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Functional Analysis of Outer Membrane Vesicles Shed by Legionella Pneumophila

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Functional Analysis of Outer Membrane Vesicles Shed by Legionella Pneumophila Functional analysis of outer membrane vesicles shed by Legionella pneumophila Von der Fakultät für Lebenswissenschaften der Technischen Universität Carolo-Wilhelmina zu Braunschweig zur Erlangung des Grades eines Doktors der Naturwissenschaften (Dr. rer. nat.) genehmigte D i s s e r t a t i o n von Jens Jäger aus Bad Hersfeld 1. Referent: Professor Dr. Michael Steinert 2. Referentin: Professor Dr. Petra Dersch 3. Referent: Professor Dr. Ulrich Dobrindt eingereicht am: 08.07.2013 mündliche Prüfung (Disputation) am: 20.08.2013 Druckjahr 2013 Vorveröffentlichungen der Dissertation Teilergebnisse aus dieser Arbeit wurden mit Genehmigung der Fakultät für Lebenswissenschaften, vertreten durch den Mentor der Arbeit, in folgenden Beiträgen vorab veröffentlicht: Publikationen Juli C, Sippel M, Jäger J, Thiele A, Weiwad M, Schweimer K, Rösch P, Steinert M, Sotriffer CA, Holzgrabe U. Pipecolic acid derivatives as small-molecule inhibitors of the Legionella MIP protein. J Med Chem. 2011 Jan 13;54(1):277-83. PubMed PMID: 21142106. Shevchuk O*, Jäger J*, Steinert M. Virulence properties of the Legionella pneumophila cell envelope. Front Microbiol. 2011;2:74. PubMed PMID: 21747794. (*: equal contributions) Jäger J, Steinert M. Enrichment of outer membrane vesicles shed by Legionella pneumophila. Methods Mol Biol. 2013;954:225-30. PMID: 23150399. Jäger J*, Marwitz S*, Tiefenau J, Rasch J, Shevchuk O, Kugler C, Goldmann T, Steinert M. Human lung tissue explants reveal novel interactions in Legionella pneumophila infections. In revision. (*: equal contributions) Jäger J, Keese S, Steinert M, Schromm A. Fusion of Legionella pneumophila outer membrane vesicles with eukaryotic membrane systems. In preparation. Tagungsbeiträge Jäger J, Schromm A, Steinert M. Functional analysis of outer membrane vesicles shed by Legionella pneumophila - Fusion with eukaryotic membranes? 1st Public Retreat of the Helmholtz International Graduate School for Infection Research, Quedlinburg, November 2010. Jäger J, Marwitz S, Goldmann T, Steinert M. Ex vivo infection of human lung tissue with Legionella pneumophila. 2nd Public Retreat of the Helmholtz International Graduate School for Infection Research, Goslar, June 2011. I Jäger J, Marwitz S, Tiefenau J, Goldmann T, Steinert M. Human lung tissue explants to study Legionella pneumophila infections. 64th Annual Meeting of the German Society of Hygiene and Microbiology (DGHM), Hamburg, October 2012. Jäger J, Marwitz S, Tiefenau J, Rasch J, Goldmann T, Steinert M. Human lung tissue explants to study Legionella pneumophila infections. 3rd Public Retreat of the Helmholtz International Graduate School for Infection Research, Bad Bevensen, October 2012. Posterbeiträge Jäger J, Steinert M. Functional analysis of outer membrane vesicles shed by Legionella pneumophila. 3rd PhD symposium of the Helmholtz Centre for Infection Research, Braunschweig, December 2009. Jäger J, Schromm A, Steinert M. Fusion studies of Legionella pneumophila outer membrane vesicles and eukaryotic membranes. 4th PhD symposium of the Helmholtz Centre for Infection Research, Braunschweig, December 2010. Jäger J, Krüger S, Galka F, Steinert M. Interaction of Legionella pneumophila outer membrane vesicles with host cells and bacteria. 63rd Annual Meeting of the German Society of Hygiene and Microbiology (DGHM), Essen, September 2011. Jäger J, Tiefenau J, Marwitz S, Goldmann T, Steinert M. Ex vivo infection of human lung tissue with Legionella pneumophila. 5th PhD symposium of the Helmholtz Centre for Infection Research, Braunschweig, December 2011. Jäger J, Krüger S, Steinert M. Interactions of Legionella pneumophila outer membrane vesicles with host cells and bacteria. Annual Conference of the Association of General and Applied Microbiology (VAAM), Tübingen, March 2012. II “I am not young enough to know everything. “ J. M. Barrie III Danksagung Mein erster Dank gilt Prof. Dr. Michael Steinert für die Möglichkeit, diese Arbeit in seiner Arbeitsgruppe zu erstellen, für die Motivation, die Diskussionen, die Anregungen – vielen Dank für alles! Außerdem danke ich Prof. Dr. Petra Dersch für die Übernahme des Zweitgutachtens sowie ihr und Prof. Dr. Susanne Häußler für die konstruktive Mitarbeit in meinem Thesis-Komitee. Prof. Dr. André Fleißner danke ich für die Übernahme des Prüfungsvorsitzes. Ich bedanke mich bei der Helmholtz International Graduate School for Infection Research für die Unterstützung sowie in besonderem Maße bei der Stiftung der Deutschen Wirtschaft für die ideelle und materielle Förderung. Riesige Mengen Backwaren, unvergessliche Mensabesuche, ausgedehnte Harzwanderungen: Besonders dankbar bin ich der AG Steinert – Barbara, Can, Chris, Corina, Dennis, Elena, Freddi, Gabi, Hannes, Jana, Janine, Julia, Olga, Qunxiu, Simon, Simone, Steffi – für Rat und Tat, für die grandiose Stimmung sowie für eine wichtige Einsicht: Genie und Wahnsinn liegen nah beieinander. Am Rande des Letzteren habe ich mich in Borstel öfters bewegt – dank Jana und Janine habe ich aber immer noch die Kurve gekriegt. Keinesfalls auslassen möchte ich die anderen „Mibis“. Ihr habt zu der fantastischen Arbeitsatmosphäre beigetragen – danke an Euch alle! Ich möchte auch den Studenten der AG Steinert danken, die mit guter Laune und Unterstützung im Labor zum Gelingen dieser Arbeit beigetragen haben. Und ich bedanke mich herzlich bei PD Dr. Andra Schromm und PD Dr. Torsten Goldmann vom Forschungszentrum Borstel dafür, dass sie mit ihrem Wissen und ihren Erfahrungen dazu beigetragen haben, dass diese Zusammenarbeit erfolgreich verlaufen ist. Ihren Arbeitsgruppen für Immunbiophysik bzw. Experimentelle Pathologie, hier im Besonderen Sebastian Marwitz, danke ich für die Unterstützung bei den Versuchen und Messungen. Schließlich möchte ich meinen Eltern, meinen Freunden und meiner Familie für die Unterstützung danken. Ihr seid die Besten! IV V Jens Jäger Table of contents Table of contents 1 Summary .................................................................................................... 1 2 Zusammenfassung .................................................................................... 2 3 Introduction ................................................................................................ 3 3.1 Legionella pneumophila: physiology and virulence ................................... 3 3.2 Intracellular life cycle ................................................................................ 4 3.2.1 Adhesion and uptake ............................................................................ 4 3.2.2 Modification of the Legionella-containing vacuole by secreted proteins................................................................................................. 5 3.2.3 Release from the LCV and the host cell ............................................... 8 3.3 Legionnaires‟ disease and Pontiac fever .................................................. 8 3.3.1 Host response to L. pneumophila infections ......................................... 9 3.4 Bacterial outer membrane vesicles ......................................................... 11 3.4.1 Functions of bacterial OMVs .............................................................. 13 3.4.2 L. pneumophila OMVs ........................................................................ 15 3.5 Model systems to study L. pneumophila infections ................................. 17 3.6 Aim of this work ...................................................................................... 20 4 Materials and methods ............................................................................ 21 4.1 Consumables and chemicals .................................................................. 21 4.2 Cell lines ................................................................................................. 21 4.3 Bacterial strains ...................................................................................... 21 4.4 Bacteriology ............................................................................................ 21 4.4.1 Cultivation of L. pneumophila ............................................................. 21 4.4.2 Cryoconservation of bacterial strains ................................................. 22 4.4.3 Enrichment of OMVs .......................................................................... 23 4.5 Biophysical methods ............................................................................... 23 4.5.1 Förster resonance energy transfer (FRET) spectroscopy .................. 24 V Jens Jäger Table of contents 4.5.2 Fourier-transform infrared (IR) spectroscopy ...................................... 25 4.6 Cell culture .............................................................................................. 25 4.6.1 Cryoconservation of mammalian cell lines ......................................... 25 4.6.2 Activation of macrophage-like cells .................................................... 26 4.6.3 Assessment of intracellular replication ............................................... 26 4.7 Human lung tissue explants .................................................................... 27 4.7.1 Infection of tissue samples ................................................................. 27 4.7.2 CFU determination from infected tissue samples ............................... 28 4.7.3 Histological analysis ........................................................................... 28 4.7.4 Immunohistochemistry.......................................................................
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