X-ray structure of putative acyl-ACP desaturase DesA2 from Mycobacterium tuberculosis H37Rv

Genome sequencing showed that two proteins in Mycobacterium tuberculosis H37Rv contain the metal binding motif (D/E)X(2)HX(approximately 100)(D/E)X(2)H characteristic of the soluble diiron enzyme superfamily. These putative acyl-ACP desaturase genes desA1 and desA2 were cloned from genomic DNA and expressed in Escherichia coli BL21(DE3). DesA1 was found to be insoluble, but in contrast, DesA2 was a soluble protein amenable to biophysical characterization. Here, we report the 2.0 A resolution X-ray structure of DesA2 determined by multiple anomalous dispersion (MAD) phasing from a Se-met derivative and refinement against diffraction data obtained on the native protein. The X-ray structure shows that DesA2 is a homodimeric protein with a four-helix bundle core flanked by five additional helices that overlay with 192 structurally equivalent amino acids in the structure of stearoyl-ACP Delta9 desaturase from castor plant with an rms difference 1.42 A. In the DesA2 crystals, one metal (likely Mn from the crystallization buffer) was bound in high occupancy at the B-site of the conserved metal binding motif, while the A-site was not occupied by a metal ion. Instead, the amino group of Lys-76 occupied this position. The relationships between DesA2 and known diiron enzymes are discussed.     

Mycobacterium tuberculosis ESAT-6 Exhibits a Unique Membrane-interacting Activity That Is Not Found in Its Ortholog from Non-pathogenic Mycobacterium smegmatis

Mycobacterium tuberculosis ESAT-6 (MtbESAT-6) reportedly shows membrane/cell-lysis activity, and recently its biological roles in pathogenesis have been implicated in rupture of the phagosomes for bacterial cytosolic translocation. However, molecular mechanism of MtbESAT-6-mediated membrane interaction, particularly in relation with its biological functions in pathogenesis, is poorly understood. In this study, we investigated the pH-dependent membrane interaction of MtbESAT-6, MtbCFP-10, and the MtbESAT-6/CFP-10 heterodimer, by using liposomal model membranes that mimic phagosomal compartments. MtbESAT-6, but neither MtbCFP-10 nor the heterodimer, interacted with the liposomal membranes at acidic conditions, which was evidenced by release of K⁺ ions from the liposomes. Most importantly, the orthologous ESAT-6 from non-pathogenic Mycobacterium smegmatis (MsESAT-6) was essentially inactive in release of K⁺. The differential membrane interactions between MtbESAT-6 and MsESAT-6 were further confirmed in an independent membrane leakage assay using the dye/quencher pair, 8-aminonapthalene-1,3,6 trisulfonic acid (ANTS)/p-xylene-bis-pyridinium bromide (DPX). Finally, using intrinsic and extrinsic fluorescence approaches, we probed the pH-dependent conformational changes of MtbESAT-6 and MsESAT-6. At acidic pH conditions, MtbESAT-6 underwent a significant conformational change, which was featured by an increased solvent-exposed hydrophobicity, while MsESAT-6 showed little conformational change in response to acidification. In conclusion, we have demonstrated that MtbESAT-6 possesses a unique membrane-interacting activity that is not found in MsESAT-6 and established the utility of rigorous biochemical approaches in dissecting the virulence of M. tuberculosis.     

Structure and function of the transketolase from Mycobacterium tuberculosis and comparison with the human enzyme

The transketolase (TKT) enzyme in Mycobacterium tuberculosis represents a novel drug target for tuberculosis treatment and has low homology with the orthologous human enzyme. Here, we report on the structural and kinetic characterization of the transketolase from M. tuberculosis (TBTKT), a homodimer whose monomers each comprise 700 amino acids. We show that TBTKT catalyses the oxidation of donor sugars xylulose-5-phosphate and fructose-6-phosphate as well as the reduction of the acceptor sugar ribose-5-phosphate. An invariant residue of the TKT consensus sequence required for thiamine cofactor binding is mutated in TBTKT; yet its catalytic activities are unaffected, and the 2.5 Å resolution structure of full-length TBTKT provides an explanation for this. Key structural differences between the human and mycobacterial TKT enzymes that impact both substrate and cofactor recognition and binding were uncovered. These changes explain the kinetic differences between TBTKT and its human counterpart, and their differential inhibition by small molecules. The availability of a detailed structural model of TBTKT will enable differences between human and M. tuberculosis TKT structures to be exploited to design selective inhibitors with potential antitubercular activity.     

Insight from the structural molecular model of cytidylate kinase from Mycobacterium tuberculosis

Mycobacterium tuberculosis is a gram-positive bacterium causes tuberculosis in human. H37Rv strain is a pathogenic strain utilized for tuberculosis research. The cytidylate mono-phosphate (CMP) kinase of Mycobacterium tuberculosis belongs to the family nucleoside mono-phosphate kinase (NMK), this enzyme is required for the bacterial growth. Therefore, it is important to study the structural and functional features of this enzyme in the control of the disease. Hence, we developed the structural molecular model of the CMP kinase protein from Mycobacterium tuberculosis by homology modeling using the software MODELLER (9v10). Based on sequence similarity with protein of known structure (template) of Mycobacterium smegmatis (PDB ID: 3R20) was chosen from protein databank (PDB) by using BLASTp. The energy of constructed models was minimized and the qualities of the models were evaluated by PROCHECK and VERRIFY-3D. Resulted Ramachandran plot analysis showed that conformations for 100.00% of amino acids residues are within the most favored regions. A possible homologous deep cleft active site was identified in the Model using CASTp program. Amino acid composition and polarity of that protein was observed by CLC-Protein Workbench tool. Expasy''s Prot-param server and CYC_REC tool were used for physiochemical and functional characterization of the protein. Studied of secondary structure of that protein was carried out by computational program, ProFunc. The structure is finally submitted in Protein Model Database. The predicted model permits initial inferences about the unexplored 3D structure of the CMP kinase and may be promote in relational designing of molecules for structure-function studies.     

Sublineages of Beijing Strain of Mycobacterium tuberculosis in Sri Lanka

Strains of the Beijing/W genotype of Mycobacterium tuberculosis have been responsible for large outbreaks of tuberculosis around the world, sometimes involving multi-drug resistance. It has been shown that more recently evolved Beijing sublineages are prone to cause outbreaks. Furthermore Beijing is the single predominant cluster in Sri Lanka. The present study identifies that recently evolved sublineages of Beijing strains are present in the study population. The majority of Beijing isolates (92.85%) were pan-susceptible. However, these findings may have important implications for the control and prevention of tuberculosis in Sri Lanka.     

Analysis of DevR regulated genes in Mycobacterium tuberculosis

The DevRS two component system of Mycobacterium tuberculosis is responsible for its dormancy in host and becomes operative under hypoxic condition. It is experimentally known that phosphorylated DevR controls the expression of several downstream genes in a complex manner. In the present work we propose a theoretical model to show role of binding sites in DevR mediated gene expression. Individual and collective role of binding sites in regulating DevR mediated gene expression has been shown via modeling. Objective of the present work is twofold. First, to describe qualitatively the temporal dynamics of wild type genes and their known mutants. Based on these results we propose that DevR controlled gene expression follows a specific pattern which is efficient in describing other DevR mediated gene expression. Second, to analyze behavior of the system from information theoretical point of view. Using the tools of information theory we have calculated molecular efficiency of the system and have shown that it is close to the maximum limit of isothermal efficiency.     

Mycobacterium tuberculosis Rv2536 protein implicated in specific binding to human cell lines

The gene encoding the Mycobacterium tuberculosis Rv2536 protein is present in the Mycobacterium tuberculosis complex (as assayed by PCR) and transcribed (as determined by RT-PCR) in M. tuberculosis H37Rv, M. tuberculosis H37Ra, M. bovis BCG, and M. africanum strains. Rabbits immunized with synthetic polymer peptides from this protein produced antibodies specifically recognizing a 25-kDa band in mycobacterial sonicate. U937 and A549 cells were used in binding assays involving 20-amino-acid-long synthetic peptides covering the whole Rv2536 protein sequence. Peptide 11207 (161DVFSAVRADDSPTGEMQVAQY180) presented high specific binding to both types of cells; the binding was saturable and presented nanomolar affinity constants. Cross-linking assays revealed that this peptide specifically binds to 50 kDa U937 cell membrane and 45 kDa A549 cell membrane proteins.     

Aptamer from whole-bacterium SELEX as new therapeutic reagent against virulent Mycobacterium tuberculosis

Worldwide, tuberculosis (TB) remains the most frequent and important infectious disease causing morbidity and death. One-third of the world's population is infected with Mycobacterium tuberculosis (MTB), the etiologic agent of TB. Because of the global health problems of TB, the development of potent new anti-TB drugs without cross-resistance with known antimycobacterial agents is urgently needed. In this study, we have applied a Systematic Evolution of Ligands by Exponential Enrichment (SELEX) process to identify a single aptamer (NK2) that binds to virulent strain M. tuberculosis (H37Rv) with high affinity and specificity. We have found that this aptamer improves CD4(+)T cells to produce IFN-gamma after binding to H37Rv. The different component between H37Rv and BCG was identified as some membrane protein. Moreover, the survival rates of mice challenged with i.v. H37Rv have been prolonged after treatment with single injection of aptamer NK2. The bacterial numbers were significantly lower in the spleen of mice treated with aptamer NK2. The histopathological examination of lung biopsy specimens showed lesser pulmonary alveolar fusion and swelling in the presence of the aptamer. These results suggest that aptamer NK2 has inhibitory effects on M. tuberculosis and can be used as antimycobacterial agent.     

Insights from the docking and molecular dynamics simulation of the Phosphopantetheinyl transferase (PptT) structural model from Mycobacterium tuberculosis

A great challenge is posed to the treatment of tuberculosis due to the evolution of multidrug-resistant (MDR) and extensively drugresistant (XDR) strains of Mycobacterium tuberculosis in recent times. The complex cell envelope of the bacterium contains unusual structures of lipids which protects the bacterium from host enzymes and escape immune response. To overcome the drug resistance, targeting “drug targets” which have a critical role in growth and virulence factor is a novel approach for better tuberculosis treatment. The enzyme Phosphopantetheinyl transferase (PptT) is an attractive drug target as it is primarily involved in post translational modification of various types-I polyketide synthases and assembly of mycobactin, which is required for lipid virulence factors. Our in silico studies reported that the structural model of M.tuberculosis PptT characterizes the structure-function activity. The refinement of the model was carried out with molecular dynamics simulations and was analyzed with root mean square deviation (RMSD), and radius of gyration (Rg). This confirmed the structural behavior of PptT in dynamic system. Molecular docking with substrate coenzyme A (CoA) identified the binding pocket and key residues His93, Asp114 and Arg169 involved in PptT-CoA binding. In conclusion, our results show that the M.tuberculosis PptT model and critical CoA binding pocket initiate the inhibitor design of PptT towards tuberculosis treatment.     

Structure and function of Rv0130, a conserved hypothetical protein from Mycobacterium tuberculosis

A large fraction of the Mycobacterium tuberculosis genome codes for proteins of unknown function. We here report the structure of one of these proteins, Rv0130, solved to a resolution of 1.8 å. The Rv0130 monomer features a single hotdog fold composed of a highly curved β-sheet on top of a long and a short α-helix. Two monomers in turn pack to form a double-hotdog-folded homodimer, similar to a large group of enzymes that use thiol esters as substrates. Rv0130 was found to contain a highly conserved R-specific hydratase motif buried deeply between the two monomers. Our biochemical studies show that the protein is able to hydrate a short trans-2-enoyl-coenzyme A moiety with a k _cat of 1.1 × 10² sec⁻¹. The importance of the side chains of D40 and H45 for hydratase activity is demonstrated by site-directed mutagenesis. In contrast to many hotdog-folded proteins, a proline residue distorts the central helix of Rv0130. This distortion allows the creation of a long, curved tunnel, similar to the substrate-binding channels of long-chain eukaryotic hydratase 2 enzymes.     

Cloning and expression of multiple integral membrane proteins from Mycobacterium tuberculosis in Escherichia coli

Seventy integral membrane proteins from the Mycobacterium tuberculosis genome have been cloned and expressed in Escherichia coli. A combination of T7 promoter-based vectors with hexa-His affinity tags and BL21 E. coli strains with additional tRNA genes to supplement sparsely used E. coli codons have been most successful. The expressed proteins have a wide range of molecular weights and number of transmembrane helices. Expression of these proteins has been observed in the membrane and insoluble fraction of E. coli cell lysates and, in some cases, in the soluble fraction. The highest expression levels in the membrane fraction were restricted to a narrow range of molecular weights and relatively few transmembrane helices. In contrast, overexpression in insoluble aggregates was distributed over a broad range of molecular weights and number of transmembrane helices.     

Potential of Mycobacterium tuberculosis resuscitation-promoting factors as antigens in novel tuberculosis sub-unit vaccines

Novel vaccines are needed to control tuberculosis (TB), the bacterial infectious disease that together with malaria and HIV is worldwide responsible for high levels of morbidity and mortality. TB can result from the reactivation of an initially controlled latent infection by Mycobacterium tuberculosis (Mtb). Mtb proteins for which a possible role in this reactivation process has been hypothesized are the five homologs of the resuscitation-promoting factor of Micrococcus luteus, namely Mtb Rv0867c (rpfA), Rv1009 (rpfB), Rv1884c (rpfC), Rv2389c (rpfD) and Rv2450c (rpfE). Analysis of the immune recognition of these 5 proteins following Mtb infection or Mycobacterium bovis BCG vaccination of mice showed that Rv1009 (rpfB) and Rv2389c (rpfD) are the most antigenic in the tested models. We therefore selected rpfB and rpfD for testing their vaccine potential as plasmid DNA vaccines. Elevated cellular immune responses and modest but significant protection against intra-tracheal Mtb challenge were induced by immunization with the rpfB encoding DNA vaccine. The results indicate that rpfB is the most promising candidate of the five rpf-like proteins of Mtb in terms of its immunogenicity and protective efficacy and warrants further analysis for inclusion as an antigen in novel TB vaccines.     

Mycothiol/Mycoredoxin 1-dependent Reduction of the Peroxiredoxin AhpE from Mycobacterium tuberculosis

Mycobacterium tuberculosis (M. tuberculosis), the pathogen responsible for tuberculosis, detoxifies cytotoxic peroxides produced by activated macrophages. M. tuberculosis expresses alkyl hydroxyperoxide reductase E (AhpE), among other peroxiredoxins. So far the system that reduces AhpE was not known. We identified M. tuberculosis mycoredoxin-1 (MtMrx1) acting in combination with mycothiol and mycothiol disulfide reductase (MR), as a biologically relevant reducing system for MtAhpE. MtMrx1, a glutaredoxin-like, mycothiol-dependent oxidoreductase, directly reduces the oxidized form of MtAhpE, through a protein mixed disulfide with the N-terminal cysteine of MtMrx1 and the sulfenic acid derivative of the peroxidatic cysteine of MtAhpE. This disulfide is then reduced by the C-terminal cysteine in MtMrx1. Accordingly, MtAhpE catalyzes the oxidation of wt MtMrx1 by hydrogen peroxide but not of MtMrx1 lacking the C-terminal cysteine, confirming a dithiolic mechanism. Alternatively, oxidized MtAhpE forms a mixed disulfide with mycothiol, which in turn is reduced by MtMrx1 using a monothiolic mechanism. We demonstrated the H₂O₂-dependent NADPH oxidation catalyzed by MtAhpE in the presence of MR, Mrx1, and mycothiol. Disulfide formation involving mycothiol probably competes with the direct reduction by MtMrx1 in aqueous intracellular media, where mycothiol is present at millimolar concentrations. However, MtAhpE was found to be associated with the membrane fraction, and since mycothiol is hydrophilic, direct reduction by MtMrx1 might be favored. The results reported herein allow the rationalization of peroxide detoxification actions inferred for mycothiol, and more recently, for Mrx1 in cellular systems. We report the first molecular link between a thiol-dependent peroxidase and the mycothiol/Mrx1 pathway in Mycobacteria.     

Structural bioinformatics study of EPSP synthase from Mycobacterium tuberculosis

The shikimate pathway is an attractive target for herbicides and antimicrobial agent development because it is essential in algae, higher plants, bacteria, and fungi, but absent from mammals. Homologues to enzymes in the shikimate pathway have been identified in the genome sequence of Mycobacterium tuberculosis. Among them, the EPSP synthase was proposed to be present by sequence homology. Accordingly, in order to pave the way for structural and functional efforts towards anti-mycobacterial agent development, here we describe the molecular modeling of 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase isolated from M. tuberculosis that should provide a structural framework on which the design of specific inhibitors may be based on. Significant differences in the relative orientation of the domains in the two models result in "open" and "closed" conformations. The possible relevance of this structural transition in the ligand biding is discussed.     

Crystal Structure of Mycobacterium tuberculosis Polyketide Synthase 11 (PKS11) Reveals Intermediates in the Synthesis of Methyl-branched Alkylpyrones

PKS11 is one of three type III polyketide synthases (PKSs) identified in Mycobacterium tuberculosis. Although many PKSs in M. tuberculosis have been implicated in producing complex cell wall glycolipids, the biological function of PKS11 is unknown. PKS11 has previously been proposed to synthesize alkylpyrones from fatty acid substrates. We solved the crystal structure of M. tuberculosis PKS11 and found the overall fold to be similar to other type III PKSs. PKS11 has a deep hydrophobic tunnel proximal to the active site Cys-138 to accommodate substrates. We observed electron density in this tunnel from a co-purified molecule that was identified by mass spectrometry to be palmitate. Co-crystallization with malonyl-CoA (MCoA) or methylmalonyl-CoA (MMCoA) led to partial turnover of the substrate, resulting in trapped intermediates. Reconstitution of the reaction in solution confirmed that both co-factors are required for optimal activity, and kinetic analysis shows that MMCoA is incorporated first, then MCoA, followed by lactonization to produce methyl-branched alkylpyrones.     

Comparative genomic analysis of Mycobacterium tuberculosis clinical isolates

Background

Due to excessive antibiotic use, drug-resistant Mycobacterium tuberculosis has become a serious public health threat and a major obstacle to disease control in many countries. To better understand the evolution of drug-resistant M. tuberculosis strains, we performed whole genome sequencing for 7 M. tuberculosis clinical isolates with different antibiotic resistance profiles and conducted comparative genomic analysis of gene variations among them.

Results

We observed that all 7 M. tuberculosis clinical isolates with different levels of drug resistance harbored similar numbers of SNPs, ranging from 1409–1464. The numbers of insertion/deletions (Indels) identified in the 7 isolates were also similar, ranging from 56 to 101. A total of 39 types of mutations were identified in drug resistance-associated loci, including 14 previously reported ones and 25 newly identified ones. Sixteen of the identified large Indels spanned PE-PPE-PGRS genes, which represents a major source of antigenic variability. Aside from SNPs and Indels, a CRISPR locus with varied spacers was observed in all 7 clinical isolates, suggesting that they might play an important role in plasticity of the M. tuberculosis genome. The nucleotide diversity (Л value) and selection intensity (dN/dS value) of the whole genome sequences of the 7 isolates were similar. The dN/dS values were less than 1 for all 7 isolates (range from 0.608885 to 0.637365), supporting the notion that M. tuberculosis genomes undergo purifying selection. The Л values and dN/dS values were comparable between drug-susceptible and drug-resistant strains.

Conclusions

In this study, we show that clinical M. tuberculosis isolates exhibit distinct variations in terms of the distribution of SNP, Indels, CRISPR-cas locus, as well as the nucleotide diversity and selection intensity, but there are no generalizable differences between drug-susceptible and drug-resistant isolates on the genomic scale. Our study provides evidence strengthening the notion that the evolution of drug resistance among clinical M. tuberculosis isolates is clearly a complex and diversified process.

Electronic supplementary material

The online version of this article (doi: 10.1186/1471-2164-15-469) contains supplementary material, which is available to authorized users.     

Origin of asymmetry in adenylyl cyclases: structures of Mycobacterium tuberculosis Rv1900c

Rv1900c, a Mycobacterium tuberculosis adenylyl cyclase, is composed of an N-terminal alpha/beta-hydrolase domain and a C-terminal cyclase homology domain. It has an unusual 7% guanylyl cyclase side-activity. A canonical substrate-defining lysine and a catalytic asparagine indispensable for mammalian adenylyl cyclase activity correspond to N342 and H402 in Rv1900c. Mutagenic analysis indicates that these residues are dispensable for activity of Rv1900c. Structures of the cyclase homology domain, solved to 2.4 A both with and without an ATP analog, form isologous, but asymmetric homodimers. The noncanonical N342 and H402 do not interact with the substrate. Subunits of the unliganded open dimer move substantially upon binding substrate, forming a closed dimer similar to the mammalian cyclase heterodimers, in which one interfacial active site is occupied and the quasi-dyad-related active site is occluded. This asymmetry indicates that both active sites cannot simultaneously be catalytically active. Such a mechanism of half-of-sites-reactivity suggests that mammalian heterodimeric adenylyl cyclases may have evolved from gene duplication of a primitive prokaryote-type cyclase, followed by loss of function in one active site.     

Molecular model of shikimate kinase from Mycobacterium tuberculosis

Tuberculosis (TB) resurged in the late 1980s and now kills approximately 3 million people a year. The reemergence of tuberculosis as a public health threat has created a need to develop new anti-mycobacterial agents. The shikimate pathway is an attractive target for herbicides and anti-microbial agents development because it is essential in algae, higher plants, bacteria, and fungi, but absent from mammals. Homologs to enzymes in the shikimate pathway have been identified in the genome sequence of Mycobacterium tuberculosis. Among them, the shikimate kinase I encoding gene (aroK) was proposed to be present by sequence homology. Accordingly, to pave the way for structural and functional efforts towards anti-mycobacterial agents development, here we describe the molecular modeling of M. tuberculosis shikimate kinase that should provide a structural framework on which the design of specific inhibitors may be based.     

Probing mechanisms of resistance to the tuberculosis drug isoniazid: Conformational changes caused by inhibition of InhA, the enoyl reductase from Mycobacterium tuberculosis

The frontline tuberculosis drug isoniazid (INH) inhibits InhA, the NADH-dependent fatty acid biosynthesis (FAS-II) enoyl reductase from Mycobacterium tuberculosis (MTB), via formation of a covalent adduct with NAD(+) (the INH-NAD adduct). Resistance to INH can be correlated with many mutations in MTB, some of which are localized in the InhA cofactor binding site. While the InhA mutations cause a substantial decrease in the affinity of InhA for NADH, surprisingly the same mutations result in only a small impact on binding of the INH-NAD adduct. Based on the knowledge that InhA interacts in vivo with other components of the FAS-II pathway, we have initiated experiments to determine whether enzyme inhibition results in structural changes that could affect protein-protein interactions involving InhA and how these ligand-induced conformational changes are modulated in the InhA mutants. Significantly, while NADH binding to wild-type InhA is hyperbolic, the InhA mutants bind the cofactor with positive cooperativity, suggesting that the mutations permit access to a second conformational state of the protein. While cross-linking studies indicate that enzyme inhibition causes dissociation of the InhA tetramer into dimers, analytical ultracentrifugation and size exclusion chromatography reveal that ligand binding causes a conformational change in the protein that prevents cross-linking across one of the dimer-dimer interfaces in the InhA tetramer. Interestingly, a similar ligand-induced conformational change is also observed for the InhA mutants, indicating that the mutations modulate communication between the subunits without affecting the two conformational states of the protein that are present.