UMLS:C0026918 - FACTA Search

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Query: UMLS:C0026918 (Mycobacterium)
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The murine chromosome 1 gene Lsh/Ity/Bcg (candidate Nramp) regulates macrophage activation for antimicrobial activity against Salmonella typhimurium, Leishmania donovani, and Mycobacterium spp. To determine early events in the activation pathway, the ability of mycobacterial lipoarabinomannan (LAM) to induce early gene (KC and JE) expression in macrophages from susceptible (S) C57BL/10ScSn (Lshs) and congenic resistant (R) B10.L-Lshr mice was investigated. Stimulation with 1.8 microgram of arabinofuranosyl-terminated LAM (AraLAM) per ml resulted in similar kinetics for KC or JE expression in S and R macrophages. However, whereas JE/glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA ratios remained equivalent, R macrophages consistently showed enhanced KC/GAPDH ratios within 30 to 40 min of stimulation compared with S macrophages. Significant differences in KC/GAPDH ratios were observed throughout the peak period (0.5 to 6 h) of the KC response and with doses of AraLAM ranging from 0.01 to 2.5 micrograms/ml. Heavily mannosylated LAM from virulent Mycobacterium tuberculosis Erdman, in doses of up to 2.5 micrograms/ml, failed to stimulate KC or JE in S or R macrophages. Gamma interferon alone (25 U/ml) stimulated equivalent JE expression in S and R macrophages and synergized with AraLAM to enhance JE in both. In contrast, AraLAM-induced KC expression was inhibited in the presence of gamma interferon. Agonist/inhibitor studies were undertaken to determine the signal transduction pathways mediating KC expression. The protein kinase C (PKC) inhibitor Calphostin C (200 nM) inhibited AraLAM-induced KC by 34% +/- 4% in S macrophages and 43% +/- 5% in R macrophages; the cyclic AMP-dependent PKA inhibitor KT5720 (2 microM) inhibited AraLAM-induced KC by 33% +/- 4% (S) and 25% +/- 5% (R). A role for Ca2+ was indicated because ionophore alone stimulated KC expression and synergized with AraLAM to give a dramatically enhanced response. Induction of KC was also inhibited by (i) blocking constitutive nitric oxide (NO) production by preincubation of macrophages with NG-monomethyl-L-arginine (400 microM) (48% +/- 8% [S] and 40% +/- 11% [R]) and (ii) incubation of macrophages with the cyclic GMP-dependent kinase inhibitor KT5823 (4 microM) (65% +/- 4% [S] and 72% +/- 6% [R]). The manner in which these PKC-, PKA-, and Ca(2+)-dependent, NO-mediated cyclic GMP-dependent kinase signal transduction pathways may relate to function of the candidate Lsh/Ity/Bcg gene Nramp is discussed.
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PMID:Induction of early-response genes KC and JE by mycobacterial lipoarabinomannans: regulation of KC expression in murine macrophages by Lsh/Ity/Bcg (candidate Nramp). 813 24

Mycobacterium avium is an intracellular pathogen that has evolved to be a frequent cause of disseminated infection in immunocompromised patients. Although these bacilli are readily phagocytized, they are able to survive and even multiply within human macrophages. The process whereby mycobacteria circumvent the lytic functions of the macrophages is currently not well understood, but this is a key aspect in the pathogenicity of all pathogenic mycobacteria. Previously, we identified a gene in M. avium, designated mig (for macrophage-induced gene), the expression of which is induced when the bacilli grow in human macrophages (G. Plum and J. E. Clark-Curtiss, Infect. Immun. 62:476-483, 1994). In the present study we show that (i) the nucleotide sequence of the mig gene has an open reading frame of 295 amino acids with a strong bias for mycobacterial codon usage, (ii) the mig gene also codes for a putative signal peptide of 19 amino acid residues, (iii) mig is induced by acidity to be expressed as an early-secreted 30-kDa protein, and (iv) the Mig protein exhibits an AMP-binding domain signature. However, beyond this motif which is common to enzymes that activate a large variety of substrates, no homologies to known sequences are found. We also show that (v) Mycobacterium smegmatis strains expressing the Mig protein have a limited advantage for survival in macrophages. These findings may be concordant with a role of the mig gene in the virulence of M. avium.
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PMID:Cloning, sequencing, and expression of the mig gene of Mycobacterium avium, which codes for a secreted macrophage-induced protein. 935 32

MTD (GEN-PROBE AMPLIFIED MYCOBACTERIUM TUBERCULOSIS DIRECT TEST) for Mycobacterium tuberculosis, and Amplicor Mycobacterium for Mycobacteria (AMP-M. tb for M. tuberculosis, AMP-M. av for M. avium and AMP-M. in for M. intracellulare) were used for the detection of relevant Mycobacterium. Their sensitivity and specificity were evaluated. Total 244 clinical specimens including 164 sputa were examined by the above two tests. The results were compared with those obtained by the conventional methods. Of 244 samples, number of the M. tuberculosis positive samples by microscopy, cultural test, MTD and AMP-M. tb were 32, 33, 38 and 35, respectively. Among 33 culture positive samples, 25 were MTD positive and 26 were AMP-M. tb positive. Therefore, sensitivity of MTD and AMP-M. tb were 75.8% and 78.8%, and their specificity were 93.8% and 95.7%, respectively. When only sputa were used for the tests as the clinical specimens, both sensitivity of MTD and AMP-M. tb were increased to 94.4%. For MAC, positive samples of M. avium complex by culture, M. avium by AMP-M. av and M. intracellulare by AMP-M. in were 13, 16, and 8, respectively. Sensitivity and specificity of AMP-M. av/M. in were 100% and 95.2%, respectively. Clinical findings of the patients whose MTD tests were positive but negative by culture were reexamined. Three of 9 specimens were also positive in AMP-M. tb. From the records of the isolations of tubercle bacilli or other important pathogens from the other kind of clinical specimens, smear tests and patients' response to tuberculosis chemotherapy, four of 9 specimens were confirmed as true positive, three were suspected as positive, and two other specimens were false positive which might be caused by contamination. From these observations, it could be concluded that MTD and AMP-M. tb are more sensitive than conventional culture method, and MTD is more sensitive than AMP-M. tb but needs more careful treatment to avoid the contamination.
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PMID:[Evaluations of MTD and Amplicor Mycobacterium for direct detection of Mycobacteria from clinical specimens]. 1038 32

The role of salicylic acid in iron metabolism was examined in two wild-type strains (mc(2)155 and NCIMB 8548) and three mutant strains (mc(2)1292 [lacking exochelin], SM3 [lacking iron-dependent repressor protein IdeR] and S99 [a salicylate-requiring auxotroph derived in this study]) of Mycobacterium smegmatis. Synthesis of salicylate in SM3 was derepressed even in the presence of iron, as was synthesis of the siderophores exochelin, mycobactin, and carboxymycobactin. S99 was dependent on salicylate for growth and failed to grow with the three ferrisiderophores, suggesting that salicylate fulfills an additional function(s) other than being a precursor of mycobactin and carboxymycobactin. Salicylic acid at 100 microgram/ml repressed the formation of a 29-kDa cell envelope protein (putative exochelin receptor protein) in S99 grown both iron deficiently and iron sufficiently. In contrast, synthesis of this protein was affected only under iron-limited conditions in the parent strain, mc(2)155, and remained unaltered in SM3, suggesting an interaction between the IdeR protein and salicylate. Thus, salicylate may also function as a signal molecule for recognition of cellular iron status. Growth of all strains and mutants with p-aminosalicylate (PAS) at 100 microgram/ml increased salicylate accumulation between three- and eightfold under both iron-limited and iron-sufficient growth conditions and decreased mycobactin accumulation by 40 to 80% but increased carboxymycobactin accumulation by 50 to 55%. Thus, although PAS inhibited salicylate conversion to mycobactin, presumptively by blocking salicylate AMP kinase, PAS also interferes with the additional functions of salicylate, as its effect was heightened in S99 when the salicylate concentration was minimal.
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PMID:Mutational analysis of a role for salicylic acid in iron metabolism of Mycobacterium smegmatis. 1062 69

Mycobacterium smegmatis DSM 43756 inactivates rifampin by ribosylation. To study this process of rifampicin, all possible inactivated forms of the antibiotic were extracted and purified. Structural studies showed the presence of a new inactivation product, designated RIP-TAp(23-phosphoribosyl-rifampin). Formation of 23-(O-ADP-ribosyl)rifampin (RIP-TAs) is the first step, followed by removal of AMP to give rise to the newly identified compound. Lastly, dephosphorylation leads to formation of 23-ribosyl-rifampin (RIP-Mb). Feeding experiments with the ADP-ribosylated antibiotic obtained from the cell homogenates of an Escherichia coli strain carrying the cloned M. smegmatis gene confirmed this rifampin inactivation process.
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PMID:Identification and characterization of a new intermediate in the ribosylative inactivation pathway of rifampin by Mycobacterium smegmatis. 1064 83

A new class of glutamate dehydrogenase (GDH) is reported. The GDH of Streptomyces clavuligerus was purified to homogeneity and characterized. It has a native molecular mass of 1,100 kDa and exists as an alpha(6) oligomeric structure composed of 183-kDa subunits. GDH, which requires AMP as an essential activator, shows a maximal rate of catalysis in 100 mm phosphate buffer, pH 7.0, at 30 degrees C. Under these conditions, GDH displayed hyperbolic behavior toward ammonia (K(m), 33 mm) and sigmoidal responses to changes in alpha-ketoglutarate (S(0.5) 1.3 mm; n(H) 1.50) and NADH (S(0.5) 20 microm; n(H) 1.52) concentrations. Aspartate and asparagine were found to be allosteric activators. This enzyme is inhibited by an excess of NADH or NH(4)(+), by some tricarboxylic acid cycle intermediates and by ATP. This GDH seems to be a catabolic enzyme as indicated by the following: (i) it is NAD-specific; (ii) it shows a high value of K(m) for ammonia; and (iii) when S. clavuligerus was cultured in minimal medium containing glutamate as the sole source of carbon and nitrogen, a 5-fold increase in specific activity of GDH was detected compared with cultures provided with glycerol and ammonia. GDH has 1,651 amino acids, and it is encoded by a DNA fragment of 4,953 base pairs (gdh gene). It shows strong sequence similarity to proteins encoded by unidentified open reading frames present in the genomes of species belonging to the genera Mycobacterium, Rickettsia, Pseudomonas, Vibrio, Shewanella, and Caulobacter, suggesting that it has a broad distribution. The GDH of S. clavuligerus is the first member of a class of GDHs included in a subfamily of GDHs (large GDHs) whose catalytic requirements and evolutionary implications are described and discussed.
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PMID:A new class of glutamate dehydrogenases (GDH). Biochemical and genetic characterization of the first member, the AMP-requiring NAD-specific GDH of Streptomyces clavuligerus. 1092 16

Lon (La) proteases are multimeric enzymes that are activated by ATP and Mg(2+) ions and stimulated by unfolded proteins such as alpha-casein. The peptidase activity of the Lon protease from Mycobacterium smegmatis (Ms-Lon) is dependent upon both its concentration and that of Mg(2+). Addition of alpha-casein partially substitutes for Mg(2+) in activating the enzyme. In chemical dissociation experiments, higher concentrations of urea were required to inhibit Ms-Lon's catalytic activities after an addition of alpha-casein. Analytical ultracentrifugation was used to directly probe the effect of activators of peptidase activity on Ms-Lon self-association. Sedimentation velocity experiments reveal that Ms-Lon monomers are in a reversible equilibrium with oligomeric forms of the protein and that the self-association reaction is facilitated by Mg(2+) ions but not by AMP-PNP or ATP gamma S. NaCl at 100 mM facilitates oligomerization and stimulates peptidase activity at suboptimal concentrations of MgCl(2). Sedimentation equilibrium analysis shows that Ms-Lon associates to a hexamer at 50 mM Tris and 10 mM MgCl(2), at pH 8.0 and 20 degrees C, and that the assembly reaction is Mg(2+) dependent; the mole fraction of hexamer decreases with decreasing MgCl(2) to undetectable levels in 10 mM EDTA. The analysis of experiments conducted at a series of initial protein and MgCl(2) concentrations yields two assembly models: dimer <--> tetramer <--> hexamer and timer <--> hexamer, equally consistent with the data. Limited trypsin digestion, CD, and tryptophan fluorescence suggest only minor changes in secondary and tertiary structure upon Mg(2+)-linked oligomerization. These results show that activation of Ms-Lon peptidase activity requires oligomerization and that Ms-Lon self-association reaction is facilitated by its activator, Mg(2+), and stimulator, unfolded protein.
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PMID:Mg2+-linked oligomerization modulates the catalytic activity of the Lon (La) protease from Mycobacterium smegmatis. 1147 99

Pantothenate synthetase (EC 6.3.2.1), encoded by the panC gene, catalyzes the essential ATP-dependent condensation of D-pantoate and beta-alanine to form pantothenate in bacteria, yeast and plants. Pantothenate synthetase from Mycobacterium tuberculosis was expressed in E. coli, purified to homogeneity, and found to be a homodimer with a subunit molecular mass of 33 kDa. Initial velocity, product, and dead-end inhibition studies showed the kinetic mechanism of pantothenate synthetase to be Bi Uni Uni Bi Ping Pong, with ATP binding followed by D-pantoate binding, release of PP(i), binding of beta-alanine, followed by the release of pantothenate and AMP. Michaelis constants were 0.13, 0.8, and 2.6 mM for D-pantoate, beta-alanine, and ATP, respectively, and the turnover number, k(cat), was 3.4 s(-1). The formation of pantoyl adenylate, suggested as a key intermediate by the kinetic mechanism, was confirmed by (31)P NMR spectroscopy of [(18)O]AMP produced from (18)O transfer using [carboxyl-(18)O]pantoate. Single-turnover reactions for the formation of pyrophosphate and pantothenate were determined using rapid quench techniques, and indicated that the two half-reactions occurred with maximum rates of 1.3 +/- 0.3 and 2.6 +/- 0.3 s(-)(1), respectively, consistent with pantoyl adenylate being a kinetically competent intermediate in the pantothenate synthetase reaction. These data also suggest that both half-reactions are partially rate-limiting. Reverse isotope exchange of [(14)C]-beta-alanine into pantothenate in the presence of AMP was observed, indicating the reversible formation of the pantoyl adenylate intermediate from products.
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PMID:Steady-state and pre-steady-state kinetic analysis of Mycobacterium tuberculosis pantothenate synthetase. 1166 27

2-Methyladenosine (methyl-ado) has demonstrated selective activity against Mycobacterium tuberculosis, which indicates that differences in the substrate preferences between mycobacterial and human purine metabolic enzymes can be exploited to develop novel drugs for the treatment of mycobacterial diseases. Therefore, in an effort to better understand the reasons for the anti-mycobacterial activity of methyl-ado, its metabolism has been characterized in Mycobacterium smegmatis. In a wild-type strain, methyl-ado was phosphorylated by adenosine kinase to methyl-AMP, which was further converted to methyl-ATP and incorporated into RNA. In contrast, a mutant strain of M. smegmatis was isolated that was resistant to methyl-ado, deficient in adenosine kinase activity and was not able to generate methyl-ado metabolites in cells treated with methyl-ado. These results indicated that phosphorylated metabolites of methyl-ado were responsible for the cytotoxic activity of this compound. Methyl-ado was not a substrate for either adenosine deaminase or purine-nucleoside phosphorylase from M. smegmatis. Treatment of M. smegmatis with methyl-ado resulted in the inhibition of ATP synthesis, which indicated that a metabolite of methyl-ado inhibited one of the enzymes involved in de novo purine synthesis. These studies demonstrated the importance of adenosine kinase in the activation of methyl-ado to toxic metabolites in M. smegmatis.
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PMID:The metabolism of 2-methyladenosine in Mycobacterium smegmatis. 1178 21

Phosphatidylinositol is an essential component of mycobacteria, and phosphatidylinositol-based lipids such as phosphatidylinositolmannosides, lipomannan, and lipoarabinomannan are major immunomodulatory components of the Mycobacterium tuberculosis cell wall. Inositol monophosphatase (EC 3.1.3.25) is a crucial enzyme in the biosynthesis of free myo-inositol from inositol-1-phosphate, a key substrate for the phosphatidylinositol synthase in mycobacteria. Analysis of the M. tuberculosis genome suggested the presence of four M. tuberculosis gene products that exhibit an inositol monophosphatase signature. In the present report, we have focused on SuhB, which possesses the highest degree of homology with human inositol monophosphatase. SuhB gene was cloned into an E. coli expression vector to over-produce a His-tagged protein, which was purified and characterized. SuhB required divalent metal ions for functional inositol monophosphatase activity, with Mg(2+) being the strongest activator. Inositol monophosphatase activity catalyzed by SuhB was inhibited by the monovalent cation lithium (IC(50) = 0.9 mM). As anticipated, inositol-1-phosphate was the preferred substrate (K(m) = 0.177 +/- 0.025 mM; k(cat) = 3.6 +/- 0.2 s(-)(1)); however, SuhB was also able to hydrolyze a variety of polyol phosphates such as glucitol-6-phosphate, glycerol-2-phosphate, and 2'-AMP. To provide further insight into the structure-function relationship of SuhB, different mutant proteins were generated (E83D, D104N, D107N, W234L, and D235N). These mutations almost completely abrogated inositol monophosphatase activity, thus underlining the importance of these residues in inositol-1-phosphate dephosphorylation. We also identified L81 as a key residue involved in sensitivity to lithium. The L81A mutation rendered SuhB inositol monophosphatase activity 10-fold more resistant to inhibition by lithium (IC(50) = 10 mM). These studies provide the first steps in the delineation of the biosynthesis of the key metabolite inositol in M. tuberculosis.
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PMID:Purification and biochemical characterization of Mycobacterium tuberculosis SuhB, an inositol monophosphatase involved in inositol biosynthesis. 1191 86

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