According to the World Health Organization (WHO), M. tuberculosis, the causative agent of TB, accounts for approximately 1.7 million deaths annually. Further contributing causes to the worldwide TB incidence, is the widespread unavailability and ineffectiveness of TB vaccines, time consuming diagnostic methods and unsuccessful treatment approaches. Research for better characterising mycobacteria in general, or other Mycobacterium species, may help us to better understand M. tuberculosis and TB disease mechanisms, which will in turn lower the future TB disease prevalence, as this may lead to the development of better treatments, diagnostics and vaccines. Mycobacteria use various secretion pathways, including the ESX- or type VII secretion (T7S) system, to ensure transport across the complex cell wall. The genome of M. tuberculosis has five copies of a gene cluster known as the ESX gene cluster region, which is associated with virulence and viability of mycobacteria. The ESX-3 gene cluster is thought to be essential for growth of M. tuberculosis and proposed to be involved in iron / zinc homeostasis. Mycobacteria synthesise siderophores, which are proposed to be involved in the uptake of iron over their cell wall. M. tuberculosis are known to produce two types of siderophores, namely: carboxymycobactins and mycobactins. Loots and colleagues, however illustrated, that ESX-3 knockouts, show signs of iron overload, despite the absence of the mycobactins induced by knocking out the ESX-3 gene cluster. It was hypothesised, that this overload occurs due to an increase in exochelin synthesis, another iron uptake protein not associated with ESX-3, overcompensating for the perceived iron depletion in the knockout organism. A Metabolomics research approach was subsequently used in this study, to generate new information in order to better characterise the role of iron on the metabolism of these organisms, and additionally confirm the role of ESX-3 in iron uptake. In this study, we firstly determined the most optimal extraction conditions for this metabolomics investigation. Two extraction methods were subsequently investigated and compared, considering their repeatability and their respective capacities to extract those compounds which best differentiate the M. smegmatis ESX-3 knockouts and wild-type parent strains. Considering the results generated, the total metabolome method was chosen for further analyses, for the following reasons: 1) it is simpler, 2) faster, 3) showed better repeatability, 4) extracts those compounds best differentiating the compared groups and 5) has been previously described for metabolomics analyses characterising ESX-3 gene functionality, hence potentially allowing us to compare results to that previously generated and published data. Subsequently, we used the chosen extraction method, followed by GCxGC-TOFMS analysis of the separately cultured M. smegmatis wild-type sample extracts, cultured in normal, low and high iron conditions, to determine the influence of varying iron concentrations on the metabolome of this organism, by metabolomics comparisons of these groups. Following this, an identical research approach was used to compare the metabolome of a M. smegmatis ESX-3 knock-out strain, to that of a M. smegmatis wild type parent strain, both cultured in normal / standardised iron concentrations. Considering the results generated when comparing the metabolome of a M. smegmatis ESX-3 knock-out strain to that of a M. smegmatis wild type parent strain, the altered metabolome of the M. smegmatis ESX-3 knockouts correlated well to that of the M. smegmatis wild type cultured in elevated iron growth conditions. This suggests ESX-3 is involved in iron uptake, and that knocking out the ESX-3 gene cluster of M. smegmatis does in fact result in a metabolome profile suggesting iron overload, as was proposed by Loots et al (2012), most probably due the exochelins overcompensating for the absence of mycobactins, in M. smegmatis ESX-3 knockouts. / MSc (Biochemistry) North-West University, Potchefstroom Campus 2013.

According to the World Health Organization (WHO), M. tuberculosis, the causative agent of TB, accounts for approximately 1.7 million deaths annually. Further contributing causes to the worldwide TB incidence, is the widespread unavailability and ineffectiveness of TB vaccines, time consuming diagnostic methods and unsuccessful treatment approaches. Research for better characterising mycobacteria in general, or other Mycobacterium species, may help us to better understand M. tuberculosis and TB disease mechanisms, which will in turn lower the future TB disease prevalence, as this may lead to the development of better treatments, diagnostics and vaccines. Mycobacteria use various secretion pathways, including the ESX- or type VII secretion (T7S) system, to ensure transport across the complex cell wall. The genome of M. tuberculosis has five copies of a gene cluster known as the ESX gene cluster region, which is associated with virulence and viability of mycobacteria. The ESX-3 gene cluster is thought to be essential for growth of M. tuberculosis and proposed to be involved in iron / zinc homeostasis. Mycobacteria synthesise siderophores, which are proposed to be involved in the uptake of iron over their cell wall. M. tuberculosis are known to produce two types of siderophores, namely: carboxymycobactins and mycobactins. Loots and colleagues, however illustrated, that ESX-3 knockouts, show signs of iron overload, despite the absence of the mycobactins induced by knocking out the ESX-3 gene cluster. It was hypothesised, that this overload occurs due to an increase in exochelin synthesis, another iron uptake protein not associated with ESX-3, overcompensating for the perceived iron depletion in the knockout organism. A Metabolomics research approach was subsequently used in this study, to generate new information in order to better characterise the role of iron on the metabolism of these organisms, and additionally confirm the role of ESX-3 in iron uptake. In this study, we firstly determined the most optimal extraction conditions for this metabolomics investigation. Two extraction methods were subsequently investigated and compared, considering their repeatability and their respective capacities to extract those compounds which best differentiate the M. smegmatis ESX-3 knockouts and wild-type parent strains. Considering the results generated, the total metabolome method was chosen for further analyses, for the following reasons: 1) it is simpler, 2) faster, 3) showed better repeatability, 4) extracts those compounds best differentiating the compared groups and 5) has been previously described for metabolomics analyses characterising ESX-3 gene functionality, hence potentially allowing us to compare results to that previously generated and published data. Subsequently, we used the chosen extraction method, followed by GCxGC-TOFMS analysis of the separately cultured M. smegmatis wild-type sample extracts, cultured in normal, low and high iron conditions, to determine the influence of varying iron concentrations on the metabolome of this organism, by metabolomics comparisons of these groups. Following this, an identical research approach was used to compare the metabolome of a M. smegmatis ESX-3 knock-out strain, to that of a M. smegmatis wild type parent strain, both cultured in normal / standardised iron concentrations. Considering the results generated when comparing the metabolome of a M. smegmatis ESX-3 knock-out strain to that of a M. smegmatis wild type parent strain, the altered metabolome of the M. smegmatis ESX-3 knockouts correlated well to that of the M. smegmatis wild type cultured in elevated iron growth conditions. This suggests ESX-3 is involved in iron uptake, and that knocking out the ESX-3 gene cluster of M. smegmatis does in fact result in a metabolome profile suggesting iron overload, as was proposed by Loots et al (2012), most probably due the exochelins overcompensating for the absence of mycobactins, in M. smegmatis ESX-3 knockouts. / MSc (Biochemistry) North-West University, Potchefstroom Campus 2013.

No description available.

Emergence of drug resistance is a major problem in the treatment of many diseases including tuberculosis. To tackle the problem, it is essential to obtain a global perspective of the molecular mechanisms by which bacteria acquire drug resistance. Systems biology approaches therefore become necessary. This work aims to understand pathways to drug resistance and strategies for inhibition of the resistant strains by using a combination of experimental genomics and computational molecular systems approaches. Laboratory evolution of Mycobacterium smegmatis MC2 155 by treatment with isoniazid (INH), a front-line anti-tubercular drug, resulted in a drug-resistant strain (4XR), capable of growth even at about 10-times the minimum inhibitory concentration of the drug. Whole genome sequence of the 4XR was determined, which indicated only 31 variations in the whole genome, including 3 point mutations, 17 indels and 11 frame-shifts. Two mutations were in proteins required for the pharmacological action of the drug, albeit in regions distant from the drug binding site. The variations however were insufficient to explain the observed resistance to isoniazid. For a better understanding of the global changes associated with drug resistance, whole genome-wide gene expression data was obtained for the resistant strain and compared with that of the WT strain. 716 genes were found to be differentially regulated in 4XR, spanning different biochemical, signaling and regulatory pathways. From this, some explanations for the emergence of drug resistance were obtained, such as the up-regulation of the enzymes in the mycolic acid biosynthesis pathway and also of the drug efflux pumps. In addition, enrichment analysis indicated that up-regulated genes belong to functional categories of response to stress, carbohydrate metabolism, oxidation-reduction process, ion transport, signaling as well as lipid metabolism. The differential gene regulations seemed to be partially responsible for conferring the phenotype to the organism. Alterations in the metabolic pathways in 4XR were characterized using the phenotypic microarray technology, which experimentally scanned the respiratory ability of the resistant bacteria under 280 different nutrient conditions and 96 different inhibitors. Phenotypic gain, where the resistant strain grows significantly better than the wild type and phenotypic loss, where the growth of the resistant strain is compromised as compared to the sensitive strains were derived from the comparison of the phenotypic responses. Differences in survival ability and growth rates in different nutrient sources in the resistant phenotype as compared to the wild type were observed, suggesting rewiring in the metabolic network of the drug-resistant strain. In particular, the pathways of central carbon metabolism and amino acid biosynthesis exhibit significant differences. The strain-specific metabolic pathway differences may guide in devising strategies to tackle the drug-resistant strains selectively and in a rational manner. Scanning electron microscopy indicated the morphology of the drug-resistant strains to be significantly altered, as compared to the control drug-sensitive strain. It is well-known that isoniazid acts by inhibiting mycolic acid biosynthesis. The pathway turns out to be a target for many other anti-tubercular drugs also, since mycolic acids are major components of the cell wall. It is therefore important to understand what changes occur in the mycolic acid and the associated pathways in the drug-resistant variety so that strategies to tackle the latter can be chosen more judiciously. The lipidome of the cell wall was therefore quantitatively characterized by mass spectrometric analyses, which indeed confirmed that the 4XR strain has a significantly different composition profile. Among the six categories of lipids, the members of the glycerophospolipids category were abundant while the fatty acyls, polyketides and saccharolipids were lower in the 4XR strain as compared to the WT. The lipidomic data derived from the cell wall of INH-resistant strain shows that it results in the mycolic acid pathway function restoration, which would otherwise be lost upon drug exposure in the sensitive strain. Understanding the precise changes that occur in the lipidome in the drug-resistant strains is expected to be useful in developing new ways to tackle resistance. Next, to understand the implications of altered gene expression profiles, protein-protein interaction networks are constructed at a genome-scale that captures various structural and functional associations mediated by proteins in the mycobacterial cell. Using transcriptome data of 4XR, a response network is computed. Using an algorithm previously developed in the laboratory, the networks have been mined to identify highest differential activity paths and possible mechanisms that are deployed by the cells leading to drug resistance. Known resistance mechanisms such as efflux, cytochromes, SOS, are all seen to constitute the highest activities for achieving drug resistance in 4XR. Interestingly, such paths are seen to form a well-connected subnet, indicating such differential activities to be orchestrated. This clearly shows that multiple mechanisms are simultaneously active in the 4XR and may together generate drug resistance. Mechanisms of detoxification and antioxidant responses are seen to predominate in the 4XR subnet. Overall the analysis provides a shortlist of strategies for targeting the drug resistant strain. Next, the phenotypic microarray platform was used for screening for growth in Msm in the presence of various drugs. Data analysis and clustering resulted in identification of conditions that lead to phenotypic gain or loss in the 4XR as well as those that lead to differential susceptibility to various drugs. Drugs such as cephalosporins, tobramycin, aminotriazole, phenylarsine oxide, vancomycin and oxycarboxin were also found to inhibit growth in the resistant strain selectively. In other words, the 4XR is found to be collaterally sensitive to these drugs. The top-net formed by the highest differential activity paths, identified from the network described earlier has already indicated the involvement of proteins that generate antioxidant responses. Insights from the two methods, first from the targeted approach and second, from the phenotypic discovery approach were combined together to select only those compounds to which the 4XR strain was collaterally sensitive and targeted proteins responsible for antioxidant responses. These compounds are vancomycin, phenylarsine oxide, ebselen and clofazimine. These were further tested against the virulent M. tuberculosis H37Rv strain in a collaborator‘s laboratory. 3 of these compounds such as vancomycin, ebselen and phenylarsine oxide were found to be highly active in combinations with isoniazid against all tested Mtb strains, showed high levels of inhibition against H37Rv and 3 different single drug resistant, MDR and XDR strains. Moreover, they were observed to be highly potent when given in combinations. Clofazimine on the other hand, in combination with isoniazid showed activity but no significant synergy in the virulent drug-resistant strains of M. tuberculosis though synergistic to the sensitive strain. Thus, experiments with M. tuberculosis provide empirical proof that four different compounds, all capable of blocking antioxidant responses, are capable of inhibiting growth of single-, multiple- and extremely-drug-resistant clinical isolates of M. tuberculosis. Using transcriptome data from literature for M. tuberculosis exposed to six different drugs, similar drug specific response networks were constructed. These networks indicate differences in the cellular response to different drugs. Interestingly, the analysis suggests that different drug targets and hence different drugs could trigger drug resistance to various extents, leading to the possibility of prioritizing drug targets based on their resistance evolvability. An earlier study from the laboratory suggested the concept of target-co-target pairs, where-in the co-target could be a key protein in mediating drug resistance for that particular drug and hence for its target protein. Top ranked hubs in multiple drug specific networks such as PolA, FadD1, CydA, a monoxygenase and GltS, can possibly serve as co-targets. Simultaneous inhibition of the co-target along with the primary target could lower the chances of emergence of drug resistance. Such analyses of drug specific networks provide insights about possible routes of communication in the cell leading to drug resistance and strategies to inhibit such communication to retard emergence of drug resistance. Since mutations in the target proteins are known to form an important mechanism by which resistant strains emerge, an understanding of the nature of mutations in different drug targets and how they achieve resistance is crucial. Sequence as well as structural bases for the resistance from known drug-resistant mutants in different drug targets is deciphered and then positions amenable to such mutations are predicted in each target. Mutational indices of individual residues in each target structure are computed based on sequence conservation. Saturated mutagenesis is performed in silico and structural stability analysis of the target proteins has been carried out. Critical insights were obtained in terms of which amino acid positions are prone to acquiring mutations. This in turn suggests interactions that are not desirable, thus can be translated into guidelines for modifying the existing drugs as well as for designing new drugs. Finally, the work presented here describes application of the systems biology approaches to understand the underlying mechanisms of drug resistance, which has provided insights for drug discovery on multiple fronts though target identification, target prioritization and identification of co-targets. In particular, the work has led to a rational exploration of collateral drug sensitivity and cross-resistance of the drug-resistant strain to other compounds. Combinations of such compounds with isoniazid were first identified in the M. smegmatis model system and later tested to hold good for the virulent M. tuberculosis strain, in a collaborative study. The combinations were found to be active against three different clinical drug-resistant isolates of M. tuberculosis. Therefore, this study not only reveals the global view of resistance mechanisms but also identifies synergistic combinations of promising drug candidates based on the learnt mechanisms, demonstrating a possible route to exploring drug repurposing. The combinations are seen to work at a much reduced dosage as compared to the conventional tuberculosis drug regimens, indicating that the toxicity and any associated adverse effects may be greatly reduced, suggesting that the combinations may have a high chance to succeed in the next steps of the drug discovery pipeline.

No description available.

No description available.

Bacterial stationary phase is an interesting biological system to study, as the organism undergoes several metabolic changes during this period and new molecules are generated to support its survival. The stationary phase of mycobacteria has been extensively studied since the discovery of Mycobacterium tuberculosis, the causative agent of tuberculosis. The stationary phase of mycobacteria adds further complication as many antibacterial drugs become less effective. The M. tuberculosis infects the alveolar macrophages and dendritic cells or monocytes recruited from peripheral blood. Macrophages are supposed to provide an initial barrier against the bacterial infection, but fails. Mycobacteria have evolved several strategies to survive and set up an initial residence within these cells and grow actively inside the host. The host immune system tries to limit the bacterial growth and confines the organism to a latent state in which the organism can persist indefinitely, known as granuloma stage. During latency or granuloma stage mycobacteria can retain the ability to resume the growth in the future. Mycobacteria must adapt to a highly dynamic and challenging environment because the interior environment of granuloma is devoid of or in low level of oxygen, depleted nutrient, high carbon dioxide, and possess increased levels of aliphatic organic acids and hydrolytic enzymes. The survival of a bacterium in less nutrient supply or in depleted oxygen is important for its long-¬term persistence inside the host under harsh environmental conditions. Mycobacterium smegmatis is the closest non-¬pathogenic homologue of M. tuberculosis, and has been used widely as a model system to study gene regulation under such conditions. In these harsh environmental conditions bacteria need to sense the external environment to modulate their gene expression. More importantly, each individual cell should communicate with its neighbours, and the response takes place in a concerted manner, which is termed as quorum sensing. Thus, the quorum sensing is a cell-¬cell signaling process that allow the bacteria to monitor the presence of other bacteria in their surroundings by producing and responding to small signaling molecules, which are known as autoinducers. It is a density dependent phenomenon and regulates the expression of the genes in response to fluctuation in cell¬-population density. A minimum threshold level of autoinducers is necessary to detect the signal and respond to it. Quorum sensing enables bacteria to behave like multicellular organisms and controls group activities like biofilm formation, sporulation, bioluminescence, virulence, and pigment production, etc (Bassler, 1999; Camilli & Bassler, 2006; Fuqua et al., 1996; Miller & Bassler, 2001). In Gram-¬negative bacteria, small-¬molecules, which are known as autoinducers are produced. They are acyl homoserine lactones (AHLs), which are derived from S¬adenosyl methionine (SAM) and particular fatty acyl carrier protein by LuxI¬type AHL synthases (Fuqua et al., 1996). In Gram-¬positive bacteria small peptides autoinducers, 5¬12 amino acids long, play an active role in communication. These oligopeptides are post--translationally modified by the incorporation of lactone and thiolactone rings, lanthionines and isoprenyl groups. These oligopeptide autoinducers are detected by membrane-¬bound two-¬component signaling proteins, and signal transduction occurs by a phosphorylation cascade (Camilli & Bassler, 2006; More et al., 1996; Novick, 2003; Zhang et al., 2002). In bacteria, the cyclic adenosine monophosphate (cAMP), and guanosine pentaphosphate and/or tetraphosphate ((p)ppGpp) are well known second messengers, which play important role in relaying extracellular information, but recently cyclic diguanosine monophosphate (c-¬di¬-GMP) is being studied most comprehensively as a nucleotide-¬based second messenger. C-¬di¬-GMP was first discovered in Gluconacetobacter xylinus as a positive allosteric regulator of cellulose synthase (Ross et al., 1987; Tal et al., 1998; Weinhouse et al., 1997). The in vivo level of c-¬di-¬GMP in bacterial cell is maintained by the balance between diguanylate cyclase and phosphodiesterase activities. The GGDEF and EAL amino acids sequence are the signature motif for GGDEF and EAL domain protein within its active site, respectively. The GGDEF domain protein is involved in synthesis of c-¬di-¬GMP and the EAL domain protein is involved in the hydrolysis of c-¬di-¬GMP, and the majority of these proteins contain additional signal input domains (Paul et al., 2004; Ross et al., 1987; Ryjenkov et al., 2005; Tal et al., 1998). M. smegmatis has a single bi-¬functional protein having both the domains, GGDEF and EAL, for the diguanylate cyclase (DGC) and phosphodiesterase (PDE¬A) activities. In addition to GGDEF and EAL domain, one sensory domain, GAF, is also there at the N-terminal of MSMEG_2196 in M. smegmatis. In the present investigation, studies have been carried out to understand the regulation of c-¬di-¬GMP in M. smegmatis at protein and gene level. The entire study on mycobacterial MSMEG_2196 (msdgc¬1) can be broadly divided into five parts; the first part will cover the identification and biochemical characterization of MSDGC¬1 protein, responsible for the regulation of in vivo c-¬di-¬GMP concentration in M. smegmatis, and the presence of GGDEF¬EAL domain containing proteins in various mycobacterial species. The second part will cover the structure function relationship as a function of substrate, GTP and product, c-¬di-GMP, molecule using fluorescence spectroscopy as a tool, and the mutational and structural studies, which leads to the identification of a novel structural motif. The third part will cover the characterization of msdgc¬1 gene knockout and complementation studies in great detail. The fourth part will comprise in vivo and in vitro promoter characterization and regulation of the msdgc¬1 gene under nutritional starvation. The last chapter will cover the characterization of novel synthetic glycolipids, which are working as a growth and biofilm inhibitors in mycobacteria, and can be used as a new drug candidates. Chapter 1 outlines the signal transduction and quorum sensing mechanism, and small molecule signaling modules in brief. The importance of the study started with a brief introduction about the historical aspect of tuberculosis, the current scenario of the treatment of tuberculosis. The urgent need for new drug targets and drugs will be discussed. The important role of the novel second messenger, c-¬di¬-GMP has been explained in greater details in both Gram-¬positive and Gram-¬negative bacteria, and the information available on the different cellular targets has been documented. Chapter 2 describes the identification and biochemical characterization of M. smegmatis MSMEG_2196 protein. The domain architecture and individual domain role have been studied. The MSMEG_2196 proteins consist of three domains, GAF, GGDEF and EAL in tandem, and individual role of each domain has been studied. The diguanylate cyclases containing GGDEF and phosphodiesterases containing EAL domains have been identified as the enzymes involved in the regulation of in vivo cellular concentration of c-¬di-¬GMP. GAF domain has been identified as a metal binding domain in other bacteria and may be playing a role in the regulation of synthesis and hydrolysis activities of c-¬di¬-GMP. The identification, cloning expression and purification of MSMEG_2196 and MSMEG_2774 have been discussed. We have reported that mycobacterial MSDGC¬1 protein has dual activity, which means that it can synthesize and hydrolyse c¬-di-¬GMP; and also full-¬length protein is necessary for its either of the activities. The synthesis and hydrolysis products, c-¬di-¬GMP and pGpG, of MSDGC¬1 protein have been identified and characterized using radiolabelled alpha [α¬32P]GTP and Matrix Assisted Laser Desorption/Ionization mass spectrometry (MALDI). The effects of temperature and pH on the activities of MSDGC¬1 have been studied. The circular dichroism studies show that the MSDGC¬1 protein is predominantly α¬helical in nature, and secondary structure does not alter upon GTP binding. The kinetic parameters for MSDGC¬1 protein have been calculated as a function of substrate, GTP. The protein, MSDGC¬1, exist as a monomer and a dimer in solution. The MSDGC¬1 protein has four cysteines, and we have shown here using mass spectrometric analysis that none of the cysteines is involved in the disulphide linkage. Chapter 3 deals with the structure-¬function relationship as a function of GTP and c¬-di-GMP molecules using fluorescence spectroscopy as a tool. In order to do so we have generated several cysteine mutants using site directed mutagenesis, and protein was labelled with thiol-¬specific fluorophores. The labelled protein was checked for its DGC and PDE¬A activities and specificity of labelling was confirmed using MALDI and radiometric analysis. The Fluorescence Resonance Energy Transfer (FRET) has been carried out to observe domain-¬domain interaction as a function of GTP and c¬-di-¬GMP. The bioinformatics, structural, and mutational analysis suggest that cysteine at 579 position is important for DGC and PDE¬A activities, and may be involved in the formation of a novel structural motif, GCXXXQGF, which is necessary for synthesis and degradation of c-¬di-¬GMP. Chapter 4 describes the construction of a deletion mutation of MSMEG_2196 gene in M. smegmatis. The strategy for the construction of the knockout strain has been shown and confirmation of the knockout event has been carried out using PCR and Southern hybridization. The effect of deletion of msdgc¬1 has been studied in great detail, and it was noticed that biofilm formation is not affected, but long-¬term survival is significantly compromised. It is hypothesized here that c-¬di¬-GMP is involved in the regulation of cell population density in mycobacteria. We have successfully detected the c-¬di¬-GMP in the total nucleotide extract using HLPC coupled with MALDI, and we have shown here that level of c-¬di-¬GMP increases many fold in the stationary phase of growth under nutritional starvation. Chapter 5 deals with the identification and characterization of the promoter element of msdgc¬1 in M. smegmatis. The study was undertaken to understand the mechanism of regulation at promoter level. We have observed here that msdgc¬1 promoter is starvation induced, and expression of msdgc¬1 increases many fold in the stationary phase under nutritional starvation. We have also tried to establish the link between the ppGpp and c-di¬-GMP signalling, and possible role of c-¬di-¬GMP in the regulation of cell population density have been discussed. Further, the +1 transcription start site has been identified using primer extension method. The putative ¬10 hexamer region for the RNA polymerase binding has been identified and confirmed using site-¬directed mutagenesis. It was found to be TCGATA, which is 14 bp upstream from the +1 transcription start site. The msdgc-1 promoter is specific for mycobacteria and does not function in E. coli. Moreover, we have identified the sigma factors, which regulate the msdgc¬1 promoter in growth phase dependent manner. Chapter 6 begins with the screening of synthetic glycolipids as a novel drug candidate. The different glycolipids have been tested for their effect on growth, biofilm formation, and sliding motility of M. smegmatis, and we have screened few of them, which were found to be effective in inhibiting the microbial growth, biofilm formation, and sliding motility. Chapter 7 summarizes the work presented in this thesis. Appendix: The protein sequences of MSDGC¬1 and MSDGC¬2, and the multiple sequence alignments of MSDGC¬1 protein have been documented. The FORTRAN program, which was used to calculate spectral overlap integral J, and the diagrams of the plasmids used in this study have been provided.

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.

The discovery of cyclic AMP (cAMP), nearly 50 years ago by Sutherland radically altered the appreciation of metabolic regulation. Since then the presence of cAMP and its tremendous physiological impact has been demonstrated in many prokaryotic systems. In fact, virulence mechanisms of several pathogens known today exploit cAMP dependent pathways. Interestingly the genome of Mycobacterium tuberculosis H37Rv, the causative agent of tuberculosis, encodes as many as 16 adenylyl cyclases (enzymes that convert ATP to 3’, 5’-cAMP) and 10 cyclic-nucleotide binding proteins. Recent reports show that bacterial-derived cAMP manipulates host signaling for bacterial survival, suggesting an important role for cAMP in the pathogenesis of M. tuberculosis. A large number of non-pathogenic species of mycobacteria also share and conserve several of these cAMP metabolism genes, suggesting that cAMP is not only important for pathogenesis but also may play a critical physiological role in the genus. The work carried out in this thesis aims at a better understanding of the role of cAMP by studying the adenylyl cyclases and cyclic AMP receptor proteins (CRPs) from Mycobacterium smegmatis, a non-pathogenic member of the genus. Intracellular cAMP levels in a cell are precisely maintained by modulating the activities of the adenylyl cyclases (cAMP synthesising enzymes), the phosphodiesterases (cAMP hydrolysing enzymes) and the secretion machinery, if any. To assess the role of cAMP in mycobacteria, cAMP levels were measured in M. smegmatis during growth and under various stress conditions. The results show that cAMP levels peak at log phase of growth and decline thereafter. Under acidic conditions or on perturbing the cell-wall, cellular cAMP levels are altered, which indicate a possible role of cAMP in stress adaptation. Earlier work in our laboratory has led to the identification of multiple adenylyl cyclases in the mycobacterial genomes. These cyclases are similar in sequence to the mammalian enzymes and several of them have been shown to be active in vitro displaying a diverse range of biochemical properties. The M. smegmatis genome encodes 10 adenylyl cyclase-like genes. In order to understand the role of cAMP in M. smegmatis, individual cyclases were analysed for their biochemical properties and physiological functions. The work presented in this thesis is concerned with the functional characterization of MSMEG_3578 and MSMEG_3780, two of the several adenylyl cyclases from M. smegmatis. MSMEG_3578 encodes for a protein that comprises two transmembrane domains, an extracellular receptor-like domain, a membrane anchoring HAMP domain and an intracellular cyclase domain. The cyclase domain is closely related to mammalian cyclases but lacks the canonical residues that are critical for the catalysis of class III cyclases. Interestingly, the stop codon of this gene overlaps with the start codon of the downstream gene, MSMEG_3579 (a putative cyclic nucleotide gated mechanosensitive ion channel), suggesting a functional link between the two genes. The conservation of this gene pair across the mycobacterial genus indicates the importance of this putative receptor-effector pair in the physiology of mycobacteria. Additionally, microarray analysis by various groups have shown that this gene pair in Mycobacterium tuberculosis is differentially regulated in conditions that mimic stress the bacteria may experience during infection. In order to ascertain the physiological role of MSMEG_3578, a knock-out M. smegmatis strain was generated and tested for growth and cAMP defects. The knock-out strain showed growth and cAMP profiles similar to the wild-type strain. Over-expression of MSMEG_3578 in M. smegmatis resulted in a significant rise in cAMP levels. Interestingly, over-expression of the MSMEG_3578 adenylyl cyclase in E. coli did not lead to an elevation in cAMP levels indicating that other mycobacterial proteins may be required for the activity of MSMEG_3578 in vivo. In agreement with this, the purified adenylyl cyclase domain of MSMEG_3578 was found to be biochemically inactive in vitro. Additionally, the over-expressing strain has altered colony morphology as compared to the wild type strain. Perturbation of cAMP levels by over-expression of other cyclases also leads to a similar colony morphology phenotype, indicating the phenotype to be controlled by cAMP in general rather than by a specific cyclase in the cell. MSMEG_3780 is a highly conserved, biochemically active adenylyl cyclase, speculated to play a house-keeping function in M. smegmatis. Its orthologs from M. tuberculosis (Rv1647) and M. leprae (ML1399) have been biochemically characterized earlier in our laboratory. To unravel the role of this gene in vivo, the ∆MSMEG_3780 strain was tested for growth and cAMP defects under various conditions. The deletion strain did not show any difference in growth rate or morphology when compared to the wild-type strain. However it showed a reduction in intracellular cAMP levels at the log-phase of growth. Reintroduction of the MSMEG_3780 gene in the deletion strain restored cAMP to wild-type levels, thus indicating a crucial role for this adenylyl cyclase in the maintenance of intracellular cAMP levels during logarithmic growth. In order to investigate the regulation of the MSMEG_3780 gene, its promoter activity was tested under various stress-conditions. Acid-stress specifically resulted in the repression of the MSMEG_3780 promoter activity, a condition which also leads to a decrease in cAMP levels in the cells. Further evidence for the susceptibility of the MSMEG_3780 gene to acid-stress was obtained when the ∆MSMEG_3780 strain failed to reduce intracellular cAMP content upon sustained acid-stress conditions. Since Rv1647 shares similar biochemical properties with MSMEG_3780 and can also heterodimerize with the MSMEG_3780 protein in vitro, it was interesting to test whether the M. tuberculosis ortholog could functionally complement MSMEG_3780. To assess this, a complement strain was generated that contained the Rv1647 gene under the control of MSMEG_3780 promoter, integrated into the genome of ∆MSMEG_3780 strain. Rv1647 protein was able to restore the cAMP phenotype seen on acid stress as well as the cAMP levels in the mutant strain at the log phase of growth. This study indicated the role of cAMP and MSMEG_3780 in acid adaptation and also suggested a non-redundancy of adenylyl cyclases in mycobacteria, where different individual cyclases may have unique functions in the cells. Since Rv1647 could complement the cAMP defective phenotype in ∆MSMEG_3780, this suggests functional parallels between the proteins from the two species. Bacterial adaptation to environmental stress is brought about by a rapid change in its gene expression profile. Cyclic AMP plays an important role by binding to and activating a transcriptional factor, cAMP receptor protein or CRP. We have identified two CRPs from M. smegmatis, viz., MSMEG_0539 and MSMEG_6189 that possess high similarity at the amino acid level (78% overall sequence identity). The CRP ortholog from M. tuberculosis, Rv3676 has been characterized structurally, biochemically and functionally earlier. Western blot and RT-PCR analyses showed that CRPs in M. smegmatis were present during all phases of growth. Both the CRPs were cloned, expressed and shown to bind cAMP. Since the DNA binding domains of Rv3676 and the two M. smegmatis CRPs are nearly identical, the CRPs from M. smegmatis were predicted to bind similar target sequences. Interestingly, a CRP site was identified in the promoter of the MSMEG_3780 gene, suggesting a possible feed-forward or feed-back loop, where the enzymatic product of the adenylyl cyclase now governs its own gene expression. We performed Electrophoretic Mobility Gel Shift Assays (EMSAs) with M. smegmatis lysates to show that CRP binds to the MSMEG_3780 promoter at the CRP site. Subsequent Chromatin Immunoprecipitation (ChIP) assays confirmed that CRP binding to the MSMEG_3780 promoter occurred in vivo. To investigate the role of CRP in the regulation of the MSMEG_3780 gene, luciferase reporter assays with the wild-type and CRP site mutant promoters were carried out. Results suggest that CRP regulates the MSMEG_3780 gene under osmotic stress. However, CRP did not play any role in basal expression of MSMEG_3780 during growth. To assess which CRP of the two is functionally linked to the MSMEG_3780 promoter, we carried out a footprint assay with purified CRPs. It was intriguing to note that both the CRPs were in fact able to bind the promoter albeit under different conditions. Whereas MSMEG_6189 bound the promoter both in the presence and absence of cAMP, MSMEG_0539 bound the promoter only in the presence of cAMP. MSMEG_6189 thus deviates from the accepted CRP paradigm that seeks an absolute requirement of cAMP for specific DNA binding. The present study identifies cAMP as an important stress signal in M. smegmatis. Using MSMEG_3780 as a model gene, the role of cAMP in mycobacteria was studied. The two divergent CRPs that were characterized may function and dictate cAMP-mediated and perhaps independent functions in cells. Taken together, our results provide compelling evidence for the important role of cAMP in the general physiology and stress adaptation in M. smegmatis.

Pathogenic bacteria such as M.tuberculosis have evolved several mechanisms to aid their intracellular survival and subvert host defenses. One of the contributing factors is thought to be the production and secretion of large amount of cAMP, Mycobacterial genomes encode a large number of adenylyl cyclases distinct in their structure and regulatory mechanisms. The roles of these enzymes in the physiology and pathogenesis of virulent mycobacteria are only now being elucidated. The roles of phosphodiesterases (PDEs), which serve to lower cAMP levels through degradation are, however, relatively unexplored. The Rv0805 gene was previously shown to code for an active phosphodiesterase from Mycobacterium tuberculosis. Bioinformatics analysis revealed that orthologs of Rv0805 were found even in eukaryotes. Biochemical and structural characterization of Rv0805 revealed that it was a class III cAMP phosphodiesterase. Comparative genomics identified a close ortholog of Rv0805 in M. leprae (ML2210). The genome of M. leprae Encodes only 1,604 predicted proteins and possesses the highest number of pseudogenes, 1,116. The retention of a functional PDE, the ortholog of Rv0805, in the minimal genome of M. leprae is indicative of its importance in cellular physiology. Biochemical characterization of proteins from M. leprae and use of heterologous hosts will help understand this human pathogen better, since there are no tools currently available to genetically manipulate this bacterium. Sequence analysis of ML2210 revealed the presence of conserved motifs and residues known to be critical for catalysis and unique to class III phosphodiesterases. ML2210 shares 83% sequence identity with Rv0805 and 24% sequence identity with the phosphodiesterase from E. coli (cpdA). In vitro biochemical characterization of ML2210 using non-nucleotide colorigenic and cyclic nucleotide substrates revealed that it was an enzymatically active phosphodiesterase. Kinetic parameters of ML2210 with respect ot colorigenic substrates revealed that its catalytic properties were similar to that of Rv0805. However, with respect to hydrolysis of 3’, 5’-cAMP, ML2210 was catalytically more efficient than Rv0805, suggesting that in spite of being orthologs, these enzymes have evolved distinct specificities at their active site. A parallel of monoclonal antibodies raised to Rv0805 was also used understand the differences in the biochemical properties of Rv0805 and ML2210 better. It was observed that only one monoclonal antibody was able to recognize ML2210 by ELISA and not by Western blot analysis. These results revealed that conformational differences between ML2210 and Rv0805 exist. Over-expression of ML2210 in M. smegmatis resulted in a modest decrease in intracellular cAMP levels. Despite the absence of a predicted transmembrane region or a membrane-targeting signal, ML2210 localized to the cell envelop fraction upon over expression in M. smegmatis. Moreover, like Rv0805, over-expression of ML2210 also resulted in perturbation of the cell wall of M. smegmatis, arguing for additional cellular roles of this protein. Orthologs of Rv0805 or ML2210 are found only in slow growing mycobacteria suggesting that other cyclic nucleotide phosphodiesterases could regulate cAMP levels in fast growing mycobacteria like M. smegmatis. Since BLAST results did not retrieve an ortholog of Rv0805 or ML2210, COG1409 (COG database) containing Rv0805 was examined for the presence of other mycobacterial phosphodiesterases. Bioinformatics analysis identified Rv2795c as another PDE from M. tuberculosis. Sequence analysis of Rv2795c revealed the presence of all the motifs conserved in the class III PDEs but Rv2795c shared only 22% sequence identity with Rv0805 and 19% sequence identity with CpdA. Importantly, an ortholog of Rv2795c was identified in M. leprae. Interestingly. Rv2795c and its orthologs branched away from Rv0805, making it phylogenetically distinct and hence warranting further characterization. Recombinant, purified MSMEG_2647 (the Rv2795c ortholog from M. smegmatis) was able to hydrolyze cyclic nucleotides and other phosphodiester substrates in vitro. The Km for colorigenic substrates was higher when compared to the Km of ML2210 or Rv0805 for these substrates. However, the kinetic parameters of MSMEG_2647 for cyclic nucleotides were comparable to those of ML2210 or Rv0805. MSMEG_2647 was a metal dependent enzyme and among the panel of metals tested, Mn2+ supported the highest in vitro catalytic activity of MSMEG_2647. Zn2+ inhibited the catalytic activity of MSMEG_2647. In order to gain insight into the catalysis of MSMEG_2647, the end products of cAMP hydrolysis by MSMEG_2647 were analysed using reverse phase HPLC. The assay revealed that the end products of cyclic nucleotide hydrolysis by MSMEG_2647 were different when compared to the end products of hydrolysis of the same substrates by Rv0805 or ML2210. This suggests differences in the architecture of the active site residues of the mycobacterial MPEs. A mutational anlaysis of the active site residues in MSMEG_2647 was carried out to identify residues involved in substrate recognition and metal coordination. Although Rv0805 and MSMEG_2647 shared only a 22% sequence identity, MSMEG_2647 displayed strict conservation in the core MPE motifs. Mutation of the active residues N97 and H98 in Rv0805 had led to an abrogation of its catalytic activity. However, corresponding mutations of N76A and H77A in MSMEG_2647, did not lead to a loss in its catalytic activity. A third mutation known to be important for the catalytic activity of Rv0805 (D19) was incorporated. The corresponding residue at D19 position was mutated to an alanine. The catalytic activity of MSMEG_2647D19AN76AH77A mutant was abrogated, suggesting that while the core MPE motifs are conserved between mycobacterial PDEs, differences in the ensemble of the active site residues contributing to their catalytic activity exist. Thus, at least two biochemically diverse PDE clades are found in mycobacterial species. In order to decipher the function of MSMEG_2647, its expression was monitored during the growth of M. Smegmatis. The promoter of MSMEG_2647 displayed maximum activity during the logarithmic phase of M. smegmatis growth after which its activity declined as M. smegmatis entered the stationary phase. However in contrast to this, the transcript corresponding to msmeg_2647 mRNA was found at both logarithmic and stationary phases. The MSMEG_2647 protein was also detected at both logarithmic and stationary phases of M. smegmatis. These results suggest that additional factors may contribute to the stability of msmeg_2647 mRNA and protein levels. Localization studies of MSMEG_2647 revealed that MSMEG_2647 was present in the cytosol as well as in the cell envelope fractions. Interestingly, over-expression of MSMEG_2647 did not result in a significant increase in PDE activity in various subcellular fractions, suggesting tight regulation on the in vivo activity in various subcellular fractions, suggesting tight regulation on the in vivo activity of MSMEG_2647. In addition, over-expression of MSMEG_2647 in M. smegmatis led to only a modest decrease in cAMP levels in M. smegmatis. These results suggested additional roles of MSMEG_2647 in the biology of mycobacteria. Overexpression of MSMEG_2647 peturbed the integrity of cell wall as assessed by the use of lipophillic indicators of cell growth, crystal violet and malachite green, and a cell wall targeting antibiotic, isoniazid. Analyzing the gene neighborhood of MSMEG_2647 provided an insight into its putative function. It was observed that the stop codon of msmeg_2647 overlapped with the start codon of msmeg_2648 and stop codon of msmeg-2648 overlapped with the start codon of msmeg_2649. RT PCR was carried out at logarhtimic and stationary phases of M. smegmatis growth, which revealed that a polycistronic mRNA was being transcribed. These results confirmed that msmeg_2647, msmeg_2648 and msmeg_2649 were a part of an operon. Interestingly, these three genes as a gene cluster were confined to only those actinobacteria that produced mycolic acids. An operon often encodes products that form multiprotein complexes and operate in a common pathway. Since there were a part of an operon, a GST pull-down approach was employed to test if MSMEG_2647, MSMEG_2648 and MSMEG_2649 could physically interact. It was observed that MSMEG_2647 interacted with MSMEG_2648 and MSMEG_2649. MSMEG_2648 in turn interacted with MSMEG_2649. A role for MSMEG_2647 as a scaffold recruiting MSMEG_2648 and MSMEG_2649 is therefore proposed. In turn, a complex formation with these proteins may regulate the activity of MSMEG_2647. Attempts to generate a knock out of msmeg_2647 in M. smegmatis by homologous recombination were not successful suggesting either the gene was essential or a polar effect on msmeg_2648(an essential gene for the viability of M. smegmatis) may not allow msmeg_2647 to be deleted from the genome of M. smegmatis. In summary, this study has identified and characterized two new phosphodiesterases from mycobacteria, one from the pathogenic mycobacterium, M. leprae and the other, a PDE from M. smegmatis that is conserved in all species of mycobacteria. Several, key biochemical differences were observed using biochemical and biological approaches. It appears that the cellular roles of mycobacterial phsophodiesterases may extend beyond cAMP hydrolysis, with these proteins not only regulating cell wall properties but also acting as scaffolding proteins in the cell.