Laxman S. Meena

Survival mechanisms of pathogenic Mycobacterium tuberculosis H37Rv.

Mycobacterium tuberculosis H37Rv is a highly successful pathogen and its success fully relies on its ability to utilize macrophages for its replication and, more importantly, the macrophage should remain viable to host the Mycobacterium. Despite the fact that these phagocytes are usually very effective in internalizing and clearing most of the bacteria, M. tuberculosis H37Rv has evolved a number of very effective survival strategies, including: (a) the inhibition of phagosome–lysosome fusion; (b) the inhibition of phagosome acidification; (c) the recruitment and retention of tryptophan‐aspartate containing coat protein on phagosomes to prevent their delivery to lysosomes; and (d) the expression of members of the host‐induced repetitive glycine‐rich protein family of proteins. However, the mechanisms by which M. tuberculosis H37Rv enters the host cell, circumvents host defenses and spreads to neighboring cell are not completely understood. Therefore, a better understanding of host–pathogen interaction is essential if the global tuberculosis pandemic is ever to be controlled. This review addresses some of the pathogenic strategies of the M. tuberculosis H37Rv that aids in its survival and pathogenicity.

Expression of cholera toxin B subunit in transgenic tomato plants

Cholera toxin, secreted by Vibrio cholerae, consists of A and B subunits. The latter binds to GM1-ganglioside receptors as a pentamer (∼55 kDa). Tomato plants were transformed with the gene encoding cholera toxin B subunit (ctxB) along with an endoplasmic reticulum retention signal (SEKDEL) under the control of the CaMV 35S promoter via Agrobacterium-mediated transformation. PCR and Southern analysis confirmed the presence of the ctxB gene in transformed tomato plants. Northern analysis showed the presence of the ctxB-specific transcript. Immunoblot assays of the plant-derived protein extract showed the presence of cholera toxin subunit B (CTB) with mobility similar to purified CTB from V. cholerae. Both tomato leaves and fruits expressed CTB at levels up to 0.02 and 0.04% of total soluble protein, respectively. The GM1-ELISA showed that the plant-derived CTB bound specifically to GM1-ganglioside receptor, suggesting that it retained its native pentameric form. This study forms a basis for exploring the utility of CTB to develop tomato-based edible vaccines against cholera.

Expression of toxin co-regulated pilus subunit A (TCPA) of Vibrio cholerae and its immunogenic epitopes fused to cholera toxin B subunit in transgenic tomato (Solanum lycopersicum)

For protection against cholera, it is important to develop efficient vaccine capable of inducing anti-toxin as well as anti-colonizing immunity against Vibrio cholerae infections. Earlier, expression of cholera toxin B subunit (CTB) in tomato was reported by us. In the present investigation, toxin co-regulated pilus subunit A (TCPA), earlier reported to be an antigen capable of providing anti-colonization immunity, has been expressed in tomato. Further, to generate more potent combinatorial antigens, nucleotides encoding P4 or P6 epitope of TCPA were fused to cholera toxin B subunit gene (ctxB) and expressed in tomato. Presence of transgenes in the tomato genome was confirmed by PCR and expression of genes was confirmed at transcript and protein level. TCPA, chimeric CTB-P4 and CTB-P6 proteins were also expressed in E. coli. TCPA protein expressed in E. coli was purified to generate anti-TCPA antibodies in rabbit. Immunoblot and GM1-ELISA verified the synthesis and assembly of pentameric chimeric proteins in fruit tissue of transgenic tomato plants. The chimeric protein CTB-P4 and CTB-P6 accumulated up to 0.17 and 0.096% of total soluble protein (TSP), respectively, in tomato fruits. Whereas expression of TCPA, CTB-P4 and CTB-P6 in E. coli can be utilized for development of conventional vaccine, expression of these antigens which can provide both anti-toxin as well as anti-colonization immunity, has been demonstrated in plants, in a form which is potentially capable of inducing immune response against cholera infection.

Expression of accessory colonization factor subunit A (ACFA) of Vibrio cholerae and ACFA fused to cholera toxin B subunit in transgenic tomato (Solanum lycopersicum)

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.

Biosynthesis and Virulent Behavior of Lipids Produced by Mycobacterium tuberculosis: LAM and Cord Factor: An Overview

Mycobacterium tuberculosis is the causative agent of tuberculosis disease, which has developed a myriad of exceptional features contributing to its survival within the hostile environment of host cell. Unique cell wall structure with high lipid content plays an imperative role in the pathogenicity of mycobacteria. Cell wall components of MTB such as lipoarabinomannan and Trehalose dimycolate (cord factor) are virulent in nature apart from its virulence genes. Virulent effect of these factors on host cells reduces host cell immunity. LAM has been known to inhibit phagosome maturation by inhibiting the Ca2+/calmodulin phosphatidyl inositol-3-kinase hvps34 pathways. Moreover, TDM (Trehalose dimycolate) also inhibits fusion between phospholipid vesicles and migration of polymorphonuclear neutrophils. The objective of this paper is to understand the virulence of LAM and cord factor on host cell which might be helpful to design an effective drug against tuberculosis.

An overview to understand the role of PE_PGRS family proteins in Mycobacterium tuberculosis H37Rv and their potential as new drug targets

Tuberculosis has long been the scourge of humanity, claiming millions of lives. The family of PE_PGRS gene has been attributed to the Mycobacterium tuberculosis pathogenesis over the past few decades. The gene of PE_PGRS family proteins are most often clustered in a region of the genome often as overlapping genes and role in cell surface markers, adhesion and invasion of defense cells of the host (macrophage and dendritic cells). The proline–glutamic acid (PE) domain is responsible for the cellular localization of these proteins on bacterial cells. This gene family shows immense genetic variability in terms of multiple insertion–deletions and single‐nucleotide polymorphisms as seen in PE_PGRS9, PE_PGRS17, PE_PGRS18, and PE_PGRS33. In spite of variability, there are indications of shared epitopes in these proteins. Few of these gene sequences that have been studied from evolutionary perspective show indication of positive selection and also landmarks of recent evolutionary events. Many of these proteins show calcium‐binding motifs and consequently seen to be responsible in inhibition of phagolysosome formation via a calmodulin‐kinase‐dependent pathway. A number of PE_PGRS genes were tested for its expression with different growth conditions in vitro and in vivo, among which the contrast in expressivity was seen vividly in PE_PGRS16 (upregulated) and PE_PGRS26 (downregulated) in bacteria persisting in macrophages. Similarly, PE_PGRS33 has been indicated in macrophagial necrosis by a tumor necrosis factor‐α‐induced pathway. These PE_PGRS family genes may be an interesting subject for research and development. Their fibronectin‐binding and calcium‐binding property may be strongly implicated in immunopathogenesis of virulent M. tuberculosis strain. In this review, an attempt has been made to evaluate and present data for better understanding of in vivo pathogen functions, for understanding the physiological significance of PE_PGRS gene family, and their potential as new drug targets.

Biochemical characterization of an S-adenosyl-l-methionine-dependent methyltransferase (Rv0469) of Mycobacterium tuberculosis.

Abstract 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.

Cloning and characterization of GTP-binding proteins of Mycobacterium tuberculosis H37Rv

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.

Expression and characterization of Rv0447c product, potentially the methyltransferase involved in tuberculostearic acid biosynthesis in Mycobacterium tuberculosis

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.

Elucidation of Mg2+ binding activity of adenylate kinase from Mycobacterium tuberculosis H37Rv using fluorescence studies

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.