CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / A interleucina-6 (IL-6) à uma importante citocina que exerce papel fundamental na imunopatogÃnese de diversas doenÃas infecciosas. O objetivo deste estudo foi investigar o nÃvel de produÃÃo sistÃmica de IL-6 e aferir o papel funcional do polimorfismo -174 G>C do gene dessa citocina em indivÃduos diagnosticados como portadores de infecÃÃo latente pelo Mycobacterium tuberculosis (ILTB). Para controle, foram utilizados dois grupos de comparaÃÃo: um deles composto por portadores de tuberculose pulmonar ativa (TB) e o outro formado por indivÃduos saudÃveis, doadores de sangue. O grupo ILTB foi composto por 15 indivÃduos, selecionados dentre os contactantes de portadores de TB pulmonar ativa, atendidos no Hospital SÃo Josà de DoenÃas Infecciosas e no Centro de SaÃde da FamÃlia AnastÃcio MagalhÃes. O grupo TB foi formado por 38 pacientes com TB pulmonar ativa, procedentes do Hospital de Messejana, Hospital de Maracanaà e Hospital Geral Dr. CÃsar Cals. O grupo de indivÃduos saudÃveis contava com 63 doadores voluntÃrios de sangue do Centro de Hematologia e Hemoterapia do CearÃ. A dosagem sÃrica de IL-6 foi realizada por meio de um ensaio imunoenzimÃtico (ELISA), com kit especÃfico fornecido pela Invitrogen Corporation. Para purificaÃÃo do DNA, foi utilizado o kit GFX Genomic Blood DNA Purification, da GE Healthcare. O polimorfismo -174G&#61614;C do gene da IL â 6 foi tipificado pela tÃcnica de reaÃÃo em cadeia da polimerase (PCR), utilizando-se iniciadores de sequÃncia especÃfica (PCR-SSP) (One-Lambda). As medianas de concentraÃÃes sÃricas de IL-6 para os grupos ILTB, TB e saudÃveis foram de, respectivamente, 1,7 pg/mL, 4,3 pg/mL e 0,5 pg/mL (p < 0,0001). Nos trÃs grupos estudados, o genÃtipo encontrado com maior frequÃncia foi o G/G [ILTB = (80%); TB = (58,9%); saudÃveis = (62,8%)]. Em conclusÃo, podemos inferir que a IL-6 deve desempenhar um papel importante na manutenÃÃo do estado de latÃncia, haja vista que sua concentraÃÃo, nos indivÃduos com ILTB, foi 3,4 vezes maior que no grupo saudÃvel. Ademais, constatamos que, na populaÃÃo estudada, o polimorfismo -174G&#61614;C nÃo se mostrou funcional no Ãmbito da infecÃÃo latente pelo Mycobacterium tuberculosis. / Interleukin-6 (IL-6) is an important cytokine involved in the pathogenesis of multiple infectious diseases. The aim of this study was to investigate the levels of IL-6 production and to correlate to the -174G>C polymorphism at the IL-6 gene in latent infection with M. tuberculosis (ILTB). As controls, two groups were used. One of them with active pulmonary tuberculosis (TB) patients and the other with healthy blood donors. ILTB group was composed by 15 individuals, selected among active pulmonary TB contacts seen at the Hospital SÃo Josà de DoenÃas Infecciosas and the Centro de SaÃde da FamÃlia AnastÃcio MagalhÃes. TB group had 38 patients with active pulmonary disease seen at the Hospital de Messejana, Hospital de Maracanaà and the Hospital Geral Dr. CÃsar Cals. The third group was composed by 63 healthy blood donors from the Centro de Hematologia e Hemoterapia do CearÃ. Serum levels of IL-6 were measured by an ELISA using specific kits from Invitrogen Corporation. For DNA purification a GFX Genomic Blood DNA Purification kit (GE Healthcare) was used. The -174G&#61614;C polymorphism was analyzed by a SSP-PCR method using One-Lambda kits. Median values of serum levels of IL-6 from ILTB, TB and healthy groups were, respectively, 1.7 pg/mL, 4.3 pg/mL and 0.5 pg/mL (p < 0.0001). For the three studied group, the most frequent genotype found was the G/G (ILTB = 80%; TB = 58.9%; saudÃveis = 62.8%). In conclusion, it is possible to consider that IL-6 should play an important role in the maintenance of latent infection state as its concentrations were 3.4 fold higher in ILTB group than that of healthy controls. Moreover, the -174G&#61614;C polymorpism was not functional in the ILTB group.

Mycobacterium tuberculosis is one of the most successful human pathogens, estimated to have infected close to one-third of the global human population. In order to survive within its host, M. tuberculosis utilises multiple signalling strategies, one of them being synthesis and secretion of universal second messenger cAMP. This process is enabled by the presence of sixteen predicted adenylyl cyclases in the genome of M. tuberculosis H37Rv, ten of which have been characterised in vitro. The synthesized cAMP is recognised by ten putative cAMP-binding proteins in which the cyclic AMP-binding domain is associated with a variety of enzymatic domains. The cAMP signal can be extinguished by degradation by phosphodiesterase’s, secretion into the extracellular milieu or via sequestration of the nucleotide by upregulation of a high-affinity cAMP-binding protein. Of the sixteen adenylyl cyclases (ACs) encoded by M. tuberculosis H37Rv, a subset of multidomain adenylyl cyclases remain poorly characterised, primarily due to challenges associated with studying these in vitro. The adenylyl cyclase domain in these proteins is associated with an NB-ARC domain (nucleotide binding domain common to APAF-1, plant R proteins and CED-4), a TPR domain (tetratricopeptide repeat) and an LuxR-type HTH motif (helix-turn-helix). This architecture places these multidomain mycobacterial ACs within a larger group of STAND (Signal transduction ATPase’s with numerous domains) proteins, and hence they will be referred to as STAND ACs. The STAND proteins are a recently recognised class of multidomain ATPases which integrate a variety of signals prior to activation. Activation is accompanied by formation of large oligomeric signalling hubs which facilitate downstream signalling events. While most STAND proteins have a single effector domain followed by an NB-ARC domain and a scaffolding domain, the STAND ACs distinguish themselves by retaining two effector domains, the AC domain and the HTH domain, at the N- and C- termini respectively. The cyclase, NB-ARC, TPR and HTH domains have widely divergent taxonomic distributions making the presence of these four domains in a single polypeptide rare. In fact, proteins with cyclase-NB-ARC-TPR-HTH (C-A-T-H) domain organisation were found to be encoded almost exclusively by slow growing mycobacterial species, a clade that harbours most mycobacterial pathogens, such as M. tuberculosis and M. leprae. Notably, one of the STAND ACs, Rv0386, is the only mycobacterial AC shown till date to be required for virulence of M. tuberculosis in mice. Using phylogenetic, the evolutionary underpinnings of this domain architecture were examined. The STAND ACs appear to have most likely evolved via a domain gain event from a cyclase-ATPase-TPR progenitor encoded by a strain ancestral to M. marina. Subsequently, the genes duplicated and diverged, sometimes leading to frameshift mutations splitting the cyclase domain from the C-terminal domains. Consequently, M. tuberculosis encodes for three ‘full-length’ STAND ACs, namely, Rv0386, Rv1358 and Rv2488c and one split STAND AC. The split STAND AC is made up of Rv0891c, containing the AC domain, and Rv0890c, containing the NB-ARC, TPR and HTH domains. rv0891c and rv0890c were found to be expressed as an operatic transcript, though they were translationally uncoupled. Pertinently, M. Canetti, an early-branching species of the M. tuberculosis complex, contains an orthologue of Rv0891c and Rv0890c where all four domains are present in a single polypeptide. Sequence analysis of the four STAND ACs in M. tuberculosis allowed predictions of significant divergence in function. These proteins showed high sequence conservation in their HTH domains, with substantial sequence divergence in their TPR, NB-ARC and AC domains. Biochemical analysis on the AC domains revealed that Rv0891c and Rv2488c possessed poor or no AC activity, respectively. On the other hand, the cyclase domain of Rv0386 could catalyse cAMP synthesis. Moreover, for both Rv0891c and Rv0386, presence of the C-terminal domains potentiated adenylyl cyclase activity, suggestive of allosteric regulation within the STAND AC module. Studies on Rv0891c also revealed that the protein could inhibit the adenylyl cyclase activity of Rv0386 in trans. This result thus provided a novel mechanism by which proteins harbouring poorly active/inactive adenylyl cyclase domains could contribute to cAMP levels, by acting as inhibitors of other adenylyl cyclases. The STAND ACs were found to be inactive ATPases. Additionally, incubation with nucleotides did not stimulate oligomerisation of these proteins, unlike what has been shown for several other STAND proteins. However, mutations in the NB-ARC domain perturbed the basal oligomeric state of these proteins, indicating that the NB-ARC domain can influence self- association. A subset of NB-ARC domain mutants also showed increased adenylyl cyclase activity, reiterating the inter-domain cross-talk in the STAND ACs. Since the AC activity of these proteins was meagre, the properties of the HTH domain were examined, as an alternative effector domain. Genomic SELEX was performed using the TPR-HTH domains of Rv0890c, and revealed a set of sequences that bound to this protein, though they lacked common sequence features. Further analysis revealed that Rv0890c bound to DNA in a sequence-independent manner, through the HTH domain. This binding was cooperative with multiple protein units engaging in DNA-binding. Due to the cooperative nature of binding and the lack of sequence preference, Rv0890c appeared coat the DNA molecule. This was further proved by the ability of Rv0890c to protect DNA from DNaseI-mediated degradation, and the requirement for long DNA sequences to form stable DNA-protein complexes. Studies also revealed that Rv0890c interacted with RNA and ssDNA. In fact, the protein as purified from heterologously expressing E. coli cells was bound to RNA. RNA-binding by a LuxR-type HTH has not been reported previously, providing a new function for this class of HTHs. Interestingly, nucleic acid-binding by a fusion Rv0891c-Rv0890c protein, similar to the one encoded in M. canetti, was shown to stimulate adenylyl cyclase activity. This was likely due to a relief of inhibitory interactions between the TPR-HTH and the AC domains, on DNA-binding. Given the high sequence similarity between the HTH domains of the STAND ACs, they were expected to bind to DNA in an identical manner. Indeed, the HTH domains of Rv0386 and Rv1358 engaged with DNA with an identical affinity as Rv0890c. Sequence comparisons in the HTH domain enabled identification of conserved basic residues, of which one, R850 was essential for nucleic acid-binding. Surprisingly however, Rv0386 and Rv1358 did not exhibit RNA-binding, pointing towards functional divergence of Rv0890c from its paralogues. Since the HTH domains of the STAND ACs were highly conserved, it was possible that the ability to bind to RNA was instead dictated by the adjacent TPR modules. To examine this possibility, TPR domains were swapped between Rv0890c and Rv0386. Interestingly, both the chimeric proteins showed a reduced ability to bind to DNA, while showing a complete absence of RNA- binding. These results suggested that the TPR domains were critical in modulating nucleic acid-binding. Moreover, the effect of the TPR domain was context-dependent, since the presence of non-cognate TPR domains hampered nucleic acid-binding. However, the ability to bind to RNA was not solely governed by the TPR domain since the Rv0890cTPR-Rv0386HTH chimeric protein did not show RNA-binding, in spite of containing a permissive TPR domain. To further dissect the molecular requirements for RNA-binding, the conservation of basic residues between the HTH domains of Rv0890c versus Rv1358 and Rv0386 was examined. Interestingly the HTH domain Rv0890c contained two additional positively charged residues over Rv1358 and Rv0386. Mutations of these abolished RNA-binding by Rv0890c. Thus the evolution of two basic residues permit Rv0890c to diverge in its nucleic acid-binding properties, a possible example of defunctionalisation following gene duplication. In summary, this thesis attempts to understand the evolution and functions of the STAND ACs, a group of pathogenically relevant and uniquely mycobacterial multidomain proteins. Phylogenetic analysis revealed an expansion of this gene family in slow growing mycobacteria. Biochemical characterisation showed that following gene duplication, the resulting proteins diverge both in their ability to synthesize cAMP and in their association with nucleic acids. Studies on these proteins also revealed novel mechanisms of regulation of mycobacterial cAMP levels. Additionally, these proteins exhibited indiscriminate binding to DNA/nucleic acids indicating that they may be responsible for global functions in the cell which extend beyond cAMP synthesis.

Tuberculosis, caused by the infection with Mycobacterium tuberculosis remained as a major global health challenge with one third of world population being infected by this pathogen. M. tuberculosis can persist for decades in infected individuals in the latent state as an asymptomatic disease and can emerge to cause active disease at a later stage. Thus, pathways and the mechanisms that are involved in the maintenance of genome integrity appear to be important for M. tuberculosis survival, persistence and pathogenesis. Helicases are ubiquitous enzymes known to play a key role in DNA replication, repair and recombination. However, role of helicases in providing selective advantage for M. tuberculosis survival and genome maintenance is obscure. Therefore, understanding the role of various helicases could provide insights into the M. tuberculosis survival, persistence and pathogenesis in humans. This information could be useful in considering helicases as a novel therapeutic target as well as developing effective vaccines. The research focus of my thesis has been to understand the role of helicases in safeguarding the M. tuberculosis genome from various genotoxic stresses. The major focus of the current study has been addressed towards understanding the role of M. tuberculosis RecG (MtRecG) helicase in recombinational repair and in remodeling stalled replication forks. This study highlights the importance of RecG helicase in the maintenance of genome integrity via DNA repair, recombination and in remodeling the stalled replication forks in M. tuberculosis. The thesis has been divided into following sections as follows: Chapter I: General introduction that describes the causes and consequences of replication stress and DNA repair pathways in M. tuberculosis The genome is susceptible to various types of damage induced by exogenous as well as endogenous DNA damaging agents. Unrepaired or misrepaired DNA lesions can lead to gross chromosomal rearrangements and ultimately cell death. Thus, organisms have evolved with efficient DNA damage response machinery to cope up with deleterious effects of genotoxic agents. Accurate transmission of genetic information requires error-free duplication of chromosomal DNA during every round of cell division. Defects associated with replication are considered as a major source of genome instability in all organisms. Normal DNA replication is hampered when the fork encounters road blocks that have the potential to stall or collapse a replication fork. The types of lesions that potentially block replication fork include lesions on the template DNA, various secondary structures, R-loops, or DNA bound proteins. To understand the DNA damage induced replication stress and the role of fork remodeling enzymes in the repair of stalled replication forks and its restart, chapter I of the thesis has been distributed into multiple sections as follows: Briefly, initial portion of the chapter describes overall replication process in prokaryotes highlighting the importance of coordinated replisome assembly and disassembly during initiation and termination. Later section discusses about various types of exogenous and endogenous DNA damages leading to replication fork stalling. Subsequent section of chapter I provide detailed description and mechanism of various repair pathways cell operates to repair such damages. Chapter I further summarizes causes of stalled replication forks majorly including template lesions, natural impediments like DNA secondary structures and DNA-protein cross links. Subsequent section discusses various pathways of replication restart that include essential role of primosomal proteins in reloading replisome machinery at stalled replication forks. Subsequent section of chapter I provide a comprehensive description of replication fork reversal (RFR) and mechanism of replication restart. RFR involves unwinding of blocked forks via simultaneous unwinding and annealing of parental and daughter strands to generate Holliday junction (HJ) intermediate. Genetic and biochemical studies highlighted the importance of RecG, RuvAB and RecA proteins in driving RFR reaction in E. coli. Hence, in the subsequent chapter, the functional role of RecG, RuvAB and RecA in replication-recombination processes has been discussed. Last section of the chapter devotes completely to M. tuberculosis, its genome dynamics and the various pathways of mycobacterial DNA repair. M. tuberculosis experiences substantial DNA damage inside host macrophages owing to the acidic environment, reactive oxygen species (ROS) and reactive nitrogen intermediates (RNI) which are sufficient enough to cause replication stress. To gain insights into the role of M. tuberculosis RecG helicase in DNA repair, recombination and in remodeling the stalled replication forks the following objectives were laid for my PhD thesis: 1 To understand the functional role of M. tuberculosis RecG (MtRecG) in DNA repair and recombination. 2 To investigate the distinct role(s) of MtRecG, MtRuvAB and MtRecA in remodeling the stalled replication forks. Chapter II: Evidence for the role of Mycobacterium tuberculosis RecG helicase in DNA repair and recombination In order to survive and replicate in a variety of stressful conditions during its life cycle, M. tuberculosis must possess mechanisms to safeguard the integrity of the genome. Although DNA repair and recombination related genes are thought to play key roles in the repair of damaged DNA in all organisms, so far only a few of them have been functionally characterized in the tubercle bacillus. Helicases are one such ubiquitous enzyme involved in all DNA metabolic transaction pathways for maintenance of genome stability. To understand the role of M. tuberculosis RecG (MtRecG) helicase in recombination and repair, we carried out functional and biochemical studies. In our study, we show that M. tuberculosis RecG expression was induced in response to different genotoxic agents. Strikingly, expression of M. tuberculosis RecG in Escherichia coli ∆recG mutant strain provided protection against MMC, MMS and UV-induced cell death. Purified M. tuberculosis RecG exhibited higher binding affinity for the Holliday junction (HJ) as compared to a number of canonical recombinational DNA repair intermediates. Notably, although MtRecG binds at the core of the mobile and immobile HJs, and with higher binding affinity for the immobile junction, branch migration and resolution was evident only in the case of the mobile junction. Furthermore, immobile HJs stimulate MtRecG ATPase activity less efficiently as compared to the mobile HJs. In addition to HJ substrates, MtRecG exhibited binding affinity for a variety of branched DNA structures including three-way junctions, replication forks, flap structures, forked duplex and a D-loop structures, but demonstrated strong unwinding activity on replication fork and flap DNA structures. Altogether, these results support that MtRecG plays an important role in processes related to DNA metabolism under normal as well as in stress conditions. Chapter III: Mycobacterium tuberculosis RecG but not RuvAB or RecA is efficient at remodeling the stalled replication forks: Implications for multiple mechanisms of replication restart in mycobacteria Aberrant DNA replication, defects in the protection and restart of stalled replication forks are a major cause of genome instability in all organisms. Replication fork reversal is emerging as an evolutionarily conserved physiological response for restart of stalled forks. Escherichia coli RecG, RuvAB and RecA proteins have been shown to reverse the model replication fork structures in vitro. However, the pathways and the mechanisms by which Mycobacterium tuberculosis, a slow growing human pathogen responds to different types of replication stress and DNA damage is unclear. In our study, we show that M. tuberculosis RecG rescues E. coli ∆recG cells from replicative stress. The purified M. tuberculosis RecG (MtRecG) and RuvAB (MtRuvAB) proteins catalyze fork reversal of model replication fork structures with and without leading strand ssDNA gap. Interestingly, SSB suppresses the MtRecG and MtRuvAB mediated fork reversal with substrates that contain lagging strand gap. Notably, our comparative studies with fork structures containing template damage and template switching mechanism of lesion bypass reveal that MtRecG but not MtRuvAB or MtRecA is proficient in driving the fork reversal. Finally, unlike MtRuvAB, we find that MtRecG drives efficient reversal of forks when fork structures are tightly bound by protein. These results provide direct evidence and valuable insights into the underlying mechanism of MtRecG catalyzed replication fork remodeling and restart pathways in vivo.

The current, grim world-TB scenario, with TB being the single largest infectious disease killer, warrants a more effective approach to tackle the deadly pathogen, Mycobacterium tuberculosis. The deadly synergy of this pathogen with HIV and the emergence of drugresistant strains of the organism present a challenge for disease treatment (Russell et al., 2010). Thus, there is a pressing need for newer drugs with faster killing-kinetics which can claim both the actively-multiplying and latent forms of this pathogen causing the oldest known disease to man. This thesis entitled “Mechanistic and Regulatory Aspects of the Mycobacterium tuberculosis Dephosphocoenzyme A Kinase” describes one such potential drug target, which holds promise in future drug development, in detail. The development of efficacious antimycobacterials now requires previously unexplored pathways of the pathogen and cofactor biosynthesis pathways present a good starting point. Therefore, the mycobacterial Coenzyme A (CoA) biosynthesis was chosen for investigation, with the last enzyme of this pathway, dephosphocoenzyme A kinase (CoaE) which was shown to be essential for M. tuberculosis survival, as the focus of the present study (Sassetti et al., 2003). This thesis presents a detailed biochemical and biophysical characterization of the enzymatic mechanism of mycobacterial CoaE, highlighting several hitherto-unknown, unique features of the enzyme. Mutagenic studies described herein have helped identify the critical residues of the kinase involved in substrate recognition, binding and catalysis. Further, a role has been assigned to the UPF0157 domain of unknown function found in the mycobacterial CoaE as well as in several organisms throughout the living kingdom. Detailed insights into the regulatory characteristics of this enzyme from this work further our current understanding of the regulation of the universal CoA biosynthetic pathway and call for the attribution of a greater role to the last enzyme in pathway regulation than has been previously accredited. The thesis begins with a survey of the current literature available on tuberculosis and where we stand today in our fight against this dreaded pathogen. Chapter 1 details the characteristic features of the causative organism M. tuberculosis, briefly describing its unique genome and the cellular envelope which the organism puts forward as a tough shield to its biology. This is followed by a brief description of the infection cycle in the host, the pathogen-host interplay in the lung macrophages, the deadly alliance of the disease with HIV and our current drug arsenal against tuberculosis. Further, emphasizing on the need for newer, faster-acting anti-mycobacterials, Chapter 1 presents the rationale for choosing the mycobacterial coenzyme A biosynthetic pathway as an effective target for newer drugs. A detailed description of our current understanding of the five steps constituting the pathway follows, including a comparison of all the five enzymatic steps between the human host and the pathogen. This chapter also sets the objectives of the thesis, describing the choice of the last enzyme of the mycobacterial CoA biosynthesis, dephosphocoenzyme A kinase, for detailed investigation. As described in Chapter 1, the mycobacterial CoaE is vastly different from its human counterpart in terms of its domain organization and regulatory features and is therefore a good target for future drug development. In this thesis, Rv1631, the probable mycobacterial dephosphocoenzyme A kinase annotated in the Tuberculist database (http://genolist.pasteur.fr/TubercuList), has been unequivocally established as the last enzyme of the tubercular CoA biosynthesis through several independent assays detailed in Chapter 2. The gene was cloned from the mycobacterial genomic DNA, expressed in E. coli and the corresponding recombinant protein purified via a single-step affinity purification method. The mechanistic details of the enzymatic reaction phosphorylating dephosphocoenzyme A (DCoA) to the ubiquitous cofactor, Coenzyme A, have been described in this chapter which presents a detailed biochemical and biophysical characterization of the mycobacterial enzyme, highlighting its novel features as well as unknown properties of this class of enzymes belonging to the Nucleoside Tri-Phosphate (NTP) hydrolase superfamily. The kinetics of the reaction have been biochemically elucidated via four separate assays and the energetics of the enzyme-substrate and enzymeproduct interactions have been detailed by isothermal titration Calorimetry (ITC). Further details on the phosphate donor specificity of the kinase and the order of substrate binding to the enzyme provide a complete picture of the enzymatic mechanism of the mycobacterial dephosphocoenzyme A kinase. Following on the leads generated in Chapter 2 on the unexpected strong binding of CTP to the enzyme but its inability to serve as a phosphate donor to CoaE, enzymatic assays described in Chapter 3 helped in the identification of a hitherto unknown, novel regulator of the last enzyme of CoA biosynthesis, the cellular metabolite CTP. This chapter outlines the remarkable interplay between the regulator, CTP and the leading substrate, dephosphocoenzyme A, possibly employed by the cell to modulate enzymatic activity. The interesting twist to the regulatory mechanisms of CoaE added by the involvement of various oligomeric forms of the enzyme and the influence of the regulator and the leading substrate on the dynamic equilibrium between the trimer and the monomer is further detailed. This reequilibration of the oligomeric states of the enzyme effected by the ligands and its role in activity regulation is further substantiated by the fact that CoaE oligomerization is not cysteine-mediated. Further, the effects of the cellular metabolites on the enzyme have been corroborated by limited proteolysis, CD and fluorescence studies which helped elucidate the conformational changes effected by CTP and DCoA on the enzyme. Thus, the third chapter discusses the novel regulatory features employed by the pathogen to regulate metabolite flow through a critical biosynthetic pathway. Results presented in this chapter highlight the fact that greater importance should be attributed to the last step of CoA biosynthesis in the overall pathway regulation mechanisms than has been previously accorded. The availability of only three crystal structures for a critical enzyme like dephosphocoenzyme A kinase (those from Escherichia. coli, Haemophilus influenzae and Thermus thermophilus) is indeed surprising (Obmolova et al., 2001; O’Toole et al., 2003; Seto et al., 2005). In search of a structural basis for the dynamic regulatory interplay between the leading substrate, DCoA and the regulator, CTP, a computational approach was adopted. Interestingly, the mycobacterial enzyme, unlike its other counterparts from the prokaryotic kingdom, is a bi-domain protein of which the C-terminal domain has no assigned function. Thus both the N- and C-terminal domains were independently modeled, stitched together and energy minimized to generate a three-dimensional picture of the mycobacterial dephosphocoenzyme A kinase, as described in Chapter 4. Ligand-docking analyses and a comprehensive analysis of the interactions of each ligand with the enzyme, in terms of the residues interacted with and the strength of the interaction, presented in this chapter provide interesting insights into the CTP-mediated regulation of CoaE providing a final confirmation of the enzymatic inhibition effected by CTP. These homology modeling and ligand-docking studies reveal that CTP binds the enzyme at the site overlapping with that occupied by the leading substrate, thereby potentially obscuring the active site and preventing catalysis. Further, very close structural homology of the modeled full-length enzyme to uridylmonophosphate/cytidylmonophosphate kinases, deoxycytidine kinases and cytidylate kinases from several different sources, with RMSD values in the range of 2.8-3 Å further lend credence to the strong binding of CTP detailed in Chapter 2 and the regulation of enzymatic activity described in Chapter 3. Computational analyses on the mycobacterial CoaE detailed in this chapter further threw up some interesting features of dephosphocoenzyme A kinases, such as the universal DXD motif in these enzymes, which appears to play a crucial role in catalysis as has been assessed in the next chapter. It is interesting to note that the P-loop-containing nucleoside monophosphate kinases (NMPK), with which the dephosphocoenzyme A kinases share significant homology, have three catalytic domains, the nucleotide-binding domain, the acceptor substrate-binding domain and the lid domain. Computational analyses detailed in Chapter 4 including the structural and sequential homology studies, helped in the delineation of the three domains in the mycobacterial enzyme as well as highly conserved residues potentially involved in crucial roles for substrate binding and catalysis. Therefore important residues from all three domains of the mycobacterial CoaE were chosen for mutagenesis to study their contributions to catalysis. Conservative and non-conservative replacements of these residues detailed in Chapter 5 helped in the identification of crucial residues involved in phosphate donor, ATP binding (Lys14 and Arg140); leading substrate, DCoA binding (Leu113); stabilization of the phosphoryl transfer reaction (Asp32 and Arg140) and catalysis (Asp32). Thus, the results reported here present a first attempt to identify the previously unknown functional roles of highly conserved residues in dephosphocoenzyme A kinases. Chapter 5 also delineates the dependence of this kinase on the divalent cation, magnesium, for catalysis, describing a comparison of the kinetic activity by the wild type and the mutants, in the presence and absence of Mg2+. Therefore, this chapter presents a thorough molecular dissection of the roles played by crucial amino acids of the protein and the results herein can serve as a good starting point for targeted drug development approaches. As described above, another unusual characteristic of the mycobacterial CoaE is the fact that it carries a domain of unknown function, UPF0157, C-terminal to the N-terminal dephosphocoenzyme A kinase domain. The function of this unique C-terminal domain carried by the mycobacterial CoaE has been explored in Chapter 6. The failure of the Nterminal domain (NTD) to be expressed and purified in the soluble fraction in the absence of a domain at its C-terminus (either the mycobacterial CoaE CTD or GST from the pETGEXCT vector) pointed out a possible chaperonic activity for the CTD. A universal chaperonic activity by this domain in the cell was ruled out by carrying out established chaperone assays with insulin, abrin and -crystallin. In order to delineate the CTD sequence involved in the NTD-specific chaperoning activity, deletion mutagenesis helped establish the residues 35-50 (KIACGHKALRVDHIG) of the CTD in the N-terminal domain-specific assistance in folding. Chapter 6 further details the several other potential roles of the mycobacterial CTD probed, including the 4’-phosphopantethienyl transfer, SAM-dependent methyltransferase activity, activation of the NTD via phospholipids among others. Thus the results presented in this chapter are a first attempt at investigating the role of this domain found in several unique architectures in several species across the living kingdom. Chapter 7 is an attempt to stitch together and summarize the results presented in all the preceding chapters, giving an overview of our present understanding of the mycobacterial CoaE and its novel features.

Introduction Nucleotide excision repair (NER) is a highly conserved pathway involved in repair of a wide variety of structurally unrelated DNA lesions. One of the well characterized NER systems is from E. coli which involves UvrABC nucleases. NER consists of two related sub-pathways: global genomic repair (GGR), which removes lesions from the overall genome, and transcription coupled repair (TCR), which removes lesions from the transcribed strand of active genes. Bulky DNA lesions such as cyclobutane pyrimidine photodimers (CPD) induced by UV irradiation block RNA polymerase (RNAP) during transcription. In bacteria, a gene product of mfd called transcription repair coupling factor (TRCF) or Mfd is required for TCR. Bacterial Mfd interacts with the stalled RNAP, displaces it from the DNA and recruits NER proteins at the site of damage. Mfd, thus contributes to the faster repair of the transcribed strand compared to the non-transcribed strand for similar kind of lesions. Intracellular pathogens like M. tuberculosis are constantly exposed to a variety of stress conditions inside the host, mainly due to host defense systems and antibiotic treatments. It is therefore, extremely important for bacteria to have DNA damage repair and reversal mechanisms that can efficiently counteract these effects. However, very little is known about DNA repair systems in M. tuberculosis compared to other bacteria. Sequencing of M. tuberculosis genome revealed the presence of NER associated genes including a putative mfd. Additionally, due to the high GC content of genome as well as the DNA damage prone host environment, the transcription in M. tuberculosis may encounter the problems, which are not apparent in other bacteria. Therefore, the gene like mfd may play very important role in physiology of M. tuberculosis. In the present study, we describe the biochemical and functional characterization of Mfd from M. tuberculosis (MtbMfd) and discuss its unusual properties. Biochemical characterization of MtbMfd Genome analysis of M. tuberculosis as well as the sequence alignment studies revealed that MtbMfd is 1234 amino acids long multifunctional protein having various domains specialized for different functions. Cloning of Mtbmfd was carried out by reconstructing the full length gene from three PCR amplified fragments using genomic DNA as a template. Complementation study using Mtbmfd suggested that the gene of interest complements E. coli counterpart and increases survival of UV irradiated cells. To further characterize the function of Mtbmfd, a road block reporter assay was performed, which indicates that the MtbMfd interacts with stalled E. coli RNAP and displaces it from the site of transcription resulting in low reporter gene activity. The MtbMfd protein was expressed and purified by using various chromatographic techniques, and confirmed by mass spectrometry. In addition to full length protein, a number of truncated MtbMfd constructs were generated and purified to homogeneity. Mfd is a motor protein and requires ATP hydrolysis in order to translocate along DNA. The signature motifs of superfamily 2 helicases / ATPases are present at the C-terminal of Mfd along with translocase motif which is highly homologous to motif present in RecG helicase. To analyze the kinetics of ATP hydrolysis of MtbMfd and its truncated proteins, ATPase reactions were carried out using γ32P-ATP as a tracer. Wild-type MtbMfd exhibited ATPase activity, which was stimulated ~1.5 fold in presence of dsDNA. The mutant MtbMfd (D778A), which harbors mutation in one of the key residues of Walker B motif of the ATPase domain showed negligible ATPase activity indicating the importance of residue D778 for ATP hydrolysis. While the C-terminal domain (CTD) comprising amino acids 600 to 1234 showed elevated ATPase activity, the N-terminal domain (NTD) containing the first 500 amino acid residues was able to bind ATP but deficient in hydrolysis. Deletion of 184 amino acids from the C-terminal end of MtbMfd (MfdΔC) increased the ATPase activity by ~10-fold compared to full-length MtbMfd. The translocase activity of MtbMfd was measured by an oligonucleotide displacement assay and it was found that full length MtbMfd and CTD have a very weak translocase activity whereas, MfdΔC exhibited efficient translocation along DNA in ATP dependent manner. These results provide a direct correlation between translocase and ATPase activity of MtbMfd, and suggest possibly an auto-regulatory function for the extreme C-terminus of MtbMfd. Oligomeric status of MtbMfd was determined using various techniques including gel filtration chromatography and it was found that MtbMfd exists as monomer and hexamer in solution. The monomer showed increased ATPase activity and susceptibility to proteases compared to the hexameric form. MfdΔC, on the other hand, was predominantly monomer in solution implicating importance of the extreme C-terminal region in oligomerization of protein. Taken together, the biochemical evidence suggests that monomeric MtbMfd is an active form and oligomerization provides stability to the protein. One important finding of the present study is the binding of ATP to NTD of MtbMfd. All Mfd NTDs resemble UvrB and possesses the degenerate ATPase motifs. Indeed, on the basis of sequence and structural similarities, it has been suggested that Mfds have evolved from UvrB incorporating an additional translocase activity. UvrB has a cryptic ATPase activity while the NTD of Mfd may have lost the activity as it possesses degenerate Walker motifs. In contrast, NTD of MtbMfd binds ATP but is hydrolysis deficient. A closer comparison of the amino acid sequences in the Walker A motif reveal that conserved K 45 of UvrB has been replaced by R in case of NTD of MtbMfd. It has been shown previously that mutation of K 45 to A, D and R led to a loss of ATPase activity of UvrB. Thus, MtbMfd seems to be a natural mutant of UvrB. Since NTD harbors an intact UvrA interacting domain, when it is expressed it may sequester the cellular pool of UvrA leading to dominant negative phenotype. When UV survival assays were carried out, cells expressing NTD showed hyper-sensitivity to UV light – a typical characteristic of NER deficiency. In addition, in vitro NER assay clearly suggested that NTD sequesters pool of UvrA inside the cell and blocks both GGR and TCR which further affects the mutation frequency of bacterial cells. Influence of MtbMfd on elongation state of RNAP The movement of RNAP along the template during transcription elongation is not uniform and is interrupted due to various factors. To overcome transcription elongation interruptions, a number of proteins viz. Mfd, Gre and Nus act on RNAP and modify its activity. RNAP displacement and transcript release experiments showed that MtbMfd influenced the elongating RNAP by more than one way. MtbMfd displaced stalled RNAP, which was blocked by NTP starvation on T7A1 promoter based template in a concentration and time-dependent manner. RNAP displacement activity of MtbMfd was shown to depend on ATP or dATP hydrolysis. On the other hand nucleotides like ADP, GTP, CTP and ATPγS did not support the RNAP displacement activity. However, in presence of ATPγS, MtbMfd was able to bind stalled complex but unable to displace RNAP suggesting that ATP or dATP hydrolysis is important for MtbMfd function. On the other hand, MtbMfd did not affect initiating RNAP when σ factor was still bound suggesting that upstream DNA is necessary for Mfd function. To assay RNA or transcript release activity of MtbMfd after transcription complex disruption, immobilized transcription complex assay was carried out. Immobilized stalled complex was generated by UTP and CTP starvation on biotinylated T7A1 promoter based template which can be affixed to temporary pellet in presence of streptavidin beads. It was found that MtbMfd released RNA into a supernatant fraction in a concentration-dependent manner suggesting that MtbMfd releases transcript after ternary complex disruption. MtbMfd released transcript in an energy-dependent manner and both ATP and dATP supported the activity, which allows the complete separation of RNA release from RNA synthesis inside the cell. An ATPase mutant of MtbMfd (MfdD778A) failed to release transcript, which further supported that ATP hydrolysis is important for MtbMfd function. Since both Mfds and RNAPs are evolutionary conserved proteins, to analyze the effect of MtbMfd on other bacterial RNAPs, displacement and release assays were carried out. Stalled complexes were generated using EcoRNAP (E. coli), MsRNAP (M. smegmatis) and MtbRNAP (M. tuberculosis) on T7A1 promoter based template. It was observed that MtbMfd was able to displace all the three RNAPs from stalled elongation complex as well as released transcript with varying efficiency. MtbMfd showed optimal displacement and release activity in presence of mycobacterial RNAPs. Transcription elongation complexes adopt various conformations and exist as different isomerized states during elongation. In an active elongation complex the 3'-OH polymerizing end of transcript aligns with an active centre of the RNAP. However, one of the most common and intrinsic properties of RNAP is backtracking or reverse translocation, which leads to misalignment of 3'-OH polymerizing end from an active centre of the polymerase. It is of interest to know if backtracking affects MtbMfd function. It is likely that complexes blocked by lesions inside the cell might tend to backtrack, and different translocational isomers possibly have different sensitivities to MtbMfd action which may illuminate the overall mechanism of MtbMfd. Backtracking of RNAP was induced on +20 and +39 stalled complexes and the effect of MtbMfd was analyzed in presence of NTPs in the reaction. It was found that arrested or backtracked complexes were restored to the forward position by the activity of MtbMfd in presence of NTP resulting into productive elongation. These results suggest that arrested RNAP again resumes transcription if conditions are favorable; otherwise, MtbMfd further assists RNAP to dissociate which leads to release of transcript. Anti-backtracking activity of MtbMfd might have important function in cellular metabolism and it has been speculated that Mfd could play more general role during transcription apart from repair. To explore the role of MtbMfd as a transcription factor and effect of MtbMtb on transcription processes in the mycobacteria, a variety of T7A1 promoter based templates were generated. These templates were derived from genes of M. tuberculosis and E. coli having varying GC content (39-81 %). The rationale behind this experiment is that the high GC content of mycobacteria and the template derived from mycobacterial genes may pose as sequence dependent structural constraints and hence block the RNAP during transcription. By anti-backtracking activity of MtbMfd these paused complexes may get relieved, leading to efficient transcription by RNAP which may lead to the formation of more full length transcript. To analyze the effect of MtbMfd, purified templates of different GC content were incubated with RNAP and MtbMfd to carry out in vitro transcription. Although, in case of multiple rounds of transcription, multiple pauses were observed even in presence of MtbMfd. However, in presence MtbMfd around 1.5 - 2 fold increased full-length transcripts were observed suggesting that MtbMfd assisted RNAP during elongation to overcome sequence dependent pause. To avoid multiple pauses that are likely to occur due to the initiation of multiple round of transcription, and trailing effect of RNAP itself, single round of transcriptions were carried out in presence of heparin. Sequence specific pauses were observed with increasing GC percentage in template suggesting that indeed high GC content contributes to transcription pause. At the same time, MtbMfd in the reaction increased the amount of full length transcript by 1.5 - 2.0 fold probably by pushing paused RNAP forward to resume elongation. Taken together, this study investigates the biochemical properties of MtbMfd and its mechanism of action. In addition, it explores the importance of the coupling of transcription to repair in M. tuberculosis as well as the overall proof reading mechanism of transcription elongation in the GC rich genome of mycobacteria.

M. Tech. (Department of Health Sciences, Faculty of Applied and Computer Sciences), Vaal University of Technology. / Tuberculosis (TB) remains one of the most difficult infectious diseases to control in the world today. The disease spreads easily in overcrowded, badly ventilated places and among people who are undernourished. Trends in the incidence of TB together with the development of multi-drug (MDR-TB) and extensively drug resistant (XDR-TB) strains of TB raises the need to intensify the search for more efficient drugs to combat this disease. Herbal remedies used in traditional medicine provide an interesting and largely unexplored source for the discovery of potentially new drugs for infections such as TB. The aim of the study was to evaluate the in vitro antimycobacterial activity of synthesized compounds from medicinal plants against Mycobacterium tuberculosis (M. tuberculosis). About 40 synthesized compounds isolated from South African medicinal plants were screened against H37RV using microplate alamar blue assay (MABA). Identified active compounds were screened against resistant strains of M. tuberculosis (MDR, XDR and pre-XDR) and sensitive clinical isolates of TB. Cytotoxicity and synergistic drug combination studies were done on active compounds to validate their toxicity and synergy levels. Cytotoxicity was done by sulforhodamine assay (SRB) against the C2C12 cell line. Only six compounds showed activity against M. tuberculosis with minimum inhibitory concentration (MIC) below 10μg/ml. The results obtained indicated that the cytotoxicity effects of the three compounds on C2C12 cells demonstrated marginal toxicity except for MVB 282/61215 which showed a high toxicity at the lowest concentration of 0.156μg/ml with over 100% viable cells at the highest concentration (5μg/ml). MVB 282/61271 had the highest percentage cell viability (65%) at the lowest concentration. Only two compounds had a higher potency evoking a bigger response at low concentrations with treated cells still viable after 3 days of incubation with the compound which was comparable with the treatment of isoniazid (INH). Synergistic activity of the six compounds was less in INH combination as compared to the rifampicin’s (RIF) combination. The results demonstrated that the synergistic interaction between the compounds and RIF could the antituberculosis acitivity. In conclusion the synergistic effects with RIF translate to lower dosing requirements of the compounds and the potential to combat multidrug resistant TB. In deed there is no doubt that natural products, with their range of interesting chemical structures and powerful antimycobacterial effects are certain to remain important participants in the development of new generations of antimycobacterial drugs.

Pathogenic Mycobacteria are among the most unrelenting pathogens known to mankind as one-third of the world population is latently infected with Mycobacterium tuberculosis, the causative agent of pulmonary tuberculosis. Despite many species of mycobacteria elicits robust host T cell responses as well as production of cytokines like interferon-γ (IFN- γ) that are essential for the control of infection, the mounted immune response contain, but does not eliminate the infection. One potential mechanism by which mycobacteria may achieve a state of long-term persistence amid a robust host immune response is by modulating the signaling cascades leading to macrophage activation. Activation of proinflammatory responses by the host macrophages upon infection with mycobacteria requires the involvement of a variety of signaling events. Studies have indicated that macrophages infected with pathogenic mycobacteria produce significantly less tumor necrosis factor (TNF)-α and other proinflammatory molecules compared with infection with nonpathogenic mycobacteria, which likely play a role in enhancing mycobacterial survival in vivo. Furthermore, macrophages infected with mycobacteria become refractory to many cytokines including IFN-γ and modulation of host cell signaling responses is critical for the suppression of a generalized inflammatory response which might influence the persistence of mycobacteria within the host. In this context, Suppressor of cytokine signaling (SOCS) 3, a member of SOCS family function as negative regulators of multiple cytokine and toll like receptor induced signaling. The SOCS3 has been shown to specifically inhibit signaling by IFN-γ, IL-6 family of cytokines and can act as a negative regulator of inflammatory responses. In this regard, many species of mycobacteria including M. bovis BCG triggers the inducible expression of SOCS3. Further, it has been suggested that M. bovis BCG triggered SOCS3 and SOCS1 proteins leads to the inhibition of IFN- γ stimulated JAK/STAT signaling in macrophages. Albeit JAK/STAT signaling pathway is generally believed to be involved, STAT-independent signals are suggested to take part in the induction of SOCS proteins in many systems signifying the involvement of multiple signal pathways in regulation of SOCS expression. Further little is known about the early, receptor proximal signaling mechanisms underlying mycobacteria-mediated induction of SOCS3. Albeit mycobacteria reside within phagolysosomes of the infected macrophages, many cell wall antigens like LAM, PIM, TDM, PE family antigens etc are released and traffic out of the mycobacterial phagosome into endocytic compartments as well as can gain access to the extra cellular environment in the form of exocytosed vesicles. In this context, PIM represent a variety of phosphatidyl-myo-inositol mannosides (PIM) 1-6 containing molecules and are integral component of the mycobacterial envelope. PIM are suggested to be the common anchor of LM and LAM as PIM, LM, and LAM originate from identical biosynthetic pathway. PIM are present in virulent M. tuberculosis H37Rv as well as in M. bovis BCG and a number of biological functions have been recently credited to PIM2. PIM2 is suggested to trigger the activation of cells via Toll like receptor (TLR)-2 and stimulation resulted in activation of NF-κB, AP-1, and mitogen-activated protein (MAP) kinases. PIM2 induces proinflammatory stimuli such as TNF-α and IL-12 in murine and human macrophages in a TLR2 dependent manner. PIM exhibited pulmonary granuloma-forming activities as well as was shown to be responsible for the recruitment of NKT cells to granulomas. Accordingly, mycobacterial envelope antigen PIM2 could initiate or affect the inflammatory responses similar to mycobacteria bacilli. In this perspective, we explored whether M. bovis BCG or novel cell surface antigens like PIM2 or Rv0978c, a PE-PGRS protein with unknown function can contribute to M. bovis BCG triggered molecular signaling events leading to SOCS3 expression in macrophages. Our studies clearly demonstrated that M. bovis BCG can trigger SOCS3 expression in macrophages. The inception of signaling by M. bovis BCG is TLR2-MyD88 dependent, but not TLR4 dependent. The perturbation of TLR2 signaling and the downregulation of MyD88 resulted in significant decrease in SOCS3 expression implicating the role of TLR2-MyD88 axis in M. bovis BCG triggered signaling. Experiments with cycloheximide and neutralizing antibodies to IL-10 evinced that M. bovis BCG triggered SOCS3 expression is a primary response and requires direct activation of signaling cascades. In the current study, we show for the first time that infection of macrophages with M. bovis BCG activates NOTCH1 signaling events, which leads to expression of SOCS3. The perturbation of NOTCH signaling in infected macrophages either by siRNA mediated down regulation of NOTCH1 or RBP-Jk or by inhibition with pharmacological inhibitor gamma secretase-I, resulted in the marked reduction in the expression of SOCS3. Further, the enforced expression of the NOTCH1 intracellular domain (NICD) in RAW264.7 macrophages induces the expression of SOCS3, which can be further potentiated by M. bovis BCG. Furthermore, the inhibition of TLR2 signaling by a TLR2 dominant-negative construct resulted in inhibition of NOTCH1 activation. Additionally, our results demonstrates for the first time that physical association of TLR2 with both Phosphoinositide-3 Kinase (PI3K) and NOTCH1, which suggest the significant role of TLR2 triggering by of M. bovis BCG in the activation of PI3K and NOTCH1. More importantly, signaling perturbations data suggest the involvement of cross-talk among the members of PI3K and MAPK cascades with NOTCH1 signaling in SOCS3 expression. In addition, SOCS3 expression requires the NOTCH1 mediated recruitment of CSL/RBP-Jk and Nuclear Factor-B (NF-B) to the SOCS3 promoter. A number of biological functions triggered by mycobacteria are often attributed to many of the cell wall antigens. As part of our current investigation, we explored whether two novel cell wall associated antigens namely PIM2 and a PE-PGRS antigen, Rv0978c could play as significant or crucial cell wall ingredients which imparts ability to M. bovis BCG to trigger activation of NOTCH signaling leading to SOCS3 expression. Akin to M. bovis BCG, PIM2 activates NOTCH1 signaling resulting NICD formation which leads to the expression of SOCS3 in a TLR2-MyD88 dependent manner. PIM2 mediated NOTCH1 activation, both directly influences the SOCS3 expression by serving as coactivator in RBP-Jk complex and indirectly triggers SOCS3 expression by activating PI3K-MAPK-NF-κB cascade. One important outcome of the genome sequencing project of M. tuberculosis was the discovery of two new multigene families designated PE and PPE, named for the Pro-Glu (PE) and Pro-Pro-Glu (PPE) motifs near the N-terminus of their gene products. Many PE and PPE proteins are composed only of PE or PPE homologous domains. However, in other proteins, the PE domain is often linked to a unique domain of various lengths that is rich in alanine and glycine amino acids, termed the PGRS domain (PE-PGRS subfamily). PE family genes were suggested to play roles in the virulence of the pathogen and many members of PE family proteins are reported be localized on the surface of M. tuberculosis bacilli. Some of the PE proteins may play a role in immune evasion and antigenic variation or may be linked to virulence. Additionally, it has been suggested that the PE-PGRS subfamily of PE genes is enriched in genes with a high probability of being essential for M. tuberculosis. The uniqueness of the PE genes is further illustrated by the fact that these genes are restricted to mycobacteria. However, despite their abundance in mycobacteria, very little is known regarding the expression or the functions of PE family genes. In this context, we have chosen to study Rv0978c as a typical member of PE-PGRS family based on the following observations. Rv0978c was upregulated in TB bacilli upon infection of macrophages. Rv0978c was demonstrated to be a member of a group of genes called in vivo-expressed genomic island, which were shown to be upregulated in M. tuberculosis bacilli during infection of mice. Rv0978c was also shown to be upregulated, at least eightfold, in human brain microvascular endothelial cell-associated M. tuberculosis infection, suggesting a role for endothelial cell invasion and intracellular survival. In the current investigation, we have demonstrated that Rv0978c is hypoxia responsive gene based on promoter analysis and upregulated in M. tuberculosis during the infection of macrophages. Further, Rv0978c is associated with cell wall and is exposed outside the surface of the bacterium suggesting the possible access to intracellular compartments of the infected macrophages. In this perspective, our results clearly demonstrate that Rv0978c triggers SOCS3 expression by activating PI3K-ERK1/2-NF-B cascade in mouse macrophages. Additionally, Rv0978c elicited humoral antibody reactivities in a panel of human sera or in cerebrospinal fluid samples obtained from different clinical categories of tuberculosis patients. DNA immunizations experiments in mice clearly suggested that Rv0978c is an immunodominant antigen demonstrating significant T cell and humoral reactivites. These observations clearly advocate that Rv0978c protein is expressed in vivo during active infection with M. tuberculosis and that the Rv0978c is immunogenic. These results clearly describe the cross-talk of NOTCH1 signaling with signaling pathways like PI3K and MAPK pathways during infection of macrophages with M. bovis BCG eventually resulting in regulation of specific gene expressions, such as SOCS3. These observations lead to a possibility of differential effects of NOTCH1 signaling activated upon infection by an intracellular bacillus, which could be involved in modulation of macrophage functions depending on a local immunological milieu. Taken together, our findings suggest that, induction of Suppressors of Cytokine Signaling 3 molecule by M. bovis BCG or by its cell wall antigens represents a crucial immune subversion mechanism in order to suppress or attenuate host responses to cytokines to generate the conditions that favor survival of the mycobacteria.

More human lives have been lost to tuberculosis than to any other disease and despite the availability of effective short course chemotherapy (DOTS) as well as the Bacilli Calmette Guerin (BCG) vaccine, tuberculosis continues to claim more than a million lives annually. Mycobacterium tuberculosis (M. tuberculosis), the causative agent of tuberculosis, is one of the most successful and scientifically challenging pathogens of all time. However in the last two decades, the ability to perform molecular genetic analysis of M. tuberculosis has resulted in powerful new research tools, while the availability of the complete genome sequence has provided us with a wealth of new information and understanding of the biology of this major pathogen. One of the major challenges, however, is to analyze the properties and functions of those genes that are unique to M. tuberculosis genome. The identification and characterization of such genes which impart various survival strategies employed by M. tuberculosis for successful infection will be of particular significance. One of the important outcomes from the complete genome sequence of M. tuberculosis is the discovery of two multigene families designated PE (99 members) and PPE (69 members) named respectively for the Pro-Glu (PE) and Pro-Pro-Glu (PPE) motifs near the N-terminus of their gene products. In addition to these motifs, proteins of the PE family possess highly homologous N-terminal domains of approximately 100 amino acids (PE domain), whereas the PPE proteins possess a highly homologous N-terminal domain of about 180 amino acids (PPE domain). Although the PE and PPE families of mycobacterial proteins are the focus of intense research, no precise function has so far been unraveled for any member of these families. The current study focuses on Rv3097c gene of M. tuberculosis, a PE family gene that was bioinformatically predicted to be a triacylglycerol hydrolase (lipase). In order to decipher the role of the PE domain, we have carried out functional characterization of the Rv3097c gene (also named lipY) as it was, initially, the only known PE protein for which an enzymatic function (i.e. lipase activity) had been predicted. Further, to understand the function of PE family proteins, an important question that needs to be answered is; whether the PE domain of different PE family proteins has similar or different functions? In this context, our studies were focused on studying the functional role of the PE domain in LipY, as outlined below. In general, the in vivo function and subcellular localization of any protein are integrally connected. PE domain has been reported to be essential for cell wall localization of PE_PGRS33, another PE family protein. Therefore we investigated the subcellular localization of LipY and the influence of the PE domain on subcellular localization of LipY. LipY and a truncated form of LipY lacking the PE domain [LipY(ΔPE)] were expressed in mycobacteria(M. smegmatis and M. bovis BCG). Subcellular fractionation and western blot demonstrated that both LipY and LipY(ΔPE) were predominantly detected in the cell wall fraction, indicating that LipY is localized to the cell wall and the PE domain of LipY was not required for translocation of LipY to cell wall. This result is in contrast to the findings for PE_PGRS33, where the absence of the PE domain caused the cell wall associated protein to localize to the cytosol. Furthermore, immuno-electron microscopy of M. bovis BCG expressing LipY(ΔPE) clearly showed a cell surface localization of LipY(ΔPE). These results signify that the function of the PE domain might not always be similar amongst different PE family proteins. In order to further investigate the role of the PE domain in LipY, we studied the lipase activity of LipY and the influence of the PE domain on lipase activity. Bioinformatic analysis confirmed the presence of a lipase domain containing a GDSAG active site motif characteristic of lipases. Overexpression of LipY in mycobacteria (M. smegmatis and M. bovis BCG) resulted in a significant reduction in the pool of triacylglycerols (TAG), consistent with the lipase activity of this enzyme. Interestingly, this reduction was more pronounced in mycobacteria overexpressing LipY(ΔPE), suggesting that the presence of the PE domain diminishes the lipase activity of LipY. In vitro lipase assays also confirmed LipY(ΔPE) as a more efficient lipase compared to the wild-type LipY. Together these results suggest that the PE domain of LipY might be involved in the modulation of lipase activity. Surprisingly, M. marinum, another pathogenic mycobacteria, possesses a protein homologous to LipY, termed LipYmar, in which the PE domain is substituted by a PPE domain. The overexpression of LipYmar in M. smegmatis significantly reduced the TAG pool suggesting that it is a triacylglycerol hydrolase/lipase. Interestingly, similar to the removal of the PE domain of LipY, this reduction in the TAG pool was further pronounced when the PPE domain of LipYmar was removed. This suggests that PE and PPE domains might share similar functional roles in modulating the enzymatic activities of these lipase homologs. In order to assess the in vivo relevance of LipY expression during M. tuberculosis infection, we examined the humoral immune responses against LipY in sera derived from various clinical categories of tuberculosis patients. The presence of specific antibodies against any protein is suggestive of expression of the protein during infection and could potentially be used to differentiate between healthy individuals and infected patients (serodiagnosis of tuberculosis). The cell wall localization suggested that LipY may be accessible for interaction with the host immune system during infection. Moreover, humoral responses were observed against LipY in mice immunized with DNA constructs expressing LipY, indicating that LipY could be an effective B-cell antigen. Accordingly, a strong humoral response against LipY and LipY(ΔPE) was observed in tuberculosis patients compared to healthy individuals, suggesting that LipY is expressed during infection by clinical strains of M. tuberculosis and might represent an immunodominant antigen of M. tuberculosis with potential use in serodiagnosis of tuberculosis.

Mycobacterium tuberculosis has one ribosomal RNA operon. The survival of this bacillus thus depends on a transcription mechanism that can effectively couple gene expression to changes in the environment. σ factors are transcription proteins that bind to the RNA polymerase (RNAP) and dictate gene expression. Extra Cytoplasmic Function σ factors (ECF) are a subset of σ factors that coordinate environment-induced changes in transcription. The environment specific binding of ECF σ factors to the RNAP presents an effective mechanism for the bacillus to modulate gene expression. ECF σ factors, in turn, are regulated by their interaction with an anti-σ factor. The active σ factor is released from this complex upon specific cellular or environmental stimuli. The aim of this study was to understand the structural and mechanistic aspects of σ factor activation. Towards this goal, two ECF σ factors, σC and σL, were examined. Structural and biophysical studies on M. tuberculosis σC provided a novel insight into ECF σ factor regulation. Inter-domain interactions in σC were sufficient to occlude the DNA recognition regions even in the absence of an interacting protein. The structure of M. tuberculosis σL in complex with the anti-σ factor RslA provides a structural basis to rationalize the release of active σL under oxidative stress. The other chapters of this thesis include a description of the structure and biochemical features of a hypothetical protein Rv2704 that is co-transcribed with the primary σ factor σA. In an effort to understand the collaboration-competition-redundancy model of prokaryotic σ factors, we performed a computational analysis of this system compiling experimental data from the E. coli and B. subtilis model systems. These results are also presented in this thesis. Put together, the structural and biochemical characteristics of the σ factors presented in this thesis suggest substantial variations in the regulatory mechanisms of the M. tuberculosis σ factors when compared to the canonical E. coli or B. subtilis model systems. This thesis is organized as follows: Chapter 1: The introductory chapter of this thesis is organized to frame the pertinent mechanistic issues involved in the σ factor-regulatory protein interactions in the context of the underlying biology of M. tuberculosis. The first part of this chapter provides an overview of σ factors and a summary of the classification of these proteins and their roles in different prokaryotes. The latter part of this chapter is a summary of the pathogen M. tuberculosis in terms of its genetic composition, gene expression as well as aspects of virulence and pathogenecity. Chapter 2: This chapter describes the characterization of the ECF σ factor, σC. Here we report the structure of an ECF σ factor σC from M. tuberculosis. σC is essential for the lethality of M. tuberculosis in a mouse model of infection. Our studies suggest that M. tuberculosis σC differs from the canonical ECF σ factors as it has an N-terminal domain comprising of 126 amino acids that precedes the σC2 and σC4 domains. In an effort to understand the regulatory mechanism of this protein, the crystal structures of the σC2 and C4 domains of σC were determined. These promoter recognition domains are structurally similar to the corresponding domains of E. coli σA despite the low sequence similarity. Fluorescence experiments using the intrinsic tryptophan residues of σC2 as well as surface plasmon resonance measurements reveal that the σC2 and σC4 domains interact with each other. Mutational analysis suggests that the Pribnow box-binding region of σC2 is involved in this inter-domain interaction. Interactions between the promoter recognition domains in M. tuberculosis σC are thus likely to regulate the activity of this protein even in the absence of an anti-σ factor. Chapter 3 provides an account of the regulatory features of the ECF σ factor, σL. ECF σ factors are often regulated by their interactions with an anti-σ factor that can sense diverse environmental stimuli. Transcriptional responses to changes in the oxidation state are particularly important for M. tuberculosis as it adapts to the environment of the host alveoli and macrophages. Here we demonstrate that the protein RslA binds Zinc and can sequester σL in a reducing environment. Our data suggests that the cytosolic domain at the N-terminus of RslA alone is involved in binding σL. Under oxidizing conditions, the σL/RslA complex undergoes substantial conformational rearrangements that coincide with the release of the Zinc cofactor. In the absence of Zinc, the affinity of RslA for σL reduces by ca 8 fold compared to the holo form. The CXXC motif of RslA acts as a redox sensor. In response to oxidative stimuli, the proximal cysteines in this motif can form a disulfide bond with the release of the bound Zn2+ ion. This observation could be rationalized based on the crystal structure of the σL4/RslA complex. Put together, RslA is a distinct variant of the Zinc binding anti-σ factor (ZAS) family. The structural and biophysical parameters that control σL/RslA interactions demonstrate how variations in the rate of Zinc release and associated conformational changes in RslA could regulate the release of free σL in a measured response to oxidative stress. Chapter 4 is based on the biochemical and structural characterization of a hypothetical protein Rv2704. The gene for M. tuberculosis Rv2704 is located in the same operon as the principal σ factor σA. The biochemical and structural features of Rv2704 were thus examined to identify its role, if any, in the regulation of σA. This protein is a trimer in solution and adopts a chorismate mutase-like fold. The crystal structure reveals that Rv2704 is a member of the functionally diverse YjgF family of proteins. The important structural differences between Rv2704 and other YjgF proteins lie in the arrangement of secondary structural elements and the putative functional clefts between the subunit interface. Although Rv2704 does not interact with σA in vitro, the structural similarities to the YjgF family suggests that this protein could interact with a variety of metabolites, potentially influencing its function. Chapter 5 of this thesis is based on a computational analysis of σ factors. Four conformational segments of σ factors, referred to as σ1, σ2, σ3 and σ4 interact with specific regions of promoter DNA. ECF σ factors are a subset of σ factors that coordinate environment-induced transcription. ECF σ factors are minimalist σ factors with two DNA binding domains viz., σ2 and σ4 that recognize the –10 and –35 promoter elements and are unable to interact with either upstream-activating regions or the extended –10 element of the promoter. There are several ECF σ factors in a typical bacterium often characterized by substantial overlap in function. Here we present an analysis of B. subtilis ECF σ factors and their cognate promoters to understand functional overlap and redundancy in this class of proteins. As expected, conserved bases in the –10 element appear more critical for promoter selectivity than the –35 element. However, we note distinct conformational features in the –35 promoter interaction with the helix-turn-helix (HTH) motif when compared to a data-set of known HTH-DNA complexes. Furthermore, we note differences in –35 element interaction between σ factors that act alone and those that overlap in function. The σ factor promoter interactions were then examined vis-à-vis the estimated cellular concentration of these proteins and their affinity to bind the core RNAP. Put together, this analysis suggests that while the cellular protein concentration dictates the choice of an ECF σ factor to form a complex with the RNAP, conformational features of the –35 element serve to select potential collaborative members, a subset of which eventually initiate transcription. Collaborative arrangements and functional redundancy in ECF σ factors are thus possible within the limits placed by these two parameters. Chapter 6 is a summary of the work reported in this thesis and the conclusions that can be drawn based on these studies. The appendix section of this thesis comprises of technical details that were not included in the main text of this thesis. Appendix I describes the initial characterization of the M. tuberculosis σD/anti-σD complex. Appendix II provides the experimental protocols as well as some of the supplementary data to the work reported in Chapters 2-5 of this thesis.

Tee PhD thesis presents the study of the response of Mycobacterium tuberculosis, the causative agent of tuberculosis, upon prolonged exposure to lethal concentrations of the first line anti-tuberculosis drug, rifampicin. The study shows that prolonged exposure to lethal concentration of rifampicin causes cell death initially by several log orders of cells, followed by a persistence phase, from where rifampicin resisters emerge carrying mutation in the RRDR locus of the rpoB gene. This phenomenon was found to occur even when the drug concentration is well above MBC levels. Luria-Delbruck experiment, in a modified format, showed that the resisters emerged as fresh mutants, and not due to the growth of pre-existing natural resisters to rifampicin. The per sister cells, which showed high levels of hydroxyl radical generation, were found to have thickened outer layer, unlike the mid-log phase cells, which restricts the permeability of a fluorescent-conjugate of rifampicin, 5-FAM-rifampicin, 10-fold less in per sister cells, as compared to mid-log phase cells. The thickened outer layer has high negative surface charge and is hydrophilic in nature. It is proposed that the hydrophilic-natured thickened outer layer might have restricted the permeability of lethal concentrations of hydrophobic-natured rifampicin. This, in turn, might have ensured the presence of sub-lethal concentration rifampicin inside the per sisters, which in turn might have generated the hydroxyl radical that caused mutagenesis to generate rifampicin resisters. The Chapter 1 is the Introduction to the thesis presented in 4 parts – Part 1.1, 1.2, 1.3 and 1.4, introducing briefly the history of antibiotics, antibiotic resistance, antibiotic persistence and a brief history of tuberculosis respectively. It is concluded with a rationale behind the present study. The Chapter 2 presents the entire Materials and Methods used in the experiments described in the thesis. The Chapter 3 presents the data on the response of M. tuberculosis to rifampicin upon extended exposure. Rifampicin exposure of susceptible M. tuberculosis H37Ra cells showed a decrease in their CFU/ml followed by a persistence phase wherein the CFU/ml remained constant. However, prolonged exposure of rifampicin even at higher concentrations showed regrowth in the culture, which was found to be due to the emergence of rifampicin-resistant bacteria. Screening of rifampicin-resistant mutants showed point mutations in the rifampicin resistance determining region (RRDR) of the rpoB gene in all the mutants. In parallel, using trans formants of M. tuberculosis expressing unstable GFP under the respective native ribosomal RNA promoter, the metabolic status of the per sister cells was determined. When actively growing highly fluorescing cells were exposed to lethal concentration of rifampicin, their metabolism diminished, as illustrated by the decrease in their fluorescence during persistence phase, followed by the emergence of a sub-population of bacteria which were again metabolically active. In order to verify whether the rifampicin resisters are freshly formed mutants or have come from the naturally existing resisters, Luria-Delbruck fluctuation test was performed in a modified manner. The number of rifampicin resisters that emerged from the persistent phase was found to vary amongst different cultures from different days and different times of exposure, showing fluctuation. However, the addition of theorem before persistence phase almost abolished the generation of the rifampicin-resistant bacilli, indicating the role of hydroxyl radical in the emergence of rifampicin resisters. The generation of hydroxyl radical in mycobacteria exposed to rifampicin was confirmed using electron para-magnetic resonance spectrometry (EPR), with the spin trap agent, 5,5- Dimethyl-1-pyrroline N-oxide (DMPO) specific for hydroxyl radical. An increase in the formation DMPO-OH adduct in the persistence phase cells was observed, in comparison to mid-log phase cells. Exposure to theorem significantly diminished the adduct formation. The persistence phase cells also showed significantly high levels of signal specific to the hydroxyl-specific fluorescent dye, hydroxyphenyl fluorescein (HPF), as compared to the mid-log phase cells. In addition we have determined the oxidative stress in the bacilli upon rifampicin exposure using a redox biosensor (Mrx1-roGFP) which also showed high oxidative stress in the persistent phase. These observations confirmed the presence of high levels of oxidative stress and hydroxyl radical in the rifampicin persistent cells, in comparison with mid-log phase cells. Whole genome sequencing of the four independently isolated rifampicin resistant M. tuberculosis showed genome wide mutations having less common mutations with respect to the wild type genome, indicative of the occurrence of random mutagenesis. In addition mutation frequencies were comparable between the samples with respect to the wild type sample. About 69% of the mutations were A-C or T-G, followed by A-T or T-A, which is known to be due to oxidative stress in the cells. Variations in the colony morphology were also observed on the persistent phase cells, indicating the occurrence of mutagenesis in the bacterial genome during rifampicin treatment. The Chapter 4 is on the morphological and ultrastructural studies on rifampicin-exposed M. tuberculosis cells. Transmission electron micrographs of rifampicin per sisters showed significant thickening of the outermost capsular layer (OL), as compared to the mid-log phase cells. This observation was verified by staining the cells with ruthenium red, which specifically stains anionic polysaccharide of the OL. Zeta potential (ZP) measurement of the surface charge of the persistent cells showed high negative ZP, as compared to the mid-log phase cells. The negative ZP value was found to gradually increase during the course of rifampicin treatment and to decrease during the regrowth phase. Hexadecane assay showed larger proportion of per sister cells being retained in the aqueous phase, as compared to the mid-log phase cells. This indicated the higher hydrophilicity of the per sister cells, which was in agreement with the higher surface negative charge of the cells. The permeability of rifampicin per sister cells to 5-FAM-rifampicin (rifampicin conjugated to 5-carboxy fluorescein, which is as hydrophobic as rifampicin) was found to be 10-fold less than that of the mid-log phase cells. However, removal of the thick OL by bead beating (BB) with 4 mm glass beads significantly improved the permeability of per sister cells to 5-FAM-rifampicin. On the contrary, no difference in the 5-FAM-rifampicin uptake was observed in mid-log phase cells, with or without BB. These observations implied that the thick hydrophilic-natured OL with high negative surface charge may be playing a significant role in limiting the permeability of hydrophobic-natured rifampicin entry into the persisted cells. This in turn may ensure the presence of sub-lethal concentration of rifampicin inside the persisted cells that are exposed to lethal concentration of the antibiotic. Exposure of bacteria to sub-lethal concentration of antibiotics has been reported to generate oxidative stress in the bacteria, leading to mutagenesis. A model has been proposed based on these observations in which the persistent mycobacteria are protected from lethal concentrations of the rifampicin by the thick OL which in turn ensures sub-lethal intracellular antibiotic concentration, leading to the generation of hydroxyl radical mediated mutagenesis and thereby emergence of rifampicin resisters. This thesis is concluded with the list of salient findings, publications and references.