The studies reported in this thesis address, firstly, aspects of ribosome recycling in eubacteria, and secondly, a preliminary characterization of an EFG-like locus from Mycobacterium smegmatis. A hitherto unsuspected role of the ribosome recycling factor in governing the fidelity of initiation has been discovered during the course of this work. A summary of the relevant literature is presented in chapter 1. Section I of the ‘General Introduction’ provides a brief review of the current understanding of protein biosynthesis, with a special emphasis on ribosome recycling and the fidelity of translation initiation. Section II provides a brief introduction to mycobacterial translation, and known deviations from the E. coli prototype are highlighted. This is followed by three chapters containing experimental work, as summarized below. (i) Role of elongation factor G in governing specificity of ribosome recycling In eubacteria and the eukaryotic organelles, the post-termination ribosome complexes are recycled by the combined action of ribosome recycling factor (RRF) and elongation factor G (EFG). Earlier studies both from our laboratory and other laboratories have revealed the existence of specific interactions between RRF and EFG that are crucial for ribosome recycling, using ribosomes from E. coli and factors from both E. coli and heterologous sources such as Mycobacterium tuberculosis, Thermus thermophilus etc. In this study, to further understand the mechanism of ribosome recycling, we employed polysomes from both E. coli and M. smegmatis and monitored ribosome recycling in in vitro assays using RRF and EFG from both these sources; in addition, in vivo assays were performed in E. coli using either temperature-sensitive strains or strains carrying a deletion in frr (encoding RRF) or fusA (encoding EFG) genes. It was found that, in E. coli, RRF from Mycobacterium tuberculosis and M. smegmatis function with MtuEFG or MsmEFG but not with EcoEFG. In vitro assays revealed that the mycobacterial EFGs facilitate recycling of both the mycobacterial and E. coli polysomes not only with mycobacterial RRFs but also with EcoRRF. In contrast, although EcoEFG binds to mycobacterial polysomes, carries out GTP hydrolysis and is reported to sustain translocation on mycobacterial ribosomes, its activity in recycling mycobacterial polysomes was undetectable with EcoRRF, as well as with the mycobacterial RRFs. Such an observation allowed us to infer that EFG establishes specific interactions with the ribosome that are crucial for ribosome recycling but not for translocation, suggesting that translocation and ribosome recycling are distinct functions of EFG. In addition, a number of EFG chimeras generated by swapping corresponding domains between Msm- and Eco-EFGs were analyzed for their ability to sustain translocation and/or ribosome recycling in E. coli and M. smegmatis, using a combination of in vivo (for E. coli) and in vitro (for both E. coli and M. smegmatis) approaches. Our observations reveal that a dual set of specific interactions of EFG with RRF and ribosome is essential for ribosome recycling. While the RRF-EFG specific interactions are predominantly localized to the domains IV and V of EFG, the EFG-ribosome specific interactions that are crucial for ribosome recycling are not localized to a specific region of EFG but are found throughout the molecule. Our novel observations also emphasize the importance of using ribosomes from heterologous sources to understand the mechanism of this crucial process. (ii) Impact of rRNA methylations on ribosome recycling and fidelity of initiation in Escherichia coli Ribosomal RNA (rRNA) contains a number of modified nucleosides in functionally important regions including the intersubunit bridge regions; however, very little is known about the role of these rRNA modifications in ribosome function. As the activity of ribosome recycling factor (RRF) in separating the large and the small subunits of the ribosome involves disruption of the intersubunit bridges, we investigated the impact of rRNA methylations on ribosome recycling. The isolation of a folD122 mutant strain of E. coli with a deficiency in rRNA methylations, as well as the availability of E. coli strains deficient for various individual methyltransferases that modify specific rRNA residues, provided us with a genetic tool to assay the role of rRNA methylations in ribosome recycling. We observed that deficiency of rRNA methylations, especially at positions 1518 and 1519 of 16S rRNA near the interface with the 50S subunit and in the vicinity of the IF3 binding site, adversely affects the efficiency of RRF-mediated ribosome recycling. In addition, a compromise in the RRF activity was found to afford increased initiation with a mutant tRNAfMet wherein the three consecutive G-C base pairs (29GGG31:39CCC41), a highly conserved feature of the initiator tRNAs, were mutated to those found in the elongator tRNAMet (29UCA31:39ψGA41). This observation has allowed us to uncover a new role of RRF as a factor that contributes to fidelity of initiator tRNA selection on the ribosome. In addition, it was also found that IF3 and rRNA methylations, both of which are known to affect fidelity of initiation, exert their effects through distinct mechanisms, despite the proximity of a cluster of methylated rRNA residues to the IF3 binding site on the 30S subunit. (iii) Characterization of the role of EFG2, an EFG-like locus in Mycobacterium smegmatis Several bacteria, including various species of mycobacteria (with the exception of Mycobacterium leprae) contain a second EFG-like locus, denoted as fusA2, which shows considerable homology to fusA (encoding EFG). A comparison of the sequences of EFG and EFG2 from various bacteria reveals that EFG2 contains a GTPase domain and domains with significant homology to EFG domains IV and V, suggesting that it may function as an elongation factor. With the single exception of a recent study on Thermus thermophilus EFG2, this class of EFG-like protein factors has not been studied so far. Hence, it was of interest to characterize EFG2. In the current study, EFG2 from M. smegmatis was characterized both by in vitro biochemical assays as well as by in vivo experiments targeted to investigate the biological significance of EFG2 in mycobacteria. It was found that, unlike EFG, MsmEFG2 could not sustain either translocation or ribosome recycling in E. coli. Despite the fact that the purified MsmEFG2 could bind guanine nucleotides, it lacked the ribosome-dependent GTPase activity characteristic of EFG and other translation GTPases, suggesting that it was unlikely to function as an elongation factor. However, EFG2 was found to be expressed in stationary phase cultures of M. smegmatis. To understand the biological significance of EFG2, fusA2 was disrupted in M. smegmatis. The viability of the M. smegmatis mc2155 fusA2::kan derivative indicates that MsmfusA2 is a non-essential gene. While disruption of the fusA2 gene (encoding EFG2) in M. smegmatis does not appear to affect its growth and survival in log phase or stationary phase or under hypoxic conditions, preliminary experiments indicate that disruption of fusA2 confers a fitness disadvantage to M. smegmatis when competed against M. smegmatis mc2155 (with wild type fusA2 locus).

FtsZ, the principal cell-division protein, polymerizes in GTP-dependent manner in vitro (Bramhill and Thompson, 1994; Mukherjee and Lutkenhaus, 1994; Rivas et al., 2000). FtsZ polymerization at the mid-cell site of bacterium leads to formation of a guiding scaffold, the Z-ring, for bacterial cytokinesis (Bi and Lutkenhaus, 1991; Sun and Margolin, 1998). GTP-induced polymerization process of FtsZ can be monitored in vitro Using 90º light scattering (Mukherjee and Lutkenhaus, 1999) and polymers formed can be visualized using transmission electron microscopy (Lu and Erickson, 1998) or quntitated in terms of the amount of FtsZ polymer pelleted during ultracentrifugation (Mukherjee and Lutkenhaus, 1998). The research work presented in this thesis focused on structure-function correlative analysis of Mycobacterium tuberculosis FtsZ(MtFtsZ0 and FtsZ proteins of Mycobacterium leprae (M1FtsZ), Mycobacterium smegmatis(MsFtsZ), and Streptomyces coelicolor (ScFtsZ) (as it is from Actinomycetes family to which mycobacteria belong) in vitro. It was initiated with investigation on the biochemical properties of Mycobacterium leprae FtsZ (M1FtsZ) in vitro. In comparison with those of MtFtsZ. Subsequently, the role of C-terminal stretch of amino acid residues of MtFtsZ in polymerization was investigated. Finally, a comparative analysis of the biochemical properties of MtFtsZ, MsFtsZ, and ScFtsZ was carried out in order to find out whether a correlation exists between the time taken by the FtsZ of a bacterium to polymerise and the generation time of the organism. The thesis is presented in five chapters. First Chapter gives an exhaustive introduction on the structure-function aspects of FtsZ. Second Chapter deals with materials used in this research work and details of various experimental methods [cloning and expression of FtsZ (White et. Al., 2000), decision and point mutagenesis, preparation of His-tag free MtFtsZ and M1FtsZ by thrombin cleavage method, 90º light scattering (Mukherjee and Lutkenhaus, 1999), White, et al., 2000), transmission electron microscopy (Lu and Erickson, 1998), pelleting assay for polymeric FtsZ (Mukherjee and Lutkenhaus, 1998), GTP-binding by UV-crosslinking (RayChaudhuri and Park, 1992; de Boer et al.,) GTPase assay(RayChaudhuri and Park, 1992); de Boer et al., 1992), Circular Dichroism (Saxena and Wetlaufer, 1971) and ANS fluorescence emission spectroscopy (Semisotnov, et al., 1991)]. The Chapters three to five contain all the data related to the research work, the outlines of which are given below. Chapter 3. Biochemical Characterisation of FtsZ Protein of Mycobacterium leprae In Comparison with the Biochemical Properties of FtsZ Protein of Mycobacteriulm tulberculosis In Vitro The major finding in this part of thesis work is on the demonstration that single reciprocal point mutation partially revives polymerization-inactive M1FtsZ and Inactivates polymerization-active MtFtsZ in vitro. In brief, soluble, recombinant M1FtsZ did not show detectable polymerization in vitro, in contrast to MtFtsZ, which showed appreciable polymerization, under standard conditions, when monitored using 90º light scattering assay and transmission electron microscopy. This was a surprising result, as M1FtsZ and MtFtsZ has 96% protein sequence identity. Mutation f T172 in the N-terminal domain of M1FtsZ to A172, as it exists in MtFtsZ, showed dramatic levels of polymerization in vitro. Reciprocal mutation of A172 in MtFtsZ to T172, as it exists in M1FtsZ, abolished polymerization in vitro. Further, M1FtsZ showed weak GTPase activity, in contrast to MtFtsZ, which showed appreciable GTPase activity. While T172A mutation enhanced GTPase activity of MtFtsZ in vitro. Circular dichroism spectroscopy and ANS fluorescence emission spectroscopy showed that there were no major secondary or tertiary structural changes in these point mutants. These observations demonstrate that the residue at position 172 plays a critical role in the polymerization of M1FtsZ and MtFtsZ, without appreciably affecting their respective GTpPase activity. Further, this result might have implications on evolution of a slow polymerizing FtsZ in slow growing bacteria. Further details of evolution related questions are addressed in Chapter 5. Chapter 4. Role of Carboxy Terminal Residues in the Biochemical Properties of FtsZ Protein of Mycobacterium tuberculosis In Vitro The major finding in this part of thesis work is the demonstration that the C-terminal end residues are critically required for polymerization of MtFtsZ in vitro, which is in direct contrast to the dispensability of C-terminal residues of Escherichia coli FtsZ(EcFtsZ), Bacillus subtilis FtsZ (BsFtsZ), and Pseudomonas aeruginosa (PaFtsZ) for polymerization. FtsZ protein from several bacterial species namely, Methanococcus jannaschii (MjFtsZ), Bacillus subtillis(BsFtsZ), Pseudomonas aeruginosa (PaFtsZ), and Aquifex aeolicus (AaFtsZ) (Lowe and Amos, 1998; Oliva et al., 2007), and Mycobacterium tuberculosis H37Rv (mtFtsZl Leung et al., 2004), whose crystal structures have been solved so far, were found to possess an N-terminal domain and a C-terminal domain that were connected to each other through a helix. The extreme C-terminal portion of all these FtsZ proteins is constituted by an unstructured tail (Lowe and Amos, 1998; Oliva et al., 2007l Leung et al., 2004), which is not found in the respective crystal structure of the protein. We examined whether C-terminal residues of soluble recombinant FtsZ of Mycobacterium tuberculosis (mtFtsZ) have any role in MtFtsZ polymerization in vitro. Deletion of C-terminal 66 residues (313-379) was found to abolish polymerization. Replacement of the C-terminal 66 residues with the extreme C-terminal 13-residue stretch (DDDDVDVPPFMRR) did not restore polymerization. Although the terminal R in DDDDVDVPPFMRR is dispensable for full-length MtFtsZ polymerization, the terminal R in DDDDVDVPPFMR is indispensable for polymerization. Neither replacement of this R, in the terminal R deletion mutant DDDDVDVPPFMR, with K/H/D/A residues enabled polymerization. GTP binding and GTPase activities of the mutants were partially affected. The indispensable nature of C-terminal residues for MtFtsZ polymerization in vitro is contrary to the dispensability of the equivalent extreme C-terminal residues of Escherichi coli, Pseudomonas aeruginosa, and Bacillus subtilis FtsZ (Wang et. Al., 1997; Cordell et al., 2003; Singh et al., 2007) for in vitro polymerization. The essentiality of C-terminal extreme residues of BtFtsZ for polymerization offers direction to design anti MtFtsZ polymerization agents. Chapter 5. An attempt to find correlation between Biochemical properties of FtsZ and Generation Time of the Bacterium The clue that there might be a correlation between FtsZ polymeristion and generation time of the bacterium came from the observation mentioned in chapter 3. The presence of polymerization-aversive T172 in the FtsZ of extremely slow-growing M. leprae 913.5 days generation time, Levy, 1970) and polymerization-favouring A172 in the FtsZ of M. tuberculosis(18hrs generation time, Patterson and Youmans, 1970). For a bacterium, which has short generation time, it might be conducive to have an FtsZ that will also polymerise fast. Conversely, for a bacterium, which has long generation, it might be conducive to have an FtsZ molecule that will polymerise slow. In this respect, a preliminary comparative study was carried out between the generation time of bacterial species, E. coli, Mycobacterium smegmatis, Streptomyces coelicolor, M leprae, and M. tuhberculosis and their respective FtsZ (EcFtsZ, MsFtsZ, M1FtsZ and MtFtsZ). Detailed biochemical characterization of EcFtsZ and MtFtsZ has already been reported in the literature. In this thesis work, biochemical characterisation of M1FtsZ(Chapter 3), ScFtsZ and MsFtsZ (in this Chapter) were carried out. E. coli, which has a generation time of 18-55 min(labrum, 1953), possesses FtsZ (EcFtsZ) that reaches steady state of polymerization in about 10 sec under standard conditions in vitro (Beamhill and Thompson, 1994), using 90º light scattering assay (Mukherjee and Lukenhaus, 1999). On the other hand, M. tuberculosis, which has a generation time of 18hrs in vivo (Patterson and Youmans, 1970) and 24 hrs in vitro (Hiriyanna and Ramakrishnan, 1986) possesses FtsZ (MtFtsZ) that reaches steady state of polymerization in about 6 min post-addiction of GTP in vitro (White et al., 2000). Further, M. leprae, which takes 13.5 days tp divide once in vivo (levy, 1970), possesses an FtsZ (M1FtsZ) that does not even show polymerization under standard conditions in vitro (Chapter 3 of this thesis). The organisms Mycobacterium smegmatis and Streptomyces coelicolor have generation times that fall in between those of the other three organisms mentioned above. While M. smegmatis divides once in 2-3 hrs (Husson, 1998), S. coelicolor has a variable generation time depending on growth condition, which can be as fast as once in 2.31 hours, depending upon growth conditions (Cox, 2004). We found ScFtsZ and MsFtsZ takes around 4 min to reach polymerization saturation after addition of GTP, EcFtsZ( 10 sec), MtFtsZ (10 min) and M1FtsZ (dose not polymerise in vitro) seem to indicate that there exists a correlation between polymerization saturation after addition of GTP, EcFtsZ (10sec), MtFtsZ (10 min) and M1FtsZ (does not polymerise in vitro) seem to indicate that there exists a correlation between polymerization saturation time and the generation time of the respective bacterium. But when we compared polymerization time of ScFtsZ and MsFtsZ (4 min both case) with MtFtsZ ( 6 min), we found that there is no linear correlation with generation time of these bacteria and the time taken by their FtsZ to reach steady state of polymerization. Many more bacterial FtsZ proteins need to be characterized to conclusively state wthether there exist a correlation between generation time of bacteria and the time taken for their FtsZ to reach steady state of polymeristion. Such correlation would simply reveal the fact that the primary structure of an FtsZ protein might have evolved to suit the generation time of the bacterium.

Under hostile conditions, bacteria elicit stress response. Such stress response is regulated by a secondary messenger called (p)ppGpp. (p)ppGpp is involved in wide range of functions such as GTP homeostasis, biofilm formation and cell growth. Its regulation and mode of action is not well understood. This work has been initiated with an aim to gain insights into the molecular basis of stress response. (p)ppGpp was discovered on the chromatogram of cell extract from starved E. coli cells. (p)ppGpp is synthesized and hydrolyzed by Rel/SpoT in Gram negative bacteria (such as E. coli), and by bifunctional enzyme called Rel in Gram positive bacteria (such as Mycobacteria). The obvious question that comes in our mind is how bifunctional Rel enzyme decides on synthesis or hydrolysis in Gram positive bacteria such as Mycobacterium? In our laboratory, it has been shown that N-terminal domain of Rel shows unregulated (p)ppGpp synthesis implying regulatory role of C-terminal domain. Also, concurrent increase in anisotropy of Rel C-terminal domain with the increase in concentration of pppGpp has been observed indicating the binding of pppGpp to the C-terminal domain. We performed Isothermal Calorimetry experiment to confirm that pppGpp binds with C-terminal domain of Rel enzyme. For identification of the binding region, small molecule analogue 8-azido-pppGpp has been synthesized. This analogue is UV-crosslinked with C-terminal domain of Rel and specificity of the interaction has been determined by gel based crosslinking experiments. Crosslinked protein has been subjected to the ingel¬trypsin digestion and analyzed by mass spectrometry. We identified two crosslinked peptides in the mass spectra of trypsin digest in case of the crosslinked protein where identity of the parent peptide is confirmed by MS-MS analysis. Site directed mutagenesis has been carried out based on the conservation of residues in the crosslinked peptides. Isothermal Calorimetry analysis has been done where Rel C-terminal domain mutants are titrated with pppGpp in order to detect any defect in binding due to the mutations. Mutations leading to the reduced binding affinity of pppGpp to Rel C-terminal domain have been introduced in the full length Rel protein and activity assays are carried out so as to evaluate the effects of mutations on synthesis and hydrolysis activity. In mutants, synthesis activity is found to be increased with the concomitant reduction in hydrolysis activity. This indicates the feedback loop where pppGpp binds to Rel C-terminal domain to regulate it own synthesis and hydrolysis. In E. coli, pppGpp binds to RNA polymerase and modulates the transcription. The region where it binds is controversial. In addition, whether ppGpp and pppGpp have different binding site on RNA polymerase is not known. The latter question becomes important in the light of evidence where differential regulation of transcription by ppGpp and pppGpp have been indicated. We found that ppGpp and pppGpp have an overlapping binding site on RNA polymerase. The 8-azido-ppGpp has been mapped on β and β’ subunits whereas binding site of 8-azido-pppGpp has been located on the β’ subunit. We observed that the 8-azido¬pppGpp labels RNA polymerase more efficiently than ppGpp. pppGpp can compete out ppGpp as illustrated by DRaCALA assay and gel based crosslinking experiment. However, the RNAP from B. subtilis does not bind to (p)ppGpp. (p)ppGpp is ubiquitous in bacteria but absent in mammals. Thus, blocking (p)ppGpp synthesis would impede the survival of bacteria without having any effect on humans. Recently, Relacin compound has been synthesized by another group in order to inhibit (p)ppGpp synthesis. The limitations of this compound are the requirement of high concentration (5mM) for inhibition and low permeability across the membrane. Taking hints from the latter compound, we acetylated the nd 2’, 3’ and 5’ position of ribose ring and benzoylated the 2position of guanine moiety in guanosine molecule. We observed significant inhibition of in vitro pppGpp synthesis and biofilm formation. More studies will be conducted in near future to test these compounds for their plausible functions. In collaboration with Prof. Jayaraman (Organic Chemistry, IISc), many artificial glycolipids are synthesized and tested for biological function. We observed that synthetic glycolipids exhibit a profound effect as inhibitors of the key mycobacterial functions. These analogs impede biofilm formation and can plausibly affect long term survival. Glycolipid analogs can compete with natural glycolipids, thus may help in understanding their functions. Our past and recent studies have showed that the synthetic glycolipids act as inhibitors of mycobacterial growth, sliding motility and biofilm formation. The major lacuna of these glycolipid inhibitors is the requirement of high concentration. Their inhibitions at nanomolar concentrations remain to be achieved. Issues surrounding the thick, waxy mycobacterial cell wall structures will continue to be the focus in manifold approaches to mitigate detrimental effects of mycobacterial pathogens. In chapter 1, introduction to the research work has been written and role of (p)ppGpp and its functions have been discussed. In chapter 2, novel binding site of pppGpp on Rel C-terminal domain and its regulatory role have been discussed. In chapter 3, differential binding of ppGpp and pppGpp to RNA polymerase has been discussed. In chapter 4, studies on natural and synthetic analogues of pppGpp have been presented. In chapter 5, synthetic glycolipids studies have been described. Chapter 6 summarizes all the chapters.

DNA repair proteins in mycobacteria and their physiological importance Mycobacterium tuberculosis, the causative organism of tuberculosis, resides in the host macrophages where it is subjected to a plethora of stresses like reactive oxygen species (ROS) and reactive nitrogen intermediate(RNI) which are generated as a part of the host’s primary immune response. These stresses can damage the cellular components of the pathogen including DNA and its precursors. Two common damages to DNA and its precursors caused by ROS and RNI are oxidation of guanine to 8-oxo-guanine and deamination of cytosine to uracil. Mycobacteria, which are known to have high G+C content, must be more susceptible to such damages, and are thus equipped with the mechanisms to counteract these damages. One such mechanism is to hydrolyse the 8-oxo-dGTP into 8-oxo-dGMP to avoid its incorporation in the DNA during its synthesis. This job is done by a protein called MutT.In mycobacteria four homologs of MutT, namely MutT1, MutT2, MutT3 and MutT4 have been annotated. The second mechanism deals with the repair of uracil residues present in DNA which are generated by deamination of cytosines or incorporation of dUTP during DNA synthesis. This is taken care of by a protein called uracil DNA glycosylase (UDG) which excises uracil by cleaving the N-C1’ glycosidic bond between the uracil and the deoxyribose sugar in a DNA repair pathway called the base excision repair (BER). In this study, the biochemical properties and physiological role of mycobacterial MutT2 and, MSMEG_0265 (MsmUdgX), a novel uracil DNA glycosylase superfamily protein, have been investigated. I.Biochemical characterization of MutT2 from mycobacteria and its antimutator role. Nucleotide pool, the substrate for DNA synthesis is one of the targets of ROS which is generated in the macrophage upon Mycobacterium tuberculosis infection. Thus, the pathogen is at increased risk of accumulating oxidised guanine nucleotides such as 8-oxo-dGTP and 8-oxo-GTP. By hydrolysing the damaged guanine nucleotides before their incorporation into nucleic acids, MutT proteins play a critical role inallowing organisms to avoid their deleterious effects. Mycobacteria possess several MutT proteins. Here, we have purified recombinantM. tuberculosisMutT2 (MtuMutT2) andM. smegmatisMutT2 (MsmMutT2) proteins as representative of slow and fast growing mycobacteria, for the purpose of biochemical characterization. UnlikeEscherichia coliMutT, which hydrolyzes 8-oxo-dGTP and 8-oxo-GTP, the mycobacterial proteins hydrolyze not only 8-oxo-dGTP and 8-oxo-GTP but also dCTP and 5-methyl-dCTP. Determination of kinetic parameters (KmandVmax) revealed thatwhileMtuMutT2 hydrolyzes dCTP nearly four times better than it does 8-oxo-dGTP,MsmMutT2 hydrolyzes them almost equally well. Also,MsmMutT2 is about 14 times more efficient thanMtuMutT2 in its catalytic activity of hydrolyzing 8-oxo-dGTP.Consistent with these observations,MsmMutT2 but notMtuMutT2 rescuesE. colifor MutT deficiency by decreasing both themutation frequency and A to C mutations (a hallmark of MutT deficiency). We discuss these findings in the context of the physiological significance of MutT proteins. II.Understanding the biochemical properties of MSMEG_0265 (MsmUdgX), a novel uracil DNA glycosylase superfamily protein Uracil DNA glycosylases (UDGs) are base excision repair enzymes which excise uracil from DNA by cleaving the N-glycosidic bond. UDGs are classified into 6 different families based on their two functional motifs, i. e.,motif A and motif B. In mycobacteria, there are two uracil DNA glycosylases, Ung and UdgB which belong to Family 1 and Family 5, respectively. In this study, based on the presence of the two functional motifs, we have discovered yet another uracil DNA glycosylase in M. smegmatis, which we have called MsmUdgX.The motif A and motif B of this protein indicate that it does not belong to any of the UDG families already classified but has highest similarity with Family 4 UDGs. Homologs of this protein are also present in several other organisms like M. avium, Streptomyces ceolicolor, Rhodococcus etc., but absent in M. tuberculosis, archaea and eukaryotes. Activity assays of this protein show that unlike other UDGs, MsmUdgX does not excise uracil, but forms a tight complex with uracil containing single stranded (ss) and double stranded (ds) DNAs, as observed by a shifted band in 8M urea-PAGE as well as SDS-PAGE. It also does not recognize other modified nucleotides that we investigated, in DNA. The protein binds to uracil-DNA in a wide range of pH and the minimum substrate required for its binding is pNUNN. Like Family 4 UDG, the protein has Fe-S cluster but it is not as thermostable as the Family 4 UDGs. Addition of different metal ions does not affect its binding property, and even the presence of M. smegmatis cell free extract does not diminish its binding activity. Since this protein binds specifically to uracil in DNA, an application of the protein for detection of uracil in the genomic DNA is proposed. III. Elucidation of the role of KRRIH loop in MsmUdgX by mutational analysis MsmUdgX is a novel uracil DNA glycosylase superfamily protein which has the highest homology to Family 4 UDGs. However, alignment of MsmUdgX amino acid sequence with that of Family 4 UDGs shows that there is an extra stretch of amino acids which is unique to this group of proteins. This stretch, defined by AGGKRRIH is absent in all Family 4 UDGs and the region KRRIH of the strtch is quite conserved amongst all UdgX proteins. Homology modelling of MsmUdgX, using a Family 4 UDG (TthUdgA) shows that this extra stretch of amino acids forms an outloop near the enzyme active site. Another unique difference between MsmUdgX and Family 4 UDGs is in the motif A where MsmUdgX has GEQPG and the Family 4 UDGs haveGE(A/G)PG. Our work on MsmUdgX has shown that, unlike other UDGs, this protein does not excise uracils, but forms a tight complex with the uracil containing DNA. This unique tight uracil binding property as well as KRRIH amino acid stretch has not been observed for any uracil DNA glycosylase superfamily proteins. So, to gain insight into the role of KRRIH and glutamine (Q) of motif A in MsmUdgX family of proteins, site directed mutagenesis was done in this region and we observed that mutation of His109 of the KRRIH loop to serine (S) leads to a gain of uracil excision activity, whereas changing the R107 to S, ‘RRIH’ to ‘SSAS’ or deleting the loop altogether leads to loss of its complex formation activity. Further, mutation of H109 to other amino acids like G, Q and A also shows uracil excision activity. Mutation of the glutamine in the motif A to alanine so that it is exactly similar to that of Family 4 UDGs, does not affect its uracil binding activity. This observation indicates that the KRRIH loop has an important role in the tight binding and/or uracil excision activity of MsmUdgX. Crystal structure of MsmUdgX in complex with uracil-DNA oligo and MsmUdgX H109S mutants are being studied.IV. Physiological importance of MsmUdgX in M. smegmatis MsmUdgX is a uracil DNA glycosylase superfamily protein which binds tightly to uracil (in DNA) without excising it. To elucidate its role in M. smegmatis, knockout of udgX was generated. Growth comparison of the wild type and the ΔudgX strains does not show any growth differences under the conditions tested. However, overexpression of MsmUdgX in recA deficient strains of E. coli as well as M. smegmatis leads to their retarded growth. Retarded grown is also observed in strains deficient in other DNA repair proteins that work in conjunction with RecA. These observations indicate that repair/release of MsmUdgX-uracil DNA complex might be a RecA dependent process.

One of Africa’s neglected tropical diseases, African Trypanosomiasis, is not only fatal but also has a crippling impact on economic development. Heat shock proteins play a wide range of roles in the cell and they are required to assist the parasite as it moves from a cold blooded insect vector to a warm blooded mammalian host. The expression of heat shock proteins increases during these heat shock conditions, and this is considered to play a role in differentiation of these vector-borne parasites. Heat shock protein 70 (Hsp70) is an important molecular chaperone that is involved in protein homeostasis, Hsp40 acts as a co-chaperone and stimulates its intrinsically weak ATPase activity. In silico analysis of the T. brucei genome has revealed the existence of 12 Hsp70 proteins and 65 Hsp40 proteins to date. A novel Hsp70, TbHsp70.c, was recently identified in T. brucei. Different from the prototypical Hsp70, TbHsp70.c contains an acidic substrate binding domain and lacks the C-terminal EEVD motif. By implication the substrate range and mechanism by which the substrates are recognized may be novel. The ability of a Type I Hsp40, Tbj2, to function as a co-chaperone of TbHsp70.c was investigated. The main objective of this study was to biochemically characterize TbHsp70.c and its partnership with Tbj2 to further enhance our knowledge of parasite biology. TbHsp70.c and Tbj2 were heterologously expressed and purified and both proteins displayed chaperone activities in their ability to suppress aggregation of thermolabile MDH. TbHsp70.c also suppressed aggregation of rhodanese. ATPase assays revealed that the ATPase activity of TbHsp70.c was stimulated by Tbj2. The targeted inhibition of the function of heat shock proteins is emerging as a tool to combat disease. The small molecule modulators quercetin and methylene blue are known to inhibit the ATPase activity of Hsp70. However, methylene blue did not significantly inhibit the ATPase activity of TbHsp70.c; while quercetin, did inhibit the ATPase activity. In vivo heat stress experiments indicated an up-regulation of the expression levels of TbHsp70.c. RNA interference studies showed partial knockdown of TbHsp70.c with no detrimental effect on the parasite. Fluorescence microscopy studies of TbHsp70.c showed a probable cytoplasmic subcellular localization. In this study both TbHsp70.c and Tbj2 demonstrated chaperone activity and Tbj2 possibly functions as a co-chaperone of TbHsp70.c.

Maintenance of the levels of nucleoside triphosphates (NTPs) as well as their corresponding deoxy derivatives (dNTPs) is crucial to all growth and developmental processes. The enzyme nucleoside diphosphate kinase (NDK) utilises an autophosporylated enzyme intermediate to catalyse the transfer of 5’ terminal phosphate from NTPs (mostly ATP) to nucleoside diphosphates (NDPs) via a reversible mechanism as given below. N1TP + NDK ↔N1DP+ −NDK-His* (1) N2DP + NDK-His* P ↔N2TP + NDK−His. (2) In the γ-phosphoryl group transfer, the highly conserved His 117 active site residue becomes autocatalytically phosphorylated, in the enzyme intermediate (NDK-H*). This phosphoryl group is transferred to ribo-or deoxyribonucleotides (N2DP) in a substrate non-specific manner. In addition to its fundamental role in nucleotide metabolism, NDP kinase is also involved in a number of cellular regulatory functions such as growth and developmental control, tumor metastasis suppression, signal transduction and so on. From mycobacterial genera, NDK of Mycobacterium tuberculosis (MtNDK) has been crystallised, structure was solved and biochemical functions were elucidated. However, there has not been any such study on the NDK of Mycobacterium smegmatis, except on the possible interaction with other proteins which modulates the NTP synthesising activity of MsNDK, towards specific NTPs. M. smegmatis, being a saprophytic, fast growing and non-pathogenic mycobacterium that is widely used as an experimental model mycobacterial system to study various biological processes in mycobacteria, it was thought appropriate to study NDK from this organism. The outcome of current study is presented in five chapters. The First Chapter gives a detailed introduction on the structural and functional aspects of NDK from diverse organisms, from bacteria to humans. Chapter 2. Molecular Cloning, Expression and Characterisation of Biochemical Activities of Nucleoside Diphosphate Kinase from Mycobacterium smegmatis mc 155 The research work starts with the molecular cloning, overexpression, purification, and characterisation of biochemical activities of recombinant MsNDK protein. In brief, ndk gene from M. smegmatis (Msndk) has been cloned, efficiently overexpressed as a soluble 6xHis-tagged recombinant protein, purified through affinity chromatography, and its biochemical characterisation for ATPase, GTPase and NTP synthesising activities have been demonstrated. Catalytic mutant of MsNDK, MsNDK-H117Q, was generated using site-directed mutagenesis approach and H117 was shown to be essential for the catalytic activity. Further experiments revealed that it is the same H117 that is required for mediating autophosphorylation as well, which is an intermediate in the transphosphorylation reaction of NDK. Chapter 3. Characterisation of Oligomerisation Property of M. smegmatis Nucleoside Diphosphate Kinase: the Possible Role of Hydrogen Bond and Hydrophobic Interactions The present study revealed that presence of homodimer of MsNDK could be observed in the presence of heat and SDS. Chemical cross-linking experiments revealed that MsNDK forms dimer, tetramer and hexamer. Homology modeling of MsNDK on the MtNDK crystal structure supported the existence of hexamer as three homodimers. Gln 17, Ser 24 and Glu 27 were found to be positioned at the dimer interface. Mutations on these residues did not abolish the stability of the respective mutant dimers in the presence of SDS and heat. Modeled structure of MsNDK revealed the existence of hydrophobic interactions at the dimer interface. In silico approach helped in mapping the existence of hydrophobic interactions at the dimer interface as two consecutive β-strands. Exposure of hydrophobic residues, using organic solvent methanol, abolished the dimer completely, indicating the vital role of hydrophobic interactions in the dimer stability. In solution, the native MsNDK was found to be a hexamer. Chapter 4. Mycobacterial Nucleoside Diphosphate Kinase Functions as GTPase Activating Protein for Mycobacterial Cytokinetic Protein FtsZ In Vitro Mammalian, plant, and bacterial NDKs can function as GTPase activating protein (GAP) for small G proteins namely, p21 Ras, Rad, and Rho-GTPases in animals and Pra1, Pra2, and GPA1 in Arabidopsis thaliana in vitro. We examined whether NDK of M. tuberculosis (MtNDK) can function as GAP in vitro for the cytokinetic protein FtsZ of Mycobacterium tuberculosis (MtFtsZ), which is a protein with a classical G-protein fold, possessing GTP-binding and GTPase activities (like G proteins). Both MtNDK and MsNDK could function as GAP for MtFtsZ and FtsZ of M. smegmatis (MsFtsZ) respectively in vitro. Similarly, MtNDK could function as GAP for MsFtsZ and reciprocally MsNDK could function as GAP from MtFtsZ. Interaction of NDK with respective FtsZ could be observed. Physiological implications of GAP activity of NDK on FtsZ are discussed. Chapter 5. Transcriptional Analyses of Nucleoside Diphosphate Kinase Gene of Mycobacterium smegmatis mc 155 Although there are studies on the structural and functional aspects of NDK, there are not many studies available on the transcriptional analysis of nucleoside diphosphate kinase (NDK) gene expression in general and nothing in particular in mycobacterial systems. Therefore we studied the transcriptional analysis of expression of Msndk gene, in order to map the Transcriptional Start Site (TSS), identification of promoter elements, and elucidated of transcriptional activity of the promoters. Expression of Msndk gene was analysed in exponential growth phase and under two different stress conditions wherein DNA replication gets arrested. Hydroxy Urea (HU), which reduce dNTP pools by inhibiting ribonucleotide reductase and Phenethyl Alcohol (PEA), which affects membrane structure resulting in DNA replication arrest, were used. Two transcripts and their promoter elements were mapped and their promoter activities were demonstrated. The profile of transcripts was found to be identical under the three different conditions examined.

For survival and successful propagation, every organism has to maintain the genomic integrity of the cell. The information content, in the form of nucleotide bases, is constantly threatened by endogenous agents and environmental pollutants. In particular, pathogenic mycobacteria are constantly exposed to DNA-damaging assaults such as reactive oxygen species (ROS) and reactive nitrogen intermediate (RNI), in their habitat which is inside host macrophage. In addition, the genome of Mycobacterium tuberculosis makes it more susceptible for guanine oxidation and cytosine deamination as it is G-C rich. Therefore DNA repair mechanisms are extremely important for the mycobacterium. An important enzyme involved in DNA repair is uracil-DNA glycosylase (Ung). To access the genomic information, during repair as well as DNA replication and recombination, dsDNA must unwind to form single stranded (ss) intermediates. ssDNA is more prone to chemical and nuclease attacks that can produce breaks or lesions and can also inappropriately self associate. In order to preserve ssDNA intermediates, cells have evolved a specialized class of ssDNA-binding proteins (SSB) that associate with ssDNA with high affinity. As part of a major programme on mycobacterial proteins in this laboratory, structural studies on mycobacterial uracil-DNA glycosylase (Ung) and single-stranded DNA binding protein (SSB) have been carried out. The structures were solved using the well-established techniques of protein X-ray crystallography. The hanging drop vapour diffusion and microbatch methods were used for crystallization in all cases. X-ray intensity data were collected on a MAR Research imaging plate mounted on a Rigaku RU200 X-ray generator. The data were processed using the HKL program suite. The structures were solved by the molecular replacement method using the program PHASER and AMoRe. Structure refinements were carried out using the programs CNS and REFMAC. Model building was carried out using COOT. PROCHECK, ALIGN, INSIGHT and NACCESS were used for structure validation and analysis of the refined structures. MD simulations were performed using the software package GROMACS v 3.3.1. Uracil-DNA glycosylase (UNG), a repair enzyme involved in the excision of uracil from DNA, from mycobacteria differs from UNGs from other sources, particularly in the sequence in the catalytically important loops. The structure of the enzyme from Mycobacterium tuberculosis (MtUng) in complex with a proteinaceous inhibitor (Ugi) has been determined by X-ray analysis of a crystal containing seven crystallographically independent copies of the complex. This structure provides the first geometric characterization of a mycobacterial UNG. A comparison of the structure with those of other UNG proteins of known structure shows that a central core region of the molecule is relatively invariant in structure and sequence, while the N- and C-terminal tails exhibit high variability. The tails are probably important in folding and stability. The mycobacterial enzyme exhibits differences in UNG-Ugi interactions compared with those involving UNG from other sources. The MtUng-DNA complex modelled on the basis of the known structure of the complex involving the human enzyme indicates a domain closure in the enzyme when binding to DNA. The binding involves a larger burial of surface area than is observed in binding by human UNG. The DNA-binding site of MtUng is characterized by the presence of a higher proportion of arginyl residues than is found in the binding site of any other UNG of known structure. In addition to the electrostatic effects produced by the arginyl residues, the hydrogen bonds in which they are involved compensate for the loss of some interactions arising from changes in amino-acid residues, particularly in the catalytic loops. The results arising from the present investigation represent unique features of the structure and interaction of mycobacterial Ungs. To gain further insights, the structure of Mycobacterium tuberculosis Ung (MtUng) in its free form was also determined. Comparison with appropriate structures indicate that the two domain enzyme slightly closes up when binding to DNA while it slightly opens up when binding to its proteinaceous inhibitor Ugi. The structural changes on complexation in the catalytic loops reflect the special features of their structure in the mycobacterial protein. A comparative analysis of available sequences of the enzyme from different sources indicates high conservation of amino acid residues in the catalytic loops. The uracil binding pocket in the structure is occupied by a citrate ion. The interactions of the citrate ion with the protein mimic those of uracil in addition to providing insights into other possible interactions that inhibitors could be involved in. SSB is an essential accessory protein required during DNA replication, repair and recombination, and various other DNA transactions. Eubacteral single stranded DNA binding (SSB) proteins constitute an extensively studied family of proteins. The variability in the quaternary association in these tetrameric proteins was first demonstrated through the X-ray analysis of the crystal structure of Mycobacterium tuberculosis SSB (MtSSB) and Mycobacterium smegmatis (MsSSB) in this laboratory. Subsequent studies on these proteins elsewhere have further explored this variability, but attention was solely concentrated on the variability in the relative orientation of the two dimers that constitute the tetramer. Furthermore, the effect of this variability on the properties of the tetrameric molecule was not adequately addressed. In order to further explore this variability and strengthen structural information on mycobacterial SSBs in particular, and on SSB proteins in general, the crystal structures of two forms of Mycobacterium leprae single stranded DNA-binding protein (MlSSB) has been determined. Comparison of the structures with other eubacterial SSB structures indicates considerable variation in their quaternary association although the DNA binding domains in all of them exhibit the same OB-fold. This variation has no linear correlation with sequence variation, but it appears to correlate well with variation in protein stability. Molecular dynamics simulations have been carried out on tetrameric molecules derived from the two forms and the prototype E. coli SSB and the individual subunits of both the proteins. The X-ray studies and molecular dynamics simulations together yield information on the relatively rigid and flexible regions of the molecule and the effect of oligomerization on flexibility. The simulations provide insights into the changes in the subunit structure on oligomerization. They also provide insights into the stability and time evolution of the hydrogen bonds/water-bridges that connect two pairs of monomers in the tetramer. In continuation of our effort to understand structure-function relationships of mycobacterial SSBs, the structure of MsSSB complexed with a 31-mer polydeoxy-cytidine single stranded DNA (ssDNA) was determined. The mode of ssDNA binding in the MsSSB is different from the modes in the known structures of similar complexes of the proteins from E. coli (EcSSB) and Helicobacter pylori (HpSSB). The modes in the EcSSB and HpSSB also exhibit considerable differences between them. A comparison of the three structures reveals the promiscuity of DNA-binding to SSBs from different species in terms of symmetry and the path followed by the bound DNA chain. It also reveals commonalities within the diversity. The regions of the protein molecule involved in DNA-binding and the nature of the residues which interact with the DNA, exhibit substantial similarities. The regions which exhibit similarities are on the central core of the subunit which is unaffected by tetramerisation. The variable features of DNA binding are associated with the periphery of the subunit, which is involved in oligomerization. Thus, there is some correlation between variability in DNA-binding and the known variability in tetrameric association in SSBs. In addition to the work on Ung and SSB, the author was involved in X-ray studies on crystals of horse methemoglobin at different levels of hydration, which is described in the Appendix of the thesis. The crystal structure of high-salt horse methaemoglobin has been determined at environmental relative humidities (r.h.) of 88, 79, 75 and 66%. The molecule is in the R state in the native and the r.h. 88% crystals. At r.h.79% the molecule appears to move towards the R2 state. The crystal structure at r.h.66% is similar, but not identical, to that at r.h.75%. Thus variation in hydration leads to variation in the quaternary structure. Furthermore, partial dehydration appears to shift the structure from the R state to the R2 state. This observation is in agreement with the earlier conclusion that the changes in protein structure that accompany partial dehydration are similar to those that occur during protein action. A part of the work presented in the thesis has been reported in the following publications. 1. Singh, P., Talawar, R.K., Krishna, P.D., Varshney, U. & Vijayan, M. (2006). Overexpression, purification, crystallization and preliminary X-ray analysis of uracil N-glycosylase from Mycobacterium tuberculosis in complex with a proteinaceous inhibitor. Acta Crystallogr. F62, 1231-1234. 2. Kaushal, P.S., Talawar, R.K., Krishna, P.D., Varshney, U. & Vijayan, M. (2008). Unique features of the structure and interactions of mycobacterial uracil-DNA glycosylase: structure of a complex of the Mycobacterium tuberculosis enzyme in comparison with those from other sources. Acta Crystallogr. D64, 551-560. 3. Kaushal, P.S., Sankaranarayanan, R. & Vijayan, M. (2008). Water-mediated variability in the structure of relaxed-state haemoglobin. Acta Crystallogr. F64, 463-469.

One key to the pathogenic potential of the mycobacteria lies in their capacity to resist destruction by infected macrophages and dendritic cells. Robust host immune responses during mycobacterial infection often involve a potent CD4, CD8 and gamma delta T cell mediated effector responses including lysis of mycobacteria infected host cells, secretion of variety of cytokines like IFN-γ etc. However, pathogenic mycobacteria survives for prolonged periods in the phagasomes of infected macrophages within the host in an asymptomatic, latent state and can reactivate years later if the host’s immune system wanes. One of the most devastating consequences of infection with mycobactreia is the formation of caseating granulomas followed by tissue destruction with liquefaction causing cavity formation. Pathogenic mycobacteria reside in these granulomas, which are formed by the accumulation of monocytes, epithelioid and foamy macrophages as well as cytolytic lymphocytes including CD8 T cells around the infection focus. In this regard, rigid balance as well as modulation of inflammatory immune responses by the host upon infection of pathogenic microbes is one of the crucial steps not only in controlling the spread of pathogen from the site of infection to reminder of host organs, but also in mounting an effective memory response so that future exposures/infections by similar pathogen can be effectively controlled. Significantly, despite this complex host response, it remains unclear, that why the immune response controls mycobacteria but does not eradicate infection. Both human and mouse studies have provided ample evidence that even in the face of an adequate immune response, mycobacteria are able to persist inside macrophages. These findings have suggested series of survival strategies employed by Mycobacterium sp. during its infection of host macrophages/dendritic cells which include, blockade of phagosome-lysosome fusion, secretion of ROI antagonistic proteins like superoxide dismutase & catalase, inhibition of processing of its antigens for presentation to T cells, decrease in secretion of proinflammatory cytokines by inducing secretion of immunosuppressive cytokines like IL-10 and TGF-β etc. In view of above-mentioned observations, graulomas in response to pathogenic mycobacterial infections have long been considered host-protective structures formed to contain infection. In this perspective, Matrix metalloproteinase-9 (MMP-9), an important member of Zn2+ and Ca2+ dependent endopeptidases, participates in a significant manner in several aspects of host immune responses to mycobacterial infection such as graunloma formation, matrix (ECM) reorganization, lymphocytes trafficking and infiltrations, inflammation etc. MMP-9 is expressed at various clinical categories of tuberculosis disease like active cavitary tuberculosis, meningitis and pleuritis. Notably, in case of pulmonary tuberculosis, breakdown of ECM by MMP-9 forms an integral part of the granuloma formation. Importantly, Mycobacterium tuberculosis infection in MMP-9 deficient mice revealed defective bacterial proliferation, reduced bacterial burden and reduced lung macrophages recruitment compared to wild-type, in addition, to reduced ability to initiate or maintain well-formed granulomas. In this context, we explored the signaling events modulated by Mycobacterium bovis bacillus Calmette-Gue´rin (BCG) or its novel cell wall antigens during induced expression of MMP-9 or SPI6 in macrophages. Our studies clearly demonstrate that NO, a product of iNOS activity, is responsible for M. bovis BCG-triggered activation of Notch1 in macrophages through direct regulation of Jagged1 expression as well as in generation of activated Notch1. We present the evidence that iNOS activity is a critical factor in TLR2 mediated Notch1 activation as macrophages derived from iNOS knockout (iNOS-/-), but not from wild-type (WT) mice failed to activate Jagged1 expression as well as Notch1 signaling upon M. bovis BCG infection. The loss of TLR2-mediated Jagged1 expression or Notch1 activation in iNOS-/-macrophages could be rescued by treatment with NO donor 3-morpholinosydnonimine (SIN1) or S-nitroso-Nacetylpenicillamine (SNAP). Signaling perturbations strongly implicated the role for cross talk among members of Notch1-PI3 Kinase and MAPK cascades in M. bovis BCG-TLR2– mediated activation of Notch1 target genes MMP-9 or Hes1. Chromatin immunoprecipitation experiments demonstrate that M. bovis BCG’s ability to trigger increased binding of CSL/RBP-Jk to MMP-9 promoter was severely compromised in macrophages derived from iNOS-/-mice compared to WT mice. These results are consistent with the observation that NO-triggered Notch1 signaling-mediated CSL/RBP-Jk recruitment has a positive regulatory role in M. bovis BCG-induced MMP-9 transcription. We show the correlative evidence that this mechanism operates in vivo by immunohistochemical expression analysis of activated Notch1 or its target gene products Hes1 or MMP-9 in brains of WT or iNOS-/-mice that were intracerebrally infected with M. bovis BCG. Further, activation of Notch1 signaling in vivo could be demonstrated only in granulomatous lesions in brains derived from human patients with tuberculous meningitis (TBM) as opposed to healthy individuals, validating the role of Notch1 signaling in mycobacterial pathogenesis. Briefly, we have identified NO as the pathological link between TLR2 and Notch1 signaling, which regulates the relative abundance of various immunopathological parameters including MMP-9 in macrophages. Synopsis Despite mycobacteria elicits robust host T cell responses as well as production of NO, ROI or 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. These findings clearly advocate roles for mycobacteria mediated various immune evasion strategies to modulate the signaling cascades thus leading to macrophage activation. Importantly, TLR2 triggering by mycobacteria elicits the activation of divers sets of anti or pro-apototic genes expression, a balance of which will have strong bearing on the overall cell-fate decisions across many cell types. In this regard, a novel granzyme B inhibitor, SPI6/PI9, can exhibit robust resistance to various cells including dendritic cells or tumor cells from lysis by CD8 cytotoxic T cells (CTL). SPI6/PI9 predominantly functions by inhibiting Granzyme B, an effector protease of cytotoxic granules released by CTL upon its TCR recognition of infected cells such as macrophages, dendritic cells etc. In this context, current investigation attempted to investigate molecular details involved in M. bovis BCG triggered SPI6 expression as well as the involvement of TLR2NO-Notch1 signaling axis in driving induced expression of SPI6, akin to that of MMP-9 expression. We demonstrate that M. bovis BCG trigger SPI6 expression in macrophages and requires critical participation of TLR2-MyD88 dependent NO-Notch1 signaling events. More importantly, signaling perturbations data suggest the involvement of cross talk among the members of PI3 Kinase and MAPK cascades with Notch1 signaling in SPI6 expression. In addition, SPI6 expression requires the Notch1 mediated recruitment of CSL/RBP-Jk and NF-κB to the SPI6 promoter. Functional studies strongly attribute critical involvement of SPI6 and MMP-9 in imparting protection to M.bovis BCG infected macrophages from lysis effectuated by CTL. Macrophages are principal mediators of initiation as well as activation of host inflammatory responses to pathogenic mycobacterial infection. Albeit mycobacteria reside within phagolysosomes of the infected macrophages, envelope glycoconjugates like Lipoarabinomannan (LAM), phosphatidyl-myo-inositol mannosides (PIM), Trehalose 6,6′dimycolate (TDM; cord factor) etc. are released and traffic out of the mycobacterial phagosome into endocytic compartments as well as can gain access to the extracellular environment in the form of exocytosed vesicles. In this perspective, PIM represent a variety of phosphatidyl-myo-inositol mannosides (PIM) 1-6 containing molecules and are integral component of the mycobacterial envelope. A number of biological functions have been credited to PIM2. PIM2 was shown to trigger TLR2 mediated activation of macrophages that resulted in activation of NF-κB, AP-1, and mitogen-activated protein (MAP) kinases. In addition to pulmonary granuloma-forming activities, PIM2 was shown to recruit NKT cells into granulomas. Further, surface associated PIM was suggested to act as adhesins mediating attachment of M. tuberculosis bacilli to non-phagocytic cells. Accordingly, mycobacterial envelope antigen PIM2 could initiate or affect the inflammatory responses similar to mycobacteria bacilli. In this perspective, we explored whether novel cell surface antigen PIM2 similar to whole M. bovis BCG bacilli can contribute to molecular signaling events leading to MMP-9 expression in macrophages. Our current study provides the evidence that PIM2 driven activation of signaling cascades triggers the expression of MMP-9. TLR stimulation by various agonists has been shown to activate Notch signaling resulting in modulation of diverse target genes involved in pro-inflammatory responses in macrophages. In this regard we demonstrated that PIM2 induced expression of MMP-9 involved Notch1 upregulation and activation of Notch1 signaling pathway in a TLR2-MyD88 manner. Enforced expression of the cleaved Notch1 in macrophages induced the expression of MMP-9. Further, PIM2 triggered significant p65 nuclear factor-κB (NF-κB) nuclear translocation that was dependent on activation of PI3 Kinase or Notch1 signaling. Furthermore, MMP-9 expression requires Notch1 mediated recruitment of Suppressor of Hairless (CSL) and NFκB to MMP-9 promoter. Taken together, our observations clearly describe involvement of TLR2/iNOS in activating Notch1 and PI3 Kinase signaling during infection of macrophages with M. bovis BCG, thus effectuating the regulation of specific effector gene expressions, such as SPI6 and MMP-9. These results clearly describe the cross talk of Notch1 signaling with PI3 Kinase and MAPK pathways, thus leading to differential effects of Notch1 signaling. Overall, we believe that our work will extend the current understanding of inflammatory parameters associated with host-mycobacteria interactions which might lead to better design as well as evaluation of therapeutic potential of novel agents targeted at diverse mycobacterial diseases.

Mycobacteria are multifaceted pathogens capable of causing both acute disease as well as an asymptomatic latent infection. Protective immunity against pathogenic mycobacteria depends principally on cell-mediated immunity executed by efficient anti-infectious functions of type 1 T helper (Th1) subset of CD4+ T cells. The polarization of Th1 responses is orchestrated by IL-12 secreted by antigen presenting cells (APCs) such as macrophages and dendritic cells (DCs). A hallmark of Th1 type CD4+ T cells is the production of IFN-γ that activates plethora of innate cell-mediated immunity. It is well known that cytokines such as IFN-γ, IL-12 and TNF-α are required for control of mycobacterial infection in humans as well as in mice. However, it remains unclear that why the immune response controls mycobacteria, but does not eradicate infection suggesting critical roles for series of survival strategies employed by pathogenic mycobacteria. In general, these evasion strategies include blockade of phagosome-lysosome fusion, secretion of ROI antagonistic proteins like superoxide dismutase & catalase, inhibition of processing of its antigens for presentation to T cells, induced secretion of immunosuppressive cytokines like IL-10 and TGF-β etc. that ultimately suppress the secretion of IL-12 and IFN-γ from APCs and T cells respectively, culminating in a skewed Th1/Th2 balance towards unprotective Th2 responses. Th2 cells secrete IL-4, IL-5, IL-9, IL-10 and IL-13 but are deficient in clearing intracellular infections including pathogenic mycobacteria. This eventually leads to inhibition of host’s immuno-protective responses with concomitant increase in the vulnerability to chronic mycobacterial infection. In this intricate process, modulation of cyclooxygenase-2 (COX-2) levels, a key enzyme catalyzing the rate-limiting step in the inducible production of prostaglandin E2 (PGE2), by mycobacteria like Mycobacterium bovis BCG assumes critical importance in influencing the overall host immune response. PGE2, an immunosuppressive member of prostaglandin family, is known to restrain production of IL-12, as well as reactive oxygen intermediates. PGE2-mediated inhibition of IL-12R, diminishes IL-12 responsiveness of macrophages and dendritic cells. PGE2 also inhibits the secretion of IFN-γ, which is important in activating T cells and macrophages. In contrast, PGE2 promotes IL-10 production by macrophages, dendritic cells and Th1-to-Th2 shift of acquired immune responses by inhibiting IL-2 and enhancing IL-4 production. Albeit, mitogen-activated protein kinase (MAPK) and nuclear factor-kappa B (NF-κB) signaling pathways are generally believed to be involved, little is known about the signaling molecules playing significant roles upstream of MAPK and NF-κB pathways during mycobacteria triggered COX-2 expression. Further, information on early receptor proximal signaling mechanisms essential during mycobacteria mediated induction of COX-2 remains scanty. In this regard, signaling cascade triggered upon recognition of mycobacterial components by pattern recognition receptors (PRR) signify as critical event in overall regulation of cell fate decisions. PRR like Toll like receptor (TLR2) and nucleotide-binding oligomerization domain 2 (NOD2) are two nonredundant recognition mechanisms of pathogenic mycobacteria. Several components of mycobacteria have been identified as being responsible for TLR2-dependent activation including 19-kDa lipoprotein, lipomannan etc.; while NOD2 recognizes mycobacterial peptidoglycans through its interaction with muramyl dipeptide (MDP). Interestingly, although mycobacteria reside within phagolysosomes of the infected macrophages, many cell wall antigens like lipoarabinomannan (LAM), phosphatidyl-myo-inositol mannosides (PIM), trehalose 6,6′-dimycolate (TDM; cord factor), PE/PPE family proteins etc., are released and traffic out of the mycobacterial phagosome platform into endocytic compartments. Importantly, these antigens could gain access to the extracellular environment in the form of exocytosed vesicles. In this perspective, PIM represents a variety of phosphatidyl-myo-inositol mannosides (PIM) 1-6 containing molecules and are integral component of the mycobacterial envelope. Further, PIM2 is a known TLR2 agonist and reported to activate NF-κB, AP-1, and MAPK suggesting that mycobacterial envelope antigen PIM2 could modulate the inflammatory responses similar to mycobacteria bacilli. In this context, we explored the signaling events modulated by M. bovis BCG, and role for TLR2 and NOD2 in this intricate process, to trigger the expression of COX-2 in macrophages. Our studies demonstrated that M. bovis BCG triggered TLR2-dependent signaling leads to COX-2 expression and PGE2 secretion in vitro in macrophages and in vivo in mice. Further, the presence of PGE2 could be demonstrated in sera or CSF of tuberculosis patients. Similarly, mycobacterial TLR2 agonist PIM2 and NOD2 ligand MDP triggered COX-2 expression in macrophages. The induced COX-2 expression in macrophages either by M. bovis BCG or PIM2 or MDP was dependent on NF-κB activation, which was in turn mediated by iNOS/NO and Wnt-β-Catenin dependent participation of the members of Notch1-PI3K signaling cascade. Importantly, loss of iNOS activity either in iNOS null macrophages or by pharmacological intervention in wild type macrophages severely abrogated M. bovis BCG ability to trigger the generation of Notch1 intracellular domain (NICD) as well as activation of PI3K signaling cascade. On contrary, treatment of macrophages with SIN-1, an NO donor, resulted in a rapid increase in generation of NICD, activation of PI3K pathway as well as the expression of COX-2. Interestingly, pharmacological inhibition as well as siRNA mediated knockdown of Wnt-β-Catenin signaling compromised ability of M. bovis BCG to induce activation of Notch1-PI3K signaling and drive COX-2 expression. Concomitantly, activation of Wnt-β-Catenin signaling by LiCl triggered activation of Notch1 and PI3K pathway as well as COX-2 expression. Stable expression of NICD in RAW 264.7 macrophages resulted in augmented expression of COX-2. Further, signaling perturbation experiments suggested involvement of the cross-talk of Notch1 with PI3K signaling cascade. In this perspective, we propose TLR2 and NOD2 as two major receptors involved in mycobacteria mediated activation of Notch1PI3K signaling, and the activation of iNOS/NO and Wnt-β-Catenin signaling axis as obligatory early receptor proximal signaling events during mycobacteria induced COX-2 expression in macrophages. Functional characterization of mycobacterial antigens that are potent modulators of host immune responses to pathogens by virtue of induced expression of COX-2 assumes critical importance for deciphering pathogenesis of mycobacterial diseases as well as to identify novel therapeutic targets to combat the disease. In this context, a group of novel antigens carried by M. tuberculosis that are expressed upon infection of macrophages belong to PE and PPE family of proteins. Ten percent of the coding capacity of M. tuberculosis genome is devoted to the PE and PPE gene family members, exemplified by the presence of Pro-Glu (PE) and Pro-Pro-Glu (PPE) motifs near the N-terminus of their gene products. Many members of the PE family exhibit multiple copies of polymorphic guanine-cytosine– rich sequences (PGRS) at the C-terminal end, which are designated as the PE_PGRS family of proteins. A number of PE/PPE proteins associate with the cell wall and are known to induce strong T & B cell responses in humans. However information related to effects of PE/PPE antigens on the maturation and functions of human dendritic cells and eventual modulation of T cell responses as well as underlying signaling events remains obscure. Our results demonstrated that two cell wall associated/secretory PE_PGRS proteins PE_PGRS 17, PE_PGRS 11 and PPE family protein PPE 34 recognize TLR2, induce maturation and activation of human dendritic cells and enhance the ability of dendritic cells to stimulate CD4+ T cells. In addition, tuberculosis patients were found to have a high frequency of T cells specific to PE_PGRS and PPE antigens. We further found that PE/PPE proteins-mediated activation of dendritic cells involves participation of ERK1/2, p38 MAPK and NF-κB signaling pathways. While, PE_PGRS antigens-matured dendritic cells secreted high amounts of inflammatory cytokine IL-12, PPE 34 triggered maturation of dendritic cells was associated with secretion of high amounts of anti-inflammatory cytokine IL-10 but not the Th1-polarizing cytokine IL-12. Consistent with these results, PPE 34-matured dendritic cells favored secretion of IL-4, IL-5 and IL-10 from CD4+ T cells and contributed to Th2 skewed cytokine balance ex vivo in healthy individuals and in patients with pulmonary tuberculosis. Interestingly, PPE 34-skewed Th2 immune response involved induced expression of COX-2 in dendritic cells. Our results suggest that by inducing differential maturation and activation of human dendritic cells, PE/PPE proteins could potentially modulate the initiation of host immune responses against mycobacteria. Taken together, our observations clearly signify the potential role for TLR2 and NOD2 triggering by M. bovis BCG in activating receptor proximal Notch1-PI3K signaling during induced COX-2/PGE2 expression which represents a crucial immune subversion mechanism employed by mycobacteria in order to suppress or attenuate host immune responses. Further, differential maturation of human dendritic cells by PE_PGRS and PPE antigens as well as their ability to stimulate CD4+ T cells towards Th1 and Th2 phenotype respectively, improves our understanding about host-mycobacteria interactions and clearly paves a way towards the development of novel combinatorial therapeutics.

The global re-emergence of TB and other mycobacterial infections have underscored the need for a thorough investigation of the biology of the causative agent, Mycobacterium tuberculosis, at the molecular level. The peculiar features of the bacterium such as slow growth rate, dormancy, unique cell wall composition and resistance towards phagocytosis by macrophages demands a detailed understanding of different essential molecular processes including transcription in this genus. Sequencing of several mycobacterial genomes provided an impetus for understanding the gene function and regulation of this formidable pathogen. Transcriptional regulation is one of the major mechanisms controlling gene expression. While a number of transcription units, promoters, sigma factors, and gene functions were identified and characterized, key features of transcription process are yet to be understood. The current study aims to understand some of the facets of transcription initiation and elongation in mycobacteria. The thesis is divided into five chapters. Chapter 1 introduces the bacterial transcription process. It starts with the description of the central molecule in transcription -the RNA polymerase (RNAP) and its catalytic mechanism. In the next section, each step of the transcription initiation, elongation and termination has been discussed. The mechanistic details as well as the different cellular factors involved in the regulation of the transcription have been discussed. The final part gives an overview of the transcription machinery of the mycobacteria, describing the promoter specificity and regulation of different sigma factors and other transcription factors known till date in mycobacteria. The scope and the objectives of the thesis are presented at the end of this chapter. In Chapter 2, a method of purification of RNAP from mycobacteria for optimized promoter -polymerase interactions is described. In vitro transcription analysis is important to understand the mechanism of transcription. Various assays for the analysis of initiation, elongation and termination form the basis for better understanding of the process. Purified RNAP with high specific activity is necessary to carry out a variety of these specific reactions. The RNAP purified from Mycobacterium smegmatis from exponential phase showed low σA-promoter specificity in promoter -polymerase interaction studies. This is due to the presence of a large number of sigma factors during exponential phase and under-representation of σA required for house - keeping transcription. In vivo reconstitution of RNAP holoenzyme with σA and its purification procedure which resulted in a holoenzyme with stoichiometric σA content is described in this chapter. The reconstituted holoenzyme showed enhanced promoter -specific binding and transcription activity compared to the enzyme isolated using standard procedure. Chapter 3 is aimed at the comparison of promoter - specific events during transcription initiation in mycobacteria. DNA -protein interactions that occur during transcription initiation play an important role in regulating gene expression. To initiate transcription, RNAP binds to promoters in a sequence -specific fashion. This is followed by a series of steps governed by the equilibrium binding and kinetic rate constants, which in turn determine the overall efficiency of the transcription process. The first detailed kinetic analysis of promoter - RNAP interactions during transcription initiation in the σA-dependent promoters PrrnAPCL1, PrrnB and Pgyr of M. smegmatis are presented in this chapter. The promoters show comparable equilibrium binding affinity but differ significantly in open complex formation, kinetics of isomerization and promoter clearance. Furthermore, the two rrn promoters exhibit varied kinetic properties during transcription initiation and appear to be subjected to different modes of regulation. In addition to the distinct kinetic patterns, each one of the house -keeping promoters studied has its own rate-limiting step in the initiation pathway, indicating the differences in their regulation. Moving the focus of the thesis from transcription initiation to elongation, a transcript cleavage factor of M. tuberculosis has been characterized in Chapter 4. After initiation of transcription, a number of proteins participate during elongation and termination by modifying the properties of the RNAP. Gre proteins are one such class of transcription elongation factors which are conserved across bacteria. They regulate transcription by binding near the secondary channel of RNAP, projecting their N-terminal coiled-coil domain into the active center and stimulating hydrolysis of the newly synthesized RNA by RNAP in the backtracked elongation complexes. Rv1080c is a putative gre factor homolog (MtbGre) present in M. tuberculosis.The protein enhanced the efficiency of promoter clearance by lowering the abortive transcription and also rescued the arrested and paused elongation complexes efficiently in the GC rich mycobacterial template. The Gre factor of M. smegmatis encoded by the gene MSMEG_5263 also showed biochemical properties similar to the M. tuberculosis protein. Although the mycobacterial Gre is similar in domain organization and shared the key residues for catalysis and RNAP interaction with Escherichia coli Gre proteins, it could not complement the E. coli strain deficient in Gre factors. Moreover, MtbGre failed to rescue E. coli RNAP stalled elongation complexes, indicating the importance of specific protein - protein interactions for transcript cleavage. Decrease in the level of MtbGre also reduced the bacterial survival by several fold indicating its essential role in mycobacteria and suggesting that a single Gre copes up with the burden of transcription fidelity of the genome. Chapter 5 describes the studies carried out to identify Gre factor homologs in mycobacteria and deciphering their function during transcription. Gre factors are members of a growing family of proteins which regulate RNAP through secondary channel. Apart from the Gre factor, putative members of this class of proteins are identified in both M. smegmatis and M. tuberculosis.The closest homologue of the canonical Gre factor of M. tuberculosis in its genome is Rv3788. The protein has Gre factor like domain organization and possess the key acidic residues required for transcript cleavage activity and the putative hydrophobic RNAP interacting residues in the C-terminus similar to MtbGre. Despite having these common features, Rv3788 did not stimulate transcript cleavage. In contrast, it turns out to be a transcription inhibitor by preventing the binding of NTPs to the enzyme. The transcription inhibition is not promoter specific, and is mediated by its binding to RNAP through the secondary channel with its N-terminus coiled coil domain. Like M. tuberculosis, the fast growing non-pathogenic mycobacteria M. smegmatis also has an ORF (MSMEG_6292) which is homologous to its canonical Gre factor and it interacts with RNAP in a similar manner. However, this protein did not exert any transcript cleavage or inhibitory activities but could compete with the Gre factor for binding to RNAP. The Gre factor homologs in mycobacteria may be involved in regulation by inhibiting transcription or by blocking the RNAP secondary channel from other RNAP active site modulators.