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The Relevance of Ribonuclease III in Pathogenic Bacteria

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The Relevance of Ribonuclease III in Pathogenic Bacteria The Relevance of Ribonuclease III in Pathogenic Bacteria Ana Margarida Teixeira Saramago Dissertation presented to obtain the Ph.D degree in Biology Instituto de Tecnologia Química e Biológica Universidade Nova de Lisboa Oeiras, March, 2014 Financial Support from Fundação para a Ciência e Tecnologia (FCT) – Ph.D: grant - SFRH/BD/65607/2009 Work performed at: Control of Gene Expression Laboratory Instituto de Tecnologia Química e Biológica Av. da República (EAN) 2781-901 Oeiras – Portugal Tel: +351-21-4469548 Fax:+351-21-4469549 Supervisor : Professora Doutora Cecília Maria Pais de Faria de Andrade Arraiano – Investigadora Coordenadora, Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa. (Head of the Laboratory of Control of Gene Expression, where the work of this Dissertation was performed) Co-supervisor : Doutora Susana Margarida Lopes Domingues – Investigadora Pós-Doutorada, Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa. (Post-doc Fellow in the Laboratory of Control of Gene Expression, where the work of this Dissertation was performed) President of the Jury : Doutor Carlos José Rodrigues Crispim Romão – Professor Catedrático Aposentado do Instituto de Tecnologia Química e Biológica da Universidade Nova de Lisboa, por delegação. iii Examiners: Professora Doutora Mónica Amblar Esteban – Investigadora Principal do Instituto de Salud Carlos III, Madrid (Principal Examiner). Professor Doutor Arsénio do Carmo Sales Mendes Fialho – Professor Associado com Agregação, Instituto Superior Técnico, Universidade Técnica de Lisboa (Principal Examiner). Professor Doutor Miguel Agostinho Sousa Pinto Torres Fevereiro – Investigador Principal do Instituto Nacional de Investigação Agrária e Veterinária - INIAV Professora Doutora Maria Teresa Ferreira de Oliveira Barreto Goulão Crespo – Investigadora Principal, IBET - Instituto de Biologia Experimental e Tecnológica “A journey of a thousand miles begins with a single step.” -Anonymous v Acknowledgements Acknowledgements I want express my gratitude to all that supported me in the realization of my Ph.D. and dedicate to them several words: - To Instituto de Tecnologia Química e Biológica (ITQB) for hosting me, for providing good work conditions, and to give me the excellent opportunity to develop exceptional scientific projects, even with a surrounding environment of economic crisis. Once more, we proved that we (Portuguese people) are plastic and creative people who do not let themselves go down easily. I also would like to thank to the past and present ITQB directors, namely Professor Doutor José Artur Martinho Simões, Professor Doutor Luís Paulo Rebelo, and currently Professor Doutor Cláudio Soares. - To Fundação para a Ciência e Tecnologia (FCT) for financial support that made my Ph.D. possible. for - To my Supervisor Professora Cecília Maria Arraiano for supporting me during these past years, and for continuing to support me. I would like to thank you for encouraging my research and for allowing me to grow as a research scientist. I hope that I could be as energic and enthusiastic as Professor Cecília, and to someday be able to command an audience as well as she can. I am also thankful for the excellent example she has provided as a successful woman and professor. - This thesis would not have been possible without the help and patience of my co-supervisor, good friend (and officemate) Susana Domingues. Thank you for being so supportive, to give me the freedom to pursue ideas without objection, to trust on my capacities, to share your knowledge, and for challenging my mind to bring me to another level. I also want to acknowledge for all the crazy and funny moments!! Laugh is the best medicine!! ix - I also would like to thank to Sandra Viegas, my friend and officemate, for all the good advices, support and good friendship. She has been precious on both academic and at personal level, for which I am extremely grateful!! - To Inês Silva and Andreia Aires, also my very good and special friends! I want to thank you for being wonderful persons, for sharing so many pleasant moments (never forgetting the professional side), and for being such good friends. - To Rute Matos for the friendship and for giving me the pleasure to work with her! I hope that we continue to work together! - To may labmate Igor Ruiz de los Mozos for being an exceptional friend and hard worker, and for all the fun and crazy times during my last year of Ph.D… and to Nabila Haddad for being an extremely nice and helpful person!! I will never forget you both!! - To the members of the CMA group that have contributed greatly to my personal and professional time. The group has been a source of friendships as well as good advices, fun and team spirit. My greatest thanks to Inês Silva, Sandra Viegas, Susana Domingues, Andreia Aires, Cátia Bárria, Susana Barahona, Patrícia Apura, Ricardo Moreira, Rute Matos, Teresa Pinto (thank you for helping me to rescue my polyacrylamide gels), Ricardo Santos, Raquel Santos, Lisete Galego, Clémentine Dressaire, Michal Malecki, Filipa Reis, Vânia Pobre, Zé Andrade, Patrick Freire and Ana Barbas! - To Iñigo Lasa, Alejandro Toledo-Arana and Igor Ruiz de los Mozos from Instituto de Agrobiotecnología, Pamplona, Spain, and to Nabila Haddad and Hervé Prévost from LUNAM Université, Nantes, France for their collaboration and precious help in this Doctoral work. I hope that we can continue our collaboration. - I want to thank to Professor Arsénio Fialho, Doutora Mónica Amblar, Doutora Teresa Crespo and Doutor Miguel Fevereiro for letting my defense be an enjoyable moment, and for your brilliant comments and suggestions. x Acknowledgements - I thank to my good friends and Ph.D. colleagues for all the fun, friendship, and for all the memorable moments altogether! - I also want to express my greatest gratitude to my biggest friends who are always by my side (they know who they are) for their unconditional friendship, support and encouragement throughout all the important moments of my life. - I also want to express my gratitude for my two lovely feline friends, Pantufa and Lucky for their unconditional love. - Lastly, I would like to thank to my mom who raised me with an unconditional love, and supported me in all my pursuits. Without her continuous support and encouragement I never would have been able to achieve my goals! To my dad for all the motivation and also for being always by my side! And to my family for all the love and encouragement. At the end of my thesis, it is a pleasant task to express my gratitude to all who contributed in many ways to the success of this study and made it an unforgettable experience for me. Thank you!! xi Table of contents Table of Contents ABSTRACT ................................................................................................... XVII RESUMO ..................................................................................................... XXIII LIST OF PUBLICATIONS ...................................................................... XXXI DISSERTATION OUTLINE ............................................................... XXXVII ABBREVIATIONS........................................................................................ XLI CHAPTER 1 ........................................................................................................ 1 CHAPTER 2 ..................................................................................................... 43 CHAPTER 3 ..................................................................................................... 81 CHAPTER 4 ................................................................................................... 119 CHAPTER 5 ................................................................................................... 191 CHAPTER 6 ................................................................................................... 231 APPENDIX .................................................................................................... 247 xv Abstract Abstract Ribonucleases (RNases) are key factors in the control of all biological processes, since they modulate the stability of RNA transcripts, allowing rapid changes in gene expression. Some RNases are up-regulated under stress situations and are involved in virulence processes in pathogenic microorganisms. RNases also control the levels of regulatory RNAs, which play very important roles in cell physiology. In general, RNA degradation starts with an internal cleavage performed by an endoribonuclease. This is followed by degradation from one extremity by one or more exoribonucleases. RNase E has been considered the main enzyme that catalyses the initial cleavage event in Gram-negative bacteria. In Gram- positives, this degradation step has been attributed to its functional equivalents: RNase Y and RNases J1/2. However, the ubiquitous RNase III-like enzymes, which cleave the RNA molecule internally within double-stranded segments, are also emerging as important players in the control of bacterial mRNA stability. The first objective of this doctoral work was to perform a functional and biochemical analysis of RNase III from Campylobacter jejuni. This microorganism is a foodborne pathogen for which there is little information regarding ribonucleases. However, there are hints that they can be instrumental in the virulence mechanisms of this bacterium. Namely, Polynucleotide phosphorylase (PNPase) was shown to allow survival of C. jejuni in cold conditions, and to have a
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