%% This BibTeX bibliography file was created using BibDesk. %% http://bibdesk.sourceforge.net/ %% Created for Ralf Stephan at 2010-03-04 18:57:28 +0100 %% Saved with string encoding Unicode (UTF-8) @article{Deb:2006if, Abstract = {Twenty-four putative lipase/esterase genes of Mycobacterium tuberculosis H37Rv were expressed in Escherichia coli and assayed for long-chain triacylglycerol (TG) hydrolase activity. We show here that the product of Rv3097c (LIPY) hydrolyzed long-chain TG with high specific activity. LIPY was purified after solubilization from inclusion bodies; the enzyme displayed a K(m) of 7.57 mM and V(max) of 653.3 nmol/mg/min for triolein with optimal activity between pH 8.0 and pH 9.0. LIPY was inhibited by active serine-directed reagents and was inactivated at temperatures above 37 degrees C. Detergents above their critical micellar concentrations and divalent cations inhibited the activity of LIPY. The N-terminal half of LIPY showed sequence homology with the proline glutamic acid-polymorphic GC-rich repetitive sequences protein family of M. tuberculosis. The C-terminal half of LIPY possesses amino acid domains homologous with the hormone-sensitive lipase family and the conserved active-site motif GDSAG. LIPY shows low sequence identity with the annotated lipases of M. tuberculosis and with other bacterial lipases. We demonstrate that hypoxic cultures of M. tuberculosis, which had accumulated TG, hydrolyzed the stored TG when subjected to nutrient starvation. Under such conditions, lipY was induced more than all lipases, suggesting a central role for it in the utilization of stored TG. We also show that in the lipY-deficient mutant, TG utilization was drastically decreased under nutrient-deprived condition. Thus, LIPY may be responsible for the utilization of stored TG during dormancy and reactivation of the pathogen.}, Author = {Deb, Chirajyoti and Daniel, Jaiyanth and Sirakova, Tatiana D and Abomoelak, Bassam and Dubey, Vinod S and Kolattukudy, Pappachan E}, Date-Added = {2010-03-04 18:47:41 +0100}, Date-Modified = {2010-03-04 18:52:42 +0100}, Doi = {10.1074/jbc.M505556200}, Journal = {J Biol Chem}, Journal-Full = {The Journal of biological chemistry}, Mesh = {Amino Acid Sequence; Cloning, Molecular; Enzyme Induction; Lipase; Molecular Sequence Data; Mycobacterium tuberculosis; Triglycerides}, Month = {Feb}, Number = {7}, Pages = {3866-75}, Pmc = {PMC1523426}, Pmid = {16354661}, Pst = {ppublish}, Title = {A novel lipase belonging to the hormone-sensitive lipase family induced under starvation to utilize stored triacylglycerol in Mycobacterium tuberculosis}, Volume = {281}, Year = {2006}, Bdsk-Url-1 = {http://dx.doi.org/10.1074/jbc.M505556200}} @article{Camacho:1999ud, Abstract = {Tuberculosis remains the greatest cause of death worldwide due to a single pathogen. In order to identify the genes required for the pathogenicity of Mycobacterium tuberculosis, a functional genomic approach was developed. A library of signature-tagged transposon mutants of this bacterium was constructed and screened for those affected in their multiplication within the lungs of mice. From 1927 mutants tested, 16 were attenuated for their virulence. The insertions harboured by the selected mutants were mapped on the M. tuberculosis genome and most of the mutated loci appeared to be involved in lipid metabolism or transport across the membrane. Four independent mutations identified a cluster of virulence genes located on a 50 kb chromosomal region. These genes might be involved in the production of phthiocerol and phenolphthiocerol derivatives, a group of molecules restricted to eight mycobacterial species, seven of them being either strict or opportunistic pathogens. The interaction of five mutant strains with mouse bone marrow macrophages was investigated. These five mutants were still able to multiply in this cell type. However, in three cases, there was a growth defect in comparison with the wild-type strain. The other two strains exhibited no clear difference from the virulent strain, MT103, in this model. This study, which is the first global research of virulence factors of M. tuberculosis, opens the way to a better understanding of the molecules that are key players in the interaction of this pathogen with its host.}, Author = {Camacho, L R and Ensergueix, D and Perez, E and Gicquel, B and Guilhot, C}, Date-Added = {2010-03-04 18:35:58 +0100}, Date-Modified = {2010-03-04 18:35:58 +0100}, Journal = {Mol Microbiol}, Journal-Full = {Molecular microbiology}, Mesh = {Animals; Bone Marrow Cells; DNA Transposable Elements; Gene Library; Genes, Bacterial; Lung; Macrophages; Mice; Mice, Inbred BALB C; Multigene Family; Mutagenesis, Insertional; Mycobacterium tuberculosis; Plasmids; Tuberculosis; Virulence}, Month = {Oct}, Number = {2}, Pages = {257-67}, Pmid = {10564470}, Pst = {ppublish}, Title = {Identification of a virulence gene cluster of Mycobacterium tuberculosis by signature-tagged transposon mutagenesis}, Volume = {34}, Year = {1999}} @article{Danilchanka:2008bd, Abstract = {The impermeability of the outer membrane in combination with drug efflux are major determinants of the natural drug resistance of mycobacteria. beta-Lactams are the most widely used antibiotics for treatment of bacterial infections. However, it is unknown how beta-lactams enter Mycobacterium tuberculosis and whether efflux pumps exist that can export these drugs out of the cell. To identify the molecular mechanisms of M. tuberculosis resistance to beta-lactams, a library of 7,500 transposon mutants was generated in the model organism Mycobacterium bovis BCG. Thirty-three unique insertion sites were determined that conferred medium or high-level (> or =2,000 microg/ml) resistance to ampicillin. Three mutants in sulfolipid synthesis or transport were highly resistant to ampicillin, indicating an indirect effect of the lipid composition on the outer membrane permeability of M. bovis BCG to ampicillin. Mutants with insertions in genes encoding surface molecules such as PPE proteins or lipoarabinomannan were also completely resistant to ampicillin, thus suggesting a lack of transport across the outer membrane. Insertion of the transposon in front of bcg0231 increased transcription of the gene and concomitantly the resistance of M. bovis BCG to ampicillin, streptomycin, and chloramphenicol by 32- to 64-fold. Resistance to vancomycin and tetracycline was increased four- to eightfold. Bcg0231 and Rv0194 are almost identical ATP-binding cassette transporters. Expression of rv0194 significantly reduced accumulation of ethidium bromide and conferred multidrug resistance to Mycobacterium smegmatis. Both effects were abrogated in the presence of the efflux pump inhibitor reserpine. These results demonstrate that Rv0194 is a novel multidrug efflux pump of M. tuberculosis.}, Author = {Danilchanka, Olga and Mailaender, Claudia and Niederweis, Michael}, Date-Added = {2010-03-04 18:31:05 +0100}, Date-Modified = {2010-03-04 18:31:50 +0100}, Doi = {10.1128/AAC.00298-08}, Journal = {Antimicrob Agents Chemother}, Journal-Full = {Antimicrobial agents and chemotherapy}, Keywords = {single protein, Rv0194}, Mesh = {ATP-Binding Cassette Transporters; Ampicillin Resistance; Bacterial Proteins; Base Sequence; DNA, Bacterial; Drug Resistance, Multiple, Bacterial; Ethidium; Genes, Bacterial; Humans; Membrane Transport Proteins; Models, Biological; Molecular Sequence Data; Mutagenesis, Insertional; Mycobacterium bovis; Mycobacterium smegmatis; Mycobacterium tuberculosis; Transcription, Genetic; Tuberculosis, Multidrug-Resistant; beta-Lactam Resistance; beta-Lactamases; beta-Lactams}, Month = {Jul}, Number = {7}, Pages = {2503-11}, Pmc = {PMC2443884}, Pmid = {18458127}, Pst = {ppublish}, Title = {Identification of a novel multidrug efflux pump of Mycobacterium tuberculosis}, Volume = {52}, Year = {2008}, Bdsk-Url-1 = {http://dx.doi.org/10.1128/AAC.00298-08}} @article{Chubb:1998mw, Abstract = {Proteins secreted by Mycobacterium tuberculosis may play a key role in virulence and may also constitute antigens that elicit the host immune response. However, the M. tuberculosis protein export machinery has not been characterized. A library of M. tuberculosis H37Rv genomic DNA fragments ligated into a signal sequence selection vector that contained a leaderless beta-lactamase gene and an upstream Tac promoter was constructed. Transformation of Escherichia coli with the M. tuberculosis DNA library and selection on plates containing 50-100 micrograms ampicillin ml-1 resulted in the identification of 15 Ampr clones out of a total of 14,000 transformants. Twelve of the beta-lactamase gene fusions conferred high levels of Ampr (up to 1 mg ampicillin ml-1); insert sizes ranged from 350 to 3000 bp. Of ten inserts that were completely sequenced, two were identified as fragments of the genes for M. tuberculosis antigens 85A and 85C, which are the major secreted proteins of this pathogen. Seven of the remaining inserts were > or = 97\% identical to hypothetical ORFs in the M. tuberculosis genome, one of which encoded a protein with 35\% identity to a low-affinity penicillin-binding protein (PBP) from Streptomyces clavuligerus. Four of the seven hypothetical ORFs encoded putative exported proteins with one or more membrane interaction elements, including lipoprotein attachment sites and type I and II transmembrane (TM) segments. All of the inserts encoded typical signal sequences, with the exception of a possible type II membrane protein. It is concluded that expression of beta-lactamase gene fusions in E. coli provides a useful system for the identification and analysis of M. tuberculosis signal-sequence-encoding genes.}, Author = {Chubb, A J and Woodman, Z L and da Silva Tatley, F M and Hoffmann, H J and Scholle, R R and Ehlers, M R}, Date-Added = {2010-03-04 18:27:03 +0100}, Date-Modified = {2010-03-04 18:27:03 +0100}, Journal = {Microbiology}, Journal-Full = {Microbiology (Reading, England)}, Mesh = {Amino Acid Sequence; Bacterial Proteins; Electrophoresis, Polyacrylamide Gel; Escherichia coli; Genes, Bacterial; Genetic Vectors; Immunoblotting; Molecular Sequence Data; Mycobacterium tuberculosis; Polymerase Chain Reaction; Protein Sorting Signals; Sequence Alignment; Transformation, Bacterial; beta-Lactamases}, Month = {Jun}, Pages = {1619-29}, Pmid = {9639933}, Pst = {ppublish}, Title = {Identification of Mycobacterium tuberculosis signal sequences that direct the export of a leaderless beta-lactamase gene product in Escherichia coli}, Volume = {144 ( Pt 6)}, Year = {1998}} @article{Gold:2008qc, Abstract = {A screen of a genomic library from Mycobacterium tuberculosis (Mtb) identified a small, unannotated open reading frame (MT0196) that encodes a 4.9-kDa, cysteine-rich protein. Despite extensive nucleotide divergence, the amino acid sequence is highly conserved among mycobacteria that are pathogenic in vertebrate hosts. We synthesized the protein and found that it preferentially binds up to six Cu(I) ions in a solvent-shielded core. Copper, cadmium and compounds that generate nitric oxide or superoxide induced the gene's expression in Mtb up to 1,000-fold above normal expression. The native protein bound copper within Mtb and partially protected Mtb from copper toxicity. We propose that the product of the MT0196 gene be named mycobacterial metallothionein (MymT). To our knowledge, MymT is the first metallothionein of a Gram-positive bacterium with a demonstrated function.}, Author = {Gold, Ben and Deng, Haiteng and Bryk, Ruslana and Vargas, Diana and Eliezer, David and Roberts, Julia and Jiang, Xiuju and Nathan, Carl}, Date-Added = {2010-03-04 18:19:40 +0100}, Date-Modified = {2010-03-04 18:20:33 +0100}, Doi = {10.1038/nchembio.109}, Journal = {Nat Chem Biol}, Journal-Full = {Nature chemical biology}, Keywords = {single protein, Rv0186A}, Mesh = {Amino Acid Sequence; Binding Sites; Cloning, Molecular; Copper; Gene Expression Regulation, Bacterial; Metallothionein; Molecular Sequence Data; Mycobacterium tuberculosis; Sequence Alignment}, Month = {Oct}, Number = {10}, Pages = {609-16}, Pmc = {PMC2749609}, Pmid = {18724363}, Pst = {ppublish}, Title = {Identification of a copper-binding metallothionein in pathogenic mycobacteria}, Volume = {4}, Year = {2008}, Bdsk-Url-1 = {http://dx.doi.org/10.1038/nchembio.109}} @article{Manganelli:1999ss, Abstract = {The ability of Mycobacterium tuberculosis to adapt to different environments in the infected host is essential for its pathogenicity. Consequently, this organism must be able to modulate gene expression to respond to the changing conditions it encounters during infection. In this paper we begin a comprehensive study of M. tuberculosis gene regulation, characterizing the transcript levels of 10 of its 13 putative sigma factor genes. We developed a real-time RT-PCR assay using a family of novel fluorescent probes called molecular beacons to quantitatively measure the different mRNAs. Three sigma factor genes were identified that have increased mRNA levels after heat shock, two of which also responded to detergent stress. In addition, we also identified a sigma factor gene whose mRNA increased after mild cold shock and a second that responded to conditions of low aeration.}, Author = {Manganelli, R and Dubnau, E and Tyagi, S and Kramer, F R and Smith, I}, Date-Added = {2010-03-04 18:08:25 +0100}, Date-Modified = {2010-03-04 18:08:25 +0100}, Journal = {Mol Microbiol}, Journal-Full = {Molecular microbiology}, Mesh = {Bacterial Proteins; Culture Media; DNA-Directed RNA Polymerases; Gene Expression Regulation, Bacterial; Mycobacterium tuberculosis; RNA, Messenger; Reverse Transcriptase Polymerase Chain Reaction; Sigma Factor; Time Factors}, Month = {Jan}, Number = {2}, Pages = {715-24}, Pmid = {10027986}, Pst = {ppublish}, Title = {Differential expression of 10 sigma factor genes in Mycobacterium tuberculosis}, Volume = {31}, Year = {1999}} @article{Uchida:2007fc, Abstract = {Mycobacterium tuberculosis causes a variety of clinical outcomes determined by host as well as bacterial factors. M. tuberculosis disrupted in the mce1 operon causes increased mortality in immunocompetent mice. This operon is negatively regulated by mce1R (Rv0165c). We studied the role of mce1R in infection outcome in mice. At 5 x 10(4) tail vein infectious dose, the median survival time (MST) of mice infected with the mce1R mutant M. tuberculosis H37Rv was 293 days, while mice infected with the wild-type H37Rv survived more than 350 days (P < 0.0001). At a higher dose (5 x 10(6)), the MST of mutant-infected mice was 32 days, compared with 127 days for wild type-infected mice (P < 0.0001). With either tail vein or aerosol infection, mutant-infected mice developed larger granulomatous lesions in their lungs than mice infected with the wild type. Mutant-infected mice were unable to control the bacterial burden in the first 4 weeks of infection, but even after achieving control later, these mice succumbed to granulomatous pneumonia. These observations suggest that the early deregulated expression of the mce1 operon products determines later granulomatous tissue response. mce1 operon may homeostatically regulate the cell wall architecture in vivo that elicits a steady-state granuloma tissue response permitting M. tuberculosis to establish a long-term infection.}, Author = {Uchida, Yujiro and Casali, Nicola and White, Amy and Morici, Lisa and Kendall, Lon V and Riley, Lee W}, Date-Added = {2010-03-04 17:53:03 +0100}, Date-Modified = {2010-03-04 17:54:03 +0100}, Doi = {10.1111/j.1462-5822.2006.00870.x}, Journal = {Cell Microbiol}, Journal-Full = {Cellular microbiology}, Keywords = {single protein, Rv0165c}, Mesh = {Animals; Bacterial Proteins; Cytokines; Enzyme-Linked Immunosorbent Assay; Female; Granuloma; Immunohistochemistry; Lung; Mice; Mice, Inbred BALB C; Mutation; Mycobacterium tuberculosis; Operon; Tail; Time Factors; Tuberculosis; Veins}, Month = {May}, Number = {5}, Pages = {1275-83}, Pmid = {17223927}, Pst = {ppublish}, Title = {Accelerated immunopathological response of mice infected with Mycobacterium tuberculosis disrupted in the mce1 operon negative transcriptional regulator}, Volume = {9}, Year = {2007}, Bdsk-Url-1 = {http://dx.doi.org/10.1111/j.1462-5822.2006.00870.x}} @article{Beresford:2007rq, Abstract = {Bacterial pathogens have developed sophisticated mechanisms of evading the immune system to survive in infected host cells. Central to the pathogenesis of Mycobacterium tuberculosis is the arrest of phagosome maturation, partly through interference with PtdIns signalling. The protein phosphatase MptpB is an essential secreted virulence factor in M. tuberculosis. A combination of bioinformatics analysis, enzyme kinetics and substrate-specificity characterization revealed that MptpB exhibits both dual-specificity protein phosphatase activity and, importantly, phosphoinositide phosphatase activity. Mutagenesis of conserved residues in the active site signature indicates a cysteine-based mechanism of dephosphorylation and identifies two new catalytic residues, Asp165, essential in catalysis, and Lys164, apparently involved in substrate specificity. Sequence similarities with mammalian lipid phosphatases and a preference for phosphoinositide substrates suggests a potential novel role of MptpB in PtdIns metabolism in the host and reveals new perspectives for the role of this phosphatase in mycobacteria pathogenicity.}, Author = {Beresford, Nicola and Patel, Sumayya and Armstrong, Jane and Sz{\"o}or, Bal{\'a}zs and Fordham-Skelton, Anthony P and Tabernero, Lydia}, Date-Added = {2010-03-04 17:46:10 +0100}, Date-Modified = {2010-03-04 17:46:44 +0100}, Doi = {10.1042/BJ20070670}, Journal = {Biochem J}, Journal-Full = {The Biochemical journal}, Keywords = {single protein, Rv0153c}, Mesh = {Amino Acid Sequence; Bacterial Proteins; Binding Sites; Fungi; Kinetics; Lipid Metabolism; Molecular Sequence Data; Mutagenesis; Mycobacterium tuberculosis; Phosphatidylinositols; Phosphorylation; Protein Structure, Secondary; Protein Tyrosine Phosphatases; Substrate Specificity; Virulence Factors}, Month = {Aug}, Number = {1}, Pages = {13-8}, Pmc = {PMC1948985}, Pmid = {17584180}, Pst = {ppublish}, Title = {MptpB, a virulence factor from Mycobacterium tuberculosis, exhibits triple-specificity phosphatase activity}, Volume = {406}, Year = {2007}, Bdsk-Url-1 = {http://dx.doi.org/10.1042/BJ20070670}} @article{Singh:2003kh, Abstract = {Protein tyrosine kinases and tyrosine phosphatases from several bacterial pathogens have been shown to act as virulence factors by modulating the phosphorylation and dephosphorylation of host proteins. The identification and characterization of two tyrosine phosphatases namely MptpA and MptpB from Mycobacterium tuberculosis has been reported earlier. MptpB is secreted by M. tuberculosis into extracellular mileu and exhibits a pH optimum of 5.6, similar to the pH of the lysosomal compartment of the cell. To determine the role of MptpB in the pathogenesis of M. tuberculosis, we constructed a mptpB mutant strain by homologous recombination and compared the ability of parent and the mutant strain to survive intracellularly. We show that disruption of the mptpB gene impairs the ability of the mutant strain to survive in activated macrophages and guinea pigs but not in resting macrophages suggesting the importance of its role in the host-pathogen interaction. Infection of guinea pigs with the mutant strain resulted in a 70-fold reduction in the bacillary load of spleens in infected animals as compared with the bacillary load in animals infected with the parental strain. Upon reintroduction of the mptpB gene into the mutant strain, the complemented strain was able to establish infection and survive in guinea pigs at rates comparable to the parental strain. These observations demonstrate a role of MptpB in the pathogenesis of M. tuberculosis.}, Author = {Singh, Ramandeep and Rao, Vivek and Shakila, H and Gupta, Radhika and Khera, Aparna and Dhar, Neeraj and Singh, Amit and Koul, Anil and Singh, Yogendra and Naseema, M and Narayanan, P R and Paramasivan, C N and Ramanathan, V D and Tyagi, Anil K}, Date-Added = {2010-03-04 17:42:17 +0100}, Date-Modified = {2010-03-04 17:43:07 +0100}, Journal = {Mol Microbiol}, Journal-Full = {Molecular microbiology}, Keywords = {single protein, Rv0153c}, Mesh = {Animals; Bacterial Proteins; Cells, Cultured; Genetic Complementation Test; Guinea Pigs; Host-Parasite Interactions; Macrophages; Mice; Mutation; Mycobacterium tuberculosis; Protein Tyrosine Phosphatases; Recombinant Fusion Proteins; Recombination, Genetic; Spleen; Tuberculosis; Virulence}, Month = {Nov}, Number = {3}, Pages = {751-62}, Pmid = {14617138}, Pst = {ppublish}, Title = {Disruption of mptpB impairs the ability of Mycobacterium tuberculosis to survive in guinea pigs}, Volume = {50}, Year = {2003}} @article{Koul:2000cq, Abstract = {Two genes with sequence homology to those encoding protein tyrosine phosphatases were cloned from genomic DNA of Mycobacterium tuberculosis H(37)Rv. The calculated molecular masses of these two putative tyrosine phosphatases, designated MPtpA and MPtpB, were 17. 5 and 30 kDa, respectively. MPtpA and MPtpB were expressed as glutathione S-transferase fusion proteins in Escherichia coli. The affinity-purified proteins dephosphorylated the phosphotyrosine residue of myelin basic protein (MBP), but they failed to dephosphorylate serine/threonine residues of MBP. The activity of these phosphatases was inhibited by sodium orthovanadate, a specific inhibitor of tyrosine phosphatases, but not by okadaic acid, an inhibitor of serine/threonine phosphatases. Mutations at the catalytic site motif, cysteine 11 of MPtpA and cysteine 160 of MPtpB, abolished enzyme activity. Southern blot analysis revealed that, while mptpA is present in slow-growing mycobacterial species as well as fast-growing saprophytes, mptpB was restricted to members of the M. tuberculosis complex. These phosphatases were present in both whole-cell lysates and culture filtrates of M. tuberculosis, suggesting that these proteins are secreted into the extracellular medium. Since tyrosine phosphatases are essential for the virulence of several pathogenic bacteria, the restricted distribution of mptpB makes it a good candidate for a virulence gene of M. tuberculosis.}, Author = {Koul, A and Choidas, A and Treder, M and Tyagi, A K and Drlica, K and Singh, Y and Ullrich, A}, Date-Added = {2010-03-04 17:38:25 +0100}, Date-Modified = {2010-03-04 17:38:25 +0100}, Journal = {J Bacteriol}, Journal-Full = {Journal of bacteriology}, Mesh = {Amino Acid Sequence; Bacterial Proteins; Blotting, Western; Catalytic Domain; Cloning, Molecular; Gene Expression; Genes, Bacterial; Molecular Sequence Data; Mycobacterium smegmatis; Mycobacterium tuberculosis; Phosphotyrosine; Protein Tyrosine Phosphatases; Recombinant Fusion Proteins; Sequence Homology, Amino Acid}, Month = {Oct}, Number = {19}, Pages = {5425-32}, Pmc = {PMC110985}, Pmid = {10986245}, Pst = {ppublish}, Title = {Cloning and characterization of secretory tyrosine phosphatases of Mycobacterium tuberculosis}, Volume = {182}, Year = {2000}} @article{Kendall:2007gb, Abstract = {The Mycobacterium tuberculosis TetR-type regulator Rv3574 has been implicated in pathogenesis as it is induced in vivo, and genome-wide essentiality studies show it is required for infection. As the gene is highly conserved in the mycobacteria, we deleted the Rv3574 orthologue in Mycobacterium smegmatis (MSMEG_6042) and used real-time quantitative polymerase chain reaction and microarray analyses to show that it represses the transcription both of itself and of a large number of genes involved in lipid metabolism. We identified a conserved motif within its own promoter (TnnAACnnGTTnnA) and showed that it binds as a dimer to 29 bp probes containing the motif. We found 16 and 31 other instances of the motif in intergenic regions of M. tuberculosis and M. smegmatis respectively. Combining the results of the microarray studies with the motif analyses, we predict that Rv3574 directly controls the expression of 83 genes in M. smegmatis, and 74 in M. tuberculosis. Many of these genes are known to be induced by growth on cholesterol in rhodococci, and palmitate in M. tuberculosis. We conclude that this regulator, designated elsewhere as kstR, controls the expression of genes used for utilizing diverse lipids as energy sources, possibly imported through the mce4 system.}, Author = {Kendall, Sharon L and Withers, Mike and Soffair, Catherine N and Moreland, Nicole J and Gurcha, Sudagar and Sidders, Ben and Frita, Rosangela and Ten Bokum, Annemieke and Besra, Gurdyal S and Lott, J Shaun and Stoker, Neil G}, Date-Added = {2010-03-03 19:26:45 +0100}, Date-Modified = {2010-03-03 19:26:45 +0100}, Doi = {10.1111/j.1365-2958.2007.05827.x}, Journal = {Mol Microbiol}, Journal-Full = {Molecular microbiology}, Mesh = {Amino Acid Motifs; Bacterial Proteins; Base Sequence; Conserved Sequence; DNA, Intergenic; Dimerization; Gene Expression Regulation, Bacterial; Genes, Bacterial; Lipid Metabolism; Molecular Sequence Data; Mycobacterium smegmatis; Mycobacterium tuberculosis; Oligonucleotide Array Sequence Analysis; Promoter Regions, Genetic; Protein Binding; Regulon; Repressor Proteins; Sequence Deletion; Transcription, Genetic; Up-Regulation}, Month = {Aug}, Number = {3}, Pages = {684-99}, Pmc = {PMC1995591}, Pmid = {17635188}, Pst = {ppublish}, Title = {A highly conserved transcriptional repressor controls a large regulon involved in lipid degradation in Mycobacterium smegmatis and Mycobacterium tuberculosis}, Volume = {65}, Year = {2007}, Bdsk-Url-1 = {http://dx.doi.org/10.1111/j.1365-2958.2007.05827.x}} @article{Lee:2009jl, Abstract = {Methionine sulphoxide reductases (Msr) reduce methionine sulphoxide to methionine and protect bacteria against reactive oxygen intermediates (ROI) and reactive nitrogen intermediates (RNI). Many organisms express both MsrA, active against methionine-(S)-sulphoxide, and MsrB, active against methionine-(R)-sulphoxide. Mycobacterium tuberculosis (Mtb) expresses MsrA, which protects DeltamsrA-Escherichia coli from ROI and RNI. However, the function of MsrA in Mtb has not been defined, and it is unknown whether Mtb expresses MsrB. We identified MsrB as the protein encoded by Rv2674 in Mtb and confirmed the distinct stereospecificities of recombinant Mtb MsrA and MsrB. We generated strains of Mtb deficient in MsrA, MsrB or both and complemented the mutants. Lysates of singly deficient strains displayed half as much Msr activity as wild type against N-acetyl methionine sulphoxide. However, in contrast to other bacteria, single mutants were no more vulnerable than wild type to killing by ROI/RNI. Only Mtb lacking both MsrA and MsrB was more readily killed by nitrite or hypochlorite. Thus, MsrA and MsrB contribute to the enzymatic defences of Mtb against ROI and RNI.}, Author = {Lee, Warren L and Gold, Benjamin and Darby, Crystal and Brot, Nathan and Jiang, Xiuju and de Carvalho, Luiz Pedro S and Wellner, Daniel and St John, Gregory and Jacobs, Jr, William R and Nathan, Carl}, Date-Added = {2010-03-03 19:21:15 +0100}, Date-Modified = {2010-03-03 19:21:15 +0100}, Doi = {10.1111/j.1365-2958.2008.06548.x}, Journal = {Mol Microbiol}, Journal-Full = {Molecular microbiology}, Mesh = {Amino Acid Sequence; Animals; Bacterial Proteins; Gene Expression Regulation, Bacterial; Genes, Bacterial; Genetic Complementation Test; Hypochlorous Acid; Mice; Mice, Inbred C57BL; Molecular Sequence Data; Mutagenesis, Insertional; Mycobacterium tuberculosis; Nitrites; Oxidoreductases; Reactive Nitrogen Species; Reactive Oxygen Species; Recombinant Proteins; Sequence Alignment; Substrate Specificity}, Month = {Feb}, Number = {3}, Pages = {583-93}, Pmid = {19040639}, Pst = {ppublish}, Title = {Mycobacterium tuberculosis expresses methionine sulphoxide reductases A and B that protect from killing by nitrite and hypochlorite}, Volume = {71}, Year = {2009}, Bdsk-Url-1 = {http://dx.doi.org/10.1111/j.1365-2958.2008.06548.x}} @article{St-John:2001zt, Abstract = {Inducible nitric oxide synthase (iNOS) plays an important role in host defense. Macrophages expressing iNOS release the reactive nitrogen intermediates (RNI) nitrite and S-nitrosoglutathione (GSNO), which are bactericidal in vitro at a pH characteristic of the phagosome of activated macrophages. We sought to characterize the active intrabacterial forms of these RNI and their molecular targets. Peptide methionine sulfoxide reductase (MsrA; EC ) catalyzes the reduction of methionine sulfoxide (Met-O) in proteins to methionine (Met). E. coli lacking MsrA were hypersensitive to killing not only by hydrogen peroxide, but also by nitrite and GSNO. The wild-type phenotype was restored by transformation with plasmids encoding msrA from E. coli or M. tuberculosis, but not by an enzymatically inactive mutant msrA, indicating that Met oxidation was involved in the death of these cells. It seemed paradoxical that nitrite and GSNO kill bacteria by oxidizing Met residues when these RNI cannot themselves oxidize Met. However, under anaerobic conditions, neither nitrite nor GSNO was bactericidal. Nitrite and GSNO can both give rise to NO, which may react with superoxide produced by bacteria during aerobic metabolism, forming peroxynitrite, a known oxidant of Met to Met-O. Thus, the findings are consistent with the hypotheses that nitrite and GSNO kill E. coli by intracellular conversion to peroxynitrite, that intracellular Met residues in proteins constitute a critical target for peroxynitrite, and that MsrA can be essential for the repair of peroxynitrite-mediated intracellular damage.}, Author = {St John, G and Brot, N and Ruan, J and Erdjument-Bromage, H and Tempst, P and Weissbach, H and Nathan, C}, Date-Added = {2010-03-03 19:15:27 +0100}, Date-Modified = {2010-03-03 19:16:07 +0100}, Doi = {10.1073/pnas.161295398}, Journal = {Proc Natl Acad Sci U S A}, Journal-Full = {Proceedings of the National Academy of Sciences of the United States of America}, Keywords = {single protein, Rv0137c}, Mesh = {Bacterial Proteins; Escherichia coli; Genetic Complementation Test; Glutathione; Macrophages; Methionine; Mycobacterium tuberculosis; Nitrates; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Nitrites; Nitroso Compounds; Oxidative Stress; Oxidoreductases; Phenotype; Recombinant Fusion Proteins; S-Nitrosoglutathione}, Month = {Aug}, Number = {17}, Pages = {9901-6}, Pmc = {PMC55550}, Pmid = {11481433}, Pst = {ppublish}, Title = {Peptide methionine sulfoxide reductase from Escherichia coli and Mycobacterium tuberculosis protects bacteria against oxidative damage from reactive nitrogen intermediates}, Volume = {98}, Year = {2001}, Bdsk-Url-1 = {http://dx.doi.org/10.1073/pnas.161295398}} @article{Gurvitz:2008il, Abstract = {We report on the identification of Mycobacterium tuberculosis HtdZ (Rv0130), representing a novel 3-hydroxyacyl-thioester dehydratase. HtdZ was picked up by the functional complementation of Saccharomyces cerevisiae htd2Delta cells lacking the dehydratase of mitochondrial type II fatty acid synthase. Mutant cells expressing HtdZ contained dehydratase activity, recovered their respiratory ability, and partially restored de novo lipoic acid synthesis.}, Author = {Gurvitz, Aner and Hiltunen, J Kalervo and Kastaniotis, Alexander J}, Date-Added = {2010-03-03 19:08:22 +0100}, Date-Modified = {2010-03-03 19:08:58 +0100}, Doi = {10.1128/JB.00016-08}, Journal = {J Bacteriol}, Journal-Full = {Journal of bacteriology}, Keywords = {single protein, Rv0130}, Mesh = {Bacterial Proteins; Enoyl-CoA Hydratase; Genetic Complementation Test; Mutation; Mycobacterium tuberculosis; Organisms, Genetically Modified; Oxygen Consumption; Saccharomyces cerevisiae}, Month = {Jun}, Number = {11}, Pages = {4088-90}, Pmc = {PMC2395046}, Pmid = {18375556}, Pst = {ppublish}, Title = {Identification of a novel mycobacterial 3-hydroxyacyl-thioester dehydratase, HtdZ (Rv0130), by functional complementation in yeast}, Volume = {190}, Year = {2008}, Bdsk-Url-1 = {http://dx.doi.org/10.1128/JB.00016-08}} @article{Johansson:2006qo, Abstract = {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 {\aa}. The Rv0130 monomer features a single hotdog fold composed of a highly curved beta-sheet on top of a long and a short alpha-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 x 10(2) 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.}, Author = {Johansson, Patrik and Castell, Alina and Jones, T Alwyn and B{\"a}ckbro, Kristina}, Date-Added = {2010-03-03 19:02:13 +0100}, Date-Modified = {2010-03-03 19:03:14 +0100}, Doi = {10.1110/ps.062309306}, Journal = {Protein Sci}, Journal-Full = {Protein science : a publication of the Protein Society}, Keywords = {single protein, Rv0130}, Mesh = {Bacterial Proteins; Conserved Sequence; Crystallography, X-Ray; Hydro-Lyases; Kinetics; Molecular Structure; Mutagenesis, Site-Directed; Mycobacterium tuberculosis; Protein Conformation}, Month = {Oct}, Number = {10}, Pages = {2300-9}, Pmc = {PMC2242379}, Pmid = {16963641}, Pst = {ppublish}, Title = {Structure and function of Rv0130, a conserved hypothetical protein from Mycobacterium tuberculosis}, Volume = {15}, Year = {2006}, Bdsk-Url-1 = {http://dx.doi.org/10.1110/ps.062309306}} @article{Puech:2002ye, Abstract = {Mycobacterium tuberculosis produces a series of major secreted proteins, the fibronectin-binding proteins (Fbps), also known as the antigen 85 complex, that are believed to play an essential role in the pathogenesis of tuberculosis through their mycoloyltransferase activity required for maintaining the integrity of the bacterial cell envelope. Four different fbp genes are found in the genome of M. tuberculosis, but the reason for the existence of these Fbps sharing the same substrate specificity in vitro in mycobacteria is unknown. We have shown previously that, in the heterologous host, Corynebacterium glutamicum, FbpA, FbpB and FbpC can all add mycoloyl residues to the cell wall arabinogalactan and that, in M. tuberculosis, the cell wall mycoloylation decreases by 40\% when fbpC is knocked out. To investigate whether the remaining 60\% mycoloylation came from the activity of FbpA and/or FbpB, fbpA- and fbpB-inactivated mutant strains were biochemically characterized and compared with the previously studied fbpC-disrupted mutant. Unexpectedly, both mutants produced normally mycoloylated cell walls. Overproduction of FbpA, FbpB or FbpC, but not FbpD, in the fbpC-inactivated mutant strain of M. tuberculosis restored both the cell wall-linked mycolate defect and the outer cell envelope permeability barrier property. These results are consistent with all three enzymes being involved in cell wall mycoloylation and FbpC playing a more critical role than the others or, alternatively, FbpC is able to compensate for FbpA and FbpB in ways that these enzymes cannot compensate for FbpC, pointing to a partial redundancy of Fbps. In sharp contrast, FbpD does not appear to be an active mycoloyltransferase enzyme, as it cannot complement the fbpC-inactivated mutant. Most importantly, application of Smith degradation to the cell walls of transformants demonstrated that the multiple Fbp enzymes are redundant rather than specific for the various arabinogalactan mycoloylation regions. Neither FbpA nor FbpB attaches mycoloyl residues to specific sites but, like FbpC, each enzyme transfers mycoloyl residues onto the four sites present in the arabinogalactan non-reducing end hexaarabinosides.}, Author = {Puech, Virginie and Guilhot, Christophe and Perez, Esther and Tropis, Marielle and Armitige, Lisa Y and Gicquel, Brigitte and Daff{\'e}, Mamadou}, Date-Added = {2010-03-03 18:23:25 +0100}, Date-Modified = {2010-03-03 18:23:25 +0100}, Journal = {Mol Microbiol}, Journal-Full = {Molecular microbiology}, Mesh = {Adhesins, Bacterial; Amino Acid Sequence; Bacterial Proteins; Biological Transport; Carrier Proteins; Cell Wall; Chromatography, Thin Layer; Corynebacterium; Galactans; Glycerol; Membrane Transport Proteins; Molecular Sequence Data; Mutation; Mycobacterium tuberculosis; Mycolic Acids; Sequence Alignment; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization}, Month = {May}, Number = {4}, Pages = {1109-22}, Pmid = {12010501}, Pst = {ppublish}, Title = {Evidence for a partial redundancy of the fibronectin-binding proteins for the transfer of mycoloyl residues onto the cell wall arabinogalactan termini of Mycobacterium tuberculosis}, Volume = {44}, Year = {2002}} @article{Kremer:2002fu, Abstract = {AIMS: The antigen 85 complex (Ag85) from Mycobacterium tuberculosis consists of three abundantly secreted proteins (FbpA, FbpB and FbpC2) which play a key role in the pathogenesis of tuberculosis and also exhibit cell wall mycolyltransferase activity. A related protein with similarity to the Ag85 complex was recently annotated in the M. tuberculosis genome as FbpC1. An investigation was carried out to determine whether FbpC1 may also possess mycolyltransferase activity, a characteristic feature of the Ag85 complex. METHODS AND RESULTS: Heterologous expression of FbpA, FbpC1 and FbpC2 was performed in Escherichia coli. Recombinant proteins were purified under non-denaturating conditions and used in an in vitro mycolyltransferase assay. CONCLUSIONS: In contrast to FbpA and FbpC2, recombinant FbpC1 did not possess in vitro mycolyltransferase activity and was not recognized by two monoclonal antibodies to the native Ag85. SIGNIFICANCE AND IMPACT OF THE STUDY: Mycolyltransferase activity is restricted to FbpA, FbpbB and FbpC2 only; the actual function of FbpC1 remains to be established.}, Author = {Kremer, L and Maughan, W N and Wilson, R A and Dover, L G and Besra, G S}, Date-Added = {2010-03-03 18:20:09 +0100}, Date-Modified = {2010-03-03 18:20:09 +0100}, Journal = {Lett Appl Microbiol}, Journal-Full = {Letters in applied microbiology}, Mesh = {Acyltransferases; Amino Acid Sequence; Antigens, Bacterial; Bacterial Proteins; Molecular Sequence Data; Multienzyme Complexes; Mycobacterium tuberculosis; Recombinant Proteins; Sequence Alignment; Sequence Analysis, Protein}, Number = {4}, Pages = {233-7}, Pmid = {11940150}, Pst = {ppublish}, Title = {The M. tuberculosis antigen 85 complex and mycolyltransferase activity}, Volume = {34}, Year = {2002}} @article{Wilson:1999lh, Abstract = {Tuberculosis is a chronic infectious disease that is transmitted by cough-propelled droplets that carry the etiologic bacterium, Mycobacterium tuberculosis. Although currently available drugs kill most isolates of M. tuberculosis, strains resistant to each of these have emerged, and multiply resistant strains are increasingly widespread. The growing problem of drug resistance combined with a global incidence of seven million new cases per year underscore the urgent need for new antituberculosis therapies. The recent publication of the complete sequence of the M. tuberculosis genome has made possible, for the first time, a comprehensive genomic approach to the biology of this organism and to the drug discovery process. We used a DNA microarray containing 97\% of the ORFs predicted from this sequence to monitor changes in M. tuberculosis gene expression in response to the antituberculous drug isoniazid. Here we show that isoniazid induced several genes that encode proteins physiologically relevant to the drug's mode of action, including an operonic cluster of five genes encoding type II fatty acid synthase enzymes and fbpC, which encodes trehalose dimycolyl transferase. Other genes, not apparently within directly affected biosynthetic pathways, also were induced. These genes, efpA, fadE23, fadE24, and ahpC, likely mediate processes that are linked to the toxic consequences of the drug. Insights gained from this approach may define new drug targets and suggest new methods for identifying compounds that inhibit those targets.}, Author = {Wilson, M and DeRisi, J and Kristensen, H H and Imboden, P and Rane, S and Brown, P O and Schoolnik, G K}, Date-Added = {2010-03-03 18:17:27 +0100}, Date-Modified = {2010-03-03 18:17:27 +0100}, Journal = {Proc Natl Acad Sci U S A}, Journal-Full = {Proceedings of the National Academy of Sciences of the United States of America}, Mesh = {Antitubercular Agents; Base Sequence; DNA Primers; Gene Expression Regulation, Bacterial; Genes, Bacterial; Isoniazid; Mycobacterium tuberculosis; Nucleic Acid Hybridization; Reverse Transcriptase Polymerase Chain Reaction}, Month = {Oct}, Number = {22}, Pages = {12833-8}, Pmc = {PMC23119}, Pmid = {10536008}, Pst = {ppublish}, Title = {Exploring drug-induced alterations in gene expression in Mycobacterium tuberculosis by microarray hybridization}, Volume = {96}, Year = {1999}} @article{De-Smet:2000ff, Abstract = {Trehalose is present as a free disaccharide in the cytoplasm of mycobacteria and as a component of cell-wall glycolipids implicated in tissue damage associated with mycobacterial infection. To obtain an overview of trehalose metabolism, we analysed data from the Mycobacterium tuberculosis genome project and identified ORFs with homology to genes encoding enzymes from three trehalose biosynthesis pathways previously characterized in other bacteria. Functional assays using mycobacterial extracts and recombinant enzymes derived from these ORFs demonstrated that mycobacteria can produce trehalose from glucose 6-phosphate and UDP-glucose (the OtsA-OtsB pathway) from glycogen-like alpha(1-->4)-linked glucose polymers (the TreY-TreZ pathway) and from maltose (the TreS pathway). Each of the pathways was found to be active in both rapid-growing Mycobacterium smegmatis and slow-growing Mycobacterium bovis BCG. The presence of a disrupted treZ gene in Mycobacterium leprae suggests that this pathway is not functional in this organism. The presence of multiple biosynthetic pathways indicates that trehalose plays an important role in mycobacterial physiology.}, Author = {De Smet, K A and Weston, A and Brown, I N and Young, D B and Robertson, B D}, Date-Added = {2010-03-03 18:12:43 +0100}, Date-Modified = {2010-03-03 18:12:43 +0100}, Journal = {Microbiology}, Journal-Full = {Microbiology (Reading, England)}, Mesh = {Genome, Bacterial; Glucans; Glucose-6-Phosphate; Maltose; Mycobacterium; Mycobacterium tuberculosis; Osmotic Pressure; Recombinant Proteins; Sequence Analysis, DNA; Sequence Homology, Amino Acid; Trehalose}, Month = {Jan}, Pages = {199-208}, Pmid = {10658666}, Pst = {ppublish}, Title = {Three pathways for trehalose biosynthesis in mycobacteria}, Volume = {146 ( Pt 1)}, Year = {2000}} @article{Skeiky:1999pi, Abstract = {Culture filtrate proteins (CFP) of Mycobacterium tuberculosis have been shown to contain immunogenic components that elicit at least partial protective immunity against Mycobacterium infection. To clone genes encoding some of the immunogenic proteins, we made a high-titer rabbit anti-CFP serum and used it to screen an M. tuberculosis genomic expression library in Escherichia coli. In this paper, we describe the molecular cloning of two new protein components of CFP and identified them as members of the serine protease gene family. Their open reading frames contain N-terminal hydrophobic secretory signals consistent with their detection in CFP. The predicted molecular masses of the mature, fully processed forms of both antigens are approximately 32 kDa, in agreement with their observed sizes on immunoblots of CFP probed with polyclonal rabbit antisera made to the recombinant proteins. Thus, these proteins have been designated MTB32A and MTB32B. Interestingly, and despite 66\% amino acid sequence homology between the two proteins, polyclonal rabbit antisera made to each of the recombinant proteins were found to be specific for the respective immunizing antigens. The recombinant proteins were also evaluated in in vitro assays with donor peripheral blood mononuclear cells (PBMC) from healthy purified protein derivative (PPD)-positive individuals of diverse ethnic backgrounds. MTB32A but not MTB32B stimulated PBMC from healthy PPD-positive donors but not from PPD-negative donors to proliferate and secrete gamma interferon. MTB32A is encoded by a single-copy gene which is present in both virulent and avirulent strains of the M. tuberculosis complex and the BCG strain of Mycobacterium bovis but absent in the environmental mycobacterial species tested. In addition, nucleotide sequence comparison of mtb32a of the avirulent H37Ra strain and the virulent Erdman strain, as well as with the corresponding sequences (identified in the databases) of strain H37Rv and the clinical isolate CSU93, revealed 100\% identity. MTB32A, therefore, represents a candidate for inclusion in subunit vaccine development. Finally, the possible role of MTB32 serine proteases as a virulence factor(s) during Mycobacterium spp. infection is discussed.}, Author = {Skeiky, Y A and Lodes, M J and Guderian, J A and Mohamath, R and Bement, T and Alderson, M R and Reed, S G}, Date-Added = {2010-03-03 18:09:07 +0100}, Date-Modified = {2010-03-03 18:09:07 +0100}, Journal = {Infect Immun}, Journal-Full = {Infection and immunity}, Mesh = {Amino Acid Sequence; Animals; Antigens, Bacterial; Bacterial Proteins; Base Sequence; Blotting, Western; Cloning, Molecular; Conserved Sequence; Humans; Interferon-gamma; Lymphocyte Activation; Molecular Sequence Data; Mycobacterium tuberculosis; Rabbits; Serine Endopeptidases}, Month = {Aug}, Number = {8}, Pages = {3998-4007}, Pmc = {PMC96687}, Pmid = {10417166}, Pst = {ppublish}, Title = {Cloning, expression, and immunological evaluation of two putative secreted serine protease antigens of Mycobacterium tuberculosis}, Volume = {67}, Year = {1999}} @article{Domenech:2001mi, Abstract = {Mycobacterial genomics has uncovered a novel regulatory gene, oxyS, belonging to the LysR family. There is extensive similarity in the DNA-binding domain of OxyS with that of OxyR, the oxidative stress response protein of many bacteria. Since the oxyR gene of Mycobacterium tuberculosis has been multiply inactivated during evolution it was conceivable that some of its functions could be effected by OxyS. It is shown here that OxyS is produced at low levels and that there are at least three different oxyS alleles present in clinical isolates of M. tuberculosis that are susceptible or resistant to isoniazid. Overproduction or depletion of OxyS did not affect susceptibility to isoniazid but increasing the concentration of the regulator lowered levels of the alkyl hydroperoxide reductase, AhpC, and rendered the tubercle bacillus more susceptible to organic hydroperoxides.}, Author = {Domenech, P and Honor{\'e}, N and Heym, B and Cole, S T}, Date-Added = {2010-03-03 18:00:02 +0100}, Date-Modified = {2010-03-03 18:00:02 +0100}, Journal = {Microbes Infect}, Journal-Full = {Microbes and infection / Institut Pasteur}, Mesh = {Amino Acid Sequence; Antitubercular Agents; Bacterial Proteins; Drug Resistance, Microbial; Gene Expression Regulation, Bacterial; Humans; Isoniazid; Molecular Sequence Data; Mycobacterium tuberculosis; Oxidative Stress; Peroxidases; Peroxides; Peroxiredoxins; Repressor Proteins; Transcription Factors}, Month = {Jul}, Number = {9}, Pages = {713-21}, Pmid = {11489419}, Pst = {ppublish}, Title = {Role of OxyS of Mycobacterium tuberculosis in oxidative stress: overexpression confers increased sensitivity to organic hydroperoxides}, Volume = {3}, Year = {2001}} @article{Gold:2001qa, Abstract = {In this work, we characterize genes in Mycobacterium tuberculosis that are regulated by IdeR (iron-dependent regulator), an iron-responsive DNA-binding protein of the DtxR family that has been shown to regulate iron acquisition in Mycobacterium smegmatis. To identify some of the genes that constitute the IdeR regulon, we searched the M. tuberculosis genome for promoter regions containing the consensus IdeR/DxR binding sequence. Genes preceded by IdeR boxes included a set encoding proteins necessary for iron acquisition, such as the biosynthesis of siderophores (mbtA, mbtB, mbtI), aromatic amino acids (pheA, hisE, hisB-like) and others annotated to be involved in the synthesis of iron-storage proteins (bfrA, bfrB). Some putative IdeR-regulated genes identified in this search encoded proteins predicted to be engaged in the biosynthesis of lipopolysaccharide (LPS)-like molecules (rv3402c), lipids (acpP) and peptidoglycan (murB). We analysed four promoter regions containing putative IdeR boxes, mbtA-mbtB, mbI, rv3402c and bfrA-bfd, for interaction with IdeR and for iron-dependent expression. Gel retardation experiments and DNase footprinting analyses with purified IdeR showed that IdeR binds to these IdeR boxes in vitro. Analysis of the promoters by primer extension indicated that the IdeR boxes are located near the -10 position of each promoter, suggesting that IdeR acts as a transcriptional repressor by blocking RNA polymerase binding. Using quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) coupled to molecular beacons, we showed that mRNA levels of mbtA, mbtB, mbtI, rv3402c and bfd are induced 14- to 49-fold in cultures of M. tuberculosis starved for iron, whereas mRNA levels of bfrA decreased about threefold. We present evidence that IdeR not only acts as a transcriptional repressor but also functions as an activator of bfrA. Three of the IdeR- and iron-repressed genes, mbtB, mbtI and rv3402c, were induced during M. tuberculosis infection of human THP-1 macrophages.}, Author = {Gold, B and Rodriguez, G M and Marras, S A and Pentecost, M and Smith, I}, Date-Added = {2010-03-03 17:48:36 +0100}, Date-Modified = {2010-03-03 17:48:49 +0100}, Journal = {Mol Microbiol}, Journal-Full = {Molecular microbiology}, Keywords = {nopdf}, Mesh = {Bacterial Proteins; Base Sequence; Gene Expression Regulation, Bacterial; Humans; Iron; Macrophages; Molecular Sequence Data; Mycobacterium tuberculosis; Repressor Proteins; Transcription, Genetic; Tuberculosis; Virulence}, Month = {Nov}, Number = {3}, Pages = {851-65}, Pmid = {11722747}, Pst = {ppublish}, Title = {The Mycobacterium tuberculosis IdeR is a dual functional regulator that controls transcription of genes involved in iron acquisition, iron storage and survival in macrophages}, Volume = {42}, Year = {2001}} @article{McAdam:2002kl, Abstract = {A library of Mycobacterium tuberculosis insertional mutants was generated with the transposon Tn5370. The junction sequence between the transposon and the mycobacterial chromosome was determined, revealing the positions of 1329 unique insertions, 1189 of which were located in 351 different ORFs. Transposition was not completely random and examination of the most susceptible genome regions revealed a lower-than-average G+C content ranging from 54 to 62 mol\%. Mutants were obtained in all of the recognized M. tuberculosis functional protein-coding gene classes. About 30\% of the disrupted ORFs had matches elsewhere in the genome that suggested redundancy of function. The effect of gene disruption on the virulence of a selected set of defined mutants was investigated in a severe combined immune deficiency (SCID) mouse model. A range of phenotypes was observed in these mutants, the most notable being the severe attenuation in virulence of a strain disrupted in the Rv1290c gene, which encodes a protein of unknown function. The library described in this study provides a resource of defined mutant strains for use in functional analyses aimed at investigating the role of particular M. tuberculosis genes in virulence and defining their potential as targets for new anti-mycobacterial drugs or as candidates for deletion in a rationally attenuated live vaccine.}, Author = {McAdam, Ruth A and Quan, Selwyn and Smith, Debbie A and Bardarov, Stoyan and Betts, Joanna C and Cook, Fiona C and Hooker, Elizabeth U and Lewis, Alan P and Woollard, Peter and Everett, Martin J and Lukey, Pauline T and Bancroft, Gregory J and Jacobs Jr, Jr, William R and Duncan, Ken}, Date-Added = {2010-03-03 17:44:08 +0100}, Date-Modified = {2010-03-03 17:44:08 +0100}, Journal = {Microbiology}, Journal-Full = {Microbiology (Reading, England)}, Mesh = {Animals; DNA Transposable Elements; Disease Models, Animal; Gene Library; Humans; Mice; Mice, SCID; Mutagenesis, Insertional; Mutation; Mycobacterium tuberculosis; Open Reading Frames; Tuberculosis, Pulmonary; Virulence}, Month = {Oct}, Number = {Pt 10}, Pages = {2975-86}, Pmid = {12368431}, Pst = {ppublish}, Title = {Characterization of a Mycobacterium tuberculosis H37Rv transposon library reveals insertions in 351 ORFs and mutants with altered virulence}, Volume = {148}, Year = {2002}} @article{Quadri:1998fu, Abstract = {BACKGROUND: Many pathogenic bacteria secrete iron-chelating siderophores as virulence factors in the iron-limiting environments of their vertebrate hosts to compete for ferric iron. Mycobacterium tuberculosis mycobactins are mixed polyketide/nonribosomal peptides that contain a hydroxyaryloxazoline cap and two N-hydroxyamides that together create a high-affinity site for ferric ion. The mycobactin structure is analogous to that of the yersiniabactin and vibriobactin siderophores from the bacteria that cause plague and cholera, respectively. RESULTS: A ten-gene cluster spanning 24 kilobases of the M. tuberculosis genome, designated mbtA-J, contains the core components necessary for mycobactin biogenesis. The gene products MbtB, MbtE and MbtF are proposed to be peptide synthetases, MbtC and MbtD polyketide synthases, MbtI an isochorismate synthase that provides a salicylate activated by MbtA, and MbtG a required hydroxylase. An aryl carrier protein (ArCP) domain is encoded in mbtB, and is probably the site of siderophore chain initiation. Overproduction and purification of the mbtB ArCP domain and MbtA in Escherichia coli allowed validation of the mycobactin initiation hypothesis, as sequential action of PptT (a phosphopantetheinyl transferase) and MbtA (a salicyl-AMP ligase) resulted in the mbtB ArCP domain being activated as salicyl-S-ArCP. CONCLUSIONS: Mycobactins are produced in M. tuberculosis using a polyketide synthase/nonribosomal peptide synthetase strategy. The mycobactin gene cluster has organizational homologies to the yersiniabactin and enterobactin synthetase genes. Enzymatic targets for inhibitor design and therapeutic intervention are suggested by the similar ferric-ion ligation strategies used in the siderophores from Mycobacteria, Yersinia and E. coli pathogens.}, Author = {Quadri, L E and Sello, J and Keating, T A and Weinreb, P H and Walsh, C T}, Date-Added = {2010-03-03 17:39:35 +0100}, Date-Modified = {2010-03-03 17:39:47 +0100}, Journal = {Chem Biol}, Journal-Full = {Chemistry \& biology}, Keywords = {nopdf}, Mesh = {Bacterial Proteins; Carrier Proteins; Chorismic Acid; Chromatography, High Pressure Liquid; Cloning, Molecular; Coenzyme A; Cyclohexenes; DNA, Bacterial; Escherichia coli; Iron; Mass Spectrometry; Multigene Family; Mycobacterium tuberculosis; Oxazoles; Peptide Synthases; Siderophores}, Month = {Nov}, Number = {11}, Pages = {631-45}, Pmid = {9831524}, Pst = {ppublish}, Title = {Identification of a Mycobacterium tuberculosis gene cluster encoding the biosynthetic enzymes for assembly of the virulence-conferring siderophore mycobactin}, Volume = {5}, Year = {1998}} @article{Rickman:2005dz, Abstract = {Deletion of gene Rv3676 in Mycobacterium tuberculosis coding for a transcription factor belonging to the cAMP receptor protein (CRP) family caused growth defects in laboratory medium, in bone marrow-derived macrophages and in a mouse model of tuberculosis. Transcript profiling of M. tuberculosis grown in vitro identified 16 genes with significantly altered expression in the mutant compared with the wild type. Analysis of the DNA sequences upstream of the corresponding open reading frames revealed that 12 possessed sequences related to a consensus CRP binding site that could represent the sites of action of Rv3676. These included rpfA, lprQ, whiB1 and ahpC among genes with enhanced expression in the wild type, and Rv3616c-Rv3613c, Rv0188 and lipQ among genes exhibiting enhanced expression in the mutant. The activity of an rpfA::lacZ promoter fusion was lowered in the Rv3676 mutant and by mutation of the predicted Rv3676 binding site. Moreover, the product of Rv3676 (isolated as a TrxA fusion protein) interacted specifically with the rpfA promoter, and binding was inhibited by mutation of the Rv3676 site. Although Rv3676 retains four of the six amino acid residues that bind cAMP in Escherichia coli CRP addition of cAMP did not enhance Rv3676 binding at the rpfA promoter in vitro. In summary, it has been shown that Rv3676 is a direct regulator of rpfA expression, and because rpfA codes for a resuscitation promoting factor this may implicate Rv3676 in reactivation of dormant M. tuberculosis infections.}, Author = {Rickman, Lisa and Scott, Colin and Hunt, Debbie M and Hutchinson, Thomas and Men{\'e}ndez, M Carmen and Whalan, Rachael and Hinds, Jason and Colston, M Joseph and Green, Jeffrey and Buxton, Roger S}, Date-Added = {2010-03-03 17:32:34 +0100}, Date-Modified = {2010-03-03 17:32:56 +0100}, Doi = {10.1111/j.1365-2958.2005.04609.x}, Journal = {Mol Microbiol}, Journal-Full = {Molecular microbiology}, Keywords = {nopdf}, Mesh = {Aconitate Hydratase; Amino Acid Sequence; Animals; Artificial Gene Fusion; Bacterial Proteins; Colony Count, Microbial; Cyclic AMP Receptor Protein; Disease Models, Animal; Female; Gene Deletion; Gene Expression Regulation, Bacterial; Genes, Bacterial; Genes, Reporter; Lac Operon; Lung; Macrophages; Mice; Mice, Inbred BALB C; Molecular Sequence Data; Mycobacterium tuberculosis; Oligonucleotide Array Sequence Analysis; Regulatory Sequences, Nucleic Acid; Spleen; Transcription, Genetic; Tuberculosis; Virulence; beta-Galactosidase}, Month = {Jun}, Number = {5}, Pages = {1274-86}, Pmid = {15882420}, Pst = {ppublish}, Title = {A member of the cAMP receptor protein family of transcription regulators in Mycobacterium tuberculosis is required for virulence in mice and controls transcription of the rpfA gene coding for a resuscitation promoting factor}, Volume = {56}, Year = {2005}, Bdsk-Url-1 = {http://dx.doi.org/10.1111/j.1365-2958.2005.04609.x}} @article{Gurvitz:2009fv, Abstract = {We report on Mycobacterium tuberculosis Rv0241c and Rv3389c, representing two physiologically functional 3-hydroxyacyl-thioester dehydratases (Htd). These enzymes are potentially entrained in type 2 fatty acid synthase (FASII). Mycobacterial FASII is involved in the synthesis of mycolic acids, which are the major constituents of the protective layer around the pathogen, shielding it from noxious chemicals and the host's immune system. Mycolic acids are additionally associated with the virulence and resilience of M. tuberculosis. Here, Rv0241c and Rv3389c, which are distinct from the previously identified heterodimers Rv0635-Rv0636 (HadAB) and Rv0636-Rv0637 (HadBC) but also the homodimer Rv0130 (HtdZ), were identified by expressing the corresponding candidate open reading frames in Saccharomyces cerevisiae htd2Delta cells lacking mitochondrial 3-hydroxyacyl-acyl carrier protein dehydratase activity, followed by scoring for phenotype rescue. The htd2Delta mutant fails to produce sufficient levels of lipoic acid and does not respire or grow on nonfermentable carbon sources. Soluble protein extracts made from mutant htd2Delta cells expressing mitochondrially targeted Rv0241c or Rv3389c contained 3-hydroxyacyl-thioester hydratase activity. Moreover, mutant yeast cells expressing Rv0241c or Rv3389c were able to recover their respiratory growth on glycerol medium and efficiently reduce 2,3,5-triphenyltetrazolium chloride. Additionally, expression of mitochondrial Rv0241c or Rv3389c in htd2Delta cells also restored de novo lipoic acid synthesis to 92 and 40\% of the level in the wild-type strain, respectively. We propose naming Rv0241c and Rv3389c as HtdX and HtdY, respectively, and discuss the implications of our finding with reference to Rv0098, a candidate mycobacterial FabZ homologue with intrinsic thioesterase and hydratase activities that lacks the eukaryotic-like hydratase-2 motif.}, Author = {Gurvitz, Aner and Hiltunen, J Kalervo and Kastaniotis, Alexander J}, Date-Added = {2010-03-03 17:30:00 +0100}, Date-Modified = {2010-03-03 17:30:00 +0100}, Doi = {10.1128/JB.01046-08}, Journal = {J Bacteriol}, Journal-Full = {Journal of bacteriology}, Mesh = {Bacterial Proteins; Cloning, Molecular; Gene Deletion; Gene Expression; Genetic Complementation Test; Glycerol; Hydro-Lyases; Mitochondrial Proteins; Mycobacterium tuberculosis; Recombinant Proteins; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Tetrazolium Salts; Thioctic Acid}, Month = {Apr}, Number = {8}, Pages = {2683-90}, Pmc = {PMC2668405}, Pmid = {19136596}, Pst = {ppublish}, Title = {Heterologous expression of mycobacterial proteins in Saccharomyces cerevisiae reveals two physiologically functional 3-hydroxyacyl-thioester dehydratases, HtdX and HtdY, in addition to HadABC and HtdZ}, Volume = {191}, Year = {2009}, Bdsk-Url-1 = {http://dx.doi.org/10.1128/JB.01046-08}} @article{Wang:2007bs, Abstract = {Rv0098 is part of an operon, Rv0096-Rv0101, from Mycobacterium tuberculosis (Mtb) that is essential for Mtb's survival in mouse macrophages. This operon also contains an acyl carrier protein and one of the only two nonribosomal peptide synthases in Mtb. Rv0098 is annotated in the genome as a hypothetical protein and was proposed to be an acyl-coenzyme A (CoA) dehydratase. The structure of Rv0098, together with subsequent biochemical analysis, indicated that Rv0098 is a long-chain fatty acyl-CoA thioesterase (FcoT). However, FcoT lacks a general base or a nucleophile that is always found in the catalytic site of type II and type I thioesterases, respectively. The active site of Mtb FcoT reveals the structural basis for its substrate specificity for long-chain acyl-CoA and allows us to propose a catalytic mechanism for the enzyme. The characterization of Mtb FcoT provides a putative function of this operon that is crucial for Mtb pathogenicity.}, Author = {Wang, Feng and Langley, Robert and Gulten, Gulcin and Wang, Lei and Sacchettini, James C}, Date-Added = {2010-03-03 17:26:28 +0100}, Date-Modified = {2010-03-03 17:26:56 +0100}, Doi = {10.1016/j.chembiol.2007.04.005}, Journal = {Chem Biol}, Journal-Full = {Chemistry \& biology}, Keywords = {single protein, Rv0098}, Mesh = {Acyl Coenzyme A; Amino Acid Sequence; Binding Sites; Catalysis; Crystallization; Data Interpretation, Statistical; Kinetics; Models, Molecular; Mutagenesis; Mycobacterium tuberculosis; Operon; Palmitoyl-CoA Hydrolase; Structure-Activity Relationship; Substrate Specificity}, Month = {May}, Number = {5}, Pages = {543-51}, Pmid = {17524985}, Pst = {ppublish}, Title = {Identification of a type III thioesterase reveals the function of an operon crucial for Mtb virulence}, Volume = {14}, Year = {2007}, Bdsk-Url-1 = {http://dx.doi.org/10.1016/j.chembiol.2007.04.005}} @article{Parish:2003ij, Abstract = {Two-component regulatory systems have been widely implicated in bacterial virulence. To investigate the role of one such system in Mycobacterium tuberculosis, a strain was constructed in which the senX3-regX3 system was deleted by homologous recombination. The mutant strain (Tame15) showed a growth defect after infection of macrophages and was attenuated in both immunodeficient and immunocompetent mice. Competitive hybridization of total RNA from the wild-type and mutant strains to a whole-genome microarray was used to identify changes in gene expression resulting from the deletion. One operon was highly up-regulated in the mutant, indicating that regX3 probably has a role as a repressor of this operon. Other genes which were up- or down-regulated were also identified. Many of the genes showing down-regulation are involved in normal growth of the bacterium, indicating that the mutant strain is subject to some type of growth slow-down or stress. Genes showing differential expression were further grouped according to their pattern of gene expression under other stress conditions. From this analysis 50 genes were identified which are the most likely to be controlled by RegX3. Most of these genes are of unknown function and no obvious motifs were found upstream of the genes identified. Thus, it has been demonstrated that the senX3-regX3 two-component system is involved in the virulence of M. tuberculosis and a number of genes controlled by this system have been identified.}, Author = {Parish, Tanya and Smith, Debbie A and Roberts, Gretta and Betts, Joanna and Stoker, Neil G}, Date-Added = {2010-03-03 17:20:20 +0100}, Date-Modified = {2010-03-03 17:20:20 +0100}, Journal = {Microbiology}, Journal-Full = {Microbiology (Reading, England)}, Mesh = {Animals; Bacterial Proteins; Cell Line; Gene Deletion; Gene Expression Regulation, Bacterial; Genes, Bacterial; Genes, Regulator; Humans; Macrophages; Mice; Mice, Inbred BALB C; Mice, SCID; Mycobacterium tuberculosis; Phosphotransferases; Promoter Regions, Genetic; Regulon; Tuberculosis; Virulence}, Month = {Jun}, Number = {Pt 6}, Pages = {1423-35}, Pmid = {12777483}, Pst = {ppublish}, Title = {The senX3-regX3 two-component regulatory system of Mycobacterium tuberculosis is required for virulence}, Volume = {149}, Year = {2003}} @article{Park:2003hc, Abstract = {Unlike many pathogens that are overtly harmful to their hosts, Mycobacterium tuberculosis can persist for years within humans in a clinically latent state. Latency is often linked to hypoxic conditions within the host. Among M. tuberculosis genes induced by hypoxia is a putative transcription factor, Rv3133c/DosR. We performed targeted disruption of this locus followed by transcriptome analysis of wild-type and mutant bacilli. Nearly all the genes powerfully regulated by hypoxia require Rv3133c/DosR for their induction. Computer analysis identified a consensus motif, a variant of which is located upstream of nearly all M. tuberculosis genes rapidly induced by hypoxia. Further, Rv3133c/DosR binds to the two copies of this motif upstream of the hypoxic response gene alpha-crystallin. Mutations within the binding sites abolish both Rv3133c/DosR binding as well as hypoxic induction of a downstream reporter gene. Also, mutation experiments with Rv3133c/DosR confirmed sequence-based predictions that the C-terminus is responsible for DNA binding and that the aspartate at position 54 is essential for function. Together, these results demonstrate that Rv3133c/DosR is a transcription factor of the two-component response regulator class, and that it is the primary mediator of a hypoxic signal within M. tuberculosis.}, Author = {Park, Heui-Dong and Guinn, Kristi M and Harrell, Maria I and Liao, Reiling and Voskuil, Martin I and Tompa, Martin and Schoolnik, Gary K and Sherman, David R}, Date-Added = {2010-03-03 17:10:26 +0100}, Date-Modified = {2010-03-03 17:11:16 +0100}, Journal = {Mol Microbiol}, Journal-Full = {Molecular microbiology}, Keywords = {single protein, Rv3133c}, Mesh = {Anoxia; Aspartic Acid; Bacterial Proteins; Gene Expression Regulation, Bacterial; Gene Targeting; Genes, Reporter; Humans; Mycobacterium tuberculosis; Oligonucleotide Array Sequence Analysis; Oxygen; Protein Binding; Regulatory Sequences, Nucleic Acid; Transcription Factors; Tuberculosis}, Month = {May}, Number = {3}, Pages = {833-43}, Pmc = {PMC1992516}, Pmid = {12694625}, Pst = {ppublish}, Title = {Rv3133c/dosR is a transcription factor that mediates the hypoxic response of Mycobacterium tuberculosis}, Volume = {48}, Year = {2003}} @article{Sherman:2001tg, Abstract = {Unlike many pathogens that are overtly toxic to their hosts, the primary virulence determinant of Mycobacterium tuberculosis appears to be its ability to persist for years or decades within humans in a clinically latent state. Since early in the 20th century latency has been linked to hypoxic conditions within the host, but the response of M. tuberculosis to a hypoxic signal remains poorly characterized. The M. tuberculosis alpha-crystallin (acr) gene is powerfully and rapidly induced at reduced oxygen tensions, providing us with a means to identify regulators of the hypoxic response. Using a whole genome microarray, we identified >100 genes whose expression is rapidly altered by defined hypoxic conditions. Numerous genes involved in biosynthesis and aerobic metabolism are repressed, whereas a high proportion of the induced genes have no known function. Among the induced genes is an apparent operon that includes the putative two-component response regulator pair Rv3133c/Rv3132c. When we interrupted expression of this operon by targeted disruption of the upstream gene Rv3134c, the hypoxic regulation of acr was eliminated. These results suggest a possible role for Rv3132c/3133c/3134c in mycobacterial latency.}, Author = {Sherman, D R and Voskuil, M and Schnappinger, D and Liao, R and Harrell, M I and Schoolnik, G K}, Date-Added = {2010-03-03 17:08:20 +0100}, Date-Modified = {2010-03-03 17:08:20 +0100}, Doi = {10.1073/pnas.121172498}, Journal = {Proc Natl Acad Sci U S A}, Journal-Full = {Proceedings of the National Academy of Sciences of the United States of America}, Mesh = {Aerobiosis; Anaerobiosis; Bacterial Proteins; Crystallins; Gene Expression Regulation, Bacterial; Kinetics; Mycobacterium tuberculosis; Time Factors}, Month = {Jun}, Number = {13}, Pages = {7534-9}, Pmc = {PMC34703}, Pmid = {11416222}, Pst = {ppublish}, Title = {Regulation of the Mycobacterium tuberculosis hypoxic response gene encoding alpha -crystallin}, Volume = {98}, Year = {2001}, Bdsk-Url-1 = {http://dx.doi.org/10.1073/pnas.121172498}} @article{Braibant:2000kl, Abstract = {We have undertaken the inventory and assembly of the typical subunits of the ABC transporters encoded by the complete genome of Mycobacterium tuberculosis. These subunits, i.e. the nucleotide binding domains (NBDs), the membrane-spanning domains (MSDs) and the substrate binding proteins (SBPs), were identified on the basis of their characteristic stretches of amino acids and/or conserved structure. A total of 45 NBDs present in 38 proteins, of 47 MSDs present in 44 proteins and of 15 SBPs were found to be encoded by M. tuberculosis. Analysis of transcriptional clusters and searches of homology between the identified subunits of the transporters and proteins characterized in other organisms allowed the reconstitution of at least 26 complete (including at least one NBD and one MSD) and 11 incomplete ABC transporters. Sixteen of them were unambiguously classified as importers whereas 21 were presumed to be exporters. By searches of homology with already known transporters from other organisms, potential substrates (peptides, macrolides, carbohydrates, multidrugs, antibiotics, iron, anions) could be attributed to 30 of the ABC transporters identified in M. tuberculosis. The ABC transporters have been further classified in nine different sub-families according to a tree obtained from the clustering of their NBDs. Contrary to Escherichia coli and similarly to Bacillus subtilis, there is an equal representation of extruders and importers. Many exporters were found to be potentially implicated in the transport of drugs, probably contributing to the resistance of M. tuberculosis to many antibiotics. Interestingly, a transporter (absent in E. coli and in B. subtilis) potentially implicated in the export of a factor required for the bacterial attachment to the eukaryotic host cells was also identified. In comparison to E. coli and B. subtilis, there is an under-representation of the importers (with the exception of the phosphate importers) in M. tuberculosis. This may reflect the capacity of this bacterium to synthesize many essential compounds and to grow in the presence of few external nutrients. The genes encoding the ABC transporters occupy about 2.5\% of the genome of M. tuberculosis.}, Author = {Braibant, M and Gilot, P and Content, J}, Date-Added = {2010-03-03 17:02:25 +0100}, Date-Modified = {2010-03-03 17:02:43 +0100}, Journal = {FEMS Microbiol Rev}, Journal-Full = {FEMS microbiology reviews}, Keywords = {nopdf}, Mesh = {ATP-Binding Cassette Transporters; Amino Acid Sequence; Amino Acids; Anions; Anti-Bacterial Agents; Artificial Gene Fusion; Bacterial Proteins; Binding Sites; Carbohydrate Metabolism; Genome, Bacterial; Iron; Macrolides; Membranes; Molecular Sequence Data; Mycobacterium tuberculosis; Nucleotides; Peptides}, Month = {Oct}, Number = {4}, Pages = {449-67}, Pmid = {10978546}, Pst = {ppublish}, Title = {The ATP binding cassette (ABC) transport systems of Mycobacterium tuberculosis}, Volume = {24}, Year = {2000}} @article{Varrot:2005oq, Abstract = {The genomes of various Mycobacterium tuberculosis strains encode proteins that do not appear to play a role in the growth or survival of the bacterium in its mammalian host, including some implicated in plant cell wall breakdown. Here we show that M. tuberculosis H37Rv does indeed possess a functional cellulase. The x-ray crystal structure of this enzyme, in ligand complex forms, from 1.9 to 1.1A resolution, reveals a highly conserved substrate-binding cleft, which affords similar, and unusual, distortion of the substrate at the catalytic center. The endoglucanase activity, together with the existence of a putative membrane-associated crystalline polysaccharide-binding protein, may reflect the ancestral soil origin of the Mycobacterium or hint at a previously unconsidered environmental niche.}, Author = {Varrot, Annabelle and Leydier, Sabine and Pell, Gavin and Macdonald, James M and Stick, Robert V and Henrissat, Bernard and Gilbert, Harry J and Davies, Gideon J}, Date-Added = {2010-03-03 16:27:35 +0100}, Date-Modified = {2010-03-03 16:28:52 +0100}, Doi = {10.1074/jbc.C500142200}, Journal = {J Biol Chem}, Journal-Full = {The Journal of biological chemistry}, Keywords = {single protein, Rv0062}, Mesh = {Binding Sites; Catalysis; Cellulases; Cellulose; Crystallization; Crystallography, X-Ray; Models, Molecular; Molecular Structure; Mycobacterium tuberculosis; Phylogeny; Polysaccharides; Protein Structure, Secondary; Soil Microbiology; Substrate Specificity; beta-Glucans}, Month = {May}, Number = {21}, Pages = {20181-4}, Pmid = {15824123}, Pst = {ppublish}, Title = {Mycobacterium tuberculosis strains possess functional cellulases}, Volume = {280}, Year = {2005}, Bdsk-Url-1 = {http://dx.doi.org/10.1074/jbc.C500142200}} @article{Covert:2001nx, Abstract = {The complete sequencing of the Mycobacterium tuberculosis genome offers a unique opportunity to fully elucidate the biology of this human pathogen. One aspect of significant importance is the definition of T cell antigens. This report describes the development and implementation of a proteomic approach to defining such antigens. Large quantities of subcellular protein fractions of M. tuberculosis were resolved by two-dimensional liquid phase electrophoresis (2-D LPE), resulting in 355 and 299 fractions of culture filtrate and cytosolic proteins, respectively. Analysis of these fractions against splenocytes of C57Bl/6 mice infected with M. tuberculosis resulted in the identification of 37 fractions that stimulated a dominant T cell response, as measured by the production of interferon-gamma. Additionally, when the 2-D LPE fractions were assayed against splenocytes harvested at 10 and 40 days post infection significant changes in the T cell response were observed. Molecular characterization of the proteins contained in each of the 38 immunodominant fractions by liquid chromatography-mass spectrometry and liquid chromatography-tandem mass spectrometry resulted in the identification of 30 individual proteins. Many of these represented previously defined antigens. However 17 of these proteins were novel T cell antigens. The data presented demonstrate that proteomics offers a rapid and facile approach for elucidation of immunodominant T cell antigens of pathogenic bacteria.}, Author = {Covert, B A and Spencer, J S and Orme, I M and Belisle, J T}, Date-Added = {2010-03-03 16:19:21 +0100}, Date-Modified = {2010-03-03 16:19:37 +0100}, Doi = {10.1002/1615-9861(200104)1:4<574::AID-PROT574>3.0.CO;2-8}, Journal = {Proteomics}, Journal-Full = {Proteomics}, Keywords = {nopdf}, Mesh = {Animals; Antigens, Bacterial; Bacterial Proteins; Electrophoresis, Gel, Two-Dimensional; Genome, Bacterial; Humans; Interferon-gamma; Mice; Mice, Inbred C57BL; Mycobacterium tuberculosis; Proteome; T-Lymphocytes}, Month = {Apr}, Number = {4}, Pages = {574-86}, Pmid = {11681210}, Pst = {ppublish}, Title = {The application of proteomics in defining the T cell antigens of Mycobacterium tuberculosis}, Volume = {1}, Year = {2001}, Bdsk-Url-1 = {http://dx.doi.org/10.1002/1615-9861(200104)1:4%3C574::AID-PROT574%3E3.0.CO;2-8}} @article{Brooks:2001cr, Abstract = {The repair of DNA damage is expected to be particularly important to intracellular pathogens such as Mycobacterium tuberculosis, and so it is of interest to examine the response of M. tuberculosis to DNA damage. The expression of recA, a key component in DNA repair and recombination, is induced by DNA damage in M. tuberculosis. In this study, we have analyzed the expression following DNA damage in M. tuberculosis of a number of other genes which are DNA damage inducible in Escherichia coli. While many of these genes were also induced by DNA damage in M. tuberculosis, some were not. In addition, one gene (ruvC) which is not induced by DNA damage in E. coli was induced in M. tuberculosis, a result likely linked to its different transcriptional arrangement in M. tuberculosis. We also searched the sequences upstream of the genes being studied for the mycobacterial SOS box (the binding site for LexA) and assessed LexA binding to potential sites identified. LexA is the repressor protein responsible for regulating expression of these SOS genes in E. coli. However, two of the genes which were DNA damage inducible in M. tuberculosis did not have identifiable sites to which LexA bound. The absence of binding sites for LexA upstream of these genes was confirmed by analysis of LexA binding to overlapping DNA fragments covering a region from 500 bp upstream of the coding sequence to 100 bp within it. Therefore, it appears most likely that an alternative mechanism of gene regulation in response to DNA damage exists in M. tuberculosis.}, Author = {Brooks, P C and Movahedzadeh, F and Davis, E O}, Date-Added = {2010-03-03 16:15:17 +0100}, Date-Modified = {2010-03-03 16:15:17 +0100}, Doi = {10.1128/JB.183.15.4459-4467.2001}, Journal = {J Bacteriol}, Journal-Full = {Journal of bacteriology}, Mesh = {Bacterial Proteins; DNA Damage; DNA Helicases; DNA-Binding Proteins; Endodeoxyribonucleases; Escherichia coli Proteins; Genes, Bacterial; Mycobacterium tuberculosis; Operon; SOS Response (Genetics); Serine Endopeptidases}, Month = {Aug}, Number = {15}, Pages = {4459-67}, Pmc = {PMC95339}, Pmid = {11443079}, Pst = {ppublish}, Title = {Identification of some DNA damage-inducible genes of Mycobacterium tuberculosis: apparent lack of correlation with LexA binding}, Volume = {183}, Year = {2001}, Bdsk-Url-1 = {http://dx.doi.org/10.1128/JB.183.15.4459-4467.2001}} @article{Saxena:2008dq, Abstract = {Molecular mechanisms involved in maintaining the latent infection of Mycobacterium tuberculosis are least understood. We have applied principles of in vivo expression technology (IVET) to identify upregulated genes in an in vitro simulated condition of anaerobic persistence likely to be encountered by the pathogen in lung granulomas. A promoter library of M. tuberculosis constructed in plasmid pLL192 was subjected to hypoxic condition (dissolved oxygen <1\%) in a controlled fermenter. On the basis of green fluorescent protein fluorescence and kanamycin resistance the upregulated promoters were selected, identified by nucleotide sequence and the genes were confirmed by RT-PCR. The upregulated genes include Rv0050 (penicillin binding protein), Rv1511 (GDP-d-mannose dehydratase), Rv1489, Rv2257, Rv2258 (all conserved hypothetical proteins), Rv0467 (isocitrate lyase) and Rv2031c (alpha-crystalline homolog). The involvement of the last four genes in latency has been suggested before. The functional role of Rv0050 and Rv1511 may be important in determining cell wall characteristics controlling permeability of nutrients and antibiotics.}, Author = {Saxena, Alka and Srivastava, Vikas and Srivastava, Ranjana and Srivastava, Brahm S}, Date-Added = {2010-03-03 11:36:58 +0100}, Date-Modified = {2010-03-03 11:36:58 +0100}, Doi = {10.1016/j.tube.2008.01.003}, Journal = {Tuberculosis (Edinb)}, Journal-Full = {Tuberculosis (Edinburgh, Scotland)}, Mesh = {Anaerobiosis; Gene Expression Regulation, Bacterial; Genes, Bacterial; Genes, Reporter; Green Fluorescent Proteins; Kanamycin Resistance; Luminescent Agents; Mycobacterium bovis; Mycobacterium tuberculosis; Reverse Transcriptase Polymerase Chain Reaction; Up-Regulation}, Month = {Nov}, Number = {6}, Pages = {518-25}, Pmid = {18434250}, Pst = {ppublish}, Title = {Identification of genes of Mycobacterium tuberculosis upregulated during anaerobic persistence by fluorescence and kanamycin resistance selection}, Volume = {88}, Year = {2008}, Bdsk-Url-1 = {http://dx.doi.org/10.1016/j.tube.2008.01.003}} @article{Bhakta:2002bh, Abstract = {The product of the gene ponA present in cosmid MTCY21D4, one of the collection of clones representing the genome of Mycobacterium tuberculosis, has been named penicillin-binding protein 1* (PBP1*), by analogy to the previously characterized PBP1* of M. leprae. This gene has been overexpressed in Escherichia coli. His(6)-tagged PBP1* localizes to the membranes of induced E. coli cells. Its susceptibility to degradation upon proteinase K digestion of spheroplasts from E. coli expressing the protein supports the view that the majority of the protein translocates to the periplasmic side of the membrane. Recombinant PBP1* binds benzylpenicillin and several other beta-lactams, notably cefotaxime, with high affinity. Truncation of the N-terminal 64 amino acid residues results in an expressed protein present exclusively in inclusion bodies and unable to associate with the membrane. The C-terminal module encompassing amino acids 272-663 can be extracted from inclusion bodies under denaturing conditions using guanidine/HCl and refolded to give a protein fully competent in penicillin-binding. Deletion of Gly(95)-Gln(143) results in the expression of a protein, which is localized in the cytosol. The soluble derivative of PBP1* binds benzylpenicillin with the same efficiency as the full-length protein. This is the first report of a soluble derivative of a class A high-molecular-mass PBP.}, Author = {Bhakta, Sanjib and Basu, Joyoti}, Date-Added = {2010-03-03 11:32:24 +0100}, Date-Modified = {2010-03-03 11:33:07 +0100}, Journal = {Biochem J}, Journal-Full = {The Biochemical journal}, Keywords = {single protein, Rv0050}, Mesh = {Amino Acid Sequence; Anti-Bacterial Agents; Bacterial Proteins; Blotting, Western; Carrier Proteins; Cell Membrane; Cytosol; Endopeptidase K; Escherichia coli; Gene Deletion; Glutamine; Glycine; Hexosyltransferases; Kinetics; Molecular Sequence Data; Muramoylpentapeptide Carboxypeptidase; Mycobacterium tuberculosis; Open Reading Frames; Penicillin-Binding Proteins; Penicillins; Peptidyl Transferases; Protein Binding; Protein Structure, Tertiary; Recombinant Proteins}, Month = {Feb}, Number = {Pt 3}, Pages = {635-9}, Pmc = {PMC1222347}, Pmid = {11802794}, Pst = {ppublish}, Title = {Overexpression, purification and biochemical characterization of a class A high-molecular-mass penicillin-binding protein (PBP), PBP1* and its soluble derivative from Mycobacterium tuberculosis}, Volume = {361}, Year = {2002}} @article{Graham:1999qf, Abstract = {A widely applicable, positive cDNA selection method was developed to identify RNAs synthesized by Mycobacterium tuberculosis in response to phagocytosis by cultured human primary macrophages. cDNAs for sigE and sigH (alternative sigma factors), aceA (isocitrate lyase), ponA (class I penicillin-binding protein), pks2 (polyketide synthase), uvrA (UvrABC endonuclease), and ctpV (putative cation transporter) were obtained from macrophage-grown bacteria. cDNAs for ORFs Rv3070, Rv3483c, Rv0903c (encoding a putative bacterial two-component transcriptional activator), and Rv0170 of the mce1 virulence operon also were obtained from phagocytized bacilli. cDNAs for these genomic regions were not obtained from approximately 1, 000-fold more bacteria grown in laboratory broth. Methods described here, which have identified M. tuberculosis genes expressed in response to host interaction, will allow the study of gene expression in a variety of microorganisms, including expression resulting from interaction with human tissues in natural disease states.}, Author = {Graham, J E and Clark-Curtiss, J E}, Date-Added = {2010-03-03 11:26:31 +0100}, Date-Modified = {2010-03-03 11:26:31 +0100}, Journal = {Proc Natl Acad Sci U S A}, Journal-Full = {Proceedings of the National Academy of Sciences of the United States of America}, Mesh = {Bacterial Proteins; Cells, Cultured; DNA, Complementary; Humans; Macrophages; Mycobacterium tuberculosis; Phagocytosis; RNA, Bacterial; RNA, Messenger; Sigma Factor; Trans-Activators}, Month = {Sep}, Number = {20}, Pages = {11554-9}, Pmc = {PMC18072}, Pmid = {10500215}, Pst = {ppublish}, Title = {Identification of Mycobacterium tuberculosis RNAs synthesized in response to phagocytosis by human macrophages by selective capture of transcribed sequences (SCOTS)}, Volume = {96}, Year = {1999}} @article{Talaat:2004ve, Abstract = {Infection with Mycobacterium tuberculosis causes the illness tuberculosis with an annual mortality of approximately 2 million. Understanding the nature of the host-pathogen interactions at different stages of tuberculosis is central to new strategies for developing chemotherapies and vaccines. Toward this end, we adapted microarray technology to analyze the change in gene expression profiles of M. tuberculosis during infection in mice. This protocol provides the transcription profile of genes expressed during the course of early tuberculosis in immune-competent (BALB/c) and severe combined immune-deficient (SCID) hosts in comparison with growth in medium. The microarray analysis revealed clusters of genes that changed their transcription levels exclusively in the lungs of BALB/c, SCID mice, or medium over time. We identified a set of genes (n = 67) activated only in BALB/c and not in SCID mice at 21 days after infection, a key point in the progression of tuberculosis. A subset of the lung-activated genes was previously identified as induced during mycobacterial survival in a macrophage cell line. Another group of in vivo-expressed genes may also define a previously unreported genomic island. In addition, our analysis suggests the similarity between mycobacterial transcriptional machinery during growth in SCID and in broth, which questions the validity of using the SCID model for assessing mycobacterial virulence. The in vivo expression-profiling technology presented should be applicable to any microbial model of infection.}, Author = {Talaat, Adel M and Lyons, Rick and Howard, Susan T and Johnston, Stephen Albert}, Date-Added = {2010-03-03 11:19:24 +0100}, Date-Modified = {2010-03-03 11:19:24 +0100}, Doi = {10.1073/pnas.0306023101}, Journal = {Proc Natl Acad Sci U S A}, Journal-Full = {Proceedings of the National Academy of Sciences of the United States of America}, Mesh = {Animals; Gene Expression Profiling; Genome, Bacterial; Kinetics; Mice; Mice, Inbred BALB C; Mice, SCID; Mycobacterium tuberculosis; Oligonucleotide Array Sequence Analysis; Transcription, Genetic; Tuberculosis}, Month = {Mar}, Number = {13}, Pages = {4602-7}, Pmc = {PMC384793}, Pmid = {15070764}, Pst = {ppublish}, Title = {The temporal expression profile of Mycobacterium tuberculosis infection in mice}, Volume = {101}, Year = {2004}, Bdsk-Url-1 = {http://dx.doi.org/10.1073/pnas.0306023101}} @article{Bachhawat:1999ly, Abstract = {1L-myo-inositol (inositol) is vital for the biogenesis of mycothiol, phosphatidylinositol and glycosylphosphatidylinositol anchors linked to complex carbohydrates in Mycobacterium tuberculosis. All these cellular components are thought to play important roles in host-pathogen interactions and in the survival of the pathogen within the host. However, the inositol biosynthetic pathway in M. tuberculosis is not known. To delineate the pathways for inositol formation, we employed a unique combination of tertiary structure prediction and yeast-based functional assays. Here, we describe the identification of the gene for mycobacterial INO1 that encodes inositol-1-phosphate synthase distinct in many respects from the eukaryotic analogues.}, Author = {Bachhawat, N and Mande, S C}, Date-Added = {2010-03-03 11:14:51 +0100}, Date-Modified = {2010-03-03 11:15:53 +0100}, Doi = {10.1006/jmbi.1999.2980}, Journal = {J Mol Biol}, Journal-Full = {Journal of molecular biology}, Keywords = {nopdf, single protein}, Mesh = {Amino Acid Sequence; Binding Sites; Genes, Bacterial; Models, Molecular; Molecular Sequence Data; Mycobacterium tuberculosis; Myo-Inositol-1-Phosphate Synthase; NADP; Protein Conformation; Sequence Homology, Amino Acid}, Month = {Aug}, Number = {3}, Pages = {531-6}, Pmid = {10448034}, Pst = {ppublish}, Title = {Identification of the INO1 gene of Mycobacterium tuberculosis H37Rv reveals a novel class of inositol-1-phosphate synthase enzyme}, Volume = {291}, Year = {1999}, Bdsk-Url-1 = {http://dx.doi.org/10.1006/jmbi.1999.2980}} @article{Walters:2006zr, Abstract = {Two-component signal transduction systems (2-CS) play an important role in bacterial pathogenesis. In the work presented here, we have studied the effects of a mutation in the Mycobacterium tuberculosis (Mtb) PhoPR 2-CS on the pathogenicity, physiology and global gene expression of this bacterial pathogen. Disruption of PhoPR causes a marked attenuation of growth in macrophages and mice and prevents growth in low-Mg2+ media. The inability to grow in THP-1 macrophages can be partially overcome by the addition of excess Mg2+ during infection. Global transcription assays demonstrate PhoP is a positive transcriptional regulator of several genes, but do not support the hypothesis that the Mtb PhoPR system is sensing Mg2+ starvation, as is the case with the Salmonella typhimurium PhoPQ 2-CS. The genes that were positively regulated include those found in the pks2 and the msl3 gene clusters that encode enzymes for the biosynthesis of sulphatides and diacyltrehalose and polyacyltrehalose respectively. Complementary biochemical studies, in agreement with recent results from another group, indicate that these complex lipids are also absent from the phoP mutant, and the lack of these components in its cell envelope may indirectly cause the mutant's high-Mg2+ growth requirement. The experiments reported here provide functional evidence for the PhoPR 2-CS involvement in Mtb pathogenesis, and they suggest that a major reason for the attenuation observed in the phoP mutant is the absence of certain complex lipids that are known to be important for virulence.}, Author = {Walters, Shaun B and Dubnau, Eugenie and Kolesnikova, Irina and Laval, Francoise and Daffe, Mamadou and Smith, Issar}, Date-Added = {2010-03-03 11:11:57 +0100}, Date-Modified = {2010-03-03 11:12:17 +0100}, Doi = {10.1111/j.1365-2958.2006.05102.x}, Journal = {Mol Microbiol}, Journal-Full = {Molecular microbiology}, Keywords = {nopdf}, Mesh = {Animals; Anti-Bacterial Agents; Bacterial Proteins; Drug Resistance, Bacterial; Gene Expression Regulation, Bacterial; Lipids; Macrophages; Magnesium; Metals, Heavy; Mice; Mutation; Mycobacterium tuberculosis; Oligonucleotide Array Sequence Analysis; Oxidative Stress; Sequence Deletion; Signal Transduction; Virulence}, Month = {Apr}, Number = {2}, Pages = {312-30}, Pmid = {16573683}, Pst = {ppublish}, Title = {The Mycobacterium tuberculosis PhoPR two-component system regulates genes essential for virulence and complex lipid biosynthesis}, Volume = {60}, Year = {2006}, Bdsk-Url-1 = {http://dx.doi.org/10.1111/j.1365-2958.2006.05102.x}} @article{Manca:1997ys, Abstract = {Proteins that are actively secreted by Mycobacterium tuberculosis serve as major targets of immune responses in the infected host. To identify and purify novel proteins in the filtrates of M. tuberculosis cultures, a bacteriophage lambda library of M. tuberculosis H37Rv DNA was immunoscreened by using an anti-culture filtrate rabbit antiserum. Of 20 positive clones isolated, 6 were analyzed and found to express the genes for two known components of the early culture filtrate, the secreted 45/47-kDa antigen complex and the KatG protein, and two novel genes. Here we report the molecular cloning and nucleotide sequence of one of the new genes encoding a culture filtrate protein of 310 amino acid (aa) residues. We called this gene mtc28. The deduced polypeptide sequence contained an NH2-terminal, highly hydrophobic 32-aa region having properties of a secretion signal peptide. The putative 278-aa mature MTC28 protein was characterized at its NH2 and COOH termini by a high content of proline and alanine residues organized in an (AP)n motif. Thus, MTC28 is a new member of a group of proline-rich antigens found in M. tuberculosis and Mycobacterium leprae. As shown by DNA hybridization experiments, the mtc28 gene was present only in species of the M. tuberculosis complex. Purified recombinant MTC28 antigen evoked strong delayed-type hypersensitivity and antibody responses in guinea pigs immunized with Mycobacterium bovis BCG, but not in guinea pigs immunized with Mycobacterium avium. The strong immunological activity of MTC28 and the absence of B- and T-cell epitopes cross-reactive with a common environmental mycobacterial species, such as M. avium, make this novel antigen an attractive reagent for immunodiagnosis of tuberculosis.}, Author = {Manca, C and Lyashchenko, K and Colangeli, R and Gennaro, M L}, Date-Added = {2010-03-03 11:08:51 +0100}, Date-Modified = {2010-03-03 11:09:22 +0100}, Journal = {Infect Immun}, Journal-Full = {Infection and immunity}, Keywords = {single protein, Rv0040c}, Mesh = {Amino Acid Sequence; Animals; Antigens, Bacterial; Bacterial Proteins; Base Sequence; Cloning, Molecular; Molecular Sequence Data; Mycobacterium tuberculosis; Proline; Rabbits; Sequence Analysis}, Month = {Dec}, Number = {12}, Pages = {4951-7}, Pmc = {PMC175714}, Pmid = {9393781}, Pst = {ppublish}, Title = {MTC28, a novel 28-kilodalton proline-rich secreted antigen specific for the Mycobacterium tuberculosis complex}, Volume = {65}, Year = {1997}} @article{Malen:2008vn, Abstract = {Culture filtrates of Mycobacterium tuberculosis H37Rv highly enriched with secreted proteins were used to identify antigens recognized by a serum pool from tuberculosis patients. Two different approaches were used to separate the culture filtrate protein mixture: (i) proteins were fractionated according to their hydrophobicity using an HPLC-C18 chromatography column followed by separation based on their molecular mass by SDS-PAGE and subsequent immunoblotting or (ii) proteins were separated by two-dimensional gel electrophoresis, based on their isoelectric point and their molecular mass. Twenty serologically reactive proteins were ultimately identified by both methods, including four novel antigens. Further, to estimate the immunogenicity of the identified culture filtrate proteins, the relative antibody quantities were measured using Image master software. Our results show that the antibodies against proteins belonging to the antigen 85 complex were the most abundant in the serum of patients with active tuberculosis. The most immunogenic proteins in terms of high antibody-to-protein-ratio were Rv3881c and three lipoproteins Rv0934 (the 38 kDa antigen), Rv0932c (pstS2), and Rv3006 (LppZ). Rv 3881c [corrected] is located close to the RD1 region and is also present in BCG. [corrected] The proteins from the M. tuberculosis H37Rv culture filtrate are strong candidates to be evaluated for improvement of the serodiagnostic tests of tuberculosis.}, Author = {M{\aa}len, H and S{\o}fteland, T and Wiker, H G}, Date-Added = {2010-03-03 11:02:58 +0100}, Date-Modified = {2010-03-03 11:03:15 +0100}, Doi = {10.1111/j.1365-3083.2007.02064.x}, Journal = {Scand J Immunol}, Journal-Full = {Scandinavian journal of immunology}, Keywords = {nopdf}, Mesh = {Antibodies, Bacterial; Antigens, Bacterial; Cells, Cultured; Chromatography, High Pressure Liquid; Electrophoresis, Polyacrylamide Gel; Filtration; Humans; Image Processing, Computer-Assisted; Immunoblotting; Mycobacterium tuberculosis; Tuberculosis, Pulmonary}, Month = {Mar}, Number = {3}, Pages = {245-52}, Pmid = {18208443}, Pst = {ppublish}, Title = {Antigen analysis of Mycobacterium tuberculosis H37Rv culture filtrate proteins}, Volume = {67}, Year = {2008}, Bdsk-Url-1 = {http://dx.doi.org/10.1111/j.1365-3083.2007.02064.x}} @article{Mattow:2001kx, Abstract = {A proteome approach, combining high-resolution two-dimensional electrophoresis (2-DE) with mass spectrometry, was used to compare the cellular protein composition of two virulent strains of Mycobacterium tuberculosis with two attenuated strains of Mycobacterium bovis Bacillus Calmette-Guerin (BCG), in order to identify unique proteins of these strains. Emphasis was given to the identification of M. tuberculosis specific proteins, because we consider these proteins to represent putative virulence factors and interesting candidates for vaccination and diagnosis of tuberculosis. The genome of M. tuberculosis strain H37Rv comprises nearly 4000 predicted open reading frames. In contrast, the separation of proteins from whole mycobacterial cells by 2-DE resulted in silver-stained patterns comprising about 1800 distinct protein spots. Amongst these, 96 spots were exclusively detected either in the virulent (56 spots) or in the attenuated (40 spots) mycobacterial strains. Fifty-three of these spots were analyzed by mass spectrometry, of which 41 were identified, including 32 M. tuberculosis specific spots. Twelve M. tuberculosis specific spots were identified as proteins, encoded by genes previously reported to be deleted in M. bovis BCG. The remaining 20 spots unique for M. tuberculosis were identified as proteins encoded by genes that are not known to be missing in M. bovis BCG.}, Author = {Mattow, J and Jungblut, P R and Schaible, U E and Mollenkopf, H J and Lamer, S and Zimny-Arndt, U and Hagens, K and M{\"u}ller, E C and Kaufmann, S H}, Date-Added = {2010-03-03 10:55:42 +0100}, Date-Modified = {2010-03-03 10:56:12 +0100}, Doi = {10.1002/1522-2683(200108)22:14<2936::AID-ELPS2936>3.0.CO;2-S}, Journal = {Electrophoresis}, Journal-Full = {Electrophoresis}, Keywords = {nopdf}, Mesh = {Bacterial Proteins; Electrophoresis, Gel, Two-Dimensional; Gene Deletion; Gene Expression Profiling; Genes, Bacterial; Genome, Bacterial; Mycobacterium bovis; Mycobacterium tuberculosis; Proteome; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Subtraction Technique; Virulence}, Month = {Aug}, Number = {14}, Pages = {2936-46}, Pmid = {11565788}, Pst = {ppublish}, Title = {Identification of proteins from Mycobacterium tuberculosis missing in attenuated Mycobacterium bovis BCG strains}, Volume = {22}, Year = {2001}, Bdsk-Url-1 = {http://dx.doi.org/10.1002/1522-2683(200108)22:14%3C2936::AID-ELPS2936%3E3.0.CO;2-S}} @article{Huang:2006uq, Abstract = {Mycolic acids are generated in Mycobacterium tuberculosis as a result of the interaction of two fatty acid biosynthetic systems: type I fatty acid synthase (FAS) and type II fatty acid synthase. Acyl carrier protein (ACP) is a small, acidic protein in type II FAS systems. It plays a central role in mycolic acid biosynthesis by transferring the acyl groups from one enzyme to another for the completion of the fatty acid synthesis cycle. The nature of the proper recognition between ACPs and its many interactive proteins is not understood. Here, we report the over-expression, purification, and characterization of two putative ACPs: Rv0033 and Rv1344 in M. tuberculosis. In order to study the role of the conserved residues and the conformation of whole protein, some site-directed mutations of recombinant Acp1344 were made and the 3D structure of Acp1344 was modeled.}, Author = {Huang, Yishu and Ge, Jing and Yao, Yongchao and Wang, Qingzhong and Shen, Hongbo and Wang, Honghai}, Date-Added = {2010-03-03 10:53:02 +0100}, Date-Modified = {2010-03-03 10:53:02 +0100}, Doi = {10.1016/j.bbrc.2006.01.178}, Journal = {Biochem Biophys Res Commun}, Journal-Full = {Biochemical and biophysical research communications}, Mesh = {Acyl Carrier Protein; Amino Acid Sequence; Bacterial Proteins; Blotting, Western; Circular Dichroism; Cloning, Molecular; Kinetics; Models, Molecular; Molecular Sequence Data; Molecular Weight; Mutagenesis, Site-Directed; Mycobacterium tuberculosis; Protein Structure, Tertiary; Sequence Alignment}, Month = {Apr}, Number = {2}, Pages = {618-24}, Pmid = {16487939}, Pst = {ppublish}, Title = {Characterization and site-directed mutagenesis of the putative novel acyl carrier protein Rv0033 and Rv1344 from Mycobacterium tuberculosis}, Volume = {342}, Year = {2006}, Bdsk-Url-1 = {http://dx.doi.org/10.1016/j.bbrc.2006.01.178}} @article{Chopra:2003fk, Abstract = {The regulation of cellular processes by the modulation of protein phosphorylation/dephosphorylation is fundamental to a large number of processes in living organisms. These processes are carried out by specific protein kinases and phosphatases. In this study, a previously uncharacterized gene (Rv0018c) of Mycobacterium tuberculosis, designated as mycobacterial Ser/Thr phosphatase (mstp), was cloned, expressed in Escherichia coli, and purified as a histidine-tagged protein. Purified protein (Mstp) dephosphorylated the phosphorylated Ser/Thr residues of myelin basic protein (MBP), histone, and casein but failed to dephosphorylate phospho-tyrosine residue of these substrates, suggesting that this phosphatase is specific for Ser/Thr residues. It has been suggested that mstp is a part of a gene cluster that also includes two Ser/Thr kinases pknA and pknB. We show that Mstp is a trans-membrane protein that dephosphorylates phosphorylated PknA and PknB. Southern blot analysis revealed that mstp is absent in the fast growing saprophytes Mycobacterium smegmatis and Mycobacterium fortuitum. PknA has been shown, whereas PknB has been proposed to play a role in cell division. The presence of mstp in slow growing mycobacterial species, its trans-membrane localization, and ability to dephosphorylate phosphorylated PknA and PknB implicates that Mstp may play a role in regulating cell division in M. tuberculosis.}, Author = {Chopra, Puneet and Singh, Bhuminder and Singh, Ramandeep and Vohra, Reena and Koul, Anil and Meena, Laxman S and Koduri, Harshavardhan and Ghildiyal, Megha and Deol, Parampal and Das, Taposh K and Tyagi, Anil K and Singh, Yogendra}, Date-Added = {2010-03-03 10:17:01 +0100}, Date-Modified = {2010-03-03 10:18:19 +0100}, Journal = {Biochem Biophys Res Commun}, Journal-Full = {Biochemical and biophysical research communications}, Keywords = {single protein, Rv0018c}, Mesh = {Amino Acid Sequence; Bacterial Proteins; Enzyme Activation; Molecular Sequence Data; Mycobacterium tuberculosis; Phosphoprotein Phosphatases; Phosphorylation; Protein-Serine-Threonine Kinases; Sequence Alignment; Sequence Analysis, Protein; Substrate Specificity; Tissue Distribution}, Month = {Nov}, Number = {1}, Pages = {112-20}, Pmid = {14575702}, Pst = {ppublish}, Title = {Phosphoprotein phosphatase of Mycobacterium tuberculosis dephosphorylates serine-threonine kinases PknA and PknB}, Volume = {311}, Year = {2003}} @article{Chaba:2002oq, Abstract = {A eukaryotic-type protein serine/threonine kinase, PknA, was cloned from Mycobacterium tuberculosis strain H37Ra. Sequencing of the clone indicated 100\% identity with the published pknA sequence of M. tuberculosis strain H37Rv. PknA fused to maltose-binding protein was expressed in Escherichia coli; it exhibited a molecular mass of approximately 97 kDa. The fusion protein was purified from the soluble fraction by affinity chromatography using amylose resin. In vitro kinase assays showed that the autophosphorylating ability of PknA is strictly magnesium/manganese-dependent, and sodium orthovanadate can inhibit this activity. Phosphoamino-acid analysis indicated that PknA phosphorylates at serine and threonine residues. PknA was also able to phosphorylate exogenous substrates, such as myelin basic protein and histone. A comparison of the nucleotide-derived amino-acid sequence of PknA with that of functionally characterized prokaryotic serine/threonine kinases indicated its possible involvement in cell division/differentiation. Protein--protein interaction studies revealed that PknA is capable of phosphorylating at least a approximately 56-kDa soluble protein from E. coli. Scanning electron microscopy showed that constitutive expression of this kinase resulted in elongation of E. coli cells, supporting its regulatory role in cell division.}, Author = {Chaba, Rachna and Raje, Manoj and Chakraborti, Pradip K}, Date-Added = {2010-03-03 09:22:14 +0100}, Date-Modified = {2010-03-03 10:25:11 +0100}, Journal = {Eur J Biochem}, Journal-Full = {European journal of biochemistry / FEBS}, Keywords = {single protein, Rv0015c}, Mesh = {Bacterial Proteins; Cell Division; Escherichia coli; Mycobacterium tuberculosis; Phosphorylation; Protein-Serine-Threonine Kinases; Recombinant Fusion Proteins; Substrate Specificity}, Month = {Feb}, Number = {4}, Pages = {1078-85}, Pmid = {11856348}, Pst = {ppublish}, Title = {Evidence that a eukaryotic-type serine/threonine protein kinase from Mycobacterium tuberculosis regulates morphological changes associated with cell division}, Volume = {269}, Year = {2002}} @article{Av-Gay:1999nx, Abstract = {PknB is a member of the newly discovered eukaryotic-like protein serine/threonine kinase (PSTK) family of proteins. The pknB gene was cloned and expressed in Escherichia coli. The active recombinant protein was purified and shown to be reactive with antiphosphoserine antibodies, as well as with antibodies to the phosphorylated eukaryotic Ser/Thr kinases mitogen-activated protein kinase kinase 3 and 6, P38, and Creb. In vitro kinase assays demonstrated that PknB is a functional kinase that is autophosphorylated on serine/threonine residues and is also able to phosphorylate the peptide substrate myelin basic protein. Analysis of pknB expression in Mycobacterium tuberculosis indicates the presence of pknB mRNA in (i) organisms grown in vitro in bacteriological media, (ii) a murine macrophage in vitro infection model, and (iii) in vivo alveolar macrophages from a patient with tuberculosis.}, Author = {Av-Gay, Y and Jamil, S and Drews, S J}, Date-Added = {2010-03-03 09:16:59 +0100}, Date-Modified = {2010-03-03 10:25:44 +0100}, Journal = {Infect Immun}, Journal-Full = {Infection and immunity}, Keywords = {single protein, Rv0014c}, Mesh = {Animals; Bacterial Proteins; Cell Line; Cloning, Molecular; Humans; Macrophages, Alveolar; Mice; Mycobacterium tuberculosis; Phosphorylation; Protein-Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Tuberculosis}, Month = {Nov}, Number = {11}, Pages = {5676-82}, Pmc = {PMC96941}, Pmid = {10531215}, Pst = {ppublish}, Title = {Expression and characterization of the Mycobacterium tuberculosis serine/threonine protein kinase PknB}, Volume = {67}, Year = {1999}} @article{Marmiesse:2004cr, Abstract = {To better understand the biology and the virulence determinants of the two major mycobacterial human pathogens Mycobacterium tuberculosis and Mycobacterium leprae, their genome sequences have been determined recently. In silico comparisons revealed that among the 1439 genes common to both M. tuberculosis and M. leprae, 219 genes code for proteins that show no similarity with proteins from other organisms. Therefore, the latter 'core' genes could be specific for mycobacteria or even for the intracellular mycobacterial pathogens. To obtain more information as to whether these genes really were mycobacteria-specific, they were included in a focused macro-array, which also contained genes from previously defined regions of difference (RD) known to be absent from Mycobacterium bovis BCG relative to M. tuberculosis. Hybridization of DNA from 40 strains of the M. tuberculosis complex and in silico comparison of these genes with the near-complete genome sequences from Mycobacterium avium, Mycobacterium marinum and Mycobacterium smegmatis were undertaken to answer this question. The results showed that among the 219 conserved genes, very few were not present in all the strains tested. Some of these missing genes code for proteins of the ESAT-6 family, a group of highly immunogenic small proteins whose presence and number is variable among the genomically highly conserved members of the M. tuberculosis complex. Indeed, the results suggest that, with few exceptions, the 'core' genes conserved among M. tuberculosis H37Rv and M. leprae are also highly conserved among other mycobacterial strains, which makes them interesting potential targets for developing new specific anti-mycobacterial drugs. In contrast, the genes from RD regions showed great variability among certain members of the M. tuberculosis complex, and some new specific deletions in Mycobacterium canettii, Mycobacterium microti and seal isolates were identified and further characterized during this study. Together with the distribution of a particular 6 or 7 bp micro-deletion in the gene encoding the polyketide synthase pks15/1, these results confirm and further extend the revised phylogenetic model for the M. tuberculosis complex recently presented.}, Author = {Marmiesse, Magali and Brodin, Priscille and Buchrieser, Carmen and Gutierrez, Christina and Simoes, Nathalie and Vincent, Veronique and Glaser, Philippe and Cole, Stewart T and Brosch, Roland}, Date-Added = {2010-03-03 09:12:16 +0100}, Date-Modified = {2010-03-03 09:12:16 +0100}, Journal = {Microbiology}, Journal-Full = {Microbiology (Reading, England)}, Mesh = {Antigens, Bacterial; Bacterial Proteins; Base Sequence; Computational Biology; Genetic Variation; Molecular Probe Techniques; Molecular Sequence Data; Multigene Family; Mycobacterium tuberculosis; Phylogeny; Polymerase Chain Reaction; Restriction Mapping; Sequence Deletion}, Month = {Feb}, Number = {Pt 2}, Pages = {483-96}, Pmid = {14766927}, Pst = {ppublish}, Title = {Macro-array and bioinformatic analyses reveal mycobacterial 'core' genes, variation in the ESAT-6 gene family and new phylogenetic markers for the Mycobacterium tuberculosis complex}, Volume = {150}, Year = {2004}} @article{Wong:1999dq, Abstract = {Iron plays a critical role in the pathophysiology of Mycobacterium tuberculosis. To gain a better understanding of iron regulation by this organism, we have used two-dimensional (2-D) gel electrophoresis, mass spectrometry, and database searching to study protein expression in M. tuberculosis under conditions of high and low iron concentration. Proteins in cellular extracts from M. tuberculosis Erdman strain grown under low-iron (1 microM) and high-iron (70 microM) conditions were separated by 2-D polyacrylamide gel electrophoresis, which allowed high-resolution separation of several hundred proteins, as visualized by Coomassie staining. The expression of at least 15 proteins was induced, and the expression of at least 12 proteins was decreased under low-iron conditions. In-gel trypsin digestion was performed on these differentially expressed proteins, and the digestion mixtures were analyzed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry to determine the molecular masses of the resulting tryptic peptides. Partial sequence data on some of the peptides were obtained by using after source decay and/or collision-induced dissociation. The fragmentation data were used to search computerized peptide mass and protein sequence databases for known proteins. Ten iron-regulated proteins were identified, including Fur and aconitase proteins, both of which are known to be regulated by iron in other bacterial systems. Our study shows that, where large protein sequence databases are available from genomic studies, the combined use of 2-D gel electrophoresis, mass spectrometry, and database searching to analyze proteins expressed under defined environmental conditions is a powerful tool for identifying expressed proteins and their physiologic relevance.}, Author = {Wong, D K and Lee, B Y and Horwitz, M A and Gibson, B W}, Date-Added = {2010-03-03 08:46:32 +0100}, Date-Modified = {2010-03-03 08:46:32 +0100}, Journal = {Infect Immun}, Journal-Full = {Infection and immunity}, Mesh = {Aconitate Hydratase; Amino Acid Sequence; Antigens, Bacterial; Bacterial Proteins; Crystallins; Culture Media, Conditioned; Electrophoresis, Gel, Two-Dimensional; Heat-Shock Proteins; Iron; Molecular Sequence Data; Mycobacterium tuberculosis; Oxidoreductases; Peptide Elongation Factor Tu; Peptidylprolyl Isomerase; Phosphoenolpyruvate Carboxykinase (ATP); Recombinant Fusion Proteins; Repressor Proteins; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization}, Month = {Jan}, Number = {1}, Pages = {327-36}, Pmc = {PMC96314}, Pmid = {9864233}, Pst = {ppublish}, Title = {Identification of fur, aconitase, and other proteins expressed by Mycobacterium tuberculosis under conditions of low and high concentrations of iron by combined two-dimensional gel electrophoresis and mass spectrometry}, Volume = {67}, Year = {1999}} @article{Weldingh:1998bh, Abstract = {Culture filtrate from Mycobacterium tuberculosis contains molecules which promote high levels of protective immunity in animal models of subunit vaccination against tuberculosis. We have used two-dimensional electrophoresis for analysis and purification of six novel M. tuberculosis culture filtrate proteins (CFPs): CFP17, CFP20, CFP21, CFP22, CFP25, and CFP28. The proteins were tested for recognition by M. tuberculosis-reactive memory cells from different strains of inbred mice and for their capacity to induce a skin test response in M. tuberculosis-infected guinea pigs. CFP17, CFP20, CFP21 and CFP25 induced both a high gamma interferon release and a strong delayed-type hypersensitivity response, and CFP21 was broadly recognized by different strains of inbred mice. N-terminal sequences were obtained for the six proteins, and the corresponding genes were identified in the Sanger M. tuberculosis genome database. In parallel we established a two-dimensional electrophoresis reference map of short-term culture filtrate components and mapped novel proteins as well as already-known CFP.}, Author = {Weldingh, K and Rosenkrands, I and Jacobsen, S and Rasmussen, P B and Elhay, M J and Andersen, P}, Date-Added = {2010-03-03 08:43:35 +0100}, Date-Modified = {2010-03-03 08:43:35 +0100}, Journal = {Infect Immun}, Journal-Full = {Infection and immunity}, Mesh = {Amino Acid Sequence; Animals; Antigens, Bacterial; Bacterial Proteins; Cells, Cultured; Chromosome Mapping; Cloning, Molecular; Electrophoresis, Gel, Two-Dimensional; Female; Gene Expression; Genes, Bacterial; Guinea Pigs; Mice; Mice, Inbred C57BL; Molecular Sequence Data; Mycobacterium tuberculosis; Sequence Analysis}, Month = {Aug}, Number = {8}, Pages = {3492-500}, Pmc = {PMC108378}, Pmid = {9673225}, Pst = {ppublish}, Title = {Two-dimensional electrophoresis for analysis of Mycobacterium tuberculosis culture filtrate and purification and characterization of six novel proteins}, Volume = {66}, Year = {1998}} @article{Prakash:2005qf, Abstract = {IdeR (iron-dependent regulator) is a key regulator of virulence factors and iron acquisition systems in Mycobacterium tuberculosis. Despite the wealth of information available on IdeR-regulated genes of M.tuberculosis, there is still an underlying possibility that there are novel genes/pathways that have gone undetected, the identification of which could give new insights into understanding the pathogenesis of M.tuberculosis. We describe an in silico approach employing the positional relative entropy method to identify potential IdeR binding sites in the upstream sequences of all the 3919 ORFs of M.tuberculosis. While many of the predictions made by this approach overlapped with the ones already identified by microarray experiments and binding assays, pointing to the accuracy of our method, a few genes for which there has been no evidence for IdeR regulation were additionally identified. Our results have implications on the iron-dependent regulatory mechanism of M.tuberculosis vis-a-vis the activity of urease operon and novel transcription regulators and transporters.}, Author = {Prakash, Prachee and Yellaboina, Sailu and Ranjan, Akash and Hasnain, Seyed E}, Date-Added = {2010-03-03 08:41:11 +0100}, Date-Modified = {2010-03-03 08:41:11 +0100}, Doi = {10.1093/bioinformatics/bti375}, Journal = {Bioinformatics}, Journal-Full = {Bioinformatics (Oxford, England)}, Mesh = {Bacterial Proteins; Binding Sites; Computer Simulation; DNA, Bacterial; Gene Expression Regulation, Bacterial; Models, Biological; Models, Chemical; Mycobacterium tuberculosis; Open Reading Frames; Protein Binding; Repressor Proteins; Sequence Analysis, DNA; Sequence Analysis, Protein}, Month = {May}, Number = {10}, Pages = {2161-6}, Pmid = {15746274}, Pst = {ppublish}, Title = {Computational prediction and experimental verification of novel IdeR binding sites in the upstream sequences of Mycobacterium tuberculosis open reading frames}, Volume = {21}, Year = {2005}, Bdsk-Url-1 = {http://dx.doi.org/10.1093/bioinformatics/bti375}} @article{Mattow:2006ve, Abstract = {Pathogenic mycobacteria persist and replicate within phagosomes of host phagocytes by inhibiting phagosome maturation at an early endosome stage. The molecular basis for this behavior is not understood. To identify proteins of Mycobacterium tuberculosis unique to the intraphagosomal phase, mycobacteria were purified from phagosomes of infected murine bone marrow-derived macrophages and analyzed by high-resolution 2-DE and MS. Protein patterns of intraphagosomally grown M. tuberculosis were compared with those of broth-cultured mycobacteria. The analysis revealed 11 mycobacterial proteins exclusively detected in intraphagosomal mycobacteria. Some of these proteins are involved in metabolism and cell envelope synthesis, such as the lipid carrier protein Rv1627c, and the conserved hypothetical protein Rv1130 that shows homology to a virulence-associated protein of Legionella pneumophila. The relevance of these proteins as factors enabling intracellular survival of M. tuberculosis is being discussed.}, Author = {Mattow, Jens and Siejak, Frank and Hagens, Kristine and Becher, D{\"o}rte and Albrecht, Dirk and Krah, Alexander and Schmidt, Frank and Jungblut, Peter R and Kaufmann, Stefan H E and Schaible, Ulrich E}, Date-Added = {2010-03-03 08:36:52 +0100}, Date-Modified = {2010-03-03 09:43:37 +0100}, Doi = {10.1002/pmic.200500547}, Journal = {Proteomics}, Journal-Full = {Proteomics}, Keywords = {nopdf}, Mesh = {Bacterial Proteins; Electrophoresis, Gel, Two-Dimensional; Macrophages; Mycobacterium tuberculosis; Phagosomes; Proteomics; Silver Staining; Virulence Factors}, Month = {Apr}, Number = {8}, Pages = {2485-94}, Pmid = {16548060}, Pst = {ppublish}, Title = {Proteins unique to intraphagosomally grown Mycobacterium tuberculosis}, Volume = {6}, Year = {2006}, Bdsk-Url-1 = {http://dx.doi.org/10.1002/pmic.200500547}} @article{Mattow:2003ly, Abstract = {A comprehensive analysis of culture supernatant (CSN) proteins of Mycobacterium tuberculosis H37Rv was accomplished by combination of two-dimensional electrophoresis (2-DE), mass spectrometry, and N-terminal sequencing by Edman degradation. Analytical 2-DE gels resolved approximately 1250 protein spots from CSN of M. tuberculosis H37Rv, 381 of which were identified by mass spectrometry and/or Edman degradation. This study revealed 137 different proteins, 42 of which had previously been described as secreted. Comparative proteome analysis of CSN from virulent M. tuberculosis H37Rv and attenuated Mycobacterium bovis BCG Copenhagen identified 39 M. tuberculosis-specific spots containing 27 different proteins, representing candidate antigens for novel vaccines and diagnostics in tuberculosis. These included five proteins encoded by open reading frames absent from M. bovis BCG, e.g., early secretory antigen target (Esat6), as well as 22 novel differential proteins, such as acetyl-CoA C-acetyltransferase (Rv0243) and two putative Esat6-like proteins (Rv1198, Rv1793).}, Author = {Mattow, Jens and Schaible, Ulrich E and Schmidt, Frank and Hagens, Kristine and Siejak, Frank and Brestrich, Gordon and Haeselbarth, Gisela and M{\"u}ller, Eva-Christina and Jungblut, Peter R and Kaufmann, Stefan H E}, Date-Added = {2010-03-03 08:34:38 +0100}, Date-Modified = {2010-03-03 09:43:11 +0100}, Doi = {10.1002/elps.200305601}, Journal = {Electrophoresis}, Journal-Full = {Electrophoresis}, Keywords = {nopdf}, Mesh = {Bacterial Proteins; Culture Media, Conditioned; Electrophoresis, Gel, Two-Dimensional; Mass Spectrometry; Mycobacterium bovis; Mycobacterium tuberculosis; Proteomics; Sequence Analysis, Protein}, Month = {Oct}, Number = {19-20}, Pages = {3405-20}, Pmid = {14595687}, Pst = {ppublish}, Title = {Comparative proteome analysis of culture supernatant proteins from virulent Mycobacterium tuberculosis H37Rv and attenuated M. bovis BCG Copenhagen}, Volume = {24}, Year = {2003}, Bdsk-Url-1 = {http://dx.doi.org/10.1002/elps.200305601}} @article{Manjunatha:2001zr, Abstract = {DNA gyrase is an essential type II topoisomerase found in bacteria. We have previously characterized DNA gyrase from Mycobacterium tuberculosis and Mycobacterium smegmatis. In this study, several monoclonal antibodies were generated against the gyrase A subunit (GyrA) of M. smegmatis. Three, MsGyrA:C3, MsGyrA:H11 and MsGyrA:E9, were further analyzed for their interaction with the enzyme. The monoclonal antibodies showed high degree of cross-reactivity with both fast-growing and slow-growing mycobacteria. In contrast, none recognized Escherichia coli GyrA. All the three monoclonal antibodies were of IgG1 isotype falling into two distinct types with respect to epitope recognition and interaction with the enzyme. MsGyrA:C3 and MsGyrA:H11 IgG, and their respective Fab fragments, inhibited the DNA supercoiling activity catalyzed by mycobacterial DNA gyrase. The epitope for the neutralizing monoclonal antibodies appeared to involve the region towards the N-terminus (residues 351-415) of the enzyme in a conformation-dependent manner. These monoclonal antibodies would serve as valuable tools for structure-function analysis and immunocytological studies of mycobacterial DNA gyrase. In addition, they would be useful for designing peptide inhibitors against DNA gyrase.}, Author = {Manjunatha, U H and Mahadevan, S and Visweswariah, S S and Nagaraja, V}, Date-Added = {2010-03-02 19:55:08 +0100}, Date-Modified = {2010-03-03 10:26:27 +0100}, Journal = {Eur J Biochem}, Journal-Full = {European journal of biochemistry / FEBS}, Keywords = {single protein, Rv0006}, Mesh = {Antibodies, Monoclonal; Antibody Specificity; Blotting, Western; DNA Gyrase; DNA Topoisomerases, Type II; DNA, Superhelical; Electrophoresis, Polyacrylamide Gel; Epitope Mapping; Escherichia coli; Immunoglobulin Fab Fragments; Immunoglobulin G; Mycobacterium; Nucleic Acid Conformation; Protein Conformation}, Month = {Apr}, Number = {7}, Pages = {2038-46}, Pmid = {11277926}, Pst = {ppublish}, Title = {Monoclonal antibodies to mycobacterial DNA gyrase A inhibit DNA supercoiling activity}, Volume = {268}, Year = {2001}} @article{Gu:2003ys, Abstract = {Mycobacterium tuberculosis is an infectious microorganism that causes human tuberculosis. The cell membranes of pathogens are known to be rich in possible diagnostic and therapeutic protein targets. To compliment the M. tuberculosis genome, we have profiled the membrane protein fraction of the M. tuberculosis H37Rv strain using an analytical platform that couples one-dimensional SDS gels to a microcapillary liquid chromatography-nanospray-tandem mass spectrometer. As a result, 739 proteins have been identified by two or more distinct peptide sequences and have been characterized. Interestingly, approximately 450 proteins represent novel identifications, 79 of which are membrane proteins and more than 100 of which are membrane-associated proteins. The physicochemical properties of the identified proteins were studied in detail, and then biological functions were obtained by sorting them according to Sanger Institute gene function category. Many membrane proteins were found to be involved in the cell envelope, and those proteins with energy metabolic functions were also identified in this study.}, Author = {Gu, Sheng and Chen, Jin and Dobos, Karen M and Bradbury, E Morton and Belisle, John T and Chen, Xian}, Date-Added = {2010-03-02 19:46:37 +0100}, Date-Modified = {2010-03-02 19:46:37 +0100}, Doi = {10.1074/mcp.M300060-MCP200}, Journal = {Mol Cell Proteomics}, Journal-Full = {Molecular \& cellular proteomics : MCP}, Mesh = {Amino Acid Sequence; Bacterial Proteins; Cell Membrane; Chromatography, Liquid; Electrophoresis, Polyacrylamide Gel; Membrane Proteins; Molecular Sequence Data; Mycobacterium tuberculosis; Proteomics; Spectrometry, Mass, Electrospray Ionization; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Subcellular Fractions}, Month = {Dec}, Number = {12}, Pages = {1284-96}, Pmid = {14532352}, Pst = {ppublish}, Title = {Comprehensive proteomic profiling of the membrane constituents of a Mycobacterium tuberculosis strain}, Volume = {2}, Year = {2003}, Bdsk-Url-1 = {http://dx.doi.org/10.1074/mcp.M300060-MCP200}} @article{Stewart:2002vn, Abstract = {Regulation of the expression of heat-shock proteins plays an important role in the pathogenesis of Mycobacterium tuberculosis. The heat-shock response of bacteria involves genome-wide changes in gene expression. A combination of targeted mutagenesis and whole-genome expression profiling was used to characterize transcription factors responsible for control of genes encoding the major heat-shock proteins of M. tuberculosis. Two heat-shock regulons were identified. HspR acts as a transcriptional repressor for the members of the Hsp70 (DnaK) regulon, and HrcA similarly regulates the Hsp60 (GroE) response. These two specific repressor circuits overlap with broader transcriptional changes mediated by alternative sigma factors during exposure to high temperatures. Several previously undescribed heat-shock genes were identified as members of the HspR and HrcA regulons. A novel HspR-controlled operon encodes a member of the low-molecular-mass alpha-crystallin family. This protein is one of the most prominent features of the M. tuberculosis heat-shock response and is related to a major antigen induced in response to anaerobic stress.}, Author = {Stewart, Graham R and Wernisch, Lorenz and Stabler, Richard and Mangan, Joseph A and Hinds, Jason and Laing, Ken G and Young, Douglas B and Butcher, Philip D}, Date-Added = {2010-03-02 19:22:56 +0100}, Date-Modified = {2010-03-02 19:22:56 +0100}, Journal = {Microbiology}, Journal-Full = {Microbiology (Reading, England)}, Mesh = {Bacterial Proteins; Base Sequence; DNA-Binding Proteins; Gene Expression Profiling; Gene Expression Regulation, Bacterial; Heat-Shock Proteins; Heat-Shock Response; Humans; Molecular Sequence Data; Mutation; Mycobacterium tuberculosis; Oligonucleotide Array Sequence Analysis; Regulon; Repressor Proteins; Transcription, Genetic}, Month = {Oct}, Number = {Pt 10}, Pages = {3129-38}, Pmid = {12368446}, Pst = {ppublish}, Title = {Dissection of the heat-shock response in Mycobacterium tuberculosis using mutants and microarrays}, Volume = {148}, Year = {2002}} @article{Lamichhane:2003kx, Abstract = {We describe a postgenomic in silico approach for identifying genes that are likely to be essential and estimate their proportion in haploid genomes. With the knowledge of all sites eligible for mutagenesis and an experimentally determined partial list of nonessential genes from genome mutagenesis, a Bayesian statistical method provides reasonable predictions of essential genes with a subsaturation level of random mutagenesis. For mutagenesis, a transposon such as Himar1 is suitable as it inserts randomly into TA sites. All of the possible insertion sites may be determined a priori from the genome sequence and with this information, data on experimentally hit TA sites may be used to predict the proportion of genes that cannot be mutated. As a model, we used the Mycobacterium tuberculosis genome. Using the Himar1 transposon, we created a genetically defined collection of 1,425 insertion mutants. Based on our Bayesian statistical analysis using Markov chain Monte Carlo and the observed frequencies of transposon insertions in all of the genes, we estimated that the M. tuberculosis genome contains 35\% (95\% confidence interval, 28\%-41\%) essential genes. This analysis further revealed seven functional groups with high probabilities of being enriched in essential genes. The PE-PGRS (Pro-Glu polymorphic GC-rich repetitive sequence) family of genes, which are unique to mycobacteria, the polyketide/nonribosomal peptide synthase family, and mycolic and fatty acid biosynthesis gene families were disproportionately enriched in essential genes. At subsaturation levels of mutagenesis with a random transposon such as Himar1, this approach permits a statistical prediction of both the proportion and identities of essential genes of sequenced genomes.}, Author = {Lamichhane, Gyanu and Zignol, Matteo and Blades, Natalie J and Geiman, Deborah E and Dougherty, Annette and Grosset, Jacques and Broman, Karl W and Bishai, William R}, Date-Added = {2010-03-02 19:19:51 +0100}, Date-Modified = {2010-03-02 19:19:51 +0100}, Doi = {10.1073/pnas.1231432100}, Journal = {Proc Natl Acad Sci U S A}, Journal-Full = {Proceedings of the National Academy of Sciences of the United States of America}, Mesh = {Anti-Bacterial Agents; Base Sequence; DNA, Bacterial; Genes, Bacterial; Genetic Techniques; Genome, Bacterial; Genomics; Multigene Family; Mutagenesis, Insertional; Mycobacterium tuberculosis; Open Reading Frames}, Month = {Jun}, Number = {12}, Pages = {7213-8}, Pmc = {PMC165855}, Pmid = {12775759}, Pst = {ppublish}, Title = {A postgenomic method for predicting essential genes at subsaturation levels of mutagenesis: application to Mycobacterium tuberculosis}, Volume = {100}, Year = {2003}, Bdsk-Url-1 = {http://dx.doi.org/10.1073/pnas.1231432100}} @article{Mawuenyega:2005uq, Abstract = {Trends in increased tuberculosis infection and a fatality rate of approximately 23\% have necessitated the search for alternative biomarkers using newly developed postgenomic approaches. Here we provide a systematic analysis of Mycobacterium tuberculosis (Mtb) by directly profiling its gene products. This analysis combines high-throughput proteomics and computational approaches to elucidate the globally expressed complements of the three subcellular compartments (the cell wall, membrane, and cytosol) of Mtb. We report the identifications of 1044 proteins and their corresponding localizations in these compartments. Genome-based computational and metabolic pathways analyses were performed and integrated with proteomics data to reconstruct response networks. From the reconstructed response networks for fatty acid degradation and lipid biosynthesis pathways in Mtb, we identified proteins whose involvements in these pathways were not previously suspected. Furthermore, the subcellular localizations of these expressed proteins provide interesting insights into the compartmentalization of these pathways, which appear to traverse from cell wall to cytoplasm. Results of this large-scale subcellular proteome profile of Mtb have confirmed and validated the computational network hypothesis that functionally related proteins work together in larger organizational structures.}, Author = {Mawuenyega, Kwasi G and Forst, Christian V and Dobos, Karen M and Belisle, John T and Chen, Jin and Bradbury, E Morton and Bradbury, Andrew R M and Chen, Xian}, Date-Added = {2010-03-02 19:12:53 +0100}, Date-Modified = {2010-03-02 19:12:53 +0100}, Doi = {10.1091/mbc.E04-04-0329}, Journal = {Mol Biol Cell}, Journal-Full = {Molecular biology of the cell}, Mesh = {Automation; Cell Membrane; Cell Wall; Computational Biology; Cytosol; Databases, Protein; Fatty Acids; Genome; Lipid Metabolism; Models, Biological; Models, Statistical; Mycobacterium tuberculosis; Protein Array Analysis; Proteins; Proteome; Proteomics; Software; Spectrometry, Mass, Electrospray Ionization; Subcellular Fractions}, Month = {Jan}, Number = {1}, Pages = {396-404}, Pmc = {PMC539182}, Pmid = {15525680}, Pst = {ppublish}, Title = {Mycobacterium tuberculosis functional network analysis by global subcellular protein profiling}, Volume = {16}, Year = {2005}, Bdsk-Url-1 = {http://dx.doi.org/10.1091/mbc.E04-04-0329}} @article{Sassetti:2003fk, Abstract = {Despite over a century of research, tuberculosis remains a leading cause of infectious death worldwide. Faced with increasing rates of drug resistance, the identification of genes that are required for the growth of this organism should provide new targets for the design of antimycobacterial agents. Here, we describe the use of transposon site hybridization (TraSH) to comprehensively identify the genes required by the causative agent, Mycobacterium tuberculosis, for optimal growth. These genes include those that can be assigned to essential pathways as well as many of unknown function. The genes important for the growth of M. tuberculosis are largely conserved in the degenerate genome of the leprosy bacillus, Mycobacterium leprae, indicating that non-essential functions have been selectively lost since this bacterium diverged from other mycobacteria. In contrast, a surprisingly high proportion of these genes lack identifiable orthologues in other bacteria, suggesting that the minimal gene set required for survival varies greatly between organisms with different evolutionary histories.}, Author = {Sassetti, Christopher M and Boyd, Dana H and Rubin, Eric J}, Date-Added = {2010-03-02 19:05:23 +0100}, Date-Modified = {2010-03-03 09:43:51 +0100}, Journal = {Mol Microbiol}, Journal-Full = {Molecular microbiology}, Keywords = {nopdf}, Mesh = {DNA Transposable Elements; Evolution, Molecular; Genes, Bacterial; Mutagenesis; Mycobacterium leprae; Mycobacterium tuberculosis}, Month = {Apr}, Number = {1}, Pages = {77-84}, Pmid = {12657046}, Pst = {ppublish}, Title = {Genes required for mycobacterial growth defined by high density mutagenesis}, Volume = {48}, Year = {2003}} @article{Provvedi:2009ye, Abstract = {In order to gain additional understanding of the physiological mechanisms used by bacteria to maintain surface homeostasis and to identify potential targets for new antibacterial drugs, we analysed the variation of the Mycobacterium tuberculosis transcriptional profile in response to inhibitory and subinhibitory concentrations of vancomycin. Our analysis identified 153 genes differentially regulated after exposing bacteria to a concentration of the drug ten times higher than the MIC, and 141 genes differentially expressed when bacteria were growing in a concentration of the drug eightfold lower than the MIC. Hierarchical clustering analysis indicated that the response to these different conditions is different, although with some overlap. This approach allowed us to identify several genes whose products could be involved in the protection from antibiotic stress targeting the envelope and help to confer the basal level of M. tuberculosis resistance to antibacterial drugs, such as Rv2623 (UspA-like), Rv0116c, PE20-PPE31, PspA and proteins related to toxin-antitoxin systems. Moreover, we also demonstrated that the alternative sigma factor sigma(E) confers basal resistance to vancomycin, once again underlining its importance in the physiology of the mycobacterial surface stress response.}, Author = {Provvedi, Roberta and Boldrin, Francesca and Falciani, Francesco and Pal{\`u}, Giorgio and Manganelli, Riccardo}, Date-Added = {2010-03-02 11:50:45 +0100}, Date-Modified = {2010-03-02 11:50:45 +0100}, Doi = {10.1099/mic.0.024802-0}, Journal = {Microbiology}, Journal-Full = {Microbiology (Reading, England)}, Mesh = {Anti-Bacterial Agents; Bacterial Proteins; Culture Media; Dose-Response Relationship, Drug; Drug Resistance, Bacterial; Gene Expression Profiling; Gene Expression Regulation, Bacterial; Heat-Shock Response; Humans; Molecular Sequence Data; Mycobacterium tuberculosis; Oligonucleotide Array Sequence Analysis; Vancomycin}, Month = {Apr}, Number = {Pt 4}, Pages = {1093-102}, Pmid = {19332811}, Pst = {ppublish}, Title = {Global transcriptional response to vancomycin in Mycobacterium tuberculosis}, Volume = {155}, Year = {2009}, Bdsk-Url-1 = {http://dx.doi.org/10.1099/mic.0.024802-0}} @article{Thum:2009fu, Abstract = {The Mycobacterium tuberculosis cmk gene, predicted to encode a CMP kinase (CMK), was cloned and expressed, and its product was purified to homogeneity. Steady-state kinetics confirmed that M. tuberculosis CMK is a monomer that preferentially phosphorylates CMP and dCMP by a sequential mechanism. A plausible role for CMK is discussed.}, Author = {Thum, Caroline and Schneider, Cristopher Z and Palma, Mario S and Santos, Di{\'o}genes S and Basso, Luiz A}, Date-Added = {2010-03-02 11:50:05 +0100}, Date-Modified = {2010-03-03 10:26:57 +0100}, Doi = {10.1128/JB.01337-08}, Journal = {J Bacteriol}, Journal-Full = {Journal of bacteriology}, Keywords = {single protein, Rv1712}, Mesh = {Amino Acid Sequence; Cloning, Molecular; Deoxycytidine Monophosphate; Gene Expression; Kinetics; Molecular Sequence Data; Mycobacterium tuberculosis; Nucleoside-Phosphate Kinase; Phosphorylation; Recombinant Proteins; Sequence Alignment}, Month = {Apr}, Number = {8}, Pages = {2884-7}, Pmc = {PMC2668428}, Pmid = {19181797}, Pst = {ppublish}, Title = {The Rv1712 Locus from Mycobacterium tuberculosis H37Rv codes for a functional CMP kinase that preferentially phosphorylates dCMP}, Volume = {191}, Year = {2009}, Bdsk-Url-1 = {http://dx.doi.org/10.1128/JB.01337-08}} @article{Be:2008lh, Abstract = {BACKGROUND: Tuberculosis of the central nervous system (CNS) is a serious, often fatal disease primarily affecting young children. It develops after hematogenous dissemination and subsequent invasion of the CNS by Mycobacterium tuberculosis. The microbial determinants involved in CNS disease are poorly characterized. METHODS: Hematogenously disseminated M. tuberculosis infection was simulated in BALB/c mice by intravenous challenge. Bacteria were recovered using standard culture techniques. Host immune response to M. tuberculosis infection was assessed by histopathological and cytokine profile analysis. By means of a pooled infection with genotypically defined M. tuberculosis mutants, bacterial genes required for invasion or survival were determined in the CNS and lung tissue. RESULTS: M. tuberculosis were detected in whole mouse brains as early as 1 day after intravenous infection and at all time points assessed thereafter. No significant immune response was elicited in the infected brain tissue, compared with extensive inflammation in the infected lung tissue at the same time point. We identified mutants for 5 M. tuberculosis genes (Rv0311, Rv0805, Rv0931c, Rv0986, and MT3280) with CNS-specific phenotypes, absent in lung tissue. CONCLUSIONS: We have identified CNS-specific M. tuberculosis genes involved in the pathogenesis of tuberculosis. Further characterization of these genes will help in understanding the microbial pathogenesis of CNS tuberculosis.}, Author = {Be, Nicholas A and Lamichhane, Gyanu and Grosset, Jacques and Tyagi, Sandeep and Cheng, Qi-Jian and Kim, Kwang Sik and Bishai, William R and Jain, Sanjay K}, Date-Added = {2010-03-02 11:48:13 +0100}, Date-Modified = {2010-03-02 11:48:13 +0100}, Doi = {10.1086/592447}, Journal = {J Infect Dis}, Journal-Full = {The Journal of infectious diseases}, Mesh = {Animals; Central Nervous System; Cytokines; Disease Models, Animal; Female; Genes, Bacterial; Mice; Mice, Inbred BALB C; Mutation; Mycobacterium tuberculosis; Tuberculosis, Central Nervous System}, Month = {Nov}, Number = {10}, Pages = {1520-8}, Pmid = {18956986}, Pst = {ppublish}, Title = {Murine model to study the invasion and survival of Mycobacterium tuberculosis in the central nervous system}, Volume = {198}, Year = {2008}, Bdsk-Url-1 = {http://dx.doi.org/10.1086/592447}} @article{Malen:2007ff, Abstract = {Proteins secreted by Mycobacterium tuberculosis play an essential role in the pathogenesis of tuberculosis. The culture filtrates of M. tuberculosis H37Rv made by Sadamu Nagai (Japan), are considerably enriched for secreted proteins compared to other culture filtrates. Complementary approaches were used to identify the secreted proteins in these culture filtrates: (i) 2-DE combined with MALDI-TOF MS and (ii) LC coupled MS/MS. Peptides derived from a total of 257 proteins were identified of which 144 were identified by more than one peptide. Several members of the immunologically important early secretory antigenic target-6 (ESAT-6) family of proteins were found to be major components. The majority of the identified proteins, 159 (62\%), were predicted to be exported through the general secretory pathway. We experimentally verified that the signal peptides, which mediate translocation through the cell membrane, had been removed in 41 of the identified proteins, and in 35 of those, there was an AXA motif N-terminally to the cleavage site, showing that this motif is important for the recognition and cleavage of signal peptides in mycobacteria. A large fraction of the secreted proteins were unknown, suggesting that we have mapped an unexplored part of the exported proteome of M. tuberculosis. complement.}, Author = {M{\aa}len, Hiwa and Berven, Frode S and Fladmark, Kari E and Wiker, Harald G}, Date-Added = {2010-03-02 11:46:58 +0100}, Date-Modified = {2010-03-02 11:46:58 +0100}, Doi = {10.1002/pmic.200600853}, Journal = {Proteomics}, Journal-Full = {Proteomics}, Mesh = {Amino Acid Sequence; Bacterial Proteins; Electrophoresis, Gel, Two-Dimensional; Molecular Sequence Data; Molecular Weight; Mycobacterium tuberculosis; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization}, Month = {May}, Number = {10}, Pages = {1702-18}, Pmid = {17443846}, Pst = {ppublish}, Title = {Comprehensive analysis of exported proteins from Mycobacterium tuberculosis H37Rv}, Volume = {7}, Year = {2007}, Bdsk-Url-1 = {http://dx.doi.org/10.1002/pmic.200600853}} @article{Stewart:2005pi, Abstract = {The pathogenesis of mycobacterial infection is associated with an ability to interfere with maturation of the phagosomal compartment after ingestion by macrophages. Identification of the mycobacterial components that contribute to this phenomenon will allow rational design of novel approaches to the treatment and prevention of tuberculosis. Microarray-based screening of a transposon library was used to identify mutations that influence the fate of Mycobacterium bovis bacille Calmette-Gu{\'e}rin (BCG) following uptake by macrophages. A screen based on bacterial survival during a 3-d infection highlighted genes previously implicated in growth of Mycobacterium tuberculosis in macrophages and in mice, together with a number of other virulence genes including a locus encoding virulence-associated membrane proteins and a series of transporter molecules. A second screen based on separation of acidified and non-acidified phagosomes by flow cytometry identified genes involved in mycobacterial control of early acidification. This included the KefB potassium/proton antiport. Mutants unable to control early acidification were significantly attenuated for growth during 6-d infections of macrophages. Early acidification of the phagosome is associated with reduced survival of BCG in macrophages. A strong correlation exists between genes required for intracellular survival of BCG and those required for growth of M. tuberculosis in mice. In contrast, very little correlation exists between genes required for intracellular survival of BCG and those that are up-regulated during intracellular adaptation of M. tuberculosis. This study has identified targets for interventions to promote immune clearance of tuberculosis infection. The screening technologies demonstrated in this study will be useful to the study of pathogenesis in many other intracellular microorganisms.}, Author = {Stewart, Graham R and Patel, Janisha and Robertson, Brian D and Rae, Aaron and Young, Douglas B}, Date-Added = {2010-03-02 11:45:53 +0100}, Date-Modified = {2010-03-02 11:45:53 +0100}, Doi = {10.1371/journal.ppat.0010033}, Journal = {PLoS Pathog}, Journal-Full = {PLoS pathogens}, Month = {Nov}, Number = {3}, Pages = {269-78}, Pmc = {PMC1291353}, Pmid = {16322769}, Pst = {ppublish}, Title = {Mycobacterial mutants with defective control of phagosomal acidification}, Volume = {1}, Year = {2005}, Bdsk-Url-1 = {http://dx.doi.org/10.1371/journal.ppat.0010033}} @article{Siroy:2008mi, Abstract = {Mycobacteria contain an outer membrane composed of mycolic acids and a large variety of other lipids. Its protective function is an essential virulence factor of Mycobacterium tuberculosis. Only OmpA, which has numerous homologs in Gram-negative bacteria, is known to form channels in the outer membrane of M. tuberculosis so far. Rv1698 was predicted to be an outer membrane protein of unknown function. Expression of rv1698 restored the sensitivity to ampicillin and chloramphenicol of a Mycobacterium smegmatis mutant lacking the main porin MspA. Uptake experiments showed that Rv1698 partially complemented the permeability defect of the M. smegmatis porin mutant for glucose. These results indicated that Rv1698 provides an unspecific pore that can partially substitute for MspA. Lipid bilayer experiments demonstrated that purified Rv1698 is an integral membrane protein that indeed produces channels. The main single channel conductance is 4.5 +/- 0.3 nanosiemens in 1 M KCl. Zero current potential measurements revealed a weak preference for cations. Whole cell digestion of recombinant M. smegmatis with proteinase K showed that Rv1698 is surface-accessible. Taken together, these experiments demonstrated that Rv1698 is a channel protein that is likely involved in transport processes across the outer membrane of M. tuberculosis. Rv1698 has single homologs of unknown functions in Corynebacterineae and thus represents the first member of a new class of channel proteins specific for mycolic acid-containing outer membranes.}, Author = {Siroy, Axel and Mailaender, Claudia and Harder, Daniel and Koerber, Stephanie and Wolschendorf, Frank and Danilchanka, Olga and Wang, Ying and Heinz, Christian and Niederweis, Michael}, Date-Added = {2010-03-02 11:41:32 +0100}, Date-Modified = {2010-03-03 10:27:08 +0100}, Doi = {10.1074/jbc.M800866200}, Journal = {J Biol Chem}, Journal-Full = {The Journal of biological chemistry}, Keywords = {single protein, Rv1698}, Mesh = {Anti-Bacterial Agents; Bacterial Outer Membrane Proteins; Cell Membrane; Endopeptidase K; Escherichia coli; Glucose; Lipid Bilayers; Models, Biological; Mutation; Mycobacterium bovis; Mycobacterium smegmatis; Mycobacterium tuberculosis; Point Mutation; Porins; Protein Structure, Secondary}, Month = {Jun}, Number = {26}, Pages = {17827-37}, Pmc = {PMC2440620}, Pmid = {18434314}, Pst = {ppublish}, Title = {Rv1698 of Mycobacterium tuberculosis represents a new class of channel-forming outer membrane proteins}, Volume = {283}, Year = {2008}, Bdsk-Url-1 = {http://dx.doi.org/10.1074/jbc.M800866200}} @article{Lavollay:2008qa, Abstract = {Our understanding of the mechanisms used by Mycobacterium tuberculosis to persist in a "dormant" state is essential to the development of therapies effective in sterilizing tissues. Gene expression profiling in model systems has revealed a complex adaptive response thought to endow M. tuberculosis with the capacity to survive several months of combinatorial antibiotic treatment. We show here that this adaptive response may involve remodeling of the peptidoglycan network by substitution of 4-->3 cross-links generated by the D,D-transpeptidase activity of penicillin-binding proteins by 3-->3 cross-links generated by a transpeptidase of L,D specificity. A candidate gene, previously shown to be upregulated upon nutrient starvation, was found to encode an L,D-transpeptidase active in the formation of 3-->3 cross-links. The enzyme, Ldt(Mt1), was inactivated by carbapenems, a class of beta-lactam antibiotics that are poorly hydrolyzed by the M. tuberculosis beta-lactamases. Ldt(Mt1) and carbapenems may therefore represent a target and a drug family relevant to the eradication of persistent M. tuberculosis.}, Author = {Lavollay, Marie and Arthur, Michel and Fourgeaud, Martine and Dubost, Lionel and Marie, Arul and Veziris, Nicolas and Blanot, Didier and Gutmann, Laurent and Mainardi, Jean-Luc}, Date-Added = {2010-03-02 11:41:14 +0100}, Date-Modified = {2010-03-03 10:29:37 +0100}, Doi = {10.1128/JB.00239-08}, Journal = {J Bacteriol}, Journal-Full = {Journal of bacteriology}, Keywords = {single protein, Rv0116c}, Mesh = {Anti-Bacterial Agents; Models, Biological; Mycobacterium tuberculosis; Peptidoglycan; Peptidyl Transferases; Spectrometry, Mass, Electrospray Ionization; beta-Lactams}, Month = {Jun}, Number = {12}, Pages = {4360-6}, Pmc = {PMC2446752}, Pmid = {18408028}, Pst = {ppublish}, Title = {The peptidoglycan of stationary-phase Mycobacterium tuberculosis predominantly contains cross-links generated by L,D-transpeptidation}, Volume = {190}, Year = {2008}, Bdsk-Url-1 = {http://dx.doi.org/10.1128/JB.00239-08}} @article{Slayden:2006kl, Abstract = {In Mycobacterium tuberculosis the mechanism of septum formation and regulation of cell division remains undefined. In other bacterial species FtsZ polymerization and septum formation are influenced through protein interactions in addition to transcriptional regulation, and the combination of these provides tight regulation of this process. However, homologues of proteins known to affect FtsZ assembly have not been identified and substantiated in M. tuberculosis. This suggests that M. tuberculosis may possess unique processes for regulation of septum formation. To begin to address this poorly understood aspect of M. tuberculosis physiology, FtsZ inhibitors were used to block cell division and the effects on bacterial morphology and the transcriptional response were examined. Inhibition of septum formation prevented cell division and led to bacterial filamentation. Microarray-based transcriptional profiling allowed the evaluation of multiple metabolic processes in response to inhibition of septum formation and when coupled with bioinformatics provided a means to identify regulatory elements and other gene products that probably influence septum formation.}, Author = {Slayden, Richard A and Knudson, Dennis L and Belisle, John T}, Date-Added = {2010-03-02 11:40:14 +0100}, Date-Modified = {2010-03-02 11:40:14 +0100}, Doi = {10.1099/mic.0.28762-0}, Journal = {Microbiology}, Journal-Full = {Microbiology (Reading, England)}, Mesh = {Bacterial Proteins; Cell Cycle; Cell Division; Cytoskeletal Proteins; Gene Expression Regulation, Bacterial; Humans; Microscopy, Electron; Mycobacterium tuberculosis; Oligonucleotide Array Sequence Analysis; Transcription, Genetic}, Month = {Jun}, Number = {Pt 6}, Pages = {1789-97}, Pmid = {16735741}, Pst = {ppublish}, Title = {Identification of cell cycle regulators in Mycobacterium tuberculosis by inhibition of septum formation and global transcriptional analysis}, Volume = {152}, Year = {2006}, Bdsk-Url-1 = {http://dx.doi.org/10.1099/mic.0.28762-0}} @article{Rachman:2006fu, Abstract = {As one of the world's most successful intracellular pathogens, Mycobacterium tuberculosis, the causative agent of human tuberculosis, is responsible for two to three million deaths annually. The pathogenicity of M. tuberculosis relies on its ability to survive and persist within host macrophage cells during infection. It is of central importance, therefore, to identify genes and pathways that are involved in the survival and persistence of M. tuberculosis within these cells. Utilizing genome-wide DNA arrays we have identified M. tuberculosis genes that are specifically induced during macrophage infection. To better understand the cellular context of these differentially expressed genes, we have also combined our array analyses with computational methods of protein network identification. Our combined approach reveals certain signatures of M. tuberculosis residing within macrophage cells, including the induction of genes involved in DNA damage repair, fatty acid degradation, iron metabolism, and cell wall metabolism.}, Author = {Rachman, Helmy and Strong, Michael and Schaible, Ulrich and Schuchhardt, Johannes and Hagens, Kristine and Mollenkopf, Hans and Eisenberg, David and Kaufmann, Stefan H E}, Date-Added = {2010-03-02 11:39:55 +0100}, Date-Modified = {2010-03-02 11:39:55 +0100}, Doi = {10.1016/j.micinf.2005.09.011}, Journal = {Microbes Infect}, Journal-Full = {Microbes and infection / Institut Pasteur}, Mesh = {Amino Acids; Bacterial Proteins; Biological Transport; Cell Membrane; Cell Wall; Cluster Analysis; DNA Repair; Gene Expression Profiling; Gene Expression Regulation, Bacterial; Iron; Lipid Metabolism; Mycobacterium tuberculosis; Up-Regulation}, Month = {Mar}, Number = {3}, Pages = {747-57}, Pmid = {16513384}, Pst = {ppublish}, Title = {Mycobacterium tuberculosis gene expression profiling within the context of protein networks}, Volume = {8}, Year = {2006}, Bdsk-Url-1 = {http://dx.doi.org/10.1016/j.micinf.2005.09.011}} @article{Manjunatha:2006dz, Abstract = {PA-824 is a promising new compound for the treatment of tuberculosis that is currently undergoing human trials. Like its progenitors metronidazole and CGI-17341, PA-824 is a prodrug of the nitroimidazole class, requiring bioreductive activation of an aromatic nitro group to exert an antitubercular effect. We have confirmed that resistance to PA-824 (a nitroimidazo-oxazine) and CGI-17341 (a nitroimidazo-oxazole) is most commonly mediated by loss of a specific glucose-6-phosphate dehydrogenase (FGD1) or its deazaflavin cofactor F420, which together provide electrons for the reductive activation of this class of molecules. Although FGD1 and F420 are necessary for sensitivity to these compounds, they are not sufficient and require additional accessory proteins that directly interact with the nitroimidazole. To understand more proximal events in the reductive activation of PA-824, we examined mutants that were wild-type for both FGD1 and F420 and found that, although these mutants had acquired high-level resistance to PA-824 (and another nitroimidazo-oxazine), they retained sensitivity to CGI-17341 (and a related nitroimidazo-oxazole). Microarray-based comparative genome sequencing of these mutants identified lesions in Rv3547, a conserved hypothetical protein with no known function. Complementation with intact Rv3547 fully restored sensitivity to nitroimidazo-oxazines and restored the ability of Mtb to metabolize PA-824. These results suggest that the sensitivity of Mtb to PA-824 and related compounds is mediated by a protein that is highly specific for subtle structural variations in these bicyclic nitroimidazoles.}, Author = {Manjunatha, Ujjini H and Boshoff, Helena and Dowd, Cynthia S and Zhang, Liang and Albert, Thomas J and Norton, Jason E and Daniels, Lacy and Dick, Thomas and Pang, Siew Siew and Barry, 3rd, Clifton E}, Date-Added = {2010-03-02 11:39:37 +0100}, Date-Modified = {2010-03-03 10:30:12 +0100}, Doi = {10.1073/pnas.0508392103}, Journal = {Proc Natl Acad Sci U S A}, Journal-Full = {Proceedings of the National Academy of Sciences of the United States of America}, Keywords = {single protein, Rv3547}, Mesh = {Bacterial Proteins; DNA Transposable Elements; Drug Resistance, Bacterial; Genome, Bacterial; Glucosephosphate Dehydrogenase; Molecular Sequence Data; Molecular Structure; Mutation; Mycobacterium tuberculosis; Nitroimidazoles; Oxazines; Phenotype}, Month = {Jan}, Number = {2}, Pages = {431-6}, Pmc = {PMC1326169}, Pmid = {16387854}, Pst = {ppublish}, Title = {Identification of a nitroimidazo-oxazine-specific protein involved in PA-824 resistance in Mycobacterium tuberculosis}, Volume = {103}, Year = {2006}, Bdsk-Url-1 = {http://dx.doi.org/10.1073/pnas.0508392103}} @article{Arcus:2005fv, Abstract = {PIN-domains (homologues of the pilT N-terminal domain) are small protein domains of approximately 140 amino acids. They are found in a diverse range of organisms and recent evidence from bioinformatics, biochemistry, structural biology and microbiology suggest that the majority of the prokaryotic PIN-domain proteins are the toxic components of toxin-antitoxin (TA) operons. Several microorganisms have a large cohort of these operons. For example, the genome of Mycobacterium tuberculosis encodes 48 PIN-domain proteins, of which 38 are thought to be involved in TA interactions. This large array of PIN-domain TA operons raises questions as to their evolutionary origin and contemporary functional significance. We suggest that the evolutionary origin of genes encoding mycobacterial PIN-domain TA operons is linked to the mobile gene pool, but that TA operons can become resident within the chromosome of host cells from where they might be recruited to fulfil a variety of roles associated with retardation of cell growth and persistence in stressful environments.}, Author = {Arcus, Vickery L and Rainey, Paul B and Turner, Susan J}, Date-Added = {2010-03-02 11:36:37 +0100}, Date-Modified = {2010-03-02 11:36:37 +0100}, Doi = {10.1016/j.tim.2005.06.008}, Journal = {Trends Microbiol}, Journal-Full = {Trends in microbiology}, Mesh = {Adenosine Triphosphatases; Amino Acid Sequence; Antitoxins; Bacterial Proteins; Models, Molecular; Molecular Motor Proteins; Molecular Sequence Data; Mycobacterium tuberculosis; Operon; Protein Structure, Tertiary; Sequence Alignment}, Month = {Aug}, Number = {8}, Pages = {360-5}, Pmid = {15993073}, Pst = {ppublish}, Title = {The PIN-domain toxin-antitoxin array in mycobacteria}, Volume = {13}, Year = {2005}, Bdsk-Url-1 = {http://dx.doi.org/10.1016/j.tim.2005.06.008}} @article{Xiong:2005bs, Abstract = {Because many membrane-associated proteins represent potential drug targets, diagnostic probes, and components of vaccines, we have chosen to study the membrane proteins of Mycobacterium tuberculosis H37Rv. To remove cytosolic proteins and facilitate access to the integral membrane proteins, membrane fractions of M. tuberculosis H37Rv were intensely washed with 5 M urea and high pH carbonate solution. One-dimensional SDS-PAGE, followed by enzymatic hydrolysis and nanoLC electrospray ionization MS/MS, proved to be the most efficient way to identify the proteins contained within the membrane fraction. Here we report 349 protein identifications in total, validated by at least two tryptic peptide matches and MOWSE scores greater than 75. Of those 349 proteins, 100 are integral membrane proteins with at least one predicted transmembrane alpha helix (excluding the possible signal sequence). 84 M. tuberculosis H37Rv proteins, including 42 integral membrane proteins, are described for the first time.}, Author = {Xiong, Ying and Chalmers, Michael J and Gao, Fei Philip and Cross, Timothy A and Marshall, Alan G}, Date = {2005 May-Jun}, Date-Added = {2010-03-02 11:36:00 +0100}, Date-Modified = {2010-03-02 11:36:00 +0100}, Doi = {10.1021/pr0500049}, Journal = {J Proteome Res}, Journal-Full = {Journal of proteome research}, Mesh = {Bacterial Proteins; Chromatography, High Pressure Liquid; Electrophoresis, Polyacrylamide Gel; Membrane Proteins; Mycobacterium tuberculosis; Proteomics; Spectrometry, Mass, Electrospray Ionization; Trypsin; Urea}, Number = {3}, Pages = {855-61}, Pmid = {15952732}, Pst = {ppublish}, Title = {Identification of Mycobacterium tuberculosis H37Rv integral membrane proteins by one-dimensional gel electrophoresis and liquid chromatography electrospray ionization tandem mass spectrometry}, Volume = {4}, Year = {2005}, Bdsk-Url-1 = {http://dx.doi.org/10.1021/pr0500049}} @article{Rengarajan:2005ij, Abstract = {Macrophages are central to host defense against microbes, but intracellular pathogens have evolved to evade their antimicrobial functions. Mycobacterium tuberculosis (MTB) has successfully exploited macrophages as its primary niche in vivo, but the bacterial genome-wide requirements that promote its intracellular survival remain undefined. Here we comprehensively identify the MTB genes required for survival by screening for transposon mutants that fail to grow within primary macrophages. We identify mutants showing decreased growth in macrophage environments that model stages of the host immune response. By systematically analyzing several biologically relevant data sets, we have been able to identify putative pathways that could not be predicted by genome organization alone. In one example, phosphate transport, requiring physically unlinked genes, was found to be critical for MTB growth in macrophages and important for establishing persistent infection in lungs. Remarkably, the majority of MTB genes found by this analysis to be required for survival are constitutively expressed rather than regulated by macrophages, revealing the host-adapted lifestyle of an evolutionarily selected intracellular pathogen.}, Author = {Rengarajan, Jyothi and Bloom, Barry R and Rubin, Eric J}, Date-Added = {2010-03-02 11:35:32 +0100}, Date-Modified = {2010-03-02 11:35:32 +0100}, Doi = {10.1073/pnas.0503272102}, Journal = {Proc Natl Acad Sci U S A}, Journal-Full = {Proceedings of the National Academy of Sciences of the United States of America}, Mesh = {Adaptation, Physiological; Animals; Biological Transport; Gene Expression Profiling; Gene Expression Regulation, Bacterial; Genes, Bacterial; Genome, Bacterial; Macrophages; Mice; Mice, Inbred C57BL; Mycobacterium tuberculosis; Operon; Organ Specificity; Phosphates}, Month = {Jun}, Number = {23}, Pages = {8327-32}, Pmc = {PMC1142121}, Pmid = {15928073}, Pst = {ppublish}, Title = {Genome-wide requirements for Mycobacterium tuberculosis adaptation and survival in macrophages}, Volume = {102}, Year = {2005}, Bdsk-Url-1 = {http://dx.doi.org/10.1073/pnas.0503272102}} @article{Dubnau:2005hc, Abstract = {Using a promoter trap, we have identified 56 Mycobacterium tuberculosis genes preferentially expressed in the mouse lung. Quantitative real-time PCR showed that RNA levels of several genes were higher from bacteria growing in mouse lungs than from broth cultures. These results support the current hypothesis that Mycobacterium tuberculosis utilizes fatty acids as a carbon source in the mouse lung.}, Author = {Dubnau, Eugenie and Chan, John and Mohan, V P and Smith, Issar}, Date-Added = {2010-03-02 11:35:00 +0100}, Date-Modified = {2010-03-02 11:35:00 +0100}, Doi = {10.1128/IAI.73.6.3754-3757.2005}, Journal = {Infect Immun}, Journal-Full = {Infection and immunity}, Mesh = {Animals; Antigens, Bacterial; Bacterial Proteins; Drug Resistance, Bacterial; Isoniazid; Lung; Mice; Mycobacterium tuberculosis; Oxidoreductases; Reverse Transcriptase Polymerase Chain Reaction; Sequence Analysis, DNA}, Month = {Jun}, Number = {6}, Pages = {3754-7}, Pmc = {PMC1111836}, Pmid = {15908407}, Pst = {ppublish}, Title = {responses of mycobacterium tuberculosis to growth in the mouse lung}, Volume = {73}, Year = {2005}, Bdsk-Url-1 = {http://dx.doi.org/10.1128/IAI.73.6.3754-3757.2005}} @article{Hisert:2004tg, Abstract = {Tuberculosis (TB) is characterized by lifetime persistence of Mycobacterium tuberculosis. Despite the induction of a vigorous host immune response that curtails disease progression in the majority of cases, the organism is not eliminated. Subsequent immunosuppression can lead to reactivation after a prolonged period of clinical latency. Thus, while it is clear that protective immune mechanisms are engaged during M. tuberculosis infection, it also appears that the pathogen has evolved effective countermechanisms. Genetic studies with animal infection models and with patients have revealed a key role for the cytokine gamma interferon (IFN-gamma) in resistance to TB. IFN-gamma activates a large number of antimicrobial pathways. Three of these IFN-gamma-dependent mechanisms have been implicated in defense against M. tuberculosis: inducible nitric oxide synthase (iNOS), phagosome oxidase (phox), and the phagosome-associated GTPase LRG-47. In order to identify bacterial genes that provide protection against specific host immune pathways, we have developed the strategy of differential signature-tagged transposon mutagenesis. Using this approach we have identified three M. tuberculosis genes that are essential for progressive M. tuberculosis growth and rapid lethality in iNOS-deficient mice but not in IFN-gamma-deficient mice. We propose that these genes are involved in pathways that allow M. tuberculosis to counter IFN-gamma-dependent immune mechanisms other than iNOS.}, Author = {Hisert, Katherine B and Kirksey, Meghan A and Gomez, James E and Sousa, Alexandra O and Cox, Jeffery S and Jacobs, Jr, William R and Nathan, Carl F and McKinney, John D}, Date-Added = {2010-03-02 11:34:40 +0100}, Date-Modified = {2010-03-02 11:34:40 +0100}, Doi = {10.1128/IAI.72.9.5315-5321.2004}, Journal = {Infect Immun}, Journal-Full = {Infection and immunity}, Mesh = {Animals; Bacterial Proteins; DNA Transposable Elements; Female; Humans; Interferon-gamma; Lung; Male; Mice; Mice, Inbred C57BL; Mutagenesis; Mutation; Mycobacterium tuberculosis; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Tuberculosis, Pulmonary}, Month = {Sep}, Number = {9}, Pages = {5315-21}, Pmc = {PMC517420}, Pmid = {15322028}, Pst = {ppublish}, Title = {Identification of Mycobacterium tuberculosis counterimmune (cim) mutants in immunodeficient mice by differential screening}, Volume = {72}, Year = {2004}, Bdsk-Url-1 = {http://dx.doi.org/10.1128/IAI.72.9.5315-5321.2004}} @article{Daniel:2004kl, Abstract = {Mycobacterium tuberculosis enters the host by inhalation of an infectious aerosol and replicates in the alveolar macrophages until the host's immune defense causes bacteriostasis, which leads the pathogen to go into nonreplicative drug-resistant dormancy. The dormant pathogen can survive for decades till the host's immune system is weakened and active tuberculosis develops. Even though fatty acids are thought to be the major energy source required for the persistence phase, the source of fatty acids used is not known. We postulate that the pathogen uses triacylglycerol (TG) as a storage form of fatty acids. Little is known about the biosynthesis of TG in M. tuberculosis. We show that 15 mycobacterial genes that we identified as putative triacylglycerol synthase (tgs) when expressed in Escherichia coli showed TGS activity, and we report some basic catalytic characteristics of the most active enzymes. We show that several tgs genes are induced when the pathogen goes into the nonreplicative drug-resistant state caused by slow withdrawal of O(2) and also by NO treatment, which is known to induce dormancy-associated genes. The gene (Rv3130c) that shows the highest TGS activity when expressed in E. coli shows the highest induction by hypoxia and NO treatment. Biochemical evidence shows that TG synthesis and accumulation occur under both conditions. We conclude that TG may be a form of energy storage for use during long-term dormancy. Therefore, TG synthesis may be an appropriate target for novel antilatency drugs that can prevent the organism from surviving dormancy and thus assist in the control of tuberculosis.}, Author = {Daniel, Jaiyanth and Deb, Chirajyoti and Dubey, Vinod S and Sirakova, Tatiana D and Abomoelak, Bassam and Morbidoni, Hector R and Kolattukudy, Pappachan E}, Date-Added = {2010-03-02 11:34:16 +0100}, Date-Modified = {2010-03-02 11:34:16 +0100}, Doi = {10.1128/JB.186.15.5017-5030.2004}, Journal = {J Bacteriol}, Journal-Full = {Journal of bacteriology}, Mesh = {Acyltransferases; Anaerobiosis; Bacterial Proteins; Culture Media; Diacylglycerol O-Acyltransferase; Enzyme Induction; Escherichia coli; Gene Expression Regulation, Bacterial; Mycobacterium tuberculosis; Nitric Oxide; Triglycerides}, Month = {Aug}, Number = {15}, Pages = {5017-30}, Pmc = {PMC451596}, Pmid = {15262939}, Pst = {ppublish}, Title = {Induction of a novel class of diacylglycerol acyltransferases and triacylglycerol accumulation in Mycobacterium tuberculosis as it goes into a dormancy-like state in culture}, Volume = {186}, Year = {2004}, Bdsk-Url-1 = {http://dx.doi.org/10.1128/JB.186.15.5017-5030.2004}} @article{Cowley:2004oq, Abstract = {The function of the Mycobacterium tuberculosis eukaryotic-like protein serine/threonine kinase PknG was investigated by gene knock-out and by expression and biochemical analysis. The pknG gene (Rv0410c), when cloned and expressed in Escherichia coli, encodes a functional kinase. An in vitro kinase assay of the recombinant protein demonstrated that PknG can autophosphorylate its kinase domain as well as its 30 kDa C-terminal portion, which contains a tetratricopeptide (TPR) structural signalling motif. Western analysis revealed that PknG is located in the cytosol as well as in mycobacterial membrane. The pknG gene was inactivated by allelic exchange in M. tuberculosis. The resulting mutant strain causes delayed mortality in SCID mice and displays decreased viability both in vitro and upon infection of BALB/c mice. The reduced growth of the mutant was more pronounced in the stationary phase of the mycobacterial growth cycle and when grown in nutrient-depleted media. The PknG-deficient mutant accumulates glutamate and glutamine. The cellular levels of these two amino acids reached approximately threefold of their parental strain levels. Higher cellular levels of the amine sugar-containing molecules, GlcN-Ins and mycothiol, which are derived from glutamate, were detected in the DeltapknG mutant. De novo glutamine synthesis was shown to be reduced by 50\%. This is consistent with current knowledge suggesting that glutamine synthesis is regulated by glutamate and glutamine levels. These data support our hypothesis that PknG mediates the transfer of signals sensing nutritional stress in M. tuberculosis and translates them into metabolic adaptation.}, Author = {Cowley, Siobhan and Ko, Mary and Pick, Neora and Chow, Rayken and Downing, Katrina J and Gordhan, Bhavna G and Betts, Joanna C and Mizrahi, Valerie and Smith, Debbie A and Stokes, Richard W and Av-Gay, Yossef}, Date-Added = {2010-03-02 11:33:40 +0100}, Date-Modified = {2010-03-03 10:30:51 +0100}, Doi = {10.1111/j.1365-2958.2004.04085.x}, Journal = {Mol Microbiol}, Journal-Full = {Molecular microbiology}, Keywords = {single protein, Rv0410c}, Mesh = {Animals; Bacterial Proteins; Blotting, Western; Cell Membrane; Cloning, Molecular; Cytoplasm; Escherichia coli; Gene Deletion; Gene Expression Regulation, Bacterial; Genes, Bacterial; Glutamic Acid; Glutamine; Mice; Mice, Inbred BALB C; Mice, SCID; Mutagenesis, Insertional; Mycobacterium tuberculosis; Protein-Serine-Threonine Kinases; Recombinant Proteins; Tuberculosis; Virulence}, Month = {Jun}, Number = {6}, Pages = {1691-702}, Pmid = {15186418}, Pst = {ppublish}, Title = {The Mycobacterium tuberculosis protein serine/threonine kinase PknG is linked to cellular glutamate/glutamine levels and is important for growth in vivo}, Volume = {52}, Year = {2004}, Bdsk-Url-1 = {http://dx.doi.org/10.1111/j.1365-2958.2004.04085.x}} @article{Walburger:2004nx, Abstract = {Pathogenic mycobacteria resist lysosomal delivery after uptake into macrophages, allowing them to survive intracellularly. We found that the eukaryotic-like serine/threonine protein kinase G from pathogenic mycobacteria was secreted within macrophage phagosomes, inhibiting phagosome-lysosome fusion and mediating intracellular survival of mycobacteria. Inactivation of protein kinase G by gene disruption or chemical inhibition resulted in lysosomal localization and mycobacterial cell death in infected macrophages. Besides identifying a target for the control of mycobacterial infections, these findings suggest that pathogenic mycobacteria have evolved eukaryotic-like signal transduction mechanisms capable of modulating host cell trafficking pathways.}, Author = {Walburger, Anne and Koul, Anil and Ferrari, Giorgio and Nguyen, Liem and Prescianotto-Baschong, Cristina and Huygen, Kris and Klebl, Bert and Thompson, Charles and Bacher, Gerald and Pieters, Jean}, Date-Added = {2010-03-02 11:33:22 +0100}, Date-Modified = {2010-03-03 10:32:05 +0100}, Doi = {10.1126/science.1099384}, Journal = {Science}, Journal-Full = {Science (New York, N.Y.)}, Keywords = {single protein, Rv0410c}, Mesh = {Amides; Animals; Cell Line; Cyclic GMP-Dependent Protein Kinases; Enzyme Inhibitors; Gene Deletion; Lysosomes; Macrophages; Mice; Mycobacterium bovis; Mycobacterium smegmatis; Mycobacterium tuberculosis; Phagosomes; Signal Transduction; Thiophenes; Vacuoles}, Month = {Jun}, Number = {5678}, Pages = {1800-4}, Pmid = {15155913}, Pst = {ppublish}, Title = {Protein kinase G from pathogenic mycobacteria promotes survival within macrophages}, Volume = {304}, Year = {2004}, Bdsk-Url-1 = {http://dx.doi.org/10.1126/science.1099384}} @article{Shimono:2003cr, Abstract = {An estimated one-third of the world's population is latently infected with Mycobacterium tuberculosis, the etiologic agent of tuberculosis. Here, we demonstrate that, unlike wild-type M. tuberculosis, a strain of M. tuberculosis disrupted in the mce1 operon was unable to enter a stable persistent state of infection in mouse lungs. Instead, the mutant continued to replicate and killed the mice more rapidly than did the wild-type strain. Histological examination of mouse lungs infected with the mutant strain revealed diffusely organized granulomas with aberrant inflammatory cell migration. Murine macrophages infected ex vivo with the mutant strain were reduced in their ability to produce tumor necrosis factor alpha, IL-6, monocyte chemoattractant protein 1, and nitric oxide (NO), but not IL-4. The mce1 mutant strain complemented with the mce1 genes stimulated tumor necrosis factor alpha and NO production by murine macrophages at levels stimulated by the wild-type strain. These observations indicate that the mce1 operon mutant is unable to stimulate T helper 1-type immunity in mice. The hypervirulence of the mutant strain may have resulted from its inability to stimulate a proinflammatory response that would otherwise induce organized granuloma formation and control the infection without killing the organism. The mce1 operon of M. tuberculosis may be involved in modulating the host inflammatory response in such a way that the bacterium can enter a persistent state without being eliminated or causing disease in the host.}, Author = {Shimono, Nobuyuki and Morici, Lisa and Casali, Nicola and Cantrell, Sally and Sidders, Ben and Ehrt, Sabine and Riley, Lee W}, Date-Added = {2010-03-02 11:32:56 +0100}, Date-Modified = {2010-03-03 10:35:39 +0100}, Doi = {10.1073/pnas.2433882100}, Journal = {Proc Natl Acad Sci U S A}, Journal-Full = {Proceedings of the National Academy of Sciences of the United States of America}, Keywords = {single protein, Rv0169}, Mesh = {Animals; Bacterial Proteins; Cytokines; Female; Lung; Macrophages; Mice; Mice, Inbred BALB C; Mycobacterium bovis; Mycobacterium tuberculosis; Operon; Tuberculosis; Virulence}, Month = {Dec}, Number = {26}, Pages = {15918-23}, Pmc = {PMC307668}, Pmid = {14663145}, Pst = {ppublish}, Title = {Hypervirulent mutant of Mycobacterium tuberculosis resulting from disruption of the mce1 operon}, Volume = {100}, Year = {2003}, Bdsk-Url-1 = {http://dx.doi.org/10.1073/pnas.2433882100}} @article{Majlessi:2003dq, Abstract = {Here we describe the identification of a new CD8(+)-T-cell epitope, the GYAGTLQSL nonamer, shared by the TB10.3 and TB10.4 proteins of the Mycobacterium tuberculosis ESAT-6 family. Cytotoxic T cells from mycobacterium-infected mice efficiently recognized this epitope. GYAGTLQSL-specific T-cell hybridomas, which were able to recognize Mycobacterium bovis BCG-infected macrophages, were generated and now allow investigation of mycobacterial-antigen processing through the major histocompatibility complex class I pathway.}, Author = {Majlessi, Laleh and Rojas, Marie-J{\'e}sus and Brodin, Priscille and Leclerc, Claude}, Date-Added = {2010-03-02 11:32:37 +0100}, Date-Modified = {2010-03-02 11:32:37 +0100}, Journal = {Infect Immun}, Journal-Full = {Infection and immunity}, Mesh = {Amino Acid Sequence; Animals; Antigens, Bacterial; Bacterial Proteins; CD8-Positive T-Lymphocytes; Epitopes, T-Lymphocyte; Histocompatibility Antigens Class I; Hybridomas; Immunization; Macrophages; Mice; Mice, Inbred BALB C; Molecular Sequence Data; Mycobacterium bovis; Mycobacterium tuberculosis; T-Lymphocytes, Cytotoxic; Tuberculosis, Pulmonary}, Month = {Dec}, Number = {12}, Pages = {7173-7}, Pmc = {PMC308897}, Pmid = {14638811}, Pst = {ppublish}, Title = {CD8+-T-cell responses of Mycobacterium-infected mice to a newly identified major histocompatibility complex class I-restricted epitope shared by proteins of the ESAT-6 family}, Volume = {71}, Year = {2003}} @article{Casali:2002bh, Abstract = {The mce1A gene of Mycobacterium tuberculosis was initially identified by its ability to promote uptake of Escherichia coli into HeLa cells. It was subsequently shown that this activity was confined to a 58-amino-acid region of the protein. A 72-amino-acid fragment (InvX) incorporating this active peptide was expressed in E. coli as a fusion to the AIDA (adhesin involved in diffuse adherence) autotransporter translocator, and its stable expression on the surface of the bacterium was demonstrated. Recombinant E. coli expressing InvX-AIDA showed extensive association with HeLa cells, and InvX was shown to be sufficient for internalization. Uptake was found to be both microtubule and microfilament dependent and required the Rho family of GTPases. Thus, the E. coli AIDA system facilitated both the qualitative and quantitative analysis of the functional domain of a heterologous protein.}, Author = {Casali, Nicola and Konieczny, Marc and Schmidt, M Alexander and Riley, Lee W}, Date-Added = {2010-03-02 11:32:09 +0100}, Date-Modified = {2010-03-03 10:35:57 +0100}, Journal = {Infect Immun}, Journal-Full = {Infection and immunity}, Keywords = {single protein, Rv0169}, Mesh = {Adhesins, Escherichia coli; Bacterial Adhesion; Bacterial Proteins; Escherichia coli; Hela Cells; Humans; Microscopy, Electron; Microscopy, Fluorescence; Mycobacterium tuberculosis; Peptides; Recombinant Fusion Proteins}, Month = {Dec}, Number = {12}, Pages = {6846-52}, Pmc = {PMC133103}, Pmid = {12438361}, Pst = {ppublish}, Title = {Invasion activity of a Mycobacterium tuberculosis peptide presented by the Escherichia coli AIDA autotransporter}, Volume = {70}, Year = {2002}} @article{Rodriguez:2002qf, Abstract = {The mycobacterial IdeR protein is a metal-dependent regulator of the DtxR (diphtheria toxin repressor) family. In the presence of iron, it binds to a specific DNA sequence in the promoter regions of the genes that it regulates, thus controlling their transcription. In this study, we provide evidence that ideR is an essential gene in Mycobacterium tuberculosis. ideR cannot normally be disrupted in this mycobacterium in the absence of a second functional copy of the gene. However, a rare ideR mutant was obtained in which the lethal effects of ideR inactivation were alleviated by a second-site suppressor mutation and which exhibited restricted iron assimilation capacity. Studies of this strain and a derivative in which IdeR expression was restored allowed us to identify phenotypic effects resulting from ideR inactivation. Using DNA microarrays, the iron-dependent transcriptional profiles of the wild-type, ideR mutant, and ideR-complemented mutant strains were analyzed, and the genes regulated by iron and IdeR were identified. These genes encode a variety of proteins, including putative transporters, proteins involved in siderophore synthesis and iron storage, members of the PE/PPE family, a membrane protein involved in virulence, transcriptional regulators, and enzymes involved in lipid metabolism.}, Author = {Rodriguez, G Marcela and Voskuil, Martin I and Gold, Benjamin and Schoolnik, Gary K and Smith, Issar}, Date-Added = {2010-03-02 11:31:47 +0100}, Date-Modified = {2010-03-03 10:36:59 +0100}, Journal = {Infect Immun}, Journal-Full = {Infection and immunity}, Keywords = {single protein, Rv2711}, Mesh = {Bacterial Proteins; Gene Expression; Hydrogen Peroxide; Iron; Mycobacterium tuberculosis; Oxidative Stress; Repressor Proteins; Siderophores}, Month = {Jul}, Number = {7}, Pages = {3371-81}, Pmc = {PMC128082}, Pmid = {12065475}, Pst = {ppublish}, Title = {ideR, An essential gene in mycobacterium tuberculosis: role of IdeR in iron-dependent gene expression, iron metabolism, and oxidative stress response}, Volume = {70}, Year = {2002}} @article{Dubnau:2002ve, Abstract = {We identified Mycobacterium tuberculosis genes preferentially expressed during infection of human macrophages using a promoter trap adapted for this pathogen. inhA encodes an enoyl-acyl carrier protein reductase that is required for mycolic acid biosynthesis (A. Quemard et al., Biochemistry 34:8235-8241, 1995) and is a major target for isoniazid (INH) in mycobacterial species (A. Banerjee et al., Science 263:227-230, 1994). Since overexpression of inhA confers INH resistance in Mycobacterium smegmatis (Banerjee et al., Science 263:227-230, 1994), we designed a promoter trap based on this gene. A library of clones, containing small fragments of M. tuberculosis DNA cloned upstream of inhA in a plasmid vector, was electroporated into M. tuberculosis, and the resulting culture was used to infect the human monocytic THP-1 cell line. Selection was made for clones surviving INH treatment during infection but retaining INH sensitivity on plates. The DNA upstream of inhA was sequenced in each clone to identify the promoter driving inhA expression. Thirteen genes identified by this method were analyzed by quantitative reverse transcription-PCR (R. Manganelli et al., Mol. Microbiol. 31:715-724, 1999), and eight of them were found to be differentially expressed from cultures grown in macrophages compared with broth-grown cultures. Several of these genes are presumed to be involved in fatty acid metabolism; one potentially codes for a unique DNA binding protein, one codes for a possible potassium channel protein, and the others code for proteins of unknown function. Genes which are induced during infection are likely to be significant for survival and growth of the pathogen; our results lend support to the view that fatty acid metabolism is essential for the virulence of M. tuberculosis.}, Author = {Dubnau, Eugenie and Font{\'a}n, Patricia and Manganelli, Riccardo and Soares-Appel, Sonia and Smith, Issar}, Date-Added = {2010-03-02 11:31:22 +0100}, Date-Modified = {2010-03-02 11:31:22 +0100}, Journal = {Infect Immun}, Journal-Full = {Infection and immunity}, Mesh = {Bacterial Proteins; Cell Line; Cloning, Molecular; Gene Expression; Genes, Bacterial; Genetic Engineering; Humans; Macrophages; Mycobacterium tuberculosis; Oxidoreductases; Promoter Regions, Genetic; Reverse Transcriptase Polymerase Chain Reaction}, Month = {Jun}, Number = {6}, Pages = {2787-95}, Pmc = {PMC127980}, Pmid = {12010964}, Pst = {ppublish}, Title = {Mycobacterium tuberculosis genes induced during infection of human macrophages}, Volume = {70}, Year = {2002}} @article{Betts:2002ly, Abstract = {The search for new TB drugs that rapidly and effectively sterilize the tissues and are thus able to shorten the duration of chemotherapy from the current 6 months has been hampered by a lack of understanding of the metabolism of the bacterium when in a 'persistent' or latent form. Little is known about the condition in which the bacilli survive, although laboratory models have shown that Mycobacterium tuberculosis can exist in a non-growing, drug-resistant state that may mimic persistence in vivo. Using nutrient starvation, we have established a model in which M. tuberculosis arrests growth, decreases its respiration rate and is resistant to isoniazid, rifampicin and metronidazole. We have used microarray and proteome analysis to investigate the response of M. tuberculosis to nutrient starvation. Proteome analysis of 6-week-starved cultures revealed the induction of several proteins. Microarray analysis enabled us to monitor gene expression during adaptation to nutrient starvation and confirmed the changes seen at the protein level. This has provided evidence for slowdown of the transcription apparatus, energy metabolism, lipid biosynthesis and cell division in addition to induction of the stringent response and several other genes that may play a role in maintaining long-term survival within the host. Thus, we have generated a model with which we can search for agents active against persistent M. tuberculosis and revealed a number of potential targets expressed under these conditions.}, Author = {Betts, Joanna C and Lukey, Pauline T and Robb, Linda C and McAdam, Ruth A and Duncan, Ken}, Date-Added = {2010-03-02 11:31:04 +0100}, Date-Modified = {2010-03-02 11:31:04 +0100}, Journal = {Mol Microbiol}, Journal-Full = {Molecular microbiology}, Mesh = {Adaptation, Physiological; Bacterial Proteins; Cell Membrane; Electrophoresis, Gel, Two-Dimensional; Energy Metabolism; Gene Expression Profiling; Gene Expression Regulation, Bacterial; Lipids; Models, Biological; Mycobacterium tuberculosis; Oligonucleotide Array Sequence Analysis; Oxygen; Protein Biosynthesis; RNA, Bacterial; Ribosomes; Rifampin; Transcription, Genetic}, Month = {Feb}, Number = {3}, Pages = {717-31}, Pmid = {11929527}, Pst = {ppublish}, Title = {Evaluation of a nutrient starvation model of Mycobacterium tuberculosis persistence by gene and protein expression profiling}, Volume = {43}, Year = {2002}} @article{Chitale:2001vn, Abstract = {The ability to gain entry and resist the antimicrobial intracellular environment of mammalian cells is an essential virulence property of Mycobacterium tuberculosis. A purified recombinant protein expressed by a 1362 bp locus (mce1) in the M. tuberculosis genome promoted uptake into HeLa cells of polystyrene latex microspheres coated with the protein. N-terminus deletion constructs of Mce1 identified a domain located between amino acid positions 106 and 163 that was needed for this cell uptake activity. Mce1 contained hydrophobic stretches at the N-terminus predictive of a signal sequence, and colloidal gold immunoelectron microscopy indicated that the corresponding native protein is expressed on the surface of the M. tuberculosis organism. The complete M. tuberculosis genome sequence revealed that it contained four homologues of mce (mce1, mce2, mce3, mce4) and that they were all located within operons composed of genes arranged similarly at different locations in the chromosome. Recombinant Mce2, which had the highest level of identity (67\%) to Mce1, was unable to promote the association of microspheres with HeLa cells. Although the exact function of Mce1 is still unknown, it appears to serve as an effector molecule expressed on the surface of M. tuberculosis that is capable of eliciting plasma membrane perturbations in non-phagocytic mammalian cells.}, Author = {Chitale, S and Ehrt, S and Kawamura, I and Fujimura, T and Shimono, N and Anand, N and Lu, S and Cohen-Gould, L and Riley, L W}, Date-Added = {2010-03-02 11:28:58 +0100}, Date-Modified = {2010-03-03 10:37:32 +0100}, Journal = {Cell Microbiol}, Journal-Full = {Cellular microbiology}, Keywords = {single protein, Rv0169}, Mesh = {Antibodies, Bacterial; Bacterial Proteins; Cell Membrane; Escherichia coli; Genes, Bacterial; Hela Cells; Humans; Immunoblotting; Microscopy, Immunoelectron; Microspheres; Mycobacterium tuberculosis; Open Reading Frames; Operon; Recombinant Proteins}, Month = {Apr}, Number = {4}, Pages = {247-54}, Pmid = {11298648}, Pst = {ppublish}, Title = {Recombinant Mycobacterium tuberculosis protein associated with mammalian cell entry}, Volume = {3}, Year = {2001}} @article{Kawai:2000kx, Abstract = {An enzyme with both inorganic polyphosphate [poly(P)]- and ATP-dependent NAD kinase activities was isolated from Micrococcus flavus. The enzyme was a dimer consisting of 34 kDa subunits, and was named poly(P)/ATP-NAD kinase. Internal amino acid sequences of the enzyme showed homologies with some function-unknown proteins released on the GenBank database. Among such proteins, hypothetical Rv1695 protein (Accession No. Z98268-16), which was encoded by a gene named "Rv1695" on genomic DNA of Mycobacterium tuberculosis H37Rv, was proposed to be poly(P)-dependent NAD kinase. By cloning and expression in Escherichia coli, Rv1695 was shown to encode poly(P)/ATP-NAD kinase and named ppnk. The ppnk product, recombinant-poly(P)/ATP-NAD kinase (Ppnk) was purified and characterized. The enzyme was a tetramaer consisting of 35 kDa subunits when expressed in E. coli. Poly(P)/ATP-NAD kinases of M. flavus and Ppnk of M. tuberculosis H37Rv specifically and completely phosphorylated NAD by utilizing commercially available poly(P)s and nucleoside triphosphates as phosphoryl donors.}, Author = {Kawai, S and Mori, S and Mukai, T and Suzuki, S and Yamada, T and Hashimoto, W and Murata, K}, Date-Added = {2010-03-02 11:28:33 +0100}, Date-Modified = {2010-03-03 10:37:53 +0100}, Doi = {10.1006/bbrc.2000.3433}, Journal = {Biochem Biophys Res Commun}, Journal-Full = {Biochemical and biophysical research communications}, Keywords = {single protein, Rv1695}, Mesh = {Amino Acid Sequence; Bacterial Proteins; Micrococcus; Molecular Sequence Data; Mycobacterium tuberculosis; Phosphotransferases (Alcohol Group Acceptor); Polyphosphates}, Month = {Sep}, Number = {1}, Pages = {57-63}, Pmid = {11006082}, Pst = {ppublish}, Title = {Inorganic Polyphosphate/ATP-NAD kinase of Micrococcus flavus and Mycobacterium tuberculosis H37Rv}, Volume = {276}, Year = {2000}, Bdsk-Url-1 = {http://dx.doi.org/10.1006/bbrc.2000.3433}} @article{Rindi:1999fk, Abstract = {An mRNA differential display (DD) assay was developed to compare gene expression between Mycobacterium tuberculosis H37Rv and its avirulent mutant H37Ra. The DD protocol made use of an oligo(dT) to prime reverse-transcriptase (RT)-dependent transcription of poly-A tailed mRNAs and a PCR amplification of the RT products by using ten 12-base arbitrary primers in all their pair combinations. This analysis yielded 745 and 708 bands, including 52 and 15 differentially generated bands, in the strains H37Rv and H37Ra, respectively. Six cDNAs that appeared to be expressed in H37Rv, but not in H37Ra, were reamplified and cloned and at least 10 inserts were sequenced for each cloned cDNA. After resolving discrepant results, 6 inserts were found highly homologous to M. tuberculosis H37Rv genes. Three of these, i.e., genes Rv2770c, Rv1345, and Rv0288, coding respectively for a member of the PPE protein family, a probable polyketide synthase, and a member of the protein family containing ESAT-6, have been predictively associated to immunological or pathogenetic aspects of M. tuberculosis infection; the other genes, i.e., Rv2336, Rv1320c, and Rv2819c, code for proteins with unknown functions. These results show that mRNA DD methodology can represent a potential tool for investigation of M. tuberculosis gene expression.}, Author = {Rindi, L and Lari, N and Garzelli, C}, Date-Added = {2010-03-02 11:26:01 +0100}, Date-Modified = {2010-03-02 11:26:01 +0100}, Doi = {10.1006/bbrc.1999.0591}, Journal = {Biochem Biophys Res Commun}, Journal-Full = {Biochemical and biophysical research communications}, Mesh = {Base Sequence; Cloning, Molecular; DNA Primers; Genes, Bacterial; Mycobacterium tuberculosis; RNA, Messenger; Virulence}, Month = {Apr}, Number = {1}, Pages = {94-101}, Pmid = {10222241}, Pst = {ppublish}, Title = {Search for genes potentially involved in Mycobacterium tuberculosis virulence by mRNA differential display}, Volume = {258}, Year = {1999}, Bdsk-Url-1 = {http://dx.doi.org/10.1006/bbrc.1999.0591}} @comment{BibDesk Smart Groups{ conditions comparison 3 key Keywords value single protein version 1 conjunction 0 group name no single protein conditions comparison 2 key Keywords value single protein version 1 conjunction 0 group name single protein }}