuilloley, Emeline Bouffartigues, Koki Shioya, Amar Madi, Gwendoline Gicquel, Nicole Orange, Annelise Chapalain, Olivier Lesouhaitier, Department of Bio-engineering Sciences, Microbial Interactions, Laboratoire de Microbiologie du Froid – Signaux et Micro-Environnement (LMDF-SME), Université de Rouen Normandie (UNIROUEN), Normandie Université (NU)-Normandie Université (NU), Laboratoire de Biotechnologie et Chimie Marines (LBCM), Université de Bretagne Sud (UBS)-Université de Brest (UBO)-Institut Universitaire Européen de la Mer (IUEM), Institut de Recherche pour le Développement (IRD)-Institut national des sciences de l'Univers (INSU - CNRS)-Université de Brest (UBO)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche pour le Développement (IRD)-Institut national des sciences de l'Univers (INSU - CNRS)-Centre National de la Recherche Scientifique (CNRS), Institut Armand Frappier (INRS-IAF), Institut National de la Recherche Scientifique [Québec] (INRS)-Réseau International des Instituts Pasteur (RIIP), TheREx, Techniques de l'Ingénierie Médicale et de la Complexité - Informatique, Mathématiques et Applications, Grenoble - UMR 5525 (TIMC-IMAG), VetAgro Sup - Institut national d'enseignement supérieur et de recherche en alimentation, santé animale, sciences agronomiques et de l'environnement (VAS)-Institut polytechnique de Grenoble - Grenoble Institute of Technology (Grenoble INP )-Centre National de la Recherche Scientifique (CNRS)-Université Joseph Fourier - Grenoble 1 (UJF)-VetAgro Sup - Institut national d'enseignement supérieur et de recherche en alimentation, santé animale, sciences agronomiques et de l'environnement (VAS)-Institut polytechnique de Grenoble - Grenoble Institute of Technology (Grenoble INP )-Centre National de la Recherche Scientifique (CNRS)-Université Joseph Fourier - Grenoble 1 (UJF), Laboratory of Microbial Interactions, Vrije Universiteit Brussel (VUB)-Flanders institute of biotechnology-DEPARTMENT OF MOLECULAR AND CELLULAR INTERACTIONS, and This work was supported by the Communauté d'Agglomération d'Evreux, France (doctoral fellowship to L.F.-B.), European FEDER funds to the LBCM and to the LMDF-SME, and the Ministère de la Recherche et de la Technologie, France (postdoctoral fellowship to K.S.).
Subjects: [SDV.BIO]Life Sciences [q-bio]/Biotechnology, MESH: Virulence, Quinolones, medicine.disease_cause, MESH: Quorum Sensing, Virulence factor, Type three secretion system, Chicory, chemistry.chemical_compound, MESH: Reverse Transcriptase Polymerase Chain Reaction, MESH: Animals, [INFO.INFO-BT]Computer Science [cs]/Biotechnology, MESH: Bacterial Secretion Systems, MESH: Bacterial Proteins, Bacterial Secretion Systems, 0303 health sciences, MESH: Gene Expression Regulation, Bacterial, Virulence, Reverse Transcriptase Polymerase Chain Reaction, MESH: Pseudomonas Infections, Quorum Sensing, MESH: Plant Leaves, Infectious Diseases, Pseudomonas aeruginosa, OprF, Porin, MESH: Pseudomonas aeruginosa, Pseudomonas aeruginosa, MESH: Caco-2 Cells, Virulence Factors, Immunology, Biology, Microbiology, 03 medical and health sciences, Pyocyanin, Bacterial Proteins, MESH: Caenorhabditis elegans, medicine, Animals, Humans, Secretion, Pseudomonas Infections, Caenorhabditis elegans, 030304 developmental biology, MESH: Virulence Factors, MESH: Quinolones, MESH: Humans, 030306 microbiology, Gene Expression Regulation, Bacterial, Molecular Pathogenesis, Plant Leaves, chemistry, MESH: Chicory, Parasitology, Caco-2 Cells, Exotoxin
Abstract: OprF is a general outer membrane porin of Pseudomonas aeruginosa , a well-known human opportunistic pathogen associated with severe hospital-acquired sepsis and chronic lung infections of cystic fibrosis patients. A multiphenotypic approach, based on the comparative study of a wild-type strain of P. aeruginosa , its isogenic oprF mutant, and an oprF -complemented strain, showed that OprF is required for P. aeruginosa virulence. The absence of OprF results in impaired adhesion to animal cells, secretion of ExoT and ExoS toxins through the type III secretion system (T3SS), and production of the quorum-sensing-dependent virulence factors pyocyanin, elastase, lectin PA-1L, and exotoxin A. Accordingly, in the oprF mutant, production of the signal molecules N -(3-oxododecanoyl)- l -homoserine lactone and N -butanoyl- l -homoserine lactone was found to be reduced and delayed, respectively. Pseudomonas quinolone signal (PQS) production was decreased, while its precursor, 4-hydroxy-2-heptylquinoline (HHQ), accumulated in the cells. Taken together, these results show the involvement of OprF in P. aeruginosa virulence, at least partly through modulation of the quorum-sensing network. This is the first study showing a link between OprF, PQS synthesis, T3SS, and virulence factor production, providing novel insights into virulence expression.
Published: 2011

26. SecA Localization and SecA-Dependent Secretion Occurs at New Division Septa in Group B Streptococcus

Author: Elise Caliot, Sara Brega, Shaynoor Dramsi, Patrick Trieu-Cuot, Biologie des Bactéries pathogènes à Gram-positif, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), and Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)
Subjects: Amino Acid Motifs, Immunofluorescence, lcsh:Medicine, Fluorescent Antibody Technique, MESH: Amino Acid Sequence, Substrate Specificity, MESH: Membrane Transport Proteins, MESH: Recombinant Proteins, MESH: Amino Acid Motifs, chemistry.chemical_compound, Cell Wall, Sortase, MESH: Animals, Bacterial Physiology, lcsh:Science, MESH: Bacterial Secretion Systems, Bacterial Secretion Systems, MESH: Fluorescent Antibody Technique, MESH: Bacterial Proteins, Peptide sequence, Adenosine Triphosphatases, 0303 health sciences, Multidisciplinary, Streptococci, Recombinant Proteins, Bacterial Pathogens, Bacterial Biochemistry, Protein Transport, Sortase A, Medicine, Infectious diseases, MESH: Cell Division, Group B streptococcal infection, MESH: Protein Sorting Signals, Cell Division, Subcellular Fractions, Research Article, Signal peptide, MESH: Protein Transport, MESH: Mutation, Molecular Sequence Data, Immunology, Bacterial diseases, Protein Sorting Signals, Biology, Microbiology, Streptococcus agalactiae, 03 medical and health sciences, MESH: Cell Wall, Bacterial Proteins, MESH: Bacterial Capsules, MESH: Adenosine Triphosphatases, Animals, Secretion, Amino Acid Sequence, Bacterial Capsules, Secretory pathway, 030304 developmental biology, SecA Proteins, MESH: Molecular Sequence Data, 030306 microbiology, lcsh:R, Cell Membrane, Membrane Transport Proteins, Bacteriology, MESH: Streptococcus agalactiae, [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, Molecular biology, MESH: Extracellular Space, Membrane protein, chemistry, MESH: Subcellular Fractions, Mutation, Immunologic Techniques, lcsh:Q, MESH: Substrate Specificity, Peptidoglycan, Extracellular Space, SEC Translocation Channels, MESH: Cell Membrane
Abstract: International audience; Exported proteins of Streptococcus agalactiae (GBS), which include proteins localized to the bacterial surface or secreted into the extracellular environment, are key players for commensal and pathogenic interactions in the mammalian host. These proteins are transported across the cytoplasmic membrane via the general SecA secretory pathway and those containing the so-called LPXTG sorting motif are covalently attached to the peptidoglycan by sortase A. How SecA, sortase A, and LPXTG proteins are spatially distributed in GBS is not known. In the close relative Streptococcus pyogenes, it was shown that presence of the YSIRKG/S motif (literally YSIRKX3Gx2S) in the signal peptide (SP) constitutes the targeting information for secretion at the septum. Here, using conventional and deconvolution immunofluorescence analyses, we have studied in GBS strain NEM316 the localization of SecA, SrtA, and the secreted protein Bsp whose signal peptide contains a canonical YSIRKG/S motif (YSLRKykfGlaS). Replacing the SP of Bsp with four other SPs containing or not the YSIRKG/S motif did not alter the localized secretion of Bsp at the equatorial ring. Our results indicate that secretion and cell wall-anchoring machineries are localized at the division septum. Cell wall- anchored proteins displayed polar (PilB, Gbs0791), punctuate (CspA) or uniform distribution (Alp2) on the bacterial surface. De novo secretion of Gbs0791 following trypsin treatment indicates that it is secreted at the septum, then redistributed along the lateral sides, and finally accumulated to the poles. We conclude that the ±YSIRK SP rule driving compartimentalized secretion is not true in S. agalactiae.
Published: 2013

27. The Shigella flexneri Type Three Secretion System Effector IpgD Inhibits T Cell Migration by Manipulating Host Phosphoinositide Metabolism

Author: Elisabetta Frigimelica, Armelle Phalipon, Philippe J. Sansonetti, Daniel Scott-Algara, Katharina Nothelfer, Cristina Rodrigues, Vincenzo Di Bartolo, Christoph Konradt, Andrea Puhar, Wilmara Salgado-Pabón, Pathogénie Microbienne Moléculaire, Institut Pasteur [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM), Biologie Cellulaire des Lymphocytes (BIOCELLY), Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Régulation des Infections Rétrovirales, Institut Pasteur [Paris], Dynamique des Interactions Hôte-Pathogène - Dynamics of Host-Pathogen Interactions, Chaire Microbiologie et Maladies infectieuses, Collège de France (CdF (institution)), K.N. is a fellow from the Pasteur-Paris University International Doctoral Program. A. Puhar was supported first by EMBO long-term fellowship and is presently a Marie Curie fellow. W.S.-P. is funded by the Pasteur Foundation and the Philips Foundation. P.J.S. is a HHMI Foreign Scholar. The research leading to these results has received funding from the Institut Pasteur Transversal Research Program (PTR n° 251) and from the European Community's PEOPLE Seventh Framework Program under grant agreement EIMID IAPP - PIAP-GA-2008-217768., We warmly thank C. Parsot (Unité de Pathogénie Microbienne Moléculaire, Institut Pasteur) for very helpful discussion, M.I. Thoulouze and A. Alcover (Unité de Biologie Cellulaire des Lymphocytes, Institut Pasteur) for sharing with us their expertise in T cells, G. Chicanne and B. Payrastre (INSERM U858, I2MR, CHU Rangueil) for their expertise in PI metabolism, and M. Arpin (Institut Curie, Paris, France) for her advice on the ERM proteins. From the G5 Dynamique des Interactions Hôte-Pathogènes, Institut Pasteur, L. Audry and A. Bobard were helpful in providing tools for the injection assay, and J. Enninga for some experimental design, discussion, and critical reading of the manuscript. T. Balla (National Institute of Child Health and Human Development, National Institutes of Health [NIH]) kindly sent PLCδ1PH-RFP construct. We also thank Benoit Marteyn for his very efficient contribution in performing Shigella infection in the rabbit ileal model and processing samples for immunohistochemistry. We are also grateful to colleagues from the IMAGOPOLE platform of the Institut Pasteur. C.K. was supported by fellowships from the European Consortium PATHOGENOMICS, the Institut Pasteur Transversal Research Program n° 251, and the Howard Hughes Medical Institute (HHMI). E.F. holds a fellowship from the European Initiative for basic research in Microbiology and Infectious Diseases (EIMID Program)., Institut Pasteur [Paris] (IP)-Institut National de la Santé et de la Recherche Médicale (INSERM), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Institut Pasteur [Paris] (IP), and Collège de France - Chaire Microbiologie et Maladies infectieuses
Subjects: CD4-Positive T-Lymphocytes, Phosphatidylinositol 4,5-Diphosphate, MESH: Bacterial Secretion Systems, MESH: CD4-Positive T-Lymphocytes / immunology, Cancer Research, MESH: Phosphatidylinositols / metabolism, Fluorescent Antibody Technique, MESH: Phosphoric Monoester Hydrolases / metabolism, Phosphatidylinositols, medicine.disease_cause, Shigella flexneri, Type three secretion system, Cell Movement, Shigella, Phosphorylation, MESH: Shigella flexneri / metabolism, Bacterial Secretion Systems, MESH: Fluorescent Antibody Technique, 0303 health sciences, biology, Effector, MESH: Bacterial Proteins / metabolism, MESH: DNA-Binding Proteins / metabolism, MESH: CD4-Positive T-Lymphocytes / cytology, DNA-Binding Proteins, [SDV.MP]Life Sciences [q-bio]/Microbiology and Parasitology, MESH: Phosphatidylinositol 4,5-Diphosphate / deficiency, Host-Pathogen Interactions, Chemokines, Shigellosis, MESH: Chemokines / immunology, Blotting, Western, MESH: Shigella flexneri / genetics, Microbiology, Cell Line, 03 medical and health sciences, Bacterial Proteins, MESH: CD4-Positive T-Lymphocytes / metabolism, Immunology and Microbiology(all), Virology, medicine, Humans, MESH: Blotting, Western, Molecular Biology, MESH: Cell Movement / immunology, Dysentery, Bacillary, 030304 developmental biology, MESH: Transcription Factors / metabolism, MESH: Humans, MESH: Phosphorylation, 030306 microbiology, Cell Membrane, MESH: Host-Pathogen Interactions, MESH: CD4-Positive T-Lymphocytes / microbiology, biochemical phenomena, metabolism, and nutrition, biology.organism_classification, medicine.disease, MESH: Dysentery, Bacillary / metabolism, Phosphoric Monoester Hydrolases, MESH: Cell Line, Cell culture, T cell migration, Parasitology, MESH: Phosphatidylinositol 4,5-Diphosphate / metabolism, MESH: Cell Membrane / chemistry, Transcription Factors, MESH: Dysentery, Bacillary / genetics
Abstract: International audience; Shigella, the Gram-negative enteroinvasive bacterium that causes shigellosis, relies on its type III secretion system (TTSS) and injected effectors to modulate host cell functions. However, consequences of the interaction between Shigella and lymphocytes have not been investigated. We show that Shigella invades activated human CD4(+) T lymphocytes. Invasion requires a functional TTSS and results in inhibition of chemokine-induced T cell migration, an effect mediated by the TTSS effector IpgD, a phosphoinositide 4-phosphatase. Remarkably, IpgD injection into bystander T cells can occur in the absence of cell invasion. Upon IpgD-mediated hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP(2)), the pool of PIP(2) at the plasma membrane is reduced, leading to dephosphorylation of the ERM proteins and their inability to relocalize at one T cell pole upon chemokine stimulus, likely affecting the formation of the polarized edge required for cell migration. These results reveal a bacterial TTSS effector-mediated strategy to impair T cell function.
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27 results on '"MESH: Bacterial Secretion Systems"'

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

2. RD5-mediated lack of PE_PGRS and PPE-MPTR export in BCG vaccine strains results in strong reduction of antigenic repertoire but little impact on protection

3. Analysis of Bacterial Pilus Assembly by Shearing and Immunofluorescence Microscopy

4. Pathogenic Leptospira interrogans Exoproteins Are Primarily Involved in Heterotrophic Processes

5. Injection of T3SS effectors not resulting in invasion is the main targeting mechanism of Shigella toward human lymphocytes

6. Tracking Proteins Secreted by Bacteria: What's in the Toolbox?

7. Camelid nanobodies used as crystallization chaperones for different constructs of PorM, a component of the type IX secretion system from Porphyromonas gingivalis

8. Biological Diversity and Evolution of Type IV Secretion Systems

9. Structural Similarity of Secretins from Type II and Type III Secretion Systems

10. A Specific Polymorphism in Mycobacterium tuberculosis H37Rv Causes Differential ESAT-6 Expression and Identifies WhiB6 as a Novel ESX-1 Component

11. The Rate of Folding Dictates Substrate Secretion by the Escherichia coli Hemolysin Type 1 Secretion System

12. Biogenesis and structure of a Type VI secretion membrane core complex

13. Cytosolic Access of Mycobacterium tuberculosis: Critical Impact of Phagosomal Acidification Control and Demonstration of Occurrence In Vivo

14. Structure of a type IV secretion system

15. Membrane and chaperone recognition by the major translocator protein PopB of the type III secretion system of Pseudomonas aeruginosa

16. A safe bacterial microsyringe for in vivo antigen delivery and immunotherapy

17. The type II secretion system – a dynamic fiber assembly nanomachine

18. Structural insights into Helicobacter pylori oncoprotein CagA interaction with β1 integrin

19. Large-scale study of the interactions between proteins involved in type IV pilus biology in Neisseria meningitidis: characterization of a subcomplex involved in pilus assembly

20. Unique biofilm signature, drug susceptibility and decreased virulence in Drosophila through the Pseudomonas aeruginosa two-component system PprAB

21. ESX-1 dependent impairment of autophagic flux by Mycobacterium tuberculosis in human dendritic cells

22. Molecular interface of S100A8 with cytochrome b558 and NADPH oxidase activation

23. Pilotin-secretin recognition in the type II secretion system of Klebsiella oxytoca

24. Outer Membrane Targeting of Secretin PulD Protein Relies on Disordered Domain Recognition by a Dedicated Chaperone

25. Full virulence of Pseudomonas aeruginosa requires OprF

26. SecA Localization and SecA-Dependent Secretion Occurs at New Division Septa in Group B Streptococcus

27. The Shigella flexneri Type Three Secretion System Effector IpgD Inhibits T Cell Migration by Manipulating Host Phosphoinositide Metabolism

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27 results on '"MESH: Bacterial Secretion Systems"'

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