REGULATORY CASCADES INVOLVING CYCLIC DI-GMP SIGNALLING IN PSEUDOMONAS AERUGINOSA JOANA ALVES MOSCOSO Regulatory cascades involving cyclic di-GMP signalling in Pseudomonas aeruginosa Joana Alves Moscoso Imperial College London Department of Life Sciences This thesis is presented for the degree of Doctor of Philosophy of Imperial College London and Diploma of Imperial College London 2013. The copyright of this thesis rests with the author and is made available under a Creative Commons Attribution Non-Commercial No Derivatives licence. Researchers are free to copy, distribute or transmit the thesis on the condition that they attribute it, that they do not use it for commercial purposes and that they do not alter, transform or build upon it. For any reuse or redistribution, researchers must make clear to others the licence terms of this work. 2 Candidate’s Declaration I, Joana Alves Moscoso, hereby confirm that this thesis represents my own work and that any external contributions to the research are duly acknowledged. Joana Alves Moscoso 3 Abstract Cyclic di-GMP (c-di-GMP) has emerged as a bacterial second messenger that regulates a variety of cellular processes, in particular those associated with the switch between a motile and a sessile lifestyle. At low levels of c-di-GMP the motile lifestyle is favoured whereas at high levels of c-di-GMP the formation of biofilms is promoted. In Pseudomonas aeruginosa, over 50 genes encoding proteins involved in the synthesis, hydrolysis or sensing of c-di-GMP are found and many remain uncharacterized. Herein, an analysis of a collection of mutants was performed and supported the idea that the sophisticated c-di-GMP network operates at high specificity. In addition to c-di-GMP, P. aeruginosa has a regulatory pathway, the Gac pathway, that is known to control the bacterium lifestyle switch. By investigating a particular mutant affected in this pathway, a link between the Gac pathway and c-di-GMP was established. Furthermore, the Gac pathway not only influences biofilm formation, but it is also crucial in determining the bacterium mode of infection. In other words, biofilm formation correlates to a chronic mode of infection where the bacterium has an active type VI secretion system (T6SS), and a motile phenotype correlates to an acute infection where the type III secretion system (T3SS) is active. Interestingly, by artificially modulating the levels of c-di-GMP it was demonstrated that c-di- GMP regulation goes beyond the control of the motile/sessile phenotypes and is able to inversely regulate the T3SS and T6SS. Finally, the link between c-d-GMP and the Gac pathway was consolidated by showing that the Gac system impacts the expression of a few c-di-GMP related proteins and one protein, SadC, was identified as a central component of the network. Overall this work largely contributed to reconcile two independent concepts involved in the regulation of the P. aeruginosa lifestyle. 4 Acknowledgements First of all, I would like to thank my supervisor Alain Filloux, for being always very supportive and for having accepted me in his group. The last 4 years working with you have been a real pleasure and the way you manage the team and our expectations are a real inspiration to me. I feel that I have come a long way and learned a lot, both professionally and at the personal level, throughout my PhD. Overall, this journey has been a good one and I am truly thankful for your mentoring, patience, encouragement and understanding. Secondly, I would like to thank my parents who are always there for me and have been absolutely essential in my pursuit of happiness and professional fulfillment. You have set the standard high and it is that ambition that you passed on that keeps me going. I feel that I have made the right choices up to now and that is only possible because you have given me the tools to choose freely but consciensciously, in a right balance between being able to express myself and respecting who we are as a family. Obrigada Papa! Obrigada Mama! You have been amazing! I would also like to thank my brother, Frederico, who I know will always be there for me and who gave me the two sweetest things in my life so far: my nephew Carlinhos and my niece Clarinha. And because you are a grown up now, thank you to you Carlinhos as well (you better improve your English, by the way, so that you can understand this properly!). You have changed my life when I was only 15 years old and I still cannot define if I am more of an aunt to you or an older sister! Anyway, I love you to pieces. Now, I would like to thank my collaborators, those who replied to my requests and those who have accommodated me in their labs. On the three occasions that I went away to learn a new technique, first in Nottingham, then in Basel and finally in Marseille, I had an absolutely great time and always felt welcome and well supervised. Thank you again Stephan and Mary, Tina, Jutta and Ursi, and Christopher for your time. In addition, I would like to take this opportunity to also thank my previous “bosses” because without them I would not be here today. I will always be grateful to Victoria Shingler and Lisandro Bernardo for their mentoring in Sweden, and I will always remember Naresh Verma for hosting me in Australia. To my colleagues in the Filloux’s group, thank you for the constant support, brainstorming moments and great atmosphere. This group is indeed something special and I always felt “home” at work which is very valuable to me and I treasure that feeling a lot. Many people came in and out during these 4 years, but a special thanks goes to Nicolas Barraud who was fantastic in my early days, Helga 5 Mikkelsen who has a brain like no other, Melissa Sivanesson who has the sweetest heart, Nadine Lossi who shared with me great moments, Abdou Hachani who brings the “wow” factor to everything around him, Cerith Jones who has been a constant presence, Daniela Muhl who you can always count on, Kailyn Hui who is the magician that gets things done and Eleni Manoli who brings some shine to the office and lab. A few words go also to the new people in the group, Luke Allsopp and Sara Planamente, and the other people in the floor, especially those from the group of Gad Frankel. I will also remember with kindness my students Evelina Musial and Qi Pan, and the visiting PhD student from Spain, Isabel Cortés, with whom I enjoyed working and shared good moments. Finally, I would like to thank my dearest friends. You make my life fun and bearable! Without you I would have gone nuts a very long time ago! Thank you, thank you, thank you! With a big smile! In London, special thanks go to Antonio Santos, Lena Barrera, Veronique Peiffer, and the crowds from the LUSO team and then the PARSUK team. We will keep in touch for sure! In Portugal and around the world, thanks to my friends from Valença, specially Angela Pinheiro, my friends from Porto, specially Ana Oliveira and Joana Torres, my friend Karina Herrera, my friend Karsten Oelkers, my “friend” Ricardo Azevedo, my “sister” Susana Martins, and last but definitely not least, my friend Sofia Koch. I would like to also thank Heather Combe and Niki Gounaris at Imperial College and finally, to FCT (Fundação para a Ciência e Tecnologia) for financing my PhD. 6 Table of Contents Candidate’s Declaration .......................................................................................................................... 3 Abstract ................................................................................................................................................... 4 Acknowledgements ................................................................................................................................. 5 Index of Figures ..................................................................................................................................... 10 Index of Tables ...................................................................................................................................... 12 Abbreviations ........................................................................................................................................ 13 1) Introduction .................................................................................................................................. 17 1.1 Pseudomonas aeruginosa, an opportunistic pathogen .............................................................. 18 1.1.1 Acute versus chronic infections ........................................................................................... 19 1.1.2 Biofilm development: from formation to dispersal ............................................................. 20 1.1.3 Extracellular appendages and exopolysaccharides ............................................................. 23 1.1.4 Secretion of proteins across the membrane ....................................................................... 27 1.2 Regulatory cascades in P. aeruginosa ......................................................................................... 31 1.2.1 Regulation by direct binding to effector proteins ............................................................... 32 1.2.2 Regulation by phosphorylation ............................................................................................ 35 1.2.3 Regulation by phosphorylation and methylation ................................................................ 45 1.3 Cyclic nucleotide signalling in P. aeruginosa .............................................................................