Dissertation zur Erlangung des Doktorgrades der Fakultät fur Chemie und Pharmazie der Ludwig-Maximilians-Universität München Antibiotics and translation Overcoming emerging bacterial resistance to old and new antimicrobials Agata Lucyna Starosta aus Rzeszow, Polen 2011 Erklärung Diese Dissertation wurde im Sinne von § 13 Abs. 3 bzw. 4 der Promotionsordnung vom 29. Januar 1998 (in der Fassung der Sechsten Änderungssatzung von 16. August 2010) von Herrn Prof. Dr. Roland Beckmann betreut. Ehrenwörtliche Versicherung Diese Dissertation wurde selbstständig, ohne unerlaubte Hilfe erarbeitet. München, am 24.11.2011 Agata Lucyna Starosta Dissertation eingereicht am 24.11.2011 1. Gutachter: Herr Prof. Dr. Roland Beckmann 2. Gutachter: Herr Prof. Dr. Klaus Förstemann Mündliche Prüfung am 31.01.2012 2 Agata L. Starosta List of contents List of contents Acknowledgements .................................................................................................................................. 5 List of original publications .................................................................................................................... 6 Contribution report ................................................................................................................................. 8 Abbreviations ........................................................................................................................................ 10 Summary ................................................................................................................................................ 11 1 Introduction ........................................................................................................................................ 12 1.1 Traditional antibiotics .................................................................................................................. 13 1.1.1 Introduction to antibiotics .................................................................................................... 13 1.1.2 Targets for antibiotic action ................................................................................................. 14 1.1.2.1 Inhibition of cell wall synthesis ..................................................................................... 14 1.1.2.2 Inhibition of DNA replication ........................................................................................ 16 1.1.2.3 Inhibition of RNA synthesis ........................................................................................... 16 1.1.2.4 Inhibition of protein synthesis ....................................................................................... 16 1.1.3 Protein synthesis ................................................................................................................... 17 1.1.3.1 Inhibitors of the small ribosomal subunit .................................................................. 22 1.1.3.2 Inhibitors of the large ribosomal subunit ................................................................... 23 1.1.3.2.1 Inhibitors of the peptidyl-transferase center ...................................................... 23 1.1.3.2.2 Inhibitors of the progressing nascent polypeptide chain .................................... 24 1.1.3.2.3 Inhibitors of the GTPase-associated center (GAC) ............................................ 24 1.1.4 Mechanism of cell death induced by bactericidal antibiotics .............................................. 25 1.2 Antibiotics used in these studies .................................................................................................. 27 1.2.1 Hygromycin A ....................................................................................................................... 27 1.2.2 Macrolides ............................................................................................................................ 29 1.2.3 Thiopeptides ......................................................................................................................... 31 1.2.4 Orthosomycins ...................................................................................................................... 34 1.2.5 Fusidic acid .......................................................................................................................... 35 1.3 Alternative antimicrobials targeting virulence ............................................................................ 36 1.3.1 Definition of virulence .......................................................................................................... 36 1.3.2 Colonization ......................................................................................................................... 36 1.3.3 Biofilm .................................................................................................................................. 36 1.3.4 Quorum sensing .................................................................................................................... 37 3 Agata L. Starosta List of contents 1.3.5 Motility ................................................................................................................................. 37 1.3.6 Secretion systems .................................................................................................................. 37 1.3.7 Elongation factor P .............................................................................................................. 38 2 Objectives of these studies .................................................................................................................. 41 3 Cumulative thesis ............................................................................................................................... 43 3.1 Hygromycin A ............................................................................................................................. 43 3.1.1 Paper 1 .................................................................................................................................. 43 3.1.2 Paper 2 .................................................................................................................................. 43 3.2 Macrolides ................................................................................................................................... 47 3.2.1 Paper 3 .................................................................................................................................. 47 3.2.2 Paper 4 .................................................................................................................................. 47 3.3 Thiopeptides ................................................................................................................................ 50 3.3.1 Paper 5 .................................................................................................................................. 50 3.3.2 Paper 6 .................................................................................................................................. 50 3.4 Orthosomycins ............................................................................................................................. 52 3.5 Fusidic acid.................................................................................................................................. 53 3.5.1 Paper 7 .................................................................................................................................. 53 3.6 Elongation factor P ...................................................................................................................... 54 3.6.1 Paper 8 .................................................................................................................................. 54 4 Conclusions ........................................................................................................................................ 58 5 References .......................................................................................................................................... 59 4 Agata L. Starosta Ackowledgements Acknowledgements This would not be possible without the support of many people that I had a pleasure of meeting during my PhD studies. First of all I would like to thank Dr. Daniel Wilson for giving me an opportunity to work in his group in the Gene Center . I am grateful for the trust and support I received from him during all those years. I appreciate each and every advice, discussion, criticism and praise I ever received from him. That was a lesson I will never forget. I would like to thank Prof. Roland Beckmann for all the expertise, good advices and support as well as for providing us with a great scientific environment without which this work could not happen. I am grateful to all our collaborators for having fruitful time collecting all the data: to Prof. B. Cooperman for great work on thiopeptides; Prof. A. Bogdanov for macrolide story; Prof. K. Reynolds for hygromycin A studies; Prof. C. Spahn for first and hopefuly not the last Nature paper; Prof. G. Dinos for hosting me in Greece and support in experiments; and last but not least, to Prof. J. Remme for great work on EF-P. I would also like to thank people, whom I had a pleasure to have conducted the experiments with: Aleksandra Mikolajka, Alexandra Dönhöfer, Viktorija Karpenko, Gemma Atkinson and Vidya Dhote. Special thanks to Lauri