DETOXIFICATION OF GLUTATHIONE AND NITROSOGLUTATHIONE BY THIOREDOXIN SYSTEM OF MYCOBACTERIUM TUBERCULOSIS by RODGOUN ATTARIAN B.Sc., Tehran Azad University, 2002 A THESIS SUBMITTED IN PARTIAL FULLFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Experimental Medicine) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) July 2009 © Rodgoun Attarian, 2009 ABSTRACT Tuberculosis is the leading cause of mortality due to a single pathogenic infection. Its etiological agent, Mycobacterium tuberculosis infects, resides and multiplies within human alveolar macrophages. It is exposed to reactive oxygen intermediates and reactive nitrogen intermediates (RNI) such as nitric oxide (NO) produced within phagosomes and granulomas against invading pathogens. Therefore, proliferation of M. tuberculosis within the host depends on its strategies to counteract the onslaught of these intermediates. One example is recruitment of the thioredoxin system as one of the most proficient pathways for protection against oxidative stress, since it dominates peroxide detoxification pathways. A burst of NO within macrophages parallels the production of glutathione (GSH) to protect the host against NO toxicity. The macrophage GSH pool reduces NO to S- nitrosoglutathione (GSNO). Both glutathione disulphide (GSSG) and GSNO possess mycobactericidal activities in vitro. This thesis is focused on characterizing the role of M. tuberculosis thioredoxin system in detoxification of antimycobacterial compounds produced within the host such as GSSG and GSNO, due to the intrinsic capacity of the system to universally reduce disulfide bonds and reduce GSNO in humans. By performing NADPH oxidation assays and HPLC analysis we demonstrate that M. tuberculosis thioredoxin redox cascade is a general reduction system able to efficiently reduce the low molecular weight disulfides GSSG and MSSM, and dissimilate GSNO. We also investigated the cellular pathways in which thioredoxin of M. tuberculosis participates. Here, we present an analysis of the thioredoxin-linked M. tuberculosis proteome by using a substrate trapping assay and mass spectrometry. We have identified eleven proteins associated with TrxC, implicating the involvement of thioredoxin in distinct cellular processes in this pathogen. The findings described in this thesis elucidate a novel function for the thioredoxin system of M. tuberculosis. We demonstrate that this system serves as a detoxification pathway against mycobactericidal compounds such as GSSG and GSNO. Overall, the data presented here establishes that M. tuberculosis thioredoxin has pleiotropic roles and is involved in a spectrum of processes from metabolic pathways to gene expression and signal transduction. ii Table of Contents Abstract ........................................................................................................................ ii Table of Contents .........................................................................................................iii List of Tables................................................................................................................. v List of Figures .............................................................................................................. vi List of Abbreviations................................................................................................... vii Acknowledgements....................................................................................................... ix Dedication...................................................................................................................... x Chapter One: Introduction........................................................................................... 1 1.1 Tuberculosis: Past, Present, Future ..................................................................... 1 1.2 M. tuberculosis: A Successful Human Pathogen................................................. 3 1.3 Host Defense Mechanisms ................................................................................. 4 1.4 Redox Control Strategies in Mycobacteria.......................................................... 7 1.5 Low-molecular-weight Thiols ............................................................................ 8 1.5.1 Mycothiol: The Unique Mycobacterial Anti-oxidant............................... 10 1.6 Thioredoxins ................................................................................................... 11 1.7 Thioredoxin Systems in Mycobacteria.............................................................. 15 1.8 Glutathione and S-nitrosoglutathione are Toxic to Mycobacteria...................... 17 Chapter Two: Working Hypothesis and Specific Aims............................................. 19 2.1 Hypothesis ....................................................................................................... 20 2.2 Specific Aims................................................................................................... 20 Chapter Three: Experimental .................................................................................... 21 3.1 Materials .......................................................................................................... 21 3.2 Methods ........................................................................................................... 23 3.2.1 Gene Cloning ......................................................................................... 23 3.2.2 Site-directed Mutagenesis....................................................................... 24 3.2.3 Protein Production.................................................................................. 25 3.2.4 NADPH Oxidation Assays ..................................................................... 26 3.2.5 HPLC Analysis....................................................................................... 29 iii 3.2.6 Determination of Kinetic Parameters ...................................................... 31 3.2.7 Proteomic Analysis................................................................................. 31 3.2.7.1 Substrate Trapping Assay............................................................ 31 3.2.7.2 Silver Staining ............................................................................ 33 3.2.7.3 In-Gel Trypsin Digestion ............................................................ 33 3.2.7.4 Mass Spectrometry Analysis ....................................................... 34 Chapter Four: Results ................................................................................................ 36 4.1 Generation of a Catalytic Defective TrxC......................................................... 36 4.2 Purification of Recombinant Proteins ............................................................... 37 4.3 Reductase Activity of Mycobacterial Thioredoxin System................................ 38 4.3.1 NADPH Oxidation ................................................................................ 38 4.3.2 Analysis of Product Formation by HPLC................................................ 41 4.4 Enzyme Kinetic Studies.................................................................................... 47 4.5 Thioredoxin-Associated M. tuberculosis Proteome........................................... 51 Chapter Five: Discussion ............................................................................................ 55 Chapter Six: Future Work ......................................................................................... 63 Bibliography................................................................................................................ 67 iv LIST OF TABLES Table 1 Plasmids and Oligonucleotides Used in This Work ...................................... 21 Table 2 PCR Program for Site-directed Mutagenesis ................................................. 24 Table 3 NADPH Oxidation by Thioredoxin System Towards H2O2 and DTNB ......... 40 Table 4 Reductase Activity of Thioredoxin System Towards Candidate Substrates.... 41 Table 5 Steady-State Kinetic Parameters of Substrate Conversion ............................. 50 Table 6 Identification of Thioredoxin Targets in M. tuberculosis Lysates Using Substrate Trapping Procedure ....................................................................... 53 Table 7 Determination of Predicted Peptides According to Masses Obtained from MS Analysis ................................................................................................. 54 Table 8 Comparison of Kinetic Parameters of Different Thioredoxin Systems........... 59 v LIST OF FIGURES Figure 1 ROIs and RNIs Generation in Intracellular Milieu of Macrophages ............. 5 Figure 2 Major LMW Thiols Serving as Intracellular Redox Buffer ........................... 9 Figure 3 Mycothiol: The Major LMW Thiol in Actinomycetes.................................. 10 Figure 4 General Oxidoreductase Activities of the Thioredoxin System.................... 13 Figure 5 Genomic Context of trxB2 (Rv3913) and trxC (Rv3914).............................. 15 Figure 6 Plasmid Maps of Expression Vectors Used in This Work .......................... 23 Figure 7 NADPH-driven Route of H2O2 and DTNB Reduction in Redox Cascade.... 27 Figure 8 Proposed Route of NADPH-dependent Reduction of LMW Thiols............. 28 Figure 9 GS-mB Calibration Curve .........................................................................