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Detoxification of glutathione and nitrosoglutathione by thioredoxin system of Mycobacterium tuberculosis Attarian, Rodgoun 2009

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   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  ii 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.  iii  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  iv  3.2.6 Determination of Kinetic Parameters ...................................................... 31  3.2.7 Proteomic Analysis................................................................................. 31  Substrate Trapping Assay............................................................ 31  Silver Staining ............................................................................ 33  In-Gel Trypsin Digestion ............................................................ 33  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               v 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  vi 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 .......................................................................... 30 Figure 10   Schematic of Proteomic Analysis ............................................................... 32 Figure 11   TrxC Site-directed Mutagenesis.................................................................  36 Figure 12   Purified Recombinant Redox Proteins ........................................................ 37 Figure 13   HPLC Profile of GS-mB Generated by Thioredoxin System from GSNO... 43 Figure 14   HPLC Profile of GS-mB Generated by Thioredoxin System from GSSG ... 44 Figure 15   HPLC Profile of MS-mB Generated by Thioredoxin System from MSSM.. 45 Figure 16   Rate of Thiol Production by the Thioredoxin System Quantified by HPLC  46 Figure 17   Kinetic Analysis of the Thioredoxin System by Lineweaver-Burk Plots ..... 49 Figure 18   Substrate Trapping Assay ........................................................................... 52 Figure 19   Proposed Mechanism of Direct Reaction Between TrxC and GSNO........... 58 Figure 20   Proposed Mechanisms of Nitrosylated Cysteine Breakdown....................... 63           vii ABBREVIATIONS  ATP Adenosine Tri Phosphate BCCDC BCG British Columbia Centre for Disease Control Bacillus Calmette-Guerin CL3 CoA Containment Level 3 CoEnzyme A dATP 2’-deoxy-adenosine-5’-triphosphate dCTP 2’-deoxy-cytosine-5’-triphosphate dGTP 2’-deoxy-guanosine-5’-triphosphate DTNB 5, 5’-dithiobis-(2-nitrobenzoic acid) DTT Dithiotreitol dTTP 2’-deoxy-thymidine-5’-Triphosphate EDTA Ethylenediaminetetraacetic acid FAD Flavine Adenine Dinucleotide gorA Glutathione Reductase A grx Glutaredoxin GSH Reduced Glutathione GS-mB Bimane derivative of Glutathione GSSG Glutathione Disulfide (Oxidized Glutathione) HIV Human Immunodeficiency Virus HNO Nitroxyl HPLC High Performance (Pressure) Liquid Chromatography iNOS Inducible Nitric Oxide Synthase IPTG Isopropyl β-D-1-thiogalactopyranoside KatG Catalase-Peroxidase G LB Luria-Bertani LMW Low-Molecular-Weight MALDI-TOF Matrix Assisted Laser Desorption/Ionization-Time Of Flight mBBr Monobromobimane MDR Multi Drug Resistant  viii MSA Methane Sulfonic Acid MSH Mycothiol MS-mB Bimane derivative of Mycothiol MSSM Mycothione (Oxidized Mycothiol- Mycothiol disulfide) MWCO Molecular Weight Cut Off NADPH Nicotinamide Adenine Dinucleotide Phosphate NEM N-Ethylmaleamide NI-NTA Nitrile Triacetic Acid NO- Nitric Oxide O2- Superoxide ONOO_ Peroxinitrite Phox NADPH Phagocyte Oxidase PMSF Phenylmethanesulphonylfluoride RNI Reactive Nitrogen Intermediate ROI Reactive Oxygen Intermediate SDS-PAGE Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis SodA Superoxide Dismutase TB Tuberculosis TFA Trifluoroacetic Acid Tpx Thiol Peroxidase Tris Tris-(hydroxymethyl)aminomethane TrxC Thioredoxin C TrxR Thioredoxin Reductase B2 WHO World Health Organization XDR Extensively Drug Resistant       ix ACKNOWLEDGEMENTS  I would like to acknowledge and extend my sincerest gratitude to the following persons who have made this thesis possible.  Dr. Yossef Av-Gay, for giving me the great opportunity to complete my master studies at his laboratory. I am deeply honored to have studied under his supervision.  Dr. Horacio Bach, for his invaluable advice and profound impact on my research skills.  Dr. Zakaria Hmama, Dr. Charles Thompson, and Dr. Ted Steiner, for being my Supervisory Committee Members.  Dr. William Ramey, for his endless guidance and support.  Jerry Newton, for providing us with HPLC materials at the most critical time.  Mary Ko, for the technical support.  Chelsea Bennie, for providing us with the bacterial clones.  Past and present members of the Av-Gay Lab.                                                      THANKS EVERYONE               x DEDICATION This thesis is dedicated to my parents, Dania and Houshang, and my sister Roxanne, for their belief, support and patience during the years I have been away from them and home.     1 CHAPTER 1: INTRODUCTION 1.1 TUBERCULOSIS: PAST, PRESENT AND FUTURE Mycobacterium tuberculosis was identified by Robert Koch as the causative agent of “consumption” in 1882, known as tuberculosis (TB) today.  TB was a disease first mentioned in the Greek literature around 460 BC, and described by Hippocrates as the most common disease of that time (Kaufmann, 2003). M. tuberculosis has infected humans since ancient times, with evidence of tubercular decay found in the prehistoric spines of Egyptian mummies dating back to 3000 BC (Drancourt & Raoult, 2005).  TB is the leading cause of mortality worldwide due to a single bacterial infection. M. tuberculosis is one of the most devastating pathogens, which infects 8 million people annually, and is responsible for an estimated 2 million deaths per year (WHO, 2007). According to WHO, there are an estimated of 9.2 million new cases of TB (139 per 100,000 population) per year, and approximately one third of the world’s population has been exposed to this pathogen (WHO, 2008). Primary exposure to M. tuberculosis will lead to either immediate development of active TB or a latent asymptomatic disease state, which occurs in most cases. Approximately 10% of healthy immunocompetent individuals who are latently infected will develope active TB within their lifetimes. However, immunocompromised individuals are more susceptible to active TB development. For instance, in HIV–positive individuals, this risk is as high as 90%. India, China, Indonesia, South Africa and Nigeria have the highest absolute numbers of cases. The African region has the highest incidence rate per capita (363 per 100,000 population). Overall, 60% of all TB cases occur in Africa and South East Asia, and there is an alarming trend of co-infection with HIV in regions mentioned above. In addition to high  2 incidence rates, these areas are associated with extreme poverty, malnutrition, poor hygiene, and other environmental factors that lead to immunosuppression (WHO, 2008). Therefore, the high incidence of TB in these areas and also in AIDS patients has led to an increased spread of TB. Current TB treatment requires a combination of three or more of the first-line antibiotics: isoniazid, rifampicin, pyrazinamide, streptomycin, and ethambutol (Janin, 2007). The drastic rise in number of TB cases is associated with the emergence of multidrug resistant (MDR) and extensive or extreme drug resistant (XDR) strains. MDR strains no longer respond to the first line antibiotics, including isoniazid and rifampicin, and second line anti-TB drugs such as fluoroquinolones, ethionamide and cycloserine are used to treat them. XDR strains are resistant to three or more of these drugs (Janin, 2007). XDR strains pose a major threat for the world as a whole since they are highly virulent, and leave patients untreatable with currently available anti-TB drugs (WHO, 2008). The pathogenesis of M. tuberculosis still remains an expanding global health issue, mainly due to the fact that the mechanisms of pathogenesis are poorly understood, and the only available vaccine, BCG, has inconsistent efficacy. Therefore, TB is considered as a disease among major world health crises for the years to come, and this compels urgency for new therapeutic and preventive measures against TB.       3 1.2  M. TUBERCULOSIS: A SUCCESSFUL HUMAN PATHOGEN The genus Mycobacterium consists of Gram-positive bacteria, and depending on their growth rate, they are categorized into slow and fast growing groups. The slow growing strains are associated with human and animal diseases and include M. tuberculosis, M. leprae, M. avium, M. bovis, etc. Some of the fast growing Mycobacteria such as M. smegmatis and M. phlei are environmental and non-pathogenic strains. M. tuberculosis invades, resides and multiplies in alveolar macrophages. M. tuberculosis infection occurs via the respiratory tract, where inhalation of a few bacilli can establish pulmonary TB (Dannenberg, 1989). Infection can spread to almost any tissue in the body including skin, nervous system, and bones (Dannenberg, 1989). Alveolar macrophages serve as the first line of defense in the lung and engulf the bacilli by phagocytosis (Ernst, 1998). At this point, the infection is at the primary stage and macrophages signal the initiation of immune response (Kaufmann, 2001). As a result of the progress of the disease, a granuloma is developed by the host to contain the infection and to limit further dissemination of the pathogen (Qiu et al., 2001). In 90% of the cases, the immune response is able to eradicate the pathogen. In this case, the infection is contained in granulomatous lesions and immuno-competent individuals are non-infectious and asymptomatic. However, in immuno- compromised individuals, newborns, HIV-positive patients, and the aged, the primary infection most likely leads to an active disease. M. tuberculosis is able to persist in granulomas for decades in a non-replicative dormant state (Connolly et al., 2007). Upon weakening of the immune system, bacteria are released from granulomas and spread in the lungs by yet an unknown mechanism, or in some cases to the blood stream and other organs of the body (Qiu et al., 2001). Once the balance  4 between the host immune response and resistance towards M. tuberculosis fails, the mycobacterial replication is no longer contained in a localized area. This leads to progression of primary active disease and reactivation of latent infections. The ensuing widespread immune response leads to lung tissue damage, which is characteristic of tuberculosis pathology (Clark-Curtiss & Haydel, 2003, Zahrt & Deretic, 2002). 1.3 HOST DEFENSE MECHANISMS Macrophages play a central role in host defense. They are dynamic responders to extrinsic and intrinsic stimuli and are activated by factors such as interferon-gamma (IFN-γ), cytokines, viruses, and bacteria (Hoyal et al., 1998). Alveolar macrophages are in contact with airborne microorganisms and particles. Upon interaction, microorganisms are engulfed in vesicles termed phagosomes by a process termed phagocytosis (Huffman et al., 1998, Goldsmith et al., 1998, Shi et al., 1999). During phagocytosis, there is a concomitant acidification of the phagosome by vesicular proton-ATPase (Sturgill-Koszycki et al., 1994) and trafficking via endosomal pathway. This process is known as phagosome maturation, which is followed by fusion with the lysosomes (phago-lysosome fusion) (Desjardins et al., 1994, Russell, 1995). Lysosomes are acidic organelles containing proteases, which contribute to the killing and digestion of the microorganisms (Hestvik et al., 2005). Upon engulfment by macrophages, M. tuberculosis inhibits phagosome maturation and phagolysosome fusion. However, the pathogen remains enclosed in these phagocytic vacuoles, where it survives and multiplies despite the hostile surrounding environment of phagosomes. Production of reactive oxygen and reactive nitrogen intermediates (ROIs and RNIs respectively) is a major response of macrophages to inflammatory stimuli or infection  5 (Nathan & Shiloh, 2000). ROIs and RNIs include highly toxic molecules such as superoxide (.O2-), hydrogen peroxide (H2O2), nitric oxide (.NO-), and peroxynitrite (ONOO-) (Figure 1) (Gwinn & Vallyathan, 2006).                    Figure 1. ROIs and RNIs generation in intracellular milieu of macrophages. ROI including superoxide and hydrogen peroxide are generated by NADPH phagocyte oxidase and superoxide dismutase respectively. The inducible nitric oxide synthase (iNOS) generates nitric oxide from L-arginine. The reaction of nitric oxide with cysteine sulphydryls results in the formation of nitrosothiols. The interaction of superoxide and nitric oxide combine to generate the toxic intermediate peroxynitrite.     6 Oxidative/nitrosative stress is defined as deterioration in cellular function as a result of ROI and RNI production. Normally, these chemically reactive molecules are produced in the cell at basal levels as a result of metabolic processes, but they are contained and neutralized by different antioxidant systems existing in every cell (Imlay, 2008). However, upon infection ROIs/RNIs are produced at high concentrations, and they disrupt cellular homeostasis by damaging numerous components of the cell, including lipids, nucleic acids, proteins and metal cofactors. Excessive production of ROI and RNIs leads to mutagenesis, necrosis and apoptosis (Nathan & Shiloh, 2000). RNIs are generated by inducible nitric oxide synthase (iNOS), which is primarily expressed in immune cells, most notably in phagosome-containing macrophages (Davis et al., 2007, MacMicking et al., 1997). After production of NO by iNOS, it is subsequently oxidized to a series of other toxic RNIs such as nitrite, nitrogen dioxide, and nitrate. These compounds are able to inactivate biological molecules by adding a nitroso group to their functional cysteine moieties rendering enzymes and proteins functionally and structurally inactive. A particularly toxic intermediate is peroxynitrite (ONOO-) generated from the combination of NO with superoxide (Figure 1). ROIs and RNIs are produced in both granulomas and phagosomes, and play an important role in inhibiting the replication of M. tuberculosis (Clark-Curtiss & Haydel, 2003, Zahrt & Deretic, 2002, Chan et al., 1992, Nathan & Shiloh, 2000, Miller et al., 2007, Lu et al., 2002). Therefore identification of the systems that allow M. tuberculosis to survive upon infection of the host is largely focused on defining components of oxidative and nitrosative stress and their roles in infection.   7 1.4 REDOX CONTROL STRATEGIES IN MYCOBACTERIA M. tuberculosis has evolved various strategies to ensure survival within the toxic environment of both phagosomes and granulomas. The redox control system in M. tuberculosis is a complex and dynamic network of regulatory systems, consisting of transcription factors, mycothiol [a low-molecular-weight (LMW) thiol], thiol-disulfide oxidoreductases (thioredoxins) (den Hengst & Buttner, 2008), and other features. Mechanisms by which these responses are regulated in M. tuberculosis are significantly different from those observed in other bacteria (Sherman et al., 1995). For instance, in Escherichia coli, the redox-sensitive transcriptional regulator OxyR is responsible for production of enzymes that are involved in defense against oxidative stress (Christman et al., 1989). It has been reported that E. coli elicits a response to hydrogen peroxide by activating an oxyR-dependent regulon of more than 20 antioxidant genes including alkylhydroperoxide reductases (ahpC and ahpF), a catalase/peroxidase (katG), glutathione reductase (gorA), glutaredoxin 1 (grxA), a DNA-protective nucleoprotein (dps) and a thioredoxin (trxC) (Farr & Kogoma, 1991, Prieto-Alamo et al., 2000, Storz et al., 1990, Tartaglia et al., 1989, Green et al., 2000, Paget et al., 2001, Zahrt & Deretic, 2002, den Hengst & Buttner, 2008). oxyR is conserved in several mycobacterial species including M. leprae and M. tuberculosis. In M. tuberculosis, oxyR is a pseudogene, which is inactivated by multiple mutations, deletions or frameshifts (Deretic et al., 1995). However, upon oxidative and nitrosative stress, M. tuberculosis is still able to activate superoxide dismutase (sodA), katG, ahpC and trxC genes. Mutations in katG or sodA genes impaired the growth of the pathogen in murine models (Ng et al., 2004, Piddington et al., 2001). It has been shown that M. tuberculosis and M.  8 smegmatis with mutations in ahpC have increased sensitivity to peroxynitrite (Master et al., 2002). M. tuberculosis is able to mount an additional defense response towards RNIs by entering a non-replicating dormant state. High concentrations of NO may induce expression of dosR regulon, which regulates the expression of 47 genes (Wisedchaisri et al., 2005) and is recruited for survival of mycobacteria once they are in a latent state (Ohno et al., 2003, Voskuil et al., 2003). Moreover, studies from our laboratory have shown that the protein serine/threonine kinase PknH is activated in response to RNIs/ROIs and slows the in vivo growth of Mycobacteria during infection (Papavinasasundaram et al., 2005). 1.5 LOW-MOLECULAR-WEIGHT THIOLS The cytoplasm of all organisms is a highly reducing environment, which is maintained to avoid an irreversible oxidative damage and to assure the proper function of metabolic reactions. This environment is maintained by millimolar concentrations of LMW thiol compounds, which also participate in other important biological functions such as reducing cofactors and detoxification of xenobiotics (Fahey, 2001, Ritz & Beckwith, 2001, Brandes et al., 2008). LMW thiols also serve as a redox buffer to avert disulfide stress, which is a subset of oxidative stress caused by formation of unwanted disulfide bonds (Aslund & Beckwith, 1999). LMW thiols are characterized by the presence of a functional cysteine, which serves as the redox buffer. Organisms have evolved LMW thiols with diverse structures. These include, glutathione (GSH) (Figure 2) in eukaryotes and Gram-negative bacteria (Meister, 1988), trypanothione (T[SH]2) (Figure 2) in trypanosomatids (Fairlamb et al., 1985), and mycothiol in Gram-positive bacteria (Rawat & Av-Gay, 2007). Other thiol-containing  9 molecules such as coenzyme A (Figure 2) and cysteines contribute to the redox potential (Ziegler, 1985).                           Figure 2. Major LMW thiols serving as intracellular redox buffers. A. Glutathione, B. Trypanothione, C. Coenzyme A.  A. B. C.  10 GSH is a tripeptide consisting of glutamate, glycine and cysteine that plays an essential role in protecting the cell against oxygen toxicity (Anderson, 1998) by removing reactive oxygen species generated from basal metabolic activities. Other functions of GSH include the detoxification of exogenous xenobiotic agents, which is mediated by S- glutathione transferase (Mannervik & Danielson, 1988). 1.5.1 MYCOTHIOL: THE UNIQUE MYCOBACTERIAL ANTI-OXIDANT Mycobacteria are devoid of GSH, and instead they produce mycothiol (MSH) (1-D- myo-inositol-2-(N-acetyl-L-cysteinyl) amido-2-deoxy-α-D-glucopyranoside (Figure 3) (Newton et al., 1993). MSH is a pseudo-disaccharide and contains a functional cysteine group (Newton et al., 1993, Sakuda et al., 1994, Spies & Steenkamp, 1994).         Figure 3. Mycothiol: the major LMW thiol in Actinomycetes.  Several functions have been attributed to MSH beside its reducing characteristics. The primary role of MSH is to maintain the intracellular redox homeostasis. As such, it acts as an electron acceptor/donor and serves as a cofactor in detoxification reactions for  11 alkylating agents, free radicals and xenobiotics. It functions as a line of defense against ROIs (Ung & Av-Gay, 2006), stores cysteines, and detoxifies antibiotics (Steffek et al., 2003, Rawat & Av-Gay, 2007). The MSH biosynthesis pathway has been well characterized (Newton & Fahey, 2002, Newton et al., 2006). Another thiol present in actinomycetes is ergothioneine (ESH). ESH is a betaine of 2-thiol-L-histidine (Genghof & Vandamme, 1964). Unlike MSH, ESH has been detected in plants, fungi, animals and bacteria, although only fungi and actinomycetes are able to synthesize this thiol (Genghof, 1970). The amount of ESH present in actinomycetes is ten-fold lower than that of MSH and its exact function in these bacteria is still unknown (Fahey, 2001). As mentioned earlier, the antioxidant properties of both MSH and GSH are due to the presence of a functional reduced cysteine. MSH and GSH undergo oxidation to the disulfide form mycothione (MSSM) and GSSG respectively. The disulfide compounds are converted back to their reduced form by specific disulfide reductases such as mycothione reductase in the case of MSH (Newton & Fahey, 2002, Patel & Blanchard, 1999) and glutathione reductase in GSH (Karplus & Schulz, 1987). 1.6 THIOREDOXINS As mentioned earlier, the intracellular environment is maintained under reducing conditions (Gilbert, 1990). Frequently, enzymatic reactions require the formation of protein intermolecular disulfides according to the following reaction: ENZ-SH + RSSR              ENZ-SSR + RSH, or intramolecular disulfides according to: ENZ-SH2 + RSSR             ENZ-S2 + 2RSH  12 Although these reactions are chemically similar to those involved in stabilization of tertiary structures in proteins, the disulfides involved in regulation must be accessible to reduction by external thiols. One of the major ubiquitous disulfide reductases responsible for maintaining proteins in their reduced state are thioredoxins. They function in presence of thioredoxin reductases constituting a thioredoxin system.  Thioredoxins are small (8-14 kDa), heat-resistant proteins. They have multiple functions and they are involved in a plethora of cellular regulatory and metabolic pathways (Jeffery, 1999). Thioredoxins are characterized by the presence of a dithiol/disulfide active site CXXC (Holmgren, 1985). They are ubiquitously distributed and highly conserved in all organisms from Archea to humans (Eklund et al., 1991). Rapid and reversible thiol-disulfide exchange reactions control protein function via the redox state of structural or catalytic SH groups. Oxidation of a critical SH group will lead to changes in structural and functional changes in proteins. Therefore, thioredoxins have a wide range of functions in cellular physiology and pathological conditions by contributing to the maintenance of the redox state of protein thiols (Holmgren, 1985).  Oxidized thioredoxins are reduced by thioredoxin reductases (TrxR) in a NADPH- dependent reaction (Holmgren, 1985). Thioredoxin reductases are a large family of FAD- containing enzymes. The enzymatic mechanism involves the transfer of reducing equivalent from NADPH to a disulfide bond in the enzyme within the highly conserved sequence CATC via FAD. Subsequently, these electrons are transferred to a final acceptor via thioredoxins. This consecutive transfer of electrons constitutes a redox cascade (Figure 4).   13           Figure 4. General oxidoreductase activities of the thioredoxin system. The figure shows reduction of the active site in oxidized thioredoxin, Trx-S2, to a dithiol in reduced thioredoxin, Trx-(SH)2, by thioredoxin reductase (TrxR) and NADPH. Trx-(SH2) reduces protein disulfides by its general oxidoreductase activity, generating Trx-S2.  Thiol-disulfide switching is essential for maintenance of cellular proteins in their reduced form during oxidative conditions. Thiol-disulfide exchange reactions via redox active disulfides are efficient for electron transport and are used in mechanisms of essential enzymes such as ribonucleotide reductase required to provide deoxyribonucleotides for DNA synthesis and sulfate reductases essential for sulfur containing metabolite homeostasis (Black et al., 1960, Holmgren, 1985). The oxidation-reduction function of the thiol/disulfide oxidureductase activity of thioredoxins depends on two major determinants, the pKa values of the cysteine residues in the CXXC active site motif, and the standard-state redox potential. Generally Trxs have the lowest redox potential as opposed to other members of the oxidoreductase family and thus are the most reducing members of this family as well (Akif et al., 2008). For instance, the    14 redox potential of TrxA is -270mV, while the redox potential of glutaredoxins that are other members of this family range from -233 to -198 mV (Aslund et al., 1997). The oxidoreductase activity of thioredoxin system isolated from different organisms such as yeast (Black et al., 1960), E. coli (Moore et al., 1964, Laurent et al., 1964), and mammalian cells  (Engstrom et al., 1974) has been assayed towards several disulfide bond- containing substrates, particularly insulin and 5, 5’-dithiobis-(2-nitrobenzoic acid) (DTNB). Apart from its oxidoreductase activity, thioredoxin is known to play a role in a multitude of processes, which vary in different organisms (Arner & Holmgren, 2000). For instance, in Eukaryotes, thioredoxin regulates the activity of transcription factors (Schreck et al., 1992, Schenk et al., 1994, Hirota et al., 1997) such as NF-κB and AP-1 (Hayashi et al., 1993, Schenk et al., 1994). Other pathways include signal transduction, where reduced thioredoxin inactivates the apoptosis signaling kinase-1 (ASK-1) (Saitoh et al., 1998, Liu et al., 2000). In plant chloroplasts, thioredoxin regulates the light-activated Calvin cycle by reducing specific regulatory disulfides (Dai et al., 2000, Schurmann, 2003). A second regulatory mechanism driven by thioredoxin and independent of thiol redox activity is the ability to interact with other proteins to form functional protein complexes. For example, in E. coli, thioredoxin is an essential component of a protein complex required for filamentous phage assembly (Russel & Model, 1985). Thioredoxin is also an essential factor for bacteriophage T7 DNA polymerase (Richardson, 1983). In this regard, it has been shown that only the reduced form of thioredoxin binds the mentioned polymerase (Adler & Modrich, 1983, Tabor et al., 1987). In an attempt to identify thioredoxin-interacting proteins, a proteomic analysis in E. coli resulted in the identification of a total of 80 proteins found to be associated with the E.  15 ATGACC….… trxC coli thioredoxin, implicating the involvement of thioredoxin in at least 26 distinct cellular processes, including cell division, protein folding and degradation (Kumar et al., 2004). Additionally, multiple thioredoxins are found in many organisms. For instance E. coli possesses two functional thioredoxins. However, Arabidopsis thaliana possesses at least 35 thioredoxin isoforms (Collin et al., 2003). 1.7 THIOREDOXIN SYSTEMS IN MYCOBACTERIA The M. tuberculosis genome encodes three thioredoxin proteins (TrxA, TrxB1 and TrxC) and it also bears a single copy of thioredoxin reductase TrxB2 (TrxR) (Cole et al., 1998). Interestingly, only in pathogenic mycobacteria, genes encoding TrxC and TrxR are found at the same locus overlapping in one nucleotide (Wieles et al., 1995) (Figure 5).           Figure 5. Genomic context of trxB2 (Rv3913) and trxC (Rv3914). Gene organization of trxB2 and trxC. The adenosine in red represents the nucleotide overlapping both genes. Arrows represent the length and direction of the genes, while colors denote the function assigned. Intermediary metabolism are in yellow, conserved proteins are in brown, cell wall in green, and transcription factor in red.   ……CGATG trxB2  16 It has been reported that only TrxB1 and TrxC are reduced by TrxR and display oxidoreductase activity (Akif et al., 2008) by proficiently reducing disulfide bonds in the classic substrate model insulin (Holmgren, 1979). This suggests that TrxB1 and TrxC are the major cellular thioredoxins in M. tuberculosis (Akif et al., 2008). However, it appears that M. tuberculosis mutants missing any of the trx ORFs are not drastically impaired for survival in vitro, and only trxB2 turns out as an essential gene (Sassetti et al., 2003). The mechanisms by which M. tuberculosis resist oxidative killing elicited by mononuclear phagocytes are not yet clearly defined. Several mechanism have been proposed, one of which is the scavenging of free radicals produced by activated macrophages upon infection (Shinnick et al., 1995). As mentioned earlier, M. tuberculosis is devoid of GSH and synthesizes MSH (Cole et al., 1998, Rawat & Av-Gay, 2007). The involvement of thioredoxins in protection against oxidative stress and maintenance of intracellular thiol homeostasis in prokaryotes suggests that TrxC and TrxR of M. tuberculosis are most likely carrying out the same functions (Holmgren, 2000, Prinz et al., 1997, Holmgren, 1989). The system comprising TrxR and TrxC is one of the most well studied thioredoxin systems in M. tuberculosis. It has been shown to be involved in several detoxification pathways such as reducing dinitrobenzene, and hydroperoxides (Akif et al., 2008, Zhang et al., 1999). This thioredoxin system contributes to pathogen’s defense against ROIs in two ways (Manca et al., 1999, Jaeger et al., 2004). First by reducing alkyl hydroperoxide (ahpC), and keeping it catalytically active (Jaeger et al., 2004). Additionally, thioredoxin system serves as in efficient detoxification pathway against hydroperoxides and peroxynitrite once the thiol peroxidase Tpx complements the system (Jaeger et al., 2004). Tpx is part of an oxidative stress defense system that uses electrons donated by TrxC and TrxR to reduce alkyl  17 hydroperoxides (Jaeger et al., 2004). Tpx contains a redox-active intrasubunit disulfide bond, which undergoes reversible oxidation/reduction. Due to the fact that both TrxC and Tpx are consistently detectable as a major spot in proteomic analyses of M. tuberculosis cell-free filtrate (Rosenkrands et al., 2000), it is predicted that detoxification of ROIs and RNIs are predominantly mediated by the second pathway (Jaeger et al., 2004).  1.8 GLUTATHIONE AND S-NITROSOGLUTATHIONE ARE TOXIC TO             MYCOBACTERIA As mentioned earlier, macrophages generate RNIs and ROIs to combat microorganisms. These compounds are not able to discriminate between the targeted microorganisms from the producer cell. This lack of selectivity makes them highly toxic also to macrophages. ROIs and RNIs play an important role in inhibiting growth of M. tuberculosis (Chan et al., 1992, Ding et al., 1988, Nathan & Shiloh, 2000), and their toxic effect to macrophages is neutralized by GSH (Dayaram et al., 2006). Consistent with this, it has been reported that there is a GSH synthesis increase upon infection for self-protection against toxic effects of these intermediates (Dayaram et al., 2006). In addition, NO generated in phagosomes upon infection reacts with GSH to form GSNO, which probably serves as a NO donor (De Groote et al., 1995, Nikitovic & Holmgren, 1996) contributing to killing the pathogen (Venketaraman et al., 2003). In Mycobacteria, GSH and GSNO have bacteriostatic and bactericidal effects at concentrations above 5 mM (Venketaraman et al., 2003). It has been shown that in M. bovis BCG, the opp gene, which encodes an oligopeptide permease, is responsible for mycobacterial  18 uptake of GSH and GSNO (Chan et al., 1992, Green et al., 2000), resulting in increased resistance to the toxic effect of these compounds in a knockout strain of this gene (Green et al., 2000). The mechanism of GSH toxicity in Mycobacteria has not been elucidated yet, although several hypotheses have been postulated (Penninckx & Elskens, 1993). One hypothesis suggests that presence of high concentrations of GSH may result in an imbalance in mycobacterial mycothiol leading to an intracellular dysregulation of the reduction/oxidation activities (Penninckx & Elskens, 1993). In addition, according to Spallholz’s theory, GSH is an evolutionary precursor of antibiotics produced by Eukaryotes before the emergence of cellular immunity (Spallholz, 1987). This assumption is based on the structure similarities of GSH to penicillin precursors produced by the fungi Penicillium and Cephalosporium. When grown in culture, Mycobacteria are able to metabolize GSNO (at concentrations up to 2 mM) to GSH and nitrate (Vogt et al., 2003). However, when exposed to concentrations greater than 2 mM, GSNO is toxic and bactericidal (Venketaraman et al., 2003). The ability to cope with low concentrations of GSNO suggests the possible involvement of an S-nitrosothiol reductase that plays an essential role in the metabolism of S- nitrosothiols.        19 CHAPTER 2: WORKING HYPOTHESIS AND SPECIFIC AIMS Based on the information mentioned in introduction, M. tuberculosis is able to survive and multiply within toxic environment of macrophages, where it is exposed to ROIs and RNIs. One such example is the fact that compounds such as GSSG and GSNO produced by the host upon infection as part of the host defense mechanisms, are mycobactericidal. However, the successful growth of M. tuberculosis is proof that these microorganisms are able to neutralize these compounds and survive the host attack. In this thesis, we investigated the potential role of the M. tuberculosis thioredoxin system comprised of TrxC and TrxR, in detoxification of the mycobactericidal GSSG and GSNO. We hypothesized that M. tuberculosis employs the thioredoxin system to counteract the toxic effects of these molecules. We also tried to determine the capacity of this system to restore levels of MSH, as another anti-oxidant defense mechanism. Due to generalized thiol/disulfide reductase properties of thioredoxins, and the fact that they are able to reduce disulfide bonds, we proposed the question of whether they can also recruit the disulfide-bond containing mycothiol (MSSM) as the terminal electron acceptor in the redox cascade. This would elucidate whether the thioredoxin system can serve as an alternative system to regenerate levels of MSH under oxidative stress. We hope that elucidation of the role of the thioredoxin-dependent detoxification processes in mycobacteria will enable us to understand in better detail how M. tuberculosis adapts and survives within the toxic environment of macrophages.    20 In addition, in this work, we made an attempt to identify other cellular pathways in which TrxC of M. tuberculosis participates by elucidating protein targets that interact with this protein. This provides us with a better understanding of TrxC as a multifunctional protein involved in other biological pathways of M. tuberculosis, and will hopefully reveal some of the secrets of how M. tuberculosis is such a successful pathogen. 2.1 HYPOTHESIS 1) Thioredoxin system detoxifies host produced GSSG and GSNO by their reduction to GSH. 2) Thioredoxin system reduces oxidized MSSM and assists in maintenance of MSH. 2.2 SPECIFIC AIMS The specific aims of this project are: 1) To investigate reduction of GSSG, GSNO, and MSSM by the M. tuberculosis thioredoxin system. 2) To characterize the thioredoxin-associated M. tuberculosis proteome.           21 CHAPTER 3: EXPERIMENTAL 3.1 MATERIALS E. coli strain DH5α  was used for cloning purposes, and E. coli strain BL21 (D3) was used for protein expression purposes. Both strains were from Novagen, USA. They were cultured in Luria-Bertani (LB) broth purchased from Becton-Dickinson (USA), and supplemented with 100µg/ml ampicillin (Fisher, USA).  M. smegmatis mc2155 (ATCC 700084), a fast growing environmental saprophyte, was used for protein expression of TrxR. this strain was cultured in 7H9 MiddleBrook (7H9) (Becton-Dickinson, USA) broth supplemented with 0.05% Tween-80 (Fisher, USA) and 50µg/ml hygromycin (Calbiochem, USA). Pfu and DpnI were from Fermentas (USA). Oligonucleotides used in this work were synthesized by Operon Technologies, USA and detailed in Table 1.  Table 1. Plasmids and oligonucleotides used in this work. Plasmid/ oligonucleotides Characteristics Source Plasmids pALACE ace promoter, hygromycinR (Cowley et al., 2002) pET-22b PT7-based expression vector Novagen trxR/pALACE Rv3913 within AflII/ClaI restriction sites This work trxC/pET-22b Rv3914 within NdeI/HindIII restriction sites This work Tpx/pET-22b Rv1932 within NdeI/HindIII restriction sites This work  Oligonucleotides trxR-forward 5’-aaacttaagatgaccgccccgcctgt-3’ trxR-reverse 5’-aaaatatcgattcatcgttgtgctcctatca-3’ trxC-forward 5’-aattctagacatatgaccgattccgagaagt-3’ trxC-reverse  5’-aaaaagcttgttgaggttgggaacc-3’ tpx-forward 5’-aattctagacatatggcacagataaccctgc-3’ tpx-reverse 5’-aaaaagcttggcgcccagcgcg-3’ trxC-C40S-forward 5’-acatggtgtggacctagcaagatggtagcgccc-3’ trxC-C40S-reverse 5’-gggcgctaccatcttgctaggtccacaccatgt-3’  22 Reagents used in this work were from the following suppliers: Tris buffer was from Bio-Rad, USA. Glycerol, Tween-80, methanol, acetic acid, H2O2, formaldehyde, NH4HCO3, NaCl, acetonitrile, imidazole, DTT, and phosphate salts were purchased from Fisher (USA). Glucose was from Alfa Aesar (USA), and IPTG and mBBr from Invitrogen (USA). Acetamide, methane sulfonic acid (MSA), Na2CO3, AgNO3, trifluoroacetic acid, iodoacetamide, trypsin, formic acid, α-hydroxycinnamic acid, NEM, DTNB, GSH, GSNO, and EDTA were from Sigma (USA). Ampicillin was purchased from Bio Plus (USA), and hygromycin and NADPH were from Calbiochem (USA). Dialysis bags were from Spectra (USA) (MWCO 6000-8000), and Ni-NTA resin from Qiagen (USA). Complete EDTA-free protease inhibitor was purchased form Roche, Mannheim, Germany. Stage tips were from Sarstedt (UK), and C18 filter and Durapore 0.22 µM membranes from Millipore (USA). Sequazyme TM calibration mix for MALDI-TOF was from Applied Biosystems (USA). MS-mB, GS-mB and MSSM were kindly provided by Gerald Newton (University of California, San Diego). The HPLC system consisted of a Waters 717 Plus Autosampler, Farrand fluorometer (with an extinction filter at 254 nm and emission filter at 320-390 nm), a waters 1525 Binary HPLC pump, and Waters Breeze chromatography software (v. 3.30) (Waters, USA). All samples were loaded in Polly inserts WISP vials (Altech, USA), and separated on a Waters Symmetry C18 column (250 mm x 4.6 mm) (USA). All experiments were performed in triplicate. Graphs and statistical analyses were made using GraphPad Prism v. 4.0. Data are shown as means with associated standard deviations.   23 3.2 METHODS 3.2.1 GENE CLONING The genes trxB2, trxC and tpx were cloned previously in our laboratory.  Briefly, trxC and tpx were cloned into pET-22b using NdeI and HindIII restriction enzymes (Figure 6). trxB2 was cloned into the shuttle vector pALACE (hygromycinR) using AflII and ClaI restriction enzymes (Figure 6).       Figure 6. Plasmid maps of expression vectors used in this work. (A) TrxB2/pALACE over-expresses an N’ terminus 6xhis tagged TrxR. (B) TrxC/pET-22b and (C) Tpx/pET-22b over-expresses C’ terminus 6xhis tagged TrxC and Tpx respectively. Restriction sites represent the endonucleases used to clone the genes.  C.  A.  A. B.  24   3.2.2  SITE-DIRECTED MUTAGENESIS A catalytic-defective TrxC was constructed by site-directed mutagenesis using the oligonucleotide overlapping procedure (Fisher & Pei, 1997). Cysteine at position 40 was replaced by serine using the TrxC/pET-22b plasmid as DNA template (Figure 6). Oligonucleotides were designed flanking 15 bases each side from the desired point mutation and including the bases for the mutagenesis in the middle of the sequence. The mutagenesis reaction was performed in a thermocycler according to Table 2. The 50 µl reaction mix contained Pfu buffer (1X), 2 mM MgSO4, 200 µM each dATP, dCTP, dGTP, and dTTP deoxynucleoside triphosphate, 125 ng of each oligonucleotide, 5% DMSO, 50 ng of the plasmid template, and 1 unit of Pfu polymerase. 20 µl of the reaction was digested with 1 µl DpnI in order to digest non-mutated parental plasmid. 10 µl of the digestion mixture was directly transformed into E. coli strain DH5α. Plasmids were isolated from the transformants, and nucleotide sequences were determined to confirm the introduced mutations.    Table 2. PCR program for site-directed mutagenesis.  Segment Cycles Temperature [oC] Time [minute] 1 1 95 3 95 1.5 58 (annealing) 1 2 18 68 (extension) 7        25 3.2.3 PROTEIN PRODUCTION Proteins encoded by trxC/pET and tpx/pET were produced in E. coli strain BL-21 (DE3) using LB medium supplemented with 100µg/ml ampicillin. Protein coded by trxB2/pALACE was produced in M. smegmatis mc2155 using 7H9 supplemented with 0.05% Tween-80, 1% glucose, and 50 µg/ml hygromycin. For protein expression in E. coli, a starter culture was prepared by inoculating a single colony into 5 ml fresh medium and agitated in a shaker overnight at 37oC. Next day, the starter was diluted 1:100 in fresh medium and grown until an A600 of 0.6-1.00 was reached. Cells were induced with 0.1 mM IPTG overnight at room temperature. For protein expression in M. smegmatis, a starter culture was initiated by inoculating a single colony into 5 ml LB medium containing 0.05% Tween-80, 50 µg/ml of hygromycin. The culture was placed at 37oC overnight. Next day, the culture was diluted 1:100 in 7H9 medium supplemented with 0.05% Tween-80, 50 µg/ml hygromycin, and 1% glucose. The culture was placed at 37oC overnight in a shaker. Next day, the medium was changed by centrifuging the culture at 5000xg for 20 min, and cells were resuspended in 7H9 medium supplemented with 0.05% Tween-80, 50 µg/ml of hygromycin, and 0.02% acetamide. The culture was shaken at room temperature overnight. Next day, cultures were centrifuged at 5000xg for 20 min. Pelleted cells obtained after induction were processed in the same way for both E. coli and M. smegmatis by resuspension in lysis buffer (50 mM Tris- HCL pH 8, 250 mM NaCl, and 10 mM imidazole) and kept at -20oC. Protein purification was carried out by thawing the induced cells, followed by the addition of 1 mM PMSF, and sonication at a power setting of 10 for 3 bursts of 15 seconds each with 30 seconds cooling in between each burst. The total lysates were centrifuged at 13000xg for 30 min and the supernatants were applied to a Ni-NTA resin pre-equilibrated  26 with lysis buffer. After lysate loading, the resin was washed with 5 column volumes of washing buffer (50 mM Tris-HCL pH 8, 250 mM NaCl, and 20 mM imidazole). For protein purification using M. smegmatis, there was an additional washing step using washing buffer but with 65 mM imidazole. Bound proteins were eluted with elution buffer (50 mM Tris- HCL pH 8, 250 mM NaCl, and 250 mM imidazole). Eluted proteins were dialyzed overnight in 50 µM Tris-HCl pH 7.5, 5% glycerol, 1 mM DTT). Proteins were run on a 12% SDS- PAGE to check for purity, and concentrations were measured with the Bradford assay (Bradford, 1976). 3.2.4 NADPH OXIDATION ASSAYS The thioredoxin system was comprised of TrxC and TrxR. The reductase activity of this system was first verified by performing a NADPH oxidation assay towards H2O2 and DTNB (Figure 7), which are used extensively as substrates of thioredoxin systems (Jaeger et al., 2004, Zhang et al., 1999, Holmgren & Bjornstedt, 1995). The reaction mixture contained 100 mM phosphate buffer pH 7.4, 1 mM EDTA, 450 µM of NADPH, and purified proteins at a final concentration of 1 µM in a final reaction volume of 0.6 ml. 5 µM H2O2 was added to the reaction and according to the assay described by Holmgren (Holmgren, 1979). The activity of the system was determined by measuring the decrease in A340 every 10 seconds during the first minute of the reaction. The amount of NADPH oxidized was calculated as nmol/min and according to the equation Δ340 x 0.6/6.2. In this relation, 0.6 represents the reaction volume, while 6.2 is the millimolar extinction coefficient (mM-1cm-1) of NADPH. In the second assay, the disulfide compounds were replaced by DTNB (dissolved in 95% ethanol) at a final concentration of 26 µM (Holmgren, 1977), and followed by measuring the  27 increase in A412. One unit of activity was calculated according to the equation Δ412 x 0.6/13.6 x 2 (Luthman & Holmgren, 1982), where 0.6 represents the final volume of the reaction, and 13.6 is the millimolar extinction coefficient of 5-thio-2-nitrobenzoic acid (TNB) (reduction of DTNB by 1 mole of Trx-SH2 yields two moles of TNB) (Figure 7). The multiplication factor 2 corresponds to the fact that one mole of NADPH oxidized corresponds to 2 mol of sulphydryl groups. All the experiments were carried out in triplicate.                   Figure 7. NADPH-driven route of H2O2 and DTNB reduction in redox cascade. M. tuberculosis thioredoxin system transfers electrons from NADPH to final electron acceptors (adapted by Zhang et al., 1999). (A) H2O2 detoxification route. (B) DTNB reduction generating two TNB molecules.   NADPH NADP+ TrxB2-(SH)2 TrxB2-S2 TrxC-(SH)2 TrxC-S2 ROH+H2O ROOH A B 2 TNB DTNB NADPH NADP+ TrxB2-(SH)2 TrxB2-S2 TrxC-(SH)2 TrxC-S2    28 The NADPH oxidation assay towards GSSG, GSNO and MSSM (Figure 8) was performed according to the first assay described earlier. GSSG, GSNO, and MSSM were assayed at a final concentration of 26 µM.                 Figure 8. Proposed route of NADPH-dependent reduction of LMW thiols. M. tuberculosis thioredoxin system transfers electrons from NADPH to final electron acceptors. (A) GSSG reduction produces two molecules of GSH. (B) GSNO reduction produces GSH+NO. (C) MSSM reduction generates two MSH.        A B C NADPH NADP+ TrxB2-(SH)2 TrxB2-S2 TrxC-(SH)2 TrxC-S2 2 GSH GSSG NADPH NADP+ TrxB2-(SH)2 TrxB2-S2 TrxC-(SH)2 TrxC-S2 GSH + NO GSNO NADPH NADP+ TrxB2-(SH)2 TrxB2-S2 TrxC-(SH)2 TrxC-S2 2 MSH MSSM   29 3.2.5            HPLC ANALYSIS The direct formation of the reaction product GSH from GSSG and GSNO and MSH from MSSM was measured by HPLC assay. The reaction mixture contained 100 µM of phosphate buffer pH 7.4, 1mM EDTA, 450 µM of NADPH, and purified proteins at a final concentration of 1 µM in a final volume of 1 ml; each substrate was added at a concentration of 26 µM. HPLC analysis was performed based on a previously developed protocol (Newton et al., 2000). Reactions were performed at 30oC, and aliquots of 200 µl were taken at time 0, 10, and 60 min. These aliquots were treated at 60oC for 15 minutes with the fluorescent alkylating agent monobromobimane (mBBr) (2 µl of 100 mM stock) to produce the fluorescent bimane derivatives (S-conjugates) MS-mB and GS-mB from MSSM and GSSG/GSNO respectively. The treated samples were centrifuged at 14000 rpm for 3 min and subsequently acidified with 2 µl of 5 M methanesulfonic acid (MSA). The samples were then diluted 1:20 in 10 mM MSA prior to loading on the HPLC. At each time point, an aliquot of each reaction was taken for treatment with N-ethylmaleimide (NEM) (negative control) before labeling with mBBr, which binds and blocks all available thiol groups (Anderberg et al., 1998). Processed samples were then resolved on reverse phase water Symmetry C18 column (250 mm x 4.6 mm) using 0.25% glacial acetic acid pH 3.6 as solvent A, and HPLC-grade methanol as solvent B. The samples were resolved using the following program: 10% solvent B for 10 min; 18% solvent B at 15 minutes, 27% solvent B at 22 minutes, 90% solvent B at 24 minutes, 10% solvent B at 34 minutes. Flow rate was constant at 1ml/minute. Injection volume was 50 µl. The MS-mB peak appears at a retention time of 21± 0.5 minutes, and GS-mB peak at 19±  30 0.5 minutes. Fluorescence was read on a Farrand fluorometer with an excitation filter at 254 nm and an emission filter at 320-390 nm. The fluorometer was set at x0.01 for sensitivity and 3 seconds for response time. Peaks were manually integrated and analyzed using the Waters Breeze chromatography software. The absolute level of products formed was calculated by transformation of the areas obtained using a calibration curve prepared for GS-mB (Figure 9).            Figure 9. GS-mB calibration curve. A calibration curve was prepared by plotting the area obtained by HPLC analysis against known concentrations of GSH. Reactions were tagged with mBBr to produce the fluorescent GS-mB product. Shown is the mean ± S.D. of three independent experiments.       0.0 2.5 5.0 7.5 10.0 12.5 0 1 2 3 4 5 GS-mB [µM]  31 3.2.6  DETERMINATION OF KINETIC PARAMETERS Kinetic parameters were calculated by using 26, 40 and 57 µM of each substrate. Reactions were stopped after 10 minutes, and processed accordingly. The Lineweaver-Burk (Lineweaver & Burk, 1934) plot was obtained by plotting the inverse of enzymatic rate versus the inverse of substrate concentration. KM is the absolute value of the inverse of X- intercept and VMAX is the inverse of Y-intercept. KM = |1/X-intercept|, VMAX = 1/ Y-intercept.  3.2.7  PROTEOMIC ANALYSIS  SUBSTRATE TRAPPING ASSAY The catalytic-defective thioredoxin Cys40Ser was used to trap interacting proteins in M. tuberculosis following the scheme shown in Figure 10. Lysates were available in our laboratory and prepared in a CL3 facility (BCCDC, Vancouver). Briefly, lysates were obtained from a M. tuberculosis culture (A600=1.2) grown at 37oC in rolling bottles, and cells were harvested at 6000rpm for 10 min. Cell pellets were washed twice, resuspended and lysed in 50 mM HEPES (pH 7.5), 1 mM DTT, 5% glycerol in presence of complete EDTA- free protease inhibitor with glass beads in a Ribolyser at a speed of 6.5 for 2x 25 sec. Lysates were centrifuged at 13000xg for 10 min at 4oC and the supernatant was filtered through a low-binding 0.22 µM membrane filter. Protein concentrations were determined by Bradford assay. 120 µg of recombinant parental or Cys40Ser mutated thioredoxins were mixed with 1 ml of M. tuberculosis lysates containing 2 mg of total cellular proteins overnight at 4oC in a rocker. Next day, the mixture was purified by affinity chromatography by loading the mixture on a Ni-NTA resin as described in section 3.2.3. Eluted fractions were precipitated in  32 10% trichloroacetic acid and placed on ice for 3 hours. Precipitated proteins were re- suspended in sample buffer after centrifugation at 4000xg for 30 min.                   Figure 10. Schematic of proteomic analysis. * Substrate trapping assay  M. tuberculosis Lysate Incubation* with TrxC Cys40Ser Separation by SDS-PAGE Silver staining to detect protein bands In Gel Tryptic Digestion Mass Spectrometry Analysis  33  SILVER STAINING Samples were loaded onto 12% SDS-PAGE and silver stained. The method used for silver staining was compatible for mass spectrometry (MS) analysis and was a modified version of the Morrissey protocol (Morrissey, 1981). All the steps of the silver staining procedure were performed at room temperature and the gel was agitated on an orbital shaker at 40 spm. After resolving the proteins, gels were fixed in fixative solution (50% ethanol, 10% acetic acid) for one hour followed by washing (x3) with Milli-Q water for 20 min each. The gel was placed in DTT solution (5 mg/L of deionized water) for 20 min after the water was drained off. Then, the gels were incubated in 0.2% AgNO3 for 30 min, rinsed with deionized water for 5 min, and developed in Na2CO3 solution (138 g Na2CO3, 2 ml formaldehyde in 4L deionized water) for 5-10 min until a homogeneous brown color began to develop. The development step was stopped by addition of a stop solution (3% acetic acid) for 10 min. Exposure of M. tuberculosis lysate to the Ni-NTA resin was used as negative control. Bands specific for the mutated Cys40Ser TrxC were excised from the gel and processed for MS analysis.  IN-GEL TRYPSIN DIGESTION Bands observed on the SDS-PAGE after silver staining were excised and cut out into smaller pieces of about 1-2 mm in dimension. These pieces were washed in a solution containing 20% acetonitrile and 1 M NH4HCO3 for 1 hour. This was followed by another washing step in a solution containing 50% methanol and 5% acetic acid for 1 hour. Alternating washes were repeated until all the stain was out of the gel pieces. The gel pieces  34 were then dehydrated in pure acetonitrile for 5 min, and were completely dried at ambient temperature in a vacuum centrifuge. Then, they were re-hydrated in 100 mM NH4HCO3 for 30 min, followed by another step of dehydration. In the next step, gel pieces were rehydrated in a reducing solution containing 10 mM DTT in 100 mM NH4HCO3 for 30 min. After the reduction process, the excess of DTT solution was removed and an alkylating solution containing 100 mM iodoacetamide in 100 mM NH4HCO3 was added for 30 min. The alkylating step was followed by dehydration with pure acetonitrile, and rehydration with 100 mM NH4HCO3, and again, another step of dehydration. Finally, gel pieces were rehydrated in 50 mM NH4HCO3 containing 20 mg/ml trypsin. The digestion with trypsin was carried out at 37oC overnight. Next day, 30 µl of 50 mM NH4HCO3 was added for 10 min to elute the peptides. The elution was aspirated and transferred into another tube. To recover more peptides, 30 µl of formic acid was added to the gel pieces for 10 min and repeated twice. All three elutions were combined together and vacuum evaporated to a final volume of approximately 20 µl.  MASS SPECTROMETRY ANALYSIS The samples were prepared for MS analysis according to Stage tip purification protocol kindly provided by the Molecular Biophysics Laboratory at UBC. Firstly, the pH of the samples was adjusted to ≤2, to facilitate the binding of the peptides in the sample to the C18 membrane used for concentration and desalting. The acidification was carried out by adding 10 µl of sample buffer (1% TFA and 5% acetonitrile), and a drop of the sample was tested on pH strips. Tips containing C18 membranes were wetted by adding 20 µl of methanol on the top of the membrane following an equilibration with 20 µl of 0.5% acetic  35 acid using a syringe inserted into the tip. 20 µl of the sample buffer was loaded on the membrane and then the samples containing the peptides were loaded. The tip was washed with 20 µl sample buffer, and peptides were eluted with 10 µl of elution buffer (0.5% acetic acid and 80% acetonitrile). Eluted samples were placed in a speed vacuum centrifuge and dried for 30 min. The samples were re-suspended in 3.2 µl of sample buffer. 1 µl of this solution was mixed with 1 µl of the matrix α-cyano-4-hydroxycinnamic acid prepared in the same sample buffer. Samples were spotted on a 100 spot tray. Calibration mix number 2 was used for MALDI-TOF calibration purposes. A Voyager-DE STR Workstation MALDI-TOF located at Molecular Biophysics Laboratory, UBC, was used to analyze the spotted samples.                 36 CHAPTER 4: RESULTS 4.1  GENERATION OF A CATALYTIC-DEFECTIVE TRXC Like all thioredoxins M. tuberculosis thioredoxin C possesses a highly conserved CXXC catalytic motif with cysteines at position 37 and 40 (Figure 11) (Hall et al., 2006). It has been shown that a mutation in either of these cysteines renders thioredoxin unable to transfer electrons to the final electron acceptor (Stumpp et al., 1999). Therefore, we constructed a mutated TrxC by replacing cysteine with serine at position 40, and generated a catalytic- defective TrxC.              A)        B)           Figure 11. TrxC site-directed mutagenesis. (A) Replacement of cysteine with serine at position 40 in the active site of TrxC. (B) 3D structure image of TrxC. Cysteine at position 37 is shown in red, while the mutated residue is shown in blue. Image was generated by Cn3D software (NCBI, 2008).     35T W C G P C K M42       35T W C G P S K M42   37 4.2 PURIFICATION OF RECOMBINANT PROTEINS trxB2, trxC and tpx were previously cloned into expression vectors as shown in (Figure 6), by another lab member. Recombinant TrxR was over-expressed as soluble protein (35 kDa), and contained as N-terminal 6xHis fusion (5 kDa). TrxR was purified using Ni-NTA affinity chromatography, and resolved on 15% SDS-PAGE. The purified protein migrated at the expected size of 40 kDa (Figure 12). Recombinant TrxC (12 kDa), TrxC-Cys40Ser (12 kDa), and Tpx (16.8 kDa) contained C-terminal 6xHis fusions. When his-tag size was added to the protein molecular weight, they migrated according to the expected sizes: TrxC (17 kDa), TrxC-Cys40Ser (17 kDa), and Tpx (22 kDa) on the gel (Figure 12).              Figure 12. Purified recombinant redox proteins. Elution fractions corresponding to purifications by affinity chromatography using Ni-NTA resin were resolved on 15% SDS-PAGE and Commassie blue stained. Lanes: M) Marker, 1) wt TrxC, 2) C40S mutated TrxC, 3) Tpx, 4) TrxR, 5) uninduced culture (representative). Molecular weight standards are indicated on left side.     M             1                 2                3                 4         5    66-     45-    35-     25-   18.4-  14.4-   38 4.3 REDUCTASE ACTIVITY OF MYCOBACTERIAL THIOREDOXIN             SYSTEM  TrxR and TrxC form an active thioredoxin system. As described earlier, the thioredoxin system transfers electrons from NADPH to a terminal electron acceptor. Therefore, we tested the capacity of M. tuberculosis TrxR and TrxC (thioredoxin system) to reduce various substrates (Figure 7) by NADPH consumption assay and analytical determination of the reduced product using HPLC assay. The thioredoxin system acts with Tpx to detoxify hydroperoxide and peroxynitrite substrates (Jaeger et al., 2004) and these thioredoxins perform a better antioxidant function when combined with peroxyredoxins/thiol peroxidases (Kang et al., 1998, Nordberg & Arner, 2001). Therefore, we also tested the activity of TrxR and TrxC supplemented with Tpx towards the same substrates. 4.3.1 NADPH OXIDATION DTNB (Figure 7) and H2O2 have been used as synthetic model substrates for in vitro biochemical analyses of various thioredoxin systems (Kang et al., 1998, Nordberg & Arner, 2001, Holmgren & Bjornstedt, 1995, Zhang et al., 1999).  Oxidation of NADPH by the thioredoxin system in presence of DTNB and H2O2 will verify that our thioredoxin system performs in accordance with previously published studies (Kang et al., 1998, Nordberg & Arner, 2001, Holmgren & Bjornstedt, 1995, Zhang et al., 1999). Therefore, we checked whether our thioredoxin system is able to transfer electrons to these two substrates as positive controls in an NADPH-dependent manner. According to the results listed in Table 3, the thioredoxin system was able to oxidize NADPH indicating that electrons were transferred to both substrates. The activity of  39 thioredoxin system was calculated as nmol of NADPH oxidized per minute as detailed in Materials and Methods. Results listed in Table 3 show that the thioredoxin system oxidized 12 nmol of NADPH per minute when DTNB was used as the substrate. Addition of Tpx to the system, increased oxidized NADPH to 18 nmol per minute representing an approximately 40 % increase in the rate of NADPH oxidation. As expected, TrxR alone slightly oxidized NADPH (Holmgren, 1979, Zhang et al., 1999), while other individual enzymes did not show any activity. The Cys40Ser TrxC was not able to transfer electrons in the redox cascade of thioredoxin system to the final DTNB acceptor indicating that the flow of electrons was blocked. Similarly, the thioredoxin system was able to oxidize 3.9 nmol of NADPH per minute when H2O2 was used as a substrate. An increase of 40% in the NADPH oxidation was measured when Tpx was added to the system as 5.3 nmol of NADPH per minute was oxidized. No activities were measured when either individual enzymes or combination of TrxR and Tpx were tested. The mutated TrxC blocked the transfer of electrons in the redox cascade to the terminal acceptor H2O2.                  40 Table 3. NADPH oxidation by thioredoxin system towards H2O2 and DTNB.  Substrate* Protein     DTNB          H2O2 TrxR 1.8±0.3 N.D. TrxC N.D. N.D. Tpx N.D. N.D. TrxR + TrxC 12.2±0.6 3.9±0.15 TrxR + Cys40Ser TrxC  1.9±0.2 N.D. TrxR + Tpx 2.3±0.06 N.D. TrxC + Tpx N.D. N.D. TrxR + TrxC + Tpx 18.2±0.8 5.3±0.11 TrxR+ Cys40Ser TrxC + Tpx 2.5±0.07 N.D.  *Values represent nmoles of NADPH oxidized/min.  Shown is the mean (± S.D.) of three independent experiments. N.D.=No detected.  Once the performance of the thioredoxin system was confirmed, we proceeded to check whether the candidate substrates GSSG and GSNO might serve as substrates for the M. tuberculosis thioredoxin system. Results listed in Table 4 showed oxidation of NADPH by the systems towards both substrates. When GSSG was used as substrate, the system oxidized 4.3 nmol of NADPH per minute, whereas GSNO resulted in 3.6 nmol of NADPH oxidation. Upon addition of Tpx to the thioredoxin system, a 50% reduction in the NADPH oxidation was measured in both substrates; since 2.9 and 1.9 nmol of NADPH was oxidized towards GSSG and GSNO respectively. No NADPH oxidation was detectable when individual enzymes were tested, and as expected the Cys40Ser TrxC inhibited the NADPH oxidation (data not shown). In addition, since MSH is the most prevalent LMW thiol within the mycobacterial cell, we tested whether the thioredoxin system is able to reduce MSSM, which is the disulfide form of MSH. Results showed that 1.5 nmoles of NADPH per min was oxidized by the thioredoxin system. Adding Tpx to the reaction reduced the activity by 30%.  41 Results obtained from NADPH oxidation towards the candidate substrates GSSG, GSNO and MSSM suggest that these three substrates serve as substrates for M. tuberculosis thioredoxin system comprised of TrxR and TrxC.  Table 4. Reductase activity of thioredoxin system towards candidate substrates.  Substrate* Protein     GSSG          GSNO         MSSM TrxR N.D. N.D. N.D. TrxC N.D. N.D. N.D. Tpx N.D. N.D. N.D. TrxR + TrxC 4.17±0.25 3.61±0.09 1.5±0.17 TrxR + Cys40Ser TrxC  N.D. N.D. N.D. TrxR + Tpx N.D. N.D. N.D. TrxC + Tpx N.D. N.D. N.D. TrxR + TrxC + Tpx 2.1±0.2 1.9±1.5 0.46±0.036 TrxR+ Cys40Ser TrxC + Tpx N.D. N.D. N.D.  * Results are expressed as nmol of NADPH oxidized/min. Enzymes were added to each reaction at a final concentration of 1µM. Shown is the mean (± S.D.) of three independent experiments. N.D.= No detected.  4.3.2 ANALYSES OF PRODUCT FORMATION BY HPLC In the previous part, results obtained from NADPH oxidation assay only indicate the consumption of NADPH by the thioredoxin system towards candidate substrates, and does not directly monitor the formation of final products of the enzymatic reactions. Therefore, HPLC analysis was performed to directly measure formation of expected products. Reduction of GSSG and cleavage of GSNO by the thioredoxin system should yield GSH, which is subsequently tagged with mBBr forming GS-mB (Experimental Section). The HPLC chromatograms in Figure 13 and Figure 14 show a time-dependent formation of GS-  42 mB by the thioredoxin system utilizing GSNO and GSSG as substrates respectively. GS-mB standard elutes at 19± 0.5 min. Similarly, reduction of the disulfide bond in MSSM will yield MSH, which forms MS-mB upon labeling with mBBr. The chromatograms show a time-dependent formation of MS-mB, which elutes at 21± 0.5 min (Figure 15). The areas of the peak corresponding to each product were recorded and transformed to amount of products produced per minute and according to a calibration curve of GS-mB (Figure 9) and available Ms-mB calibration curve in HPLC Breeze software (Waters, USA).                 43                     Figure 13. HPLC profile of GS-mB generated by the thioredoxin system from GSNO. The formation of GS-mB (marked with an arrow) was monitored by HPLC. Shown are the reactions in presence of 26 µM GSNO. GS-mB: bimane derivative of glutathione, NEM: a specific thiol-blocking compound and Cys40Ser TrxC mutant were used as control.   m V 0 . 0 0 5 0 0 . 0 0 1 0 0 0 . 0 0 M in u t e s 0 . 0 0 2 . 0 0 4 . 0 0 6 . 0 0 8 . 0 0 1 0 . 0 0 1 2 . 0 0 1 4 . 0 0 1 6 . 0 0 1 8 . 0 0 2 0 . 0 0 2 2 . 0 0 2 4 . 0 0 2 6 . 0 0 2 8 . 0 0 3 0 . 0 0 3 2 . 0 0 3 4 . 0 0 3 6 . 0 0 GS-mB standard m V 0 . 0 0 5 0 0 . 00 1 0 0 0 . 0 0 1 5 0 0 . 0 0 2 0 0 0 . 0 0 M in u t e s 0 . 0 0 2 . 0 0 4 . 0 0 6 . 0 0 8 . 0 0 1 0 . 0 0 1 2 . 0 0 1 4 . 0 0 1 6 . 0 0 1 8 . 0 0 2 0 . 0 0 2 2 . 0 0 2 4 . 0 0 2 6 . 0 0 2 8 . 0 0 3 0 . 0 0 3 2 . 0 0 3 4 .0 0 3 6 .0 0 0’ m V 0 . 0 0 5 0 0 . 0 0 1 0 0 0 . 0 0 1 5 0 0 . 0 0 2 0 0 0 . 0 0 M in u t e s 0 . 0 0 2 . 0 0 4 . 0 0 6 . 0 0 8 . 0 0 1 0 . 0 0 1 2 . 0 0 1 4 . 0 0 1 6 . 0 0 1 8 . 0 0 2 0 . 0 0 2 2 . 0 0 2 4 . 0 0 2 6 . 0 0 2 8 . 0 0 3 0 . 0 0 3 2 . 0 0 3 4 . 0 0 3 6 . 0 0 10’ m V 0 . 0 0 5 0 0 . 0 0 1 0 0 0 . 0 0 1 5 0 0 . 0 0 2 0 0 0 . 0 0 M in u t e s 0 . 0 0 2 . 0 0 4 . 0 0 6 . 0 0 8 . 0 0 1 0 . 0 0 1 2 . 0 0 1 4 . 0 0 1 6 . 0 0 1 8 . 0 0 2 0 . 0 0 2 2 . 0 0 2 4 . 0 0 2 6 . 0 0 2 8 . 0 0 3 0 . 0 0 3 2 . 0 0 3 4 . 0 0 3 6 . 0 0 60’ m V 0 . 0 0 5 0 0 . 0 0 1 0 0 0 . 0 0 1 5 0 0 . 0 0 2 0 0 0 . 0 0 M in u t e s 0 . 0 0 2 . 0 0 4 . 0 0 6 . 0 0 8 . 0 0 1 0 . 0 0 1 2 . 0 0 1 4 . 0 0 1 6 . 0 0 1 8 . 0 0 2 0 . 0 0 2 2 . 0 0 2 4 . 0 0 2 6 . 0 0 2 8 . 0 0 3 0 . 0 0 3 2 . 0 0 3 4 . 0 0 3 6 .0 0 NEM TrxC Cys40Ser 0 . 0 0 2 . 0 0 4 . 0 0 6 . 0 0 8 . 0 0 1 0 . 0 0 1 2 . 0 0 1 4 . 0 0 1 6 . 0 0 1 8 . 0 0 2 0 . 0 0 2 2 . 0 0 2 4 . 0 0 2 6 . 0 0 2 8 . 0 0 3 0 . 0 0 3 2 . 0 0 3 4 . 0 0 3 6 .0 0 m V 0 . 0 0 5 0 0 . 0 0 1 0 0 0 . 0 0 1 5 0 0 . 0 0 2 0 0 0 . 00  44                          Figure 14. HPLC profile of GS-mB generated by the thioredoxin system from GSSG. The formation of GS-mB (marked with an arrow) was monitored by HPLC. Shown are the reactions in presence of 26 µM GSSG. GS-mB: bimane derivative of glutathione, NEM: a specific thiol-blocking compound and Cys40Ser TrxC mutant were used as control. m V 0 .0 0 5 0 0 .0 0 1 0 0 0 .0 0 M in u te s 0 .0 0 2 .0 0 4 .0 0 6 .0 0 8 .0 0 1 0 .0 0 1 2 .0 0 1 4 .0 0 1 6 .0 0 1 8 .0 0 2 0 .0 0 2 2 .0 0 2 4 .0 0 2 6 .0 0 2 8 .0 0 3 0 .0 0 3 2 .0 0 3 4 .0 0 3 6 .0 0 GS-mB standard m V 0 .0 0 5 0 0 .0 0 1 0 0 0 .0 0 1 5 0 0 .0 0 2 0 0 0 .0 0 M in u te s 0 .0 0 2 .0 0 4 .0 0 6 .0 0 8 .0 0 1 0 .0 0 1 2 .0 0 1 4 .0 0 1 6 .0 0 1 8 .0 0 2 0 .0 0 2 2 .0 0 2 4 .0 0 2 6 .0 0 2 8 .0 0 3 0 .0 0 3 2 .0 0 3 4 .0 0 3 6 .0 0 0’ m V 0 . 0 0 5 0 0 . 0 0 1 0 0 0 . 0 0 1 5 0 0 . 0 0 2 0 0 0 . 0 0 M in u t e s 0 . 0 0 2 . 0 0 4 . 0 0 6 . 0 0 8 . 0 0 1 0 . 0 0 1 2 . 0 0 1 4 . 0 0 1 6 . 0 0 1 8 . 0 0 2 0 . 0 0 2 2 . 0 0 2 4 . 0 0 2 6 . 0 0 2 8 . 0 0 3 0 . 0 0 3 2 . 0 0 3 4 . 0 0 3 6 .0 0 10’ m V 0 . 0 0 5 0 0 . 0 0 1 0 0 0 . 0 0 1 5 0 0 . 0 0 2 0 0 0 . 0 0 M in u t e s 0 . 0 0 2 . 0 0 4 . 0 0 6 . 0 0 8 . 0 0 1 0 . 0 0 1 2 . 0 0 1 4 . 0 0 1 6 . 0 0 1 8 . 0 0 2 0 . 0 0 2 2 . 0 0 2 4 . 0 0 2 6 . 0 0 2 8 . 0 0 3 0 . 0 0 3 2 . 0 0 3 4 . 0 0 3 6 . 0 0 60’ m V 0 . 0 0 5 0 0 . 0 0 1 0 0 0 . 0 0 1 5 0 0 . 0 0 2 0 0 0 . 0 0 M in u t e s 0 . 0 0 2 . 0 0 4 . 0 0 6 . 0 0 8 . 0 0 1 0 . 0 0 1 2 . 0 0 1 4 . 0 0 1 6 . 0 0 1 8 . 0 0 2 0 . 0 0 2 2 . 0 0 2 4 . 0 0 2 6 . 0 0 2 8 . 0 0 3 0 . 0 0 3 2 . 0 0 3 4 . 0 0 3 6 . 0 0 NEM m V 0 .0 0 5 0 0 .0 0 1 0 0 0 .0 0 1 5 0 0 .0 0 2 0 0 0 .0 0 M in u te s 0 .0 0 2 .0 0 4 .0 0 6 .0 0 8 .0 0 1 0 .0 0 1 2 .0 0 1 4 .0 0 1 6 .0 0 1 8 .0 0 2 0 .0 0 2 2 .0 0 2 4 .0 0 2 6 .0 0 2 8 .0 0 3 0 .0 0 3 2 .0 0 3 4 .0 0 3 6 .0 0 TrxC Cys40Ser  45                         Figure 15. HPLC profile of MS-mB generated by the thioredoxin system from MSSM. The formation of MS-mB (marked with an arrow) was monitored by HPLC. Shown are the reactions in presence of 26 µM MSSM. MS-mB: bimane derivative of mycothiol, NEM: a specific thiol-blocking compound and Cys40Ser TrxC mutant were used as control. m V 0 . 0 0 2 0 0 . 0 0 4 0 0 . 0 0 6 0 0 . 0 0 8 0 0 . 0 0 1 0 0 0 . 0 0 M in u t e s 0 . 0 0 2 . 0 0 4 . 0 0 6 . 0 0 8 . 0 0 1 0 . 0 0 1 2 . 0 0 1 4 . 0 0 1 6 . 0 0 1 8 . 0 0 2 0 . 0 0 2 2 . 0 0 2 4 . 0 0 2 6 . 0 0 2 8 . 0 0 3 0 . 0 0 3 2 . 0 0 3 4 . 0 0 3 6 .0 0 MS-mB standard m V 0 . 0 0 5 0 0 . 0 0 1 0 0 0 . 0 0 1 5 0 0 . 0 0 2 0 0 0 . 0 0 M in u t e s 0 . 0 0 2 . 0 0 4 . 0 0 6 . 0 0 8 . 0 0 1 0 . 0 0 1 2 . 0 0 1 4 . 0 0 1 6 . 0 0 1 8 . 0 0 2 0 . 0 0 2 2 . 0 0 2 4 . 0 0 2 6 . 0 0 2 8 . 0 0 3 0 . 0 0 3 2 . 0 0 3 4 . 0 0 3 6 .0 0 . 0’ m V 0 . 0 0 5 0 0 . 0 0 1 0 0 0 . 0 0 1 5 0 0 . 0 0 2 0 0 0 . 0 0 M in u t e s 0 . 0 0 2 . 0 0 4 . 0 0 6 . 0 0 8 . 0 0 1 0 . 0 0 1 2 . 0 0 1 4 . 0 0 1 6 . 0 0 1 8 . 0 0 2 0 . 0 0 2 2 . 0 0 2 4 . 0 0 2 6 . 0 0 2 8 . 0 0 3 0 . 0 0 3 2 . 0 0 3 4 . 0 0 3 6 . 0 0 10’ M in u t e s 0 . 0 0 2 . 0 0 4 . 0 0 6 . 0 0 8 . 0 0 1 0 . 0 0 1 2 . 0 0 1 4 . 0 0 1 6 . 0 0 1 8 . 0 0 2 0 . 0 0 2 2 . 0 0 2 4 . 0 0 2 6 . 0 0 2 8 . 0 0 3 0 . 0 0 3 2 . 0 0 3 4 . 0 0 3 6 . 0 0 m V 0 . 0 0 5 0 0 . 0 0 1 0 0 0 . 0 0 1 5 0 0 . 0 0 2 0 0 0 . 0 0 60’ m V 0 . 0 0 5 0 0 . 0 0 1 0 0 0 . 0 0 1 5 0 0 . 0 0 2 0 0 0 . 0 0 M in u t e s 0 . 0 0 2 . 0 0 4 . 0 0 6 . 0 0 8 . 0 0 1 0 . 0 0 1 2 . 0 0 1 4 . 0 0 1 6 . 0 0 1 8 . 0 0 2 0 . 0 0 2 2 . 0 0 2 4 . 0 0 2 6 . 0 0 2 8 . 0 0 3 0 . 0 0 3 2 . 0 0 3 4 . 0 0 3 6 . 0 0 NEM M in u t e s 0 . 0 0 2 . 0 0 4 . 0 0 6 . 0 0 8 . 0 0 1 0 . 0 0 1 2 . 0 0 1 4 . 0 0 1 6 . 0 0 1 8 . 0 0 2 0 . 0 0 2 2 . 0 0 2 4 . 0 0 2 6 . 0 0 2 8 . 0 0 3 0 . 0 0 3 2 . 0 0 3 4 . 0 0 3 6 . 0 0 m V 0 . 0 0 5 0 0 . 0 0 1 0 0 0 . 0 0 1 5 0 0 . 0 0 2 0 0 0 . 0 0 TrxC Cys40Ser  46 The thioredoxin system produced 5.33 and 4 nmol of GSH ml-1min-1 when GSSG and GSNO were used as substrates respectively (Figure 16).  This represents a 25% decrease in product formation when GSNO was used as substrate. However, when Tpx was added to the system, 4.4 and 2.7 nmol of GSH ml-1min-1 was produced for GSSG and GSNO respectively, representing a 20-30% decrease in product formation (Figure 16). The thioredoxin system generated 2.6 nmol MSH ml-1min-1 towards MSSM, representing a decrease of 50% when compared to GSSG. Addition of Tpx to the system decreased the amount of product to 1.9 nmol ml-1min-1 (Figure 16). In all cases, when Cys40Ser replaced the parental TrxC, neither GSH nor MSH formation was detected (Figure 13, 14, and 15), indicating that the electron transfer from NADPH toward the respective substrates was blocked. No statistical differences were observed when Tpx was supplemented to the reactions in presence of GSSG, GSNO or MSSM according to t-test analysis.                 47              Figure 16. Rate of thiol production by the thioredoxin system quantified by HPLC. Production of thiols was analyzed by HPLC using thioredoxin system in presence or absence of Tpx. Reactions were incubated at 37oC for 10 min in presence of 26 µM of each substrate. Reactions were stopped, acidified, labeled with mBBr, and separated in HPLC. Shown is the mean value (± SD) of three independent experiments.  4.4 ENZYME KINETIC STUDIES This part of the biochemical analysis would reveal the affinity (KM) and maximum enzyme velocity (VMax) of thioredoxin system towards GSSG, GSNO and MSSM. Furthermore, these kinetic parameters will be used to calculate additional kinetic parameters such as the turnover number and the efficiency of the thioredoxin system. The activity of the thioredoxin system towards each substrate was measured by using three different concentrations of each substrate (26, 40, and 57 µM) at a given time. The inverse of enzyme velocity versus inverse of substrate concentration (Lineweaver-Burk plot) was plotted to obtain KM and VMax values of the thioredoxin system (Figure 17). The VMax values were determined by taking the reciprocal of the X-intercept. Kinetic results listed in   48 Table 5 show that GSNO is the substrate with the highest affinity for the thioredoxin system as reflected by a KM of 3.7 µM. GSSG and GSNO showed a similar turnover number with 352 and 298 molecules of substrate converted to GSH min-1, while approximately 60 molecules of MSSM were converted to MSH min-1. When Tpx was added to the system, GSSG and GSNO showed 168 and 245 molecules of substrate converted to GSH min-1, while 46 molecules of MSSM were converted to MSH min-1.                   49                       Figure 17. Kinetic analysis of the thioredoxin system by Lineweaver-Burk plots. Lineweaver-Burk plots used to calculate the kinetic parameters for thioredoxin reductase assays in absence or presence of Tpx. (A) GSSG – Tpx, (B) GSSG + Tpx, (C) GSNO – Tpx, (D) GSNO + Tpx, (E) MSSM – Tpx, (F) MSSM + Tpx.  0 1 2 3 4 5 6 7 8 9 10 3.5 4.5 5.5 6.5 7.5 8.5 9.5 10.5 11.5 12.5 1/[GS-mB] [1/µM]  A 0 1 2 3 4 5 6 7 3.5 4.5 5.5 6.5 7.5 8.5 1/[GS-mB] [1/µM]  B 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 1 2 3 4 5 6 7 8 9 1/[GS-mB] [1/µM]  C 0 1 2 3 4 5 6 3 4 5 6 7 8 9 10 11 12 13 1/[GS-mB] [1/µM]  D E F 0.3 0.4 0.5 0.6 0.7 0.8 0.010 0.011 0.012 0.013 0.014 0.015 1/[MS-mB] [1/mM] 0.5 0.6 0.7 0.8 0.9 0.015 0.016 0.017 0.018 0.019 1/[MS-mB] [1/mM]  50  Table 5. Steady-state kinetic parameters of substrate conversion.  Substrate KM [µM] Kcat [min-1] Kcat / KM [min-1 µM-1] GSSG           TrxR+TrxC 8.8±2.7 352 40           TrxR+TrxC+Tpx 1.5±0.9 168 112 GSNO           TrxR+TrxC 3.7±0.9 298 80           TrxR+TrxC+Tpx 1.6±0.13 245 153 MSSM           TrxR+TrxC 0.8±0.1 60 75           TrxR+TrxC+Tpx  0.4±0.18 46 115                    51 4.5 THIOREDOXIN-ASSOCIATED M. TUBERCULOSIS PROTEOME   To identify proteins that interact with TrxC in the M. tuberculosis proteome, we used a substrate-trapping assay utilizing the catalytic-defective mutant Cys40Ser to trap substrate complexes. It has been shown that mutants lacking the C’-terminal cysteine of the CXXC active site motif of thioredoxins form long-lived mixed disulfide intermediates with target proteins, thus targeted proteins remain covalently linked to the mutant and become amenable to isolation and analysis (Motohashi et al., 2001). To test whether TrxCys40Ser-based trapping is capable of identifying TrxC target proteins, the recombinant mutant was exposed to a M. tuberculosis lysate and then purified by affinity chromatography. Eluted proteins were resolved on SDS-PAGE, visualized by silver staining, and detected protein bands were excised and subjected to MS analysis. According to results observed in Figure 18-lane 3, a total of seven bands were detected corresponding to the mutant Cys40Ser. Proteomic analysis of these bands (Table 6 and 7) revealed that almost all of the identified proteins contained the conserved cysteines. Two of these proteins were TrxR and Tpx, which are known proteins interacting with TrxC (Jaeger et al., 2004). In addition, we identified nine thioredoxin targets, which have not been reported yet to interact with TrxC of M. tuberculosis. These proteins included the serine/threonine kinase G involved in signal transduction (Cowley et al., 2004) and other proteins involved in a variety of metabolic pathways such as purine metabolism, citric acid cycle, carbohydrate degradation, cobalamin, ATP, and cell wall syntheses.     52                             Figure 18. Substrate trapping assay. Elution fractions corresponding to purification by affinity chromatography using Ni-NTA resin were resolved on 12% SDS-PAGE and silver stained. Lanes: 1, protein markers; 2, recombinant wt TrxC; 3, Cys40Ser TrxC mutant exposed to M. tuberculosis lysate; 4, M. tuberculosis lysate alone; 5, recombinant wt TrxC exposed to M. tuberculosis lysate.          116-     66-    45-     35-     25-       18-   14-      1          2                3              4           5 1 2 3 4 5 7 6  53 Table 6. Identification of thioredoxin targets in M. tuberculosis lysates using substrate- trapping procedures.  Spot # Accession # Description MW Coverage % 1 P65728 Protein kinase G (PknG) 81578 2.8  2 P65829 Amidophosphoribosyl transferase precursor 56162 5.7  Q10809 Probable malate-quinone oxidoreductase 53596 3.0  3 P71818 Alcohol dehydrogenase B 39748 8.9  P64691 Rv0106 43700 4  Q10692 Rv2084 41314 6  4     P71801 Glycolipid sulfotransferase 37006 8.6  5 P64178 Glyceraldehyde 3-Phosphate dehydrogenase 35956 10.0  P52214 Thioredoxin reductase 35643 3.9  6 P66952 Thiol peroxidase 16896 9.5  7 P63662 Probable ATP synthase epsilon chain 13134 3.3                       54 Table 7. Determination of predicted peptides according to masses obtained from MS analysis.  Spot # Protein Mass Peptide sequence predicted MOWSE Score* Dithiol- containing motif 1 PknG 702.4246 QVRGSR 1.33 FCWNCG110   1666.7978 GYTILTPVQTGVVYR  2 PurF 1480.8089 MLREAGAVELHVR 4.6   1639.9851 GAALGELAADDEVPVGR   Mqo 668.3202 CFANR 3.62 TCHACR376   978.6067 GFAGFPIGGR  3 AdhB 729.5508 VIITVGK 4.33 ACLVGC169   883.3494 LHVDDER   1070.5195 GDEPIVDGIR   1084.2817 CPSCQAGMR   Rv0106 642.3700 APAIDR 3.64 TCVDCV138   1086.4897 EGQDVVGNLR    Rv2084 524.2920 SVYR 3.9 CNSVCR155   1060.4510 AVTDGDVDLR  4 Rv1373 865.3563 SPTEEFR 6.96   2584.3997 GGSGDWQQFFTEAEHLRYYHR  5 Gap 574.2574 AAAEGR 12.6 CTTNCL163   2813.6069 EGPAALPWGDLGVDVVVESTG LFTNAAK    TrxB2 678.2972 FGADLR 4.74 SCATCD149   811.4835 SVTLVHR  6 Tpx 1639.8202 FCGAEGTENVMPASAFR 3.53  7 AtpC 429.5231 IAAR 1.58   * MOWSE (Molecular Weight SErch) is a method for identification of proteins from the molecular weight of peptides created by proteolytic digestion and analyzed by mass spectrometry. This score comprised the calculated peptide masses for each entry in the sequence database with the set of experimental data (Pappin et al., 1993). MOWSE uses empirically determined factors to assign a statistical weight to each individual peptide match.   55 CHAPTER 5: DISCUSSION The experiments conducted in this thesis were designed to address the hypothesis that the M. tuberculosis thioredoxin system can reduce the antimycobacterial compounds glutathione disulfide (GSSG) and its nitroso derivative S-nitrosoglutathione (GSNO). This hypothesis was propounded based on certain facts about biochemical characteristics and functions of thioredoxins. It is known that member proteins of the ubiquitous thioredoxin family are active disulfide reductases, and they maintain cellular redox homeostasis through generalized thiol/disulfide exchange reactions. In this work we tested the capacity of M. tuberculosis thioredoxin system composed of thioredoxin reductase B2 (TrxR) and TrxC to reduce the LMW thiols GSSG and GSNO produced by the host macrophage. Recombinant TrxR and TrxC were used as the thioredoxin system. This system is one of the most studied and well characterized in M. tuberculosis. It has been shown that this system is able to detoxify H2O2 and reduce DTNB (Zhang et al., 1999). The ahpC mediated detoxification pathway in M. tuberculosis is also mainly dominated by this thioredoxin system (Jaeger et al., 2004). Therefore we checked the activity of the system in M. tuberculosis towards our candidate substrates in absence or presence of thiol peroxidase Tpx. It has been reported that Tpx acts as an enhancer of the redox activity of the system, and that the TrxR, TrxC and Tpx constitute the most efficient system to protect M. tuberculosis against oxidative and nitrosative stress in situ (Kang et al., 1998, Nordberg & Arner, 2001).     56 We demonstrated by an NADPH oxidation assay that this system was able to transfer electrons to the final substrates. To confirm that as a result of the catalytic activity of the system the corresponding products were formed, we measured formation of final product GSH by HPLC analysis. Results showed that both systems were able to transform GSNO and GSSG to the final product GSH (Figure 13 and 14). When comparing the performance of the system upon addition of Tpx, a higher level of NADPH oxidation and product formation was observed towards GSSG and GSNO in absence of Tpx. This is also consistent with the higher Kcat values of the thioredoxin system, which converts more molecules of substrate to product in absence of Tpx. We suggest that the reduction in the thioredoxin system activity upon addition of Tpx can be explained by its interaction with both TrxR and TrxC. In this regard, previous work has reported that Tpx- Cys60 forms disulfide bridges with TrxR-Cys31 and TrxC-Cys37 (Trujillo et al., 2006). Since the Tpx substrate H2O2 is absent in this reaction, we suggest that there is a transient binding of Tpx with TrxC, which eventually reduces the number of available catalytic sites of TrxC, and then, decreases the activity of the thioredoxin system. It has been shown that mycobacteria are sensitive to GSH and GSNO in physiological concentration. Since GSH is mycobacteriostatic and GSNO is mycobactericidal (Venketaraman et al., 2003) oxidized glutathione has been shown to be toxic and transported inside mycobacteria and in the reducing environment of cytoplasm is eventually reduced to GSH. Our results demonstrate that thioredoxin system of M. tuberculosis can efficiently reduce and detoxify this compound. When grown in culture GSNO is decomposed and the presence of a nitrosothiol reductase has been suggested. There have been attempts to elucidate the mechanism of  57 GSNO detoxification. An M. tuberculosis enzyme with nitrosothiol reductase activity is the mycothiol-dependent formaldehyde dehydrogenase, which reduces nitrosomycothiol (Vogt et al., 2003). However, this enzyme is unable to utilize GSNO as substrate. MSNO reductase was considered to have the capacity to decompose GSNO, but it was shown that this enzyme is unable to utilize GSNO. The nitrosation of cellular thiols has attracted a lot of interest as a regulatory mechanism, since virtually all enzymes contain cysteines residues that can be subjected to S- nitrosation. For instance in activated human neutrophils a burst of NO converts intracellular GSH to GSNO as well, but it is subsequently cleaved to restore GSH. Holmgren’s group has extensively studied denitrosylation of GSNO by human thioredoxins (Nikitovic & Holmgren, 1996). This group reported a homolytic breakdown of GSNO generating GSH and NO⋅. They discuss that the reaction between thioredoxin and GSNO is operated according to the reaction mentioned in the Figure 19. They propose that the first cysteine in the active site is exposed in the hydrophobic active site surface of the thioredoxin whereas the second one is more buried, and it can act as a potent nucleophile.          58           Figure 19. Proposed mechanism of direct reaction between TrxC and GSNO (adapted by Nikitovic & Holmgren, 1996).  The following reaction summarizes the formation of final products of homolytic cleavage of GSNO catalyzed by thioredoxin: Trx-(SH)2 + GSNO + O2  Trx-S2 + GSH + NO. + O2.- The Stoyanovsky laboratory reported that denitrosation of GSNO by human thioredoxins follows a heterolytic breakdown of GSNO producing GSH and HNO (Stoyanovsky et al., 2005). However, both Holmgren and Stoyanovsky laboratories suggest that other cellular nitrosylated proteins may serve as substrates for thioredoxin system.  In this work we present evidence that the M. tuberculosis thioredoxin system catalyzes the denitrosylation of GSNO, resulting in the formation of GSH. The consequence of this breakdown and the mechanism of inactivation of NO intermediates released from GSNO inside the pathogen remains to be elucidated. Therefore, production of NO from GSNO reduction seems paradoxical as RNI, and specifically NO, are antimicrobial agents   59 generated by macrophages upon infection (MacMicking et al., 1997, Nathan & Hibbs, 1991). However, bearing in mind the short half life of NO, and our previous studies showing that mycothiol efficiently protects mycobacteria from its bactericidal effect (Miller et al., 2007), NO is considered much less toxic compared to GSNO, which upon infection is transported into the bacilli (Green et al., 2000).  The mycobacterial thioredoxin system shows higher kinetic parameters (Kcat and Kcat/KM) values compared to other thioredoxin systems in terms of substrate specificity as exemplified by human thioredoxins or the protozoan parasite Plasmodium falciparum thioredoxin systems (Table 8). This observation suggests that the mycobacterial thioredoxin system is able to efficiently reduce host and mixed sulfides such as GSNO.  Table 8.  Comparison of kinetic parameters of different thioredoxin systems.  Substrate Organism Kcat [min-1] Kcat / KM [min-1 µM-1] Reference  GSSG      M. tuberculosis 352 40 This work            Plasmodium falciparum < 0.2 < 0.00001 (Kanzok et al., 2000)   GSNO       M. tuberculosis 298 80 This work            Human TrxC 36 0.6 (Nikitovic & Holmgren, 1996)  Plasmodium falciparum 9.4 0.3 (Kanzok et al., 2000)   MSSM M. tuberculosis Thioredoxin system 60 75 This work   M. tuberculosis Mycothiol reductase 64 55 (Patel & Blanchard, 1999)  Mycothiol is the major LMW thiol in Mycobacteria, and the equivalent of glutathione in other organisms. Under oxidative stress MSH becomes oxidized and forms the oxidized  60 MSSM disulfide. Mycothiol reductase (Mtr) reduces MSSM and maintains MSH levels (Rawat & Av-Gay, 2007). However, Mtr is not an essential gene in M. tuberculosis, suggesting that other electron donors can reduce MSSM. Indeed, we have shown that this thioredoxin system is able to reduce MSSM and may contribute or compensate for Mtr function under extreme oxidative stress. Our results showed that our systems are able to reduce MSSM to MSH, and this activity is comparable to Mtr. Several proteomic analyses of thioredoxins corresponding to different organisms have been published (Kumar et al., 2004, Wong et al., 2004), but a comprehensive thioredoxin analysis of M. tuberculosis has not been reported yet. In an attempt to identify pathways in which TrxC is involved, we carried out a proteomic analysis of M. tuberculosis lysate using the Cys40Ser mutant TrxC as a substrate trapping protein. Results showed that a total of seven bands were observed to bind specifically the mutated thioredoxin but not to the parental thioredoxin or the resin used for affinity purification (Figure 18). Protein identification analysis showed that most of these proteins present a CXXC conserved motif (Table 7). Identified proteins were grouped according to the following pathways based on the functions obtained by in silico analysis: (i) Detoxification/oxidative stress response. The proteins TrxR and Tpx, known substrates of TrxC, were identified in this experiment and this finding is in agreement with previous reports (Jaeger et al., 2004, Zhang et al., 1999). The results obtained throughout our work indicate that an interaction between these proteins exists in order to transfer electrons to the final acceptor as determined by a direct product formation measured by HPLC analysis. (ii) Signal transduction. The identified serine/threonine protein kinase G mediates glutamine/glutamate metabolism in M. tuberculosis (Cowley et al., 2004).  61 Interestingly, this protein shows a double conserved CXXC motif suggesting that its activity, in part, is regulated by an oxidoreductase activity. Other thioredoxins have been implicated in regulating protein kinases. For instance, thioredoxin regulates the activation of the stress- induced mitogen-activated protein kinase-signaling cascade by binding directly to the apoptosis signal-regulating kinase in human (Shao et al., 2002). Moreover, thioredoxins have been reported to inhibit the S receptor kinase in Brassica napus (Haffani et al., 2004). (iii) Cell wall component synthesis. The identified glycolipid sulfotransferase has been reported to intervene in the synthesis of cell wall sulpholipids (Rivera-Marrero et al., 2002), which have been implicated in M. tuberculosis virulence (Goren et al., 1974, Rivera- Marrero et al., 2002). Although no evidence of interaction with M. tuberculosis thioredoxin has been reported (Rivera-Marrero et al., 2002), other sulfotransferases depend exclusively on the activity of thioredoxins. For instance, the adenosine 3’-phosphate 5’-phosphosulfate sulfotranferase from E. coli utilizes thioredoxin as a cofactor to perform its catalytic activity (Tsang & Schiff, 1978). (iv) Intermediary metabolism. Five proteins involved in purine and alcohol metabolism as well as in the citric cycle, glycolysis, and ATP biosynthesis were identified. Regarding to purine metabolism, it has been reported that a knock out strain of the amidophosphoribosyl transferase homolog in M. smegmatis impaired the survival of the mutant under oxygen-starved conditions (Keer et al., 2001). Although no data has been reported regarding to the interaction of alcohol dehydrogenase and TrxC, it has been shown that thioredoxins interacted with alcohol dehydrogenases in yeast by using a two-hybrid analysis (Brodegger et al., 2004). Two proteins related to intermediate metabolite formation in glycolysis and the citric acid cycle were identified. Glyceraldehyde 3-phosphate  62 dehydrogenase is an enzyme involved in glycolysis and catalyzes the formation of 3-phospho glyceroyl phosphate, while malate quinone oxidoreductase participates in the conversion of malate to oxaloacetate. Both enzymes function in a NAD-dependent manner, suggesting that an oxidoreductase activity is necessary to regulate their enzymatic activity. Finally, the ATP synthase epsilon has been implicated in ATP synthesis in chloroplasts and is directly modulated by thioredoxin (He et al., 2000). No reports have been published yet in M. tuberculosis. (v) Unknown function. Two proteins without an annotated function were complexed to thioredoxin. However, Rv0106 shares 79% homology to CobW in M. avium, which is involved in the synthesis of cobalamin. No defined signatures/domains were found for Rv2084.  In conclusion, in this study we show that MSSM, GSNO and GSSG are substrates of the thioredoxin system in M. tuberculosis. Our in vitro results suggest that this system may potentially be involved in the detoxification of mycobactericidal compounds such as GSSG and GSNO, which are produced by macrophages to limit mycobacterial growth within the infected host. In addition, nine new interacting proteins were identified by proteomic analysis, suggesting that thioredoxins plays an essential role in a diversity of M. tuberculosis pathways.         63 CHAPTER 6: FUTURE WORK The detoxification of GSNO by thioredoxin system of M. tuberculosis raises the question that how the breakdown products GSH and NO are further processed in the pathogen. It has been proposed that the cleavage of GSNO results in formation and release of two alternative products -NO and -HNO in either a homolytic or heterolytic cleavage (Nikitovic & Holmgren, 1996, Stoyanovsky et al., 2005) (Figure 20). These intermediates are highly reactive and render a nitrosylated cysteine in the active site of thioredoxins (Stoyanovsky et al., 2005).          Figure 20. Proposed mechanisms of nitrosylated cysteine breakdown. Nitrosylated cysteines release NO from sulfur by homolytic or heterolytic cleavage.  As a result of this cleavage, cysteines are nitrosylated and the direct cellular effects of this nitrosylation are not yet fully understood. Some targets of NO-mediated thiol modifications include enzymes such as catalases, kinases, and transcription factors (Lin et al., 2007, Sun et al., 2006).   64  In addition, there are other reactive NO-derived RNIs, including peroxynitrite, nitroxyl anion and nitrogen dioxide that contribute to damaging the cells (Stamler et al., 1992). These intermediates are toxic within the pathogen and damage critical cell processes, such as protein synthesis and DNA replication (Bundy et al., 2000, Kim et al., 1998). Moreover, it has been demonstrated that NO mediates nitration of tyrosines (Beckman, 1996), reversible binding to metal centers such as haem–iron [4Fe-4S] (Arnold et al., 1977, Cruz-Ramos et al., 2002, Nunoshiba et al., 1993), and direct formation of S-nitrosothiols in proteins (Padgett & Whorton, 1995). Therefore, it is necessary to identify nitrosylated proteins formed as the result of RNI release from GSNO breakdown. This will contribute substantially to understand the mechanisms of defense of M. tuberculosis against these compounds and eventually will conclude in a better knowledge of how this pathogen survives in macrophages under nitrosative stress.  Detection of S-nitrosylated proteins is difficult due to the lability of S-NO bond. However, the biotin switch and chemiluminiscence techniques (Jaffrey et al., 2001, Mannick & Schonhoff, 2008) can measure protein nitrosylation and then identify them directly. Depending on the individual proteins, thiol modifications lead to activation/inactivation of the respective protein function. M. tuberculosis is able to sense and response accordingly in presence of RNIs (Lin et al., 2007, Papavinasasundaram et al., 2005). This is suggestive of an organized network to cope with these toxic agents. This response is pivotal to the survival of the pathogen in human macrophages to cope with the effects of NO. Not only identification of nitrosylated proteins generated as the result of GSNO cleavage will contribute to understanding the mechanism of defense against these  65 compounds, but these markers can also serve as drug targets to design new anti-TB drugs. For instance, as mentioned earlier, the protein serine/threonine kinase H (PknH) has been shown to undergo autophosphorylation under nitrosative stress, suggesting that a network is mounted in the pathogen to resist the NO effects. We anticipate that other kinases or receptors can be involved as well in sensing the presence of NO or RNIs.  In other words, finding new inhibitors to target nitrosylated proteins involved in detoxification of RNIs, or new inhibitors targeting proteins in signaling will increase the vulnerability of M. tuberculosis in macrophages/granulomas. Therefore, in a first step, novel chemical inhibitors will be selected from available libraries, and in a second step, since our laboratory possess extensive experience knocking out genes in M. tuberculosis, the efficacy of these compounds can be compared to KO strains. It appears that a selective inhibition of the TrxC in M. tuberculosis is difficult. This assumption is based on the conserved structures of thioredoxins, which are similar in all organisms according to the overall folding patterns. This similarity makes it difficult to design drugs to distinguish between host endogenous and pathogenic thioredoxins. In addition, an inhibition of TrxC renders the probability that other thioredoxins in M. tuberculosis can compensate its activity (Akif et al., 2008). However, that is not an issue when considering TrxR as a drug target because a compensating substitute is not envisaged in M. tuberculosis. Therefore, TrxR can be targeted by inhibition, impairing the viability and virulence of the pathogen, with a minimum risk of resistance development. According to published data (Akif et al., 2005) the substantial differences in sequences, mechanisms and structures between human TrxRs and the mycobacterial TrxR render a selective inhibition possible.  66 Consequently, identification of nitrosylated proteins as the result of NO release from GSNO breakdown will enable us to have a broader knowledge of M. tuberculosis defense mechanisms under nitrosative stress. We hope that by therapeutic interference with one of the most upstream factors in the mycobacterial antioxidant defense system, the entire redox cascade will be targeted, increasing the vulnerability of the pathogen to NO derivatives upon infection.  Preliminary proteomic analysis carried out in this work revealed the involvement of thioredoxin in different pathways. Although this participation is expected, no reports have been published yet in Mycobacteria. Therefore, a more extensive proteomic analysis will provide more details of the multiple tasks performed by this protein in M. tuberculosis. More detailed information about the multiple pathways can be obtained by performing a 2D-PAGE separation of the eluted fractions, which will provide a high-resolution gel with proteins isolated as single spots. In addition, an in vitro assay is required to validate the thioredoxin- substrate interactions. 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