The Experts below are selected from a list of 7371 Experts worldwide ranked by ideXlab platform
Laurent Kremer - One of the best experts on this subject based on the ideXlab platform.
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controlling extra and intramacrophagic mycobacterium abscessus by targeting Mycolic Acid transport
Frontiers in Cellular and Infection Microbiology, 2017Co-Authors: Albertus Viljoen, Laurent Kremer, Alan P Kozikowski, Oluseye K Onajole, Jozef Stec, Jeanlouis HerrmannAbstract:Mycobacterium abscessus is a rapidly growing mycobacterium (RGM) causing serious infections especially among cystic fibrosis patients. Extremely limited therapeutic options against M. abscessus and a rise in infections with this mycobacterium require novel chemotherapies and a better understanding of how the bacterium causes infection. Different from most RGM, M. abscessus can survive inside macrophages and persist for long durations in infected tissues. We recently delineated differences in the infective programs followed by smooth (S) and rough (R) variants of M. abscessus. Unexpectedly, we found that the S variant behaves like pathogenic slow growing mycobacteria, through maintaining a block on the phagosome maturation process and by inducing phagosome-cytosol communications. On the other hand, R variant infection triggers autophagy and apoptosis, reminiscent of the way that macrophages control RGM. However, the R variant has an exquisite capacity to form extracellular cords, allowing these bacteria to rapidly divide and evade phagocytosis. Therefore, new chemotherapeutic interventions against M. abscessus need to efficiently deal with both the reservoir of intracellular bacilli and the extracellular cords. In this context, we recently identified two chemical entities that were very effective against both M. abscessus populations. Although being structurally unrelated these two chemotypes inhibit the activity of the essential Mycolic Acid transporter, MmpL3. In this Perspective, we aimed to highlight recent insights into how M. abscessus interacts with phagocytic cells and how the inhibition of Mycolic Acid transport in this pathogenic RGM could be an efficient means to control both intracellular and extracellular populations of the bacterium.
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The Molecular Genetics of Mycolic Acid Biosynthesis.
Microbiology spectrum, 2014Co-Authors: Jakub PaweŁczyk, Laurent KremerAbstract: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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phosphorylation of mycobacterial pcaa inhibits Mycolic Acid cyclopropanation consequences for intracellular survival and for phagosome maturation block
Journal of Biological Chemistry, 2012Co-Authors: Rosa Milagros Corrales, Virginie Molle, Jade Leiba, Lionel Mourey, Chantal De Chastellier, Laurent KremerAbstract: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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Phosphorylation of Mycobacterial PcaA Inhibits Mycolic Acid Cyclopropanation
Journal of Biological Chemistry, 2012Co-Authors: Rosa Milagros Corrales, Virginie Molle, Jade Leiba, Lionel Mourey, Chantal De Chastellier, Laurent KremerAbstract:Background: Mycolic Acid cyclopropanation plays an important role in mycobacterial virulence but molecular mechanisms controlling cyclopropanation remain unknown. Results: Phosphorylation of the cyclopropane synthase PcaA decreases Mycolic Acid cyclopropanation and intracellular survival and abrogates the phagosome maturation block (PMB). Conclusion: PMB modulation is dependent on mycobacterial protein phosphorylation. Significance: This study highlights Ser/Thr-dependent phosphorylation of a Mycolic Acid biosynthetic enzyme in mycobacte-rial virulence.
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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, 2011Co-Authors: Jakub Pawelczyk, Laurent Kremer, Anna Brzostek, Bozena Dziadek, Anna Rumijowskagalewicz, Marta J Fiolka, Jaroslaw DziadekAbstract: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.
Virginie Molle - One of the best experts on this subject based on the ideXlab platform.
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ser thr phosphorylation regulates the fatty acyl amp ligase activity of fadd32 an essential enzyme in Mycolic Acid biosynthesis
Journal of Biological Chemistry, 2016Co-Authors: Virginie Molle, Fabienne Bardou, Nathalie Eynard, Mathieu Miras, Alexandre Stella, Segolene Galandrin, Valerie Guillet, Gwenaelle Andreleroux, Marco Bellinzoni, Pedro M AlzariAbstract:Mycolic Acids are essential components of the mycobacterial cell envelope, and their biosynthetic pathway is a well known source of antituberculous drug targets. Among the promising new targets in the pathway, FadD32 is an essential enzyme required for the activation of the long meroMycolic chain of Mycolic Acids and is essential for mycobacterial growth. Following the in-depth biochemical, biophysical, and structural characterization of FadD32, we investigated its putative regulation via post-translational modifications. Comparison of the fatty acyl-AMP ligase activity between phosphorylated and dephosphorylated FadD32 isoforms showed that the native protein is phosphorylated by serine/threonine protein kinases and that this phosphorylation induced a significant loss of activity. Mass spectrometry analysis of the native protein confirmed the post-translational modifications and identified Thr-552 as the phosphosite. Phosphoablative and phosphomimetic FadD32 mutant proteins confirmed both the position and the importance of the modification and its correlation with the negative regulation of FadD32 activity. Investigation of the Mycolic Acid condensation reaction catalyzed by Pks13, involving FadD32 as a partner, showed that FadD32 phosphorylation also impacts the condensation activity. Altogether, our results bring to light FadD32 phosphorylation by serine/threonine protein kinases and its correlation with the enzyme-negative regulation, thus shedding a new horizon on the Mycolic Acid biosynthesis modulation and possible inhibition strategies for this promising drug target.
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Ser/Thr Phosphorylation Regulates the Fatty Acyl-AMP Ligase Activity of FadD32, an Essential Enzyme in Mycolic Acid Biosynthesis.
Journal of Biological Chemistry, 2016Co-Authors: Virginie Molle, Fabienne Bardou, Nathalie Eynard, Mathieu Miras, Alexandre Stella, Segolene Galandrin, Valerie Guillet, Marco Bellinzoni, Gwenaelle Andre-leroux, Pedro M AlzariAbstract:Mycolic Acids are essential components of the mycobacterial cell envelope, and their biosynthetic pathway is a well known source of antituberculous drug targets. Among the promising new targets in the pathway, FadD32 is an essential enzyme required for the activation of the long meroMycolic chain of Mycolic Acids and is essential for mycobacterial growth. Following the in-depth biochemical, biophysical, and structural characterization of FadD32, we investigated its putative regulation via post-translational modifications. Comparison of the fatty acyl-AMP ligase activity between phosphorylated and dephosphorylated FadD32 isoforms showed that the native protein is phosphorylated by serine/threonine protein kinases and that this phosphorylation induced a significant loss of activity. Mass spectrometry analysis of the native protein confirmed the post-translational modifications and identified Thr-552 as the phosphosite. Phosphoablative and phosphomimetic FadD32 mutant proteins confirmed both the position and the importance of the modification and its correlation with the negative regulation of FadD32 activity. Investigation of the Mycolic Acid condensation reaction catalyzed by Pks13, involving FadD32 as a partner, showed that FadD32 phosphorylation also impacts the condensation activity. Altogether, our results bring to light FadD32 phosphorylation by serine/threonine protein kinases and its correlation with the enzyme-negative regulation, thus shedding a new horizon on the Mycolic Acid biosynthesis modulation and possible inhibition strategies for this promising drug target.
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phosphorylation of mycobacterial pcaa inhibits Mycolic Acid cyclopropanation consequences for intracellular survival and for phagosome maturation block
Journal of Biological Chemistry, 2012Co-Authors: Rosa Milagros Corrales, Virginie Molle, Jade Leiba, Lionel Mourey, Chantal De Chastellier, Laurent KremerAbstract: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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Phosphorylation of Mycobacterial PcaA Inhibits Mycolic Acid Cyclopropanation
Journal of Biological Chemistry, 2012Co-Authors: Rosa Milagros Corrales, Virginie Molle, Jade Leiba, Lionel Mourey, Chantal De Chastellier, Laurent KremerAbstract:Background: Mycolic Acid cyclopropanation plays an important role in mycobacterial virulence but molecular mechanisms controlling cyclopropanation remain unknown. Results: Phosphorylation of the cyclopropane synthase PcaA decreases Mycolic Acid cyclopropanation and intracellular survival and abrogates the phagosome maturation block (PMB). Conclusion: PMB modulation is dependent on mycobacterial protein phosphorylation. Significance: This study highlights Ser/Thr-dependent phosphorylation of a Mycolic Acid biosynthetic enzyme in mycobacte-rial virulence.
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Phosphorylation of InhA inhibits Mycolic Acid biosynthesis and growth of Mycobacterium tuberculosis
Molecular Microbiology, 2010Co-Authors: Virginie Molle, James C Sacchettini, Gulcin Gulten, Catherine Vilchèze, Romain Veyron-churlet, Isabelle Zanella-cléon, William R. Jacobs, Laurent KremerAbstract:The remarkable survival ability of Mycobacterium tuberculosis in infected hosts is related to the presence of cell wall-associated Mycolic Acids. Despite their importance, the mechanisms that modulate expression of these lipids in response to environmental changes are unknown. Here we demonstrate that the enoyl-ACP reductase activity of InhA, an essential enzyme of the Mycolic Acid biosynthetic pathway and the primary target of the anti-tubercular drug isoniazid, is controlled via phosphorylation. Thr-266 is the unique kinase phosphoacceptor, both in vitro and in vivo. The physiological relevance of Thr-266 phosphorylation was demonstrated using inhA phosphoablative (T266A) or phosphomimetic (T266D/E) mutants. Enoyl reductase activity was severely impaired in the mimetic mutants in vitro, as a consequence of a reduced binding affinity to NADH. Importantly, introduction of inhA_T266D/E failed to complement growth and Mycolic Acid defects of an inhA-thermosensitive Mycobacterium smegmatis strain, in a similar manner to what is observed following isoniazid treatment. This study suggests that phosphorylation of InhA may represent an unusual mechanism that allows M. tuberculosis to regulate its Mycolic Acid content, thus offering a new approach to future anti-tuberculosis drug development.
Pedro M Alzari - One of the best experts on this subject based on the ideXlab platform.
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ser thr phosphorylation regulates the fatty acyl amp ligase activity of fadd32 an essential enzyme in Mycolic Acid biosynthesis
Journal of Biological Chemistry, 2016Co-Authors: Virginie Molle, Fabienne Bardou, Nathalie Eynard, Mathieu Miras, Alexandre Stella, Segolene Galandrin, Valerie Guillet, Gwenaelle Andreleroux, Marco Bellinzoni, Pedro M AlzariAbstract:Mycolic Acids are essential components of the mycobacterial cell envelope, and their biosynthetic pathway is a well known source of antituberculous drug targets. Among the promising new targets in the pathway, FadD32 is an essential enzyme required for the activation of the long meroMycolic chain of Mycolic Acids and is essential for mycobacterial growth. Following the in-depth biochemical, biophysical, and structural characterization of FadD32, we investigated its putative regulation via post-translational modifications. Comparison of the fatty acyl-AMP ligase activity between phosphorylated and dephosphorylated FadD32 isoforms showed that the native protein is phosphorylated by serine/threonine protein kinases and that this phosphorylation induced a significant loss of activity. Mass spectrometry analysis of the native protein confirmed the post-translational modifications and identified Thr-552 as the phosphosite. Phosphoablative and phosphomimetic FadD32 mutant proteins confirmed both the position and the importance of the modification and its correlation with the negative regulation of FadD32 activity. Investigation of the Mycolic Acid condensation reaction catalyzed by Pks13, involving FadD32 as a partner, showed that FadD32 phosphorylation also impacts the condensation activity. Altogether, our results bring to light FadD32 phosphorylation by serine/threonine protein kinases and its correlation with the enzyme-negative regulation, thus shedding a new horizon on the Mycolic Acid biosynthesis modulation and possible inhibition strategies for this promising drug target.
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Ser/Thr Phosphorylation Regulates the Fatty Acyl-AMP Ligase Activity of FadD32, an Essential Enzyme in Mycolic Acid Biosynthesis.
Journal of Biological Chemistry, 2016Co-Authors: Virginie Molle, Fabienne Bardou, Nathalie Eynard, Mathieu Miras, Alexandre Stella, Segolene Galandrin, Valerie Guillet, Marco Bellinzoni, Gwenaelle Andre-leroux, Pedro M AlzariAbstract:Mycolic Acids are essential components of the mycobacterial cell envelope, and their biosynthetic pathway is a well known source of antituberculous drug targets. Among the promising new targets in the pathway, FadD32 is an essential enzyme required for the activation of the long meroMycolic chain of Mycolic Acids and is essential for mycobacterial growth. Following the in-depth biochemical, biophysical, and structural characterization of FadD32, we investigated its putative regulation via post-translational modifications. Comparison of the fatty acyl-AMP ligase activity between phosphorylated and dephosphorylated FadD32 isoforms showed that the native protein is phosphorylated by serine/threonine protein kinases and that this phosphorylation induced a significant loss of activity. Mass spectrometry analysis of the native protein confirmed the post-translational modifications and identified Thr-552 as the phosphosite. Phosphoablative and phosphomimetic FadD32 mutant proteins confirmed both the position and the importance of the modification and its correlation with the negative regulation of FadD32 activity. Investigation of the Mycolic Acid condensation reaction catalyzed by Pks13, involving FadD32 as a partner, showed that FadD32 phosphorylation also impacts the condensation activity. Altogether, our results bring to light FadD32 phosphorylation by serine/threonine protein kinases and its correlation with the enzyme-negative regulation, thus shedding a new horizon on the Mycolic Acid biosynthesis modulation and possible inhibition strategies for this promising drug target.
Hiroyasu Onaka - One of the best experts on this subject based on the ideXlab platform.
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catenulobactins a and b heterocyclic peptides from culturing catenuloplanes sp with a Mycolic Acid containing bacterium
Journal of Natural Products, 2018Co-Authors: Shotaro Hoshino, Hiroyasu Onaka, Takayoshi Awakawa, Masahiro Ozeki, Hiroyuki Morita, Ikuro AbeAbstract:The production of two new heterocyclic peptide isomers, catenulobactins A (1) and B (2), in cultures of Catenuloplanes sp. RD067331 was significantly increased when it was cocultured with a Mycolic Acid-containing bacterium. The planar structures and absolute configurations of the catenulobactins were determined based on NMR/MS and chiral-phase GC-MS analyses. Catenulobactin B (2) displayed Fe(III)-chelating activity and moderate cytotoxicity against P388 murine leukemia cells.
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umezawamides new bioactive polycyclic tetramate macrolactams isolated from a combined culture of umezawaea sp and Mycolic Acid containing bacterium
The Journal of Antibiotics, 2018Co-Authors: Shotaro Hoshino, Shumpei Asamizu, Takayoshi Awakawa, Huiping Zhang, Fumiaki Hayashi, Masahiro Ozeki, Chin Piow Wong, Hiroyasu OnakaAbstract:New polycyclic tetramate macrolactams, Umezawamides A (1) and B (2) were isolated from a combined-culture of Umezawaea sp. RD066910 and Mycolic-Acid containing bacterium Tsukamurella pulmonis TP-B0596. Their planar structures and partial stereochemistries were determined based on the spectroscopic analysis, MMFF conformational search, and ECD calculations. Umezawamides are the first secondary metabolites isolated from the genus Umezawaea and they exhibited cytotoxicities to P388 murine leukemia cells. Furthermore, umezawamide A (1) showed growth inhibitory activity against Candida albicans.
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umezawamides new bioactive polycyclic tetramate macrolactams isolated from a combined culture of umezawaea sp and Mycolic Acid containing bacterium
The Journal of Antibiotics, 2018Co-Authors: Shotaro Hoshino, Hiroyasu Onaka, Shumpei Asamizu, Takayoshi Awakawa, Huiping Zhang, Fumiaki Hayashi, Masahiro Ozeki, Chin Piow Wong, Ikuro AbeAbstract:New polycyclic tetramate macrolactams, Umezawamides A (1) and B (2) were isolated from a combined-culture of Umezawaea sp. RD066910 and Mycolic-Acid containing bacterium Tsukamurella pulmonis TP-B0596. Their planar structures and partial stereochemistries were determined based on the spectroscopic analysis, MMFF conformational search, and ECD calculations. Umezawamides are the first secondary metabolites isolated from the genus Umezawaea and they exhibited cytotoxicities to P388 murine leukemia cells. Furthermore, umezawamide A (1) showed growth inhibitory activity against Candida albicans.
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Catenulobactins A and B, Heterocyclic Peptides from Culturing Catenuloplanes sp. with a Mycolic Acid-Containing Bacterium
2018Co-Authors: Shotaro Hoshino, Hiroyasu Onaka, Takayoshi Awakawa, Masahiro Ozeki, Hiroyuki Morita, Ikuro AbeAbstract:The production of two new heterocyclic peptide isomers, catenulobactins A (1) and B (2), in cultures of Catenuloplanes sp. RD067331 was significantly increased when it was cocultured with a Mycolic Acid-containing bacterium. The planar structures and absolute configurations of the catenulobactins were determined based on NMR/MS and chiral-phase GC-MS analyses. Catenulobactin B (2) displayed Fe(III)-chelating activity and moderate cytotoxicity against P388 murine leukemia cells
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Mycolic Acid containing bacterium stimulates tandem cyclization of polyene macrolactam in a lake sediment derived rare actinomycete
Organic Letters, 2017Co-Authors: Shotaro Hoshino, Hiroyasu Onaka, Shumpei Asamizu, Takayoshi Awakawa, Masahiro Okada, Ikuro AbeAbstract:Two novel macrolactams, dracolactams A and B, were identified from a combined-culture of Micromonospora species and a Mycolic-Acid containing bacterium (MACB). Their structures and stereochemistries were completely assigned, based on spectroscopic analyses and chemical derivatization. Both dracolactams were probably generated from a common macrolactam precursor produced by the Micromonospora species. In this combined-culture system, MACB is likely to activate cryptic oxidase genes in the Micromonospora species and induce the downstream polyene macrolactam cyclization.
Apoorva Bhatt - One of the best experts on this subject based on the ideXlab platform.
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the Mycolic Acid reductase rv2509 has distinct structural motifs and is essential for growth in slow growing mycobacteria
Molecular Microbiology, 2020Co-Authors: Asma Javid, Albel Singh, Charlotte Cooper, Steffen Schindler, Milena Hanisch, Robert L Marshall, Rainer Kalscheuer, Vassiliy N Bavro, Apoorva BhattAbstract:The final step in Mycolic Acid biosynthesis in Mycobacterium tuberculosis is catalysed by mycolyl reductase encoded by the Rv2509 gene. Sequence analysis and homology modelling indicate that Rv2509 belongs to the short-chain fatty Acid dehydrogenase/reductase (SDR) family, but with some distinct features that warrant its classification as belonging to a novel family of short-chain dehydrogenases. In particular, the predicted structure revealed a unique α-helical C-terminal region which we demonstrated to be essential for Rv2509 function, though this region did not seem to play any role in protein stabilisation or oligomerisation. We also show that unlike the M. smegmatis homologue which was not essential for growth, Rv2509 was an essential gene in slow-growing mycobacteria. A knockdown strain of the BCG2529 gene, the Rv2509 homologue in Mycobacterium bovis BCG, was unable to grow following the conditional depletion of BCG2529. This conditional depletion also led to a reduction of mature Mycolic Acid production and accumulation of intermediates derived from 3-oxo-mycolate precursors. Our studies demonstrate novel features of the mycolyl reductase Rv2509 and outline its role in mycobacterial growth, highlighting its potential as a new target for therapies.
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MmpL Genes Are Associated with Mycolic Acid Metabolism in Mycobacteria and Corynebacteria
Chemistry & biology, 2012Co-Authors: Cristian Varela, Karin Krumbach, Gurdyal S Besra, Lothar Eggeling, Doris Rittmann, Albel Singh, Kiranmai Bhatt, Apoorva BhattAbstract:Mycolic Acids are vital components of the cell wall of the tubercle bacillus Mycobacterium tuberculosis and are required for viability and virulence. While Mycolic Acid biosynthesis is studied extensively, components involved in mycolate transport remain unidentified. We investigated the role of large membrane proteins encoded by mmpL genes in Mycolic Acid transport in mycobacteria and the related corynebacteria. MmpL3 was found to be essential in mycobacteria and conditional depletion of MmpL3 in Mycobacterium smegmatis resulted in loss of cell wall mycolylation, and of the cell wall-associated glycolipid, trehalose dimycolate. In parallel, an accumulation of trehalose monomycolate (TMM) was observed, suggesting that Mycolic Acids were transported as TMM. In contrast to mycobacteria, we found redundancy in the role of two mmpL genes, in Corynebacterium glutamicum; a complete loss of trehalose-associated and cell wall bound corynomycolates was observed in an NCgl0228-NCgl2769 double mutant, but not in individual single mutants. Our studies highlight the role of mmpL genes in Mycolic Acid metabolism and identify potential new targets for anti-TB drug development.
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identification of the dehydratase component of the mycobacterial Mycolic Acid synthesizing fatty Acid synthase ii complex
Microbiology, 2007Co-Authors: Alistair K Brown, Albel Singh, Apoorva Bhatt, Elesh Saparia, Alex F Evans, Gurdyal S BesraAbstract:Mycolic Acids are vital components of the Mycobacterium tuberculosis cell wall and are essential for survival. While most components of the fatty Acid synthase-II (FAS-II) enzymic machinery that synthesizes these long chain α-alkyl, β-hydroxy fatty Acids have been identified, the gene encoding the β-hydroxyacyl-acyl carrier protein (ACP) dehydratase activity has remained elusive. Recent bioinformatics-based studies and drug inhibition experiments have identified the M. tuberculosis gene Rv0636 as a promising candidate for this role. Using a recently described, specialized transduction-based genetic tool we now demonstrate that MSMEG1341, the Mycobacterium smegmatis homologue of Rv0636, is an essential gene; null mutants of the gene could only be generated in a merodiploid strain which contained a second integrated acetamide-inducible copy of MSMEG1341. Growth of the conditional mutant in the absence of acetamide resulted in loss of Mycolic Acid biosynthesis and eventually loss of viability due to cell lysis. Null MSMEG1341 mutants could also be generated in a M. smegmatis strain containing an integrated copy of Rv0636, indicating that Rv0636 was the functional counterpart of MSMEG1341 in M. tuberculosis. Our results demonstrate that MSMEG1341 is an essential gene involved in Mycolic Acid biosynthesis and encodes the FAS-II β-hydroxyacyl-ACP dehydratase.
