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Thiol-dependent mycobacterial responses to oxidative and nitrosative stress Ung, Korine (Sim Ee) 2006

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T H I O L - D E P E N D E N T M Y C O B A C T E R I A L R E S P O N S E S T O O X I D A T I V E A N D N I T R O S A T I V E S T R E S S by K O R I N E (SIM EE) U N G B.Sc. Honors, Simon Fraser University, 2003 A THESIS S U B M I T T E D I N P A R T I A L F U L F I L L M E N T O F T H E R E Q U I R E M E N T S F O R T H E D E G R E E O F M A S T E R OF S C I E N C E in T H E F A C U L T Y OF G R A D U A T E S T U D I E S (Experimental Medicine) T H E U N I V E R S I T Y OF B R I T I S H C O L U M B I A January 2006 © Korine (Sim Ee) Ung, 2006 A B S T R A C T Mycothiol (MSH) , produced in actinomycetes including mycobacteria, is functionally analogous to glutathione (GSH) in other organisms, replacing G S H as the main systemic protectant against oxidative stress. In this work, we investigated two possible control points in the regulation of M S H in response to oxidative stress: 1) transcriptional upregulation of M S H biosynthesis mediated by Rv0485 & Rv0818 (putative transcriptional regulators located directly upstream of some M S H biosynthesis genes); and 2) maintenance of the M S H : M S = S M redox balance upon oxidative stress. To monitor the changes in redox state and total M S H levels in Mycobacterium smegmatis mc 2155 a n d M bovis B C G cultures upon exposure to diamide (a thiol-specific oxidative agent), H2O2, or gaseous nitric oxide, we performed mycothiol assays and developed a novel, modified mycothiol assay to detect M S H oxidized as M S = S M . We found that diamide and H 2 02- induced oxidative stress in M. bovis B C G induces partial depletion of M S H to the oxidized form M S = S M , while treatment with gNO does not. M. smegmatis, an environmental saprophyte, displays a greater tolerance to these oxidative stresses than M. bovis B C G , as reflected by the lesser magnitudes in changes in redox state and total M S H levels upon treatment. We also investigated gene expression of Rv0485 and Rv0818 in M. bovis B C G upon exposure to diamide and upon infection of J774A.1 murine macrophages, using quantitative real-time reverse-transcriptase P C R . We found that although expressions of Rv0485 and Rv0818 were unchanged in diamide-treated bacteria, they were increased about 8-fold in bacteria harvested 6 and 18 hours after macrophage infections, h i addition, we conducted protein-protein binding assays to investigate i f Rv0818 protein binds to the SigH R N A polymerase subunit specifically under oxidizing conditions in vitro, as would be expected i f Rv0818 is involved in the transcriptional regulation of msh biosynthesis genes upon oxidative stress. A s an addendum to this thesis, we looked at two potential GSH-dependent genes in mycobacteria, ggtA & ggtB, which code for putative gammaglutamyltranspeptidases and might have a role in the recently described phenomenon of mycobacterial sensitivity to G S H and G S N O . 11 T A B L E O F C O N T E N T S A B S T R A C T , i i T A B L E O F C O N T E N T S i i i L I S T O F T A B L E S v L I S T O F F I G U R E S v i A B B R E V I A T I O N S . . v i i i A C K N O W L E D G E M E N T S x 1.0 I N T R O D U C T I O N 1 1.1 G L O B A L I M P A C T O F T U B E R C U L O S I S 1 1.2 H O S T D E F E N S E S T R A T E G I E S 1 1.3 M Y C O B A C T E R I A L D E F E N S E S T R A T E G I E S 3 1.4 M Y C O T H I O L IS A U N I Q U E S Y S T E M I C P R O T E C T A N T 4 1.5 R E G U L A T I O N O F M Y C O T H I O L L E V E L S 8 2.0 W O R K I N G H Y P O T H E S I S & S P E C I F I C A I M S 11 2.1 H Y P O T H E S E S 1 11 2.2 S P E C I F I C A I M S 11 3.0 E X P E R I M E N T A L 12 3.1 M A T E R I A L S 12 3.2 M E T H O D S 14 3.2.1 M Y C O B A C T E R I A L C U L T U R E A N D S T R E S S T R E A T M E N T S . . . 14 3.2.2 H P L C A N A L Y S I S O F M S H A N D M S = S M L E V E L S 15 3.2.3 R T - Q P C R A S S A Y S 19 3.2.4 M A C R O P H A G E I N F E C T I O N S 21 3.2.5 P R O T E I N - P R O T E I N B I N D I N G A S S A Y S 22 4.0 R E S U L T S .'. 23 4.1 E F F E C T O F S T R E S S T R E A T M E N T S O N M. SMEGMATIS M C 2 1 5 5 . . . 23 4.1.1 D I A M I D E T R E A T M E N T 23 4.1.2 H 2 0 2 T R E A T M E N T 25 4.1.3 g N O T R E A T M E N T 27 4.1.4 R E D O X R A T I O S 29 in 4.2 E F F E C T O F S T R E S S T R E A T M E N T S O N M. BOVIS B C G 30 4.2.1 D I A M I D E T R E A T M E N T 30 4.2.2 H 2 0 2 T R E A T M E N T 32 4.2.3 g N O T R E A T M E N T 34 4.2.4 R E D O X R A T I O S . . . . . . . . 36 4.3 RV0485 A N D RV0818 38 4.3.1 IN VITRO E X P R E S S I O N I N M. BOVIS B C G 38 4.3.2 IN VIVO E X P R E S S I O N I N M. BOVIS B C G 41 4.3.2 S I G H & RV0818 P R O T E I N B I N D I N G A S S A Y S 44 5.0 D I S C U S S I O N 47 5.1 T H E M S H : M S = S M R E D O X B A L A N C E I N M Y C O B A C T E R I A 47 5.2 M S H T R A N S C R I P T I O N A L R E G U L A T I O N 52 7.0 A D D E N D U M 59 A.1 R O L E O F G L U T A T H I O N E I N H O S T C E L L D E F E N S E 59 A.2 G S H - D E P E N D E N T E N Z Y M E S I N A C T I N O B A C T E R I A 60 A.3 E X P R E S S I O N O F G G T G E N E S 69 A . 5 F U T U R E W O R K 75 iv L I S T O F T A B L E S Table Title Page 1 List of primers used for q P C R 13 2 Spreadsheet for calculation of mycothiol levels 18 A . l P S I - B L A S T results for GgtA 62 A . 2 P S I - B L A S T results for GgtB 64 A.S P S I - B L A S T results for Get 68 A.4 List of primers used for q P C R 69 V L I S T O F F I G U R E S Fig. Title Page 1 Biochemical structures of M S H & G S H 5 2 Overview o f mycothiol biosynthesis and metabolism 6 3 Genomic context o f Rv0485 & Rv0818 10 4 General oxidation reaction of diamide and thiols 12 5 Schematic of M S H and M S = S M H P L C assays 17 6 Example of a data chromatogram 17 7 Thiol analysis of M. smegmatis mc2155 diamide treatment 24 A) in 0.9% saline B) in M B 7 H 9 / A D S 8 Thiol analysis of M. smegmatis mc 155 upon H2O2 treatment 26 A ) in 0.9% saline B) in M B 7 H 9 / A D S 9 Thiol analysis o f M. smegmatis mc2155 upon g N O treatment 28 A ) in 0.9% saline B) in M B 7 H 9 / A D S 10 Redox ratios ( M S H : M S = S M ) of M. smegmatis mc2155 upon exposure to 29 oxidative and nitrosative stressors. 11 Survival and thiol analysis of M. bovis BCG upon diamide treatment 31 A ) viable count B) in 0 .9% saline C) in M B 7 H 9 / A D S 12 Survival and thiol analysis of M. bovis BCG upon H2O2 treatment 33 A ) viable count B) in 0.9% saline C) in M B 7 H 9 / A D S 13 Survival and thiol analysis of M. bovis BCG upon gNO treatment 35 A ) viable count B) in 0.9% saline C) in M B 7 H 9 / A D S 14 . Redox ratios ( M S H : M S = S M ) of M. bovis BCG upon exposure to 37 oxidative and nitrosative stressors. 15 A) Gene expressions of sigH in M. bovis BCG following 1 and 4 hour 39 treatments with 5 m M diamide 15 B) Gene expressions of M. bovis B C G Rv0485 and Rv0818 following .1 40 and 4 hour treatments-with 5 m M diamide 16 Infection of J774A.1 murine macrophages with M. bovis B C G at M O I 10 42 vi A ) Gene expressions of hspXin M. bovis BCG 6 and 18 hours post-infection B) Visualization of internalized FITC-labeled M. bovis BCG 17 Gene expressions of M. bovis B C G Rv0485 and Rv0818 following 6 and 43 18-hour infections in J774A.1 macrophages at M O I of 10 18 Putative S igH promoter binding sites 44 19 SDS gel analysis of Rv0818 and SigH proteins 45 20 Protein-protein binding assay of Rv0818 and S igH at 37°C 46 21 Protein-protein binding assay of Rv0818 and S igH at 42°C 46 22 Preliminary H P L C analysis of GlcN-Ins levels in log-phase M. smegmatis 52 mc 155 exposed to 200ppm gNO for 2 hours 23 Putative model of SigH-mediated transcription involving Rv0485 and 55 Rv0818 A.1 Phylogenetic tree of GGT- l ike enzymes in Actinobacteria 65 A.2 Simplified phylogenetic tree of Actinobacteria based on 16s r R N A 66 sequences A.3 Genomic context of ggtA & ggtB 67 A.4 Gene expressions of M. bovis B C G ggtA and-ggtB following 2hour 70 treatments with 5 m M glutathione A.5 Gene expressions of M. bovis B C G ggtA and ggtB following 6 and 18- 71 hour infections in J774A.1 macrophages at M O I of 10 A.6 Overview of the y-glutamyl cycle 73 v i i A B B R E V I A T I O N S Abbreviation Definition A D S Albumin-Dextrose-Saline (media supplement) B - M e Beta-Mercaptoethanol B S A Bovine Serum Albumin C F U Colony Forming Units D M E M Dulbecco's Modified Eagle's Medium D T T Dithiothreitol E D T A Ethylenediaminetetraacetic acid F C S Fetal Ca l f Serum FITC Fluorescein isothiocyanate G G T Gamma-glutamyltranspeptidase gNO Gaseous nitric oxide GS=SG Glutathione (oxidized disulfide form) G S H Glutathione (reduced form) . G S N O S-nitrosoglutathione H B S S Hanks' Balanced Salt Solution H P L C High pressure liquid chromatography L D A O N,N-Dimethyldodecylamine-N-oxide M B 7H10 Middlebrook 7H10 (solid media) M B 7H9 Middlebrook 7H9 (liquid media) m B B r Monobromobimane M c a Mycothiol S-conjugate amidase M E M - N E A A Minimal Essential Medium-Non-Essential Amino Acids M O I Multiplicity of infection M S = S M Mycothiol (oxidized disulfide form) M S H Mycothiol (reduced form) M S N O S-nitrosomycothiol Mtr Mycothiol disulfide reductase v i i i N E M N-ethylmaleimide O A D C Oleic acid-Albumin-Dextrose-Catalase (media supplement) ONOO" Peroxynitrite P B S Phosphate Buffered Saline ppm Parts per mil l ion q P C R Quantitative real-time reverse-transcriptase P C R RNIs Reactive nitrogen intermediates ROIs Reactive oxygen intermediates R P M I Roswell Park Memorial Institute SDS Sodium-dodecyl-sulfate W H O World Health Organization ix A C K N O W L E D G E M E N T S This thesis was made possible due to the following people... Dr. Yossef A v - G a y - for being my supervisor and giving me the opportunity to play with a dazzling assortment of pathogens & carcinogens in his laboratory. Dr. Zakaria H m a m a & Dr . Ted Steiner - for being helpful & constructive supervisory committee members Jerry Newton - for invaluable technical support at a crucial time for the H P L C assays. M a r y K o & Rayken Chow - for equally invaluable technical support for everything else. Dr. Ala-Eddine Deghmane - for technical support with the macrophage infections. Dr. Markus Kaufmann - for providing us with the SigH & Rv0818 proteins Dr. Wil l iam Bishai - for providing us with several T B mutants for continuing work in this area Past & present members of the A v - G a y lab - for being a nice bunch of people to work with. Patrice Godin - my life partner, cheerleader, occasional lab assistant, and my reason for being. T H A N K S E V E R Y O N E ! ! ! 1.0 INTRODUCTION 1.1 G L O B A L I M P A C T O F TUBERCULOSIS Tuberculosis (TB) is the leading cause of mortality worldwide due to a single infectious agent and is responsible for an estimated 3 mil l ion deaths and 8 mil l ion new cases of active disease per year. Approximately one third of the world's population wi l l have positive reactions to the tuberculin skin test, indicating prior exposure to mycobacterial species such as the live attenuated vaccine strain Mycobacterium bovis B C G or the disease-causing strain Mycobacterium tuberculosis (WHO, 2005). Upon primary exposure to M. tuberculosis bacilli , patients can either develop immediate active disease, or in most cases, harbour the pathogen in a latent, symptom-less disease state. Immuno-competent individuals who are latently-infected have a 10% risk of developing active T B in their lifetime; however, the risk increases to as much as 90% for the immuno-compromised, such as HIV-positive individuals. Over 60% of all T B cases occur in Africa and South East Asia , highlighting an alarming trend of co-infection with H I V in these regions. Associated with the high incidence rates in these areas are extreme poverty, malnutrition, poor hygiene, and other factors leading to immuno-suppression ( W H O , 2005). Once thought to be an easily eradicable disease, current problems such as inadequate understanding of the mechanisms that allow for latency in the human host, inconsistent efficacy of the B C G vaccine, lack of new and effective vaccines/drug treatments against active and latent T B , and increasing occurrences of multi-drug-resistant M. tuberculosis have ensured a place for this disease among major world health crises for years to come. 1.2 H O S T D E F E N S E STRATEGIES The most prevalent form of T B is pulmonary tuberculosis. Upon entering the lung, the naturally obligate intracellular aerobic tuberculosis bacilli are engulfed by alveolar macrophages via phagocytosis. However, phagocytosis is inhibited by mycobacterial defense mediators that prevent complete fusion of the bacilli-containing phagosome with lysosomes, thus allowing mycobacterial replication to proceed. The macrophages become activated and secrete cytokines that recruit T cells and other activated macrophages to the site of infection. This results in the formation of granulomas surrounding the bacteria. In the course of human disease, granulomas typically evolve to contain the bacilli in necrotizing regions that are low in nutrients and high in 1. concentrations of reactive oxygen/nitrogen intermediates (ROIs/RNIs) and other toxins released by the lysed macrophages, which help inhibit the replication of the bacill i . These caseous centers are enclosed by a localized body of activated macrophages and other immune cells that also serve to control spread of the pathogen. In cases of primary active disease progression and reactivation of latent infection, the balance between the host immune response and mycobacterial resistance fails, and mycobacterial replication is no longer controlled in a localized manner. The ensuing widespread immune response results in rampant lung tissue damage characteristic of the disease pathology (Clark-Curtiss and Haydel, 2003; Zahrt, 2003). A major response of macrophages to inflammatory stimuli is the production and controlled release of antimicrobial ROIs and RNIs (Nathan and Shiloh, 2000). They can wreak havoc on cellular homeostasis by damaging D N A , proteins, and other chemical moieties in a non-specific manner. Although these chemically reactive intermediates are also present in the cell at basal levels due to normal metabolic processes, at high concentrations they result in mutagenesis, necrosis, and apoptosis. In the macrophage, RNIs are generated via an inducible nitric oxide synthase (NOS2, or iNOS) . The major product nitric oxide (NO) is oxidized into a series of toxic RNIs including nitrite (NO2"), nitrogen dioxide (NO2), and nitrate (NO3") among others. One consequence of RNIs is S-nitrosylation, the formation of NO-adducts via cysteine residues, usually on proteins. These modifications tend to adversely affect protein enzymatic activity and/or structural purpose. S-nitrosylation of G S H also occurs to form the toxic S-nitrosothiol, G S N O . A particularly toxic intermediate is peroxynitrite ( O O N O ) , arising from the reaction of N O with the ROI superoxide (O2). ROIs in phagocytes are generated from molecular oxygen (O2) via phagocyte oxidase (Phox). These reduction products include superoxide, H2O2, and hydroxyl radicals (OH'). The significance of Phox and N O S 2 to host defense is illustrated by studies with gp91phox~l~ NOS2'1' mice (Shiloh et al, 1999). Unless these double knockout mice are raised with life-long antimicrobial chemotherapy under sterile conditions, they are all prone to death from spontaneous infection with commensals, even though other components of their immune response such as antimicrobial peptides, T cells, natural killer cells, neutrophils, etc, remain intact. However, phox and NOS2 play overlapping roles in host defense, as mice with single mutations in either gene do not show such high susceptibility to spontaneous infections. The sensitivity of mycobacteria to ROIs and RNIs in vitro (Laochumroonvorapong et al, 1997; Long et al, 1999), and the exacerbation of disease in both phox and NOS2 knockout mice (Cooper et al, 2000; MacMick ing et al, 1997) illustrate the importance of these intermediates in host 2 defense against mycobacteria. Interestingly, studies show that M. tuberculosis is more resistant to ONOO" than avirulent species such as M. bovis B C G and M. smegmatis ( Y u et al, 1999), illustrating that the basis of mycobacterial resistance to ROIs and RNIs is diverse both in mechanism and species specificity . 1.3 M Y C O B A C T E R I A L D E F E N S E S T R A T E G I E S Mycobacteria employ various strategies to ensure survival in the toxic environment of the granuloma and within the activated macrophage. In Escherichia coli, exposure to ROIs induces the redox-sensitive transcriptional regulator OxyR, which in turn induces the production of various enzymes to combat oxidative stress. The O x y R regulon includes oxyR itself, a glutaredoxin (grxA), a DNA-protective nucleoprotein (dps), an alkylhydroperoxide reductase (ahpC), a thioredoxin (trxC), and a catalase/peroxidase (katG), among others (Farr and Kogoma, 1991; Prieto-Alamo et al, 2000; Storz et al, 1990; Tartaglia et al, 1989). However, in several species of mycobacteria, including M. tuberculosis, the oxyR gene is rendered non-functional by deletions, missense mutations, or frameshifts (Deretic et al, 1995). Regardless of this, mycobacteria are still able to mount several varied, specific responses to both exogenous and endogenous oxidative/nitrosative stresses via functional ahpC, katG, and trxC genes. One study showed that M. tuberculosis and M. smegmatis mutants of ahpC have increased sensitivity to ONOO", but not to N O donors (Master et al, 2002). A h p C , A h p D (an adaptor protein), SucB (a dihydrolipoamide succinyltransferase), and L p d (a dihydrolipoamide dehydrogenase) may form a complex which has NADH-dependent peroxidase and O N O O " reductase activities (Bryk et al, 2002). Mutants of katG are attenuated in wild-type and NOS2" 7" mice and macrophages, but not in gp91phox''' mice and macrophages, suggesting a specific role of katG in protection of M. tuberculosis against ROIs generated by hostphox (Ng et al, 2004). Ironically, katG is also detrimental to mycobacteria in the sense that it is essential for activation of the I N H pro-drug. Another study also showed that RNIs are generated as a consequence of in vivo I N H activation by katG (Timmins et al, 2004). Mutants of sodA/C superoxide dismutase are attenuated in wi ld-type mice, show increased sensitivity to H2O2 in vitro, and allow a robust immune response in mice, including early mononuclear cell infiltration and apoptosis in infected tissues. The M. tuberculosis thioredoxin reductase and thioredoxin genes trxB2 and trxC are also known to be induced upon exposure to oxidative stress (Raman et al, 2001). In a surprising study, M. tuberculosis transposon mutants in mpa (mycobacterial proteasome ATPase, Rv2115c) andpaf 3 (proteasome accessory factor, Rv2097c) were found to be hypersusceptible to RNIs in vitro, and were also attenuated in wild-type mice. Virulence of the mutants was somewhat restored in NOS2" 7" mice, suggesting an additional role for the mycobacterial proteasome in defense against RNIs generated by host N O S 2 (Darwin et al, 2003). However, it also appears that M p a and Ka tG are among a host of mycobacterial proteins that are targets for inhibition via S-nitrosylation by RNIs (Rhee et al, 2005). Another mycobacterial response to RNIs might be to enter a state of dormancy. Low concentrations of N O may inhibit respiration and induce expression of the DosR regulon required for survival of mycobacteria in a latent state (Ohno et al, 2003; Voskui l et al, 2003). Recent work from our laboratory has shown that mutants of PknH, one of several protein serine/threonine kinases in M. tuberculosis, were less sensitive to acidified nitrite and peroxide stress in vitro compared to wild-type, and furthermore demonstrated hypersurvival in B A L B / c mice (Papavinasasundaram, 2005). This suggests that PknH may be activated in response to RNIs/ROIs, downregulating bacillary load during chronic infection, thus facilitating long-term survival within the host. The mycobacterial defenses outlined above serve as effective, specific responses to specific dangers in the host cell; however, there is also a need for effective, immediate, and universal responses to the wide spectrum of threats a bacilli may encounter during its life cycle. Low-molecular weight thiols serve such a role in all known living organisms. They maintain cellular homeostasis by ensuring a reducing environment in the cell, and also function as general-use detoxification agents not only for ROIs and RNIs generated by the immune response, but also for toxins such as antibiotics, alkylating agents, electrophiles, and other exogenous or endogenous reactive intermediates. In eukaryotes and gram-negative bacteria, glutathione is the major cellular thiol. In mycobacteria, mycothiol serves as the major systemic protectant. 1.4 M Y C O T H I O L IS A UNIQUE S Y S T E M I C P R O T E C T A N T Mycothiol (MSH) is the dominant low molecular-weight thiol, reducing agent, and storage form of cysteine, produced by mycobacteria and a number of other actinomycetes such as Micrococcus, Nocardia, and Rhodococcus. It is not found in eukaryotes and gram-negative bacteria, which use G S H as their major antioxidant thiol. O f the actinomycetes, M. smegmatis contains the highest amount of M S H , followed by M. tuberculosis and other species of mycobacteria. G S H is not found in significant levels in MSH-producing organisms, nor is M S H produced by GSH-producing organisms (Newton et al, 1996). Although functionally similar, 4 M S H possesses a slightly more complex biochemical structure than the G S H tripeptide of glutamate, cysteine and glycine (Fig. 1) (Newton and Fahey, 2002). The M S H biosynthetic pathway consists of four enzymes (MshA, M s h B , M s h C , and MshD) and is well conserved in gene sequence and functionality among pathogenic and non-pathogenic mycobacteria. With exception to the initial step of biosynthesis, the entire M S H biosynthetic pathway has been well characterized (Fig. 2}. Initially, M s h A (Rv0486), an N -acetylglucosamine transferase, catalyzes the production of GlcNAc-Ins (lD-myo-Inosityl 2-acetamido-2-deoxy-a-D-glucopyranoside) from an as yet unknown substrate (Newton et al., 2003). M s h B ( R v l 170), a deacetylase, then catalyzes the removal of acetate from GlcNAc-Ins, resulting in the compound GlcN-Ins (lD-wyo-Inosityl 2-amino-2-deoxy-a-D-glucopyranoside) (Buchmeier et al., 2003). This is followed by the ATP-dependent ligation of cysteine to G l c N -Ins via the M s h C ligase (Rv2130c), producing Cys-GlcN-Ins (lD-myo-Inosityl 2-(L-cysteinyl) amido-2-deoxy-a-D-glucopyranoside) (Rawat et al., 2002). In the final step, M s h D (Rv0819), an acetyltransferase, catalyzes the addition of acetate to Cys-GlcN-Ins from an acetyl-CoA donor to form the final product mycothiol, AcCys-GlcN-Ins (lD-myo-Inosityl 2-(N-acetyl-L-cysteinyl) amido-2-deoxy-a-D-glucopyranoside) (Koledin et al., 2002). o " N H Q HS. L - H O ^ H A O ^ C H 2 O H Q HO H O ^ r - - / A ^ M ^ M I G S H (Glutathione) HO ',, AcCys-GlcN-Ins T , , T . . . . 0 H Mycothiol gamma-L-glutamyl-L-cysteinyl-glycine Fig. 1: Biochemical structures of M S H and G S H . ( K E G G L I G A N D database, 2005; Newton and Fahey, 2002) 5 Fig. 2: Overview of mycothiol biosynthesis and metabolism. (Metabolites are shown in red and enzymes are shown in black) (MetaCyc, 2005) The antioxidant properties of G S H and M S H are due to the presence of the sulphur atom of the amino acid cysteine, functional only in its reduced (-SH) form. Both G S H and M S H can be spontaneously oxidized to their respective disulfide forms of GS=SG and M S = S M , which are then converted back to their reduced forms via specific disulfide reductases (Mtr (Rv2855) in mycobacteria) in a NADPH-dependent reaction. These thiols are also able to spontaneously or enzymatically form S-conjugates with a variety of compounds. Indeed, the molecular mechanism of MSH-dependent detoxification involves the formation of mycothiol S-conjugates (MS-R, where R is the toxin) spontaneously (Rawat et al, 2004); however, the existence of substrate-specific mycothiol S-transferases (similar to known glutathione S-transferases) has not been proven nor ruled out. These S-conjugates are subsequently cleaved by the amidase M c a (Rvl082), to result in GlcN-Ins and the modified toxin A c C y s R (Steffek et al, 2003). This mercapturic acid is then excreted from the cell (via an as yet unidentified mechanism), and GlcN-Ins is recycled back into the M S H biosynthetic pathway. M c a has been shown to be specific for the mycothiol moiety of the M S - R , and non-specific for the R-moiety. This pathway is known to be involved in detoxification of alkylating agents such as monobromobimane, electrophiles, and antibiotics such as cerulenin and rifamycin S (a derivative of a first-line T B drug) (Newton et al, 2000; Rawat et al, 2004; Steffek et al, 2003). It is worthy to note that natural and synthetic bromotyrosine-based inhibitors of M c a have been identified (Fetterolf and Bewley, 2004; Nicholas et al, 2003). 6 In addition to this versatile detoxification system, there also exist specialized M S H -dependent detoxification pathways. One such example is formaldehyde detoxification, involving a specific NAD/MSH-dependent formaldehyde dehydrogenase, M s c R (Rv2259) which hydrolyzes the S-formylmycothiol conjugate (Misset-Smits et al, 1997; Norin et al, 1997) (Fig. 2). Studies have also demonstrated nitrosothiol reductase activity in this enzyme (Vogt et al, 2003). Mutants of M s h C are non-viable in M. tuberculosis Erdman (Sareen et al, 2003), B C G , and H37Rv (Sassetti et al, 2003), demonstrating essentiality of M s h C . Other mutants of the M S H biosynthetic pathway are non-essential for growth in vitro (Sassetti et al, 2003), nevertheless these mutants severely deficient in M S H exhibit increased sensitivity to a variety of commonly used antibiotics including streptomycin, azithromycin, erythromycin, vancomycin and penicillin G , and oxidants such as menadione, plumbagin, and hydrogen peroxide (Rawat et al, 2002; Rawat et al, 2003; Rawat et al, 2004). Mtr was found to be essential for optimal growth in vitro, and while M s c R is non-essential, it is located directly upstream (without an intergenic region) of Rv2260, a conserved hypothetical protein that was found to be essential for optimal growth (Sassetti et al, 2003). Interestingly, M S H mutants are also highly resistant to isoniazid (INH), another first-line T B drug. I N H is a pro-drug that is inactive until oxidized by mycobacterial Ka tG in the presence of H2O2 to produce a reactive intermediate capable of disrupting the integrity of the mycobacterial cell wall by inhibiting mycolic acid biosynthesis. The specific role of M S H in this process has not been elucidated. Previous studies demonstrated survival of M. smegmatis in 200ppm gNO for up to 8 hours, a resistance significantly higher than other pathogenic bacteria tested (Miller, 2004). Furthermore, the role of M S H in this gNO-resistance was thought to be demonstrated in experiments where the M s h A M. smegmatis'mutant showed increased sensitivity to gNO compared to wild-type, and treatment with 200ppm gNO for 8 hours resulted in a 40% decrease in M S H levels compared to the control (Miller, 2004). However, these results have not been replicated in other attempts. Recent unpublished experiments indicate that M s h A , MshB and M s c R mutants in M. smegmatis are not significantly more sensitive than wild-type to exposure to 200ppm gNO for up to 8 hours. In fact, the M s h D mutant appears to demonstrate decreased sensitivity to gNO exposure (Av-Gay, unpublished data). Thus the role of M S H in gNO-resistance remains to be deciphered. 7 1.5 R E G U L A T I O N O F M Y C O T H I O L L E V E L S Theoretically, there are four key regulatory points in the M S H metabolic pathway (Fig. 2) at which cellular M S H levels can be governed: 1) regeneration of M S H via the detoxification pathway and M c a or MscR; 2) transcriptional regulation of the M S H biosynthesis genes; 3) maintenance of the M S H : M S = S M redox balance via Mtr; or 4) a negative feedback mechanism on the biosynthetic pathway, triggered by current cellular M S H levels. There is data to suggest that there exists a negative feedback mechanism of control via M s h B . Normal cellular concentrations of M S H range from 1 to 8 m M , while activity of M s h B on GlcNAc-Ins is inhibited by up to 99% in the presence of I m M M S H in vitro (Av-Gay, unpublished data; Newton and Fahey, 2002). While it is known that the redox status of G S H : G S = S G is -100:1 in eukaryotes (Gilbert, 1990), this has not yet been investigated with M S H : M S = S M in mycobacteria. Transcriptional regulation of M S H levels has also not been investigated in depth. Some studies on gene expression of mca, mtr, and the M S H biosynthesis genes show that they are all (with the exception of mshB), actively transcribed during log-phase growth in M. bovis B C G (Av-Gay, unpublished data; Hayward et al, 2004). mca and mtr expression was not detected during stationary phase (Hayward et al, 2004). As can be predicted from previous studies, inhibition of mtr and mshB with anti-sense oligonucleotides resulted in growth inhibition at 7 days in vitro. When M. bovis B C G is exposed to isoniazid at 75% of the minimum inhibitory concentration, mshB is induced, mtr is upregulated, and mca gene expression is not affected, suggesting depletion of M S H in part due to oxidation to M S = S M (Hayward et al, 2004). Exposure of M. bovis B C G to diamide, a thiol-specific oxidant, results in upregulation of mshA up to 2.5-fold, mshC up to 3-fold, and mshD up to 8-fold (Av-Gay, unpublished data). These observations further emphasize the importance of M S H in maintaining cellular homeostasis in virulent mycobacteria. While levels of G S H in GSH-containing organisms are controlled in part by O x y R (via regulation of glutaredoxin which participates in glutathione-glutaredoxin redox reactions), mycobacteria lack the O x y R regulator (Deretic et al, 1995). Clues to the transcriptional regulatory control of mycobacterial M S H may come from the distantly related actinomycete Streptomyces coelicolor. In S. coelicolor, the disulfide stress response is modulated by sigmaR (rjR), of the extracytoplasmic 70kDa sigma factor (ECF) family, and its anti-sigma factor Rs rA (Kang et al, 1999; Paget et al, 1998). ECFs confer specificity to the bacterial R N A polymerase holoenzyme by identifying the correct sites of transcription via binding to specific sequences, 8 usually in the -35 and -10 promoter regions; thus bacteria possess several E C F s , each specific to an environmental stress (Manganelli et al, 2004). The a R mutant in S. coelicolor has increased sensitivity to oxidants such as diamide, menadione, and plumbagin, as well as 4-fold lower levels of M S H than the wild-type, suggesting a role for CTr in the control of M S H levels in this species (Paget et al, 1998; Paget et al, 2001). However, a search of predicted a R promoter sites, did not identify any MSH-related genes (Paget et al, 2001). Some genes that are controlled by a R include the trx operon, sigR itself, and glutaredoxin-like proteins, and enzymes involved in transcriptional machinery (Paget et al, 2001). The homolog of a R in mycobacteria is sigmaH (o H ) (Rv3223c), with 65% sequence identity (with no gaps) over 83% of the protein sequence (Paget et al, 1998). The RsrA homolog, RshA, has also been identified (Song et al, 2003). There are over 15 predicted and known sigma factors in mycobacteria, varying in function and presence. c?H is present in both virulent and avirulent strains of mycobacteria, although like many genes it has mutated into a pseudogene in M. leprae (Manganelli et al, 2004). Like Rs rA and a R , RshA is shown to form a complex with CTh and inhibit aH-mediated transcription specifically under reducing conditions. The complex dissociates to allow transcription under oxidizing conditions such as exposure to H2O2 or diamide. Sensing of the redox environment is mediated by disulfide bond formation between adjacent cysteine residues in both Rs rA and RshA (Kang et al, 1999; Song et al, 2003). RshA and CTh complex formation can also be disrupted by heat shock in vitro (Song et al, 2003). a H is thought to control responses to heat and oxidation (in the form of H2O2 and diamide) as CTh mutants are more sensitive to these stresses than wild-type. However, these mutants do not display attenuated virulence in macrophages (Manganelli et al, 2002). Some genes that were not induced in the a H mutant compared to wild-type in the presence of diamide, include those coding for thioredoxin (TrxC), thioredoxin reductases (TrxB, TrxB2), cysteine transport/biosynthetic enzymes ( C y s A / M / T / W ) , a conserved hypothetical protein containing a glutaredoxin active site (Rv2466c), heat-shock proteins (DnaK, C lpB) , S igH and other sigma factors SigB and SigE (Manganelli et al, 2002; Raman et al, 2001), suggesting that their expression is aH-dependent following oxidative stress. It is interesting to note that mshA and mshD lie directly downstream of two genes annotated as putative transcriptional regulators, Rv0485 and Rv0818 respectively (Fig. 3). According to a key study of Himar l transposon mutants (in M. tuberculosis H37Rv and M. bovis B C G ) to investigate genes required for optimal growth in vitro on normal media, both Rv0485 9 and Rv0818 were found to be non-essential genes (Sassetti et al, 2003). However, in a follow-up study of genes required for survival during infection in the spleens of C57BL/6J wild-type mice, the average ratio of survival of the Rv0485 transposon mutant of M. tuberculosis H37Rv in vitro to in vivo was 2:1 over the course of 8-week infections, P=0.007 (Sassetti and Rubin, 2003). Although significant, this did not pass the authors' arbitrary cut-off for genes essential for survival during infection. This observation was also recently confirmed in another study; it was shown that a Himar l transposon mutant of Rv0485 in M. tuberculosis C D C 1551 is significantly attenuated for survival after 7 weeks in B A L B / c mouse lungs (Lamichhane et al, 2005). From this basis, it is not unreasonable to hypothesize that these genes may play a role in M S H -dependent responses of mycobacteria. J R U 0 4 8 5 J 573088 I 1 1 Rv0487 gpnl regX3 mshR Ru84S8 senX3 Rv0484c Rv8492c Genomic context of Rv0485 and Rv0818 in M tb H37Rv: From TubercuList (Institut Pasteur) 91871 < PhoT | Rv8826 - i - j 928188 Ru8817c Rv0822c 'desfll phoY2 Ru0823c Rv0825c LEGEND Coding sequences: yirulence,detox,adapt ^ 1 ip id metabol ism ^ information pathway •4 eel 1 ual 1,process — • s t a b l e RNfl IS/phage PE/PPE intermediary metabolism •™* unknown regulatory conserved • • 4 conserved in H. bovis — rRNfl • tRNfi Rv0485 Rv0818 OnpR Trans_reg_C CadC N C B I Conserved Domain matches for Rv0485 and Rv0818: R O K = R O K family transcriptional regulator domain; NagC= transcriptional regulator/sugar kinase domain; Trans_reg_C= transcriptional regulatory protein, C terminal domain; OmpR= response regulators with CheY-like receiver domain and a winged-helix DNA-binding domain; CadC= DNA-binding winged-HTH domain. F i g . 3: Genomic context of Rv0485 and Rv0818. ( N C B I - C D D , 2005; TubercuList, 2005). 10 2.0 W O R K I N G H Y P O T H E S I S & S P E C I F I C A I M S It is apparent from the above introduction that there are many aspects of thiol-dependent interactions between microbe and host, which have not been elucidated and merit further investigation. In the present thesis, we wi l l attempt to answer some of these questions as related to mycothiol metabolism. We asked the question of how mycobacteria use M S H to respond to RNIs and ROIs, and i f the redox balance of M S H : M S = S M plays an important role in these responses. We also tried to determine i f the putative transcriptional regulators Rv0485 and Rv0818 might play a role in these MSH-dependent responses. We hope that characterization of these thiol-dependent processes in mycobacteria w i l l help us understand how the pathogen M. tuberculosis readily adapts to oxidative/nitrosative stresses, and yield potential novel drug targets for disabling such systems and eliminating T B . 2.1 H Y P O T H E S E S A ) Exposure to oxidative and nitrosative stresses w i l l induce changes in total mycobacterial M S H levels and create fluxes in the basal M S H : M S = S M redox status. B) The putative transcriptional regulators Rv0485 and Rv0818 have roles in MSH-dependent responses to oxidative stress. 2.2 S P E C I F I C A I M S The specific aims of this project are to: 1) Characterize changes in total M S H levels and M S H : M S = S M redox status in response to time-course treatments with diamide, H2O2, and gNO, using M. bovis B C G and M. smegmatis as models. 2) Investigate changes in gene expressions of Rv0485 and Rv0818 o f M. bovis B C G in response to depletion of M S H via diamide treatment, and in response to macrophage infections. 11 3.0 EXPERIMENTAL 3.1 MATERIALS We chose to use M. bovis B C G as the virulent Class II pathogen substitute for M. tuberculosis H37Rv because Class III access could not be obtained. These two species exhibit similar growth characteristics and high genetic similarity, including the regions containing M S H -related genes (Cole et al, 1998; Brosch et al, 2001). M. smegmatis mc 2 155, a fast-growing environmental saprophyte, was also selected as a model organism for comparison. A l l bacterial cell cultures were obtained from the main laboratory of Dr. Yossef Av-Gay . Bacterial culture media were Difco M B 7H9 broth and M B 7H10 agar (Becton-Dickinson, U S A ) . Mueller-Hinton agar plates (with 5% sheep's blood) were purchased from Gibson (USA) . J774A.1 murine macrophages were kindly provided by Dr. Zakaria Hmama's laboratory. Cel l culture media was D M E M (Sigma, Canada). H B S S , H E P E S buffer, and M E M - N E A A solution were purchased from StemCell Technologies (Canada). Diamide ( l , l ' - A z o b i s (N,N-dimethylformamide)), is a small thiol-specific rapidly-acting oxidant commonly used in studies of G S H oxidation. This diazene dicarbonyl compound has a long half-life, is non-toxic to cells, and oxidizes low-molecular weight thiols without producing free radicals (Kosower, 1969). This compound was used as a MSH-specif ic oxidant in our studies (Fig. 4). O \ N ' "N=N' - N \ + 2 R S H N* O O H I ~ N — N -I H N \ + RS=SR O Fig. 4: General oxidation reaction of diamide and thiols. H2O2 was chosen as the oxidative stress reagent in our studies. The nitrosative stress reagent was gaseous nitric oxide (gNO). A l l reagents were provided by the main laboratory. While diamide and H2O2 were added to the bacterial cultures in liquid form, gNO was delivered via a customized regulated gas flow chamber developed in our laboratory (Ghaffari et al, 2005; Mil ler , 2004). The H P L C system consisted of a Waters 717 Plus Autosampler, Farrand Ratio-2 filter fluorometer (with an excitation filter at 254nm and emission filter at 320-390nm), a Waters 1525 12 Binary HPLC pump, and Waters Breeze Chromatography software (v. 3.30) (Waters, USA). All samples were loaded in Poly inserts for WISP vials (Alltech, USA), and separated on a Waters Symmetry C18 5.Oum column. The RNeasy Mini Kit for RNA cleanup was purchased from Qiagen (USA). RNA quantitation was performed using the Agilent 2100 Bioanalyzer, associated 2100 Expert software (v. B.01), Agilent RNA 6000 Nano Chips, and associated RNA 6000 Nano Kit reagents (Agilent, USA). The Revert Aid H-minus First Strand cDNA synthesis kit, (RNase-free) DNase I and 10X buffer, dNTPs, RNase inhibitor, and lOObp DNA Ladder Plus were purchased from Fermentas (Canada). All primers were designed using Primer Designer v. 3.0 and Clone Manager v. 4.1 (Scientific & Educational Software, USA), and synthesized by Operon Technologies Inc. They are listed in Table 1. Primers for sigA were designed by Dr. K.G. Papavinasasundaram. Table 1: List of primers used for qPCR analyses. Gene Forward Primer Reverse Primer Product Size (bpl T - A Anneal lllllllillll Rv0485 5"-(TA.U C G C A C C T C A C A G T C A - 3 5 "-A1 G C i ' C A G L G A C G T A G A T C L . - J 184 5 h T Rv0818 5 ' - T G A T C G A C G A A G G C A C C T A C - 3 5 ' - C A C G T G C A C A T C A A C A G T C C - 3 185 55°C sigA 5 ' - C T C G G T T C G C G C C T A C C T C A - 3 5 ' - G C G C T C G C T A A G C T C G G T C A - 3 130 57°C sigh 5 ' - G T A T C C G A C C G A G C A G A T C A - 3 5 ' - C C T T C G A C A T C G G C G T A G T A - 3 180 55°C hspX 5' - G A C A T T A T G G T C C G C G A T G G - 3 5 ' - T C A G T T G G T G G A C C G G A T C T - 3 237 55°C qPCR was performed using 2X DyNAmo Master Mix (with S Y B R Green dye) (Finnzymes, Finland), and samples were loaded in specialized polypropylene thin-walled tubes for qPCR (MJ Research, USA). Thermocycling and fluorescence detection were performed on the D N A Engine Opticon Continuous Fluorescence Detector and data was analyzed using Opticon Monitor Analysis software (v. 1.07) (MJ Research, USA). Purified SigH and Rv0818 protein preparations were kindly provided by Dr. Markus Kaufmann (UCLA-DOE). Full-length SigH (Rv3223c) was purified by Ni-affmity and gel filtration, contains a C-terminal histidine tag, and is stored in a buffer of 200mM NaCl, 25mM Tris/HCl pH 8.0. Full-length Rv0818 was purified by Ni-affinity, is tagged with an N-terminal histidine tag and maltose binding protein, and is stored in a buffer of 20mM Tris/HCl pH 8.0, 300mM NaCl, 10% glycerol, lOmM B-Me, ImM EDTA, lOmM D(+) maltose, and 0.2% L D A O . 13 Unless otherwise specified, chemicals and other reagents were purchased from either Sigma or Fisher Scientific (Canada). Polypropylene roller bottles, T-75 flasks, and other culture plasticware were purchased from Corning (USA) . A l l experiments were performed as biological triplicates (unless otherwise specified), with readings in at least duplicates. Graphs and statistical analyses were made using GraphPad Prism v. 2.0 (GraphPad Software). Data are shown as means with the associated standard errors of the means (SEM) , unless otherwise specified. Statistical significances were calculated using unpaired two-tailed T-tests with P = 0.05 as the threshold for statistical significance. 3.2 M E T H O D S 3.2.1 M Y C O B A C T E R I A L C U L T U R E A N D S T R E S S T R E A T M E N T S A l l M. bovis B C G cultures were grown in M B 7H9 media (0.2% v/v glycerol, 0.1% v/v Tween-80) supplemented with 10% A D S and incubated in roller bottles at 37°C, 2 rpm until stationary phase. We did not use the common supplement of O A D C because the presence of catalase would have masked the effects of oxidative stress treatments. To reduce clumping during growth we incubated cultures with additional amounts of the non-ionic detergent Tween-80 and sterile metal coils to regularly disrupt flow of the liquid culture. A s the cultures were grown under antibiotic-free conditions, we checked for possible contamination by plating on blood-agar plates and incubating them overnight at 37°C. The fast-growing M. smegmatis cultures were also grown in M B 7H9-ADS, but were kept in upright flasks at 37°C, 250rpm in a shaking incubator. Bacterial concentrations were determined from spectrophotometric Q measurements at OD600 nm and estimation of 1.0 O D / m L = 2.5 x 10 cells. Cel l viability assays were performed by serial dilution of cultures onto M B 7H10-ADS agar plates and subsequent counting of visible colonies following incubation at 37°C. To simulate specific depletion of M S H , cultures were exposed to 5 m M diamide in liquid culture. Diamide was added from a 1 M stock solution and cultures were then incubated at 37°C, 50 rpm, in the absence of light for the specified time points. The concentration of 5 m M was chosen because previous studies have demonstrated non-toxicity and shown change in the mycobacterial proteome (including induction of AhpC) at that concentration (Dosanjh et al, 2005). Oxidative stress was simulated by addition of 10mm H2O2, diluted from a 50% w/v stock solution. Previous studies have also shown non-toxicity at that concentration (Rawat et al, 2002). Cultures were incubated in the same manner as diamide-treated cultures. 14 Nitrosative stress was simulated by exposure to 200ppm gNO delivered from a customized gas chamber developed by a previous student in our laboratory (Ghaffari et al, 2005; Mil ler , 2004). In brief, the chamber facilitates temperature and humidity-controlled delivery of gases into two separate enclosed tubes. Normal medical-quality air was delivered to the control tube, while 200ppm gNO was delivered to the treatment tube. 6mL culture volumes were loaded onto 6-well cell culture plates, covered, and placed into the tubes for the specified time points. Some studies have indicated that gNO may be consumed by H E P E S and riboflavin. In addition, oxidation of H E P E S contributes to the reaction of superoxide radical and N O to form ONOO", resulting in loss of N O and additional effects on the bacteria by O N O O " (Keynes et al, 2003). This led us to test the effects of stress treatments in both bacteria grown in M B 7H9-ADS media and in bacteria resuspended in 0.9% normal saline, to rule out bacterial growth and unknown effects of components in the growth media. 3.2.2 H P L C A N A L Y S I S O F M S H A N D M S = S M L E V E L S H P L C analysis of M S H levels was performed based on a previously developed protocol (Newton et al, 2000). First, treated and control bacterial cultures were centrifuged at 4000rpm for 15 minutes. The supernatants were then discarded and the pellets were either processed immediately or stored at -80°C and processed within a few days. Initially, warm monobromobimane (mBBr) (30 to 50°C) is added to the bacterial pellet in a final concentration of 2 m M , dissolved in 50% acetonitrile/water and 2 0 m M H E P E S p H 8.0. First, the acetonitrile breaks down cell walls, and then the fluorescent S-conjugate M S - M b is formed in a rapid one-to-one reaction of M B B r with M S H . The samples are then heated at 60°C for 15 minutes. The samples are centrifuged at 14000rpm for 7 minutes to pellet out bacterial debris, and the supernatants containing M S - M B are then transferred to a new microcentrifuge tube and acidified with 2uL of 5 M methanesulfonic acid ( M S A ) to maintain a reducing environment. The samples are then diluted 1:20 in l O m M M S A prior to loading on the H P L C . The basic scheme of this assay is illustrated in Fig. 5. To detect the presence of M S H oxidized as M S = S M , we modified the assay as follows (Fig. 5): first, 0.5mL of warm (30 to 50°C) 5 m M N E M (dissolved in 50% acetonitrile/water and . 2 0 m M H E P E S , p H 8.0) was added to bind all - S H groups, including high-affinity binding to M S H . Thus the remaining mycothiol moieties in the supernatant would either be M S = S M or M S - R molecules. The samples are then incubated at 60°C for 15 minutes to inactivate the cells. 15 In the second step, 5uL of 200mM B - M e is added to inactivate excess, unbound N E M and the samples are incubated at room temperature for 10 minutes. 5uL of 200mM D T T , a strong reductant, is then added to reduce all disulfide bonds, including M S = S M , and samples are left to incubate at room temperature for 10 minutes. The reduced M S H molecules are then reacted with excess M B B r (30uL of lOOmM stock) to produce fluorescent M S - M b , incubated at 60°C for 15 minutes, then centrifuged at 14000rpm for 7 minutes. The supernatant is then isolated and acidified with 2uL of 5 M M S A , diluted 1:20 with l O m M M S A , and finally loaded on the H P L C . The samples were resolved on a reversed-phase Waters Symmetry C I 8 5.0um column using the following gradient: 90%A/10%B at 0 and 10 minutes; 82%A/18%B at 15 minutes; 73%A/27%B at 22 minutes; 10%A/90%B at 24 minutes; 90%A/10%B at 25 and 34 minutes. Solvent A is filtered 0.25% glacial acetic acid p H 3.6 and solvent B is 100% HPLC-grade methanol. Flow rate was a constant lmL/minute. Injection volume was 15uL and samples were run for 28 minutes with a 4 minute delay between samples. The M S H peak occurs between 19 to 20 minutes. A n example data chromatogram is shown in Fig. 6 with the M S H peak outlined in red. The other major peak occurring between 18 to 19 minutes is an internal "control" peak of an as yet unidentified metabolite which has always been observed in all M S H cell extracts and does not seem affect the M S H peak itself. Fluorescence was read on a Farrand Ratio-2 filter fluorometer with an excitation filter at 254nm and an emission filter at 320-390nm. The fluorometer settings were set at xO.Ol for sensitivity and 3 seconds for response time. Peaks were manually integrated and analyzed using the Waters Breeze chromatography software. Absolute M S H levels were calculated using the spreadsheet illustrated in Table 2. 7 .5uM of pure M S - M b was run as a positive standard, and samples processed with only N E M were run as "negative" controls for the M S = S M assay to correct for <100% efficiency of N E M in blocking M S H molecules. 16 MSH assay M S H excess Mbbr > MS-Mb MS=SM assay B-Me** M S H N E M * • MS=SM HPLC 500.00: 400.00: 300.00^  200.0Ch 100.00: o.ocr 18.00 20.00 Minutes excess Mbbr MS=SM • 2 x M S H -• MS-Mb * N-ethyl maleimide; blocks al l - S H groups * * B-Mercaptoethanol; inactivates N E M * * * Dithiothreitol; reduces all S=S disulfide bonds Fig . 5: Schematic of M S H and M S = S M H P L C assays. F ig . 6: Example of a data chromatogram. The M S H peak is outlined in red. 17 Mycothiol Assay - HPLC Data Assayed on May 4; run on May 5; M. bovis BCG in 0.9%saline; MSH assay only Cone. Sample Inj. of Inj. Sample Sample nmoles Sample Vol *Cells NET Conv. Vol. Sample Dil. Cone. Vol. per 109 Sample OD600 (mL) X109 Area NEM Area Factor pmol (uL) (uM) Factor (uM) (mL) nmol Cells avg stdev stderr Ohrl 1.468 2 0.73 15690887 0 15690887 253582.39 61.88 15 4.13 20 82.50 0.2 16.50 22.48 Ohrll 1.424 2 0.71 17024474 0 17024474 253582.39 67.14 15 4.48 20 89.51 0.2 17.90 25.14 Ohr III 1.454 2 0.73 13339447 0 13339447 253582.39 52.60 15 3.51 20 70.14 0.2 14.03 19.30 22.31 2.93 1.69 NI I 1.479 2 0.74 16577744 0 16577744 253582.39 65.37 15 4.36 20 87.17 0.2 17.43 23.57 NI II 1.38 2 0.69 14210423 0 14210423 253582.39 56.04 15 3.74 20 74.72 0.2 14.94 21.66 NI III 1.362 2 0.68 12317985 0 12317985 253582.39 48.58 15 3.24 20 64.77 0.2 12.95 19.02 21.42 2.29 1.32 DI I 1.41 2 0.71 2758980 0 2758980 253582.39 10.88 15 0.73 20 14.51 0.2 2.90 4.12 DI II 1.404 2 0.70 3121664 0 3121664 253582.39 12.31 15 0.82 20 16.41 0.2 3.28 4.68 DI III 1.345 2 0.67 1410104 0 1410104 253582.39 5.56 15 0.37 20 7.41 0.2 1.48 2.20 3.67 1.30 0.75 HI I 1.351 2 0.68 3677590 0 3677590 253582.39 14.50 15 0.97 20 19.34 0.2 3.87 5.73 HI II 1.395 2 0.70 875040 0 875040 253582.39 3.45 15 0.23 20 4.60 0.2 0.92 1.32 HI III 1.404 2 0.70 10047586 0 10047586 253582.39 39.62 15 2.64 20 52.83 0.2 10.57 15.05 7.37 7.01 4.05 NOl I 1.427 2 0.71 16515018 0 16515018 253582.39 65.13 15 4.34 20 86.84 0.2 17.37 24.34 NOl II 1.469 2 0.73 15436431 0 15436431 253582.39 60.87 15 4.06 20 81.16 0.2 16.23 22.10 NOl III 1.493 2 0.75 13272260 0 13272260 253582.39 52.34 15 3.49 20 69.79 0.2 13.96 18.70 21.71 2.84 1.64 * Assumption that O.D. 600nm/mL of 1.0 represents 2.5X10 cells Conversion Factor Calculation: MS-mB Standards 1 7418295 2 7762495 3 7641625 Average Area of Standard = 7607471.7 Standard Concentration (M) = 0.000002 Injection Volume of Standard (L) = 0.000015 Standard Amount (pmole) =30 Conversion Factor (Area/pmole) = 253582.39 3.2.3 R T - Q P C R ASSAYS Immediately following the stress treatments, samples were centrifuged at 4000rpm, 4°C, for 15 minutes. The supernatant was discarded, and the pellets were processed for R N A isolation. First, the pellets are resuspended in 0.5mL of washing buffer (0.5% v/v Tween-80, 0.80% N a C l , in 0.1% v/v diethylpyrocarbonate (DEPC)-treated deionized-distilled water), then transferred to a 2mL cryovial with screw-cap. They were centrifuged at 14000g, 4°C, for 1 minute. The supernatant was discarded and the pellets were resuspended in 0.4mL of lysis buffer (20mM sodium acetate p H 4.0, 0.5% SDS, I m M E D T A , in DEPC-treated water). 0.8g of Zirconia/silica beads were added to each sample, and layered with l m L of acidified phenol-chloroform (5:1, p H 4.). To lyse the cells, we then subjected them to a bead beater for 4 rounds of 30 seconds each at maximum speed, with rest intervals of 30 seconds on ice in between. The samples were then left on ice for 10 minutes to complete the solubilization. They were centrifuged at 14000g, 4°C, for 5 minutes to separate the three aqueous, organic, and beads/cellular debris phases. The upper aqueous phase (containing the R N A ) was transferred into a new microcentrifuge tube containing 600u.L of chloroform-isoamylalcohol (24:1), and mixed by inversion. The sample was then centrifuged at 14000g, 4°C, for another 5 minutes to generate the two aqueous and organic phases. The aqueous phase was again transferred to a new microcentrifuge tube containing 600uL isopropanol and 60uL 3 M sodium acetate (made with DEPC-treated water), and mixed by inversion. In some instances, genomic D N A was seen to form a visible precipitate immediately upon mixing. The samples were then left to precipitate at -80°C overnight. On the following day, the samples were thawed and centrifuged at 14000g, 4°C, for 10 minutes. The supernatant was discarded, and l m L of 70% ethanol (made with D E P C -treated water) was added to wash the R N A pellet. The sample was mixed gently by inversion and centrifuged at 14000g, 4°C, for 5 minutes. The supernatant was then carefully aspirated, and the pellet left to air-dry for 3 to 5 minutes. The pellet now containing total R N A and some genomic D N A was then resuspended in lOOuL of DEPC-treated water. In order to remove genomic D N A remnants from the sample, 40uL of the resuspended pellet was mixed with 50uL of DEPC-water, lOuL of 10X DNase I buffer, l u L (20U) of RNase inhibitor mix, and 5-10uL (10-20U) of DNase I. The sample was then left to incubate at 37°C for 1 hour. The sample was then "cleaned-up" on a column using the Qiagen RNeasy Mini -k i t and the RNeasy M i n i Protocol for R N A Cleanup (see manufacturer's instructions). In the final step, samples were eluted in 50uL of DEPC-treated water. The resulting total R N A samples were then loaded on R N A 6000 Nano chips and analyzed on the Agilent 2100 Bioanalyzer (using the 19 Prokaryotic Total R N A Nano assay) for both the quality and quantity/concentration of R N A (see manufacturer's instructions). If the R N A was not judged to be significantly degraded, lOOng - 1 ug of total R N A was then used as a template for first strand c D N A synthesis using the RevertAid kit (see manufacturer's instructions). Negative controls were the R N A templates mixed with water to the final reaction volume of 20uL, in the absence of the reverse-transcriptase enzyme. The random hexamer primers were included in the kit. The resulting c D N A was then used as templates for P C R with sigA primers. 2uL of c D N A template was added in a 20u,L reaction of O . l m M dNTP's , I X P C R buffer (without M g C l 2 ) , 2 .5mM M g C l 2 , 0 .06uM of each primer, and 0.4uL of T A Q D N A polymerase. The P C R program was as follows: initial denaturation at 95°C for 5 minutes, cycle denaturation at 94°C for 1 minute, 60°C cycle annealing for 1 minute, 72°C cycle extension for 1 minute, run for 30 cycles, 72°C final extension for 10 minutes, and hold at 4°C. To visualize the amplification products, lOuL volumes were loaded on an ethidium bromide-stained 1% agarose gel, run at 120V with a Fermentas 1Kb D N A ladder, and exposed to U V light. If there was no genomic D N A contamination, as evidenced by lack of the expected amplification product in the negative (no reverse-transcriptase) controls, the c D N A templates were then used for qPCR. The q P C R reaction was as follows: I X S Y B R Green master mix (includes dNTP's , T A Q , S Y B R Green dye, and other components), 0.06uM of each primer, and 2uL of c D N A template, in a 20u.L total reaction volume. Reactions were loaded in thin-walled P C R tubes, briefly centrifuged to remove bubbles, and amplified in the D N A Engine Opticon. The q P C R program was as follows: initial denaturation at 95°C for 10 minutes, cycle denaturation at 94°C for 10 seconds, 60°C cycle annealing for 15 seconds, 72°C cycle extension for 15 seconds, a plate read of the fluorescence at the end of the cycle, run for 40 cycles, 72°C extension for 7 minutes, a melting curve from 65°C to 95°C (read every 0.2°C and hold for 1 second between readings), 72°C final extension for 7 minutes, and hold at 4°C. The melting curve step was performed to check for specificity of the amplification product, as the S Y B R Green dye is non-specific and binds to any double-stranded nucleic acid. The fluorescence threshold for C(t) was manually set to be above background fluorescence for each assay. Quantitation of the unknowns was determined according to the amplification curves of known amounts of M. bovis B C G genomic D N A . 20 3.2.4 M A C R O P H A G E I N F E C T I O N S The J774A.1 cell-line murine macrophages were grown to confluence in T-75 flasks (-9,4 x 10 6 cells) at 37°C/5% C 0 2 , in D M E M supplemented with 5% v/v F C S , 1% v/v glutamine, 1% v/v M E M - N E A A solution, 1% v/v 1 M H E P E S buffer, 1% v/v streptomycin/penicillin, and 0.1% v/v fungisome. Cells were washed and passaged with H B S S and fresh antibiotic-containing complete D M E M until Day 0 of the infections. They were then washed and incubated in antibiotic-free complete D M E M for 24 hours prior to infection. Log-phase M. bovis B C G cultures were filtered through sterile cotton to disrupt potential clumps, then measured at OD600nm to calculate the amount needed for infections with M O I of 10:1 (bacteria to cell). The bacterial cultures were then pelleted and resuspended 3 times with cell culture medium to remove B S A from the cultures. They were resuspended in 5mL of antibiotic-free complete media per T-75 flask. The existing cell culture media was removed and replaced with the infection media, and the cells were incubated at 4°C for 30 minutes to synchronize adhesion of the bacteria. The cells were then incubated at 37°C/5% C 0 2 for 2 hours. The infection media was then removed and the cells were washed twice with H B S S to remove the non-adherent bacteria. Phagocytosis was then allowed to proceed in fresh culture media until total post-infection incubation times of 6 and 18 hours: The cells were then washed twice with H B S S to remove any attached but unphagocytosed bacteria. Cells were scrapped and resuspended in antibiotic-free media, then centrifuged at 4000rpm, 25°C, for 12 minutes. The supernatant was discarded and the pellet was then processed for R N A isolation. To evaluate the efficacy of the infection process, some cells were seeded onto glass coverslips in 24-well plates, and then infected with FITC-labeled M. bovis B C G in parallel with the other infections. To label the bacteria, they were washed once in M B 7H9 media, then resuspended in M B 7H9 with 1 ixg/mL of F ITC and incubated at 37°C for 1 hour. The labeled bacteria were then washed three times in M B 7H9 media, then resuspended in D M E M and kept in the dark until use. A t the times of harvest, the coverslips were washed twice in warm H B S S , then incubated with Cytofix Buffer ( B D Biosciences, U S A ) for 30 minutes at 37°C in the dark. They were then washed twice with H B S S and rinsed once with deionized-distilled water before being applied to 20uL of 2mg/mL Trypan Blue/0.1% (w/v) sodium azide on a clean microscope slide. The slides were dried for 20 minutes in the dark, and a thin layer of clear nail polish was applied around the edges of the coverslips to hold them in place. The slides were stored at 4°C in the dark until use. The slides were analyzed via fluorescence microscopy on an epifluorescence microscope (Zeiss 21 Axioplan II) using F ITC and red filters. Images observed through the 63x objective lens were recorded through a C C D camera via F ITC and Red fluorescence filters, then merged using Northern Eclipse v. 6.0 (Empix Imaging). 3.2.5 P R O T E I N - P R O T E I N B I N D I N G A S S A Y S S igH and Rv0818 proteins were incubated separately or in combination, in the presence of 5 m M D T T , 5 m M diamide, or under neutral conditions. In each reaction volume of 20uL, 4 u M of each protein was incubated at either 37°C or 42°C for 30 minutes. The binding buffer was 4 0 m M Tr i s /HCl p H 8.0, l O m M M g C l 2 , 0 .0 ImM E D T A , and 20% v/v glycerol, as described in (Song et al, 2003). The reactions were then run on a non-denaturing (native) discontinuous 7% polyacrylamide gel at 90V until the end of the stacking gel, then resolved at 25V for 4 hours. Gels were run on a Bio-Rad minigel apparatus (see manufacturer's instructions for gel/buffer formulations and apparatus assembly), and visualized by silver staining. The silver staining protocol was as follows: fix overnight in 50% methanol, 12% acetic acid, 0.05% formaldehyde; wash gel 3 times for 10 minutes each in 35% ethanol; sensitize gel for 2 minutes in 0.02% sodium thiosulfate; wash gel 2 times for 5 minutes each in water; stain gel for 20 minutes in 0.2% silver nitrate, 0.076% formaldehyde; wash gel 2 times for 1 minute each in water; develop gel in 6% sodium carbonate, 0.05% formaldehyde, 0.004% sodium thiosulfate; stop development for 5 minutes in 50% methanol, 12% acetic acid; store gel at 4°C in 1% acetic acid, 10% glycerol. 22 4.0 R E S U L T S 4.1 E F F E C T O F S T R E S S T R E A T M E N T S O N M. SMEGMA TIS M C 2 1 5 5 4.1.1 D I A M I D E T R E A T M E N T In our first attempts to characterize changes in total mycobacterial M S H levels in response to oxidative and nitrosative stresses, we optimized our assays by performing time-course treatments on fast-growing M. smegmatis. Our first experiments were with diamide, a MSH-specific oxidant, in order to observe how the basal M S H : M S = S M redox status is affected in a basic system where the only "known" consequence of the treatment should be the depletion of cellular M S H and corresponding accumulation of M S = S M . Since we decided to use A D S as the media supplement instead of O A D C to avoid the confounding effects of catalase, we further reasoned that there was the possibility that treatment in M B 7H9 media could mask the effects of diamide through unknown reactions or buffering with components in the media, thus the experiments were performed using both M B 7H9 media and 0.9% normal saline in order to isolate the phenomenon of M S H depletion. The results of the 1, 2, 4, and 8 hour treatments are displayed in Fig. 7 A for the treatments in saline, and Fig. 7B for the treatments in M B 7H9 media. Preliminary assays show that there is no change in viability for any of the stress treatments over the treatment period. Both the untreated controls and treated samples were found to have relatively constant levels of reduced M S H (in the range of ~ 15 to 40nmol per 10 9 cells), and low levels of oxidized M S H (MS=SM) over the period of 8 hours. We see no significant differences in reduced M S H levels over the 8 hour period, between the untreated and treated cells, in both 0.9% saline and M B 7H9 growth media. However, there are slight increases of ~ 0.09nmol and ~ 0.25nmol per 10 9 cells of oxidized M S H in the treated cells compared to the untreated at 4 and 8 hours respectively. These differences were found to be statistically significant at P < 0.05. The ~0.18nmol per 10 9 cells increase in oxidized M S H at 2 hours of diamide treatment in M B 7 H 9 is close to the statistical significance threshold with P = 0.0556. 23 A • 50-1 X c 0-1—, 1 1 1 r 0 2 4 6 8 time (hours) B 0-1-1 1 1 1 r 0 2 4 6 8 time (hours) Fig. 7. Thiol analysis of M. smegmatis mc 2155 upon diamide treatment. ( A ) M S H (reduced form) i n control (c losed squares) and 5 m M diamide-treated (c losed tr iangles) cultures, and M S H (ox id ized form) i n control (open squares) and 5 m M diamide-treated (open tr iangles) cultures in 0 .9% sal ine. (B ) Same as A , but i n M B 7 H 9 - A D S . S h o w n are the means and S E M s o f N = 3 exper iments. * denotes P < 0.05. 24 4.1.2 H 2 0 2 TREATMENT Having documented the effects of a known M S H oxidant, we then continued on to time-course treatments with H 2 0 2 , a biologically relevant form of oxidative stress. The results of the 1, 2, 4, and 8 hour treatments are displayed in Fig. 8 A for the treatments in saline, and Fig. 8B for the treatments in M B 7H9 media. Preliminary assays show that there is no change in viability for any of the stress treatments over the treatment period. Both the untreated controls and treated samples were found to have relatively constant levels of reduced M S H (in the range of ~ 15 to 40nmol per 10 9 cells), and low levels of oxidized M S H over the period of 8 hours. However, there are slight but statistically significant decreases of ~ 3 to 4 nmol of reduced M S H per 10 9 cells upon diamide treatment at the 1 and 2 hour time points in cultures in 0.9% saline (P < 0.01 for the 1 hour time point, and P < 0.05 for the 2 hour time point). This time, we see no significant differences in either reduced M S H or oxidized M S H levels over the 8 hour period, between the untreated and treated cells, in M B 7H9 growth media. 25 A 5 0 - , I c O - L - i 1 1 1 r 0 2 4 6 8 time (hours) F i g . 8. T h i o l analysis of M. smegmatis mc 2155 upon H2O2 treatment. (A) M S H (reduced form) in control (closed squares) and 1 OmM H2O2 -treated (closed triangles) cultures, and M S H (oxidized form) in control (open squares) and l O m M H2O2 -treated (open triangles) cultures in 0 .9% saline. (B) Same as A , but in M B 7 H 9 - A D S . Shown are the means and S E M s of N = 3 experiments. * denotes P < 0.05; ** denotes P < 0.01 26 4.1.3 gNO TREATMENT The next set of time-course treatments were performed with gNO, a biologically relevant form of nitrosative stress. The results of the 1, 2, 4, and 8 hour treatments are displayed in Fig. 9 A for the treatments in saline, and Fig . 9B for the treatments in M B 7H9 media. Preliminary assays show that there is no change in viability for any of the stress treatments over the treatment period. Both the untreated controls and treated samples were found to have relatively constant levels of reduced M S H (in the range of ~ 15 to 40nmol per 10 9 cells), and low levels of oxidized M S H over the period of 8 hours, as expected. We see no significant differences in reduced M S H between the untreated and treated cells in 0.9% saline, but is a slight, but statistically significant decrease of ~ 6nmol of reduced M S H per 10 9 cells in the 200ppm gNO-treated cultures in M B 7H9 growth media (P < 0.01). However, we see no statistically significant changes in the oxidized M S H levels over the 8 hour period in M B 7H9. 27 A 5 0 -X (/> 2 4 0 -0) "Si _ Q . o E c 1 0 -0- - r -0 i 2 i 4 6 - r -8 time (hours) B i 1 1 1 r 0 2 4 6 8 time (hours) 2 F i g . 9. T h i o l analysis ofM. smegmatis mc 155 upon g N O treatment. (A) M S H (reduced form) in control (closed squares) and 200ppm gNO-treated (closed triangles) cultures, and M S H (oxidized form) in control (open squares) and 200ppm gNO-treated (open triangles) cultures in 0.9% saline. (B) Same as A , but in M B 7 H 9 - A D S . Shown are the means and S E M s of N = 3 experiments. ** denotes P < 0.01 28 4.1.4 R E D O X R A T I O S The summary of the calculated redox ratios of reduced M S H to oxidized M S H (MSH:MS=SM) from these experiments are shown in Fig. 10. As can be seen, the basal redox ratio in M. smegmatis is ~200:1. However, despite the statistically significant increases in oxidized M S H levels at 4 and 8 hours of diamide treatment, and the decrease in reduced M S H at 8 hours of gNO treatment in M B 7H9 media as seen in the previous graphs, these did not result in statistically significant changes in the calculated redox ratios. The only statistically significant redox ratio difference was at the 4 hour time point of gNO treatment where the MSH:MS=MSM ratio dropped from 287 to 115:1 (P < 0.05). o 2 CO « II a: co x § •Si O CO CH 5 1000.0-100.0 10.0 1.0-4 0.1 —r-0 —r-2 4 6 time (hours) 4 1 I l l l l ( h r s ) C o n t r o l 5 I I I M D k n i i k l i - i n i n M ii.n. 2I)II|) |IIII « M ) 0 200.75 ± 42.27 200.75 ± 42.27 200.75 ± 42.27 200.75 ± 4 2 . 2 7 1 240.81 ± 5 6 . 2 2 231.11 N/A 131.50 ± 3 5 . 0 9 2 250.78 ± 3 5 . 5 6 156.48 N/A 264.05 ± 69.57 4 287.66 ± 18.36 147.40 N/A 115.47 ± 4 9 . 3 5 8 217.82 ± 6 5 . 4 4 103.86 ± 9 . 1 0 289.41 ± 17.63 199.05 ± 5 9 . 9 2 Fig. 10. Redox ratios (MSH:MS=SM) of M. smegmatis mc 2155 upon exposure to oxidative and nitrosative stressors. Control (closed squares), 5mM diamide-treated (closed triangles), lOmM H202-treated (closed circles), and 200ppm gNO-treated (closed diamonds) cultures in M B 7 H 9 - A D S . N / A = not available. Shown are the means and SEMs of N = 3 experiments. * denotes P<0.05. 29 4.2 E F F E C T O F S T R E S S T R E A T M E N T S O N M. BOVIS B C G 4.2.1 D I A M I D E T R E A T M E N T Having described the effects of diamide, oxidative, and nitrosative stress treatments on M S H levels in the non-virulent soil strain M. smegmatis, we decided to also survey the effects of these treatments on the virulent strain M. bovis B C G , as these species colonize different pathospheres and might have evolved different responses to the various stresses. The time-course treatments were performed under exactly the same conditions as with the M. smegmatis experiments. The viability assays are shown in Fig. 11 A . It was confirmed that there is no significant change in cell viability upon treatment with 5 m M diamide in M B 7H9 media for up to 8 hours. The H P L C analysis results of the 1, 2, 4, and 8 hour 5 m M diamide treatments are shown in Fig. 1 I B for the treatments in saline, and Fig. 1 I C for the treatments in M B 7H9 media. The untreated controls in both 0.9% saline and M B 7H9 were found to have relatively constant levels of reduced M S H (in the range of - 17 to 25nmol per 10 9 cells), and low levels of oxidized M S H over the period of 8 hours, as expected. The diamide-treated cells in 0.9% saline underwent a dramatic decrease in reduced M S H levels, up to 4-fold, by the first time point at 1 hour, and did not recover basal reduced M S H levels by the end of treatment (P < 0.0001 for all time points). A s there was a sudden drop by the first time point, it is plausible that the mass depletion could have occurred sooner than 1 hour after treatment. Accompanying the rapid drop in reduced M S H levels is a significant increase in oxidized M S H levels (by at least 10-fold), indicating that a significant portion of M S H is oxidized to M S = S M upon treatment with diamide. The oxidized M S H levels also did not recover to basal levels by the end of the treatment (P < 0.0001 for all time points). In contrast, upon treatment with 5 m M diamide in M B 7H9 media, there is a slower rate of M S H depletion, with significant decreases in reduced M S H levels at the 1 and 2 hour time points (P < 0.01), followed by a recovery towards the end of the treatment. The decreases at the 4 and 8 hour time points are close to the threshold of statistical significance with P = 0.0557 and P = 0.0674 respectively. A s with the cultures in 0.9% saline, the initial depletion of reduced M S H in M B 7H9 media is mirrored by an initial increase in oxidized M S H . However, as the reduced M S H levels begin to recover by the 4 hour time point, we see a corresponding decrease in oxidized M S H levels. 30 05 „ O 3-_ l 2-1-0-time (hours) c 30-i r15 0 2 4 6 8 time (hours) F i g . 11. Surv iva l and thiol analysis of M. bovis B C G upon diamide treatment. (A) Viable count of M. bovis B C G in control (closed squares) and 5 m M diamide-treated (closed triangles) cultures in M B 7H9-ADS. (B) M S H (reduced form) in control (closed squares) and 5 m M diamide-treated (closed triangles) cultures, and M S H (oxidized form) in control (open squares) and 5 m M diamide-treated (open triangles) cultures in 0.9% saline. (C) Same as B , but in M B 7H9-ADS. Shown are the means and S E M s of N = 3 experiments. ** denotes P < 0.01 31 4.2.2 H 2 0 2 T R E A T M E N T Having documented the effects of a known M S H oxidant, we then continued on to time-course treatments with H 2 0 2 , a biologically relevant form of oxidative stress. The viability assays are shown in Fig. 12A. It was confirmed that there is no significant change in cell viability upon treatment with l O m M H 2 0 2 in M B 7H9 media for up to 8 hours. The H P L C analysis results of the 1, 2, 4, and 8 hour l O m M H 2 0 2 treatments are shown in Fig. 12B for the treatments in saline, and Fig. 12C for the treatments in M B 7H9 media. A s with the diamide experiments, the untreated controls in both 0.9% saline and M B 7H9 were found to have relatively constant levels of reduced M S H (in the range of ~ 17 to 25nmol per 10 9 cells), and low levels of oxidized M S H over the period of 8 hours. The l O m M H 2 0 2 -treated cells in 0.9% saline seemed to undergo a decrease in reduced M S H levels by up to 2-fold throughout the time points, but was only statistically significant at the 1 and 2 hour time points (P < 0.05). The decrease at the 4 hour time point was close to the threshold for statistical significance with P = 0.0572. Accompanying the supposed drops in reduced M S H levels are increases in oxidized M S H levels (by at least 2-fold), but these were not found to be statistically significant, although the increase at the 4 hour time point was close to threshold at P = 0.0931. Overall, there are no statistically significant differences in the reduced M S H or oxidized M S H levels over the 8 hour period, between the untreated and l O m M H 20 2-treated cells, in M B 7 H 9 - A D S media. 32 2 4 6 time (hours) i V) 5 •o 0) o 3 30-20-o E c •15 3 o 10 i •* _». o O * . <° Q. 8 B 5 5T Q. CO time (hours) F i g . 12. Surv iva l and thiol analysis of M. bovis B C G upon H2O2 treatment. (A) Viable count of M. bovis B C G in control (closed squares) and l O m M H202-treated (closed triangles) cultures in M B 7H9-ADS. (B) M S H (reduced form) in control (closed squares) and l O m M H202-treated (closed triangles) cultures, and M S H (oxidized form) in control (open squares) and l O m M H202-treated (open triangles) cultures in 0.9% saline. (C) Same as B , but in M B 7H9-ADS. Shown are the means and S E M s of N = 3 experiments. * denotes P < 0.05 33 4.2.3 gNO TREATMENT The next set of time-course treatments were performed with gNO, a biologically relevant form of nitrosative stress. The viability assays are shown in Fig . 13 A . It was confirmed that there is no significant change in cell viability upon treatment with 200ppm gNO in M B 7H9 media for up to 8 hours. The H P L C analysis results of the 1, 2, 4, and 8 hour 200ppm gNO treatments are shown in Fig. 13B for the treatments in saline, and Fig. 13C for the treatments in M B 7H9 media. A s with the previous experiments, the untreated controls in both 0.9% saline and M B 7H9 were found to have relatively constant levels of reduced M S H (in the range of - 17 to 25nmol per 10 9 cells), and low levels of oxidized M S H over the period of 8 hours. This time we see no significant differences in reduced M S H levels over the 8 hour treatment, between the untreated and treated cells, in both 0.9% saline and M B 7H9 growth media. However, there is a slight but statistically significant decrease of - 0.4nmol per 10 9 cells in oxidized M S H levels at the 8 hour time point of gNO treatment in 0.9% saline (P < 0.05). 34 O 3--1 r-4 6 time (hours) time (hours) Fig. 13. Survival and thiol analysis of M. bovis B C G upon g N O treatment. (A) Viable count of M. bovis B C G in control (closed squares) and 200ppm gNO-treated (closed triangles) cultures in M B 7 H 9 - A D S . (B) M S H (reduced form) in control (closed squares) and 200ppm gNO-treated (closed triangles) cultures, and M S H (oxidized form) in control (open squares) and 200ppm gNO-treated (open triangles) cultures in 0.9% saline. (C) Same as B , but in M B 7 H 9 - A D S . Shown are the means and S E M s of N = 3 experiments. 35 4.2.4 R E D O X R A T I O S The summary of the calculated redox ratios of reduced M S H to oxidized M S H ( M S H : M S = S M ) from these experiments are shown in Fig. 14. A s can be seen, the basal redox ratio in M. bovis B C G is -50:1. In M. bovis B C G treated with 5 m M diamide in 0.9% saline solution, the redox ratio is significantly reduced by - 2 orders of magnitude by the first time point and does not recover by the end of the treatment (P < 0.01 for all time points). Treatment with l O m M H2O2 in 0.9% saline also drops the ratio significantly by - 1 order of magnitude at the 4 and 8 hour time points (P < 0.01), but it seems to recover slightly by the end of the 8 hour treatment. In contrast, there appears to be a significant increase in the redox ratios of the 200ppm gNO-treated samples in 0.9% saline at the 8 hour time point by - 4 fold (P < 0.05). On closer examination, this seems due to decreases in oxidized M S H levels rather than increases in reduced M S H levels. There is a similar but slight increase at the 2 hour time point (P < 0.05). 36 1000.0-3 0.1 , , , r 0 2 4 6 8 time (hours) 1 inn-lIllS) Control 5IIIM Diumiik- ID mM II,(K 200ppni g.\(> 0 60.02 ± 8.93 60.02 ±8.93 60.02 ± 8.93 60.02 ± 8.93 1 34.58 ±4.08 0.49 ± 0.09 11.58 ± 10.23 40.89 ±11.61 2 39.06 + 3.43 0.40 ± 0.06 11.38 ± 10.86 62.91 ±6.10 4 65.04 ±9.16 0.19 ±0.02 4.18 ±3.93 46.11 ± 10.24 8 47.17 ± 7.15 0.18 ±0.04 15.04 ±8.21 190.81 ±33.44 Fig. 14. Redox ratios (MSH:MS=SM) of M. bovis BCG upon exposure to oxidative and nitrosative stressors. Control (closed squares), 5mM diamide-treated (closed triangles), lOmM H202 - t rea ted (closed circles), and 200ppm gNO-treated (closed diamonds) cultures in 0.9% saline. Shown are the means and SEMs of N = 3 experiments. * denotes P < 0.05 37 4.3 RV0485 AND RV0818 4.3.1 IN VITRO EXPRESSION IN M. BOVIS BCG The roles of the putative transcriptional regulators Rv0485 and Rv0818 in mycobacterial responses to M S H depletion were investigated by monitoring changes in gene expression upon diamide exposure. M. bovis B C G cultures were treated with 5 m M diamide in M B 7H9 media for 1 and 4 hour periods, total R N A was extracted, c D N A was synthesized, and gene expressions analyzed via qPCR. A s a positive control, we assayed gene expression of sigH, previously shown to be upregulated 20-fold in log-phase M. tuberculosis exposed to 5 m M diamide for 1 hour (Manganelli et al., 2002). In our assays, sigH mas upregulated about 7-fold upon 1 hour of diamide treatment, and about 26-fold upon 4 hours of diamide treatment (Fig. 15 A ) . q P C R analyses of Rv0485 and Rv0818 revealed no change in expression upon 1 and 4 hour treatments with diamide (Fig. 15B). A s there is visible amplification of both genes in the reverse transcriptase reactions with the enzyme, and none without the enzyme, following Dnasel treatment, it is clear that even though there is no clear change in expression, Rv0485 and Rv0818 both encode bona fide genes which produce m R N A transcript in vivo. 38 1 hr 4 hrs Fig . 15: (A) Gene expressions of M. bovis B C G sigH following 1 and 4 hour treatments wi th 5 m M diamide. Data are expressed as the ratio between the amount of c D N A detected in the treatment samples and the amount of c D N A detected in the control samples, normalized to the amount of house-keeping sigA c D N A present in the samples. Shown are the means and S E M s of N=3 experiments. Also shown are the representative fluorescence curves of the samples assayed via q P C R and the representative products of amplification as visualized by EtBr staining on a 1% agarose gel. +RT denotes the positive samples from the c D N A synthesis reactions and - R T denotes the genomic D N A contamination controls where the reverse transcriptase enzyme was not added to the c D N A synthesis reaction. F i g . 15: (B) Gene expressions of M. bovis B C G Rv0485 and Rv0818 following 1 and 4 hour treatments wi th 5 m M diamide. Data are expressed as the ratio between the amount of c D N A detected in the treatment samples and the amount of c D N A detected in the control samples, normalized to the amount of house-keeping sigA c D N A present in the samples. Shown are the means and S E M s of N=3 experiments. Also shown are the representative fluorescence curves of the samples assayed via q P C R and the representative products of amplification as visualized by EtBr staining on a 1% agarose gel. +RT denotes the positive samples from the c D N A synthesis reactions and - R T denotes the genomic D N A contamination controls where the reverse transcriptase enzyme was not added to the c D N A synthesis reaction. 4.3.2 IN VIVO E X P R E S S I O N I N M. BOVIS B C G Although there is no clear change in the gene expressions of Rv0485 and Rv0818 upon fluxes in mycobacterial M S H : M S = S M redox ratios induced by exposure to diamide, it does not rule out the possibility that these genes might play a role in vivo. To help determine the in vivo roles of Rv0485 and Rv0818, gene expressions following 6 and 18 hour infections of log-phase M. bovis B C G in J774A. 1 murine macrophages was also assayed. The "negative" controls were bacteria incubated in cell medium for two hours. The success of the infection process was assessed via q P C R analyses of hspX, a 14kDa mycobacterial heat-shock protein homologous to a-crystallin, shown to be upregulated upon 6-hour infections of THP-1 macrophages w i t h M tuberculosis (Dubnau et al., 2002), and fluorescence microscopy of J774A.1 macrophages infected at an M O I of 10 with FITC-labeled M. bovis B C G . A s can be seen, hspXwas highly upregulated by about 14-fold at 6 hours post-infection and about 60-fold at 18 hours post-infection (Fig. 16a). J774A.1 macrophages infected with FITC-labeled M. bovis B C G were harvested 2 hours post-infection and stained with Trypan Blue prior to fluorescence microscopy analysis. The addition of Trypan Blue quenches any extracellular fluorescence of bacteria which may not be internalized, and can be visualized using a red fluorescence filter (Wan et al., 1993). In Fig. 16b, the merger of images captured via the FITC and red filters indicate that the mycobacteria are indeed internalized at an M O I of 10 at 2 hours post-infection. The q P C R results (Fig. 17) show that both Rv0485 and Rv0818 are upregulated upon infection of I774A.1 macrophages. Rv0485 is upregulated by about 10-fold at 6 hours post-infection, and by about 9-fold at 18 hours post-infection. Rv0818 is upregulated by a similar amount of about 7-fold at both 6 and 18 hours post-infection. 41 A B 6 hrs 18hrs Fig. 16: Infection of J774A.1 murine macrophages with M. bovis BCG at M O I 10. (A) Gene expressions of M. bovis B C G hspX following 6 and 18-hour infections. Data are expressed as the ratio between the amount of cDNA detected in the treatment samples and the amount of cDNA detected in the control samples, normalized to the amount of house-keeping sigA cDNA present in the samples. Shown are the means and SEMs of N=3 experiments. Also shown are the representative fluorescence curves of the samples assayed via qPCR and the representative products of amplification as visualized by EtBr staining on a 1 % agarose gel. +RT denotes the positive samples from the cDNA synthesis reactions and - RT denotes the genomic D N A contamination controls where the reverse transcriptase enzyme was not added to the cDNA synthesis reaction. (B) Representative image of Trypan Blue-stained J 7 7 4 A . 1 macrophages with intracellular FITC-labeled M. bovis B C G at MOI of 10 , 2 hours post-infection, as visualized via fluorescence microscopy. 4^ F i g . 17: Gene expressions of M. bovis B C G Rv0485 and Rv0818 following 6 and 18-hour infections in J774A.1 macrophages at MOI of 10. Data are expressed as the ratio between the amount of c D N A detected in the treatment samples and the amount of c D N A detected in the control samples, normalized to the amount of house-keeping sigA c D N A present in the samples. Shown are the means and S E M s of N=3 experiments. Also shown are the representative fluorescence curves of the samples assayed via q P C R and the representative products of amplification as visualized by EtBr staining on a 1% agarose gel. +RT denotes the positive samples from the c D N A synthesis reactions and - R T denotes the genomic D N A contamination controls where the reverse transcriptase enzyme was not added to the c D N A synthesis reaction. 4.3.2 S I G H & RV0818 P R O T E I N B I N D I N G A S S A Y S A s discussed previously, S igH is implicated in regulation of mycothiol levels in Streptomyces species, but has not yet been verified in mycobacteria. Preliminary analyses of the upstream regions of Rv0485 and Rv0818 reveal that there may be S igH promoter binding sites present, based on the known consensus of SigH promoter regions (Manganelli et al., 2004), shown in Fig. 18. Thus we decided to conduct protein binding assays to investigate i f Rv0818 binds to SigH, as might be expected under oxidizing conditions. Courtesy of Dr. Markus Kaufmann at the U C L A - D O E Institute of Genomics and Proteomics, we were able to obtain protein preparations of Rv0818 and SigH for use in protein binding assays. s i g H s i g B ,trxB2 Rv2466c dnaK c l p B Rv04 8 5 mshB cysQ Rv0818 C C C G C T G G C G A A j j A C C A A A G T C C G G C T T T l T G C A G T G C C T A C G C T 1 G G G T G C G G G G C G G C G A C C C G C A C G A C G A C T C A A C A T T G A | G C G C C G G T G A C C A C G ' G C I C C C G A T C T C G T G J J J A T T T G C G A C C T J g T C G G C G C C G A C G G C G C | G A T T G G T T § J G G T T C A A C C C C A T G A C A G JC G T T G T G T I P A A G A T G T G C T G G G I G G T G T G C A T G C T ' C A G T A A I G A A C A A C C G . G A C A A I mi I T M C I B I G C G G G T G C G C G G C C G T T T | C C C A A C G T G A G C C C G G C A C G G 0 G A G A A C G G C C A C G g Distance from start codon (bp) 198 29 31 42 123 37 201 216 197 182 F i g . 18: Putative S i g H promoter binding sites. These sites were predicted in the upstream regions of Rv0485, Rv0818, cysQ, and mshB, based on the known consensus of SigH promoter regions located upstream of sigH, sigB, trxB2, Rv2466c, dnaK, and clpB. The published sigH consensus sequence is S G G A A C - N 1 7 . 2 2 - S G T T S (N = any nucleotide; S = G or C) (Manganelli et al, 2002) Preliminary gel analysis of the individual protein preparations under denaturing conditions with the detergent SDS (shown in Fig. 19), reveals that there are indeed some degradation products, but the amounts are slight compared to the amounts of intact protein since the degradation products are only visible upon silver staining. In the binding reactions, full-length S igH and MBP-Rv0818 proteins were incubated separately or in combination, in the presence of 5 m M D T T (reducing), 5 m M diamide (oxidizing), or under neutral conditions, at 37°C. The reactions were then resolved on a non-denaturing gel and visualized by silver staining. The results are shown in Fig. 20. The presence of multiple bands in all the lanes containing one 44 protein sample only, suggests slight contamination of the samples with the degradation products seen in the silver-stained gel of Fig. 19. However, it appears that the majority of Rv0818 and SigH runs as single discrete bands, distinct in size from one another. A s seen in Fig . 20, under all the treatment conditions, the lanes with combined proteins contain all the bands present in the lanes containing single protein samples. Unfortunately, we did not see evidence of any bands present in the combined samples that were not already present in the individual samples, indicating that no Rv0818-SigH complexes were formed under these conditions. However, a fortuitous decision to incubate the samples at 42°C for 20 minutes prior to loading on a native gel, resulted in the discovery of a band-shift suggestive of a Rv0818-SigH complex (Fig. 21). This slight heat-shock yielded a suspected band-shift only occurring in the lane containing both Rv0818 and SigH proteins in the presence of diamide. Unfortunately, repeated trials under the exact same conditions did not produce the same result, leading us to conclude that i f a protein-protein interaction between Rv0818 and SigH does indeed occur under oxidizing and heat shock conditions, it may only be transient. M W S i g H SigH-t r Rv0818 M W S i g H S igH- t r Rv0818 F ig . 19: SDS gel analysis of Rv0818 and S i g H proteins. The gels were stained with Coomassie Blue (gel on left) and silver staining (gel on right). MW=molecular weight marker lane. SigH-tr is the truncated N-terminal portion of SigH. Rv0818 was tagged with a maltose-binding protein. 45 Neutral 5 m M DTT 5 m M diamide Rv0818 + SigH + + + Fig. 20: Protein-protein binding assay of Rv0818 and SigH at 37°C. Silver-stained native gel of Rv0818 and SigH protein binding under neutral, reducing, and oxidizing conditions at 37°C. Representative of N=3 trials. Neutral SigH **** mm 5 m M DTT SmM diamide + - + + - + + - + - + + - + + - + + Fig. 21: Protein-protein binding assay of Rv0818 and SigH at 42°C. Silver-stained native gel of Rv0818 and SigH protein binding under neutral, reducing, and oxidizing conditions at 42°C. N=l. 4 6 5.0 D I S C U S S I O N 5.1 T H E M S H : M S = S M R E D O X B A L A N C E I N M Y C O B A C T E R I A The experiments conducted in the first part of this thesis were designed to address the hypothesis that exposure to oxidative and nitrosative stresses w i l l induce changes in total mycobacterial M S H levels and create fluxes in the basal M S H : M S = S M redox status. Wi th this goal in mind, we set about to characterize changes in total M S H levels and M S H : M S = S M redox status in response to time-course treatments with diamide, H2O2, and gNO, using M. bovis B C G a n d M smegmatis as models. We found that the total levels of reduced M S H in M. smegmatis in stationary phase in vitro are generally higher (ranging up to 40nmol per 10 9 cells) than those of M. bovis B C G (ranging up to 25nmol per 10 9 cells), while the levels of oxidized M S H are much lower (usually less than lnmol per 10 9 cells) in both species. Thus, the M S H : M S = S M ratios in M. bovis B C G and M. smegmatis in stationary phase in vitro are ~ 50:1 and -200:1 respectively, which are comparable to the G S H : G S S G ratios of-100:1 found in other GSH-producing prokaryotes and eukaryotes (Gilbert, 1990). These similarities probably reflect the conservation in function of cellular disulfide reductases and related thiol-based buffering systems in nature, but it is not known why these particular ratios are favored. The extent to which these ratios are altered upon oxidative stress, are also significantly different between these two model organisms. The redox ratios in M. bovis B C G were significantly altered upon treatment with diamide and H2O2, while the ratios in M. smegmatis remained unaltered (or were altered to a lesser extent) during the treatments. The robustness of the M. smegmatis response may be explained by the fact that M. smegmatis has a higher cellular level of reduced M S H and higher basal redox ratio than M. bovis B C G (Newton et al., 1996), and thus is resistant to the amounts of oxidative and nitrosative stress we used in our assays. In vivo, this is relevant in terms of the environment in which M. smegmatis normally resides. Present in soil, M. smegmatis is in an environment composed of various stressors generated by other soil bacteria and fungi, as well as heavy metals and other pollutants. Survival under exposure to these toxins requires a robust, quick detoxification system such as M S H provides. Indeed, some MSH-producing soil-dwelling actinomycetes such as Rhodococcus are currently being employed for bioremediation of pollutants such as diesel oi l (Lee et al, 2006). In contrast, M. tuberculosis, and by extension M. bovis BCG, face a far different environment in the human host, which is not a "toxic" environment per se. The thiol systemic protectant for detoxification is perhaps superceded by other mechanisms that are 47 specific to survival inside phagocytes, such as mechanisms to inhibit phagosome-lysosome fusion and the host inflammatory response. Another possibility, is that having evolved as an environmental saprophyte, instead of a naturally obligate human pathogen, M. smegmatis has developed alternate systems of defense against oxidative and nitrosative stressors, which may have more significant roles than M S H in protecting this organism against these specific toxins. A n example of this is ahpC which is differentially expressed in mycobacteria upon oxidative stress. A h p C protein is induced in M. smegmatis upon exposure to H2O2, but is not detectable by immunological methods in M. tuberculosis H37Rv upon the same stress treatment (Dhandayuthapani et al, 1996; Sherman et al, 1995), even though b o t h M tuberculosis a n d M smegmatis mutants of ahpC have increased sensitivity to O N O O " (Master et al, 2002). However, M. tuberculosis is more resistant to ONOO" than avirulent species such as M. bovis B C G a n d M smegmatis (Yu et al, 1999), illustrating that the phenomenon of mycobacterial resistance to ROIs and RNIs is complex and diverse both in mechanism and species specificity. Upon treatment with the specific thiol oxidant diamide, the redox ratios in M. smegmatis were unaltered, although there were slight but significant increases in the total levels of oxidized M S H at the 4 and 8 hour time points in M B 7H9 media. There was no change in total reduced M S H levels of M. smegmatis in 0.9% saline. In contrast, the reduced M S H levels in M. bovis B C G in 0.9% saline dropped up to 4-fold by the first time point, mirrored by a 10-fold increase in oxidized M S H levels. This is reflected by a drop in the redox ratios of roughly two orders of magnitude and with no significant recovery by the end of the 8 hour treatment. Incubation with diamide in normal M B 7H9 growth media also induced a decrease in total reduced M S H levels in M. bovis B C G , mirrored by an increase in oxidized M S H levels, resulting in a drop of the redox ratio of - 1.5 orders of magnitude, but. we see a recovery by the end of 8 hours to a redox ratio of - 5:1. This difference due to the assay medium was even more dramatically illustrated with the addition of H2O2, which had no effect on the total reduced M S H levels or redox ratio of M. bovis B C G in M B 7H9 growth media, but caused a 1 order of magnitude drop in redox ratio of M. bovis B C G in normal saline. Similarly, M. smegmatis treated with H2O2 demonstrated slight but statistically significant decreases in reduced M S H levels in 0 .9% saline, but were unaffected when treated in M B 7H9. There are several possibilities as to why these differences were observed, one being that M B 7H9, or indeed any growth media, contains numerous compounds which may chemically neutralize the oxidants, diamide and H2O2, and/or their effects on the bacteria. O A D C , the most commonly used mycobacterial growth supplement, was replaced with A D S in our assays to eliminate the confounding effects of catalase, but there may 48 have been other components in M B 7H9 which we did not account, for (i.e. the oxidants ammonium, magnesium, zinc, and copper sulfates). The other explanation is that the proper functioning of enzymes which influence the redox ratio, such as the mycothiol disulfide reductase mtr, may depend on having suitable growth conditions. Other mycobacterial defense mechanisms such as katG, sodA/C, and the thioredoxins, may also have reduced activities while under stasis/starvation in the saline medium (Muttucumaru et al., 2004). The recovery of M S H levels in M B 7H9 media may also be influenced by the organism's ability to transcribe new copies of mtr and other genes, which is most certainly attenuated under static conditions (Muttucumaru et al, 2004). Another notable observation is that the drop in M. bovis B C G M S H levels (reduced form) upon diamide treatment in saline is also accompanied by a parallel increase in oxidized M S H levels. However, the amount of M S = S M , detected as oxidized M S H in the H P L C assay, does not correspond in a 1:1 ratio with the amount of reduced M S H that was depleted, suggesting that there is a "leak" in the system, either technical in terms of detection of M S H via the H P L C assay, or biological in terms of unknown, indirect effects of diamide on the mycobacteria, leading to the formation of S-conjugates or other forms of M S H depletion. There is no significant change in the M S H : M S = S M ratio past the first time point, suggesting that the cells rapidly reached a steady-state system where the reductase Mtr is possibly saturated (or perhaps inactive), yet the cells still have enough cellular M S H to maintain viability in static culture for up to 8 hours. In M B 7H9-A D S media, we also see a drop in reduced M S H levels, however, as these levels begin to recover by the 4 hour time point, we see a corresponding decrease in oxidized M S H levels, suggesting that in the presence of M B 7H9 growth media, the cells are able to counter the diamide threat by recycling the oxidized M S = S M , probably via Mtr. A s with the saline experiment, there appears to be a "leak" in total M S H levels, perhaps due to some unknown buffering capacity of the M B 7H9 media. Upon H2O2 treatment of M. bovis B C G in 0.9% saline, we see a rapid decrease in levels of reduced M S H accompanied by a slight increase in levels of oxidized M S H . The slight recovery of M S H at the end of the treatment period is also mirrored by a slight decrease in oxidized M S H levels, indicating that Mtr is not completely saturated in this system. This suggests that in addition to the known indirect relationship between ROIs and M S H , where oxidatively-damaged molecules/proteins can be detoxified by M S H via the formation of S-conjugates, there may also be a direct relationship where H2O2 acts like diamide and is a direct oxidant that mediates the depletion of reduced form M S H to the oxidized form M S = S M . A s with 49 the diamide experiments, the amount of M S = S M detected as oxidized M S H does not correspond in a 1:1 ratio with the amount of reduced M S H that was depleted. The "leak" here is probably due to the indirect effects of H2O2 on the cells and M S H levels, namely the generation of free hydroxyl radicals and subsequent oxidative damage of proteins and cell compounds, leading to the depletion of M S H in the form of S-conjugates and not M S = S M . A s the overall depletion of reduced M S H in cells treated with H2O2 and incubated with normal saline was less pronounced than in the cells treated with diamide, it is not that surprising to see that incubation with M B 7H9 growth media effectively masks the effects of treatment with H2O2. This masking effect could be due to the buffering capacity of the media, and/or the activation of mycobacterial defense mechanisms such as Ka tG , thioredoxins, and others discussed previously, in the presence of the growth media. As described previously, the relationship between gNO and M S H has been controversial. Data generated from our laboratory is conflicting in that some experiments demonstrate increased sensitivity of msh mutants to gNO, as well as a decrease in M S H levels upon treatment with gNO (Miller, 2004), while other experiments have shown no difference, or even decreased sensitivity of msh mutants to gNO exposure (Av-Gay, unpublished data). The data from our experiments support the latter results, but the issue is far from being solved. With M. smegmatis, we observed a slight but statistically significant decrease in total reduced M S H levels of about 6nmol per 10 9 cells upon gNO treatment in M B 7H9 media at the 8 hour time point, however there was no significant change in the M S H : M S = S M redox ratio at that time point even though there is no significant change in total oxidized M S H levels. However, an apparent increase in oxidized M S H levels at the 4 hour time point is responsible for a corresponding drop in the redox ratio which was calculated to be statistically significant. In contrast, with M. bovis B C G , there were no significant changes in total reduced M S H levels upon treatment with gNO in either 0.9% saline or M B 7H9. However, there was a slight but statistically significant drop in oxidized M S H levels at the 8 hour time point in 0.9% saline. The treatment conditions we used in our assays are comparable to the ones used in previous studies, however, one major difference is that we used stationary phase M. smegmatis whereas the first experiments (Miller , 2004) used log-phase bacteria. Both experiments are valid in their own ways, but it may not be useful to directly compare them. The mechanisms of gNO detoxification may simply be growth-phase dependent; it is perhaps MSH-mediated in the log-phase and mediated by another defense mechanism, such as the proteasome, in the stationary phase. It should be noted that the putative substrate of M S H -dependent gNO detoxification, S-nitrosomycothiol ( M S N O ) has not yet been identified in 50 mycobacterial cells, and although M s c R (involved in formaldehyde detoxification in mycobacteria) has been demonstrated to have nitrosothiol reductase activity in vitro, the predominant product of M S N O metabolism, MSH-sulphinamide and its hydrolysis products produced in vitro could not be isolated from intact cells exposed to M S N O . M s c R was shown to metabolize G S N O into GSH-sulphinamide with stoichiometric by-products of GS=SG and nitrate in vitro, thus one would predict that i f M s c R metabolizes M S N O into MSH-sulphinamide in vivo, there would be a parallel accumulation of M S = S M (Vogt et al, 2003), and not the decrease seen in our assays. One possibility for the decrease in oxidized M S H levels is that i f gNO detoxification is indeed partly MSH-mediated in this system, the effect is subtle and requires only a fraction of the bacteria's M S H supply, which is more easily "re-stocked" via the reduction of M S = S M by mtr than by ab initio biosynthesis of M S H . A s there was no observed initial increase in M S = S M levels, the depletion of M S H in this case may either be direct in the form of M S N O or indirect in the form of M S - R compounds where R represents nitrosatively-damaged proteins. A s discussed previously, the antioxidant properties of M S H are due to the presence of the sulphur atom of the amino acid cysteine, functional only in its reduced (-SH) form. M S H can either be spontaneously oxidized to M S = S M (as investigated in this thesis), or spontaneously or enzymatically form S-conjugates with a variety of compounds including alkylating agents, electrophiles, and antibiotics. These S-conjugates are subsequently cleaved by the amidase Mca , to result in GlcN-Ins and the modified toxin AcCys -R . This mercapturic acid is then excreted from the cell (via an as yet unidentified mechanism), and GlcN-Ins is recycled back into the M S H biosynthetic pathway. If M S N O is not metabolized by M s c R in vivo, and is instead metabolized by M c a via the S-conjugates detoxification pathway, then the resulting products would be A c C y S - N O (S-nitrosocysteine) and GlcN-Ins. We made preliminary attempts to infer the possible metabolism of M S N O via the S-conjugates detoxification pathway by conducting H P L C analyses of GlcN-Ins levels in log-phase M. smegmatis exposed to 200ppm gNO for 2 hours, using the protocol developed by Anderberg et al. (1998). Even though there seems to be a slight increase in GlcN-Ins levels upon gNO treatment, this was not found to be statistically significant (Fig. 22). The true role(s) of M S H in gNO-resistance among different mycobacterial species is yet to be discovered. 51 15 5 M l control gNO F i g . 22: Pre l iminary H P L C analysis of GlcN-Ins levels in log-phase M. smegmatis mc 2155 exposed to 200ppm g N O for 2 hours. Shown are the means and S E M s of N=3 experiments. In a recent publication (Newton et al., 2005), the novel, modified M S H assay used in our experiments to describe M S = S M levels, was described and validated. Their results generally confirmed our observations of the basal redox state of M. smegmatis, although their ratios ranged from 200 to 1000:1. The authors succeeded in characterizing two novel thiols (N-formyl-Cys-GlcN-Ins and N-succinyl-Cys-GlcN-Ins) and the resulting altered thiol redox state in the M. smegmatis mshD mutant. They found that the accumulation of the M s h D substrate, C y s - G l c N -Ins, in the mutant, enables the production of the novel thiols, and in its facile reaction with C o A to produce acyl-Cys-GlcN-Ins, is itself sufficient to confer protection to M. smegmatis from exposure to peroxides. However, it still appears that in M. tuberculosis, mshD is vital for survival in macrophages (Rengarajan et al, 2005). The unique role of M S H as a systemic protectant in mycobacteria, as well as its added importance as a species-specific defense mechanism among members of the pathogenic M. tuberculosis complex, make M S H metabolic pathways ideal targets for developing anti-infectives which wi l l be effective at all stages of the mycobacterial life cycle. 5.2 M S H T R A N S C R I P T I O N A L R E G U L A T I O N To determine i f the putative transcriptional regulators Rv0485 and Rv0818 have a role in MSH-dependent responses to oxidative stress, we began by investigating changes in gene expressions of Rv0485 and Rv0818 of M. bovis B C G in response to depletion of M S H via diamide treatment, and macrophage infections. We then performed protein binding assays to determine i f there are protein-protein interactions between Rv0818 and SigH. 52 Little is known about Rv0485 and Rv0818 except that they both contain conserved domains of known transcriptional regulators, and were both found to be non-essential genes for optimal growth in vitro on normal media (Sassetti et al., 2003). The observation that the transposon mutant of Rv0485 in M. tuberculosis is significantly attenuated for survival after 7 weeks in B A L B / c mouse lungs (Lamichhane et al, 2005) is intriguing, but is tempered by the observation that Rv0485 is almost immediately upstream from mshA (48 bp apart) and thus the attenuation of survival may very well be the effect of aborted transcription of mshA. Mutants of Rv0818 have not been observed, although since Rv08J8 is also directly upstream of mshD (overlapping by 4 bp), it would be difficult to elucidate the exact effect of an Rv0818 knockout. Bioinformatics analyses reveal further clues: Rv0485 is predicted to be cytoplasmic (although its basic theoretical p l suggests membrane localization) with no transmembrane domains. Rv0818 has a strong probability of being cytoplasmic as it shows homology to a known cytoplasmic transcriptional regulatory protein, afsQl , in S. coelicolor, as well as in Enterococcus and Shigella. 3D structural studies of Rv0818 are now underway at U C L A . The principal investigator of those studies, Dr. Markus Kaufmann, kindly provided us with purified Rv0818 and SigH protein for our assays. Our controls for the macrophage infection assays were bacteria incubated in cell growth medium. A n additional negative control for qPCR, with J774.1 murine macrophage c D N A , would normally have been appropriate to ensure that the amplification seen was not due to background amplification of a host cell gene instead of a mycobacterial gene. However, as our melting curve analyses and gel analyses indicated that we had single band amplification at the expected sizes for all assays, and given the specific nature of the q P C R primers, as well as the high genomic dissimilarity between eukaryotic and mycobacterial genomes, it is highly unlikely that co-amplification of host D N A or R N A occurred. Our q P C R results show that gene expressions of both Rv0485 and Rv0818 (relative to the house-keeping gene sigA) are unaffected upon 1 and 4 hour treatments with 5 m M diamide in M B 7H9 media. A s expected, sigH was upregulated about 7-fold with 1 hour of diamide treatment (comparable with 20-fold in previous studies using log-phase M. tuberculosis exposed to 5 m M diamide for 1 hour (Manganelli et al, 2002)), and about 26-fold with 4 hours of diamide treatment, indicating that the assay conditions were at least successful. However, the absence of change in gene expressions of Rv0485 and Rv0818 in this scenario does not yet rule out the possibility of these genes playing an important role in mycobacterial metabolism. It is also 53 possible that the gene expressions may be dependent on growth phase, and we might have observed changes i f log-phase bacteria were used instead of stationary-phase. We also looked at gene expressions of Rv0485 and Rv0818 (relative to the house-keeping gene sigA) upon infection (MOI of 10) of J774A.1 murine macrophages with log-phase M. bovis B C G for periods of 6 and 18 hours. The "untreated" controls in these assays were bacteria incubated in cell medium for two hours. A s expected, hspX, a 14kDa mycobacterial heat-shock protein homologous to a-crystallin, was highly upregulated by about 14-fold at 6 hours post-infection and about 60-fold at 18 hours post-infection. A s another indicator of the success of the infection process, J774A. 1 macrophages infected with FITC-labeled M. bovis B C G were harvested 2 hours post-infection and stained with Trypan Blue prior to fluorescence microscopy analysis. Trypan Blue quenches any extracellular fluorescence of bacteria which may not be internalized, and can be visualized using a red fluorescence filter. The merger of images captured via the FITC and red filters indicate that the mycobacteria are indeed internalized at an M O I of 10 at 2 hours post-infection. The q P C R results show that both Rv0485 and Rv0818 are upregulated upon infection of J774A.1 macrophages. Rv0485 is upregulated by about 10-fold at 6 hours post-infection, and by about 9-fold at 18 hours post-infection. Rv0818 is upregulated by a similar amount of about 7-fold at both 6 and 18 hours post-infection. These results suggest that Rv0485 and Rv0818 might have important roles in the mycobacterial response to macrophage infection, although given the results of the diamide experiments it is not clear i f these roles are indeed linked to mycothiol metabolism as we previously hypothesized. The conditions of our protein-binding assays were replicated from previous studies on the binding of S igH and its cognate anti-sigma factor, RshA (Song et al, 2003). S igH and Rv0818 proteins were incubated separately and in combination in either neutral, reducing (with DTT) , or oxidizing (with diamide) conditions. The preparations were then resolved on native non-denaturing gels and analyzed for bandshifts which might indicate the formation of new protein complexes. Initially, after multiple assays, we did not observe any band shifts after incubation at 37°C. However, after a fortuitous decision to incubate at 42°C prior to loading on the native gel, we observed a possible band shift in the preparation where S igH and Rv0818 proteins were incubated together in the presence of the oxidant diamide. This suggests that S igH and Rv0818 may interact with each other and are sensitive to not only oxidative stress, but also slight heat shock. S igH has also been shown to be involved in the mycobacterial heat shock response (Manganelli et al, 2002; Song et al, 2003). Although our results were intriguing, we have not been able to repeat this bandshift of SigH with Rv0818 in subsequent trials, suggesting that the 54 interaction may be only transient. Future studies would have to include in vitro transcription assays to determine i f these proteins form a true interaction for the purpose of transcription. A putative visual model of how such interactions may occur is shown in Fig. 23. As can be seen, under normal reducing conditions in the cell, SigH is bound by its anti-sigma factor RshA and is inhibited from transcription. Upon the induction of stress conditions such as infection, oxidizing conditions (i.e. diamide treatment), or heat-shock, the RshA (also an oxidative and heat stress sensor) binding with SigH is interrupted. SigH is now free to bind with the mycobacterial RNA polymerase to form a functional holoenzyme and begin transcribing from SigH-dependent promoters. We hypothesized that Rv0485 and Rv0818 are among the genes that are transcribed, and that increased expression of these genes and their protein products may facilitate further auto-transcription as well as transcription of the downstream genes mshA and mshD. It is possible that Rv0485 and Rv0818 may act as positive transcriptional regulators by increasing the efficiency of the R N A polymerase holoenzyme binding to SigH-dependent promoters. There may also be a need for other regulatory factors to stabilize this transcriptional complex i f the nature of the binding between Rv0485 or Rv0818 and SigH is transient as our results may suggest. From our results, it is clear that at both Rv0485 and Rv0818 play important roles in the mycobacterial response to macrophage infection. It is unclear if these roles are linked to M S H metabolism, as originally hypothesized, nevertheless they are still worthy targets of future research into the transcriptional regulation of MSH-related genes, and merit further investigation. + ^™ upstream... Rv0485 mshA t Infection Oxidants Heat shock Fig . 23: Putative model of SigH-mediated transcription involving Rv0485 and Rv0818. 55 6.0 FUTURE WORK In order to determine the true roles of Rv0485 and Rv0818 in M S H metabolism, it is necessary to investigate them on the protein level. Rv0485 has not yet been targeted for structural studies by the T B Structural Genomics Consortium ( U C L A - D O E ) , thus the first steps would be to clone, express and purify Rv0485 as either a M B P or His-tag containing protein. A variety of assays can then be performed with purified Rv0485 and Rv0818. We might first perform protein-protein binding assays to determine i f Rv0485 binds to S igH and forms a complex under reducing or oxidizing conditions. To test i f these proteins are true transcriptional regulators, we would have to perform electrophoretic mobility shift assays ( E M S A ) and in vitro transcription assays. E M S A ' s are simple assays which can determine i f the protein of interest forms a complex with the appropriate D N A fragment. In brief, we would incubate either Rv0485 or Rv0818 with 3 2 P-labelled D N A fragments containing the putative binding site (predicted through bioinformatics), then load the reaction on a native gel. B y comparing the lanes containing the reaction with lanes containing only the protein or only the D N A fragments, we wi l l be able to see a band shift i f there is indeed a DNA-protein interaction that occurs. DNA-protein complexes are expected to migrate more slowly through a native gel than free D N A fragments. We can test multiple conditions of binding via incubation with or without S igH, in the presence or absence of diamide, D T T , or heat-shock, etc. Competition assays using D N A fragments without the binding sites may also help to establish the specificity of the DNA-protein interaction. If the interaction is confirmed, we may proceed to a D N A footprinting assay to determine the exact position of the binding site in the complex. Basically, the assay entails incubation of the protein and D N A fragment to form a complex, the addition of DNasel to cleave the portion of the D N A fragment that is not "protected" by the binding of the protein, then the analysis of the cleavage product on a Maxam-Gilbert sequencing gel to determine the exact sequence of the protected fragment. The filter binding assay is another simple method to characterize the DNA-protein interaction. First, the 3 2P-labeled D N A fragment is incubated with the protein to form a complex, then the reaction is passed through a nitrocellulose filter. Double-stranded D N A w i l l pass through the filter, but the DNA-protein complexes w i l l be retained. B y quantitating the amount of radioactive D N A retained by the filter, we can determine the binding constant of the DNA-protein interaction. To determine i f the DNA-protein complex is able to mediate transcription, we can perform in vitro transcription assays. In brief, the R N A polymerase holoenzyme is reconstituted via incubation of the protein with S igH and E. coli core R N A polymerase ( if the mycobacterial version is unavailable). The holoenzyme is then incubated with the D N A fragment containing the 5' end of 56 the gene of interest and upstream sequences including the putative binding site, and with P-labelled dUTP, along with the other unlabelled dNTPs. After purification of the unincorporated radioactive dNTPs, the resulting reactions are then run on a denaturing polyacrylamide gel to visualize the presence of the new radioactive transcripts i f transcription does indeed occur. The biological effects of Rv0485 and Rv0818 can also be elucidated through the generation of single-knockout mutants. One effective method for generating mutants is via specialized transducing mycobacteriophages (Bardarov et ah, 2002). We have begun preliminary work on this. In brief, first an allelic exchange substrate (AES) is constructed by cloning the left and right flanking regions of the gene of interest into the E. coli cosmid p Y U B 8 5 4 . This cosmid contains an origin of replication for E. coli, a unique Pad restriction digestion site, a unique X-cos site to allow in vitro packaging into phage X heads, and a selectable marker gene for hygromycin resistance flanked by multiple cloning sites for directional cloning of the flanking regions into either side of the hygromycin resistance gene. In the second step, the A E S is cloned into the conditionally replicating mycobacteriophage shuttle phasmid vector phAE87 via the unique Pad site. The phasmid is then packaged into X phages heads using an in vitro X packaging reaction kit, transduced into E. coli, and plated onto media containing hygromycin. The successful hygromycin-resistant colonies are then pooled and their cosmid D N A is purified. M. smegmatis cells are then electroporated with the purified cosmids and plated for phage plaques at 30°C. This is the permissive temperature that allows for propagation of the mycobacteriophages. The plaques are then purified and used to infect the recipient mycobacteria (such as M. bovis B C G ) at the non-permissive temperature of 37°C. Since phage replication is restricted at this point, there wi l l be accumulation of abortive transductants. This leads to allelic exchange via double crossover of the flanking regions of the gene of interest with the flanking regions of the A E S containing the hygromycin resistance gene. The successful knockout colonies can then be selected for on hygromycin-containing media. The generation of single-knockout mutants for Rv0485 and Rv0818 w i l l enable us to determine the biological effects of these genes via the same kinds of assays described in this thesis. A s a first step, it would be necessary to assay the expressions of the downstream genes mshA and mshD to rule out the possibility of a "double-knockout" via aborted transcription of those genes. If we are successful in generating true single-knockouts, we can then proceed to conventional analyses of growth characteristics, basal M S H levels, responses to oxidative and nitrosative stressors, etc. to help determine the roles of these putative transcriptional regulators in MSH-dependent responses. 57 We hope that characterization of these thiol-dependent processes in mycobacteria will help us understand how the pathogen M. tuberculosis readily adapts to oxidative/nitrosative stresses, and yield potential novel drug targets for disabling such systems and eliminating TB. 58 7.0 A D D E N D U M A.1 R O L E O F G L U T A T H I O N E I N H O S T C E L L D E F E N S E In a series of surprising studies, it was shown that mycobacteria are sensitive to G S H and G S N O in vitro and in vivo. Using a M. bovis B C G mutant of oppD in the oligopeptide permease operon, it was shown that the presence of 0 .5mM G S H (less than physiological amounts of G S H in a host cell) inhibits [ 3H]uracil incorporation in wild-type B C G by more than 50%, whereas the mutant is unaffected. It was also shown that while G S H is bacteriostatic, its derivative G S N O is bactericidal to M. bovis B C G in liquid culture (Green et al, 2000). Follow-up studies demonstrated that the oligopeptide permease mutant had significantly increased survival compared to wild-type B C G in both unstimulated and IFN-y + LPS-stimulated murine macrophages. Stimulation of these macrophages with IFN-y (interferon-gamma) cytokine and L P S (lipopolysaccharide) resulted in increase of cellular nitrite production and decrease in G S H level, suggesting formation of intracellular G S N O . Furthermore, the mutant demonstrated hypersurvival compared to wild-type in N O S 2 7 " murine macrophages (Venketaraman et al, 2003). Similar results were shown for in vitro exposure of M. tuberculosis H37Rv to G S H / G S N O and for in vivo exposure to murine macrophages (Venketaraman et al, 2005). Unfortunately, these results were not replicable in human monocyte-derived macrophages (Venketaraman et al, 2003). The mechanics of G S H and G S N O toxicity against mycobacteria are unknown. It has been proposed that the structure of G S H is similar to penicillin precursors, thus G S H might be a precursor of antibiotics pre-dating cellular immunity, for which mycobacteria might possess some rudimentary sensitivity (Spallholz, 1987). Another hypothesis is that high concentrations of another low-molecular weight thiol might throw-off the basal redox balance and activity of mycothiol and thus result in adverse cellular effects (Green et al, 2000). Intercellular transport is also an issue; there are no known G S N O or G S H transporters, and it is not clear i f these molecules are able to passively diffuse across the mycobacterial cell wal l in significant amounts. In Salmonella typhimurium and E. coli, a periplasmic gamma-glutamyltranspeptidase (GGT) cleaves the y-glutamyl moiety from G S H , and the resulting dipeptide is able to enter the cell through a dipeptide permease system (dpp) homologous to mycobacterial opp (De Groote et al, 1995). However, exposure of B C G to the dipeptide or its constituent amino acids did not result in the significant inhibition of growth associated with exposure to G S H (Green et al, 2000). Nevertheless, G G T activity on G S N O may also release an N O dipeptide which might be the 59 effective toxic species. G G T activity has been demonstrated in M. smegmatis and other mycobacterial species (Kumar et al, 1990; Shetty et al, 1981), but has not been correlated with the two putative ggt genes (ggtA, ggtB) coded for in the M. tuberculosis genome (Cole et al, 1998). There are many aspects of thiol-dependent interactions between microbe and host, which have not been elucidated and merit further investigation. In this addendum, we hypothesize that the putative y-glutamyltranspeptidases GgtA and GgtB have roles in mycobacterial responses to glutathione stress. We tried to determine i f these putative ggt genes are involved in this process by investigating changes in gene expressions of ggtA and ggtB in M. bovis B C G in response to exposure to G S H in vitro, and in response to macrophage infections. We hope that characterization of these thiol-dependent processes in mycobacteria w i l l help us understand how the pathogen M. tuberculosis reacts to the host environment, and yield potential novel drug targets for disabling such systems and eliminating T B . A.2 G S H - D E P E N D E N T E N Z Y M E S I N A C T I N O B A C T E R I A Since G S H is not present in mycobacteria, it is surprising to find that the M. tuberculosis genome contains two putative y-glutamyltranspeptidase genes, ggtA and ggtB. A s discussed previously, G G T enzyme activity has been observed in mycobacteria, but has not been definitively linked to these genes. ggtA and ggtB have not yet been shown to encode functional enzymes in vitro. A s a preliminary analysis of mycobacterial responses to host glutathione, we wondered i f there might be other GSH-dependent enzymes encoded in the genomes of mycobacteria and other related genera within Actinobacteria. Preliminary B L A S T analyses limited our queries to y -glutamyltranspeptidase and y -glutamylcyclotransferase encoding genes since other GSH-dependent enzymes such as glutathione peroxidase, G S H dehydrogenase ascorbate, G S H S-transferase and GSH-dependent reductases have several Pfam entries and thus represent broad ranges of protein domains with weak overall consensus sequences that are not amenable to B L A S T analysis. In other prokaryotes, G G T is an outer membrane protein and is the key player in the y -glutamyl cycle for the synthesis and degradation of G S H . The sequences of mammalian and bacterial G G T show regions of high similarity and generally encode a single chain precursor which is further processed into two polypeptide chains. The active site is usually located on the 60 light subunit. The ggtA and ggtB genes in M. tuberculosis are unique in relation to each other, having only 29% identity with 12% gaps within a 166-residue fragment of the 500+ residue predicted protein sequences. However, they are exactly conserved within the M. tuberculosis complex of M. tuberculosis H37Rv, M. tuberculosis C D C 1551, M. bovis, a n d M . bovis B C G . There is a single amino acid substitution in GgtB M. bovis. Both genes are annotated as non-essential genes via transposon mutagenesis analysis i n M tuberculosis H37Rv (Sassetti et al., 2003). P S I - B L A S T analyses of M. tuberculosis H37Rv GgtA against N C B I ' s non-redundant protein database, limited to Actinobacteria, and using default settings, produced no additional hits after the third iteration. The results are shown in Table A . 1. Within mycobacteria, in addition to the fully conserved ggtA genes in the species mentioned above, there is an 80% identity match, along the entire protein sequence with no gaps, to a probable Gg tA in M. avium paratuberculosis, and a 65% match, within a 44 residue segment with no gaps, to one in M. leprae (probably a non-functional pseudogene). Other hits within Actinobacteria include - 3 0 % identities, along the entire amino acid sequence, to Corynebacterium species, Kineococcus radiotolerans, Nocardia farciniq, and Streptomyces species. 61 Table A . l : Summary of P S I - B L A S T hits for GgtA: query of M. tuberculosis H37Rv GgtA against nr protein database, using default settings, limited to Actinobacteria. Scores represent 3 iterations of P S I - B L A S T analysis. Accession no. Species name \alue identity % posili\e % gaps % BAC17839.1 Corynebacterium efficiensYS-314 e-l 60 167/566 29 246/566 43 65/566 11 CAF19662.1 Corynebacterium glutamicum A T C C 13032 e-l 64 156/565 27 247/565 43 64/565 11 NP 600182.1 Corynebacterium glutamicum A T C C 13032 e-l 63 156/565 27 247/565 43 64/565 11 BAB98347.1 Corynebacterium glutamicum A T C C 13032 e-163 156/565 27 247/565 43 64/565 11 7.1' 00354684.1 Kineococcus radioiolerans SKS302I6 e-l 40 169 581 29 227/581. 39 82 581 14 NP 215287.1 M tuberculosis H37Rv, C D C 1 5 5 1 . & M . bovis AF2122/97 0 512/512 100 512/512 100 0 0 |NP 959541.1 M . avium paratuberculosis klO 0 412/510 80 454/510 89 0 0 NP 856064.1 M . bovis AF2122/97 e-155 166/566 29 241/566 42 68/566 12 AAA63131.1 M . leprae 5.E-07 29/44 65 36/44 81 0 0 NP 216910.1 M . tb H37Rv and CDC1551 e-154 166/566 29 . 241/566 42 68/566 12 YP 119809.1 Nocardia farcinia IFMI0152 e-l 59 171/565 30 249 565 44 70 565 12 ZP 00188444.1 Rubrobacter xylanophilu DSM9941 e-177 229/516 44 286/516 55 9/516 1 ZP 00186490.1 Rubrobacter xylanophilu DSM9941 e-135 159/509 31 232/509 45 45/509 8 BAC69649 1 Streptomyces averminlis \1.\-4680 e-l 69 159 601 26 242 601 40 lllBllplilifflll 103 601 17 I3AC69668.I Streptoimces avermitilib MA-4680 e-l 56 173 554 31 237 554 42 54 554 9 CAA22753.1 Streptomvccb coelicolor A3(2) e-l 69 162 608 26 235 608 38 108 608 17 C A A 16202.1 Strcptomvces coelicolor A3(2) e-153 177/579 30 233/579 40 76 579 13 CAA19618.1 Stiepiomvces coelicolor A?(2) e-l 50 172/553 31 237/5,53 42 "53/553 9 C.\.\5>~46 1 Streptomyces lincolnensis e-l 66 170/591 28 231/591 39 84 591 14 AAG42852.1 Streptomyces nogalater e-l 02 104 353 29 153 353 43 32 353 9 YP 076992.1 Symbiobacterium thermophilum IAM14863 0 269/518 51 330/518 63 14/518 2 62 P S I - B L A S T analyses with M. tuberculosis H37Rv GgtB yielded similar results, shown in Table A . 2 . M. avium paratuberculosis and M. leprae do not appear to have GgtB homologs. The hits to Corynebacterium species and N.farcinia are to the same proteins identified in the GgtA search and have greater than 50% identity with minimal gaps, suggesting that these organisms may only have GgtB-like proteins. Hits to Rubrobacter species, again with the same proteins, had slightly more percent identity than with GgtA, and hits to Streptomyces species had roughly the same percent identities as with GgtA, suggesting that these organisms only have one G G T -like protein. Overall, it seems that ggtA and ggtB are well conserved among Actinobacteria. C. glutamicum has at least two distinct GgtB-like proteins; C. effwiens and N. farcinia both have one GgtB-like protein; S. thermophilum has one GgtA-l ike protein and R. xylanophilus has two distinct GgtA-like proteins. K. radiotolerans, S.nogalator, and S. lincolnensis each have one GGT- l ike protein, although none are strongly GgtA or GgtB-like. S. avertimilis has two distinct GGT- l ike proteins, and S. coelicor has three. Segments of the GGT- l ike proteins identified in the P S I - B L A S T analyses were aligned using ClustalW, then inputted into P H Y L I P to generate a neighbour-joining phylogenetic tree, using default settings, and rooted with E. coli G G T has the outgroup. The resulting visual representation of the P S I - B L A S T analyses is shown in Fig. A . l . As can be seen, the GGT- l ike proteins in Actinobacteria tend to cluster into two groups, represented by the GgtA and GgtB proteins of Mycobacteria. In comparing this phylogeny based on GGT- l ike sequences with a phylogeny based on 16S r R N A sequences, a commonly used indicator of bacterial evolutionary relationships (Fig. A .2 - adapted from the phylogenetic tree presented in Gao and Gupta (2005)), we notice that the branching of the GgtB-l ike protein sequences (i.e. the Cornyebacteria, Nocardia, Mycobacteria-containing clade) resembles the branching for these genera in the 16S r R N A tree. This suggests that the GgtB-l ike protein sequences may have evolved according to ancestral relationships. Thus the appearance of the GgtA-like protein sequences may represent a more recent evolutionary event such as a horizontal gene transfer. 63 Table A.2: Summary of P S I - B L A S T hits for GgtB: query of M. tuberculosis H37Rv GgtB against nr protein database, using default settings, limited to Actinobacteria. Scores represent 3 iterations of P S I - B L A S T analysis. Accession no. Species name value identiu % positive % gaps % Corynebacterium 0 344/638 53 414/638 64 18/638 2 BAC17839.1 efficiensYS-314 Corynebacterium 0 319/572 55 398/572 69 9/572 1 CAF19662.1 glutamicum A T C C 13032 Corynebacterium 0 337/650 51 427/650 65 22/650 3 NP 600182.1 glutamicum A T C C 13032 Corynebacterium 0 337/650 51 427/650 65 22/650 3 BAB98347.1 glutamicum A T C C 13032 Kineococcus radiotolerans e-135 154 604 25 227/604 37 84 604 13 ZP 00354684.1 SRS30216 M tuberculosis H37Rv, e-172 165/561 29 239/561 42 68 561 12 C D C 1 5 5 1 , & M . bovis NP 215287.1 AF2122/97 M . avium paratuberculosis e-162 164/561 29 236/561 42 68/561 12 |NP 959541.1 klO NP 856064.1 M . bovis AF2122/97 0 642/643 99 643/643 100 AAA63131.1 M . leprae 0.002 10/44 22 20/44 45 2/44 4 M . tb H37Rv and 0 643/643 100 643/643 100 0 0 NP 216910.1 CDC1551 Nocardia farcinia 0 405/629 64 486/629 77 8/629 1 YP 119809.1 • IFM10152 Rubrobacter xylanophilu e-121 160/556 28 232/556 41 85/556 15 ZP 00186490.1 DSM9941 Rubrobacter xylanophilu e-151 169/581 29 253/581 43 65/581 11 ZP 00188444.1 DSM9941 Streptomyces avermitilis e-169 159 669 23 239 669 35 1 16 669 17 BAC69649.1 MA-4680 .Streptomyces avermitilis e-159* 119/611 32 283 611 46 68 611 11 BAC69668.1 MA-4680 Streptomyces coelicolor e-158 160 560 24 235/650 36 117/650 18 CAA22753.I A3(2) Streptomyces coelicolor e-149 196 610 32 270 610 44 65 610 10 CAA19618.1 A3(2) Streptomyces coelicolor e-147 157/610 - 25 227 610 37 76 610 12 C A A 16202.1 A3(2) CAA55"746.I Streptomyces lincolnensis e-147 144/619 23 216 619 34 90/619 14 A A( 142852.1 Streptomyces nogalater c-153 163/648 25 234 648 36 121 648 18 Symbiobacterium e-155 160/585 27 234/585 40 72/585 12 YP 076992.1 thermophilum IAM14863 64 E.coli.O157.H7[NP312320.1] I C.efficiens[BAC17839.1] C.glutamicum[CAF19662.1] C.glutamicum[NP600182.1] C.glutamicum[BAB98347.1] I N.farcinica[YP119809.1] M.bovis[GgtB][NP856064.1] M.tb.H37Rv[GgtB][NP216910.1] M.tb.CDC1551[GgtB][NP336945.1] R.xylanophilus[ZP00186490.1] • • S.lincolnensis[lmbA][CAA55746. I K.radiotolerans[ZP00354684.1] I S.coelicolor[CAA16202.1] I S.avermitilis[BAC69649.1] _ I S.coelicolor[CAA22753.1] I S.nogalater[AAG42852.1] I R.xylanophilus[ZP00188444.1] | • S.thermophilum[YP076992.1] I M.leprae[AAA63131.1] '— I M.aviump.paratb[GgtA][NP959541 M.tb.CDC1551[GgtA][NP335225.1] M.tb.H37Rv[GgtA][NP215287.1] M. bovis[GgtA][N P854454.1 ] I S.avertimilis[BAC69668.1] S.coelicolor[CAA19618.1] 0.1 Fig. A . l : Phylogenetic tree of G G T - l i k e proteins in Actinobacteria: generated from an 805-residue alignment (including gaps) using ClustalW, and neighbour-joining using the P H Y L I P software package. A l l settings were default unless stated otherwise. 65 E. coli .— Cornyebacteria I Nocardia I 1 Mycobacteria I I : Streptomyces I Kineococcus I Rubrobacteria Symbiobacteria F i g . A . 2 : Simplif ied phylogenetic tree of Actinobacteria based on 16s r R N A sequences. Adapted from Gao and Gupta (2005). We then queried the predicted amino acid sequences of M. tuberculosis H37Rv ggtA and ggtB against several commonly used bioinformatics tools to see what could be predicted about these putative proteins: Since there were no suitable matches to existing templates in the Swiss Protein Data Bank, the 3D structures of these proteins could not be modeled. Query against the cell localization tool P S O R T B predicted both GgtA and GgtB to be extracellular; no internal helices were found. This was supported by GlobPlot 2.1 which predicted the proteins to be mostly globular and disordered; it also predicted the presence of a signal peptide in GgtB. Query against SignalP also predicted a signal peptide in GgtB with a probability of 0.998 and a maximum cleavage site probability of 0.336 between residues 28 and 29. Analysis with P R O S I T E revealed a prokaryotic membrane lipoprotein l ipid attachment site (PS00013) on GgtB. Analysis with InterProScan revealed an additional TonB-box motif (IPR010916) at the N -terminal of GgtA, which may facilitate the interaction of GgtA with the TonB protein. In E. coli, TonB interacts with outer membrane receptor proteins to facilitate active transport of low-concentration or impermeable substrates into the periplasmic space. However, it seems that so far TonB has only been observed in gram negative bacteria and P S I - B L A S T analyses do not reveal any TonB-like proteins in Actinobacteria. Fig. A . 3 shows the genomic contexts of ggtA and ggtB in the M. tuberculosis genome. ggtA is positioned directly downstream (51 bp apart) from Rv0774c (encoding a probable conserved protein with possible lipolytic activity). ggtB is positioned amidst a possible operon containing cysH (an essential gene involved in the sulfate activation pathway of the cysteine biosynthetic pathway), Rv2393 (encoding a conserved hypothetical protein), Rv2395 (encoding a probable conserved integral membrane protein), and PEPGRS41 (of the P E - P G R S family of mycobacterial proteins). It is interesting to note that this 66 possible operon lies directly upstream of the cysA 1-cysW-cysT-subl operon which is uniquely involved in inorganic sulfate transport in M. bovis B C G (Wooff et al, 2002). Ri/0769 Rv8771 Ry0775 cypl26 361620 L J 871619 Genomic context of ggtA and ggtB in M. tb H37Rv: From TubercuList (Institut Pasteur) Ru2390c i . . cysfll LEGEM) Coding sequences: v i ru I ence, detox, adapt ^ 4 lipid metabo I i sm ••4 information pathway ^ c e l l ya 11, process ^ stable RNA — • IS/phage — * PE/PPE intermediary metabol ism • unknown regulatory conserved ~ * conserved in Fl. bouis ^ r R N f l •tRNfi ggtA ggtB 311 Ml 512 1 111 211 3 » m no m ••< G_t;Iu_LranGpept. NCBI Conserved Domain matches for ggt/l and ggtB: G_glu_transpept=gamma-glutamyltranspeptidase Fig. A.3: Genomic context of ggtA and ggtB (NCBI-CDD, 2005; TubercuList, 2005). 6 7 Glutamine cyclotransferase, the intracellular partner of GGT in the y-glutamyl cycle, is present in C. glutamicum as a 258 residue protein. PSI-BLAST analysis of this sequence against the non-redundant database of Actinobacteria proteins reveals no significant hits after only the second iteration. The results are shown in Table A. 3. GCT-like proteins appear to be present in C. diphtheriae, C. efficiens, and N. farcinia, ranging from 45% to 61% sequence identity. As all of these species (except C. diphtheriae) possess GGT -^like proteins as well, it is likely that they have functional y -glutamyl cycles. The species with only GGT-like proteins or only GCT-like proteins likely either possess non-functional remnants of this metabolic process or have evolved to use these proteins for different purposes. Table A.3: Summary of P S I - B L A S T hits for Get: query of C. glutamicum A T C C 13032 G C T against nr protein database, using default settings, limited to Actinobacteria. Scores represent 2 iterations of P S I - B L A S T analysis. Accession no. Species name 1 -value idonliiv % positive § § • § l l l l l i l % caps M*_600051.1 Corynebacterium glutamicum A T C C 13032 e-130 258/258 100 258.258 100 0 0 NP_939144.1 Corynebacterium diphtheriae N C T C 13129 e-l 22 151/262 57 178/262 67 11/262 4 NP_737507.1 Corynebacterium efficiens YS-314 e-121 167/270 61 203/270 75 14/270 5 Y P l 16851.1 Nocardia farcinia IFM10152 e-l 07 117/257 45 154/257 59 7/257 2 68 A.3 E X P R E S S I O N O F G G T G E N E S We decided to investigate the roles of ggtA and ggtB in mycobacterial responses to G S H exposure by first looking at changes in gene expression. M. bovis B C G cultures were treated with 5 m M G S H in M B 7H9 media for 2 hours, total R N A was extracted, c D N A was synthesized, and q P C R analyses were performed. A l l procedures were as described in the Methods section of this thesis. The primers used are listed Table A .4 . The results of the q P C R analyses are shown here in Fig. A.4 . As there is visible amplification of both genes in the reverse transcriptase reactions with the enzyme, and no significant amplification without the enzyme, following Dnasel treatment, it is clear that ggtA and ggtB both encode bona fide genes which produce m R N A transcript in vivo. The q P C R results indicate that ggtA is upregulated almost 3-fold while ggtB is not significantly upregulated following G S H treatment. Table A .4: L i s t of primers used for q P C R analyses. Gone forward Primer Reverse Primer Product Size (hp) 1 \nk.ii ggtA 5 ' - A C C A A C G G T G A G G A G T 1 C l'A-3 5 "-CCACTG A C C A C G A T G A C A T A - 3 241 5ft'C ggtB 5 ' - T C T T C G T T C G G C T C C T A C C A - 3 5 ' - C C A G C A T C G C C A C A A G T G T T - 3 262 57°C sigA 5 ' - C T C G G T T C G C G C C T A C C T C A - 3 5 ' - G C G C T C G C T A A G C T C G G T C A - 3 130 57°C hspX 5 ' - G A C A T T A T G G T C C G C G A T G G - 3 5 ' - T C A G T T G G T G G A C C G G A T C T - 3 237 55°C Gene expressions of ggtA and ggtB following 6 and 18-hour infection periods in J774A.1 murine macrophages were also assayed, and the results are shown in Fig . A . 5. We used the same infection samples as were used for the Rv0485/Rv0818 q P C R analyses. Our results here indicate that ggtA is upregulated at least 16-fold at 6 hours post-infection, but is not changed in expression by 18 hours post-infection. Similarly, ggtB is upregulated by about 3-fold at 6-hours post-infection, but is only slightly upregulated by about 1.5-fold by 18 hours post-infection. 69 ggtA ggtB F i g . A .4: Gene expressions of M. bovis B C G ggtA and ggtB following 2 hour treatments wi th 5 m M glutathione. Data are expressed as the ratio between the amount of c D N A detected in the treatment samples and the amount of c D N A detected in the control samples, normalized to the amount of house-keeping sigA c D N A present in the samples. Shown are the means and S E M s o f N=3 experiments. A l so shown are the representative fluorescence curves of the samples assayed via q P C R and the representative products of amplification as visualized by EtBr staining on a 1% agarose gel. +RT denotes the positive samples from the c D N A synthesis reactions and - R T denotes the genomic D N A contamination controls where the reverse transcriptase enzyme was not added to the c D N A synthesis reaction. F i g . A .5: Gene expressions of M. bovis B C G ggtA and ggtB following 6 and 18-hour infections in J774A.1 macrophages at MOI of 10. Data are expressed as the ratio between the amount of cDNA detected in the treatment samples and the amount of cDNA detected in the control samples, normalized to the amount of house-keeping sigA cDN A present in the samples. Shown are the means and SEMs of N=3 experiments. Also shown are the representative fluorescence curves of the samples assayed via qPCR and the representative products of amplification as visualized by EtBr staining on a 1 % agarose gel. +RT denotes the positive samples from the cDNA synthesis reactions and - RT denotes the genomic DNA contamination controls where the reverse transcriptase enzyme was not added to the cDNA synthesis reaction. A .4 D I S C U S S I O N In this addendum to the main thesis, we shifted our focus from looking at the mycobacterial thiol protectant, to looking at how mycobacteria respond to the threat of host thiols. Specifically, we tried to determine if the putative y-glutamyltranspeptidases GgtA and GgtB have roles in mycobacterial responses to glutathione stress by investigating changes in gene expressions of ggtA and ggtB in M. bovis B C G in response to exposure to GSH in vitro and to in vivo macrophage infections. It has been shown that M. bovis B C G and M. tuberculosis are sensitive to GSH and GSNO applied in vitro and in murine macrophages (Green et al, 2000; Venketaraman et al, 2003; Venketaraman et al, 2005). Although this phenomenon has not yet been demonstrated with human macrophages (Venketaraman et al, 2003), the mechanics of this toxicity are interesting to speculate about, and several hypotheses have been proposed. One line of reasoning is that the structure of GSH is similar to penicillin precursors, thus GSH might be a precursor of antibiotics pre-dating cellular immunity, for which mycobacteria might possess some rudimentary sensitivity (Spallholz, 1987). Another hypothesis is that high concentrations of both low-molecular weight thiols, M S H and GSH, in the cell might throw-off the basal redox balance and activity of M S H and thus result in adverse cellular effects (Green et al, 2000). The problem with that theory is that of intercellular transport. There are no known GSNO or GSH transporters, and it is not clear if these molecules are able to passively diffuse across the mycobacterial cell wall in significant amounts. However, there is a dipeptide permease system opp in mycobacteria (De Groote et al., 1995) that is homologous to the dpp system in other prokaryotes, which is able to transport Cys-Gly dipeptides through the cell membrane. However, exposure of B C G to these dipeptides or the constituent amino acids has not been found to significantly inhibit growth of mycobacteria (Green et al, 2000). Nevertheless, since M. tuberculosis has long been a naturally obligate human pathogen, it stands to reason that any sensitivity it may have to host-derived GSH in vivo, will be countered by an evolved, if not quite effective, defense mechanism. In assuming that this defense mechanism may be enzymatic, our first step was to undertake a bioinformatics search of mycobacterial genes which may code for GSH-dependent proteins. Most of our searches proved fruitless, with the exception of two: y-glutamyltranspeptidase and y -glutamylcyclotransferase. In most prokaryotes, these two enzymes function cooperatively in the y -glutamyl cycle, a key metabolic process in the degradation and 72 synthesis of cellular G S H . A n overview of the cycle is shown in Fig . A .6 . In brief, G G T is an outer membrane protein which transfers the y -glutamyl moiety from extracellular G S H (or any other y-glutamyl di- and tripeptides, including glutathione S-conjugates and GS=SG to some extent) to acceptor amino acids or dipeptides. The resulting y -glutamyl-amino-acid is then able to pass through the cell membrane (usually through a dipeptide transport system such as dpp) and become the substrate of G C T which cleaves it back into the amino acid, and 5-oxoproline which can be converted back to glutamate. These amino acids are then free to be used for other metabolic processes, including the "recycling" of glutamate to make new G S H molecules. The other product of G G T activity, the remaining Cys-Gly moiety, is degraded by cell surface dipeptidases, and then transported into the cell to be recycled into other metabolic processes, including de novo G S H synthesis (Forman et al, 1995; Meister et al, 1981). In eukaryotes and prokaryotes, the main purpose of this important metabolic process is to provide the cell with a source of free cysteine for the synthesis of G S H . In eukaryotes, G G T is found in high concentrations on the surface of epithelial tissues of organs such as the bile duct, small intestine, and kidney which export G S H into extracellular compartments in high amounts. amino acid y-glu-amino acid F i g . A.6: Overview of the y -glutamyl cycle 73 G G T activity has been demonstrated in M. smegmatis and other mycobacterial species (Kumar et al, 1990; Shetty et al, 1981), but has not been correlated with the two putative ggt genes, ggtA and ggtB. We found that ggtA and ggtB are also present in other Actinobacteria, including Streptomyces, however, the partner enzyme G C T is not found in most. Since mycobacteria do not produce any G S H , nor do they seem to encode obvious G S H biosynthesis genes, it stands to reason the presence of GSH-dependent genes (which are also not pseudogenes) in the genome may be a response to host-derived G S H . How these genes got into the mycobacterial genome is debatable, although horizontal gene transfer is certainly a possibility (Hacker and Kaper, 2000; Lorenz and Wackernagel, 1994; Ochman et al, 2000; Zubrzycki, 2004). Although M. tuberculosis is a naturally obligate intracellular pathogen, it may have inherited these ggt genes from an ancestral soil Actinobacter, which in turn may have acquired the genes through horizontal gene transfer from another GSHvContaining soil bacterium. The presence of two ggt genes with less than 30% sequence identity with each other, suggests that i f an ancestral ggt gene was acquired through horizontal gene transfer, it may have been duplicated and the copies may have evolved to different, or perhaps complementary functions. If we assume that ggtA and/or ggtB function as proper GGTs , then the lack of G C T in the genome suggests an incomplete y -glutamyl cycle. Hypothetically, this would enable mycobacteria to detoxify host G S H when inside the macrophage, and perhaps import the resulting dipeptides for use in mycobacterial metabolism, maybe in conjunction with the cysAl-cys W-cysT-subl operon for sulfate transport (Wooff et al, 2002). In this scenario, the presence o f G C T is not necessary. A s mycobacteria are still rather sensitive to physiological levels of G S H in vitro, the protective effect of ggtA and/or ggtB is likely minimal, yet may be enough justify its continued presence in the genome. However, since GSH-mediated inhibition of M. tuberculosis has not been shown for human macrophages, the protective effects of ggtA and/or ggtB may be more significant in biologically relevant scenarios. The putative detoxification of host-derived G S H may act directly against G S H as a toxic molecule to mycobacteria, and/or indirectly against G S H as an important host cell molecule. Like mycobacterial M S H , G S H is crucial as a systemic antioxidant in eukaryotic cells, but it also acts as a regulator (or "signaling molecule") of redox-sensitive proteins (Ghezzi et al. 2005); thus the putative protective effect of ggtA/B towards mycobacteria may also be directly detrimental to the host cell. 74 Our results demonstrate that ggtA is upregulated almost 3-fold while ggtB is not significantly upregulated following 2 hours of exposure to 5 m M G S H . Upon infection of J774A.1 murine macrophages, ggtA is upregulated at least 16-fold at 6 hours post-infection, but is not changed in expression by 18 hours post-infection. Similarly, ggtB is upregulated by about 3-fold at 6-hours post-infection, but is only slightly upregulated by about 1.5-fold by 18 hours post-infection. From our results, it is clear that ggtA may play a bigger role in mycobacterial responses to host-derived G S H exposure than ggtB. However, both genes seem to be worthy targets of further research into this novel aspect of the mycobacteria-host relationship. A.5 FUTURE WORK A s with Rv0485 and Rv0818, any investigations into the functions of ggtA and ggtB have to start with the preparation of purified protein. Neither gene has yet been targeted for structural studies by the T B Structural Genomics Consortium ( U C L A - D O E ) , thus the first steps would be to clone, express and purify ggtA and ggtB as either M B P or His-tag containing proteins. The first step would be to determine i f these proteins truly have G G T activity. Since G G T is a sensitive enzymatic indicator of hepatobiliary disease, tests have been developed to assay G G T activity in biological samples. In a common assay, the G G T substrate y-glutamyl-p-nitroanilide, along with glycylglycine, is reacted with the G G T containing sample at room temperature to produce the products y-glutamyl-glycylglycine and p-nitroaniline. The amount of G G T activity in the sample is proportional to the change in OD405nm of p-nitroaniline in the reaction (Szasz, 1969). Once GgtA and GgtB have been shown to have (or not have) G G T activity in vitro, it is then possible to design further experiments to characterize the in vitro kinetics of these proteins. In addition, the biological effects of GgtA and GgtB can be elucidated through the generation of single-knockout mutants, as with Rv0485 and Rv0818. 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