Laxman Meena

Serine/threonine protein kinases (STPKs) represent a burgeoning concept in prokaryotic signaling and have been implicated in a range of control mechanisms. This paper describes the enzymatic and molecular characterization of PknH, a mycobacterial STPK. After cloning and expression as a Glutathione-S-transferase fusion protein in E. coli, PknH was found to phosphorylate itself and exogenous substrates like myelin basic protein and histone. The kinase activity of PknH was inhibited by the kinase inhibitors staurosporine and H-7. The results confirmed that PknH is a transmembrane protein and is restricted to members of the Mycobacterium tuberculosis complex. In addition, transcriptional analysis of pknH in M. tuberculosis under various stress conditions revealed that exposure to low pH and heat shock decreased the level of pknH transcription significantly. This is the first report describing differential expression of a mycobacterial kinase in response to stress conditions which can indicate its ability to regulate cellular events promoting bacterial adaptation to environmental change.

Tuberculosis is the most deadly infectious disease. In particular, pulmonary tuberculosis, being the predominant one, is highly contagious. In past the 200 years, one billion tuberculosis (TB) deaths had occurred, and it is anticipated that in the next 25 years, more than 40 million people may be killed by TB unless control measures are implemented. There are various causes which increase the susceptibility to Mycobacterium tuberculosis infection; these include weakened immune system which occurs through various diseases and medications like human immunodeficiency virus (HIV)/acquired immunodeficiency syndrome (AIDS), type II diabetes, end-stage kidney disease, alcoholism and intravenous drug use, certain cancers, cancer treatment such as chemotherapy, malnutrition and very young or advanced age. Some other factors include tobacco use, which increases the risk of getting TB and dying from it. In this manuscript, the authors tried to summarize all the alterations occurring in immune system at cellular and molecular level which occur due to infection, metabolic changes and chemical exposure, which increase susceptibility to mycobacterial infection.

Tuberculostearic acid (l0-methylstearic acid, TSA) is a major constituent of mycobacterial membrane phospholipids, and its biosynthesis involves the direct methylation of oleic acid esterified as a component of phospholipids. The methyltransferases of mycobacteria were long proposed to be involved in the synthesis of methyl-branched short-chain fatty acids, but direct experimental evidence is still lacking. In this study, we identified the methyltransferase encoded by umaA in Mycobacterium tuberculosis H37Rv as a novel S-adenosyl-l-methionine (SAM)-dependent methyltransferase capable of catalyzing the conversion of olefinic double bond of phospholipid-linked oleic acid to biologically essential TSA. Therefore, UmaA, catalyzing such modifications, offer a viable target for chemotherapeutic intervention.

Adenylate kinase (AK) is a small ubiquitous enzyme that catalyzes the reversible transfer of the terminal phosphate group from adenine triphosphate (ATP): magnesium ion (Mg²⁺) to adenine monophosphate (AMP) to form two molecules of adenine diphosphate (ADP). AK thus maintains the homeostasis of adenine nucleotides in eukaryotes and prokaryotes. Because the [ATP]/[ADP] ratio is an important parameter in energy regulation in cells, Mg²⁺-activated AK has an important biological role, particularly in the case of bacteria, as imbalance in the ratio of [ATP]/[ADP] has been associated with alteration in its DNA supercoiling state. In the present study, magnesium-binding assays were carried out by systematically varying the concentrations of Mg²⁺, protein, AMP, ATP, and indicator in kinetic experiments. We report evidence that during magnesium-binding assay, the fluorescence level of the indicator "Mag-Indo-1" changes with protein concentration, suggesting that magnesium ions are binding to AK. The dual activity of AK both as nucleoside monophosphate and diphosphate kinases suggests that this enzyme may have a role in RNA and DNA biosynthesis in addition to its role in intracellular nucleotide metabolism. According to the proposed model, the magnesium-activated AK exhibits an increase in its forward reaction rate compared with the inactivated form. These findings imply that Mg²⁺ could be an important regulator in the energy signaling network in cells.

In spite of the availability of effective chemotherapy and Bacille-Calmette-Guerin (BCG) vaccine, tuberculosis remains a leading infectious killer world-wide. Many factors such as, human immunodeficiency virus (HIV) co-infection, drug resistance, lack of patient compliance with chemotherapy, delay in diagnosis, variable efficacy of BCG vaccine and various other factors contribute to the mortality due to tuberculosis. In spite of the new advances in understanding the biology of Mycobacterium tuberculosis, and availability of functional genomic tools, such as microarray and proteomics, in combination with modern approaches, no new drug has been developed in the past 30 yr. Therefore, there is an urgent need to identify new drug targets in mycobacteria and eventually, develop new drugs. The release of the complete genome sequence of M. tuberculosis has facilitated a more rational, and directional approach to search for new drug targets. In general, gene products involved in mycobacteri...

Nicotiana tabacum var. Samsun was transformed via Agrobacterium-mediated transformation with a gene encoding the cholera toxin B subunit (CTB) of Vibrio cholerae, modified to contain a sequence coding for an endoplasmic reticulum retention signal (SEKDEL), under the control of the cauliflower mosaic virus 35S promoter. Total protein from the transgenic leaf tissue was isolated and an aliquot containing 5 microg recombinant CTB was injected intradermally into Balb/c (H2K(d)) mice. CTB-specific serum IgG was detected in animals that had been administered plant-expressed or native purified CTB. A T-cell proliferation study using splenocytes and cytokine estimations in supernatants generated by in vitro stimulation of macrophages isolated from the immuno-primed animals was carried out. Inhibition of proliferation of T lymphocytes was observed in splenic T lymphocytes isolated from animals injected with either native or plant-expressed CTB. Macrophages isolated from mice immunised with native or plant-expressed CTB showed enhanced secretion of interleukin-10 but secretion of lipopolysaccharide-induced interleukin-12 and tumor necrosis factor alpha was inhibited. These studies suggest that plant-expressed protein behaved like native CTB with regards to effects on T-cell proliferation and cytokine levels, indicating the suitability of plant expression systems for the production of bacterial antigens, which could be used as edible vaccine. The transgene was found to be inherited in the progeny and was expressed to yield a pentameric form of CTB as evident by its interaction with G(M1) ganglioside.

In earlier study from our group, cholera toxin B subunit had been expressed in tomato for developing a plant-based vaccine against cholera. In the present investigation, gene for accessory colonization factor (acf) subunit A, earlier reported to be essential for efficient colonization in the intestine, has been expressed in Escherichia coli as well as tomato plants. Gene encoding for a chimeric protein having a fusion of cholera toxin B subunit and accessory colonization factor A was also expressed in tomato to generate more potent combinatorial antigen. CaMV35S promoter with a duplicated enhancer sequence was used for expression of these genes in tomato. Integration of transgenes into tomato genome was confirmed by PCR and Southern hybridization. Expression of the genes was confirmed at transcript and protein levels. Accessory colonization factor A and cholera toxin B subunit fused to this protein accumulated up to 0.25% and 0.08% of total soluble protein, respectively, in the fruits of transgenic plants. Whereas protein purified from E. coli, in combination with cholera toxin B subunit can be used for development of conventional subunit vaccine, tomato fruits expressing these proteins can be used together with tomato plants expressing cholera toxin B subunit for development of oral vaccine against cholera.

Serine/threonine protein kinases (STPKs) represent a burgeoning concept in prokaryotic signaling and have been implicated in a range of control mechanisms. This paper describes the enzymatic and molecular characterization of PknH, a mycobacterial STPK. After cloning and expression as a Glutathione-S-transferase fusion protein in E. coli, PknH was found to phosphorylate itself and exogenous substrates like myelin basic protein and histone. The kinase activity of PknH was inhibited by the kinase inhibitors staurosporine and H-7. The results confirmed that PknH is a transmembrane protein and is restricted to members of the Mycobacterium tuberculosis complex. In addition, transcriptional analysis of pknH in M. tuberculosis under various stress conditions revealed that exposure to low pH and heat shock decreased the level of pknH transcription significantly. This is the first report describing differential expression of a mycobacterial kinase in response to stress conditions which can indicate its ability to regulate cellular events promoting bacterial adaptation to environmental change.

GTP-binding proteins (G-proteins) are highly conserved signaling molecules that participate in cellular signaling and bacterial pathogenesis by regulating the activity of cognate GTPases. However, the exact role of G-proteins in the pathogenesis of Mycobacterium tuberculosis is poorly understood. The complete genome sequence of M. tuberculosis H(37)Rv, suggests the presence of several homologs of bacterial G-proteins. In the present study, three G-proteins, Era, Obg and LepA of M. tuberculosis H(37)Rv were cloned and expressed in Escherichia coli. Purified proteins showed GTP-binding and hydrolyzing activities. A point mutation in the conserved GTP-binding motif, AspXXGly (Asp to Ala) in Era (Asp-258) and Obg (Asp-212) proteins resulted in the loss of the associated activities, confirming that known key residues in well-established G-proteins are also conserved in mycobacterial homologs. This study confirms that Era, Obg and LepA of M. tuberculosis H(37)Rv possess GTPase activity and provide a platform to understand the physiological significance of these proteins in associated pathogenesis.

Adenylate kinase (AK) is a small ubiquitous enzyme that catalyzes the reversible transfer of the terminal phosphate group from adenine triphosphate (ATP): magnesium ion (Mg²⁺) to adenine monophosphate (AMP) to form two molecules of adenine diphosphate (ADP). AK thus maintains the homeostasis of adenine nucleotides in eukaryotes and prokaryotes. Because the [ATP]/[ADP] ratio is an important parameter in energy regulation in cells, Mg²⁺-activated AK has an important biological role, particularly in the case of bacteria, as imbalance in the ratio of [ATP]/[ADP] has been associated with alteration in its DNA supercoiling state. In the present study, magnesium-binding assays were carried out by systematically varying the concentrations of Mg²⁺, protein, AMP, ATP, and indicator in kinetic experiments. We report evidence that during magnesium-binding assay, the fluorescence level of the indicator "Mag-Indo-1" changes with protein concentration, suggesting that magnesium ions are binding to AK. The dual activity of AK both as nucleoside monophosphate and diphosphate kinases suggests that this enzyme may have a role in RNA and DNA biosynthesis in addition to its role in intracellular nucleotide metabolism. According to the proposed model, the magnesium-activated AK exhibits an increase in its forward reaction rate compared with the inactivated form. These findings imply that Mg²⁺ could be an important regulator in the energy signaling network in cells.

In this study, a previously uncharacterized gene (Rv0447c) of Mycobacterium tuberculosis, designated as an unknown fatty-acid methyltransferase (ufaA1), was cloned, expressed in Escherichia coli, and purified. The biochemical characterization of the purified protein (UfaA1) showed it to be a methyltransferase that catalyzes biosynthesis of the tuberculostearic acid (10-methylstearic-acid, TSA), a significant constituent lipid of the mycobacterial cell wall and a clinical marker of the disease. Here, we show that UfaA1 transfers the methyl group from S-adenosyl-l-methionine (SAM) to the double bond of oleic acid in phosphatidylethanolamine or phosphatidylcholine to produce TSA. Optimal activity was obtained between pH 7.0 and pH 8.0. The methyltransferase activity of UfaA1 was severely inhibited by S-adenosyl-l-homocysteine. The Km values for dioleyl phosphatidylethanolamine, SAM, and nicotinamide adenine dinucleotide phosphate were 14, 13, and 83 µM, respectively, with Vmax of 1.3-1.6 nmol/Min. These results identify the Rv0447c gene product of M. tuberculosis as the methyltransferase that catalyzes the biosynthesis of TSA. This provides new information in mycobacterial cell wall synthesis.