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Other Publications (115)
- Expert Opinion on Investigational Drugs
- Journal of Molecular Medicine (Berlin, Germany)
- The Biochemical Journal
- The Biochemical Journal
- The Biochemical Journal
- Chemistry & Biology
- The Journal of Biological Chemistry
- Expert Opinion on Investigational Drugs
- Microbiology (Reading, England)
- Molecular Microbiology
- Infection and Immunity
- The Journal of Biological Chemistry
- Infection and Immunity
- The Journal of Biological Chemistry
- Journal of Immunology (Baltimore, Md. : 1950)
- Biochemical and Biophysical Research Communications
- Acta Crystallographica. Section D, Biological Crystallography
- Biochemistry
- Journal of Molecular Biology
- Journal of Immunology (Baltimore, Md. : 1950)
- Tuberculosis (Edinburgh, Scotland)
- Molecular Microbiology
- Antimicrobial Agents and Chemotherapy
- The Journal of Biological Chemistry
- Molecular Microbiology
- Infection and Immunity
- The Journal of Biological Chemistry
- Journal of Bacteriology
- Cell
- Proceedings of the National Academy of Sciences of the United States of America
- Chemistry & Biology
- Molecular Microbiology
- The Journal of Biological Chemistry
- Nature Medicine
- Journal of Molecular Biology
- Antimicrobial Agents and Chemotherapy
- Journal of Immunology (Baltimore, Md. : 1950)
- FEBS Letters
- Proceedings of the National Academy of Sciences of the United States of America
- Glycoconjugate Journal
- Molecular Microbiology
- Journal of Bacteriology
- Acta Crystallographica. Section F, Structural Biology and Crystallization Communications
- PloS One
- Infection and Immunity
- The Biochemical Journal
- Antimicrobial Agents and Chemotherapy
- Proteomics
- FEBS Letters
- Protein Expression and Purification
- The Journal of Biological Chemistry
- Molecular Microbiology
- Plasmid
- Cell Host & Microbe
- Blood
- The Journal of Biological Chemistry
- Molecular Microbiology
- Journal of Bacteriology
- Structure (London, England : 1993)
- The Journal of Biological Chemistry
- The Journal of Biological Chemistry
- PloS One
- The Journal of Biological Chemistry
- The Journal of Biological Chemistry
- The Journal of Biological Chemistry
- Molecular Microbiology
- Proteomics
- Journal of the American Chemical Society
- Molecular Microbiology
- Bioorganic & Medicinal Chemistry Letters
- Molecular Microbiology
- Bioorganic & Medicinal Chemistry Letters
- Tuberculosis (Edinburgh, Scotland)
- The Journal of Biological Chemistry
- Biochemical and Biophysical Research Communications
- PloS One
- Journal of Bacteriology
- The Journal of Biological Chemistry
- Immunology Letters
- Dalton Transactions (Cambridge, England : 2003)
- Antimicrobial Agents and Chemotherapy
- The Journal of Biological Chemistry
- The Journal of Biological Chemistry
- Journal of Proteomics
- Journal of Bacteriology
- PloS One
- Dalton Transactions (Cambridge, England : 2003)
- Antimicrobial Agents and Chemotherapy
- The Journal of Biological Chemistry
- Biochemical and Biophysical Research Communications
- PloS One
- Infection, Genetics and Evolution : Journal of Molecular Epidemiology and Evolutionary Genetics in Infectious Diseases
- PloS One
- The Journal of Biological Chemistry
- MBio
- International Journal of Antimicrobial Agents
- Nature Chemical Biology
- Chemical Biology & Drug Design
- The Journal of Biological Chemistry
- Chemical Biology & Drug Design
- Proceedings of the National Academy of Sciences of the United States of America
- MBio
- Biochemical and Biophysical Research Communications
- Antimicrobial Agents and Chemotherapy
- PLoS Pathogens
- Médecine Sciences : M/S
- European Journal of Medicinal Chemistry
- FEMS Microbiology Letters
- PloS One
- Microbiology Spectrum
- The Journal of Antimicrobial Chemotherapy
- Journal of Neuroimmunology
- Alzheimer's Research & Therapy
- Molecular Microbiology
- Médecine Sciences : M/S
Articles by Laurent Kremer in JoVE
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Deciphering and Imaging Pathogenesis and Cording of Mycobacterium abscessus in Zebrafish Embryos
Audrey Bernut1,2, Christian Dupont1,2, Alain Sahuquet1, Jean-Louis Herrmann3, Georges Lutfalla1, Laurent Kremer1,2
1Dynamique des Interactions Membranaires Normales et Pathologiques, CNRS, UMR 535, Université Montpellier, 2Centre d'études d'agents Pathogènes et Biotechnologies pour la Santé, CNRS, FRE 3689, Université Montpellier, 3Unité de Formation et de Recherche des Sciences de la Santé, EA3647-EPIM, Université Versailles St Quentin
Optically transparent zebrafish embryos are widely used to study and visualize in real time the interactions between pathogenic microorganisms and the innate immune cells. Micro-injection of Mycobacterium abscessus, combined with fluorescence imaging, is used to scrutinize essential pathogenic features such as cord formation in zebrafish embryos.
Other articles by Laurent Kremer on PubMed
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Immunoregulatory Functions of Interleukin 18 and Its Role in Defense Against Bacterial Pathogens
Journal of Molecular Medicine (Berlin, Germany).
Mar, 2002 |
Pubmed ID: 11894141 Interleukin-18 (IL-18) is a proinflammatory cytokine that belongs to the IL-1 cytokine family, due to its structure, receptor family and signal transduction pathways. Similarly to IL-1beta, IL-18 is synthesized as a precursor requiring caspase-1 for cleavage into an active IL-18 molecule. However, with regard to its capacity to induce the production of Th1 cytokines and to enhance cell-mediated cytotoxicity, IL-18 is also related to IL-12. Produced mainly by antigen-presenting cells, IL-18 is a pleiotropic factor involved in the regulation of both innate and acquired immune responses, playing a key role in autoimmune, inflammatory, and infectious diseases. This review summarizes recent advances in the understanding of IL-18 structure, processing, receptor expression, and immunoregulatory functions and emphasizes the critical role of this cytokine in bacterial infections. It focuses on the participation of this cytokine in the defense against intracellular bacteria, including Listeria, Shigella, Salmonella, and Mycobacterium tuberculosis. Since this cytokine may be particularly useful in immunoprophylactic and immunotherapeutic interventions in which the cellular response is most desirable, the potential therapeutic aspects of IL-18 is also discussed.
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Ppm1, a Novel Polyprenol Monophosphomannose Synthase from Mycobacterium Tuberculosis
The Biochemical Journal.
Jul, 2002 |
Pubmed ID: 11931640 Dolichol monophosphomannose (DPM) is an ever-present donor of mannose (Man) in various eukaryotic glycosylation processes. Intriguingly, the related polyprenol monophosphomannose (PPM) is involved in the biosynthesis of lipomannan and lipoarabinomanan, key bacterial factors termed modulins that are found in mycobacteria. Based on similarities to known DPM synthases, we have identified and characterized the PPM synthase of Mycobacterium tuberculosis, now termed Mt-Ppm1. In the present study, we demonstrate that Mt-Ppm1 possesses an unusual two-domain architecture, by which the second domain is sufficient for PPM synthesis. However, when overexpressed separately in mycobacteria, domain 1 of Mt-Ppm1 appears to increase the synthesis of PPM. Interestingly, other mycobacteria such as M. smegmatis, M. avium and M. leprae produce two distinct proteins, which are similar to the two domains found in Mt-Ppm1. Using an in vitro assay, we also demonstrate that Mt-Ppm1 transfers Man from GDP-Man to a structurally diverse range of lipid monophosphate acceptors. The identification of the PPM synthase as a key enzyme in lipoarabinomannan biosynthesis now provides an attractive candidate for gene disruption to generate mutants for subsequent immunological studies. PPM synthase can also be exploited as a target for specific inhibitors of M. tuberculosis.
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Characterization of a Putative Alpha-mannosyltransferase Involved in Phosphatidylinositol Trimannoside Biosynthesis in Mycobacterium Tuberculosis
The Biochemical Journal.
May, 2002 |
Pubmed ID: 11964144 Phosphatidyl-myo-inositol mannosides (PIMs), lipomannan (LM) and lipoarabinomannan (LAM) are an important class of bacterial factors termed modulins that are found in tuberculosis and leprosy. Although their structures are well established, little is known with respect to the molecular aspects of the biosynthetic machinery involved in the synthesis of these glycolipids. On the basis of sequence similarity to other glycosyltransferases and our previous studies defining an alpha-mannosyltransferase from Mycobacterium tuberculosis, named PimB [Schaeffer, Khoo, Besra, Chatterjee, Brennan, Belisle and Inamine (1999) J. Biol. Chem. 274, 31625-31631], which catalysed the formation of triacyl (Ac(3))-PIM(2) (i.e. the dimannoside), we have identified a related gene from M. tuberculosis CDC1551, now designated pimC. The use of a cell-free assay containing GDP-[(14)C]mannose, amphomycin and membranes from Myobacterium smegmatis overexpressing PimC led to the synthesis of a new alkali-labile PIM product. Fast-atom-bombardment MS established the identity of the new enzymically synthesized product as Ac(3)PIM(3) (i.e. the trimannoside). The results indicate that pimC encodes an alpha-mannosyltransferase involved in Ac(3)PIM(3) biosynthesis. However, inactivation of pimC in Myobacterium bovis Bacille Calmette-Guérin (BCG) did not affect the production of higher PIMs, LM and LAM when compared with wild-type M. bovis BCG, suggesting the existence of redundant gene(s) or an alternate pathway that may compensate for this PimC deficiency. Further analyses, which compared the distribution of pimC in a panel of M. tuberculosis strains, revealed that pimC was present in only 22% of the clinical isolates examined.
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Mycolic Acid Biosynthesis and Enzymic Characterization of the Beta-ketoacyl-ACP Synthase A-condensing Enzyme from Mycobacterium Tuberculosis
The Biochemical Journal.
Jun, 2002 |
Pubmed ID: 12023885 Mycolic acids consist of long-chain alpha-alkyl-beta-hydroxy fatty acids that are produced by successive rounds of elongation catalysed by a type II fatty acid synthase (FAS-II). A key feature in the elongation process is the condensation of a two-carbon unit from malonyl-acyl-carrier protein (ACP) to a growing acyl-ACP chain catalysed by a beta-ketoacyl-ACP synthase (Kas). In the present study, we provide evidence that kasA from Mycobacterium tuberculosis encodes an enzyme that elongates in vivo the meromycolate chain, in both Mycobacterium smegmatis and Mycobacterium chelonae. We demonstrate that KasA belongs to the FAS-II system, which utilizes primarily palmitoyl-ACP rather than short-chain acyl-ACP primers. Furthermore, in an in vitro condensing assay using purified recombinant KasA, palmitoyl-AcpM and malonyl-AcpM, KasA was found to express Kas activity. Also, mutated KasA proteins, with mutation of Cys(171), His(311), Lys(340) and His(345) to Ala abrogated the condensation activity of KasA in vitro completely. Finally, purified KasA was highly sensitive to cerulenin, a well-known inhibitor of Kas, which may lead to the development of novel anti-mycobacterial drugs targeting KasA.
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The Methyl-branched Fortifications of Mycobacterium Tuberculosis
Chemistry & Biology.
May, 2002 |
Pubmed ID: 12031661 Mycobacterium tuberculosis continues to be the predominant global infectious agent, annually killing over three million people. Recommended drug regimens have the potential to control tuberculosis, but lack of adherence to such regimens has resulted in the emergence of resistant strains. Mycobacterium tuberculosis has an unusual cell envelope, rich in unique long-chain lipids, that provides a very hydrophobic barrier to antibiotic access. Such lipids, however, can be drug targets, as exemplified by the action of the front-line drug isoniazid on mycolic acid biosynthesis. A number of these lipids are potential key virulence factors and their structures are based on very characteristic methyl-branched long-chain acids and alcohols. This review details the history, structure, and genetic aspects of the biosynthesis of these methyl-branched components, good examples of which are the phthiocerols and the mycocerosic and mycolipenic acids.
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Structural Study of Lipomannan and Lipoarabinomannan from Mycobacterium Chelonae. Presence of Unusual Components with Alpha 1,3-mannopyranose Side Chains
The Journal of Biological Chemistry.
Aug, 2002 |
Pubmed ID: 12063260 Lipomannan (LM) and lipoarabinomannan (LAM) are major glycolipids present in the mycobacterial cell wall that are able to modulate the host immune response. In this study, we have undertaken the structural determination of these important modulins in Mycobacterium chelonae, a fast growing pathogenic mycobacterial species. One-dimensional and two-dimensional NMR spectra were used to demonstrate that LM and LAM from M. chelonae, designated CheLM and CheLAM, respectively, possess structures that differ from the ones reported earlier in other mycobacterial species. Analysis by gas chromatography/mass spectrometry of the phosphatidyl-myo-inositol anchor, which is thought to play a role in the biological functions of these lipoglycans, pointed to a high degree of heterogeneity based on numerous combinations of acyl groups on the C-1 and C-2 positions of the glycerol moiety. Characterization of the mannan core of CheLM and CheLAM revealed the presence of novel alpha1,3-mannopyranosyl side chains. This motif, which reacted specifically with the lectin from Galanthus nivalis, was found to be unique among a panel of nine mycobacterial species. Then, CheLM and CheLAM were found to be devoid of both the mannooligosaccharide cap present in Mycobacterium tuberculosis and the inositol phosphate cap present in Mycobacterium smegmatis and other fast growing species. Tumor necrosis factor-alpha and interleukin-8 production were assessed from human macrophages with LAM preparations from different species. Our results suggest that the inositol phosphate capping may represent the major cytokine-inducing component of LAMs. This work not only underlines the diversity of LAM structures among various mycobacterial species but also provides new structures that could be useful to dissect the structure-function relationships of these complex molecules.
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Current Status and Future Development of Antitubercular Chemotherapy
Expert Opinion on Investigational Drugs.
Aug, 2002 |
Pubmed ID: 12150700 Tuberculosis (TB), which kills more people than any other infectious disease, was declared a global emergency by the World Health Organization in 1993. The emergence of new Mycobacterium tuberculosis strains that are resistant to some or all current antitubercular drugs seriously hampers the control of the disease. Up to 50 million people may be infected with drug-resistant TB, with resistance being caused by inconsistent or partial treatment when patients do not comply with long-term chemotherapy. Resistance is often a corollary to HIV infection. Besides being more fatal, drug-resistant TB is more difficult and more expensive to treat. In addition to this human cost, TB also represents a significant economic burden for developing countries. Therefore, new approaches to the treatment of TB are needed. During the last few years, important efforts have been made in order to elucidate the molecular mechanism of action of antitubercular drugs and understand the genetic basis of acquired drug resistance in M. tuberculosis. The identification of novel targets requires the characterisation of biochemical pathways specific to mycobacteria. Many unique metabolic processes occur during the biosynthesis of cell wall components, including arabinogalactan and mycolic acids. In this review, the mode of action of first- and second-line agents, as well as the potentiality of some promising drugs that are still at an early stage of development will be described. Finally, some of the attractive targets offered by the mycobacterial cell wall for the rational design of new antitubercular drugs for a future and more effective control of the disease will be examined.
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Temperature-induced Changes in the Cell-wall Components of Mycobacterium Thermoresistibile
Microbiology (Reading, England).
Oct, 2002 |
Pubmed ID: 12368448 The mycobacterial cell wall consists of a core composed of peptidoglycan linked to the heteropolysaccharide arabinogalactan, which in turn is attached to mycolic acids. A variety of free lipids complements the mycolyl residues, whereas phosphatidylinositol mannosides (PIMs), lipoarabinomannan and proteins are interspersed in this framework. As a consequence, the cell envelope is extremely rich in lipids and early work has shown that the lipid content may vary with environmental conditions. To extend these studies, the influence of growth temperature on cell envelope components in Mycobacterium thermoresistibile, a temperature-resistant mycobacterial species, was investigated. Mycolic acid synthesis was reduced at 55 degrees C compared to 37 degrees C and the production of fatty acids, presumably precursors of mycolic acids, was increased. Since fatty acids are elongated by the type II fatty acid synthase complex and consequently by a mycobacterial beta-ketoacyl acyl carrier protein synthase (KasA), leading to mycolic acids, the expression level of KasA was analysed by Western blotting. KasA expression was significantly decreased at 55 degrees C over 37 degrees C. Important changes in the mycolic acid composition were observed and characterized by reduced levels of cyclopropanation and the concomitant accumulation of the cis-olefin derivatives. In addition, striking differences involved in complex lipid composition, including acylated trehaloses and trehalose dimycolate (TDM) were also observed. At 55 degrees C, M. thermoresistibile produced less TDM than at 37 degrees C, which could be explained by the down-regulation of antigen 85 (Ag85) expression as shown by Western blotting. The Ag85 complex represents a family of proteins known to catalyse the transfer of mycolates to trehalose, thereby generating TDM. Furthermore, at 55 degrees C the level of phosphatidyl-inositol hexamannoside (PIM(6)) synthesis, but not that of other PIM species, was dramatically reduced. This observation could be correlated to a decrease of mannosyltransferase activity associated with membranes prepared from cells grown at 55 degrees C as compared to 37 degrees C. Altogether, this study suggests that mycobacteria are capable of inducing important cell-wall changes in response to temperature variations, which may represent a strategy developed by the bacteria to adapt to environmental changes.
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Overexpression of InhA, but Not KasA, Confers Resistance to Isoniazid and Ethionamide in Mycobacterium Smegmatis, M. Bovis BCG and M. Tuberculosis
Molecular Microbiology.
Oct, 2002 |
Pubmed ID: 12406221 The inhA and kasA genes of Mycobacterium tuberculosis have each been proposed to encode the primary target of the antibiotic isoniazid (INH). Previous studies investigating whether overexpressed inhA or kasA could confer resistance to INH yielded disparate results. In this work, multicopy plasmids expressing either inhA or kasA genes were transformed into M. smegmatis, M. bovis BCG and three different M. tuberculosis strains. The resulting transformants, as well as previously published M. tuberculosis strains with multicopy inhA or kasAB plasmids, were tested for their resistance to INH, ethionamide (ETH) or thiolactomycin (TLM). Mycobacteria containing inhA plasmids uniformly exhibited 20-fold or greater increased resistance to INH and 10-fold or greater increased resistance to ETH. In contrast, the kasA plasmid conferred no increased resistance to INH or ETH in any of the five strains, but it did confer resistance to thiolactomycin, a known KasA inhibitor. INH is known to increase the expression of kasA in INH-susceptible M. tuberculosis strains. Using molecular beacons, quantified inhA and kasA mRNA levels showed that increased inhA mRNA levels corre--lated with INH resistance, whereas kasA mRNA levels did not. In summary, analysis of strains harbouring inhA or kasA plasmids yielded the same conclusion: overexpressed inhA, but not kasA, confers INH and ETH resistance to M. smegmatis, M. bovis BCG and M. tuberculosis. Therefore, InhA is the primary target of action of INH and ETH in all three species.
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Mycobacterium Bovis BCG Producing Interleukin-18 Increases Antigen-specific Gamma Interferon Production in Mice
Infection and Immunity.
Dec, 2002 |
Pubmed ID: 12438324 Interleukin-18 (IL-18) and IL-12 play a critical role in the expression of cell-mediated immunity involved in host defense against intracellular pathogens. Both cytokines are produced by macrophages and act in synergy to induce gamma interferon (IFN-gamma) production by T, B, and natural killer cells. In the present study, we analyzed both cellular and humoral responses upon infection with IL-18-secreting BCG of BALB/c and C3H/HeJ mice, two strains known to differ in their ability to support the growth of BCG. The cDNA encoding mature IL-18 was fused in frame with the alpha-antigen signal peptide-coding sequence, cloned downstream of the mycobacterial hsp60 promoter and expressed in BCG. IL-18 produced by the recombinant BCG strain was functional, as judged by NF-kappaB-mediated luciferase induction in a tissue culture assay. When susceptible mice were infected with IL-18-producing BCG, their splenocytes were found to produce higher amounts of Th1 cytokines after stimulation with mycobacterial antigens than the splenocytes of mice infected with the nonrecombinant BCG. This was most prominent for IFN-gamma, although the mycobacterial antigen-specific secretion of granulocyte-macrophage colony-stimulating factor and IL-10 was also augmented after infection with the recombinant BCG compared to infection with nonrecombinant BCG. In contrast, the immunoglobulin G levels in serum against mycobacterial antigens were lower when the mice were infected with IL-18-producing BCG compared to infection with nonrecombinant BCG. The IL-18 effect was delayed in BALB/c compared to C3H/HeJ mice. These results indicate that the production of IL-18 by recombinant BCG may enhance the immunomodulatory properties of BCG further toward a Th1 profile. This may be particularly useful for immunotherapeutic or prophylactic interventions in which a Th1 response is most desirable.
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Inhibition of InhA Activity, but Not KasA Activity, Induces Formation of a KasA-containing Complex in Mycobacteria
The Journal of Biological Chemistry.
Jun, 2003 |
Pubmed ID: 12654922 Isoniazid (INH) remains one of the key drugs used to control tuberculosis, with the enoyl-AcpM reductase InhA being the primary target. However, based on the observation that INH-treated Mycobacterium tuberculosis overproduces KasA, an enzyme involved in the biosynthesis of mycolic acids, and induces the formation of a covalent complex consisting of AcpM, KasA, and INH, it has been proposed that KasA represents the primary target of INH. However, the relevance of this complex to INH action remains obscure. This study was aimed at clarifying the role of InhA and KasA in relation to INH activity. By using anti-KasA antibodies we detected the KasA-containing complex in INH-treated Mycobacterium smegmatis. In addition, INH-treated cells also produced constant levels of KasA that were not sequestered in the complex and presumably were sufficient to ensure mycolic acid biosynthesis. Interestingly, a furA-lacking strain induced the complex at lower concentrations of INH compared with the control strain, whereas higher INH concentrations were necessary to induce the complex in a strain that lacks katG, suggesting that INH needs to be activated by KatG to induce the KasA-containing complex. The InhA inhibitors ethionamide and diazaborine also induced the complex; thus, its formation was not specifically relevant to INH action but was because of InhA inhibition. In addition, in vitro assays using purified InhA and KasA demonstrated that KatG-activated INH, triclosan, and diazaborine inhibited InhA but not KasA activity. Moreover, several thermosensitive InhA mutant strains of M. smegmatis constitutively expressed the KasA-containing complex. This study provides the biochemical and genetic evidence. 1) Only inhibition of InhA, but not KasA, induces the KasA-containing complex. 2) INH is not part of the complex. 3) INH does not target KasA, consistent with InhA being the primary target of INH.
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Lipomannan and Lipoarabinomannan from a Clinical Isolate of Mycobacterium Kansasii: Novel Structural Features and Apoptosis-inducing Properties
The Journal of Biological Chemistry.
Sep, 2003 |
Pubmed ID: 12829695 Although Mycobacterium kansasii has emerged as an important pathogen frequently encountered in immunocompromised patients, little is known about the mechanisms of M. kansasii pathogenicity. Lipoarabinomannan (LAM), a major mycobacterial cell wall lipoglycan, is an important virulence factor for many mycobacteria, as it modulates the host immune response. Therefore, the detailed structures of the of M. kansasii LAM (KanLAM), as well as of its biosynthetic precursor lipomannan (KanLM), were determined in a clinical strain isolated from a human immunodeficiency virus-positive patient. Structural analyses revealed that these lipoglycans possess important differences as compared with those from other mycobacterial species. KanLAM carries a mannooligosaccharide cap but is devoid of the inositol phosphate cap present in Mycobacterium smegmatis. Characterization of the mannan core of KanLM and KanLAM demonstrated the following occurrences: 1) alpha1,2-oligo-mannopyranosyl side chains, contrasting with the single mannopyranosyl residues substituting the mannan core in all the other structures reported so far; and 2) 5-methylthiopentose residues that were described to substitute the arabinan moiety from Mycobacterium tuberculosis LAM. With respect to the arabinan domain of KanLAM, succinyl groups were found to substitute the C-3 position on 5-arabinofuranosyl residues, reported to be linked to the C-2 of the 3,5-arabinofuranose in Mycobacterium bovis bacillus calmette-guerin LAM. Because M. kansasii has been reported to induce apoptosis, we examined the possibility of the M. kansasii lipoglycans to induce apoptosis of THP-1 cells. Our results indicate that, in contrast to KanLAM, KanLM was a potent apoptosis-inducing factor. This work underlines the diversity of LAM structures among various pathogenic mycobacterial species and also provides evidence of LM being a potential virulence factor in M. kansasii infections by inducing apoptosis.
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Lipomannans, but Not Lipoarabinomannans, Purified from Mycobacterium Chelonae and Mycobacterium Kansasii Induce TNF-alpha and IL-8 Secretion by a CD14-toll-like Receptor 2-dependent Mechanism
Journal of Immunology (Baltimore, Md. : 1950).
Aug, 2003 |
Pubmed ID: 12902506 Lipoarabinomannans (LAMs) are glycolipids from the mycobacterial cell wall that exhibit various biological activities, including proinflammatory and anti-inflammatory responses. However, little is known about the properties of lipomannans (LMs), considered to be precursors of LAMs. In this study, we provide evidence that LMs purified from Mycobacterium chelonae and a clinical strain of Mycobacterium kansasii stimulated mRNA expression and secretion of TNF-alpha and IL-8 from human macrophage-like differentiated THP-1 cells. In contrast to LMs, LAMs were not able to induce a significant cytokine-inducing effect. The mechanism of activation by LMs was investigated using various Abs raised against surface receptors for multiple bacterial products. The presence of anti-CD14 or anti-Toll-like receptor 2 (TLR2) Abs profoundly affected production of TNF-alpha and IL-8, suggesting that both CD14 and TLR2 participate in the LM-mediated activation process. Furthermore, stimulation of cells was dependent on the presence of the LPS-binding protein, a plasma protein that transfers glycolipids to CD14. Chemical degradation of the arabinan domain of mannose-capped LAM from M. kansasii, which presented no cytokine-eliciting effect, restored the cytokine-inducing activity at a level similar to those of LMs. These results support the hypothesis that the presence of an arabinan in LAMs prevents the interaction of these glycolipids with TLR2/CD14 receptors. In addition, we found that phosphatidylinositol dimannosides isolated from M. kansasii did not induce cytokine secretion. This study suggests that LMs isolated from different mycobacterial species participate in the immunomodulation of the infected host and that the D-mannan core of this glycolipid is essential for this function.
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Crystallization and Preliminary X-ray Diffraction Data of Mycobacterium Tuberculosis FbpC1 (Rv3803c)
Acta Crystallographica. Section D, Biological Crystallography.
Dec, 2003 |
Pubmed ID: 14646102 The heterotrimeric antigen 85 complex (Ag85) is a major component of the cell wall of Mycobacterium tuberculosis and consists of three abundantly secreted proteins (FbpA, FbpB and FbpC2). These play key roles in the pathogenesis of tuberculosis and in maintaining cell-wall integrity. A homologue of the Ag85 subunits ( approximately 40% identity) was recently annotated in the M. tuberculosis genome as FbpC1. Unlike the Ag85-complex components, FbpC1 lacks mycolyltransferase activity and its function remains to be established. In order to aid functional characterization, FbpC1 has been crystallized. At room temperature, tetragonal crystals of FbpC1 were obtained belonging to space group P4(1)2(1)2 (unit-cell parameters a = b = 109.9, c = 61.8 A), yet when frozen the crystals underwent a phase transition to orthorhombic symmetry, space group P2(1)2(1)2(1) (a = 59.9, b = 108.9, c = 109.9 A). Diffraction data complete to 1.7 A resolution were recorded at 100 K at the synchrotron.
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An FHA Phosphoprotein Recognition Domain Mediates Protein EmbR Phosphorylation by PknH, a Ser/Thr Protein Kinase from Mycobacterium Tuberculosis
Biochemistry.
Dec, 2003 |
Pubmed ID: 14690440 In bacteria, regulatory phosphorylation of proteins at serine and/or threonine residues by Ser/Thr protein kinase (STPK) is an emerging theme in prokaryotic signaling, particularly since the prediction of the occurrence of several STPKs from genome sequencing and sequence surveys. Here we show that protein PknH possesses an autokinase activity and belongs to the large STPK family found in Mycobacterium tuberculosis. Evidence is presented that PknH can also phosphorylate EmbR, a protein suspected to modulate the level of arabinosyltransferase activity involved in arabinan biosynthesis of arabinogalactan, a key molecule of the mycobacterial cell wall. Interestingly, EmbR possesses an FHA (forkhead-associated) domain, a newly described phosphoprotein recognition domain, which plays an essential role in PknH-EmbR interaction and phosphorylation of EmbR by PknH. It is demonstrated that mutation of each of three particular residues of this FHA domain, Arg312, Ser326, and Asn348, totally abolishes the PknH-mediated phosphorylation of EmbR, thus highlighting the critical role of this domain in the direct interaction between EmbR and PknH.
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The Structure of Mycobacterium Tuberculosis MPT51 (FbpC1) Defines a New Family of Non-catalytic Alpha/beta Hydrolases
Journal of Molecular Biology.
Jan, 2004 |
Pubmed ID: 14672660 Mycobacterium tuberculosis, the causative agent of tuberculosis, is known to secrete a number of highly immunogenic proteins that are thought to confer pathogenicity, in part, by mediating binding to host tissues. Among these secreted proteins are the trimeric antigen 85 (Ag85) complex and the related MPT51 protein, also known as FbpC1. While the physiological function of Ag85, a mycolyltransferase required for the biosynthesis of the cell wall component alpha,alpha'-trehalose dimycolate (or cord factor), has been identified recently, the function of the closely related MPT51 (approximately 40% identity with the Ag85 components) remains to be established. The crystal structure of M.tuberculosis MPT51, determined to 1.7 A resolution, shows that MPT51, like the Ag85 components Ag85B and Ag85C2, folds as an alpha/beta hydrolase, but it does not contain any of the catalytic elements required for mycolyltransferase activity. Moreover, the absence of a recognizable alpha,alpha'-trehalose monomycolate-binding site and the failure to detect an active site suggest that the function of MPT51 is of a non-enzymatic nature and that MPT51 may in fact represent a new family of non-catalytic alpha/beta hydrolases. Previous experimental evidence and the structural similarity to some integrins and carbohydrate-binding proteins led to the hypothesis that MPT51 might have a role in host tissue attachment, whereby ligands may include the serum protein fibronectin and small sugars.
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Toll-like Receptor 2 (TLR2)-dependent-positive and TLR2-independent-negative Regulation of Proinflammatory Cytokines by Mycobacterial Lipomannans
Journal of Immunology (Baltimore, Md. : 1950).
Apr, 2004 |
Pubmed ID: 15034058 Lipoarabinomannans (LAM) and lipomannans (LM) are integral parts of the mycobacterial cell wall recognized by cells involved in the innate immune response and have been found to modulate the cytokine response. Typically, mannosylated LAM from pathogenic mycobacteria have been reported to be anti-inflammatory, whereas phosphoinositol-substituted LAM from nonpathogenic species are proinflammatory molecules. In this study, we show that LM from several mycobacterial species, including Mycobacterium chelonae, Mycobacterium kansasii, and Mycobacterium bovis bacillus Calmette-Guérin, display a dual function by stimulating or inhibiting proinflammatory cytokine synthesis through different pathways in murine primary macrophages. LM, but none of the corresponding LAM, induce macrophage activation characterized by cell surface expression of CD40 and CD86 and by TNF and NO secretion. This activation is dependent on the presence of Toll-like receptor (TLR) 2 and mediated through the adaptor protein myeloid differentiation factor 88 (MyD88), but independent of either TLR4 or TLR6 recognition. Surprisingly, LM exerted also a potent inhibitory effect on TNF, IL-12p40, and NO production by LPS-activated macrophages. This TLR2-, TLR6-, and MyD88-independent inhibitory effect is also mediated by LAM from M. bovis bacillus Calmette-Guérin but not by LAM derived from M. chelonae and M. kansasii. This study provides evidence that mycobacterial LM bear structural motifs susceptible to interact with different pattern recognition receptors with pro- or anti-inflammatory effects. Thus, the ultimate response of the host may therefore depend on the prevailing LM or LAM in the mycobacterial envelope and the local host cell receptor availability.
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Mycobacterial Lipoarabinomannan and Related Lipoglycans: from Biogenesis to Modulation of the Immune Response
Molecular Microbiology.
Jul, 2004 |
Pubmed ID: 15228522 The cell wall component lipoarabinomannan (ManLAM) from Mycobacterium tuberculosis is involved in the inhibition of phagosome maturation, apoptosis and interferon (IFN)-gamma signalling in macrophages and interleukin (IL)-12 cytokine secretion of dendritic cells (DC). All these processes are important for the host to mount an efficient immune response. Conversely, LAM isolated from non-pathogenic mycobacteria (PILAM) have the opposite effect, by inducing a potent proinflammatory response in macrophages and DCs. LAMs from diverse mycobacterial species differ in the modification of their terminal arabinose residues. The strong proinflammatory response induced by PILAM correlates with the presence of phospho-myo-inositol on the terminal arabinose. Interestingly, recent work indicates that the biosynthetic precursor of LAM, lipomannan (LM), which is also present in the cell wall, displays strong proinflammatory effects, independently of which mycobacterial species it is isolated from. Results from in vitro assays and knock-out mice suggest that LM, like PILAM, mediates its biological activity via Toll-like receptor 2. We hypothesize that the LAM/LM ratio might be a crucial factor in determining the virulence of a mycobacterial species and the outcome of the infection. Recent progress in the identification of genes involved in the biosynthesis of LAM is discussed, in particular with respect to the fact that enzymes controlling the LAM/LM balance might represent targets for new antitubercular drugs. In addition, inactivation of these genes may lead to attenuated strains of M. tuberculosis for the development of new vaccine candidates.
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Probing the Mechanism of the Mycobacterium Tuberculosis Beta-ketoacyl-acyl Carrier Protein Synthase III MtFabH: Factors Influencing Catalysis and Substrate Specificity
The Journal of Biological Chemistry.
Sep, 2005 |
Pubmed ID: 16040614 Mycolic acids are the dominant feature of the Mycobacterium tuberculosis cell wall. These alpha-alkyl, beta-hydroxy fatty acids are formed by the condensation of two fatty acids, a long meromycolic acid and a shorter C(24)-C(26) fatty acid. The component fatty acids are produced via a combination of type I and II fatty acid synthases (FAS) with FAS-I products being elongated by FAS-II toward meromycolic acids. The beta-ketoacyl-acyl carrier protein (ACP) synthase III encoded by mtfabH (mtFabH) links FAS-I and FAS-II, catalyzing the condensation of FAS-I-derived acyl-CoAs with malonyl-acyl carrier protein (ACP). The acyl-CoA chain length specificity of mtFabH was assessed in vitro; the enzyme extended longer, physiologically relevant acyl-CoA primers when paired with AcpM, its natural partner, than with Escherichia coli ACP. The ability of the enzyme to use E. coli ACP suggests that a similar mode of binding is likely with both ACPs, yet it is clear that unique factors inherent to AcpM modulate the substrate specificity of mtFabH. Mutation of proposed key mtFabH residues was used to define their catalytic roles. Substitution of supposed acyl-CoA binding residues reduced transacylation, with double substitutions totally abrogating activity. Mutation of Arg(46) revealed its more critical role in malonyl-AcpM decarboxylation than in the acyl-CoA binding role. Interestingly, this effect was suppressed intragenically by Arg(161) --> Ala substitution. Our structural studies suggested that His(258), previously implicated in malonyl-ACP decarboxylation, also acts as an anchor point for a network of water molecules that we propose promotes deprotonation and transacylation of Cys(122).
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Identification and Structural Characterization of an Unusual Mycobacterial Monomeromycolyl-diacylglycerol
Molecular Microbiology.
Aug, 2005 |
Pubmed ID: 16091048 Systematic thin layer chromatographic (TLC) analysis of apolar lipids in Mycobacterium kansasii revealed the presence of a previously uncharacterized novel component. The product was ubiquitously found in a panel of M. kansasii clinical isolates, as well as other pathogenic and non-pathogenic mycobacterial species. TLC analysis of [(14)C]-acetate- or [(14)C]-glycerol-labelled M. kansasii cultures tentatively assigned the novel product as an unusual triacylglycerol-related lipid. Subsequent purification, followed by structural determination using (1)H-nuclear magnetic resonance (NMR) and electrospray mass spectrometry (ES/MS), led to the identification of this product as a monomeromycolyl-diacylglycerol (MMDAG). Treatment of M. kansasii with either isoniazid (INH), a well-known type II fatty acid synthase (FAS-II) and mycolic acid biosynthesis inhibitor, or tetrahydrolipstatin (THL), a drug approved for treating obesity, correlated with a reduced incorporation of [(14)C]-acetate into both mycolic acids and MMDAG. Addition of INH or THL to the cultures induced major morphological changes and, surprisingly, resulted in an increased number of lipid storage bodies, as determined by electron microscopy. The potent antimycobacterial activity of THL was confirmed against a variety of mycobacterial species, including INH-susceptible and -resistant Mycobacterium tuberculosis strains. Therefore, THL and other beta-lactones may be promising drugs for the development of new antitubercular therapy.
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LosA, a Key Glycosyltransferase Involved in the Biosynthesis of a Novel Family of Glycosylated Acyltrehalose Lipooligosaccharides from Mycobacterium Marinum
The Journal of Biological Chemistry.
Dec, 2005 |
Pubmed ID: 16257960 Members of the genus Mycobacterium are characterized by cell envelopes rich in unusual free lipids, interacting with a covalently anchored mycolyl-arabinogalactan matrix. Previous studies have shown that Mycobacterium marinum produces large amounts of a diacylglycosylphenolphthiocerol, "phenolic" glycolipid. When cultivated on liquid Sauton medium, traces of a polar lipooligosaccharide (LOS) glycolipid antigen were also previously indicated. In this study, it was found that growth of the type strain of M. marinum on solid Sauton or Middlebrook 7H10 agar gave substantial, but different, amounts of a family of four major trehalose-based LOSs. The core pentasaccharide LOS-I was a rhamnosyl diglucosyl-acylated trehalose. The heptasaccharide, LOS-II, was derived from LOS-I by adding xylose accompanied by a novel sugar (X); repeated addition of this sugar unit X gave the octasaccharide LOS-III. LOS-IV has a decasaccharide component with two additional unusual sugar units, YZ. In a recent study (Alexander, D. C., Jones, J. R., Tan, T., Chen, J. M., and Liu, J. (2004) J. Biol. Chem. 279, 18824-18833), chromatographically similar glycolipids were assigned to the family of phosphatidylinositol mannosides (PIMs) and a "PimF" (Rv1500) glycosyltransferase implicated in the conversion of a supposed "PIM5" to a "PIM7." The present study indicates that these putative PIMs are in fact members of the phosphorus-free LOS family of glycolipids and that the protein product of Rv1500, which we have now termed LosA, is a glycosyltransferase involved in transferring sugars to LOS-III to form LOS-IV of M. marinum.
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Conditional Depletion of KasA, a Key Enzyme of Mycolic Acid Biosynthesis, Leads to Mycobacterial Cell Lysis
Journal of Bacteriology.
Nov, 2005 |
Pubmed ID: 16267284 Inhibition or inactivation of InhA, a fatty acid synthase II (FASII) enzyme, leads to mycobacterial cell lysis. To determine whether inactivation of other enzymes of the mycolic acid-synthesizing FASII complex also leads to lysis, we characterized the essentiality of two beta-ketoacyl-acyl carrier protein synthases, KasA and KasB, in Mycobacterium smegmatis. Using specialized transduction for allelic exchange, null kasB mutants, but not kasA mutants, could be generated in Mycobacterium smegmatis, suggesting that unlike kasB, kasA is essential. To confirm the essentiality of kasA, and to detail the molecular events that occur following depletion of KasA, we developed CESTET (conditional expression specialized transduction essentiality test), a genetic tool that combines conditional gene expression and specialized transduction. Using CESTET, we were able to generate conditional null inhA and kasA mutants. We studied the effects of depletion of KasA in M. smegmatis using the former strain as a reference. Depletion of either InhA or KasA led to cell lysis, but with different biochemical and morphological events prior to lysis. While InhA depletion led to the induction of an 80-kDa complex containing both KasA and AcpM, the mycobacterial acyl carrier protein, KasA depletion did not induce the same complex. Depletion of either InhA or KasA led to inhibition of alpha and epoxy mycolate biosynthesis and to accumulation of alpha'-mycolates. Furthermore, scanning electron micrographs revealed that KasA depletion resulted in the cell surface having a "crumpled" appearance, in contrast to the blebs observed on InhA depletion. Thus, our studies support the further exploration of KasA as a target for mycobacterial-drug development.
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Molecular Structure of EmbR, a Response Element of Ser/Thr Kinase Signaling in Mycobacterium Tuberculosis
Proceedings of the National Academy of Sciences of the United States of America.
Feb, 2006 |
Pubmed ID: 16477027 Ser/Thr phosphorylation has emerged as a critical regulatory mechanism in a number of bacteria, including Mycobacterium tuberculosis. This problematic pathogen encodes 11 eukaryotic-like Ser/Thr kinases, yet few substrates or signaling targets have been characterized. Here, we report the structure of EmbR (2.0 A), a putative transcriptional regulator of key arabinosyltransferases (EmbC, -A, and -B), and an endogenous substrate of the Ser/Thr-kinase PknH. EmbR presents a unique domain architecture: the N-terminal winged-helix DNA-binding domain forms an extensive interface with the all-helical central bacterial transcriptional activation domain and is positioned adjacent to the regulatory C-terminal forkhead-associated (FHA) domain, which mediates binding to a Thr-phosphorylated site in PknH. The structure in complex with a phospho-peptide (1.9 A) reveals a conserved mode of phospho-threonine recognition by the FHA domain and evidence for specific recognition of the cognate kinase. The present structures suggest hypotheses as to how EmbR might propagate the phospho-relay signal from its cognate kinase, while serving as a template for the structurally uncharacterized Streptomyces antibiotic regulatory protein family of transcription factors.
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PH-dependent Pore-forming Activity of OmpATb from Mycobacterium Tuberculosis and Characterization of the Channel by Peptidic Dissection
Molecular Microbiology.
Aug, 2006 |
Pubmed ID: 16803587 Mycobacteria are characterized by an unusual cell wall that controls nutrient and small hydrophilic compound permeability. Porin-like proteins are necessary to ensure the transport of molecules into the cell. Here, we investigated the pore-forming properties of OmpATb, a porin from Mycobacterium tuberculosis, in lipid bilayers. Multi-channel experiments showed an asymmetric behaviour with channel closures at negative critical voltages (Vc) and a strong decrease in Vc at acidic pH. Single-channel experiments gave conductance values of about 850 +/- 80 pS in 1 M KCl and displayed a weak cationic selectivity in 4-8 pH range. The production and characterization of a series of truncated OmpATb proteins, showed that the central domain (OmpATb73-220) was sufficient to induce the ion channel properties of the native protein in lipid bilayers, i.e. asymmetric insertion, pH-dependent voltage closure, cationic selectivity and similar conductance values in 1 M KCl. Western blot analysis suggests that the presence of OmpATb is only restricted to certain pathogenic species. Therefore, the propensity of channels of native OmpATb to close at low pH may represent an intrinsic property allowing pathogenic mycobacteria to adapt and survive to mildly acidic conditions, such as those encountered within the macrophage phagosome.
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The Condensing Activities of the Mycobacterium Tuberculosis Type II Fatty Acid Synthase Are Differentially Regulated by Phosphorylation
The Journal of Biological Chemistry.
Oct, 2006 |
Pubmed ID: 16873379 Phosphorylation of proteins by Ser/Thr protein kinases (STPKs) has recently become of major physiological importance because of its possible involvement in virulence of bacterial pathogens. Although Mycobacterium tuberculosis has eleven STPKs, the nature and function of the substrates of these enzymes remain largely unknown. In this work, we have identified for the first time STPK substrates in M. tuberculosis forming part of the type II fatty acid synthase (FAS-II) system involved in mycolic acid biosynthesis: the malonyl-CoA::AcpM transacylase mtFabD, and the beta-ketoacyl AcpM synthases KasA and KasB. All three enzymes were phosphorylated in vitro by different kinases, suggesting a complex network of interactions between STPKs and these substrates. In addition, both KasA and KasB were efficiently phosphorylated in M. bovis BCG each at different sites and could be dephosphorylated by the M. tuberculosis Ser/Thr phosphatase PstP. Enzymatic studies revealed that, whereas phosphorylation decreases the activity of KasA in the elongation process of long chain fatty acids synthesis, this modification enhances that of KasB. Such a differential effect of phosphorylation may represent an unusual mechanism of FAS-II system regulation, allowing pathogenic mycobacteria to produce full-length mycolates, which are required for adaptation and intracellular survival in macrophages.
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X-ray Crystal Structure of Mycobacterium Tuberculosis Beta-ketoacyl Acyl Carrier Protein Synthase II (mtKasB)
Journal of Molecular Biology.
Feb, 2007 |
Pubmed ID: 17174327 Mycolic acids are long chain alpha-alkyl branched, beta-hydroxy fatty acids that represent a characteristic component of the Mycobacterium tuberculosis cell wall. Through their covalent attachment to peptidoglycan via an arabinogalactan polysaccharide, they provide the basis for an essential outer envelope membrane. Mycobacteria possess two fatty acid synthases (FAS); FAS-I carries out de novo synthesis of fatty acids while FAS-II is considered to elongate medium chain length fatty acyl primers to provide long chain (C(56)) precursors of mycolic acids. Here we report the crystal structure of Mycobacterium tuberculosis beta-ketoacyl acyl carrier protein synthase (ACP) II mtKasB, a mycobacterial elongation condensing enzyme involved in FAS-II. This enzyme, along with the M. tuberculosis beta-ketoacyl ACP synthase I mtKasA, catalyzes the Claisen-type condensation reaction responsible for fatty acyl elongation in FAS-II and are potential targets for development of novel anti-tubercular drugs. The crystal structure refined to 2.4 A resolution revealed that, like other KAS-II enzymes, mtKasB adopts a thiolase fold but contains unique structural features in the capping region that may be crucial to its preference for longer fatty acyl chains than its counterparts from other bacteria. Modeling of mtKasA using the mtKasB structure as a template predicts the overall structures to be almost identical, but a larger entrance to the active site tunnel is envisaged that might contribute to the greater sensitivity of mtKasA to the inhibitor thiolactomycin (TLM). Modeling of TLM binding in mtKasB shows that the drug fits the active site poorly and results of enzyme inhibition assays using TLM analogues are wholly consistent with our structural observations. Consequently, the structure described here further highlights the potential of TLM as an anti-tubercular lead compound and will aid further exploration of the TLM scaffold towards the design of novel compounds, which inhibit mycobacterial KAS enzymes more effectively.
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EthA, a Common Activator of Thiocarbamide-containing Drugs Acting on Different Mycobacterial Targets
Antimicrobial Agents and Chemotherapy.
Mar, 2007 |
Pubmed ID: 17220416 Many of the current antimycobacterial agents require some form of cellular activation unmasking reactive groups, which in turn will bind to their specific targets. Therefore, understanding the mechanisms of activation of current antimycobacterials not only helps to decipher mechanisms of drug resistance but may also facilitate the development of alternative activation strategies or of analogues that do not require such processes. Herein, through the use of genetically defined strains of Mycobacterium bovis BCG we provide evidence that EthA, previously shown to activate ethionamide, also converts isoxyl (ISO) and thiacetazone (TAC) into reactive species. These results were further supported by the development of an in vitro assay using purified recombinant EthA, which allowed direct assessment of the metabolism of ISO. Interestingly, biochemical analysis of [(14)C]acetate-labeled cultures suggested that all of these EthA-activated drugs inhibit mycolic acid biosynthesis via different mechanisms through binding to specific targets. This report is also the first description of the molecular mechanism of action of TAC, a thiosemicarbazone antimicrobial agent that is still used in the treatment of tuberculosis as a second-line drug in many developing countries. Altogether, the results suggest that EthA is a common activator of thiocarbamide-containing drugs. The broad specificity of EthA can now be used to improve the activation process of these drugs, which may help overcome the toxicity problems associated with clinical thiocarbamide use.
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Mycobacterial Lipomannan Induces Granuloma Macrophage Fusion Via a TLR2-dependent, ADAM9- and Beta1 Integrin-mediated Pathway
Journal of Immunology (Baltimore, Md. : 1950).
Mar, 2007 |
Pubmed ID: 17312164 Tuberculous granulomas are the sites of interaction between the host response and the tubercle bacilli within infected individuals. They mainly consist of organized aggregations of lymphocytes and macrophages (Mf). A predominant role of mycobacterial envelope glycolipids in granulomas formation has been recently emphasized, yet the signaling events interfering with granuloma cell differentiation remain elusive. To decipher this molecular machinery, we have recently developed an in vitro human model of mycobacterial granulomas. In this study, we provide evidence that the mycobacterial proinflammatory phosphatidyl-myo-inositol mannosides and lipomannans (LM), as well as the anti-inflammatory lipoarabinomannan induce granuloma formation, yet only the proinflammatory glycolipids induce the fusion of granuloma Mf into multinucleated giant cells (MGC). We also demonstrate that LM induces large MGC resembling those found in vivo within the granulomas of tuberculosis patients, and that this process is mediated by TLR2 and is dependent on the beta(1) integrin/ADAM9 cell fusion machinery. Our results demonstrate for the first time that the Mf differentiation stage specifically occurring within granulomatous structures (i.e., MGC formation) is triggered by mycobacterial envelope glycolipids, which are capable of inducing the cell fusion machinery. This provides the first characterization of the ontogeny of human granuloma MGC, thus resulting in a direct modulation by a particular mycobacterial envelope glycolipid of the differentiation process of granuloma Mf.
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The Mycobacterium Tuberculosis FAS-II Condensing Enzymes: Their Role in Mycolic Acid Biosynthesis, Acid-fastness, Pathogenesis and in Future Drug Development
Molecular Microbiology.
Jun, 2007 |
Pubmed ID: 17555433 Mycolic acids are very long-chain fatty acids representing essential components of the mycobacterial cell wall. Considering their importance, characterization of key enzymes participating in mycolic acid biosynthesis not only allows an understanding of their role in the physiology of mycobacteria, but also might lead to the identification of new drug targets. Mycolates are synthesized by at least two discrete elongation systems, the type I and type II fatty acid synthases (FAS-I and FAS-II respectively). Among the FAS-II components, the condensing enzymes that catalyse the formation of carbon-carbon bonds have received considerable interest. Four condensases participate in initiation (mtFabH), elongation (KasA and KasB) and termination (Pks13) steps, leading to full-length mycolates. We present the recent biochemical and structural data for these important enzymes. Special emphasis is given to their role in growth, intracellular survival, biofilm formation, as well as in the physiopathology of tuberculosis. Recent studies demonstrated that phosphorylation of these enzymes by mycobacterial kinases affects their activities. We propose here a model in which kinases that sense environmental changes can phosphorylate the condensing enzymes, thus representing a novel mechanism of regulating mycolic acid biosynthesis. Finally, we discuss the attractiveness of these enzymes as valid targets for future antituberculosis drug development.
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Structure of Mycobacterium Tuberculosis MtFabD, a Malonyl-CoA:acyl Carrier Protein Transacylase (MCAT)
Acta Crystallographica. Section F, Structural Biology and Crystallization Communications.
Oct, 2007 |
Pubmed ID: 17909282 Mycobacteria display a unique and unusual cell-wall architecture, central to which is the membrane-proximal mycolyl-arabinogalactan-peptidoglycan core (mAGP). The biosynthesis of mycolic acids, which form the outermost layer of the mAGP core, involves malonyl-CoA:acyl carrier protein transacylase (MCAT). This essential enzyme catalyses the transfer of malonyl from coenzyme A to acyl carrier protein AcpM, thus feeding these two-carbon units into the chain-elongation cycle of the type II fatty-acid synthase. The crystal structure of M. tuberculosis mtFabD, the mycobacterial MCAT, has been determined to 3.0 A resolution by multi-wavelength anomalous dispersion. Phasing was facilitated by Ni2+ ions bound to the 20-residue N-terminal affinity tag, which packed between the two independent copies of mtFabD.
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Thiacetazone, an Antitubercular Drug That Inhibits Cyclopropanation of Cell Wall Mycolic Acids in Mycobacteria
PloS One.
2007 |
Pubmed ID: 18094751 Mycolic acids are a complex mixture of branched, long-chain fatty acids, representing key components of the highly hydrophobic mycobacterial cell wall. Pathogenic mycobacteria carry mycolic acid sub-types that contain cyclopropane rings. Double bonds at specific sites on mycolic acid precursors are modified by the action of cyclopropane mycolic acid synthases (CMASs). The latter belong to a family of S-adenosyl-methionine-dependent methyl transferases, of which several have been well studied in Mycobacterium tuberculosis, namely, MmaA1 through A4, PcaA and CmaA2. Cyclopropanated mycolic acids are key factors participating in cell envelope permeability, host immunomodulation and persistence of M. tuberculosis. While several antitubercular agents inhibit mycolic acid synthesis, to date, the CMASs have not been shown to be drug targets.
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Functional Role of the PE Domain and Immunogenicity of the Mycobacterium Tuberculosis Triacylglycerol Hydrolase LipY
Infection and Immunity.
Jan, 2008 |
Pubmed ID: 17938218 PE and PPE proteins appear to be important for virulence and immunopathogenicity in mycobacteria, yet the functions of the PE/PPE domains remain an enigma. To decipher the role of these domains, we have characterized the triacylglycerol (TAG) hydrolase LipY from Mycobacterium tuberculosis, which is the only known PE protein expressing an enzymatic activity. The overproduction of LipY in mycobacteria resulted in a significant reduction in the pool of TAGs, consistent with the lipase activity of this enzyme. Unexpectedly, this reduction was more pronounced in mycobacteria overexpressing LipY lacking the PE domain [LipY(deltaPE)], suggesting that the PE domain participates in the modulation of LipY activity. Interestingly, Mycobacterium marinum contains a protein homologous to LipY, termed LipY(mar), in which the PE domain is substituted by a PPE domain. As for LipY, overexpression of LipY(mar) in Mycobacterium smegmatis significantly reduced the TAG pool, and this was further pronounced when the PPE domain of LipY(mar) was removed. Fractionation studies and Western blot analysis demonstrated that both LipY and LipY(deltaPE) were mainly present in the cell wall, indicating that the PE domain was not required for translocation to this site. Furthermore, electron microscopy immunolabeling of LipY(deltaPE) clearly showed a cell surface localization, thereby suggesting that the lipase may interact with the host immune system. Accordingly, a strong humoral response against LipY and LipY(deltaPE) was observed in tuberculosis patients. Together, our results suggest for the first time that both PE and PPE domains can share similar functional roles and that LipY represents a novel immunodominant antigen.
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EmbR2, a Structural Homologue of EmbR, Inhibits the Mycobacterium Tuberculosis Kinase/substrate Pair PknH/EmbR
The Biochemical Journal.
Mar, 2008 |
Pubmed ID: 17999640 EmbR is a transcriptional regulator that is phosphorylated by the cognate mycobacterial STPK (serine/threonine protein kinase) PknH. Recent studies demonstrated that PknH-dependent phosphorylation of EmbR enhances its DNA-binding activity and activates the transcription of the embCAB genes encoding arabinosyltransferases, which participate in arabinan biosynthesis. In the present study, we identified a genomic region of 4425 bp, which is present in Mycobacterium tuberculosis CDC1551, but absent from M. tuberculosis H37Rv, comprising the MT3428 gene, which is homologous with embR. Homology modelling of the MT3428 gene product illustrated its close relationship (56% identity) to EmbR, and it was hence termed EmbR2. In marked contrast with EmbR, EmbR2 was not phosphorylated by PknH, although it is a substrate of other M. tuberculosis kinases, including PknE and PknF. Tryptophan fluorescence emission of EmbR2 was monitored in the presence of three different PknH-derived phosphopeptides and demonstrated that EmbR2 binds to at least two of the threonine sites known to undergo autophosphorylation in PknH. We observed that the capacity of EmbR2 to interact physically with PknH without being phosphorylated was a result of EmbR2-mediated inhibition of kinase activity: incubation of PknH with increasing concentrations of EmbR2 led to a dose-response inhibition of the autokinase activity, similarly to O6-cyclohexylmethylguanine, a known inhibitor of eukaryotic cyclin-dependent kinases. Moreover, EmbR2 inhibited PknH-dependent phosphorylation of EmbR in a dose-dependent manner. Together, these results suggest that EmbR2 is a regulator of PknH activation, thus directly participating in the control of the PknH/EmbR pair and potentially in mycobacterial physiology/virulence of M. tuberculosis CDC1551.
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Expression, Purification and Characterisation of Soluble GlfT and the Identification of a Novel Galactofuranosyltransferase Rv3782 Involved in Priming GlfT-mediated Galactan Polymerisation in Mycobacterium Tuberculosis
Protein Expression and Purification.
Apr, 2008 |
Pubmed ID: 18248822 The arabinogalactan (AG) component of the mycobacterial cell wall is an essential branched polysaccharide which tethers mycolic acids (m) to peptidoglycan (P), forming the mAGP complex. Much interest has been focused on the biosynthetic machinery involved in the production of this highly impermeable shield, which is the target for numerous anti-tuberculosis agents. The galactan domain of AG is synthesised via a bifunctional galactofuranosyltransferase (GlfT), which utilises UDP-Galf as its high-energy substrate. However, it has proven difficult to study the protein in its recombinant form due to difficulties in recovering pure soluble protein using standard expression systems. Herein, we describe the effects of glfT co-induction with a range of chaperone proteins, which resulted in an appreciable yield of soluble protein at 5 mg/L after a one-step purification procedure. We have shown that this purified enzyme transfers [14C]Galf to a range of both beta(1-->5) and beta(1-->6) linked digalactofuranosyl neoglycolipid acceptors with a distinct preference for the latter. Ligand binding studies using intrinsic tryptophan fluorescence have provided supporting evidence for the apparent preference of this enzyme to bind the beta(1-->6) disaccharide acceptor. However, we could not detect binding or galactofuranosyltransferase activity with an n-octyl beta-d-Gal-(1-->4)-alpha-l-Rha acceptor, which mimics the reducing terminus of galactan in the mycobacterial cell wall. Conversely, after an extensive bioinformatics analysis of the H37Rv genome, further cloning, expression and functional analysis of the Rv3792 open reading frame indicates that this protein affords galactofuranosyltransferase activity against such an acceptor and paves the way for a better understanding of galactan biosynthesis in Mycobacterium tuberculosis.
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From the Characterization of the Four Serine/threonine Protein Kinases (PknA/B/G/L) of Corynebacterium Glutamicum Toward the Role of PknA and PknB in Cell Division
The Journal of Biological Chemistry.
Jun, 2008 |
Pubmed ID: 18442973 Corynebacterium glutamicum contains four serine/threonine protein kinases (STPKs) named PknA, PknB, PknG, and PknL. Here we present the first biochemical and comparative analysis of all four C. glutamicum STPKs and investigate their potential role in cell shape control and peptidoglycan synthesis during cell division. In vitro assays demonstrated that, except for PknG, all STPKs exhibited autokinase activity. We provide evidence that activation of PknG is part of a phosphorylation cascade mechanism that relies on PknA activity. Following phosphorylation by PknA, PknG could transphosphorylate its specific substrate OdhI in vitro. A mass spectrometry profiling approach was also used to identify the phosphoresidues in all four STPKs. The results indicate that the nature, number, and localization of the phosphoacceptors varies from one kinase to the other. Disruption of either pknL or pknG in C. glutamicum resulted in viable mutants presenting a typical cell morphology and growth rate. In contrast, we failed to obtain null mutants of pknA or pknB, supporting the notion that these genes are essential. Conditional mutants of pknA or pknB were therefore created, leading to partial depletion of PknA or PknB. This resulted in elongated cells, indicative of a cell division defect. Moreover, overexpression of PknA or PknB in C. glutamicum resulted in a lack of apical growth and therefore a coccoid-like morphology. These findings indicate that pknA and pknB are key players in signal transduction pathways for the regulation of the cell shape and both are essential for sustaining corynebacterial growth.
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PETPhos: a Customized Expression Vector Designed for Further Characterization of Ser/Thr/Tyr Protein Kinases and Their Substrates
Plasmid.
Sep, 2008 |
Pubmed ID: 18597845 Bacterial genomics revealed the widespread distribution of serine/threonine protein kinases (STPKs), which regulate various cellular processes. However, understanding the role of phosphorylation in prokaryotes has been hampered by the paucity of endogenous substrates identified and the restricted number of tools allowing identification and characterization of the phosphoresidues. Herein, we describe an improved vector, pETPhos, to express proteins harboring a N-terminal His-tag fusion, which can be efficiently removed using the TEV protease. One major advantage of pETPhos relies on the lack of Ser and Thr residues in the fusion tag, representing potential non-specific phosphorylation sites. The usefulness of pETPhos is illustrated by a comparative analysis in which the Mycobacterium tuberculosis protein Rv2175c, a substrate of the STPK PknL, is expressed either in a pET28 derivative or in pETPhos. Following in vitro phosphorylation with PknL, phosphoaminoacid analysis revealed the presence of phosphorylated Ser and Thr in Rv2175c expressed in the pET28 derivative. However, when expressed in pETPhos, only Thr were phosphorylated. These findings indicate that STPKs can phosphorylate Ser-containing His-tag fusions, thus conducting to misleading results. We demonstrate that pETPhos represents a valuable tool for characterization of the phosphoacceptors in bacterial STPKs, and presumably also in Tyr protein kinases, as well as in their substrates.
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TLR2-dependent Eosinophil Interactions with Mycobacteria: Role of Alpha-defensins
Blood.
Apr, 2009 |
Pubmed ID: 18978205 Peripheral blood and tissue eosinophilia are a prominent feature in allergic diseases and during helminth infections. Eosinophil recruitment also frequently occurs upon mycobacterial infections, particularly in lung granuloma. However, the mechanism by which eosinophils interact with mycobacteria remains largely unknown. Because eosinophils recently have been shown to be involved in innate immune responses, we investigated the direct interactions of eosinophils with Mycobacterium bovis BCG as a study model. We show that live BCG attracts human eosinophils and induces reactive oxygen species (ROS) synthesis, granule protein release, and tumor necrosis factor (TNF)-alpha secretion. Using anti-TLR2 neutralizing antibodies before exposure of eosinophils to BCG, we showed a critical role of TLR2 signaling in ROS and eosinophil peroxidase release. BCG-induced eosinophil activation is mediated through the p38 mitogen-activated protein (MAP) kinase and nuclear factor (NF)-kappaB pathways. In addition, a mycobacterial wall component, lipomannan, induced a TLR2-dependent eosinophil activation. In addition, we showed that eosinophils express and produce alpha-defensins upon stimulation with BCG and lipomannan and that alpha-defensins could inhibit mycobacterial growth in synergy with eosinophil cationic protein. These results suggest a role for human eosinophils as direct effectors in TLR2-mediated innate immunity against mycobacteria and confer to these cells potent cytotoxic functions through defensin and eosinophil cationic protein production.
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The Mycobacterium Tuberculosis Beta-ketoacyl-acyl Carrier Protein Synthase III Activity is Inhibited by Phosphorylation on a Single Threonine Residue
The Journal of Biological Chemistry.
Mar, 2009 |
Pubmed ID: 19074144 Mycolic acids are hallmark features of the Mycobacterium tuberculosis cell wall. They are synthesized by the condensation of two fatty acids, a C56-64-meromycolyl chain and a C24-26-fatty acyl chain. Meromycolates are produced via the combination of type I and type II fatty acid synthases (FAS-I and FAS-II). The beta-ketoacyl-acyl carrier protein (ACP) synthase III (mtFabH) links FAS-I and FAS-II, catalyzing the condensation of FAS-I-derived acyl-CoAs with malonyl-ACP. Because mtFabH represents a potential regulatory key point of the mycolic acid pathway, we investigated the hypothesis that phosphorylation of mtFabH controls its activity. Phosphorylation of proteins by Ser/Thr protein kinases (STPKs) has recently emerged as a major physiological mechanism of regulation in prokaryotes. We demonstrate here that mtFabH was efficiently phosphorylated in vitro by several mycobacterial STPKs, particularly by PknF and PknA, as well as in vivo in mycobacteria. Analysis of the phosphoamino acid content indicated that mtFabH was phosphorylated exclusively on threonine residues. Mass spectrometry analyses using liquid chromatography-electrospray ionization/tandem mass spectrometry identified Thr45 as the unique phosphoacceptor. This was further supported by complete loss of PknF- or PknA-dependent phosphorylation of a mtFabH mutant. Mapping Thr45 on the crystal structure of mtFabH illustrates that this residue is located at the entrance of the substrate channel, suggesting that the phosphate group may alter accessibility of the substrate and thus affect mtFabH enzymatic activity. A T45D mutant of mtFabH, designed to mimic constitutive phosphorylation, exhibited markedly decreased transacylation, malonyl-AcpM decarboxylation, and condensing activities compared with the wild-type protein or the T45A mutant. Together, these findings not only represent the first demonstration of phosphorylation of a beta-ketoacyl-ACP synthase III enzyme but also indicate that phosphorylation of mtFabH inhibits its enzymatic activity, which may have important consequences in regulating mycolic acid biosynthesis.
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Mycolic Acid Methyltransferase, MmaA4, is Necessary for Thiacetazone Susceptibility in Mycobacterium Tuberculosis
Molecular Microbiology.
Mar, 2009 |
Pubmed ID: 19183278 Susceptibility of Mycobacterium tuberculosis to the second-line antitubercular drug thiacetazone (TAC) requires activation by the monoxygenase, EthA. Here, we report isolation of spontaneous mutants in Mycobacterium bovis BCG that are highly resistant to TAC, but carry a functional EthA. Unexpectedly, a majority of the TAC-resistant mutants lacked keto-mycolic acids, which are long-chain fatty acids associated with the cell wall and which contribute significantly to the physiopathology of tuberculosis. Predictably, causative mutations in the above mutants were in the gene encoding methyltransferase MmaA4, which is required for synthesis of keto- and methoxy-mycolic acids. Drug-resistant phenotype of the BCG mutants was reproduced in a mmaA4, but not in a mmaA3 null mutant of M. tuberculosis CDC1551. Susceptibility to TAC could be restored by complementation with a functional mmaA4 gene. Interestingly, overexpression of MmaA4 in M. bovis BCG made it more susceptible to TAC. We provide novel mechanistic insights into antitubercular drug activation by co-ordinated actions of EthA and MmaA4. This study is the first demonstration of the participation of an enzyme linked to the synthesis of oxygenated mycolates in a drug activation process in M. tuberculosis, and highlights the interplay between mycolic acid synthesis, drug activation and mycobacterial virulence.
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The Mycobacterium Tuberculosis Ser/Thr Kinase Substrate Rv2175c is a DNA-binding Protein Regulated by Phosphorylation
The Journal of Biological Chemistry.
Jul, 2009 |
Pubmed ID: 19457863 Recent efforts have underlined the role of serine/threonine protein kinases in growth, pathogenesis, and cell wall metabolism in Mycobacterium tuberculosis. Although most kinases have been investigated for their physiological roles, little information is available regarding how serine/threonine protein kinase-dependent phosphorylation regulates the activity of kinase substrates. Herein, we focused on M. tuberculosis Rv2175c, a protein of unknown function, conserved in actinomycetes, and recently identified as a substrate of the PknL kinase. We solved the solution structure of Rv2175c by multidimensional NMR and demonstrated that it possesses an original winged helix-turn-helix motif, indicative of a DNA-binding protein. The DNA-binding activity of Rv2175c was subsequently confirmed by fluorescence anisotropy, as well as in electrophoretic mobility shift assays. Mass spectrometry analyses using a combination of MALDI-TOF and LC-ESI/MS/MS identified Thr(9) as the unique phosphoacceptor. This was further supported by complete loss of PknL-dependent phosphorylation of an Rv2175c_T9A mutant. Importantly, the DNA-binding activity was completely abrogated in a Rv2175c_T9D mutant, designed to mimic constitutive phosphorylation, but not in a mutant lacking the first 13 residues. This implies that the function of the N-terminal extension is to provide a phosphoacceptor (Thr(9)), which, following phosphorylation, negatively regulates the Rv2175c DNA-binding activity. Interestingly, the N-terminal disordered extension, which bears the phosphoacceptor, was found to be restricted to members of the M. tuberculosis complex, thus suggesting the existence of an original mechanism that appears to be unique to the M. tuberculosis complex.
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Mycobacterium Marinum Lipooligosaccharides Are Unique Caryophyllose-containing Cell Wall Glycolipids That Inhibit Tumor Necrosis Factor-alpha Secretion in Macrophages
The Journal of Biological Chemistry.
Jul, 2009 |
Pubmed ID: 19491094 Earlier studies have reported a role for lipooligosaccharides (LOSs) in sliding motility, biofilm formation, and infection of host macrophages in Mycobacterium marinum. Although a LOS biosynthetic gene cluster has recently been identified in this species, many structural features of the different LOSs (LOS-I-IV) are still unknown. This clearly hampers assessing the contribution of each LOS in mycobacterial virulence as well as structure-function-based studies of these important cell wall-associated glycolipids. In this study, we have identified an M. marinum isolate, M. marinum 7 (Mma7), which failed to produce LOS-IV but instead accumulated large amounts of LOS-III. Local genomic comparison of the LOS biosynthetic cluster established the presence of a highly disorganized region in Mma7 compared with the standard M strain, characterized by multiple genetic lesions that are likely to be responsible for the defect in LOS-IV production in Mma7. Our results indicate that the glycosyltransferase LosA alone is not sufficient to ensure LOS-IV biosynthesis. The availability of different M. marinum strains allowed us to determine the precise structure of individual LOSs through the combination of mass spectrometric and NMR techniques. In particular, we established the presence of two related 4-C-branched monosaccharides within LOS-II to IV sequences, of which one was never identified before. In addition, we provided evidence that LOSs are capable of inhibiting the secretion of tumor necrosis factor-alpha in lipopolysaccharide-stimulated human macrophages. This unexpected finding suggests that these cell wall-associated glycolipids represent key effectors capable of interfering with the establishment of a pro-inflammatory response.
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Phosphorylation of the Mycobacterium Tuberculosis Beta-ketoacyl-acyl Carrier Protein Reductase MabA Regulates Mycolic Acid Biosynthesis
The Journal of Biological Chemistry.
Apr, 2010 |
Pubmed ID: 20178986 Mycolic acids are key cell wall components for the survival, pathogenicity, and antibiotic resistance of the human tubercle bacillus. Although it was thought that Mycobacterium tuberculosis tightly regulates their production to adapt to prevailing environmental conditions, the molecular mechanisms governing mycolic acid biosynthesis remained extremely obscure. Meromycolic acids, the direct precursors of mycolic acids, are synthesized by a type II fatty acid synthase from acyl carrier protein-bound substrates that are extended iteratively, with a reductive cycle in each round of extension, the second step of which is catalyzed by the essential beta-ketoacyl-acyl carrier protein reductase, MabA. In this study, we investigated whether post-translational modifications of MabA might represent a strategy employed by M. tuberculosis to regulate mycolic acid biosynthesis. Indeed, we show here that MabA was efficiently phosphorylated in vitro by several M. tuberculosis Ser/Thr protein kinases, including PknB, as well as in vivo in mycobacteria. Mass spectrometric analyses using LC-ESI/MS/MS and site-directed mutagenesis identified three phosphothreonines, with Thr(191) being the primary phosphor-acceptor. A MabA_T191D mutant, designed to mimic constitutive phosphorylation, exhibited markedly decreased ketoacyl reductase activity compared with the wild-type protein, as well as impaired binding of the NADPH cofactor, as demonstrated by fluorescence spectroscopy. The hypothesis that phosphorylation of Thr(191) alters the enzymatic activity of MabA, and subsequently mycolic acid biosynthesis, was further supported by the fact that constitutive overexpression of the mabA_T191D allele in Mycobacterium bovis BCG strongly impaired mycobacterial growth. Importantly, conditional expression of the phosphomimetic MabA_T191D led to a significant inhibition of de novo biosynthesis of mycolic acids. This study provides the first information on the molecular mechanism(s) involved in mycolic acid regulation through Ser/Thr protein kinase-dependent phosphorylation of a type II fatty acid synthase enzyme.
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Enzymatic Hydrolysis of Trehalose Dimycolate Releases Free Mycolic Acids During Mycobacterial Growth in Biofilms
The Journal of Biological Chemistry.
Jun, 2010 |
Pubmed ID: 20375425 Mycobacterial species, like other microbes, spontaneously form multicellular drug-tolerant biofilms when grown in vitro in detergent-free liquid media. The structure of Mycobacterium tuberculosis biofilms is formed through genetically programmed pathways and is built upon a large abundance of novel extracellular free mycolic acids (FM), although the mechanism of FM synthesis remained unclear. Here we show that the FM in Mycobacterium smegmatis biofilms is produced through the enzymatic release from constitutively present mycolyl derivatives. One of the precursors for FM is newly synthesized trehalose dimycolate (TDM), which is cleaved by a novel TDM-specific serine esterase, Msmeg_1529. Disruption of Msmeg_1529 leads to undetectable hydrolytic activity, reduced levels of FM in the mutant, and retarded biofilm growth. Furthermore, enzymatic hydrolysis of TDM remains conserved in M. tuberculosis, suggesting the presence of a TDM-specific esterase in this pathogen. Overall, this study provides the first evidence for an enzymatic release of free mycolic acids from cell envelope mycolates during mycobacterial growth.
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Temperature-dependent Regulation of Mycolic Acid Cyclopropanation in Saprophytic Mycobacteria: Role of the Mycobacterium Smegmatis 1351 Gene (MSMEG_1351) in CIS-cyclopropanation of Alpha-mycolates
The Journal of Biological Chemistry.
Jul, 2010 |
Pubmed ID: 20457615 The cell envelope is a crucial determinant of virulence and drug resistance in Mycobacterium tuberculosis. Several features of pathogenesis and immunomodulation of host responses are attributable to the structural diversity in cell wall lipids, particularly in the mycolic acids. Structural modification of the alpha-mycolic acid by introduction of cyclopropane rings as catalyzed by the methyltransferase, PcaA, is essential for a lethal, persistent infection and the cording phenotype in M. tuberculosis. Here, we demonstrate the presence of cyclopropanated cell wall mycolates in the nonpathogenic strain Mycobacterium smegmatis and identify MSMEG_1351 as a gene encoding a PcaA homologue. Interestingly, alpha-mycolic acid cyclopropanation was inducible in cultures grown at 25 degrees C. The growth temperature modulation of the cyclopropanating activity was determined by high resolution magic angle spinning NMR analyses on whole cells. In parallel, quantitative reverse transcription-PCR analysis showed that MSMEG_1351 gene expression is up-regulated at 25 degrees C compared with 37 degrees C. An MSMEG_1351 knock-out strain of M. smegmatis, generated by recombineering, exhibited a deficiency in cyclopropanation of alpha-mycolates. The functional equivalence of PcaA and MSMEG_1351 was established by cross-complementation in the MSMEG_1351 knock-out mutant and also in a DeltapcaA strain of Mycobacterium bovis BCG. Overexpression of MSMEG_1351 restored the wild-type mycolic acid profile and the cording phenotype in BCG. Although the biological significance of mycolic acid cyclopropanation in nonpathogenic mycobacteria remains unclear, it likely represents a mechanism of adaptation of cell wall structure and composition to cope with environmental factors.
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Synthesis and Evaluation of Anti-tubercular Activity of New Dithiocarbamate Sugar Derivatives
Bioorganic & Medicinal Chemistry Letters.
Feb, 2011 |
Pubmed ID: 21232949 The present study was undertaken to optimize the anti-tubercular activity of 2-acetamido-2-deoxy-β-D-glucopyranosyl N,N-dimethyldithiocarbamate (OCT313, Glc-NAc-DMDC), a lead compound previously reported by us. Structural modifications of OCT313 included the replacements of the DMDC group at C-1 by pyrrolidine dithiocarbamate (PDTC) and the acetyl group at C-2 by either propyl, butyl, benzyl or oleic acid groups. The antimycobacterial activities of these derivatives were evaluated against Mycobacterium tuberculosis (MTB). Glc-NAc-pyrrolidine dithiocarbamate (OCT313HK, Glc-NAc-PDTC) exhibited the most potent anti-tubercular activity with the minimal inhibitory concentration (MIC) of 6.25-12.5 μg/ml. The antibacterial activity of OCT313HK was highly specific to MTB and Mycobacterium bovis BCG, but not against Mycobacterium avium, Mycobacterium smegmatis, Staphylococcus aureus or Escherichia coli. Importantly, OCT313HK was also effective against MTB clinical isolates, including multidrug-resistant (MDR) and extensively drug-resistant (XDR) strains. Interestingly, OCT313HK was exerted the primary bactericidal activity, and it was also exhibited the bacteriolytic activity at high concentrations. We next investigated whether the mycobacterial monooxygenase EthA, a common activator of thiocarbamide-containing anti-tubercular drugs, also activated OCT313HK. Contrary to our expectations, the anti-tubercular activity of dithiocarbamate sugar derivatives and dithiocarbamates were not dependent on ethA expression, in contrast to thiocarbamide-containing drugs. Overall, this study presents OCT313HK as a novel and potent compound against MTB, particularly promising to overcome drug resistance.
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A Mycobacterium Marinum TesA Mutant Defective for Major Cell Wall-associated Lipids is Highly Attenuated in Dictyostelium Discoideum and Zebrafish Embryos
Molecular Microbiology.
May, 2011 |
Pubmed ID: 21375593 Infection of the zebrafish with Mycobacterium marinum is regarded as a well-established experimental model to study the pathogenicity of Mycobacterium tuberculosis. Herein, a M. marinum transposon mutant library was screened for attenuated M. marinum phenotypes using a Dictyostelium discoideum assay. In one attenuated mutant, the transposon was located within tesA, encoding a putative type II thioesterase. Thin-layer chromatography analyses indicated that the tesA::Tn mutant failed to produce two major cell wall-associated lipids. Mass spectrometry and nuclear magnetic resonance clearly established the nature of missing lipids as phthioglycol diphthioceranates and phenolic glycolipids, respectively, indicating that TesA is required for the synthesis of both lipids. When injected into the zebrafish embryo bloodstream, the mutant was found to be highly attenuated, thus validating the performance and relevance of the Dictyostelium screen. Consistent with these in vivo findings, tesA::Tn exhibited increased permeability defects in vitro, which may explain its failure to survive in host macrophages. Unexpectedly, virulence was retained when bacteria were injected into the notochord. Histological and ultrastructural studies of the infected notochord revealed the presence of actively proliferating mycobacteria, leading to larval death. This work presents for the first time the notochord as a compartment highly susceptible to mycobacterial infection.
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Fatty Acyl Chains of Mycobacterium Marinum Lipooligosaccharides: Structure, Localization and Acylation by PapA4 (MMAR_2343) Protein
The Journal of Biological Chemistry.
Sep, 2011 |
Pubmed ID: 21803773 We have recently established the fine structure of the glycan backbone of lipooligosaccharides (LOS-I to LOS-IV) isolated from Mycobacterium marinum, a close relative of Mycobacterium tuberculosis. These studies culminated with the description of an unusual terminal N-acylated monosaccharide that confers important biological functions to LOS-IV, such as macrophage activation, that may be relevant to granuloma formation. It was, however, also suggested that the lipid moiety was required for LOSs to exert their immunomodulatory activity. Herein, using highly purified LOSs from M. marinum, we have determined through a combination of mass spectrometric and NMR techniques, the structure and localization of the fatty acids composing the lipid moiety. The occurrence of two distinct polymethyl-branched fatty acids presenting specific localizations is consistent with the presence of two highly related polyketide synthases (Pks5 and Pks5.1) in M. marinum and presumably involved in the synthesis of these fatty acyl chains. In addition, a bioinformatic search permitted us to identify a set of enzymes potentially involved in the biosynthesis or transfer of these lipids to the LOS trehalose unit. These include MMAR_2343, a member of the Pap (polyketide-associated protein) family, that acylates trehalose-based glycolipids in M. marinum. The participation of MMAR_2343 to LOS assembly was demonstrated using a M. marinum mutant carrying a transposon insertion in the MMAR_2343 gene. Disruption of MMAR_2343 resulted in a severe LOS breakdown, indicating that MMAR_2343, hereafter designated PapA4, fulfills the requirements for LOS acylation and assembly.
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AccD6, a Key Carboxyltransferase Essential for Mycolic Acid Synthesis in Mycobacterium Tuberculosis, is Dispensable in a Nonpathogenic Strain
Journal of Bacteriology.
Dec, 2011 |
Pubmed ID: 21984794 Acetyl coenzyme A carboxylase (ACC) is a key enzyme providing a substrate for mycolic acid biosynthesis. Although in vitro studies have demonstrated that the protein encoded by accD6 (Rv2247) may be a functional carboxyltransferase subunit of ACC in Mycobacterium tuberculosis, the in vivo function and regulation of accD6 in slow- and fast-growing mycobacteria remain elusive. Here, directed mutagenesis demonstrated that although accD6 is essential for M. tuberculosis, it can be deleted in Mycobacterium smegmatis without affecting its cell envelope integrity. Moreover, we showed that although it is part of the type II fatty acid synthase operon, the accD6 gene of M. tuberculosis, but not that of M. smegmatis, possesses its own additional promoter (P(acc)). The expression level of accD6(Mtb) placed only under the control of P(acc) is 10-fold lower than that in wild-type M. tuberculosis but is sufficient to sustain cell viability. Importantly, this limited expression level affects growth, mycolic acid content, and cell morphology. These results provide the first in vivo evidence for AccD6 as a key player in the mycolate biosynthesis of M. tuberculosis, implicating AccD6 as the essential ACC subunit in pathogenic mycobacteria and an excellent target for new antitubercular compounds. Our findings also highlight important differences in the mechanism of acetyl carboxylation between pathogenic and nonpathogenic mycobacterial species.
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Exposure of Mycobacteria to Cell Wall-inhibitory Drugs Decreases the Production of an Arabinoglycerolipid Related to Mycolyl-arabinogalactan-peptidoglycan Metabolism
The Journal of Biological Chemistry.
Feb, 2012 |
Pubmed ID: 22315220 The "cell wall core" consisting of a mycolyl-arabinogalactan-peptidoglycan (mAGP) complex represents the hallmark of the mycobacterial cell envelope. It has been the focus of intense research at both structural and biosynthetic levels during the past few decades. Because it is essential, mAGP is also regarded as a target for several anti-tubercular drugs. Herein, we demonstrate that exposure of Mycobacterium bovis BCG or Mycobacterium marinum to thiacetazone (TAC), a second-intent anti-tubercular drug, is associated with a severe decrease in the level of a major apolar glycolipid. This inhibition requires MmaA4, a methyltransferase reported to participate in the activation process of TAC. Following purification, this glycolipid was subjected to detailed structural analyses, combining gas-liquid chromatography, mass spectrometry and nuclear magnetic resonance. This allowed to identify it as a 5-O-mycolyl-β-Araf-(1→2)-5-O-mycolyl-α-Araf-(1→1)-Gro, designated Di-Mycolyl di-Arabino-Glycerol (DMAG). The presence of DMAG was subsequently confirmed in other slow-growing pathogenic species, including Mycobacterium tuberculosis. DMAG production was stimulated in the presence of exogenous glycerol. Interestingly, DMAG appears structurally identical to the terminal portion of the mycolylated arabinosyl motif of mAGP and the metabolic relationship between these two components was provided using anti-tubercular drugs such as ethambutol or isoniazid, known to inhibit the biosynthesis of arabinogalactan or mycolic acid, respectively. Finally, DMAG was identified in the cell wall of M. tuberculosis. This opens the possibility of a potent biological function for DMAG that may be important to mycobacterial pathogenesis.
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Phosphorylation of Mycobacterial PcaA Inhibits Mycolic Acid Cyclopropanation: Consequences for Intracellular Survival and for Phagosome Maturation Block
The Journal of Biological Chemistry.
Jul, 2012 |
Pubmed ID: 22621931 Pathogenic mycobacteria survive within macrophages by residing in phagosomes, which they prevent from maturing and fusing with lysosomes. Although several bacterial components were seen to modulate phagosome processing, the molecular regulatory mechanisms taking part in this process remain elusive. We investigated whether the phagosome maturation block (PMB) could be modulated by signaling through Ser/Thr phosphorylation. Here, we demonstrated that mycolic acid cyclopropane synthase PcaA, but not MmaA2, was phosphorylated by mycobacterial Ser/Thr kinases at Thr-168 and Thr-183 both in vitro and in mycobacteria. Phosphorylation of PcaA was associated with a significant decrease in the methyltransferase activity, in agreement with the strategic structural localization of these two phosphoacceptors. Using a BCG ΔpcaA mutant, we showed that PcaA was required for intracellular survival and prevention of phagosome maturation in human monocyte-derived macrophages. The physiological relevance of PcaA phosphorylation was further assessed by generating PcaA phosphoablative (T168A/T183A) or phosphomimetic (T168D/T183D) mutants. In contrast to the wild-type and phosphoablative pcaA alleles, introduction of the phosphomimetic pcaA allele in the ΔpcaA mutant failed to restore the parental mycolic acid profile and cording morphotype. Importantly, the PcaA phosphomimetic strain, as the ΔpcaA mutant, exhibited reduced survival in human macrophages and was unable to prevent phagosome maturation. Our results add new insight into the importance of mycolic acid cyclopropane rings in the PMB and provide the first evidence of a Ser/Thr kinase-dependent mechanism for modulating mycolic acid composition and PMB.
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Structural Determination and Toll-like Receptor 2-dependent Proinflammatory Activity of Dimycolyl-diarabino-glycerol from Mycobacterium Marinum
The Journal of Biological Chemistry.
Oct, 2012 |
Pubmed ID: 22798072 Although it was identified in the cell wall of several pathogenic mycobacteria, the biological properties of dimycolyl-diarabino-glycerol have not been documented yet. In this study an apolar glycolipid, presumably corresponding to dimycolyl-diarabino-glycerol, was purified from Mycobacterium marinum and subsequently identified as a 5-O-mycolyl-β-Araf-(1→2)-5-O-mycolyl-α-Araf-(1→1')-glycerol (designated Mma_DMAG) using a combination of nuclear magnetic resonance spectroscopy and mass spectrometry analyses. Lipid composition analysis revealed that mycolic acids were dominated by oxygenated mycolates over α-mycolates and devoid of trans-cyclopropane functions. Highly purified Mma_DMAG was used to demonstrate its immunomodulatory activity. Mma_DMAG was found to induce the secretion of proinflammatory cytokines (TNF-α, IL-8, IL-1β) in human macrophage THP-1 cells and to trigger the expression of ICAM-1 and CD40 cell surface antigens. This activation mechanism was dependent on TLR2, but not on TLR4, as demonstrated by (i) the use of neutralizing anti-TLR2 and -TLR4 antibodies and by (ii) the detection of secreted alkaline phosphatase in HEK293 cells co-transfected with the human TLR2 and secreted embryonic alkaline phosphatase reporter genes. In addition, transcriptomic analyses indicated that various genes encoding proinflammatory factors were up-regulated after exposure of THP-1 cells to Mma_DMAG. Importantly, a wealth of other regulated genes related to immune and inflammatory responses, including chemokines/cytokines and their respective receptors, adhesion molecules, and metalloproteinases, were found to be modulated by Mma_DMAG. Overall, this study suggests that DMAG may be an active cell wall glycoconjugate driving host-pathogen interactions and participating in the immunopathogenesis of mycobacterial infections.
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Identification of the Mycobacterium Marinum Apa Antigen O-mannosylation Sites Reveals Important Glycosylation Variability with the M. Tuberculosis Apa Homologue
Journal of Proteomics.
Oct, 2012 |
Pubmed ID: 22828516 The 45/47 kDa Apa, an immuno-dominant antigen secreted by Mycobacterium tuberculosis is O-mannosylated at multiple sites. Glycosylation of Apa plays a key role in colonization and invasion of the host cells by M. tuberculosis through interactions of Apa with the host immune system C-type lectins. Mycobacterium marinum (M.ma) a fish pathogen, phylogenetically close to M. tuberculosis, induces a granulomatous response with features similar to those described for M. tuberculosis in human. Although M.ma possesses an Apa homologue, its glycosylation status is unknown, and whether this represents a crucial element in the pathophysiology induced by M.ma remains to be addressed. To this aim, we have identified two concanavalin A-reactive 45/47 kDa proteins from M.ma, which have been further purified by a two-step anion exchange chromatography process. Advanced liquid chromatography-nanoESI mass spectrometry-based proteomic analyses of peptides, derived from either tryptic digestion alone or in combination with the Asp-N endoproteinase, established that M.ma Apa possesses up to seven distinct O-mannosylated sites with mainly single mannose substitutions, which can be further extended at the Ser/Thr/Pro rich region near the N-terminus. This opens the way to further studies focussing on the involvement and biological functions of Apa O-mannosylation using the M.ma/zebrafish model.
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The C-terminal Domain of the Virulence Factor MgtC is a Divergent ACT Domain
Journal of Bacteriology.
Nov, 2012 |
Pubmed ID: 22984256 MgtC is a virulence factor of unknown function important for survival inside macrophages in several intracellular bacterial pathogens, including Mycobacterium tuberculosis. It is also involved in adaptation to Mg(2+) deprivation, but previous work suggested that MgtC is not a Mg(2+) transporter. In this study, we demonstrated that the amount of the M. tuberculosis MgtC protein is not significantly increased by Mg(2+) deprivation. Members of the MgtC protein family share a conserved membrane N-terminal domain and a more divergent cytoplasmic C-terminal domain. To get insights into MgtC functional and structural organization, we have determined the nuclear magnetic resonance (NMR) structure of the C-terminal domain of M. tuberculosis MgtC. This structure is not affected by the Mg(2+) concentration, indicating that it does not bind Mg(2+). The structure of the C-terminal domain forms a βαββαβ fold found in small molecule binding domains called ACT domains. However, the M. tuberculosis MgtC ACT domain differs from canonical ACT domains because it appears to lack the ability to dimerize and to bind small molecules. We have shown, using a bacterial two-hybrid system, that the M. tuberculosis MgtC protein can dimerize and that the C-terminal domain somehow facilitates this dimerization. Taken together, these results indicate that M. tuberculosis MgtC does not have an intrinsic function related to Mg(2+) uptake or binding but could act as a regulatory factor based on protein-protein interaction that could be facilitated by its ACT domain.
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MmPPOX Inhibits Mycobacterium Tuberculosis Lipolytic Enzymes Belonging to the Hormone-sensitive Lipase Family and Alters Mycobacterial Growth
PloS One.
2012 |
Pubmed ID: 23029536 Lipid metabolism plays an important role during the lifetime of Mycobacterium tuberculosis, the causative agent of tuberculosis. Although M. tuberculosis possesses numerous lipolytic enzymes, very few have been characterized yet at a biochemical/pharmacological level. This study was devoted to the M. tuberculosis lipolytic enzymes belonging to the Hormone-Sensitive Lipase (HSL) family, which encompasses twelve serine hydrolases closely related to the human HSL. Among them, nine were expressed, purified and biochemically characterized using a broad range of substrates. In vitro enzymatic inhibition studies using the recombinant HSL proteins, combined with mass spectrometry analyses, revealed the potent inhibitory activity of an oxadiazolone compound, named MmPPOX. In addition, we provide evidence that MmPPOX alters mycobacterial growth. Overall, these findings suggest that the M. tuberculosis HSL family displays important metabolic functions, thus opening the way to further investigations linking the involvement of these enzymes in mycobacterial growth.
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Exposure to a Cutinase-like Serine Esterase Triggers Rapid Lysis of Multiple Mycobacterial Species
The Journal of Biological Chemistry.
Jan, 2013 |
Pubmed ID: 23155047 Mycobacteria are shaped by a thick envelope made of an array of uniquely structured lipids and polysaccharides. However, the spatial organization of these molecules remains unclear. Here, we show that exposure to an esterase from Mycobacterium smegmatis (Msmeg_1529), hydrolyzing the ester linkage of trehalose dimycolate in vitro, triggers rapid and efficient lysis of Mycobacterium tuberculosis, Mycobacterium bovis BCG, and Mycobacterium marinum. Exposure to the esterase immediately releases free mycolic acids, while concomitantly depleting trehalose mycolates. Moreover, lysis could be competitively inhibited by an excess of purified trehalose dimycolate and was abolished by a S124A mutation affecting the catalytic activity of the esterase. These findings are consistent with an indispensable structural role of trehalose mycolates in the architectural design of the exposed surface of the mycobacterial envelope. Importantly, we also demonstrate that the esterase-mediated rapid lysis of M. tuberculosis significantly improves its detection in paucibacillary samples.
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Synthesis, Antitubercular Activity and Mechanism of Resistance of Highly Effective Thiacetazone Analogues
PloS One.
2013 |
Pubmed ID: 23301038 Defining the pharmacological target(s) of currently used drugs and developing new analogues with greater potency are both important aspects of the search for agents that are effective against drug-sensitive and drug-resistant Mycobacterium tuberculosis. Thiacetazone (TAC) is an anti-tubercular drug that was formerly used in conjunction with isoniazid, but removed from the antitubercular chemotherapeutic arsenal due to toxic side effects. However, several recent studies have linked the mechanisms of action of TAC to mycolic acid metabolism and TAC-derived analogues have shown increased potency against M. tuberculosis. To obtain new insights into the molecular mechanisms of TAC resistance, we isolated and analyzed 10 mutants of M. tuberculosis that were highly resistant to TAC. One strain was found to be mutated in the methyltransferase MmaA4 at Gly101, consistent with its lack of oxygenated mycolic acids. All remaining strains harbored missense mutations in either HadA (at Cys61) or HadC (at Val85, Lys157 or Thr123), which are components of the β-hydroxyacyl-ACP dehydratase complex that participates in the mycolic acid elongation step. Separately, a library of 31 new TAC analogues was synthesized and evaluated against M. tuberculosis. Two of these compounds, 15 and 16, exhibited minimal inhibitory concentrations 10-fold lower than the parental molecule, and inhibited mycolic acid biosynthesis in a dose-dependent manner. Moreover, overexpression of HadAB HadBC or HadABC in M. tuberculosis led to high level resistance to these compounds, demonstrating that their mode of action is similar to that of TAC. In summary, this study uncovered new mutations associated with TAC resistance and also demonstrated that simple structural optimization of the TAC scaffold was possible and may lead to a new generation of TAC-derived drug candidates for the potential treatment of tuberculosis as mycolic acid inhibitors.
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Overexpression of the KdpF Membrane Peptide in Mycobacterium Bovis BCG Results in Reduced Intramacrophage Growth and Altered Cording Morphology
PloS One.
2013 |
Pubmed ID: 23577107 Membrane peptides appear as an emerging class of regulatory molecules in bacteria, which can interact with membrane proteins, such as sensor kinases. To date, regulatory membrane peptides have been completely overlooked in mycobacteria. The 30 amino-acid-long KdpF peptide, which is co-transcribed with kdpABC genes and regulated by the KdpDE two-component system, is supposed to stabilize the KdpABC potassium transporter complex but may also exhibit unsuspected regulatory function(s) towards the KdpD sensor kinase. Herein, we showed by quantitative RT-PCR that the Mycobacterium bovis BCG kdpAB and kdpDE genes clusters are differentially induced in potassium-deprived broth medium or within infected macrophages. We have overexpressed the kdpF gene in M. bovis BCG to investigate its possible regulatory role and effect on mycobacterial virulence. Our results indicate that KdpF does not play a critical regulatory role on kdp genes expression despite the fact that KdpF interacts with the KdpD sensor kinase in a bacterial two-hybrid assay. However, overexpression of kdpF results in a significant reduction of M. bovis BCG growth in both murine and human primary macrophages, and is associated with a strong alteration of colonial morphology and impaired cording formation. To identify novel KdpF interactants, a mycobacterial library was screened using KdpF as bait in the bacterial two-hybrid system. This allowed us to identify members of the MmpL family of membrane proteins, known to participate in the biosynthesis/transport of various cell wall lipids, thus highlighting a possible link between KdpF and cell wall lipid metabolism. Taken together, these data suggest that KdpF overexpression reduces intramacrophage growth which may result from alteration of the mycobacterial cell wall.
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Mycobacterium Tuberculosis Maltosyltransferase GlgE, a Genetically Validated Antituberculosis Target, is Negatively Regulated by Ser/Thr Phosphorylation
The Journal of Biological Chemistry.
Jun, 2013 |
Pubmed ID: 23609448 GlgE is a maltosyltransferase involved in the biosynthesis of α-glucans that has been genetically validated as a potential therapeutic target against Mycobacterium tuberculosis. Despite also making α-glucan, the GlgC/GlgA glycogen pathway is distinct and allosterically regulated. We have used a combination of genetics and biochemistry to establish how the GlgE pathway is regulated. M. tuberculosis GlgE was phosphorylated specifically by the Ser/Thr protein kinase PknB in vitro on one serine and six threonine residues. Furthermore, GlgE was phosphorylated in vivo when expressed in Mycobacterium bovis bacillus Calmette-Guérin (BCG) but not when all seven phosphorylation sites were replaced by Ala residues. The GlgE orthologues from Mycobacterium smegmatis and Streptomyces coelicolor were phosphorylated by the corresponding PknB orthologues in vitro, implying that the phosphorylation of GlgE is widespread among actinomycetes. PknB-dependent phosphorylation of GlgE led to a 2 orders of magnitude reduction in catalytic efficiency in vitro. The activities of phosphoablative and phosphomimetic GlgE derivatives, where each phosphorylation site was substituted with either Ala or Asp residues, respectively, correlated with negative phosphoregulation. Complementation studies of a M. smegmatis glgE mutant strain with these GlgE derivatives, together with both classical and chemical forward genetics, were consistent with flux through the GlgE pathway being correlated with GlgE activity. We conclude that the GlgE pathway appears to be negatively regulated in actinomycetes through the phosphorylation of GlgE by PknB, a mechanism distinct from that known in the classical glycogen pathway. Thus, these findings open new opportunities to target the GlgE pathway therapeutically.
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Keto-mycolic Acid-dependent Pellicle Formation Confers Tolerance to Drug-sensitive Mycobacterium Tuberculosis
MBio.
2013 |
Pubmed ID: 23653446 ABSTRACT The chronic nature of tuberculosis (TB), its requirement of long duration of treatment, its ability to evade immune intervention, and its propensity to relapse after drug treatment is discontinued are reminiscent of other chronic, biofilm-associated bacterial diseases. Historically, Mycobacterium tuberculosis was grown as a pellicle, a biofilm-like structure, at the liquid-air interface in a variety of synthetic media. Notably, the most widely administered human vaccine, BCG, is grown as a pellicle for vaccine production. However, the molecular requirements for this growth remain ill defined. Here, we demonstrate that keto-mycolic acids (keto-MA) are essential for pellicle growth, and mutants lacking in or depleted of this MA species are unable to form a pellicle. We investigated the role of the pellicle biofilm in the reduction of antibiotic sensitivity known as drug tolerance using the pellicle-defective ΔmmaA4 mutant strain. We discovered that the ΔmmaA4 mutant, which is both pellicle defective and highly sensitive to rifampicin (RIF) under planktonic growth, when incorporated within the wild-type pellicle biofilm, was protected from the bactericidal activity of RIF. The observation that growth within the M. tuberculosis pellicle biofilm can confer drug tolerance to a drug-hypersensitive strain suggests that identifying molecular requirements for pellicle growth could lead to development of novel interventions against mycobacterial infections. Our findings also suggest that a class of drugs that can disrupt M. tuberculosis biofilm formation, when used in conjunction with conventional antibiotics, has the potential to overcome drug tolerance. IMPORTANCE Two of the most important questions in tuberculosis (TB) research are (i) how does Mycobacterium tuberculosis persist in the human host for decades in the face of an active immune response and (ii) why does it take six months and four drugs to treat uncomplicated TB. Both these aspects of M. tuberculosis biology are reminiscent of infections caused by organisms capable of forming biofilms. M. tuberculosis is capable of growing as a biofilm-like structure called the pellicle. In this study, we demonstrate that a specific cell wall component, keto-mycolic acid, is essential for pellicle growth. We also demonstrate that a strain of M. tuberculosis that is both drug sensitive and pellicle defective exhibits commensal behavior and becomes drug tolerant by becoming part of a heterogeneous pellicle, a characteristic of multispecies biofilms. These observations could have important implications for identifying novel pathways for M. tuberculosis drug tolerance and the design of new modalities to rapidly treat TB.
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Increased Phagocytosis of Mycobacterium Marinum Mutants Defective in Lipooligosaccharide Production: a Structure-activity Relationship Study
The Journal of Biological Chemistry.
Jan, 2014 |
Pubmed ID: 24235141 Mycobacterium marinum is a waterborne pathogen responsible for tuberculosis-like infections in ectotherms and is an occasional opportunistic human pathogen. In the environment, M. marinum also interacts with amoebae, which may serve as a natural reservoir for this microorganism. However, the description of mycobacterial determinants in the early interaction with macrophages or amoebae remains elusive. Lipooligosaccharides (LOSs) are cell surface-exposed glycolipids capable of modulating the host immune system, suggesting that they may be involved in the early interactions of M. marinum with macrophages. Herein, we addressed whether LOS composition affects the uptake of M. marinum by professional phagocytes. Mutants with various truncated LOS variants were generated, leading to the identification of several previously uncharacterized biosynthetic genes (wbbL2, MMAR_2321, and MMAR_2331). Biochemical and structural approaches allowed resolving the structures of LOS precursors accumulating in this set of mutants. These strains with structurally defined LOS profiles were then used to infect both macrophages and Acanthamoebae. An inverse correlation between LOS completeness and uptake of mycobacteria by phagocytes was found, allowing the proposal of three mutant classes: class I (papA4), devoid of LOS and highly efficiently phagocytosed; class II, accumulating only early LOS intermediates (wbbL2 and MMAR_2331) and efficiently phagocytosed but less than class I mutants; class III, lacking LOS-IV (losA, MMAR_2319, and MMAR_2321) and phagocytosed similarly to the control strain. These results indicate that phagocytosis is conditioned by the LOS pattern and that the LOS pathway used by M. marinum in macrophages is conserved during infection of amoebae.
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Acetic Acid, the Active Component of Vinegar, is an Effective Tuberculocidal Disinfectant
MBio.
2014 |
Pubmed ID: 24570366 Effective and economical mycobactericidal disinfectants are needed to kill both Mycobacterium tuberculosis and non-M. tuberculosis mycobacteria. We found that acetic acid (vinegar) efficiently kills M. tuberculosis after 30 min of exposure to a 6% acetic acid solution. The activity is not due to pH alone, and propionic acid also appears to be bactericidal. M. bolletii and M. massiliense nontuberculous mycobacteria were more resistant, although a 30-min exposure to 10% acetic acid resulted in at least a 6-log10 reduction of viable bacteria. Acetic acid (vinegar) is an effective mycobactericidal disinfectant that should also be active against most other bacteria. These findings are consistent with and extend the results of studies performed in the early and mid-20th century on the disinfectant capacity of organic acids. IMPORTANCE Mycobacteria are best known for causing tuberculosis and leprosy, but infections with nontuberculous mycobacteria are an increasing problem after surgical or cosmetic procedures or in the lungs of cystic fibrosis and immunosuppressed patients. Killing mycobacteria is important because Mycobacterium tuberculosis strains can be multidrug resistant and therefore potentially fatal biohazards, and environmental mycobacteria must be thoroughly eliminated from surgical implements and respiratory equipment. Currently used mycobactericidal disinfectants can be toxic, unstable, and expensive. We fortuitously found that acetic acid kills mycobacteria and then showed that it is an effective mycobactericidal agent, even against the very resistant, clinically important Mycobacterium abscessus complex. Vinegar has been used for thousands of years as a common disinfectant, and if it can kill mycobacteria, the most disinfectant-resistant bacteria, it may prove to be a broadly effective, economical biocide with potential usefulness in health care settings and laboratories, especially in resource-poor countries.
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The Mycobacterium Tuberculosis Transcriptional Repressor EthR is Negatively Regulated by Serine/Threonine Phosphorylation
Biochemical and Biophysical Research Communications.
Apr, 2014 |
Pubmed ID: 24667600 Recent efforts have underlined the role of Serine/Threonine Protein Kinases (STPKs) in growth, pathogenesis and cell wall metabolism in mycobacteria. Herein, we demonstrated that the Mycobacterium tuberculosis EthR, a transcriptional repressor that regulates the activation process of the antitubercular drug ethionamide (ETH) is a specific substrate of the mycobacterial kinase PknF. ETH is a prodrug that must undergo bioactivation by the monooxygenease EthA to exert its antimycobacterial activity and previous studies reported that EthR represses transcription of ethA by binding to the ethA-ethR intergenic region. Mass spectrometry analyses and site-directed mutagenesis identified a set of four phosphoacceptors, namely Thr2, Thr3, Ser4 and Ser7. This was further supported by the complete loss of PknF-dependent phosphorylation of a phosphoablative EthR mutant protein. Importantly, a phosphomimetic version of EthR, in which all phosphosites were replaced by Asp residues, exhibited markedly decreased DNA-binding activity compared with the wild-type protein. Together, these findings are the first demonstration of EthR phosphorylation and indicate that phosphorylation negatively affects its DNA-binding activity, which may impact ETH resistance levels in M. tb.
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Phosphorylation of KasB Regulates Virulence and Acid-fastness in Mycobacterium Tuberculosis
PLoS Pathogens.
May, 2014 |
Pubmed ID: 24809459 Mycobacterium tuberculosis bacilli display two signature features: acid-fast staining and the capacity to induce long-term latent infections in humans. However, the mechanisms governing these two important processes remain largely unknown. Ser/Thr phosphorylation has recently emerged as an important regulatory mechanism allowing mycobacteria to adapt their cell wall structure/composition in response to their environment. Herein, we evaluated whether phosphorylation of KasB, a crucial mycolic acid biosynthetic enzyme, could modulate acid-fast staining and virulence. Tandem mass spectrometry and site-directed mutagenesis revealed that phosphorylation of KasB occurred at Thr334 and Thr336 both in vitro and in mycobacteria. Isogenic strains of M. tuberculosis with either a deletion of the kasB gene or a kasB_T334D/T336D allele, mimicking constitutive phosphorylation of KasB, were constructed by specialized linkage transduction. Biochemical and structural analyses comparing these mutants to the parental strain revealed that both mutant strains had mycolic acids that were shortened by 4-6 carbon atoms and lacked trans-cyclopropanation. Together, these results suggested that in M. tuberculosis, phosphorylation profoundly decreases the condensing activity of KasB. Structural/modeling analyses reveal that Thr334 and Thr336 are located in the vicinity of the catalytic triad, which indicates that phosphorylation of these amino acids would result in loss of enzyme activity. Importantly, the kasB_T334D/T336D phosphomimetic and deletion alleles, in contrast to the kasB_T334A/T336A phosphoablative allele, completely lost acid-fast staining. Moreover, assessing the virulence of these strains indicated that the KasB phosphomimetic mutant was attenuated in both immunodeficient and immunocompetent mice following aerosol infection. This attenuation was characterized by the absence of lung pathology. Overall, these results highlight for the first time the role of Ser/Thr kinase-dependent KasB phosphorylation in regulating the later stages of mycolic acid elongation, with important consequences in terms of acid-fast staining and pathogenicity.
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Design, Synthesis and Docking Studies of Some Novel (R)-2-(4'-chlorophenyl)-3-(4'-nitrophenyl)-1,2,3,5-tetrahydrobenzo[4,5] Imidazo [1,2-c]pyrimidin-4-ol Derivatives As Antitubercular Agents
European Journal of Medicinal Chemistry.
Aug, 2014 |
Pubmed ID: 24972340 Filamenting temperature-sensitive mutant (FtsZ) is a novel target for the treatment of tuberculosis. A series of (R)-2-(4'-chlorophenyl)-3-(4'-nitrophenyl)-1,2,3,5-tetrahydrobenzo[4,5] imidazo[1,2-c]pyrimidin-4-ol derivatives were designed and docked on the FtsZ protein crystal structure (PDB Id: 1RLU, resolution 2.08 Å). Compound 7t showed the highest docking score and H-bond interaction with Arg140 and Gly19. Our strategy for synthesis of (R)-2-(4'-chlorophenyl)-3-(4'-nitrophenyl)-1,2,3,5-tetrahydrobenzo[4,5]imidazo[1,2-c]pyrimidin-4-ol derivatives from o-phenylenediamine as illustrated in scheme. All the synthesized compounds were characterized by FTIR, Mass spectra, (1)H NMR, (13)C NMR, elemental analysis and purity was confirmed by HPLC and LCMS. Compound 7g was also confirmed by single crystal X-ray analysis. The in silico results are also validated with in vitro antitubercular activity of compound 7t. Compound 7b exhibited in vitro antitubercular activity 3.13 μg/mL and 4.7 μg/mL whereas compound 7t exhibited in vitro antitubercular activity 6.25 μg/mL and 9.4 μg/mL using GAST/Fe medium after week 1 and week 2 respectively against Mycobacterium tuberculosis H37Rv. Medium 7H9/ADC/Tween was found to be very less effective for in vitro antitubercular activity of all the benzimidazole derivatives. Assays for in vitro cytotoxicity against VERO cells of all the synthesized compounds was found to be very less cytotoxic.
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Overexpression of the Salmonella KdpF Membrane Peptide Modulates Expression of Kdp Genes and Intramacrophage Growth
FEMS Microbiology Letters.
Oct, 2014 |
Pubmed ID: 25197761 Membrane peptides appear as an emerging class of regulatory molecules in bacteria, which can interact with membrane proteins, including transporters and sensor kinases. The KdpF peptide, which is cotranscribed with kdpABC genes and regulated by the KdpDE two-component system, is supposed to stabilize the KdpABC potassium transporter complex but may also exhibit unsuspected regulatory function(s). The mycobacterial KdpF can interact with the KdpD histidine kinase, and kdpF overexpression has been shown to reduce intramacrophage replication of Mycobacterium bovis BCG. In this study, we investigated whether KdpF displays similar behavior in another intracellular pathogen, Salmonella enterica serovar Typhimurium. We show that Salmonella KdpF can interact with KdpD in a bacterial two-hybrid assay. We have constructed a Salmonella strain overexpressing kdpF, and we have investigated expression of the kdp regulon, as well as intramacrophage survival. We show that kdpF overexpression reduces expression of kdpA and kdpD genes under potassium limitation. Moreover, kdpF overexpression increases intramacrophage multiplication of S. Typhimurium. Hence, our results indicate that KdpF can play a regulatory role in S. Typhimurium, modulating kdp gene expression and intramacrophage survival, but in a way that differs from the one reported for M. bovis BCG.
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The Molecular Genetics of Mycolic Acid Biosynthesis
Microbiology Spectrum.
Aug, 2014 |
Pubmed ID: 26104214 Mycolic acids are major and specific long-chain fatty acids that represent essential components of the Mycobacterium tuberculosis cell envelope. They play a crucial role in the cell wall architecture and impermeability, hence the natural resistance of mycobacteria to most antibiotics, and represent key factors in mycobacterial virulence. Biosynthesis of mycolic acid precursors requires two types of fatty acid synthases (FASs), the eukaryotic-like multifunctional enzyme FAS I and the acyl carrier protein (ACP)-dependent FAS II systems, which consists of a series of discrete mono-functional proteins, each catalyzing one reaction in the pathway. Unlike FAS II synthases of other bacteria, the mycobacterial FAS II is incapable of de novo fatty acid synthesis from acetyl-coenzyme A, but instead elongates medium-chain-length fatty acids previously synthesized by FAS I, leading to meromycolic acids. In addition, mycolic acid subspecies with defined biological properties can be distinguished according to the chemical modifications decorating the meromycolate. Nearly all the genetic components involved in both elongation and functionalization of the meromycolic acid have been identified and are generally clustered in distinct transcriptional units. A large body of information has been generated on the enzymology of the mycolic acid biosynthetic pathway and on their genetic and biochemical/structural characterization as targets of several antitubercular drugs. This chapter is a comprehensive overview of mycolic acid structure, function, and biosynthesis. Special emphasis is given to recent work addressing the regulation of mycolic acid biosynthesis, adding new insights to our understanding of how pathogenic mycobacteria adapt their cell wall composition in response to environmental changes.
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