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Adaptation of Pseudomonas Aeruginosa to the Cystic Fibrosis Lung and the Role of Shear Stress in Disrupting Alginate Matrix Biofilms

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Adaptation of Pseudomonas Aeruginosa to the Cystic Fibrosis Lung and the Role of Shear Stress in Disrupting Alginate Matrix Biofilms Faculty of Science and Bio-engineering Sciences, Department of Bio-engineering Sciences, Research Group of Microbiology, Academic Year 2015-2016 Adaptation of Pseudomonas aeruginosa to the cystic fibrosis lung and the role of shear stress in disrupting alginate matrix biofilms Thesis submitted in fulfillment of the requirements for the degree of Doctor (Ph.D.) in Bio-engineering Sciences Jozef Dingemans Promotors: Em. Prof. Dr. Pierre Cornelis Prof. Dr. Daniel Charlier Co-promotors: Prof. Dr. Anne Malfroot (UZ Brussel) Dr. Rob Van Houdt (SCK•CEN) Members of the jury Promotors: Em. Prof. Dr. Pierre Cornelis (VUB) Prof. Dr. Daniel Charlier (VUB) Co-promotors: Prof. Dr. Anne Malfroot (VUB, UZ Brussel) Dr. Rob Van Houdt (SCK-CEN) President: Prof. Dr. Geert Angenon (VUB) Secretary: Prof. Dr. Wim Versees (VUB) Internal jury members: Prof. Dr. Guido Verniest (VUB) Prof. Dr. Jo Van Ginderachter (VUB) Prof. Dr. Kim Roelants (VUB) External jury members: Prof. Dr. Craig Winstanley (University of Liverpool) Prof. Dr. Sylvie Chevalier (Université de Rouen) Prof. Dr. René De Mot (KU Leuven) The work described in this dissertation was performed at: -Research Group of Microbiology, Department of Bio-engineering Sciences, Vrije Universiteit Brussel, Pleinlaan 2, 1050 Brussel -Unit of Microbiology, Institute for Health, Environment and Safety, Studiecentrum voor kernenergie (SCK•CEN), Boeretang 200, 2400 Mol This research was financially supported by: - The government agency for Innovation by Science and Technology (IWT Vlaanderen). - The Belgian Association against Cystic Fibrosis (Belgische Vereniging voor Strijd tegen Mucoviscidose). - The Flanders Interuniversity Institute for Biotechnology (VIB). Published by the Research Group Microbiology, Department of Bio-engineering Sciences, Vrije Universiteit Brussel, Pleinlaan 2, 1050 Brussel, Belgium Copyright © 2015 by Jozef Dingemans Apart from any fair dealing for the purposes of research or private study or criticism or review, this publication may not be reproduced, stored in a retrieval system, or transmitted, in any form or by any means (electronic, mechanical, photocopying, recording, scanning, or otherwise) without prior written permission of the publisher. Acknowledgements About five years ago I asked Pierre if it was possible to start a PhD in his lab. Although we have already met regularly in the scope of the microbiology courses that he taught, always in a warm atmosphere, I could never guess that my respect for him would reach these heights. Pierre, you were an excellent mentor for me in the scientific world, but also a guide in life. I could try to summarize the many conferences, dinners, movie nights, etc. that we have enjoyed together with the MICR group, but the printing costs of this thesis would accumulate to excessive amounts. Nevertheless, I want to thank you for all the freedom that you gave me in performing my research, the positive atmosphere that was omnipresent in the MICR group, and the many moments of friendship that helped both of us when skies were grey. I am convinced that the way you performed research, allowing serendipity to occur, is the right way for the VUB to continue in a world that often lacks the tolerance to allow freedom of tought and speech. One of the things that touched me most during this PhD was listening to cystic fibrosis patients and their parents during a couple of meetings (one in Paris, one in Belgium). I was astonished by the optimism that these people exhibited when telling their story. Every single day I devoted to this PhD, I felt I could contribute to the change in life quality of these people, encouraging me to bring this project to fruition. Due to the medical nature of this project, I relied on the knowledge and expertise of a great team of physiotherapists (Hanneke Eyns, Nino Galasso and Sylvie Vanlaethem) and physicians (Prof. Anne Malfroot and Dr. Julie Willekens) at UZ Brussel. In particular I want to thank my co-promotor Prof. Anne Malfroot for giving me the opportunity to attend the ECFS meeting in Lisbon, but most importantly for the many fruitful multidisciplinary meetings and collaborations. I want to conclude by thanking all CF patients that have participated in our clinical study and who provided sputum samples during the optimalisation period of this project. Next, I would like to thank all the collaborators that have contributed to this research. In all the labs I have worked during my PhD (MICR, SCK in Mol, and LabMCT in Neder-over- Heembeek), people were kind and helpful. Special thanks to Florence Bilocq for introducing me in the world of Diversilab, and to Dr. Daniel De Vos and Dr. Jean-Paul Pirnay for using the facilities at LabMCT and for the help in publishing my first 1st author paper. Also many thanks to the people of SCK. I really appreciated the help of Ilse, Wietse, Joachim, Bo, Hugo, Katinka (scanning electron microscopy), Pieter (RNAseq analysis), and especially my co- promotor Dr. Rob Van Houdt. Without your alertness, I would have missed several PhD days and I would have to ask every time I went to SCK for a new security badge! I also want to thank you for the excellent suggestions/corrections when proofreading my papers. As I have already told, the atmosphere at MICR has always been exquisite, even though we had to share the office with more than 15 people (during Prof. Jeroen Raes’ era at the VUB). I will never forget the Friday afternoon drinks in Opinio with Falk, Raul, Sarah, Youssef, and Sam! But also the nice company of the people from the lab of Prof. De Vuyst (Luc, Henning, Frédéric, Andrea, David, Marko …) The interaction between these groups has resulted in a memorable visit to the Christmas market in Aachen! This reminds me of the great opportunities the VUB offers due to its small scale to meet other groups and to collaborate. Pierre’s group has always been small, creating a cosy atmosphere in the lab that stimulated us to collaborate and perform sports after work (usually followed by a few beers in “het complex”). The strong cohesion in our group (Ken, Ye, Ameer, Wei, and myself) has always given me a warm feeling and motivated me to successfully finish this PhD. One of the key factors that created the group atmosphere was without any doubt the presence of our amazing secretary Linda! A day in the lab without Linda felt like a day without sunshine! Also after all these lovely people left the cosy VUB environment (I am often called “the last of the Mohicans”), MICR maintained a nice working environment. In particular due to the enthusiasm of Liesbeth, Charlotte, Indra, Han, and especially Karl. I think it will be difficult to find a running partner and Gin Connoisseur as enthusiastic as you, Karl! Furthermore, I want to thank Prof. Eveline Peeters for adopting me in her lab after the retirement of Pierre! Besides the MICR people, I want to express my appreciation to the people of GEVI (Prof. Jean-Pierre Hernalsteens, Sien, and Charlotte), Prof. Henri Degreve, Prof. Daniel Charlier (also for being my promotor after Pierre’s retirement), Kris, Sonya and many others. I want to conclude my gratitude to MICR by thanking my excellent thesis students Francesca, Michael, and Laila. I really enjoyed the scientific discussions that we had and the effort you put in our work! Further, I want to acknowledge all collaborators that are co-authors, including Prof. René De Mot, Dr. Maarten Ghequire (KU Leuven), Prof. Dianne Newman, and Dr. Ryan Hunter (USA), and in particular Dr. Aurélie Crabbé (Universiteit Gent)! During your stay in Arizona it was always a pleasure to Skype and brainstorm with you, advising me in my research. Great thanks to Prof. Laurence Van Melderen (ULB) and her lab too, it was always a pleasure meeting you during (and after) a day at a conference. I am also grateful to the jury members for accepting the invitation to serve on the Examination Board and for the interesting discussions we have had during the private defense. Finally, I want to thank my dear friends and family who always supported me during the more difficult moments of my PhD! Thanks to Hugo and Jens I could focus my thoughts on football when watching our beloved club RSC Anderlecht. When Wim, Loic, and Max joined, this regularly resulted in a night dancing in the Havana club. In addition, the nice excursions with Niels and Evelyne to Moroccan, Greek, and Portuguese restaurants and once in a while eating “fruits de mer” (Lobster time!) with Kurt helped me to reduce stress levels. Nevertheless, I want to express my gratitude to my parents who have always been there for me and supported me in every choice I have made so far in my life. Finally, I want to thank my lovely spouse Florien for being there for me every single moment of the day. I do realize that this PhD also put some pressure on her, but our love has passed this test with great distinction! Abstract 1 Abstract Pseudomonas aeruginosa is a Gram-negative gamma-proteobacterium that can be found in a wide range of environments including water, soil, animals, and humans. However, due to its metabolic versatility and large genome encoding multiple virulence factors, this opportunistic pathogen is able to infect patients with severe wounds, immunocompromised individuals, and most importantly cystic fibrosis (CF) patients. Chronic P. aeruginosa infections are the major cause of morbidity in CF patients due to persistent lung inflammation and the resulting irreversible lung damage. Often the initial infection is mediated by the acquisition of environmental P. aeruginosa strains, although inter-patient transmissions have also been reported. Subsequently, the initially colonizing P. aeruginosa strains adapt to the CF lung environment by undergoing extensive adaptation and switch from a planktonic to a biofilm lifestyle.
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