Biochemical Studies on Redox Regulation in Different Dormancy Models of Mycobacteria Thesis Submitted to Savitribai Phule Pune University For The Degree Of DOCTOR OF PHILOSOPHY IN BIOTECHNOLOGY By Ketaki Dilip Shurpali Research Supervisor Dr. Dhiman Sarkar Combichem Bioresource Center Organic Chemistry Division CSIR-National Chemical Laboratory Pune-411008 India October 2014 Certificate This is to certify that the work incorporated in the thesis entitled “ Biochemical Studies on Redox Regulation in Different Dormancy Models of Mycobacteria ” submitted by Ketaki Dilip Shurpali was carried out under my supervision at Combichem Bioresource Center, Organic Chemistry Division, National Chemical Laboratory, Pune-411008, Maharashtra, India. Materials obtained from other sources have been duly acknowledged in the thesis. Dr. Dhiman Sarkar (Research Guide) I Declaration by Research Scholar I hereby declare that the thesis entitled " Biochemical Studies on Redox Regulation in Different Dormancy Models of Mycobacteria ", submitted for the Degree of Doctor of Philosophy to the Savitribai Phule Pune University, has been carried out by me at Combichem Bioresource Center, Organic Chemistry Division, CSIR-National Chemical Laboratory, Pune-411008, Maharashtra, India, under the supervision of Dr. Dhiman Sarkar (Research supervisor). The work is original and has not been submitted in part or full by me for any other degree or diploma to any other University. Ketaki Dilip Shurpali (Research Scholar) II Dedications This thesis is dedicated to all those people who were always besides me in my good and bad times. First dedication goes to my father Mr. Dilip Shurpali and mother Mrs. Devyani Shurpali who instilled in me the desire to learn new things and confidence to achieve my dreams. Second, to my dear husband Kunal for never doubting my ability to achieve a goal and has always helped me by all possible means he could and my little sister Saniya who has made my journey enjoyable and colourful. Third, to my "one more" lovely parents, Mr. Prakash Pokale and Mrs. Shobha Pokale for being very supportive of every endeavour I have ventured to take especially at the end of my thesis. All these people have made my thesis possible. Ketaki Dilip Shurpali All truths are easy to understand once they are discovered; the point is to discover them. Galileo Galilei III Acknowledgements Though only my name appears on the cover of this dissertation, a great many people have contributed to its completion. I have been blessedby Almighty with some extraordinary people who have spun a web of support around me. I owe my gratitude to all those people who have made this dissertation possible and because of whom my PhD experience has been one that I will cherish forever. I am deeply indebted to my research supervisor, Dr. Dhiman Sarkar who is not only a great mentor but also a very good human being. I thank him for his limitless patience, help, encouragement and guidance he has given me till now and I wish it continues forever. Each meeting with him has added invaluable aspects to the implementation and broadened my perspective. He has guided me with his invaluable suggestions, lightened up the way when things seemed obscure and encouraged me a lot in the academic life. From him I have learned to think critically, to select problems, to solve them and to present their solutions. I thank him for giving me freedom and resources to pursue different lines of research. I will miss eating his brain for answers to my questions. It was a great pleasure for me to have a chance of working with him. I wish him continuing success in his future endeavours. I thank CSIR, New Delhi for providing me Research Fellowship to carry out my work for PhD.I consider it a great honour to have been part of the CSIR-National Chemical laboratory. This has been the best time I have spent in my life where I have learnt a lot more than just research. I am highly obliged to Director NCL, Dr. Sourav pal and our HOD, Dr. Pradeep Tripathi for their excellent leadership and an inspirational working environment they have provided in the institute. I am also thankful for the guidance and encouragement I have received from my PhD research committee members Prof. J. K. Pal, Dr. Vidya Gupta and Dr. C.G. Suresh. I also want to thank Dr. Mahesh Kulkarni and Yashwant for their help during proteomics work and Dr. Sen for teaching me how to make samples for Scanning Electron Microscopy. I also want to thank Dr. Thulasiram, Dr. M.V. Deshpande, Dr. R.A. Joshi and Dr. (Mrs.) R.R. Joshi for their help. I want to extend my thanks to all the staff members from Stores & Purchase section, Accounts section, Bill section, IV OCD office and SAC office for helping me all the time for the official work. I want to thank Dr. Mitra from NCCS for allowing me to use his Real Time PCR machine and Priyanka for helping me out in my experiment. Special thanks goes to my colleagues Upasana, Sampa, Abhishek, Shamim, Manoj, Laxman, Amar, Vijay, Nandadeep and Hardik for the help I have received countless number of times from them. I was fortunate to work with Nikita during last two years of my PhD, from whom I have learnt a lot. I specially want to thank Meghana, Manisha and Suwarna for being always positive and supportive especially during my thesis writing and helped me correct my thesis drafts. I consider it a great privilege to have been associated with such amazing "budding" scientists. Thanks to all my other friends who have always given me happiness and encouragement. I specially want to thank Herschel for always giving me right suggestions at right time. The list would be incomplete without one more person from NCL, Pooja, who made my PhD journey so joyous and I will always cherish the time we have spent together. Words fall short if I try to thank my dear husband, Kunal for the unreserved support I have received from him. His perfection and sincerity have been my inspiration. I specially want to thank my sweet little sister, Saniya for always being there for me and loving me unconditionally. I am blessed with the unconditional love I have received from my nephew, Aarya. I give all the credits to Aarya and Dad for making me forget all my tensions and worries after coming home. I take this opportunity to thank Nikhil and Trupti for being so supportive and considerate during the crucial final years of my thesis. Another important person who has made this journey joyous and easy is Uncle; I really want to thank him. The infallible love and encouragement I have received from the entire Pokale, Shurpali and Aphale families have always been my strengths. Finally, this would not have been possible without the support of my two lovely parents, Dilip & Devyani Shurpali and Prakash & Shobha Pokale and my dear husband Kunal. I feel blessed to have such a wonderful family. Ketaki Dilip Shurpali V Table of Contents CERTIFICATE I DECLARATION BY THE RESEARCH SCHOLAR II DEDICATIONS III ACKNOWLEDGEMENTS IV TABLE OF CONTENTS VI ABBREVIATIONS XII LIST OF FIGURES XIV LIST OF TABLES XVII ABSTRACT XIX PUBLICATIONS FROM THESIS XXII PUBLICATIONS OUT OF THESIS XXIII PATENTS XXIV CHAPTER 1 1 1.1 Overview of Tuberculosis 2 1.1.1 Classification of Mycobacterium tuberculosis 2 1.1.2 Characteristics of Mycobacterium tuberculosis 3 1.1.3 Characteristics of Mycobacterium smegmatis 5 1.2 Epidemiology of Tuberculosis 5 1.3 Pathogenesis of Mycobacterium tuberculosis 7 1.4 Oxidative stress 10 1.5 Strategies to combat oxidative stress 11 1.6 Role of ROS as signalling molecules 12 1.7 Latency: The root cause of TB problems 13 1.7.1 Characteristics of Latency 13 1.7.2 How to study latency? 14 VI 1.7.3 Models to study latency 15 1.7.3.1 In vitro models 15 1.7.3.1.1 Wayne model 15 1.7.3.1.2 Nutrient starvation model 16 1.7.3.1.3 Multiple stress dormancy model 16 1.7.3.2 Cell Infection Models 17 1.7.3.2.1 Vitamin C induced dormancy model 17 1.7.3.2.2 Macrophage Infection Model 17 1.7.3.3 In vivo models 17 1.8 Diagnosis of TB 18 1.8.1 Medical History 18 1.8.2 Chest X-ray 18 1.8.3 Microbiological Methods 19 1.8.3.1 Growth on selective media 19 1.8.3.2 Sputum testing for acid fast bacteria 20 1.8.3.3 PCR 20 1.8.3.4 Tuberculin skin test 21 1.9 Control & Treatment of TB 22 1.9.1 BCG Vaccine 22 1.9.2 Chemotherapy 22 1.9.2.1 Mode of action of drugs 23 1.10 Thesis Objectives 27 1.11 References 28 CHAPTER 2 41 2.1 Introduction 42 2.2 Materials & Methods 44 2.2.1 Chemicals, Media and Strains 44 VII 2.2.2 Cultivation of oxygen depletion induced dormant bacilli 44 2.2.3 Cultivation of nutrient starvation induced dormant bacilli 44 2.2.4 Assay for Total ROS estimation 45 2.2.5 Assay for superoxide production using tetrazolium salt, XTT 45 2.2.6 Detection of endogenous superoxide production by Mycobacterium smegmatis using DHE (HPLC) 46 2.2.7 Crude membrane preparation of Mycobacterium smegmatis 47 2.2.8 Extraction protocol for quantification of NAD+ and NADH redox states in active and dormant M. smegmatis 47 2.3 Results 49 2.3.1 Comparison of Total ROS in active and dormant Mycobacterium smegmatis by DCFH-DA oxidation 49 2.3.2 Detection of superoxide by active and dormant Mycobacterium smegmatis cells Using XTT 50 2.3.3 Detection of superoxide by active and dormant Mycobacterium smegmatis cells using DHE-HPLC 51 2.3.4 Effect of aeration on ROS formation in dormant culture of Mycobacterium smegmatis exposed to air 54 2.3.5 Effect of ROS modulators on superoxide production by aerobically growing Mycobacterium smegmatis 56 2.3.6 NADH oxidase as the major source of superoxide in aerobically growing Mycobacterium smegmatis 57 2.3.7 Superoxide production from membrane bound NADH oxidase in aerobically growing Mycobacterium smegmatis 59 2.3.8 Estimation of NAD +/NADH ratio in active and dormant M.