The Universal Stress Proteins of Bacteria Dominic Bradley Department of Life Sciences A thesis submitted for the degree of Doctor of Philosophy and the Diploma of Imperial College London The Universal Stress Proteins of Bacteria Declaration of Originality I, Dominic Bradley, declare that this thesis is my own work and has not been submitted in any form for another degree or diploma at any university or other institute of tertiary education. Information derived from the published and unpublished work of others has been acknowledged in the text and a list of references is given in the bibliography. 1 The Universal Stress Proteins of Bacteria Abstract Universal stress proteins (USPs) are a widespread and abundant protein family often linked to survival during stress. However, their exact biochemical and cellular roles are incompletely understood. Mycobacterium tuberculosis (Mtb) has 10 USPs, of which Rv1636 appears to be unique in its domain structure and being the only USP conserved in M. leprae. Over-expression of Rv1636 in M. smegmatis indicated that this protein does not share the growth arrest phenotype of another Mtb USP, Rv2623, suggesting distinct roles for the Mtb USPs. Purified Rv1636 was shown to have novel nucleotide binding capabilities when subjected to UV crosslinking. A range of site-directed mutants of Rv1636 were produced, including mutations within a predicted nucleotide binding motif, with the aim of identifying and characterising key residues within the Rv1636 protein. Further putative biochemical activities, including nucleotide triphosphatase, nucyleotidylyation and auto-phosphorylation were also investigated in vitro; however Rv1636 could not be shown to definitively possess these activities, raising the possibility that addition factors may be present in vivo. Bioinformatic analysis of Rv1636 has provided an in-depth look at the protein. The crystal structure of Rv1636 shows a strand swapped dimer conformation that appears to block the predicted nucleotide binding site, providing a possible reason for the low NTPase activity previously observed. Truncated Rv1636 constructs were successfully generated, in which the strand swapped dimer was disrupted, and subjected to biophysical analysis, including analytical ultracentrifugation and size exclusion chromatography combined with multi-angle light scattering. Previous Mtb single-USP mutants are known to have no phenotype under a range of stress conditions. For this reason the P. aeruginosa USP PA3309, which does possess a stress survival phenotype, was also investigated. This provided the opportunity to investigate the role of USPs and their putative nucleotide binding motif in vivo. Site-directed mutants of PA3309 were generated to investigate the role of the nucleotide binding motif in vivo. It remains to be determined if the survival defect observed for ∆PA3309 strain can be complemented with these mutants as the vector system used in these experiments proved unable to integrate into the attB site of the genome. Through the analysis of the USPs from mycobacteria and Pseudomonas, the aim was to elucidate a greater understanding of the role of Rv1636 in Mtb and the role of USPs in general. The bioinformatic and biochemical analyses of USPs, in addition to the site directed mutants generated as a result of this work, will provide a strong foundation for future studies. 2 The Universal Stress Proteins of Bacteria Acknowledgements Firstly I would pass my enormous appreciation to Huw Williams for his guidance and supervision. In the Williams’ group I express great thanks to Ben Ryall, Suzie Hingley-Wilson, Javier Levia and Kathryn Lougheed for their continued advice and support. Special mention goes to Kathryn for her meticulous reading of this thesis. I would like to show gratitude to Steve Smerdon and especially Simon Pennell for assisting with the structural studies undertaken at the National Institute for Medical Research. I am also grateful to Bob Coutts and Martin Buck for their supervision in during my MRes in Biochemical Research, which gave me my first experiences of academic research. Others to I would like acknowledge within the Imperial College Life Sciences Department include The Flying Ducks, Falcons and Dragons football team and their supporters. Members Mike, Nick, Thorsten, Luis, Rustem and Diogo brought a taste of success to the department. Ellen, Sophie, Maija, Lucy and Lei were the best supporters we could wish for. Wilson House provided a balanced environment and with four years as a sub-warden, there was always excitement waiting on returning home. From parties to 4am wake-up calls I enjoyed it all. Kudos to Mike Bluck, Laura Nicolaou, Shweta Singh and Alex Mottura, as they were brilliant and we enjoyed the challenge of keeping Wilson an awesome place to live. Through Diogo and Lara Geraldes I was afforded the opportunity to visit Portugal each year during my PhD and as a result Portugal is a country full of friends. I give credit to my kickboxing instructor Gustarve, the Scuba Diving Club, IC Tennis, IC Squash and Wilson House FC for 4 years of sporting fun and success. Finally, I would like to dedicate this thesis to my wonderful family. 3 The Universal Stress Proteins of Bacteria Table of Contents Declaration of Originality ........................................................................................................... 1 Abstract ...................................................................................................................................... 2 Acknowledgements.................................................................................................................... 3 Table of Contents .............................................................................................................. 4 List of Figures………………………………………………………………………………………………………………………11 List of Tables ............................................................................................................................ 14 Abbreviations ........................................................................................................................... 15 Chapter 1 - Introduction ....................................................................................................... 16 1.1 Scope of the Introduction ......................................................................................... 17 1.2 The Mycobacterium Genus ....................................................................................... 17 1.3 Mycobacterium tuberculosis ..................................................................................... 18 1.3.1 The Mtb Bacterium .............................................................................................. 18 1.3.2 The Mtb Cell Wall ................................................................................................. 19 1.4 Tuberculosis .............................................................................................................. 20 1.4.1 History of TB......................................................................................................... 21 1.4.2 Distribution of TB ................................................................................................. 21 1.4.3 HIV associated TB ................................................................................................. 23 1.4.4 Diagnosis of TB ..................................................................................................... 23 1.4.5 Drug Resistance and its Development in TB ........................................................ 25 1.4.6 Drug discovery ..................................................................................................... 26 1.4.7 TB Vaccine Development ..................................................................................... 26 1.5 Persistence of Mtb within the Host .......................................................................... 27 1.5.1 Survival within Macrophage ................................................................................ 28 1.5.2 The Granuloma .................................................................................................... 29 1.5.3 Latency, and Non-Replicating Persistence ........................................................... 32 1.5.4 Reactivation, Resuscitation and Disease ............................................................. 33 1.6 Models to Study Mtb ................................................................................................ 34 1.6.1 In vivo Models ...................................................................................................... 34 1.6.2 In vitro Models ..................................................................................................... 35 1.7 Stress Responses of Mtb ........................................................................................... 36 1.7.1 Reactive Oxygen Radicals .................................................................................... 37 1.7.2 Reactive Nitrogen Intermediates ......................................................................... 38 4 The Universal Stress Proteins of Bacteria 1.7.3 pH Stress .............................................................................................................. 38 1.7.4 Nutrient Starvation .............................................................................................. 39 1.7.5 Hypoxia ...............................................................................................................