Molecular, biochemical and pharmacological characterisation of Mycobacterium tuberculosis cytochrome bd- I oxidase: a putative therapeutic target Taghreed Abdulaziz Hafiz Ph.D. 2013 Molecular, biochemical and pharmacological characterisation of Mycobacterium tuberculosis cytochrome bd-I oxidase: a putative therapeutic target Thesis is submitted in accordance with the requirements of the University of Liverpool for the degree of Doctor of Philosophy By Taghreed Abdulaziz Hafiz M.Sc (University of Liverpool) June 2013 Declaration This thesis is the result of my own work. The material contained in the thesis has not been presented, nor is currently being presented, either wholly or as a part, for any other degree or other qualification. The research work was carried out in the Liverpool School of Tropical Medicine, University of Liverpool, United Kingdom. ……………………………………….. Taghreed Abdulaziz Hafiz (2013) Dedication First and foremost, I would like to dedicate this work to my lovely parents, my husband and to my son Adel and my daughter Danah I Acknowledgements I am profoundly indebted to the grace of Allah for giving me the ability, insights, ideas and strength to make this thesis possible. Thank you Allah for your blessing on my family and me. I would like to acknowledge my supervisor Dr Giancarlo Biagini for his support, encouragement, stimulating suggestions and constructive criticism throughout the time of research and writing of this thesis. I also wish to thank my co-supervisor Professor Steve Ward for his helpful suggestions and comments on my work. I would like to express my gratitude and appreciation to Dr Ashley Warman for all of the guidance and support in helping me with various aspects of my work; your advice was immeasurable and I am extremely thankful. Additionally, special thanks go to Dr Nicholas Fisher for all of his advice and help on my work. The Steve Ward group, especially Mrs Alison Ardrey, Dr Gavin Laing, Dr Darren Moss, Dr Gemma Nixon, Dr Alison Shone, Angela Travis and Mary Creegan, are all acknowledged for their support, help and ability to make the working environment very comfortable and conducive to research. I am equally grateful to my colleagues, Dr Roslaini Abd Majid, Dr Teresa Ratio and Dr Thomas Antoine for their support and help. You have all been wonderful and it was great getting to know you and working with you. My deepest gratitude to my lovely mother Rajaa Obaid and to my father Abdulaziz Hafiz; you are simply the best parents in the whole world. Without your prayers to Allah, resolute encouragement, patience and help with taking care of my kids and myself, this thesis would not have been possible. A special big thank you goes to my lovely sisters, Kholoud, Nehad, Hanan, Tahani and Deema, and to my beloved brother Mohammed for all of your prayers and support. Thank you for making my adored daughter Danah laugh and helping her to enjoy her time with you and for committing to sending me pictures of her every day. I love you all very much. My sincerest love and apology go to my son Adel (2 years and 5 months) and to my baby daughter Danah (1 year) for being busy and away from you. I am sorry. Thank you for your patience with me. I adore you. Thank you Danah for being my pretty princess; every night away from you was a big challenge and burden to bear. Finally, I wish to express my heart-felt thankfulness to my dear husband Murad Mubaraki for his love, support and help throughout my lab work and during my pregnancies of Adel and Danah. Thank you for teaching me the techniques used in my metabolomics work. Thank you for lifting the burden off my shoulders throughout my PhD and thank you for being so supportive during the hardest nights, when I had to leave Danah back in Saudi Arabia. I love you. II Publications and presentations Work in this thesis is in preparation for publication and has been presented at meetings in the following forms: Publication in preparation: Mycobacterium tuberculosis cytochrome bd oxidase; Initial characterisation of a putative therapeutic target* Taghreed A. Hafiz1*, Ashley J. Warman1*, Nicholas E. Fisher1, Gemma L. Nixon1, Neil G. Berry2, Paul M. O’Neill2, Stephen A. Ward1, and Giancarlo A. Biagini1 *Running title: Characterisation of Mtb cytochrome bd oxidase * Joint first author Presentations: Mycobacterium tuberculosis cytochrome bd oxidase: a terminal solution? Taghreed A. Hafiz1, Ashley J. Warman1, Nicholas E. Fisher1, Gemma L. Nixon1, Neil G. Berry2, Paul M. O’Neill2, Stephen A. Ward1, and Giancarlo A. Biagini1 Has been presented at the Tuberculosis 2012 Conference held, September 11-15, 2012 at the Institut Pasteur, Paris, France. III Table of content Dedication .................................................................................................................... I Acknowledgements ..................................................................................................... II Publications and presentations ................................................................................... III Table of content.......................................................................................................... IV List of Figures ............................................................................................................ XI List of Tables............................................................................................................ XV List of appendices ................................................................................................... XVI Abbreviations ........................................................................................................ XVII Abstract ................................................................................................................. XXII Chapter I ....................................................................................................................... 1 General introduction..................................................................................................... 1 1.1. Overview ....................................................................................................... 2 1.2. The global burden of tuberculosis in the world ............................................. 3 1.3. Mycobacterium tuberculosis (Mtb): .............................................................. 4 1.3.1. The pathogen of tuberculosis: biology and virulence factors ................ 4 1.3.2. Pathogenesis of tuberculosis .................................................................. 6 1.3.2.1. Life cycle of Mtb ...................................................................................... 6 1.3.2.2. Granuloma ................................................................................................ 8 1.3.2.3. Latency of Mtb ......................................................................................... 9 1.3.2.3.1. Replication state of dormant Mtb .......................................................... 9 1.3.2.3.2. Latency models ................................................................................... 10 1.3.2.4. Persister cells.......................................................................................... 12 1.3.2.5. Yin-Yang model ..................................................................................... 13 1.4. Tuberculosis drugs: ..................................................................................... 15 1.4.1. First-line tuberculosis drugs: ................................................................ 16 1.4.1.1. Isoniazid ................................................................................................. 16 1.4.1.2. Rifampicin .............................................................................................. 19 1.4.1.3. Ethambutol ............................................................................................. 20 1.4.1.4. Pyrazinamide .......................................................................................... 21 1.4.2. Second-line tuberculosis drugs ............................................................ 22 1.4.2.1. Streptomycin .......................................................................................... 23 IV 1.4.3. Metronidazole, a drug against anaerobic bacteria ................................ 23 1.4.4. Resistant tuberculosis phenotypes........................................................ 24 1.5. Mycobacterium tuberculosis and a vision for the future ............................. 29 1.5.1. The Genomic era in the study of Mtb .................................................. 29 1.5.2. Mycobacterium tuberculosis metabolism ............................................ 30 1.5.2.1. Carbohydrate, TCA cycle....................................................................... 31 1.5.2.2. Fatty acids metabolism, Glyoxylate shunt ............................................. 32 1.5.2.3. Amino acids metabolism ........................................................................ 34 1.5.3. Electron transport and respiration in Mycobacterium tuberculosis ..... 37 1.5.3.1. Electron donors ...................................................................................... 37 1.5.3.2. Quinones in Mycobacterium tuberculosis .............................................. 38 1.5.3.3. Electron Acceptors ................................................................................. 39 1.5.4. Cytochrome bd-I oxidase ....................................................................