23 results on '"Mikusova K"'
Search Results
2. Biochemical characterization of acyltransferase MSMEG_2934 from Mycobacterium smegmatis: SW04.S16–259
- Author
-
Svetlikova, Z., Kordulakova, J., and Mikusova, K.
- Published
- 2013
3. Investigation of putative ABC transporter Rv1458c/Rv1457c/Rv1456c in mycobacteria: SW04.S16–246
- Author
-
Sarkan, M., Mikusova, K., Jackson, M., and Kordulakova, J.
- Published
- 2013
4. Purification and function of mycobacterial WecA protein: SW04.S16–248
- Author
-
Huszar, S. and Mikusova, K.
- Published
- 2013
5. Synthesis of the mycobacterial arabinose donor beta-D-arabinofuranosyl-1-monophosphoryldecaprenol, development of a basic arabinosyl-transferase assay, and identification of ethambutol as an arabinosyl transferase inhibitor
- Author
-
Lee, R.E., Mikusova, K., Brennan, P.J., and Besra, G.S.
- Subjects
Organic compounds -- Synthesis ,Ethambutol -- Research ,Transferases -- Research ,Chemistry - Abstract
The preparation of the mycobacterial arabinose donor, 1-C(super 14) -beta-D-arabinofuranosyl-1-monophosphoryldecaprenol involves the formation of the beta-arabinofuranosyl allylic phosphodiester by the phosphoramidite-phosphite triester method. During the innovative process of tert-butyl dimethylsilyl arabinofuranosyl protection, the C-1 acid undergoes regioselective hydrolysis. Deprotection using ammonium fluoride gives the required product. The antimycobacterial drug ethambutol most likely restricts the arabinosyl transfer.
- Published
- 1995
6. A multitarget approach to drug discovery inhibiting Mycobacterium tuberculosis PyrG and PanK
- Author
-
Chiarelli, L, Mori, G, Orena, B, Esposito, M, Lane, T, De Jesus Lopes Ribeiro, A, Degiacomi, G, Zemanová, J, Szádocka, S, Huszár, S, Palčeková, Z, Manfredi, M, Gosetti, F, Lelièvre, J, Ballell, L, Kazakova, E, Makarov, V, Marengo, E, Mikusova, K, Cole, S, Riccardi, G, Ekins, S, Pasca, M, Chiarelli, Laurent R., Mori, Giorgia, Orena, Beatrice Silvia, Esposito, Marta, Lane, Thomas, De Jesus Lopes Ribeiro, Ana Luisa, Degiacomi, Giulia, Zemanová, Júlia, Szádocka, Sára, Huszár, Stanislav, Palčeková, Zuzana, Manfredi, Marcello, Gosetti, Fabio, Lelièvre, Joël, Ballell, Lluis, Kazakova, Elena, Makarov, Vadim, Marengo, Emilio, Mikusova, Katarina, Cole, Stewart T., Riccardi, Giovanna, Ekins, Sean, Pasca, Maria Rosalia, Chiarelli, L, Mori, G, Orena, B, Esposito, M, Lane, T, De Jesus Lopes Ribeiro, A, Degiacomi, G, Zemanová, J, Szádocka, S, Huszár, S, Palčeková, Z, Manfredi, M, Gosetti, F, Lelièvre, J, Ballell, L, Kazakova, E, Makarov, V, Marengo, E, Mikusova, K, Cole, S, Riccardi, G, Ekins, S, Pasca, M, Chiarelli, Laurent R., Mori, Giorgia, Orena, Beatrice Silvia, Esposito, Marta, Lane, Thomas, De Jesus Lopes Ribeiro, Ana Luisa, Degiacomi, Giulia, Zemanová, Júlia, Szádocka, Sára, Huszár, Stanislav, Palčeková, Zuzana, Manfredi, Marcello, Gosetti, Fabio, Lelièvre, Joël, Ballell, Lluis, Kazakova, Elena, Makarov, Vadim, Marengo, Emilio, Mikusova, Katarina, Cole, Stewart T., Riccardi, Giovanna, Ekins, Sean, and Pasca, Maria Rosalia
- Abstract
Mycobacterium tuberculosis, the etiological agent of the infectious disease tuberculosis, kills approximately 1.5 million people annually, while the spread of multidrug-resistant strains is of great global concern. Thus, continuous efforts to identify new antitubercular drugs as well as novel targets are crucial. Recently, two prodrugs activated by the monooxygenase EthA, 7947882 and 7904688, which target the CTP synthetase PyrG, were identified and characterized. In this work, microbiological, biochemical, and in silico methodologies were used to demonstrate that both prodrugs possess a second target, the pantothenate kinase PanK. This enzyme is involved in coenzyme A biosynthesis, an essential pathway for M. tuberculosis growth. Moreover, compound 11426026, the active metabolite of 7947882, was demonstrated to directly inhibit PanK, as well. In an independent screen of a compound library against PyrG, two additional inhibitors were also found to be active against PanK. In conclusion, these direct PyrG and PanK inhibitors can be considered as leads for multitarget antitubercular drugs and these two enzymes could be employed as a "double-tool" in order to find additional hit compounds.
- Published
- 2018
7. Crystal structure of membrane associated PatA from Mycobacterium smegmatis in complex with palmitate - P21 space group
- Author
-
Albesa-Jove, D., primary, Svetlikova, Z., additional, Carreras-Gonzalez, A., additional, Tersa, M., additional, Sancho-Vaello, E., additional, Cifuente, J.O., additional, Mikusova, K., additional, and Guerin, M.E., additional
- Published
- 2016
- Full Text
- View/download PDF
8. Crystal structure of membrane associated PatA from Mycobacterium smegmatis in complex with palmitate - P 42 21 2 space group
- Author
-
Albesa-Jove, D., primary, Svetlikova, Z., additional, Carreras-Gonzalez, A., additional, Tersa, M., additional, Sancho-Vaello, E., additional, Cifuente, J.O., additional, Mikusova, K., additional, and Guerin, M.E., additional
- Published
- 2016
- Full Text
- View/download PDF
9. Crystal structure of membrane associated PatA from Mycobacterium smegmatis in complex with palmitate - C 2 space group
- Author
-
Albesa-Jove, D., primary, Svetlikova, Z., additional, Carreras-Gonzalez, A., additional, Tersa, M., additional, Sancho-Vaello, E., additional, Cifuente, J.O., additional, Mikusova, K., additional, and Guerin, M.E., additional
- Published
- 2016
- Full Text
- View/download PDF
10. Crystal structure of membrane associated PatA from Mycobacterium smegmatis in complex with S-hexadecyl Coenzyme A - P21 space group
- Author
-
Albesa-Jove, D., primary, Svetlikova, Z., additional, Carreras-Gonzalez, A., additional, Tersa, M., additional, Sancho-Vaello, E., additional, Cifuente, J.O., additional, Mikusova, K., additional, and Guerin, M.E., additional
- Published
- 2016
- Full Text
- View/download PDF
11. Synthesis of the Arabinose Donor .beta.-D-Arabinofuranosyl-1-monophosphoryldecaprenol, Development of a Basic Arabinosyl-Transferase Assay, and Identification of Ethambutol as an Arabinosyl Transferase Inhibitor
- Author
-
Lee, R. E., primary, Mikusova, K., additional, Brennan, P. J., additional, and Besra, G. S., additional
- Published
- 1995
- Full Text
- View/download PDF
12. Identification of a small molecule with activity against drug-resistant and persistent tuberculosis
- Author
-
Wang, F., Sambandan, D., Halder, R., Wang, J., Batt, S. M., Weinrick, B., Ahmad, I., Yang, P., Zhang, Y., Kim, J., Hassani, M., Huszar, S., Trefzer, C., Ma, Z., Kaneko, T., Mdluli, K. E., Franzblau, S., Chatterjee, A. K., Johnson, K., Mikusova, K., Besra, G. S., Futterer, K., Jacobs, W. R., and Schultz, P. G.
- Subjects
drug resistance ,dual mechanism ,bacterial infections and mycoses - Abstract
A cell-based phenotypic screen for inhibitors of biofilm formation in mycobacteria identified the small molecule TCA1, which has bactericidal activity against both drug-susceptible and -resistant Mycobacterium tuberculosis (Mtb) and sterilizes Mtb in vitro combined with rifampicin or isoniazid. In addition, TCA1 has bactericidal activity against nonreplicating Mtb in vitro and is efficacious in acute and chronic Mtb infection mouse models both alone and combined with rifampicin or isoniazid. Transcriptional analysis revealed that TCA1 down-regulates genes known to be involved in Mtb persistence. Genetic and affinity-based methods identified decaprenyl-phosphoryl-beta-D-ribofuranose oxidoreductase DprE1 and MoeW, enzymes involved in cell wall and molybdenum cofactor biosynthesis, respectively, as targets responsible for the activity of TCA1. These in vitro and in vivo results indicate that this compound functions by a unique mechanism and suggest that TCA1 may lead to the development of a class of antituberculosis agents.
13. Investigation of putative ABC transporter Rv1458c/Rv1457c/Rv1456c in mycobacteria
- Author
-
Sarkan, M., Mikusova, K., Mary Jackson, and Kordulakova, J.
14. 2,6-Disubstituted 7-(naphthalen-2-ylmethyl)-7H-purines as a new class of potent antitubercular agents inhibiting DprE1.
- Author
-
Finger V, Kucera T, Kafkova R, Muckova L, Dolezal R, Kubes J, Novak M, Prchal L, Lakatos L, Andrs M, Hympanova M, Marek J, Kufa M, Spiwok V, Soukup O, Mezeiova E, Janousek J, Nevosadova L, Benkova M, Kitson RRA, Kratky M, Bősze S, Mikusova K, Hartkoorn R, Roh J, and Korabecny J
- Subjects
- Animals, Alcohol Oxidoreductases chemistry, Purines pharmacology, Structure-Activity Relationship, Molecular Dynamics Simulation, Bacterial Proteins metabolism, Mammals metabolism, Antitubercular Agents chemistry, Mycobacterium tuberculosis
- Abstract
Phenotypic screening of an in-house library of small molecule purine derivatives against Mycobacterium tuberculosis (Mtb) led to the identification of 2-morpholino-7-(naphthalen-2-ylmethyl)-1,7-dihydro-6H-purin-6-one 10 as a potent antimycobacterial agent with MIC
99 of 4 μM. Thorough structure-activity relationship studies revealed the importance of 7-(naphthalen-2-ylmethyl) substitution for antimycobacterial activity, yet opened the possibility of structural modifications at positions 2 and 6 of the purine core. As the result, optimized analogues with 6-amino or ethylamino substitution 56 and 64, respectively, were developed. These compounds showed strong in vitro antimycobacterial activity with MIC of 1 μM against Mtb H37 Rv and against several clinically isolated drug-resistant strains, had limited toxicity to mammalian cell lines, medium clearance with respect to phase I metabolic deactivation (27 and 16.8 μL/min/mg), sufficient aqueous solubility (>90 μM) and high plasma stability. Interestingly, investigated purines, including compounds 56 and 64, lacked activity against a panel of Gram-negative and Gram-positive bacterial strains, indicating a specific mycobacterial molecular target. To investigate the mechanism of action, Mtb mutants resistant to hit compound 10 were isolated and their genomes were sequenced. Mutations were found in dprE1 (Rv3790), which encodes decaprenylphosphoryl-β-d-ribose oxidase DprE1, enzyme essential for the biosynthesis of arabinose, a vital component of the mycobacterial cell wall. Inhibition of DprE1 by 2,6-disubstituted 7-(naphthalen-2-ylmethyl)-7H-purines was proved using radiolabelling experiments in Mtb H37 Rv in vitro. Finally, structure-binding relationships between selected purines and DprE1 using molecular modeling studies in tandem with molecular dynamic simulations revealed the key structural features for effective drug-target interaction., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)- Published
- 2023
- Full Text
- View/download PDF
15. A multitarget approach to drug discovery inhibiting Mycobacterium tuberculosis PyrG and PanK.
- Author
-
Chiarelli LR, Mori G, Orena BS, Esposito M, Lane T, de Jesus Lopes Ribeiro AL, Degiacomi G, Zemanová J, Szádocka S, Huszár S, Palčeková Z, Manfredi M, Gosetti F, Lelièvre J, Ballell L, Kazakova E, Makarov V, Marengo E, Mikusova K, Cole ST, Riccardi G, Ekins S, and Pasca MR
- Subjects
- Antitubercular Agents chemistry, Antitubercular Agents metabolism, Antitubercular Agents pharmacology, Bacterial Proteins metabolism, Computer Simulation, Humans, Models, Molecular, Mycobacterium tuberculosis enzymology, Tuberculosis drug therapy, Carbon-Nitrogen Ligases drug effects, Drug Discovery methods, Phosphotransferases (Alcohol Group Acceptor) drug effects
- Abstract
Mycobacterium tuberculosis, the etiological agent of the infectious disease tuberculosis, kills approximately 1.5 million people annually, while the spread of multidrug-resistant strains is of great global concern. Thus, continuous efforts to identify new antitubercular drugs as well as novel targets are crucial. Recently, two prodrugs activated by the monooxygenase EthA, 7947882 and 7904688, which target the CTP synthetase PyrG, were identified and characterized. In this work, microbiological, biochemical, and in silico methodologies were used to demonstrate that both prodrugs possess a second target, the pantothenate kinase PanK. This enzyme is involved in coenzyme A biosynthesis, an essential pathway for M. tuberculosis growth. Moreover, compound 11426026, the active metabolite of 7947882, was demonstrated to directly inhibit PanK, as well. In an independent screen of a compound library against PyrG, two additional inhibitors were also found to be active against PanK. In conclusion, these direct PyrG and PanK inhibitors can be considered as leads for multitarget antitubercular drugs and these two enzymes could be employed as a "double-tool" in order to find additional hit compounds.
- Published
- 2018
- Full Text
- View/download PDF
16. The phosphatidyl-myo-inositol mannosyltransferase PimA is essential for Mycobacterium tuberculosis growth in vitro and in vivo.
- Author
-
Boldrin F, Ventura M, Degiacomi G, Ravishankar S, Sala C, Svetlikova Z, Ambady A, Dhar N, Kordulakova J, Zhang M, Serafini A, Vishwas KG, Kolly GS, Kumar N, Palù G, Guerin ME, Mikusova K, Cole ST, and Manganelli R
- Subjects
- Animals, Bacterial Proteins genetics, Female, Humans, Macrophages metabolism, Macrophages microbiology, Mannosyltransferases genetics, Mice, Mice, Inbred C57BL, Mycobacterium tuberculosis genetics, Phosphatidylinositols biosynthesis, Bacterial Proteins metabolism, Mannosyltransferases metabolism, Mycobacterium tuberculosis enzymology, Mycobacterium tuberculosis growth & development, Tuberculosis microbiology
- Abstract
The cell envelope of Mycobacterium tuberculosis contains glycans and lipids of peculiar structure that play prominent roles in the biology and pathogenesis of tuberculosis. Consequently, the chemical structure and biosynthesis of the cell wall have been intensively investigated in order to identify novel drug targets. Here, we validate that the function of phosphatidyl-myo-inositol mannosyltransferase PimA is vital for M. tuberculosis in vitro and in vivo. PimA initiates the biosynthesis of phosphatidyl-myo-inositol mannosides by transferring a mannosyl residue from GDP-Man to phosphatidyl-myo-inositol on the cytoplasmic side of the plasma membrane. To prove the essential nature of pimA in M. tuberculosis, we constructed a pimA conditional mutant by using the TetR-Pip off system and showed that downregulation of PimA expression causes bactericidality in batch cultures. Consistent with the biochemical reaction catalyzed by PimA, this phenotype was associated with markedly reduced levels of phosphatidyl-myo-inositol dimannosides, essential structural components of the mycobacterial cell envelope. In addition, the requirement of PimA for viability was clearly demonstrated during macrophage infection and in two different mouse models of infection, where a dramatic decrease in viable counts was observed upon silencing of the gene. Notably, depletion of PimA resulted in complete clearance of the mouse lungs during both the acute and chronic phases of infection. Altogether, the experimental data highlight the importance of the phosphatidyl-myo-inositol mannoside biosynthetic pathway for M. tuberculosis and confirm that PimA is a novel target for future drug discovery programs., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)
- Published
- 2014
- Full Text
- View/download PDF
17. Identification of a small molecule with activity against drug-resistant and persistent tuberculosis.
- Author
-
Wang F, Sambandan D, Halder R, Wang J, Batt SM, Weinrick B, Ahmad I, Yang P, Zhang Y, Kim J, Hassani M, Huszar S, Trefzer C, Ma Z, Kaneko T, Mdluli KE, Franzblau S, Chatterjee AK, Johnsson K, Mikusova K, Besra GS, Fütterer K, Robbins SH, Barnes SW, Walker JR, Jacobs WR Jr, and Schultz PG
- Subjects
- Alcohol Oxidoreductases, Amino Acid Sequence, Animals, Antitubercular Agents administration & dosage, Antitubercular Agents chemistry, Bacterial Proteins antagonists & inhibitors, Bacterial Proteins chemistry, Bacterial Proteins genetics, Benzothiazoles administration & dosage, Benzothiazoles chemistry, Biofilms drug effects, Biofilms growth & development, Carbohydrate Epimerases antagonists & inhibitors, Carbohydrate Epimerases chemistry, Carbohydrate Epimerases genetics, Drug Resistance, Bacterial, Female, Genes, Bacterial, High-Throughput Screening Assays, Isoniazid administration & dosage, Mice, Mice, Inbred BALB C, Microbial Sensitivity Tests, Molecular Sequence Data, Mycobacterium tuberculosis enzymology, Mycobacterium tuberculosis genetics, Oxidoreductases antagonists & inhibitors, Oxidoreductases chemistry, Oxidoreductases genetics, Rifampin administration & dosage, Thiophenes administration & dosage, Thiophenes chemistry, Tuberculosis, Pulmonary microbiology, Antitubercular Agents pharmacology, Benzothiazoles pharmacology, Mycobacterium tuberculosis drug effects, Thiophenes pharmacology, Tuberculosis, Pulmonary drug therapy
- Abstract
A cell-based phenotypic screen for inhibitors of biofilm formation in mycobacteria identified the small molecule TCA1, which has bactericidal activity against both drug-susceptible and -resistant Mycobacterium tuberculosis (Mtb) and sterilizes Mtb in vitro combined with rifampicin or isoniazid. In addition, TCA1 has bactericidal activity against nonreplicating Mtb in vitro and is efficacious in acute and chronic Mtb infection mouse models both alone and combined with rifampicin or isoniazid. Transcriptional analysis revealed that TCA1 down-regulates genes known to be involved in Mtb persistence. Genetic and affinity-based methods identified decaprenyl-phosphoryl-β-D-ribofuranose oxidoreductase DprE1 and MoeW, enzymes involved in cell wall and molybdenum cofactor biosynthesis, respectively, as targets responsible for the activity of TCA1. These in vitro and in vivo results indicate that this compound functions by a unique mechanism and suggest that TCA1 may lead to the development of a class of antituberculosis agents.
- Published
- 2013
- Full Text
- View/download PDF
18. Benzothiazinones kill Mycobacterium tuberculosis by blocking arabinan synthesis.
- Author
-
Makarov V, Manina G, Mikusova K, Möllmann U, Ryabova O, Saint-Joanis B, Dhar N, Pasca MR, Buroni S, Lucarelli AP, Milano A, De Rossi E, Belanova M, Bobovska A, Dianiskova P, Kordulakova J, Sala C, Fullam E, Schneider P, McKinney JD, Brodin P, Christophe T, Waddell S, Butcher P, Albrethsen J, Rosenkrands I, Brosch R, Nandi V, Bharath S, Gaonkar S, Shandil RK, Balasubramanian V, Balganesh T, Tyagi S, Grosset J, Riccardi G, and Cole ST
- Subjects
- Amino Acid Sequence, Animals, Antitubercular Agents chemical synthesis, Antitubercular Agents chemistry, Arabinose metabolism, Cell Wall metabolism, Drug Resistance, Bacterial, Enzyme Inhibitors cerebrospinal fluid, Enzyme Inhibitors chemistry, Enzyme Inhibitors pharmacology, Enzyme Inhibitors therapeutic use, Ethambutol pharmacology, Gene Expression Regulation, Bacterial drug effects, Genes, Bacterial, Mice, Mice, Inbred BALB C, Microbial Sensitivity Tests, Molecular Sequence Data, Molecular Structure, Mycobacterium drug effects, Mycobacterium genetics, Mycobacterium tuberculosis genetics, Mycobacterium tuberculosis metabolism, Racemases and Epimerases metabolism, Spiro Compounds chemical synthesis, Spiro Compounds chemistry, Thiazines chemical synthesis, Thiazines chemistry, Tuberculosis microbiology, Antitubercular Agents pharmacology, Antitubercular Agents therapeutic use, Mycobacterium tuberculosis drug effects, Polysaccharides biosynthesis, Racemases and Epimerases antagonists & inhibitors, Spiro Compounds pharmacology, Spiro Compounds therapeutic use, Thiazines pharmacology, Thiazines therapeutic use, Tuberculosis drug therapy
- Abstract
New drugs are required to counter the tuberculosis (TB) pandemic. Here, we describe the synthesis and characterization of 1,3-benzothiazin-4-ones (BTZs), a new class of antimycobacterial agents that kill Mycobacterium tuberculosis in vitro, ex vivo, and in mouse models of TB. Using genetics and biochemistry, we identified the enzyme decaprenylphosphoryl-beta-d-ribose 2'-epimerase as a major BTZ target. Inhibition of this enzymatic activity abolishes the formation of decaprenylphosphoryl arabinose, a key precursor that is required for the synthesis of the cell-wall arabinans, thus provoking cell lysis and bacterial death. The most advanced compound, BTZ043, is a candidate for inclusion in combination therapies for both drug-sensitive and extensively drug-resistant TB.
- Published
- 2009
- Full Text
- View/download PDF
19. Molecular recognition and interfacial catalysis by the essential phosphatidylinositol mannosyltransferase PimA from mycobacteria.
- Author
-
Guerin ME, Kordulakova J, Schaeffer F, Svetlikova Z, Buschiazzo A, Giganti D, Gicquel B, Mikusova K, Jackson M, and Alzari PM
- Subjects
- Bacterial Proteins genetics, Bacterial Proteins metabolism, Catalysis, Mannosyltransferases genetics, Mannosyltransferases metabolism, Membrane Proteins genetics, Membrane Proteins metabolism, Mutagenesis, Mycobacterium smegmatis genetics, Phosphatidylinositols genetics, Phosphatidylinositols metabolism, Protein Structure, Tertiary genetics, Bacterial Proteins chemistry, Mannosyltransferases chemistry, Membrane Proteins chemistry, Models, Molecular, Mycobacterium smegmatis enzymology, Phosphatidylinositols chemistry
- Abstract
Mycobacterial phosphatidylinositol mannosides (PIMs) and metabolically derived cell wall lipoglycans play important roles in host-pathogen interactions, but their biosynthetic pathways are poorly understood. Here we focus on Mycobacterium smegmatis PimA, an essential enzyme responsible for the initial mannosylation of phosphatidylinositol. The structure of PimA in complex with GDP-mannose shows the two-domain organization and the catalytic machinery typical of GT-B glycosyltransferases. PimA is an amphitrophic enzyme that binds mono-disperse phosphatidylinositol, but its transferase activity is stimulated by high concentrations of non-substrate anionic surfactants, indicating that the early stages of PIM biosynthesis involve lipid-water interfacial catalysis. Based on structural, calorimetric, and mutagenesis studies, we propose a model wherein PimA attaches to the membrane through its N-terminal domain, and this association leads to enzyme activation. Our results reveal a novel mode of phosphatidylinositol recognition and provide a template for the development of potential antimycobacterial compounds.
- Published
- 2007
- Full Text
- View/download PDF
20. Identification of promoters recognized by RNA polymerase containing Mycobacterium tuberculosis stress-response sigma factor sigma(F).
- Author
-
Homerova D, Surdova K, Mikusova K, and Kormanec J
- Subjects
- DNA-Directed RNA Polymerases genetics, Genes, Bacterial genetics, Mycobacterium tuberculosis enzymology, Mycobacterium tuberculosis genetics, Sequence Analysis, DNA, Sigma Factor genetics, Transcription, Genetic, DNA-Directed RNA Polymerases metabolism, Mycobacterium tuberculosis physiology, Promoter Regions, Genetic physiology, Sigma Factor metabolism
- Abstract
A previously optimized Escherichia coli two-plasmid system was used to identify Mycobacterium tuberculosis promoters recognized by RNA polymerase containing the M. tuberculosis stress response sigma factor sigma(F). The method allowed the identification of five new sigma(F)-dependent promoters. Transcriptional start points of the promoters were determined by high-resolution S1-nuclease mapping using RNA prepared from E. coli containing the two-plasmid system. The promoters were confirmed by an in vitro transcription assay. The Mycobacterium smegmatis and Mycobacterium tuberculosis core RNA polymerases, after complementation with sigma(F), were able to recognize all the five promoters. All the promoters contained sequences highly similar to the sequence of the previously identified M. tuberculosis sigma(F)-dependent promoter, usfXp1. Comparison of the promoters revealed a sigma(F) consensus sequence GtTtga-N(14-18)-GGGTAT. The sigma(F)-dependent promoters may govern expression of genes encoding a transcription regulator homologous to the response regulators of bacterial two-component signal transduction systems and proteins with unknown function.
- Published
- 2007
- Full Text
- View/download PDF
21. Polymerization of mycobacterial arabinogalactan and ligation to peptidoglycan.
- Author
-
Yagi T, Mahapatra S, Mikusova K, Crick DC, and Brennan PJ
- Subjects
- Binding Sites, Cell Wall chemistry, Cell Wall metabolism, Kinetics, Models, Biological, Mycobacterium chemistry, Polymers chemistry, Solubility, Galactans chemistry, Galactans metabolism, Mycobacterium metabolism, Peptidoglycan metabolism
- Abstract
The cell wall of Mycobacterium spp. consists predominately of arabinogalactan chains linked at the reducing ends to peptidoglycan via a P-GlcNAc-(alpha1-3)-Rha linkage unit (LU) and esterified to a variety of mycolic acids at the nonreducing ends. Several aspects of the biosynthesis of this complex have been defined, including the initial formation of the LU on a polyprenyl phosphate (Pol-P) molecule followed by the sequential addition of galactofuranosyl (Galf) units to generate Pol-P-P-LU-(Galf)1,2,3, etc. and Pol-P-P-LU-galactan, catalyzed by a bifunctional galactosyltransferase (Rv3808c) capable of adding alternating 5- and 6-linked Galf units. By applying cell-free extracts of Mycobacterium smegmatis, containing cell wall and membrane fragments, and differential labeling with UDP-[14C]Galp and recombinant UDP-Galp mutase as the source of [14C]Galf for galactan biosynthesis and 5-P-[14C]ribosyl-P-P as a donor of [14C]Araf for arabinan synthesis, we now demonstrate sequential synthesis of the simpler Pol-P-P-LU-(Galf)n glycolipid intermediates followed by the Pol-P-P-LU-arabinogalactan and, finally, ligation of the P-LU-arabinogalactan to peptidoglycan. This first time demonstration of in vitro ligation of newly synthesized P-LU-arabinogalactan to newly synthesized peptidoglycan is a necessary forerunner to defining the genetics and enzymology of cell wall polymer-peptidoglycan ligation in Mycobacterium spp. and examining this step as a target for new antibacterial drugs.
- Published
- 2003
- Full Text
- View/download PDF
22. Definition of the first mannosylation step in phosphatidylinositol mannoside synthesis. PimA is essential for growth of mycobacteria.
- Author
-
Korduláková J, Gilleron M, Mikusova K, Puzo G, Brennan PJ, Gicquel B, and Jackson M
- Subjects
- Cell-Free System, Dolichol Monophosphate Mannose chemistry, Dolichol Monophosphate Mannose metabolism, Gene Expression Regulation, Bacterial, Genes, Essential, Genome, Bacterial, Glycosylation, Kinetics, Mannose metabolism, Mycobacterium smegmatis genetics, Mycobacterium tuberculosis genetics, Phosphatidylinositols biosynthesis
- Abstract
We examined the function of the pimA (Rv2610c) gene, located in the vicinity of the phosphatidylinositol synthase gene in the genomes of Mycobacterium tuberculosis and Mycobacterium smegmatis, which encodes a putative mannosyltransferase involved in the early steps of phosphatidylinositol mannoside synthesis. A cell-free assay was developed in which membranes from M. smegmatis overexpressing the pimA gene incorporate mannose from GDP-[(14)C]Man into di- and tri-acylated phosphatidylinositol mono-mannosides. Moreover, crude extracts from Escherichia coli producing a recombinant PimA protein synthesized diacylated phosphatidylinositol mono-mannoside from GDP-[(14)C]Man and bovine phosphatidylinositol. To determine whether PimA is an essential enzyme of mycobacteria, we constructed a pimA conditional mutant of M. smegmatis. The ability of this mutant to synthesize the PimA mannosyltransferase was dependent on the presence of a functional copy of the pimA gene carried on a temperature-sensitive rescue plasmid. We demonstrate here that the pimA mutant is unable to grow at the higher temperature at which the rescue plasmid is lost. Thus, the synthesis of phosphatidylinositol mono-mannosides and derived higher phosphatidylinositol mannosides in M. smegmatis appears to be dependent on PimA and essential for growth. This work provides the first direct evidence of the essentiality of phosphatidylinositol mannosides for the growth of mycobacteria.
- Published
- 2002
- Full Text
- View/download PDF
23. Interaction of human mannose-binding protein with Mycobacterium avium.
- Author
-
Polotsky VY, Belisle JT, Mikusova K, Ezekowitz RA, and Joiner KA
- Subjects
- Calcium pharmacology, Carbohydrate Conformation, Carbohydrate Sequence, Cell Wall physiology, Enzyme-Linked Immunosorbent Assay, Humans, Kinetics, Mannans chemistry, Mannose-Binding Lectins, Membrane Lipids metabolism, Molecular Sequence Data, Oligosaccharides chemistry, Recombinant Proteins metabolism, Species Specificity, Substrate Specificity, Acute-Phase Proteins metabolism, Carrier Proteins metabolism, Mannans metabolism, Mycobacterium avium physiology, Neutrophils microbiology
- Abstract
The interaction between human mannose-binding protein (MBP) and Mycobacterium avium was explored. By ELISA, calcium-dependent and mannan-inhibitable binding of human recombinant MBP (rMBP) to live M. avium was observed. Preincubation of M. avium with rMBP resulted in a 2-fold increase in uptake by human neutrophils. Mycobacterial cell wall components were assessed by ELISA for their ability to bind the carbohydrate recognition domain of rMBP. The best ligand was mannosyl-lipoarabinomannan, followed by lipomannan, phosphatidylinositol mannoside, arabinosyl-lipoarabinomannan, and dimycolated trehalose (cord factor). rMBP did not bind to partially purified lipid fractions containing glycopeptidolipids. These results are consistent with the known structural basis for rMBP ligand recognition. They suggest that MBP may play a role in host defense against M. avium by opsonizing both whole organisms and free cell wall components for internalization.
- Published
- 1997
- Full Text
- View/download PDF
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