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Staphylococcus Aureus Clpxp TECHNISCHE UNIVERSITÄT MÜNCHEN FAKULTÄT FÜR CHEMIE LEHRSTUHL FÜR ORGANISCHE CHEMIE II VIRULENCE ATTENUATION THROUGH CHEMICAL AND GENETIC MANIPULATION OF THE STAPHYLOCOCCUS AUREUS CLPXP PROTEASE DISSERTATION ZUR ERLANGUNG DES AKADEMISCHEN GRADES EINES DOKTORS DER NATURWISSENSCHAFTEN VON CHRISTIAN FETZER MÜNCHEN 2018 TECHNISCHE UNIVERSITÄT MÜNCHEN FAKULTÄT FÜR CHEMIE LEHRSTUHL FÜR ORGANISCHE CHEMIE II Virulence Attenuation through Chemical and Genetic Manipulation of the Staphylococcus aureus ClpXP Protease Christian Fetzer Vollständiger Abdruck der von der Fakultät für Chemie der Technischen Universität München zur Erlangung des akademischen Grades eines DOKTORS DER NATURWISSENSCHAFTEN (Dr. rer. nat.) genehmigten Dissertation. Vorsitzender: Prof. Dr. Michael Groll Prüfer der Dissertation: 1. Prof. Dr. Stephan A. Sieber 2. Prof. Dr. Matthias Feige Die Dissertation wurde am 07.11.2017 bei der Technischen Universität München eingereicht und durch die Fakultät für Chemie am 06.12.2017 angenommen. “… I am still confused - but on a higher level.” Enrico Fermi Danksagung An allererster Stelle möchte ich mich herzlich bei Prof. Dr. Stephan A. Sieber für die Möglichkeit bedanken, sowohl meine Master-, als auch meine Doktorarbeit an seinem Lehrstuhl angefertigt haben zu können. Sowohl die Themengebiete, als auch die große Freiheit bei der Bearbeitung empfand ich als sehr positiv. Durch die Forschung in sehr unterschiedlichen Bereichen habe ich ein sehr breites Spektrum an Methoden, Techniken und Denkweisen erlernen können, worüber ich sehr dankbar bin. Mein Dank gilt den Mitgliedern der Prüfungskommission für ihre Zeit und ihren Bemühungen bei der Bewertung der Dissertation. Tamara, Franjo, Markus und Matthias danke ich für die Korrektur der Arbeit. Dem alten AVIRU-Team, Dr. Kathrin Lorenz-Baath, Dr. Franziska Weinandy, Dr. Bianca Schwanhäußer, Heike Hofmann, Dr. Axel Pahl, Dr. Vadim Korotkov, Dr. Jan Vomacka und Ernst Bernges, möchte ich für den leichten Übergang von Studium zu Forschung und dem immer angenehmen Arbeitsklima danken. Sehr viele Ergebnisse und Experimente kommen von Kooperationspartnern aus ganz Deutschland. Ich danke Marie-Theres Vielberg und Prof. Dr. Michael Groll für die Durchführung der Kristallisationsexperimente. Dr. Katharina Rox, Dr. Jennifer Herrmann, Dr. Robert Thänert und Prof. Dr. Eva Medina vom Helmholtz-Zentrum für Infektionsforschung in Saarbrücken und Braunschweig danke ich für ihren Einsatz bei der Erstellung diverser biologischer Daten. Ein sehr großer Dank geht an Carola Seyffarth, Dr. Martin Neuenschwander und Dr. Jens Peter von Kries vom Leibniz- Forschungsinstitut für Molekulare Pharmakologie in Berlin für die Betreuung und Durchführung des HTS. Katja Bäuml und Mona Wolff bin ich sehr dankbar, dass sie nicht nur mit Studenten und Doktoranden auskommen müssen, sondern auch noch das Labor und alle Geräte am Leben halten. Bei allen meinen Praktikanten, Johanna Brüggenthies, Maximilian Biebl, Carolin Berner, Melina Vollmer und Theresa Rauh bedanke ich mich für die experimentelle Hilfe. Das wichtigste im Labor sind nette Kollegen. Viele lustige Momente, unzählige schlechte Witze, lange Abende und fachliche und außerfachliche Diskussionen haben die vergangenen vier Jahre zu einer unvergesslichen Zeit gemacht. Mein Dank gilt meinen Exil-Kollegen Kyu Myung Lee, Vadim Korotkov und Igor Pavlović, sowohl allen derzeitigen Mitglieder des AK Siebers, namentlich Nina Bach, Christina Brumer, Pavel Kielkowski, Franziska Mandl, Weining Zhao, Patrick Allihn, Dóra Balogh, Jonas Drechsel, Anja Fux, Carolin Gleißner, Thomas Gronauer, Mathias Hackl, Annabelle Hoegl, Barbara Hofbauer, Ines Hübner, Volker Kirsch, Philipp Kleiner, Elena Kunold, Markus Lakemeyer, Robert Macsics, Matthias Stahl, Stephan Hacker, Patrick Zanon, Martin Pfanzelt, Theresa Rauh und Angela Weigert Muñoz, die den Arbeitskreis zu dem machen, was er ist. Nicht zu vergessen ist die „Alte Garde“, die während meiner Zeit aus dem Arbeitskreis ausgeschieden ist, Roman Kolb, Martin Kunzmann, Johannes Kreuzer, Max Koch, Maria Dahmen, Megan Wright, Wolfgang Heydenreuter und Johannes Lehmann. Zuletzt gilt mein größter Dank meiner Familie und Tamara für die Unterstützung auf meinem Weg! Besonders Tamara danke ich für den ständigen Rückhalt. TABLE OF CONTENTS Table of Contents Table of Contents .............................................................................................................. I Summary ........................................................................................................................... V Zusammenfassung .......................................................................................................... VII Introductory Remarks ...................................................................................................... IX Introduction ...................................................................................................................... 1 1. The Antibiotic Crisis .............................................................................................. 1 2. Staphylococcus aureus.......................................................................................... 5 3. Antivirulence ......................................................................................................... 8 4. The ClpXP Protease of Staphylococcus aureus ................................................... 10 Chapter I – A Chemical Disruptor of the ClpX Chaperone Complex Attenuates the Virulence of Multidrug-Resistant Staphylococcus aureus .............................................. 15 1. Introduction ........................................................................................................ 16 2. Results and Discussion ........................................................................................ 16 2.1. High-Throughput Screen .......................................................................... 16 2.2. In vitro Characterization ........................................................................... 18 2.3. Structure-Activity Relationship Studies .................................................... 20 2.4. Efforts for in vivo/in vitro Target Validation ............................................. 22 2.4.1. Affinity-Based Protein Profiling ............................................................ 22 2.4.2. Affinity Pull-Down ................................................................................. 28 2.5. Phenotypic Effects .................................................................................... 29 3. Summary and Outlook ........................................................................................ 32 Chapter II – Small Molecule Inhibition of the Staphylococcus aureus ClpXP Complex .. 35 1. Introduction ........................................................................................................ 36 2. Results and Discussion ........................................................................................ 36 2.1. In vitro Characterization ........................................................................... 36 2.2. Structure-Activity Relationship Studies .................................................... 39 2.3. Effects on Production of α-Hemolysin ...................................................... 40 3. Summary and Outlook ........................................................................................ 42 Chapter III – Genetic Modifications of the clpP Gene in Staphylococcus aureus........... 43 1. Introduction ........................................................................................................ 44 2. Results and Discussion ........................................................................................ 44 I TABLE OF CONTENTS 2.1. Cloning of clpP-Shuttle Vectors ................................................................ 44 2.2. Generation and Selection of Genetically Modified S. aureus .................. 47 2.3. Phenotypic Studies ................................................................................... 50 2.3.1. Growth Characteristics ......................................................................... 50 2.3.2. Hemolysis .............................................................................................. 51 2.3.3. Whole-Proteome Analysis .................................................................... 52 3. Summary and Outlook ........................................................................................ 56 Experimental Section ...................................................................................................... 57 1. Biochemical and Microbiological Procedures .................................................... 57 1.1. Media ........................................................................................................ 57 1.2. Overexpression of Recombinant Proteins ................................................ 58 1.2.1. Oligonucleotides for Cloning of Expression Vectors ............................. 58 1.2.2. Expression of ClpP ................................................................................. 58 1.2.3. Expression of GFP-SsrA ......................................................................... 58 1.2.4. Expression of ClpX ................................................................................. 59 1.2.5. Expression of ClpC and MecA ............................................................... 60 1.3. High-Throughput Screen .........................................................................
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