The role of pneumococcal carbon metabolism in colonisation and invasive disease Ana Laura Paixão Dissertation presented to obtain the Ph.D degree in Biochemistry Instituto de Tecnologia Química e Biológica António Xavier | Universidade Nova de Lisboa Oeiras, March, 2015 The role of pneumococcal carbon metabolism in colonisation and invasive disease Ana Laura Paixão Supervisor: Dr. Ana Rute Neves Co-Supervisors: Professor Peter William Andrew and Dr. Hasan Yesilkaya Interim Co-Supervisor: Professor Adriano Oliveira Henriques (2012-2015) Dissertation presented to obtain the Ph.D degree in Biochemistry Instituto de Tecnologia Química e Biológica António Xavier | Universidade Nova de Lisboa Oeiras, March, 2015 i From left to right: Patrick Maria Franciscus Derkx, Raquel Sá Leão Domingues da Silva, Karina de Bívar Xavier, Cecília Maria Pais de Faria de Andrade Arraiano, Ana Laura Paixão, Ana Rute Neves, Adriano Oliveira Henriques and Sofia Rocha Pauleta. Oeiras, March 27th, 2015 Apoio financeiro da Fundação para a Ciência e Tecnologia e do FSE no âmbito do Quadro Comunitário de apoio, Bolsa de Doutoramento com a referência SFRH/BD/46997/2008. Cover page: Schematic representation of S. pneumoniae and catabolic pathways for galactose, mannose and N-acetylglucosamine. Background: phase contrast image of S. pneumoniae. ii Acknowledgments It was a long journey since I started my PhD, in the end of 2009. A full life experience. A path full of mixed feelings. The energy and motivation of a new beginning and the fear of failing such a challenge. The happiness of achieving the goals of this work, the surprise of reaching unexpected results and the frustration of failed experiments. The winnings and losses, laughs and tears, and the discovery of different worlds. And now? A new beginning. But before, I would like to express my gratitude to those that enriched this work and personal experience. First, I would like to thank my supervisor, Dr. Rute Neves, for the opportunity I was given to conduct my PhD in the LAB & in vivo NMR laboratory. Thank you for giving me the conditions to accomplish this work until the end, for the guidance, the scientific mentoring and the critical support in reviewing and writing this thesis. I thank the final effort to conclude this thesis in time. The encouragement to share my results in several European meetings and the opportunity to travel to amazing places. I thank my co-supervisors, Prof. Peter William Andrew and Dr. Hasan Yesilkaya, for accepting me as a PhD student. For the openness to discuss my work and reviewing the first manuscript, contributing to a fruitful collaboration. To Prof. Adriano Henriques, my “borrowed” co-supervisor, who accepted me in a very difficult period. For all the wise advices and for helping me not to give up. For contributing to my serenity in the most iii troublesome times. I am thankful for being always open to discuss my work and for teach me to do one thing at a time. To Dr. Jan-Willem Veening who received me in his lab (Molecular Genetics - Groningen Institute of Biomolecular Sciences & Biotechnology) and supervised my work during my stay in Groningen. The best work experience I ever had. Thank you for running such a nice lab, full of interesting people, who received me so well. To my dearest post-doc, Morten Kjos, the kindest person I have ever met. Always concerned with my work and my well-being while I was in Groningen. Thank you for all the support and help in the complementation assays, especially when I left Groningen and there were still experiments to be concluded. To Prof Helena Santos for the valuable discussions held in her lab meeting presentations during the first years of my PhD. To Ana Lúcia Carvalho for her tireless help. For her support and effort to conclude all the in vivo NMR experiments with me before leaving to her new professional challenge. For teaching me everything she knew about HPLC and in vivo NMR. And most importantly, for her friendship. Thank you for all the good moments in and outside the lab. To José Caldas I thank all the help, dedication and interest in the microarrays analysis. Thank you for such a fruitful collaboration. To Nuno Borges, for being always available to help both by giving technical tips to conduct my experiments and by showing openness to share lab material. iv To Luis Gafeira, for being such an enthusiastic, passionate about science, who contributes with creative ideas to the work of everyone, including mine. Always willing to help. I will remember him for being a good listener and a friend. To Paula Gaspar for sharing her knowledge and technical support. To all the past and present members of Cell Physiology and NMR group (because you are so many I won’t enumerate – I could fail someone), for being always available to help and for sharing good parties with the LAB & in vivo NMR laboratory. To Joana Oliveira for doubling my hands in the growth experiments. Thank you for the effort. To all my colleagues in the in the LAB & in vivo NMR that contributed to this experience in so many ways. To Dr. Susana Vinga and André Veríssimo for the modelling of the growth curves and statistical analysis. To Dr. Rita Ventura and Eva Lourenço for the synthesis of phosphorylated compounds. To all the other collaborators that contributed in different ways to this thesis: Dr. Tomas Kloosterman, Dr. Vitor Fernandes and Prof. Dr. Oscar Kuipers. To Dr. Mariana Pinho and her students Pedro, Pedrinho and Raquel, that moved to our laboratory without disturbing my ongoing work. v To Teresa Maio, Ana Mingote, Sónia Estêvão, Teresa Ferreira, Tiago Pais and Marta Rodrigues for contributing to the most hilarious times in and outside the lab. To all my colleagues from the 2010 PhD classes with whom I shared good moments. To Fundação para a Ciência e a Tecnologia for the financial support that made this doctoral work possible (SFRH / BD / 46997 / 2008). With the greatest gratitude, I dedicate this thesis to my parents and my husband, Gonçalo. This experience was also yours. I am grateful for your unconditional support, comprehension, love, and advices. Without them, it would have been much more difficult. Thank you for being always there when I needed the most. A special word to my siblings Tiago, Afonso and Mariana: love you guys. Thank you for sharing the most important times with me. vi Abstract Streptococcus pneumoniae is a common asymptomatic commensal of the human nasopharynx. However, it is better known as a threatening pathogen that causes serious diseases such as pneumonia, meningitis and sepsis, as well as other less severe but more prevalent infections (e.g. otitis media). With the increase of antibiotic resistance and the limited efficacy of vaccines, pneumococcal infections remain a major problem. Therefore, the discovery of new therapeutic targets and preventive drugs are in high demand. Given this panorama, much attention has been dedicated to classical aspects of virulence (e.g. toxins, capsule or cell wall components), but the knowledge of in vivo physiology and metabolism of S. pneumoniae is still limited. This is intriguing, considering that to a large extent, pneumococcal pathogenesis relies on efficient acquisition and metabolism of the nutrients required for growth and survival in the host niches. In line with this view, recent work uncovers substantial interdependencies between carbohydrate metabolism and virulence. These findings denote a far greater importance of basic pneumococcal physiology than previously imagined. However, a scarcity of data on sugar metabolism and its regulation in S. pneumoniae hampers a comprehensive understanding of the connections between these processes. The ultimate goal of this thesis is to improve our understanding of S. pneumoniae basic physiology and discover links between carbohydrate metabolism and the ability of the bacterium to colonise and cause disease. S. pneumoniae is a strictly fermentative microorganism that relies on glycolytic metabolism to obtain energy, but free monosaccharides are limited in the airways. The most abundant sugars present in the natural niche of S. pneumoniae are the glycoproteins mucins, which are generally vii composed of N-acetylglucosamine (GlcNAc), N-acetylgalactosamine (GalNAc), N-acetylneuraminic acid (NeuNAc), galactose (Gal), fucose (Fuc) and sulphated sugars linked to the protein core. Importantly, S. pneumoniae possesses a large set of extracellular glycosidases that act over glycans and release free sugars that can potentially be used for grow. Therefore, we hypothesised that the pneumococcus depends on one or multiple glycan-derived sugars to grow. To disclose prevalent pathways during growth on mucin we resorted to a transcriptome analysis comparing transcript levels during growth on the model glycoprotein porcine gastric mucin and glucose (Glc). The gene expression profile revealed Gal, Man and GlcNAc as the most probable glycan-derived sugars to be used as carbon sources by S. pneumoniae D39. Accordingly, D39 was able to grow on these sugars, but not on other host-derived glycan monosaccharides (Fuc, NeuNAc, GalNAc), as expected from its genomic potential. An in depth characterization of growth profiles on a chemically defined medium supplemented with each carbohydrate using two different sugar concentrations (30 and 10 mM) was performed. S. pneumoniae displayed a preference for GlcNAc, whereas Gal was the least preferable carbohydrate for growth, but energetically the most favourable. The inefficient Gal catabolism could be partially explained by the absence of a high affinity Gal transporter. Gal caused a remarkable metabolic shift from homolactic to a truly mixed acid fermentation. The in silico predicted pathways for the catabolism of each sugar were experimentally validated at the biochemical level by determining sugar-specific intracellular metabolites and pathway specific enzyme activities. Furthermore, mutants were generated in each pathway by inactivation of a gene encoding a pathway specific enzyme, and their analysis proved at the genetic level the biochemical analyses.