STUDIES IN MYCOBACTIN BIOSYNTHESIS by ANAXIMANDRO GOMEZ VELASCO A thesis submitted to The University of Birmingham for the degree of DOCTOR OF PHILOSOPHY School of Biosciences The University of Birmingham September 2008 University of Birmingham Research Archive e-theses repository This unpublished thesis/dissertation is copyright of the author and/or third parties.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. Abstract Tuberculosis (TB) is the leading cause of infectious disease mortality in the world by a single bacterial pathogen, Mycobacterium tuberculosis. Current TB chemotherapy remains useful in treating susceptible M. tuberculosis strains, however, the emergence of MDR-TB and XDR-TB demand the development of new drugs. Enzymes involved in mycobactin biosynthesis, low molecular weight iron chelators, do not have mammalian homologues; therefore they are considered potential targets for the development of new anti-TB drugs. The aims of this study were to identify potential inhibitors and to investigate the function of the mbtG and AmbtE and AMbtF genes during mycobactin biosynthesis. The full length of mbtB and the ArCP domain were successfully cloned and post-translationally modified by MtaA, a broad phosphopantetheinyl transferase from Stigmatella aurantiaca, using Escherichia coli. Inhibitors identified by virtual screening as well as 13 chemically synthesised PAS analogues were initially investigated in whole-cell assay against Mycobacterium bovis BCG Pasteur. Seven of these compounds had interesting growth inhibition under iron-sufficient conditions. The mbtA gene was cloned and expressed as soluble protein using Mycobacterium smegmatis mc2155. Preliminary in vitro MbtA assays provided hints of its activity, although, the KM for SAL and ATP have not been determined yet. The mbtG and ambtE genes have been cloned and expressed in E. coli to further investigate their biochemical function in mycobactin biosynthesis. Acknowledgments Undoubtedly, my parents and brothers have played a fundamental role during my life; they have always been with me both in the pitfalls and success. In fact, the first scholarship that I received was from my parents who supported me during all my studies until I completed a university degree. Without them I would never achieve what I have now. Their love and support have encouraged me to keep working hard. It is always difficult to choose what to do next after the undergraduate studies. However, my experience as a co-worker in the region hospital of my hometown, where I used to collaborate in the diagnosis of tuberculosis, determined my decision to do research and pursue a post-graduate degree in this area. My conviction that Science is a tool to unmask and understand nature for the benefit of human being as well as for the conservation of nature itself was another reason. Selecting a place to achieve these aims is not easy either. However, I started another fascinating path in my life here in Birmingham in Prof. Gurdyal S. Besra’s laboratory. I would like to express my gratitude to him for giving me an opportunity to do tuberculosis research in his laboratory. Also, I am very grateful to the Ford Foundation International Fellowship Program and the Mexican National Council for Research (CONACYT) for granting me a funded Ph D. My time in the “Besra lab” was fruitful in many senses. Firstly, I could learn several Molecular Biology and Microbiology techniques under the supervision of an excellent teacher, Dr. Lynn G. Dover. His experience and knowledge was fundamental for the development of my PhD. In addition, the support from Dr. Apoorva Bhat, Dr. Alistair K. Brown, Dr. Luke Alderwick, Dr. Usha Veeraraghavan and Dr. Raju Tatituri was also invaluable during my studies. Secondly, the great time and the joy I had, it was due to the presence of many friends (past and present): Hemza, Albel, Arun, Athina, Becki, Faye, Helen, Jess, Jiemin, Yoel, Justine, Natacha, Oona, Petr, Sara, Sid, Veemal and Vijaya. The second chapter could not be completed without the pSUMtaA construct, kindly provided by Prof. Rolf Müller from the Department of Pharmaceutical Technology, Saarland University, Germany. The invaluable help for MALDI analysis and bacterial conjugations techniques is also acknowledged to Matthias Altmeyer and Dr. Olena Perlova, respectively. Arriving and settling down in Birmingham was not simple, but the encouragement, laugh and memories of good friends during all this four years allowed me to go through: Larisa, Carlos and Tomoko. Thank you for your unconditional love and support when I needed most. This thesis is dedicated to my family. Table of Contents. Chapter 1. General introduction……………………………………………… 1 1.1 Tuberculosis, an ancient human disease……………………………………... 2 1.2 Epidemiology of TB: a persistant human worldwide disease……………….. 4 1.3 Biology of M. tuberculosis: understanding the success of the bacillus……… 8 1.3.1 Taxonomy and classification……………………………………………….. 9 1.3.2 Life style of mycobacteria………………………………………………….. 11 1.3.3 Mycobacterial genomes: insight to the bacilli……………………………… 11 1.3.4 The cell wall………………………………………………………………. 14 1.3.5 Pathology of M. tuberculosis……………………………………………….. 17 1.4 Combating the bacillus: vaccines and the drug arsenal………………………. 19 1.4.1 Vaccines……………………………………………………………………. 20 1.4.2 Drug arsenal………………………………………………………………… 21 1.4.2.1 Isoniazid………………………………………………………………….. 23 1.4.2.2 Rimfapicin……………………………………………………………….. 24 1.4.2.3 Pyrazinamide…………………………………………………………….. 24 1.4.2.4 Ethambutol………………………………………………………………. 25 1.4.2.5 Streptomycin…………………………………………………………….. 26 1.4.2.6 Ethionamide……………………………………………………………… 27 1.4.2.7 para-Amino salicylic acid………………………………………………... 27 1.4.2.8 Fluoroquinolones…………………………………………………………. 28 1.4.2.9 Aminoglycosides…………………………………………………………. 29 1.4.2.10 D-cycloserine…………………………………………………………… 29 1.4.2.11 Polypeptides……………………………………………………………. 30 1.5 Iron as an element……………………………………………………………. 31 1.5.1 Iron and its biological importance………………………………………….. 33 1.5.2 Iron in living organisms…………………………………………………….. 37 1.5.3 Iron in mammals……………………………………………………………. 37 1.5.4 Iron in bacteria……………………………………………………………… 41 1.5.4.1 Siderophores, iron and virulence…………………………………………. 41 1.5.4.2 Siderophores in bacteria…………………………………………………. 46 1.5.4.3 Structure of siderophores………………………………………………… 47 1.5.4.4 Nomenclature of siderophores…………………………………………… 50 1.5.4.5 Biosynthesis of siderophores: general mechanism……………………… 51 1.5.4.5.1 Adenylation domain…………………………………………………… 54 1.5.4.5.2 Carrier proteins ……………………………………………………….. 54 1.5.4.5.3 Phosphopantetheinylation………………………………………………. 55 1.5.4.5.4 The condensation domain………………………………………………. 58 1.5.4.5.5 The thioesterase domain………………………………………………... 59 1.5.4.5.6 Diversity of siderophore produced by NRPS…………………………... 61 1.5.4.5.7 Siderophore biosynthesis independent of NRPSs……………………… 62 1.6 The discovery and structural elucidation of mycobactins……………………. 62 1.6.1 Organisation of the mycobactin-carboxymycobactin gene clusters………... 68 1.6.2 Unified model for mycobactin-carboxymycobactin biosynthesis………….. 71 1.6.3 The mbt-2 gene cluster……………………………………………………... 77 1.7 Transport of ferri-siderophore across the membrane………………………… 81 1.7.1 Transport across the periplasm and cytoplasmic membrane……………….. 84 1.7.2 Intracellular release of iron into bacteria cell………………………………. 87 1.7.3 Iron storage in bacteria……………………………………………………... 90 1.7.4 Gene regulation by iron in bacteria………………………………………… 91 1.8 Aims of the project…………………………………………………………… 93 Chapter 2. Post-translational modification of MbtB..………………………... 96 2.1 Post-translational modification of MbtB…………………………………….. 97 2.2 Material and methods………………………………………………………… 101 2.2.2 Molecular cloning and expression of the mbtB and pptT genes of M. tuberculosis…………………………………………………………………… 102 2.2.3 Determination of expression levels of the mbtB-pET23b in E. coli C41 (DE3)………………………………………………………………………... 104 2.2.4 Preparation of E. coli C41 (DE3) chemical competent cells harbouring pptT-pCDFDuet1…………………………………………………….. 105 2.2.5 Co-overexpression of mbtB-pET23b and pptT-pCDFDuet1 in E. coli C41(DE3)………………………………………………………………. 105 2.2.5.1 Screening for in vivo post-translational modification of the carrier proteins by MALDI-Mass Spectrometry…………………………………. 106 2.2.5.2 Screening for in vivo post-translational modification of carrier proteins by HPLC………………………………………………………………… 108 2.2.6 In vitro post-translational modification of MbtB…………………………... 109 2.2.6.1 Screening for in vitro post-translational modification of MbtB………….. 110 2.2.7 Molecular cloning and expression of the ArCP domain of MbtB………….. 111 2.2.7.1 Protein purification of ArCP in chitin matrix column……………………. 113 2.2.7.2 Screening in vivo and in vitro post-translational modification of the ArCP protein………………………………………………………………. 114 2.2.8 In vivo post-translational modification of the carrier proteins of MbtB by a broad substrate phosphopantetheinyl transferase…………………….. 114 2.3 Results………………………………………………………………………... 115 2.3.1 In vivo post-translational modification of MbtB by the intrinsic phosphopantetheinyl transferase of M. tuberculosis…………….