Metabolome based reaction graphs of M.tuberculosis and M.leprae: A comparative network analysis

Background. Several types of networks, such as transcriptional, metabolic or protein-protein interaction networks of various organisms have been constructed, that have provided a variety of insights into metabolism and regulation. Here, we seek to exploit the reaction-based networks of three organisms for comparative genomics. We use concepts from spectral graph theory to systematically determine how differences in basic metabolism of organisms are reflected at the systems level and in the overall topological structures of their metabolic networks. Methodology/Principal Findings. Metabolome-based reaction networks of Mycobacterium tuberculosis, Mycobacterium leprae and Escherichia coli have been constructed based on the KEGG LIGAND database, followed by graph spectral analysis of the network to identify hubs as well as the sub-clustering of reactions. The shortest and alternate paths in the reaction networks have also been examined. Sub-cluster profiling demonstrates that reactions of the mycolic acid pathway in mycobacteria form a tightly connected sub-cluster. Identification of hubs reveals reactions involving glutamate to be central to mycobacterial metabolism, and pyruvate to be at the centre of the E. coli metabolome. The analysis of shortest paths between reactions has revealed several paths that are shorter than well established pathways. Conclusions. We conclude that severe downsizing of the leprae genome has not significantly altered the global structure of its reaction network but has reduced the total number of alternate paths between its reactions while keeping the shortest paths between them intact. The hubs in the mycobacterial networks that are absent in the human metabolome can be explored as potential drug targets. This work demonstrates the usefulness of constructing metabolome based networks of organisms and the feasibility of their analyses through graph spectral methods. The insights obtained from such studies provide a broad overview of the similarities and differences between organisms, taking comparative genomics studies to a higher dimension.

Detrimental Effects of Hypoxia-Specific Expression of Uracil DNA Glycosylase (Ung) in Mycobacterium smegmatis

Mycobacterium tuberculosis is known to reside latently in a significant fraction of the human population. Although the bacterium possesses an aerobic mode of metabolism, it adapts to persistence under hypoxic conditions such as those encountered in granulomas. While in mammalian systems hypoxia is a recognized DNA-damaging stress, aspects of DNA repair in mycobacteria under such conditions have not been studied. We subjected Mycobacterium smegmatis, a model organism, to the Wayne's protocol of hypoxia. Analysis of the mRNA of a key DNA repair enzyme, uracil DNA glycosylase (Ung), by real-time reverse transcriptase PCR (RT-PCR) revealed its downregulation during hypoxia. However, within an hour of recovery of the culture under normal oxygen levels, the Ung mRNA was restored. Analysis of Ung by immunoblotting and enzyme assays supported the RNA analysis results. To understand its physiological significance, we misexpressed Ung in M. smegmatis by using a hypoxia-responsive promoter of narK2 from M. tuberculosis. Although the misexpression of Ung during hypoxia decreased C-to-T mutations, it compromised bacterial survival upon recovery at normal oxygen levels. RT-PCR analysis of other base excision repair gene transcripts (UdgB and Fpg) suggested that these DNA repair functions also share with Ung the phenomenon of downregulation during hypoxia and recovery with return to normal oxygen conditions. We discuss the potential utility of this phenomenon in developing attenuated strains of mycobacteria.

Detection and characterisation of circulating cysticercal antigens in human cerebrospinal fluid

With biotin labelled and unlabelled immunoglobulin fraction of anticysticercal antibodies raised in rabbits, tandem-enzyme linked immunosorbent assay (T-ELISA), capture-dot immunobinding assay (C-DIA) and reverse passive haemagglutination (RPHA) tests were developed for the detection of cysticercal antigens. The sensitivity levels were respectively, 9 ng ml−1, 2 ng ml−1 and 45 ng ml−1. All three methods were of equal specificity as none of the antigens of Mycobacterium tuberculosis, Japanese encephalitis virus and Echinococcus granulosus reacted with anticysticercal IgG. Cysticercal antigens were detected in the cerebrospinal fluid (CSF) of confirmed neurocysticercosis at sensitivity levels of 91·6% by T-ELISA, 83·33% by C-DIA and 75% by RPHA and specificity levels of >93%. Western analysis of these antigens in CSF showed mainly antigens of 64–68 kDa and 24–28 kDA. By crossed immunoelectrophoresis (CIE) with an intermediate gel technique, five circulating antigens were found to be released from scolex and fluid.

Diversity in Functional Organization of Class I and Class II Biotin Protein Ligase

The cell envelope of Mycobacterium tuberculosis (M. tuberculosis) is composed of a variety of lipids including mycolic acids, sulpholipids, lipoarabinomannans, etc., which impart rigidity crucial for its survival and pathogenesis. Acyl CoA carboxylase (ACC) provides malonyl-CoA and methylmalonyl-CoA, committed precursors for fatty acid and essential for mycolic acid synthesis respectively. Biotin Protein Ligase (BPL/BirA) activates apo-biotin carboxyl carrier protein (BCCP) by biotinylating it to an active holo-BCCP. A minimal peptide (Schatz), an efficient substrate for Escherichia coli BirA, failed to serve as substrate for M. tuberculosis Biotin Protein Ligase (MtBPL). MtBPL specifically biotinylates homologous BCCP domain, MtBCCP87, but not EcBCCP87. This is a unique feature of MtBPL as EcBirA lacks such a stringent substrate specificity. This feature is also reflected in the lack of self/promiscuous biotinylation by MtBPL. The N-terminus/HTH domain of EcBirA has the selfbiotinable lysine residue that is inhibited in the presence of Schatz peptide, a peptide designed to act as a universal acceptor for EcBirA. This suggests that when biotin is limiting, EcBirA preferentially catalyzes, biotinylation of BCCP over selfbiotinylation. R118G mutant of EcBirA showed enhanced self and promiscuous biotinylation but its homologue, R69A MtBPL did not exhibit these properties. The catalytic domain of MtBPL was characterized further by limited proteolysis. Holo-MtBPL is protected from proteolysis by biotinyl-59 AMP, an intermediate of MtBPL catalyzed reaction. In contrast, apo-MtBPL is completely digested by trypsin within 20 min of co-incubation. Substrate selectivity and inability to promote self biotinylation are exquisite features of MtBPL and are a consequence of the unique molecular mechanism of an enzyme adapted for the high turnover of fatty acid biosynthesis.

Mycobacterium tuberculosis Expresses ftsE Gene Through Multiple Transcripts

Bacterial FtsE gene codes for the ATP-binding protein, FtsE, which in complex with the transmembrane protein, FtsX, participates in diverse cellular processes. Therefore, regulated expression of FtsE and FtsX might be critical to the human pathogen, Mycobacterium tuberculosis, under stress conditions. Although ftsX gene of M. tuberculosis (MtftsX) is known to be transcribed from a promoter inside the upstream gene, ftsE, the transcriptional status of ftsE gene of M. tuberculosis (MtftsE) remains unknown. Therefore, the authors initiated transcriptional analyses of MtftsE, using total RNA from M. tuberculosis cells that were grown under stress conditions, which the pathogen is exposed to, in granuloma in tuberculosis patients. Primer extension experiments showed the presence of putative transcripts, T1, T2, T3, and T4. T1 originated from the intergenic region between the upstream gene, MRA_3135, and MtftsE. T2 and T3 were found initiated from within MRA_3135. T4 was transcribed from a region upstream of MRA_3135. RT-PCR confirmed co-transcription of MRA_3135 and MtftsE. The cloned putative promoter regions for T1, T2, and T3 elicited transcriptional activity in Mycobacterium smegmatis transformants. T1, T2, and T3, but no new transcript, were present in the M. tuberculosis cells that were grown under the stress conditions, which the pathogen is exposed to in granuloma in tuberculosis patients. It showed lack of modulation of MtftsE transcripts under the stress conditions tested, indicating that ftsE may not have a stress response-specific function in M. tuberculosis.

Drug-drug co-crystals: Temperature-dependent proton mobility in the molecular complex of isoniazid with 4-aminosalicylic acid

Two drug-drug co-crystals of the anti-tuberculosis drugs isoniazid (INH), pyrazinamide (PYR) and 4-aminosalicylic acid (PAS) are reported. The first is the 1 : 1 molecular complex of INH and PAS. The second is the monohydrate of the 1 : 1 complex of PYR and PAS. The crystal structures of both co-crystals are characterized by a number of hydrogen bonded synthons. Hydrogen bonding of the COOH center dot center dot center dot N-pyridine type is found in both cases. In the INH : PAS co-crystal, there are two symmetry independent COOH center dot center dot center dot center dot N-pyridine hydrogen bonds. In one of these, the H-atom is located on the carboxylic group and is indicative of a co-crystal. In the second case, partial proton transfer occurs across the hydrogen bond, and the extent of proton transfer depends on the temperature. This is more indicative of a salt. Drug-drug co-crystals may have some bearing in the treatment of tuberculosis.

Crystal structure of Rv2118c: an AdoMet-dependent methyltransferase from Mycobacterium tuberculosis H37Rv

Rv2118c belongs to the class of conserved hypothetical proteins from Mycobacterium tuberculosis H37Rv. The crystal structure of Rv2118c in complex with S-adenosyl-Image -methionine (AdoMet) has been determined at 1.98 Å resolution. The crystallographic asymmetric unit consists of a monomer, but symmetry-related subunits interact extensively, leading to a tetrameric structure. The structure of the monomer can be divided functionally into two domains: the larger catalytic C-terminal domain that binds the cofactor AdoMet and is involved in the transfer of methyl group from AdoMet to the substrate and a smaller N-terminal domain. The structure of the catalytic domain is very similar to that of other AdoMet-dependent methyltransferases. The N-terminal domain is primarily a β-structure with a fold not found in other methyltransferases of known structure. Database searches reveal a conserved family of Rv2118c-like proteins from various organisms. Multiple sequence alignments show several regions of high sequence similarity (motifs) in this family of proteins. Structure analysis and homology to yeast Gcd14p suggest that Rv2118c could be an RNA methyltransferase, but further studies are required to establish its functional role conclusively.

A Transcript Cleavage Factor of Mycobacterium tuberculosis Important for Its Survival

After initiation of transcription, a number of proteins participate during elongation and termination modifying the properties of the RNA polymerase (RNAP). Gre factors are one such group conserved across bacteria. They regulate transcription by projecting their N-terminal coiled-coil domain into the active center of RNAP through the secondary channel and stimulating hydrolysis of the newly synthesized RNA in backtracked elongation complexes. Rv1080c is a putative gre factor (MtbGre) in the genome of Mycobacterium tuberculosis. The protein enhanced the efficiency of promoter clearance by lowering abortive transcription and also rescued arrested and paused elongation complexes on the GC rich mycobacterial template. Although MtbGre is similar in domain organization and shares key residues for catalysis and RNAP interaction with the Gre factors of Escherichia coli, it could not complement an E. coli gre deficient strain. 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 reduced the bacterial survival by several fold indicating its essential role in mycobacteria. Another Gre homolog, Rv3788 was not functional in transcript cleavage activity indicating that a single Gre is sufficient for efficient transcription of the M. tuberculosis genome.

The biological and structural characterization of Mycobacterium tuberculosis UvrA provides novel insights into its mechanism of action

Mycobacterium tuberculosis is an extremely well adapted intracellular human pathogen that is exposed to multiple DNA damaging chemical assaults originating from the host defence mechanisms. As a consequence, this bacterium is thought to possess highly efficient DNA repair machineries, the nucleotide excision repair (NER) system amongst these. Although NER is of central importance to DNA repair in M. tuberculosis, our understanding of the processes in this species is limited. The conserved UvrABC endonuclease represents the multi-enzymatic core in bacterial NER, where the UvrA ATPase provides the DNA lesion-sensing function. The herein reported genetic analysis demonstrates that M. tuberculosis UvrA is important for the repair of nitrosative and oxidative DNA damage. Moreover, our biochemical and structural characterization of recombinant M. tuberculosis UvrA contributes new insights into its mechanism of action. In particular, the structural investigation reveals an unprecedented conformation of the UvrB-binding domain that we propose to be of functional relevance. Taken together, our data suggest UvrA as a potential target for the development of novel anti-tubercular agents and provide a biochemical framework for the identification of small-molecule inhibitors interfering with the NER activity in M. tuberculosis.

Synergy between the N-terminal and C-terminal domains of Mycobacterium tuberculosis HupB is essential for high-affinity binding, DNA supercoiling and inhibition of RecA-promoted strand exchange

The occurrence of DNA architectural proteins containing two functional domains derived from two different architectural proteins is an interesting emerging research theme in the field of nucleoid structure and function. Mycobacterium tuberculosis HupB, unlike Escherichia coli HU, is a two-domain protein that, in the N-terminal region, shows broad sequence homology with bacterial HU. The long C-terminal extension, on the other hand, contains seven PAKK/KAAK motifs, which are characteristic of the histone H1/H5 family of proteins. In this article, we describe several aspects of HupB function, in comparison with its truncated derivatives lacking either the C-terminus or N-terminus. We found that HupB binds a variety of DNA repair and replication intermediates with K(d) values in the nanomolar range. By contrast, the N-terminal fragment of M. tuberculosis HupB (HupB(MtbN)) showed diminished DNA-binding activity, with K(d) values in the micromolar range, and the C-terminal domain was completely devoid of DNA-binding activity. Unlike HupB(MtbN), HupB was able to constrain DNA in negative supercoils and introduce negative superhelical turns into relaxed DNA. Similarly, HupB exerted a robust inhibitory effect on DNA strand exchange promoted by cognate and noncognate RecA proteins, whereas HupB(MtbN), even at a 50-fold molar excess, had no inhibitory effect. Considered together, these results suggest that synergy between the N-terminal and C-terminal domains of HupB is essential for its DNA-binding ability, and to modulate the topological features of DNA, which has implications for processes such as DNA compaction, gene regulation, homologous recombination, and DNA repair.

Signalling mechanisms in Mycobacteria

The importance of inter-and intracellular signal transduction in all forms of life cannot be underestimated. A large number of genes dedicated to cellular signalling are found in almost all sequenced genomes, and Mycobacteria are no exception. What appears to be interesting in Mycobacteria is that well characterized signalling mechanisms used by bacteria, such as the histidine-aspartate phosphorelay seen in two-component systems, are found alongside signalling components that closely mimic those seen in higher eukaryotes. This review will describe the important contribution made by researchers in India towards the identification and characterization of proteins involved in two-component signalling, protein phosphorylation and cyclic nucleotide metabolism. (C) 2011 Elsevier Ltd. All rights reserved.

Structural biology of Mycobacterium tuberculosis proteins: The Indian efforts

Among the many different objectives of large scale structural genomics projects are expanding the protein fold space, enhancing understanding of a model or disease-related organism, and providing foundations for structure-based drug discovery. Systematic analysis of protein structures of Mycobacterium tuberculosis has been ongoing towards meeting some of these objectives. Indian participation in these efforts has been enthusiastic and substantial. The proteins of M. tuberculosis chosen for structural analysis by the Indian groups span almost all the functional categories. The structures determined by the Indian groups have led to significant improvement in the biochemical knowledge on these proteins and consequently have started providing useful insights into the biology of M. tuberculosis. Moreover, these structures form starting points for inhibitor design studies, early results of which are encouraging. The progress made by Indian structural biologists in determining structures of M. tuberculosis proteins is highlighted in this review. (C) 2011 Elsevier Ltd. All rights reserved.

Base excision and nucleotide excision repair pathways in mycobacteria

About a third of the human population is estimated to be infected with Mycobacterium tuberculosis. The bacterium displays an excellent adaptability to survive within the host macrophages. As the reactive environment of macrophages is capable of inducing DNA damage, the ability of the pathogen to safeguard its DNA against the damage is of paramount significance for its survival within the host. Analysis of the genome sequence has provided important insights into the DNA repair machinery of the pathogen, and the studies on DNA repair in mycobacteria have gained momentum in the past few years. The studies have revealed considerable differences in the mycobacterial DNA repair machinery when compared with those of the other bacteria. This review article focuses especially on the aspects of base excision, and nucleotide excision repair pathways in mycobacteria. (C) 2011 Elsevier Ltd. All rights reserved.

Structural Annotation of Mycobacterium tuberculosis Proteome

Of the similar to 4000 ORFs identified through the genome sequence of Mycobacterium tuberculosis (TB) H37Rv, experimentally determined structures are available for 312. Since knowledge of protein structures is essential to obtain a high-resolution understanding of the underlying biology, we seek to obtain a structural annotation for the genome, using computational methods. Structural models were obtained and validated for similar to 2877 ORFs, covering similar to 70% of the genome. Functional annotation of each protein was based on fold-based functional assignments and a novel binding site based ligand association. New algorithms for binding site detection and genome scale binding site comparison at the structural level, recently reported from the laboratory, were utilized. Besides these, the annotation covers detection of various sequence and sub-structural motifs and quaternary structure predictions based on the corresponding templates. The study provides an opportunity to obtain a global perspective of the fold distribution in the genome. The annotation indicates that cellular metabolism can be achieved with only 219 folds. New insights about the folds that predominate in the genome, as well as the fold-combinations that make up multi-domain proteins are also obtained. 1728 binding pockets have been associated with ligands through binding site identification and sub-structure similarity analyses. The resource (http://proline.physics.iisc.ernet.in/Tbstructuralannotation), being one of the first to be based on structure-derived functional annotations at a genome scale, is expected to be useful for better understanding of TB and for application in drug discovery. The reported annotation pipeline is fairly generic and can be applied to other genomes as well.