UNIVERSITY OF COPENH AGEN FACULTY OF HEALTH AN D MEDICAL SCIENCES Intrinsic resistance mechanisms and CRISPR-Cas immunity of the opportunistic pathogen, Staphylococcus aureus PhD thesis by Kasper Mikkelsen Supervisor: Professor Hanne Ingmer This thesis has been submitted to the Graduate School of Health and Medical Sciences, University of Copenhagen 14 th of January, 2021 Intrinsic resistance mechanisms and CRISPR-Cas immunity of the opportunistic pathogen, Staphylococcus aureus This thesis has been submitted to the Graduate School of Health and Medical Sciences, University of Copenhagen 14th of January, 2021 by Kasper Mikkelsen Department of Veterinary and Animal Sciences University of Copenhagen, Denmark Principal supervisor: Professor Hanne Ingmer Department of Veterinary and Animal Sciences University of Copenhagen, Denmark Co-supervisor: Adjunct Professor Paal Skytt Andersen Department of Bacteria, Parasites and Fungi Statens Serum Institut, Denmark Assessment committee: Associate Professor Dorte Frees Department of Veterinary and Animal Sciences University of Copenhagen, Denmark Associate Professor Jan Martinussen Department of Biotechnology and Biomedicine Technical University of Denmark, Denmark Professor Angelika Gründling Section of Microbiology & MRC Center for Bacteriology and Infection Imperial College London, United Kingdom 2 | P a g e Acknowledgements First and foremost, I would like to thank my primary supervisor Hanne Ingmer. Thank you for your invaluable guidance and enthusiasm. You have been a supreme support through my entire PhD and always happy to help with an eternal positive mind. I always leave your office with a boost and a lot of positive energy. We’ve had a lot of ideas along the way, and you are always eager to test these new ideas, even when it’s a long shot – it could be cool if… I’m very thankful for you to accept me into your group, and I’m really looking forward to hang around for a while longer. A deep thank you to my co-supervisor Paal Skytt Andersen. You know what I don’t know (and much more) and that really helps to push things along and put them in perspective. But most of all, you’re the nicest guy and I wish you the best on Bornholm. People from islands are not the worst kind you know. Thank you Martin Vestergaard and Nina Molin Høyland-Kroghsbo for all the “non-official” supervision and help going through parts of the thesis. Thank you Martin Bojer, my whistling companion, for the great help with any lab issues. If you have problems with molecular biology, just ask Martin. And thank you to Katrine Nøhr-Meldgaard for your initial contributions to the Aux story. A deep thank you to Professor Waldemar Vollmer and Associate Professor Rikke Louise Meyer for letting me into your labs, and to Waldemars wife Daniela Vollmer for the great help during my stay in Newcastle. I would very much like to thank Professor Guoqing Xia and Ohood Alharbi for the great collaboration in the hard quest of finding the mechanism for what these hypothetical proteins do. Thank you for the great discussions and the sharing of data. A HUGE thank you goes to all my wonderful colleagues and for creating the best working environment. For all the nice beers, the petanque games, the trips, you name it. Thank you Ahlam (you know how it’s pronounced) for the laughs and company in our “own” little lab. Thank you Janine and Bolette for all the office fun, and Janine as well for the great collaborations on CRISPR. Thank you Helena for the openhearted and cake baking spirit. And thank you Anaëlle for the pastries, the coffees, the beers, the podcast suggestions, Idéfix & 3 | P a g e rosé, for criticizing my sad lunch, and for laughing at my jokes and taking them way too far. Merci. Thanks to Gitte and Vi who have always been there to help me if I needed anything. Thank you for all your help. A big thank you goes to all my friends and family! Even though it might not be everything that you have understood regarding my work, you have always been very supportive! Thank you! And a special thanks to my mom and Erik, who provided me with food and shelter during parts of the thesis writing, so I only needed to leave the computer for the daily meals. What a luxury! Last but not least I would like to thank my girlfriend, Lise. Thank you so much for taking the trip to Copenhagen with me. I know I don’t say it enough, but I am truly grateful! I know it has not always been easy, so thank you for standing by my side and being as supportive as you always are. Thank you for just being you and I can’t wait for the times to come together with you and for our new exciting adventure. 4 | P a g e Table of Contents Acknowledgements ......................................................................................................................... 3 Abstract ........................................................................................................................................... 7 Resumé ............................................................................................................................................ 8 Objectives ........................................................................................................................................ 9 Abbreviations ................................................................................................................................ 10 Introduction ................................................................................................................................... 11 Staphylococcus aureus – an ever adaptive opportunistic pathogen ................................... 11 The staphylococcal cell wall ................................................................................................. 12 Peptidoglycan biosynthesis .................................................................................................. 13 Cell wall synthesis machinery during S. aureus cell division ................................................ 15 Daughter cell segregation .................................................................................................... 16 Lipoteichoic acid synthesis, modifications and contributions to cell division ...................... 17 Intrinsic resistance and auxiliary factors ............................................................................. 19 CRISPR-Cas immunity ........................................................................................................... 21 The type III-A CRISPR system ................................................................................................ 23 Spacer adaptation ................................................................................................................ 24 The S. aureus type III-A CRISPR system and SCCmec type V (5C2&5) .................................. 25 References ............................................................................................................................ 27 Manuscript 1: ................................................................................................................................ 34 Abstract ................................................................................................................................ 36 Introduction .......................................................................................................................... 37 Results .................................................................................................................................. 39 Discussion ............................................................................................................................. 44 Acknowledgements .............................................................................................................. 45 Materials and methods ........................................................................................................ 46 References ............................................................................................................................ 47 Manuscript 2: ................................................................................................................................ 49 Abstract ................................................................................................................................ 50 Introduction .......................................................................................................................... 52 5 | P a g e Results and discussion .......................................................................................................... 54 Acknowledgements .............................................................................................................. 58 Materials and methods ........................................................................................................ 58 References ............................................................................................................................ 63 Manuscript 3: ................................................................................................................................ 77 Abstract ................................................................................................................................ 79 Introduction .......................................................................................................................... 80 Results and discussion .........................................................................................................