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Mycobacterial Glycolipids – Pathways to Synthesis and Role in Virulence

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Mycobacterial Glycolipids – Pathways to Synthesis and Role in Virulence Mycobacterial Glycolipids – Pathways to Synthesis and Role in Virulence by DEBASMITA SARKAR A thesis submitted to The University of Birmingham for the Degree of Doctor of Philosophy School of Biosciences College of Life and Environmental Sciences The University of Birmingham February 2012 Abstract Mycobacterial diseases are responsible for numerous deaths worldwide, the major pathogens being Mycobacterium tuberculosis and Mycobacterium leprae. Also, in recent years threats from opportunistic pathogens, such as Mycobacterium marinum and Mycobacterium kansasii have been on the rise. These mycobacteria possess a unique lipid-rich cell wall with an array of mycolic acids and species-specific antigenic glycolipids, like the lipooligosaccharides. Some of these solvent extractable lipids possess immunomodulatory properties and play an important role during infection. Lipooligosaccharides (LOS) are surface exposed, polar, antigenic glycolipids that are present in several mycobacterial species. This study used the opportunistic human pathogens M. marinum and M. kansasii as a model system to unravel the genes involved in the biosynthesis of LOSs in Mycobacterium. Using directed mutagenesis and transposon mutagenesis, mutant strains defective in various parts of the LOS biosynthetic pathway were isolated. Analysis of these strains helped in further delineating the pathway and understanding the role of LOSs in virulence. A part of this thesis focussed on studying mycolic acid processing and transport using Mycobacterium smegmatis as a surrogate system. Mycolic acids are the most distinctive components of the mycobacterial cell wall. While their biosynthesis has been studied in detail, processing and transport across the membrane is not well understood. This study attempted to explore the roles of the two putative type II mycolyltransferases MSMEG3437 and MSMEG5851 in mycolic acid processing. Additionally, the role of M. tuberculosis mmpL11 gene was probed as the Mtb-mmpL11 had been reported to be involved in virulence. A null i mutant of the M. smegmatis homologue, Ms-mmpL11 (MSMEG0241) was generated and analysed for the above study. Deletion mutant strains of the two putative mycolyltransferase II did not show any phenotype, suggesting that their roles are redundant in vivo. Although the Ms-mmpL gene was found to be non-essential, it was found to be involved in transport of free mycolic acids. ii Declaration The work presented in this thesis was carried out in the School of Biosciences at the University of Birmingham, U.K., B15 2TT during the period November 2008 to November 2011. The work in this thesis is original except where acknowledged by references. No portion of the work is being, or has been submitted for a degree, diploma or any other qualification at any other University. iii University of Birmingham Research Archive E-theses repository This unpublished thesis/dissertation is copyright of the author and/or third parties. The intellectual property rights of the author or third parties in respect of this work are as defined by The Copyright Designs and Patents Act 1988 or as modified by any successor legislation. Any use made of information contained in this thesis/dissertation must be in accordance with that legislation and must be properly acknowledged. Further distribution or reproduction in any format is prohibited without the permission of the copyright holder. iv Acknowledgement The last three years have been the most rewarding time of my life. I have learnt a lot and looking back, I did not even think I would do a PhD. This thesis would not have come to life without the encouragement of my family especially my father and my husband, help and support of my supervisors, both past and present, friends, and colleagues. I am grateful to Professor Gurdyal S. Besra, my supervisor for granting me the opportunity to pursue my PhD studies inspite of a very nervous telephone interview. I would like to express my gratitude to my supervisor, Dr. Apoorva Bhatt for all the support, guidance, patience and help with my experiments, for making my experience easier and achievable, for help with writing and giving me confidence in myself. I would like to express my gratitude to the Darwin Trust of Edinburgh, whose funding made this thesis possible. I would also like to thank Professor David E. Minnikin for his valuable comments and time. My time in the ‘Besra Lab’ has been very fulfilling both professionally and personally. It has not only been work but an opportunity to know and work with brilliant scientists and amazing friends. I would like to thank Sid and Luke, for teaching me numerous techniques. Thank you Albel for providing your patient assistance with many techniques and help with all the photography that appear in this thesis. A massive thanks to Natacha for teaching chemistry to a ‘biologist’ and for all the help and encouragement throughout. I want to thank Usha, Veemal, Amrita and Arun for all the help, support and encouragement when i needed it most. A big thank you to the wonderful friends I have made in amazing biologists - Vicky, Monika, Shipra, Jiemin, Oona, Helen, Becci, Cristian, Sarah, Georgina, Liz, Hemza, Talat, Rana, v Reenette and brilliant chemists Ting, Justyna, Peter and Petr and all members past and present. Thank you! I would like to thank Dr. David A. Lammas (Institute of Biomedical Research, University of Birmingham), for all the help with tissue culture. I would like to thank Dr. Aniek van der Woude, Dr. Astrid van der Sar, Prof. W. Bitter and their lab (VU Medical centre, The Netherlands) for the transposon mutant library (detailed in Chapter 4) and zebrafish infection experiments. Also, huge thanks to Dr. P. Ashton, Mr. N. G. May and Dr. N. Spencer for their technical superiority in performing the MALDI-MS and NMR. I acknowledge the help of friends and mentors, Dr. Lakshmi Ramachandran, Dr. J. Venkatraman, Dr. Kaveri Das, and many colleagues at Astrazeneca India, without their encouragement i would not have started to pursue my PhD. Finally, to my family for their unconditional love and support. I wish Dida, Dadu, Mesomoni were here today! Thank you Baba, Ma, Masimoni, Boro Masimoni, Mamon, Boro, Mejo and Choto Pisi, Thakuma for believing in me. Big thanks to my aunt and uncle in Blackpool for their help and support. I am thankful to my dear friends – Preeti, Sumedha, Naveen, Reena, Shubha, Shanti, Disha, Moumita, Chandrima, Moitree for being there always. I also express my thanks to the wonderful friends I have made in Birmingham, Sonali, Ravikiran, Manmeet, Bhaskar, Neelam, Janvi, Pratik, Panchi, Yogi, Indrani, Amshika, Amit, Saptarshi and Bharat Parivar members for giving me the much needed break away from lab and work. Thank you all, it has not been easy being away from home, but your support made it possible. Last but not the least I would like to acknowledge the love, support and encouragement of an amazing person - Nitin, my best friend, now my husband. This thesis would not have been possible without you. Thank you! vi This thesis is dedicated to my Family vii Table of contents Abstract ...................................................................................................................... i Declaration ............................................................................................................... iii University of Birmingham Research Archive ......................................................... iv Acknowledgement ..................................................................................................... v Table of contents .................................................................................................... viii List of Abbreviations ............................................................................................xxvi Published work associated ....................................................................................xxxi Chapter 1 General Introduction ............................................................................... 1 1.1 Mycobacterium species................................................................................... 2 1.2 Tuberculosis .................................................................................................. 4 1.3 Epidemiology ................................................................................................. 5 1.4 Immune response to Mycobacterium tuberculosis infection ......................... 7 1.5 Treatment .....................................................................................................11 1.5.1 Anti –TB drugs .................................................................................................... 11 1.5.2 Directly Observed Therapy, Short-course ............................................................. 15 1.6 Drug resistance .............................................................................................16 1.6.1 Multi drug resistant TB (MDR-TB)...................................................................... 17 1.6.2 Extensively drug resistant TB (XDR-TB) ............................................................. 19 viii 1.7 The Mycobacterium tuberculosis complex (MTBC).....................................20 1.8 Non-tuberculous mycobacteria ....................................................................22 1.9 Surrogate systems in tuberculosis research - ..............................................22 1.9.1 Corynebacterium glutamicum .............................................................................
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