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Formation of the Legionella-containing vacuole: Effectors targeting retrograde trafficking and microtubules

English title Formation of the Legionella-containing vacuole: Effectors targeting retrograde trafficking and microtubules
Applicant Hilbi Hubert
Number 153200
Funding scheme Project funding
Research institution Institut für Medizinische Mikrobiologie Universität Zürich
Institution of higher education University of Zurich - ZH
Main discipline Experimental Microbiology
Start/End 01.01.2015 - 31.03.2018
Approved amount 600'000.00
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All Disciplines (2)

Discipline
Experimental Microbiology
Medical Microbiology

Keywords (7)

Microtubules; Macrophage; Type IV secretion; Phosphoinositide lipids; Amoeba; Dictyostelium; Bacterial virulence

Lay Summary (German)

Lead
Legionellen (Legionella pneumophila) sind Umweltbakterien, welche eine schwere Lungenentzündung namens Legionärskrankheit auslösen können. Die Bakterien vermehren sich in freilebenden Amöben und nach dem Einatmen in speziellen Immunzellen in der Lunge. Unsere Forschung trägt zum Verständnis der Legionelleninfektion und zu möglichen neuen Therapieansätzen bei.
Lay summary

Inhalt undZiele des Forschungsprojekts

Legionellen(Legionella pneumophila) vermehren sichin Amöben und besiedeln Biofilme. Durch Inhalation von feinen Wassertröpfchen (Aerosolen)aus kontaminierten Wassersystemen, z.B. Kühltürmen oder Duschen, gelangen dieLegionellen in die menschliche Lunge. Hier vermehren sich die Bakterien in Immunzellen(Makrophagen) und lösen eine schwere Lungenentzündung, die Legionärskrankheit, aus.Die Krankheit trifft bevorzugt immungeschwächte und ältere Personen undverläuft in 5-20% der Fälle tödlich.

Legionellenvermehren sich in Wirtszellen in einem speziellen Kompartiment, einer Vakuole,wofür die Bakterien ein Proteintransportsystem und über 300 (!) verschiedene „Effektorproteine“verwenden. Diese Proteine werden in die Wirtszelle injiziert, wo siezellbiologische und biochemische Prozesse zugunsten der Bakterien modulieren. Imvorliegenden Projekt wird auf molekularer und zellulärer Ebene dieintrazelluläre Vermehrung von Legionellen untersucht. Dabei kommen biochemischesowie molekular- und zellbiologische Methoden zum Einsatz. Die erarbeitetenErkenntnisse liefern grundlegende Einsichten überdie Virulenz von Legionellen und finden möglicherweise therapeutische Anwendungenin der Klinik. Unsere Arbeiten im Bereich der Zell-Zell-Kommunikation,Gen-Regulation und Biofilm-Bildung von Legionellen können dazu beitragen, dasAuftreten der Bakterien in Wassersystemen zu kontrollieren.

Wissenschaftlicherund gesellschaftlicher Kontext des Forschungsprojekts

Infektionskrankheitenstellen ein schwerwiegendes Gesundheitsproblem dar. Dies zum einen, weil die mikrobiellenErreger kontinuierlich Resistenzen gegen Antibiotika erwerben, und zum anderen,weil immer wieder neue, bislang unbekannte Erreger auftreten. UnsereGrundlagenforschung trägt zur Kontrolle von pathogenen Bakterien der Spezies Legionella im Klinischen Umfeld und inWassersystemen bei.

Direct link to Lay Summary Last update: 28.10.2014

Responsible applicant and co-applicants

Employees

Publications

Publication
Legionella quorum sensing meets cyclic-di-GMP signaling
Hochstrasser Ramon, Hilbi Hubert (2020), Legionella quorum sensing meets cyclic-di-GMP signaling, in Current Opinion in Microbiology, 55, 9-16.
Mycobacterium marinum produces distinct mycobactin and carboxymycobactin siderophores to promote growth in broth and phagocytes
Knobloch Paulina, Koliwer‐Brandl Hendrik, Arnold Fabian M., Hanna Nabil, Gonda Imre, Adenau Sophia, Personnic Nicolas, Barisch Caroline, Seeger Markus A., Soldati Thierry, Hilbi Hubert (2020), Mycobacterium marinum produces distinct mycobactin and carboxymycobactin siderophores to promote growth in broth and phagocytes, in Cellular Microbiology, 22(5), e13163.
Divergent Evolution of Legionella RCC1 Repeat Effectors Defines the Range of Ran GTPase Cycle Targets
Swart A. Leoni, Steiner Bernhard, Gomez-Valero Laura, Schütz Sabina, Hannemann Mandy, Janning Petra, Irminger Michael, Rothmeier Eva, Buchrieser Carmen, Itzen Aymelt, Panse Vikram Govind, Hilbi Hubert (2020), Divergent Evolution of Legionella RCC1 Repeat Effectors Defines the Range of Ran GTPase Cycle Targets, in mBio, 11(2), e00405-20-e00405-20.
The structure of the Legionella response regulator LqsR reveals amino acids critical for phosphorylation and dimerization
Hochstrasser Ramon, Hutter Cedric A. J., Arnold Fabian M., Bärlocher Kevin, Seeger Markus A., Hilbi Hubert (2020), The structure of the Legionella response regulator LqsR reveals amino acids critical for phosphorylation and dimerization, in Molecular Microbiology, mmi.14477-mmi.14477.
Quorum sensing modulates the formation of virulent Legionella persisters within infected cells
Personnic Nicolas, Striednig Bianca, Lezan Emmanuelle, Manske Christian, Welin Amanda, Schmidt Alexander, Hilbi Hubert (2019), Quorum sensing modulates the formation of virulent Legionella persisters within infected cells, in Nature Communications, 10(1), 5216-5216.
Distinct Mycobacterium marinum phosphatases determine pathogen vacuole phosphoinositide pattern, phagosome maturation, and escape to the cytosol
Koliwer‐Brandl Hendrik, Knobloch Paulina, Barisch Caroline, Welin Amanda, Hanna Nabil, Soldati Thierry, Hilbi Hubert (2019), Distinct Mycobacterium marinum phosphatases determine pathogen vacuole phosphoinositide pattern, phagosome maturation, and escape to the cytosol, in Cellular Microbiology, 21(6), e13008-e13008.
PIKfyve/Fab1 is required for efficient V-ATPase and hydrolase delivery to phagosomes, phagosomal killing, and restriction of Legionella infection
Buckley Catherine M., Heath Victoria L., Guého Aurélie, Bosmani Cristina, Knobloch Paulina, Sikakana Phumzile, Personnic Nicolas, Dove Stephen K., Michell Robert H., Meier Roger, Hilbi Hubert, Soldati Thierry, Insall Robert H., King Jason S. (2019), PIKfyve/Fab1 is required for efficient V-ATPase and hydrolase delivery to phagosomes, phagosomal killing, and restriction of Legionella infection, in PLOS Pathogens, 15(2), e1007551-e1007551.
The pleiotropic Legionella transcription factor LvbR links the Lqs and c‐di‐GMP regulatory networks to control biofilm architecture and virulence
Hochstrasser Ramon, Kessler Aline, Sahr Tobias, Simon Sylvia, Schell Ursula, Gomez‐Valero Laura, Buchrieser Carmen, Hilbi Hubert (2019), The pleiotropic Legionella transcription factor LvbR links the Lqs and c‐di‐GMP regulatory networks to control biofilm architecture and virulence, in Environmental Microbiology, 21(3), 1035-1053.
Legionella -Containing Vacuoles Capture PtdIns(4) P -Rich Vesicles Derived from the Golgi Apparatus
Weber Stephen, Steiner Bernhard, Welin Amanda, Hilbi Hubert (2018), Legionella -Containing Vacuoles Capture PtdIns(4) P -Rich Vesicles Derived from the Golgi Apparatus, in mBio, 9(6), e02420-18-e02420-18.
Quantitative Imaging Flow Cytometry of Legionella -Infected Dictyostelium Amoebae Reveals the Impact of Retrograde Trafficking on Pathogen Vacuole Composition
Welin Amanda, Weber Stephen, Hilbi Hubert (2018), Quantitative Imaging Flow Cytometry of Legionella -Infected Dictyostelium Amoebae Reveals the Impact of Retrograde Trafficking on Pathogen Vacuole Composition, in Applied and Environmental Microbiology, 84(11), e00158-18-e00158-18.
Acanthamoeba and Dictyostelium as Cellular Models for Legionella Infection
Swart A. Leoni, Harrison Christopher F., Eichinger Ludwig, Steinert Michael, Hilbi Hubert (2018), Acanthamoeba and Dictyostelium as Cellular Models for Legionella Infection, in Frontiers in Cellular and Infection Microbiology, 8, 61.
The large GTPase atlastin controls ER remodeling around a pathogen vacuole
Steiner Bernhard, Weber Stephen, Kaech Andres, Ziegler Urs, Hilbi Hubert (2018), The large GTPase atlastin controls ER remodeling around a pathogen vacuole, in Communicative & Integrative Biology, e1440880-e1440880.
Legionella quorum sensing and its role in pathogen–host interactions
Personnic Nicolas, Striednig Bianca, Hilbi Hubert (2018), Legionella quorum sensing and its role in pathogen–host interactions, in Current Opinion in Microbiology, 29-35.
Formation of the Legionella -containing vacuole: phosphoinositide conversion, GTPase modulation and ER dynamics
Steiner Bernhard, Weber Stephen, Hilbi Hubert (2018), Formation of the Legionella -containing vacuole: phosphoinositide conversion, GTPase modulation and ER dynamics, in International Journal of Medical Microbiology, (1), 49-57.
Structural insights into Legionella RidL-Vps29 retromer subunit interaction reveal displacement of the regulator TBC1D5
Bärlocher Kevin, Hutter Cedric A. J., Swart A. Leoni, Steiner Bernhard, Welin Amanda, Hohl Michael, Letourneur François, Seeger Markus A., Hilbi Hubert (2017), Structural insights into Legionella RidL-Vps29 retromer subunit interaction reveal displacement of the regulator TBC1D5, in Nature Communications, 8(1), 1543-1543.
ER remodeling by the large GTPase atlastin promotes vacuolar growth of Legionella pneumophila
Steiner Bernhard, Swart Anna Leoni, Welin Amanda, Weber Stephen, Personnic Nicolas, Kaech Andres, Freyre Christophe, Ziegler Urs, Klemm Robin W, Hilbi Hubert (2017), ER remodeling by the large GTPase atlastin promotes vacuolar growth of Legionella pneumophila, in EMBO reports, 18(10), 1817-1836.
Legionella pneumophila Modulates Mitochondrial Dynamics to Trigger Metabolic Repurposing of Infected Macrophages
Escoll Pedro, Song Ok-Ryul, Viana Flávia, Steiner Bernhard, Lagache Thibault, Olivo-Marin Jean-Christophe, Impens Francis, Brodin Priscille, Hilbi Hubert, Buchrieser Carmen (2017), Legionella pneumophila Modulates Mitochondrial Dynamics to Trigger Metabolic Repurposing of Infected Macrophages, in Cell Host & Microbe, 22(3), 302-316.e7.
Inhibitors of Mycobacterium marinum virulence identified in a Dictyostelium discoideum host model
Ouertatani-Sakouhi Hajer, Kicka Sébastien, Chiriano Gianpaolo, Harrison Christopher F., Hilbi Hubert, Scapozza Leonardo, Soldati Thierry, Cosson Pierre (2017), Inhibitors of Mycobacterium marinum virulence identified in a Dictyostelium discoideum host model, in PLOS ONE, 12(7), e0181121-e0181121.
Role of the small GTPase Rap1 in signal transduction, cell dynamics and bacterial infection
Hilbi Hubert, Kortholt Arjan (2017), Role of the small GTPase Rap1 in signal transduction, cell dynamics and bacterial infection, in Small GTPases, 1-7.
Comparative Proteomics of Purified Pathogen Vacuoles Correlates Intracellular Replication of Legionella pneumophila with the Small GTPase Ras-related protein 1 (Rap1)
Schmölders Johanna, Manske Christian, Otto Andreas, Hoffmann Christine, Steiner Bernhard, Welin Amanda, Becher Dörte, Hilbi Hubert (2017), Comparative Proteomics of Purified Pathogen Vacuoles Correlates Intracellular Replication of Legionella pneumophila with the Small GTPase Ras-related protein 1 (Rap1), in Molecular & Cellular Proteomics, 16(4), 622-641.
Intra-Species and Inter-Kingdom Signaling of Legionella pneumophila
Hochstrasser Ramon, Hilbi Hubert (2017), Intra-Species and Inter-Kingdom Signaling of Legionella pneumophila, in Frontiers in Microbiology, 8, 79.
In Silico Driven Design and Synthesis of Rhodanine Derivatives as Novel Antibacterials Targeting the Enoyl Reductase InhA
Slepikas Liudas, Chiriano Gianpaolo, Perozzo Remo, Tardy Sébastien, Kranjc Agata, Patthey-Vuadens Ophélie, Ouertatani-Sakouhi Hajer, Kicka Sébastien, Harrison Christopher F., Scrignari Tiziana, Perron Karl, Hilbi Hubert, Soldati Thierry, Cosson Pierre, Tarasevicius Eduardas, Scapozza Leonardo (2016), In Silico Driven Design and Synthesis of Rhodanine Derivatives as Novel Antibacterials Targeting the Enoyl Reductase InhA, in Journal of Medicinal Chemistry, 59(24), 10917-10928.
Legionella shows a diverse secondary metabolism dependent on a broad spectrum Sfp-type phosphopantetheinyl transferase
Tobias Nicholas J., Ahrendt Tilman, Schell Ursula, Miltenberger Melissa, Hilbi Hubert, Bode Helge B. (2016), Legionella shows a diverse secondary metabolism dependent on a broad spectrum Sfp-type phosphopantetheinyl transferase, in PeerJ, 4, e2720-e2720.
Metabolism of myo -Inositol by Legionella pneumophila Promotes Infection of Amoebae and Macrophages
Manske Christian, Schell Ursula, Hilbi Hubert (2016), Metabolism of myo -Inositol by Legionella pneumophila Promotes Infection of Amoebae and Macrophages, in Applied and Environmental Microbiology, 82(16), 5000-5014.
Subversion of Retrograde Trafficking by Translocated Pathogen Effectors
Personnic Nicolas, Bärlocher Kevin, Finsel Ivo, Hilbi Hubert (2016), Subversion of Retrograde Trafficking by Translocated Pathogen Effectors, in Trends in Microbiology, (6), 450-462.
Pathway analysis using 13 C-glycerol and other carbon tracers reveals a bipartite metabolism of Legionella pneumophila Bipartite metabolism of L. pneumophila
Häuslein Ina, Manske Christian, Goebel Werner, Eisenreich Wolfgang, Hilbi Hubert (2016), Pathway analysis using 13 C-glycerol and other carbon tracers reveals a bipartite metabolism of Legionella pneumophila Bipartite metabolism of L. pneumophila, in Molecular Microbiology, 100(2), 229-246.
The α-hydroxyketone LAI-1 regulates motility, Lqs-dependent phosphorylation signalling and gene expression of Legionella pneumophilaLAI-1 regulates Lqs-dependent signalling
Schell Ursula, Simon Sylvia, Sahr Tobias, Hager Dominik, Albers Michael F., Kessler Aline, Fahrnbauer Felix, Trauner Dirk, Hedberg Christian, Buchrieser Carmen, Hilbi Hubert (2016), The α-hydroxyketone LAI-1 regulates motility, Lqs-dependent phosphorylation signalling and gene expression of Legionella pneumophilaLAI-1 regulates Lqs-dependent signalling, in Molecular Microbiology, 99(4), 778-793.
Legionella pneumophila S1P-lyase targets host sphingolipid metabolism and restrains autophagy
Rolando Monica, Escoll Pedro, Nora Tamara, Botti Joëlle, Boitez Valérie, Bedia Carmen, Daniels Craig, Abraham Gilu, Stogios Peter J., Skarina Tatiana, Christophe Charlotte, Dervins-Ravault Delphine, Cazalet Christel, Hilbi Hubert, Rupasinghe Thusitha W. T., Tull Dedreia, McConville Malcolm J., Ong Sze Ying, Hartland Elizabeth L., Codogno Patrice, Levade Thierry, Naderer Thomas, Savchenko Alexei, Buchrieser Carmen (2016), Legionella pneumophila S1P-lyase targets host sphingolipid metabolism and restrains autophagy, in Proceedings of the National Academy of Sciences, 113(7), 1901-1906.
IFNs Modify the Proteome of Legionella-Containing Vacuoles and Restrict Infection Via IRG1-Derived Itaconic Acid
Naujoks Jan, Tabeling Christoph, Dill Brian D., Hoffmann Christine, Brown Andrew S., Kunze Mareike, Kempa Stefan, Peter Andrea, Mollenkopf Hans-Joachim, Dorhoi Anca, Kershaw Olivia, Gruber Achim D., Sander Leif E., Witzenrath Martin, Herold Susanne, Nerlich Andreas, Hocke Andreas C., van Driel Ian, Suttorp Norbert, Bedoui Sammy, Hilbi Hubert, Trost Matthias, Opitz Bastian (2016), IFNs Modify the Proteome of Legionella-Containing Vacuoles and Restrict Infection Via IRG1-Derived Itaconic Acid, in PLOS Pathogens, 12(2), e1005408-e1005408.
Inter-kingdom Signaling by the Legionella Quorum Sensing Molecule LAI-1 Modulates Cell Migration through an IQGAP1-Cdc42-ARHGEF9-Dependent Pathway
Simon Sylvia, Schell Ursula, Heuer Natalie, Hager Dominik, Albers Michael F., Matthias Jan, Fahrnbauer Felix, Trauner Dirk, Eichinger Ludwig, Hedberg Christian, Hilbi Hubert (2015), Inter-kingdom Signaling by the Legionella Quorum Sensing Molecule LAI-1 Modulates Cell Migration through an IQGAP1-Cdc42-ARHGEF9-Dependent Pathway, in PLOS Pathogens, 11(12), e1005307-e1005307.
Formation of a pathogen vacuole according to Legionella pneumophila : how to kill one bird with many stones Legionella pathogen vacuole formation
Finsel Ivo, Hilbi Hubert (2015), Formation of a pathogen vacuole according to Legionella pneumophila : how to kill one bird with many stones Legionella pathogen vacuole formation, in Cellular Microbiology, (7), 935-950.
Adrenergic antagonists restrict replication of Legionella
Chiriano Gianpaolo, Kranjc Agata, Soldati Thierry, Ouertatani-Sakouhi Hajer, Finsel Ivo, Harrison Christopher F., Scapozza Leonardo, Hilbi Hubert, Kicka Sébastien (2015), Adrenergic antagonists restrict replication of Legionella, in Microbiology, 161(7), 1392-1406.
Amoebae-Based Screening Reveals a Novel Family of Compounds Restricting Intracellular Legionella pneumophila
Harrison Christopher F., Chiriano Gianpaolo, Finsel Ivo, Manske Christian, Hoffmann Christine, Steiner Bernhard, Kranjc Agata, Patthey-Vuadens Ophelie, Kicka Sébastien, Trofimov Valentin, Ouertatani-Sakouhi Hajer, Soldati Thierry, Scapozza Leonardo, Hilbi Hubert (2015), Amoebae-Based Screening Reveals a Novel Family of Compounds Restricting Intracellular Legionella pneumophila, in ACS Infectious Diseases, 1(7), 327-338.
Phosphatidylinositol 4-phosphate and phosphatidylinositol 3-phosphate regulate phagolysosome biogenesis
Jeschke Andreas, Zehethofer Nicole, Lindner Buko, Krupp Jessica, Schwudke Dominik, Haneburger Ina, Jovic Marko, Backer Jonathan M., Balla Tamas, Hilbi Hubert, Haas Albert (2015), Phosphatidylinositol 4-phosphate and phosphatidylinositol 3-phosphate regulate phagolysosome biogenesis, in Proceedings of the National Academy of Sciences, 112(15), 4636-4641.

Collaboration

Group / person Country
Types of collaboration
Prof. K. Jung/ LMU Munich Germany (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
- Research Infrastructure
Prof. B. Oppitz/ Charite University Hospital Berlin Germany (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Research Infrastructure
- Exchange of personnel
Prof. C. Hedberg/ University of Umea Sweden (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
Prof. D. Becher/ University of Greifswald Germany (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
- Research Infrastructure
Prof. V. Panse/ University of Zürich Switzerland (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
- Research Infrastructure
- Exchange of personnel
Prof. F. Letourneur/ University of Montpellier France (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
Prof. T. Soldati/ University of Geneva Switzerland (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
Prof. M. Seeger/ University of Zürich Switzerland (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Research Infrastructure
Prof. A. Flieger/ RKI Wernigerode Germany (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Research Infrastructure
- Exchange of personnel
Dr. R. Kammerer/ Paul-Scherrer Institute Switzerland (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Research Infrastructure
Prof. W. Eisenreich/ TU Munich Germany (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Research Infrastructure
Prof. L. Eichinger/ University of Cologne Germany (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Research Infrastructure
Prof. G. Griffiths/ University Oslo Norway (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
- Research Infrastructure
Prof. C. Buchrieser/ Institute Pasteur, Paris France (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
- Research Infrastructure
Prof. H. Bode/ University of Frankfurt Germany (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
Prof. R. Aebersold/ ETH Zürich Switzerland (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
- Research Infrastructure
PD Dr. A. Müller-Taubenberger/ LMU Munich Germany (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
- Research Infrastructure
Prof. A. Itzen/ TU Munich Germany (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
- Research Infrastructure
Prof. P. Cosson/ University of Geneva Switzerland (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
- Research Infrastructure

Communication with the public

Communication Title Media Place Year
Talks/events/exhibitions Legionellen - Aus der Amöbe in die Lunge German-speaking Switzerland 2018
Talks/events/exhibitions Workshop “Legionella in water heating systems” Western Switzerland 2017

Awards

Title Year
Fellowship American Academy of Microbiology 2018

Associated projects

Number Title Start Funding scheme
170811 Enhanced Resolution Confocal Laser Scanning Microscope 01.10.2017 R'EQUIP
112592 Survival of Shigella and Legionella within phagocytes 01.08.2006 SNSF Professorships
184024 Antibacterial function of mammalian lipid droplets; high-resolution characterization of LD-pathogen contact sites 01.09.2019 Early Postdoc.Mobility
125369 Legionella trafficking and host phosphoinositide metabolism 01.05.2009 Project funding
130016 Identification and characterization of novel antibacterial compounds using protozoan hosts 01.09.2010 Sinergia
175557 Virulence and communication of Legionella: Molecular determinants of pathogen-host cell interactions 01.04.2018 Project funding
175557 Virulence and communication of Legionella: Molecular determinants of pathogen-host cell interactions 01.04.2018 Project funding

Abstract

Environmental bacteria of the genus Legionella grow within free-living amoebae and, upon inhalation of contaminated aerosols, within lung macrophages, thereby causing a severe pneumonia termed “Legionnaires’ disease”. Employing a largely conserved mechanism, Legionella pneumophila replicates in protozoan and mammalian host cells in a distinct membrane-bound compartment, the “Legionella-containing vacuole” (LCV). LCVs avoid fusion with bactericidal lysosomes, but communicate with the endosomal, secretory and retrograde vesicle trafficking pathways, and eventually fuse with the endoplasmic reticulum (ER). Formation of LCVs is a complex and robust process, which involves the bacterial Icm/Dot type IV secretion system (T4SS) and the astonishing number of approximately 300 different secreted “effector proteins". These proteins are injected into host cells, where they modulate pivotal components of eukaryotic signal transduction and membrane dynamics, such as small GTPases, phosphoinositide (PI) lipids or the cytoskeleton. Yet, the molecular function of most effectors is unknown. Recent proteomics studies from our laboratory on intact purified LCVs from L. pneumophila-infected Dictyostelium amoebae or macrophages revealed the presence of many host factors implicated in vesicle trafficking, including small GTPases and PI-metabolizing enzymes. We also discovered that a number of Icm/Dot-translocated effectors bind distinct PI lipids, thereby anchoring to the LCV membrane to subvert vesicle trafficking and membrane dynamics. Thus, the effector SidM modulates the secretory pathway, SidC promotes ER interaction, RidL binds to the eukaryotic retromer complex to interfere with retrograde trafficking, and LegG1 activates the small GTPase Ran to promote microtubule polymerization, LCV motility and infection. In this application, I propose to analyze LCV formation and intracellular growth of L. pneumophila in protozoan and mammalian phagocytes. In particular, interactions of LCVs and bacterial effectors with retrograde trafficking and transport along microtubules will be investigated by addressing the following specific topics: (a) The L. pneumophila retromer interactor RidL (b) Components of retrograde trafficking and LCV formation (c) The L. pneumophila Ran activator LegG1(d) Host factors involved in LCV trafficking along microtubules
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