247 results on '"Lipomannan"'
Search Results
2. Crystal structure of the putative cell‐wall lipoglycan biosynthesis protein LmcA from Mycobacterium smegmatis.
- Author
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Patel, Onisha, Brammananth, Rajini, Dai, Weiwen, Panjikar, Santosh, Coppel, Ross L., Lucet, Isabelle S., and Crellin, Paul K.
- Subjects
- *
MYCOBACTERIUM smegmatis , *PROTEIN synthesis , *CRYSTAL structure , *CORYNEBACTERIUM glutamicum , *LIGAND binding (Biochemistry) , *GLYCOLIPIDS , *MYCOBACTERIUM tuberculosis - Abstract
The bacterial genus Mycobacterium includes important pathogens, most notably M. tuberculosis, which infects one‐quarter of the entire human population, resulting in around 1.4 million deaths from tuberculosis each year. Mycobacteria, and the closely related corynebacteria, synthesize a class of abundant glycolipids, the phosphatidyl‐myo‐inositol mannosides (PIMs). PIMs serve as membrane anchors for hyperglycosylated species, lipomannan (LM) and lipoarabinomannan (LAM), which are surface‐exposed and modulate the host immune response. Previously, in studies using the model species Corynebacterium glutamicum, NCgl2760 was identified as a novel membrane protein that is required for the synthesis of full‐length LM and LAM. Here, the first crystal structure of its ortholog in Mycobacterium smegmatis, MSMEG_0317, is reported at 1.8 Å resolution. The structure revealed an elongated β‐barrel fold enclosing two distinct cavities and one α‐helix extending away from the β‐barrel core, resembling a 'cone with a flake' arrangement. Through xenon derivatization and structural comparison with AlphaFold2‐derived predictions of the M. tuberculosis homolog Rv0227c, structural elements were identified that may undergo conformational changes to switch from 'closed' to 'open' conformations, allowing cavity access. An AlphaFold2‐derived NCgl2760 model predicted a smaller β‐barrel core with an enclosed central cavity, suggesting that all three proteins, which were collectively termed LmcA, may have a common mechanism of ligand binding through these cavities. These findings provide new structural insights into the biosynthetic pathway for a family of surface lipoglycans with important roles in mycobacterial pathogenesis. [ABSTRACT FROM AUTHOR]
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- 2022
- Full Text
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3. Impact of Methylthioxylose Substituents on the Biological Activities of Lipomannan and Lipoarabinomannan in Mycobacterium tuberculosis .
- Author
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Palčeková Z, De K, Angala SK, Gilleron M, Zuberogoitia S, Gouxette L, Soto-Ojeda M, Gonzalez-Juarrero M, Obregón-Henao A, Nigou J, Wheat WH, and Jackson M
- Subjects
- Humans, Lipopolysaccharides, Mycobacterium tuberculosis, Tuberculosis microbiology
- Abstract
Two lipoglycans, lipomannan (LM) and lipoarabinomannan (LAM), play various, albeit incompletely defined, roles in the interactions of mycobacteria with the host. Growing evidence points to the modification of LM and LAM with discrete covalent substituents as a strategy used by these bacteria to modulate their biological activities. One such substituent, originally identified in Mycobacterium tuberculosis ( Mtb ), is a 5-methylthio-d-xylose (MTX) sugar, which accounts for the antioxidative properties of LAM. The widespread distribution of this motif across Mtb isolates from several epidemiologically important lineages have stimulated interest in MTX-modified LAM as a biomarker of tuberculosis infection. Yet, several lines of evidence indicate that MTX may not be restricted to Mtb and that this motif may substitute more acceptors than originally thought. Using a highly specific monoclonal antibody to the MTX capping motif of Mtb LAM, we here show that MTX motifs not only substitute the mannoside caps of LAM but also the mannan core of LM in Mtb . MTX substituents were also found on the LM and LAM of pathogenic, slow-growing nontuberculous mycobacteria. The presence of MTX substituents on the LM and LAM from Mtb enhances the pro-apoptotic properties of both lipoglycans on LPS-stimulated THP-1 macrophages. A comparison of the cytokines and chemokines produced by resting and LPS-activated THP-1 cells upon exposure to MTX-proficient versus MTX-deficient LM further indicates that MTX substituents confer anti-inflammatory properties upon LM. These findings add to our understanding of the glycan-based strategies employed by slow-growing pathogenic mycobacteria to alter the host immune response to infection.
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- 2024
- Full Text
- View/download PDF
4. Synthesis of synthetic mannan backbone polysaccharides found on the surface of Mycobacterium tuberculosis as a vaccine adjuvant and their immunological properties.
- Author
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Wattanasiri, Chakree, Paha, Jiraporn, Ponpuak, Marisa, Ruchirawat, Somsak, and Boonyarattanakalin, Siwarutt
- Subjects
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MANNANS , *POLYSACCHARIDE synthesis , *MYCOBACTERIUM tuberculosis , *IMMUNOLOGICAL adjuvants , *TUBERCULOSIS treatment , *THERAPEUTICS - Abstract
Surface components of Mycobacterium tuberculosis (Mtb) play crucial roles in modulating host immune responses. Thorough understandings of immunological properties of the Mtb’s surface components are essential for the development of tuberculosis treatment and prevention. Unfortunately, the accessibility to the molecules on the surface of Mtb is limited by the structural complexity due to their various macromolecular nature and the hazard of culturing Mtb. In this study, we reveal a practical synthesis of lipomannan (LM) backbone polysaccharides – the core glycans found on Mtb’s surface. A rapid synthetic approach based on a controlled polymerization was developed for the chemical synthesis of mannopyranans, the core structure of LM. The size of the LM glycans can be controlled by using specific monomer concentrations in addition to stereo- and regioselectivity derived from the versatile tricyclic orthoester mannose monomer. The immunological properties of the synthesized mannopyranans were investigated and their adjuvant potential was revealed. The adjuvanticity mechanism of the synthetic mannopyranans appears to involve the NF-κB and inflammasome pathways. [ABSTRACT FROM AUTHOR]
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- 2017
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5. Structural and Functional Characterization of Phosphatidylinositol-Phosphate Biosynthesis in Mycobacteria
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Surajit Banerjee, Khuram U. Ashraf, Carla D. Jorge, Helena Santos, Cristina G. Timóteo, Oliver B. Clarke, Vasileios I. Petrou, Meagan Belcher Dufrisne, and Filippo Mancia
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Models, Molecular ,Protein Conformation ,Context (language use) ,Article ,Mycobacterium ,Substrate Specificity ,Phosphotransferase ,Mycobacterium tuberculosis ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,Bacterial Proteins ,Phosphatidylinositol Phosphates ,Structural Biology ,Phosphatidylinositol ,Molecular Biology ,030304 developmental biology ,Mycobacterium kansasii ,0303 health sciences ,Lipomannan ,Lipoarabinomannan ,biology ,Cytidine ,CDP-Diacylglycerol-Inositol 3-Phosphatidyltransferase ,biology.organism_classification ,chemistry ,Biochemistry ,Biocatalysis ,lipids (amino acids, peptides, and proteins) ,030217 neurology & neurosurgery - Abstract
In mycobacteria, phosphatidylinositol (PI) acts as a common lipid anchor for key components of the cell wall, including the glycolipids phosphatidylinositol mannoside, lipomannan, and lipoarabinomannan. Glycolipids in Mycobacterium tuberculosis, the causative agent of tuberculosis, are important virulence factors that modulate the host immune response. The identity-defining step in PI biosynthesis in prokaryotes, unique to mycobacteria and few other bacterial species, is the reaction between cytidine diphosphate-diacylglycerol and inositol-phosphate to yield phosphatidylinositol-phosphate, the immediate precursor to PI. This reaction is catalyzed by the cytidine diphosphate-alcohol phosphotransferase phosphatidylinositol-phosphate synthase (PIPS), an essential enzyme for mycobacterial viability. Here we present structures of PIPS from Mycobacterium kansasii with and without evidence of donor and acceptor substrate binding obtained using a crystal engineering approach. PIPS from Mycobacterium kansasii is 86% identical to the ortholog from M. tuberculosis and catalytically active. Functional experiments guided by our structural results allowed us to further characterize the molecular determinants of substrate specificity and catalysis in a new mycobacterial species. This work provides a framework to strengthen our understanding of phosphatidylinositol-phosphate biosynthesis in the context of mycobacterial pathogens.
- Published
- 2020
6. Polysaccharide Succinylation Enhances the Intracellular Survival of Mycobacterium abscessus
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Zuzana Palčeková, Shiva K. Angala, Mary Jackson, Michael R. McNeil, Martine Gilleron, Juan Manuel Belardinelli, and Luiz E. Bermudez
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0301 basic medicine ,Mannosides ,Lipoarabinomannan ,Lipomannan ,biology ,Chemistry ,030106 microbiology ,Mycobacterium abscessus ,bacterial infections and mycoses ,biology.organism_classification ,3. Good health ,Microbiology ,carbohydrates (lipids) ,Mycobacterium tuberculosis ,03 medical and health sciences ,chemistry.chemical_compound ,Succinylation ,030104 developmental biology ,Infectious Diseases ,immune system diseases ,hemic and lymphatic diseases ,lipids (amino acids, peptides, and proteins) ,Nontuberculous mycobacteria ,Phosphatidylinositol - Abstract
Lipoarabinomannan (LAM) and its biosynthetic precursors, phosphatidylinositol mannosides (PIMs) and lipomannan (LM) play important roles in the interactions of Mycobacterium tuberculosis with phago...
- Published
- 2020
7. Synthesis of glycoconjugate fragments of mycobacterial phosphatidylinositol mannosides and lipomannan
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Benjamin Cao, Jonathan M. White, and Spencer J. Williams
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fluorescently-labelled sugars ,glycoconjugates ,lipomannan ,mycobacteria ,tuberculosis ,Science ,Organic chemistry ,QD241-441 - Abstract
Mycobacterium tuberculosis, the causitive agent of tuberculosis (TB), possesses a complex cell wall containing mannose-rich glycophospholids termed phosphatidylinositol mannosides (PIMs), lipomannan (LM), and lipoarabinomannan (LAM). These glycophospholipids play important roles in cell wall function and host–pathogen interactions. Synthetic PIM/LM/LAM substructures are useful biochemical tools to delineate and dissect the fine details of mannose glycophospholipid biosynthesis and their interactions with host cells. We report the efficient synthesis of a series of azidooctyl di- and trimannosides possessing the following glycan structures: α-Man-1,6-α-Man, α-Man-1,6-α-Man-1,6-α-Man, α-Man-1,2-α-Man-1,6-α-Man and 2,6-di-(α-Man)-α-Man. The synthesis includes the use of non-benzyl protecting groups compatible with the azido group and preparation of the branched trisaccharide structure 2,6-di-(α-Man)-α-Man through a double glycosylation of a 3,4-butanediacetal-protected mannoside. The azidooctyl groups of these synthetic mannans were elaborated to fluorescent glycoconjugates and squaric ester derivatives useful for further conjugation studies.
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- 2011
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8. Mannopyranoside Glycolipids Inhibit Mycobacterial and Biofilm Growth and Potentiate Isoniazid Inhibition Activities in M. smegmatis
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Krishnagopal Maiti, Narayanaswamy Jayaraman, Avisek Mahapa, Dipankar Chatterji, and Gopal Ch Samanta
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Lipopolysaccharides ,Mycobacterium smegmatis ,Mannose ,Microbial Sensitivity Tests ,010402 general chemistry ,01 natural sciences ,Biochemistry ,Microbiology ,chemistry.chemical_compound ,Glycolipid ,Molecular Biology ,Mannan ,Lipoarabinomannan ,Lipomannan ,Molecular Structure ,biology ,010405 organic chemistry ,Organic Chemistry ,Biofilm ,biology.organism_classification ,0104 chemical sciences ,carbohydrates (lipids) ,chemistry ,Biofilms ,Molecular Medicine ,lipids (amino acids, peptides, and proteins) ,Glycolipids ,Growth inhibition - Abstract
Lipomannan and lipoarabinomannan are integral components of the mycobacterial cell wall. Earlier studies demonstrated that synthetic arabinan and arabinomannan glycolipids acted as inhibitors of mycobacterial growth, in addition to exhibiting inhibitory activities of mycobacterial biofilm. Herein, it is demonstrated that synthetic mannan glycolipids are better inhibitors of mycobacterial growth, whereas lipoarabinomannan has a higher inhibition efficiency to biofilm. Syntheses of mannan glycolipids with a graded number of mannan moieties and an arabinomannan glycolipid are conducted by chemical methods and subsequent mycobacterial growth and biofilm inhibition studies are conducted on Mycobacterium smegmatis. Growth inhibition of (73±3) % is observed with a mannose trisaccharide containing a glycolipid, whereas this glycolipid did not promote biofilm inhibition activity better than that of arabinomannan glycolipid. The antibiotic supplementation activities of glycolipids on growth and biofilm inhibitions are evaluated. Increases in growth and biofilm inhibitions are observed if the antibiotic is supplemented with glycolipids, which leads to a significant reduction of inhibition concentrations of the antibiotic.
- Published
- 2019
9. Synthetic Lipomannan Glycan Microarray Reveals the Importance of α(1,2) Mannose Branching in DC-SIGN Binding
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Harin Leelayuwapan, Somsak Ruchirawat, Siwarutt Boonyarattanakalin, Nitsara Karoonuthaisiri, and Nithinan Sawettanai
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Lipopolysaccharides ,Glycan ,Mannose ,Receptors, Cell Surface ,Plasma protein binding ,010402 general chemistry ,01 natural sciences ,chemistry.chemical_compound ,Polysaccharides ,Lectins, C-Type ,Mannan ,Lipomannan ,biology ,010405 organic chemistry ,Chemistry ,Organic Chemistry ,Microarray Analysis ,0104 chemical sciences ,carbohydrates (lipids) ,DC-SIGN ,Mannose-Binding Lectins ,Biochemistry ,biology.protein ,Cell Adhesion Molecules ,Linker ,Mannose Receptor ,Mannose receptor ,Protein Binding - Abstract
Lipomannan (LM), a glycophospholipid found on the cell surface of mycobacteria, involves the virulence and survival in host cells. However, there is little to no information on how exactly mannan alignment, including the number of mannose units and the branched motif of LM, affects protein engagement during host-pathogen interactions. In this study, we synthesized the exact substructures of the LM glycans that consist of an α(1,6) mannan core, with and without the complete α(1,2) mannose branching, and comparatively studied their protein-carbohydrate interactions. The synthetic LM glycans were equipped with a thiol linker for immobilizations on the surfaces of microarrays. As per our findings, the presence of the branching α(1,2) mannose on the LM glycans increases their binding toward the dendritic cell-specific intercellular adhesion molecule-3 grabbing non-integrin receptor. An increase in the number of mannose units on the glycans also increases the binding with the mannose receptor. Thus, the set of synthetic glycans can serve as a useful tool to study the biological activities of LM and can provide a better understanding of host-pathogen interactions.
- Published
- 2019
10. Microenvironment ofMycobacterium smegmatisCulture to Induce Cholesterol Consumption Does Cell Wall Remodeling and Enables the Formation of Granuloma-Like Structures
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Ana Cristina Doria dos Santos, Mara S.P. Arruda, Victor Hugo de Souza Marinho, Pedro Henrique de Aviz Silva, Edilene O. Silva, Barbarella de Matos Macchi, Chubert Bernardo Castro de Sena, and José Luiz Martins do Nascimento
- Subjects
0301 basic medicine ,chemistry.chemical_classification ,Lipoarabinomannan ,Lipomannan ,Article Subject ,General Immunology and Microbiology ,biology ,Chemistry ,Mycobacterium smegmatis ,lcsh:R ,030106 microbiology ,Middlebrook 7H9 broth ,lcsh:Medicine ,General Medicine ,biology.organism_classification ,General Biochemistry, Genetics and Molecular Biology ,Microbiology ,Cell wall ,03 medical and health sciences ,chemistry.chemical_compound ,030104 developmental biology ,Enzyme ,Biosynthesis ,Phosphatidylinositol - Abstract
Pathogenic species of mycobacteria are known to use the host cholesterol during lung infection as an alternative source of carbon and energy. Mycobacteria culture in minimal medium (MM) has been used as anin vitroexperimental model to study the consumption of exogenous cholesterol. Once in MM, different species of mycobacteria start to consume the cholesterol and initiate transcriptional and metabolic adaptations, upregulating the enzymes of the methylcitrate cycle (MCC) and accumulating a variety of primary metabolites that are known to be important substrates for cell wall biosynthesis. We hypothesized that stressful pressure of cultures in MM is able to induce critical adaptation for the bacteria which win the infection. To identify important modifications in the biosynthesis of the cell wall, we cultured the fast-growing and nonpathogenicMycobacterium smegmatisin MM supplemented with or without glycerol and/or cholesterol. Different from the culture in complete medium Middlebrook 7H9 broth, the bacteria when cultured in MM decreased growth and changed in the accumulation of cell wall molecules. However, the supplementation of MM with glycerol and/or cholesterol recovered the accumulation of phosphatidylinositol mannosides (PIMs) and other phospholipids but maintained growth deceleration. The biosynthesis of lipomannan (LM) and of lipoarabinomannan (LAM) was significantly modulated after culture in MM, independently of glycerol and/or cholesterol supplementation, where LM size was decreased (LM13-25KDa) and LAM increased (LAM37-100KDa), when compared these molecules after bacteria culture in complete medium (LM17-25KDaandLAM37-50KDa). These changes modified the cell surface hydrophobicity and susceptibility against H2O2. The infection of J774 macrophages withM. smegmatis,after culture in MM, induced the formation of granuloma-like structures, while supplementation with cholesterol induced the highest rate of formation of these structures. Taken together, our results identify critical changes in mycobacterial cell wall molecules after culture in MM that induces cholesterol accumulation, helping the mycobacteria to increase their capacity to form granuloma-like structures.
- Published
- 2019
11. Functional analysis and enzyme characterization of mannose-1-phosphate guanylyl transferase (ManB) from Mycobacterium tuberculosis
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Yufang Ma, Chao Wang, Shanshan Sha, Hayan Ullah, Ayaz Taj, Xiaochi Ma, Liqiu Jia, and Muhammad Haris
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Lipopolysaccharides ,Glycoconjugate ,Mannose ,Microbiology ,Phosphates ,Mycobacterium tuberculosis ,Cell membrane ,chemistry.chemical_compound ,Bacterial Proteins ,Cell Wall ,Transferases ,medicine ,Transferase ,Molecular Biology ,chemistry.chemical_classification ,Lipomannan ,Lipoarabinomannan ,biology ,General Medicine ,biology.organism_classification ,Nucleotidyltransferases ,Enzyme ,medicine.anatomical_structure ,Biochemistry ,chemistry - Abstract
Mycobacterium tuberculosis cell wall consist variety of mannose containing glycoconjugates including lipomannan (LM) and lipoarabinomannan (LAM). These lipoglycans are involved in cell wall integrity and play role in virulence of M. tuberculosis by modulating host immune response. GDP-mannose, required for the synthesis of lipoglycans, is catalyzed by enzyme Mannose-1-phosphate guanylyl transferase (ManB). The enzyme with similar function has been studied in variety of species of prokaryotes and eukaryotes. However, biological role of ManB and its enzymatic activity remains uncharacterized in M. tuberculosis. In present study, we elucidated the role of enzyme by constructing manB knockdown strain of M. tuberculosis H37Ra. The manB knockdown decreased the cell growth and also effected the morphology of M. tuberculosis by altering the permeability of cell membrane. These findings provide the understanding on ManB function and suggesting that ManB could be the potential target for novel anti-tuberculosis drug. Furthermore, we also characterized ManB enzyme by establishing 96 well plate colorimetric assay and determined the kinetic properties including initial velocity, optimum temperature, optimum pH and other kinetic parameters. Our established assay will be helpful for further high throughput screening of potential inhibitors against ManB.
- Published
- 2021
12. Controlled ring-opening polymerization of 1,2,6-tricyclic orthoesters of mannose toward size-controlled α- d-mannopyranan.
- Author
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Yongyat, Chanokpon, Ruchirawat, Somsak, and Boonyarattanakalin, Siwarutt
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RING-opening polymerization ,MYCOBACTERIUM tuberculosis ,IMMUNE response ,POLYMERIZATION ,POLYMERS - Abstract
Lipomannan (LM), one of the major components of glycans found on the cell wall of Mycobacterium tuberculosis, plays a critical role in interacting with host cells and moderating immune response. The backbone of LM is composed of α(1-6) mannopyranan, consisting of approximately 10-15 repeating mannose residues. Rapid synthesis of α(1-6) mannopyranan has been accomplished by ring-opening polymerization using 3,4- O-benzyl-β- d-mannopyranose 1,2,6-orthobenzoate ( 1) as a starting monomer. Here, we report the progress on attempts to control the size of α(1-6) mannopyranan polymer products. It is possible to prepare α- d-mannopyranan polymer products in different sizes by varying the monomer concentration. [ABSTRACT FROM AUTHOR]
- Published
- 2014
- Full Text
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13. Role of LmeA, a Mycobacterial Periplasmic Protein, in Maintaining the Mannosyltransferase MptA and Its Product Lipomannan under Stress
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Kathryn C. Rahlwes, Yasu S. Morita, and Sarah H Osman
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Lipopolysaccharides ,Mannosyltransferase ,Molecular Biology and Physiology ,glycolipids ,Mutant ,Mycobacterium smegmatis ,Microbiology ,Mannosyltransferases ,Mycobacterium ,03 medical and health sciences ,Bacterial Proteins ,Stress, Physiological ,Inner membrane ,Homeostasis ,Molecular Biology ,030304 developmental biology ,0303 health sciences ,Lipoarabinomannan ,Lipomannan ,030306 microbiology ,Chemistry ,mannose ,Cell Membrane ,Wild type ,Periplasmic space ,stress response ,QR1-502 ,Cell biology ,Biosynthetic Pathways ,Up-Regulation ,Periplasm ,Cell envelope ,biosynthesis ,Research Article - Abstract
Mycobacteria differentially regulate the cellular amounts of lipoglycans in response to environmental changes, but the molecular mechanisms of this regulation remain unknown. Here, we demonstrate that cellular lipoarabinomannan (LAM) levels rapidly decline under two stress conditions, stationary growth phase and nutrient starvation, while the levels of another related lipoglycan, lipomannan (LM), stay relatively constant. The persistence of LM under stress correlated with the maintenance of two key mannosyltransferases, MptA and MptC, in the LM biosynthetic pathway. We further showed that the stress exposures lead to the upregulation of lmeA gene expression and that the periplasmic protein LmeA plays a key role in maintaining the enzyme MptA and its product LM under stress conditions. These findings reveal new aspects of how lipoglycan biosynthesis is regulated under stress conditions in mycobacteria., The mycobacterial cell envelope has a diderm structure, composed of an outer mycomembrane, an arabinogalactan-peptidoglycan cell wall, a periplasm, and an inner membrane. Lipomannan (LM) and lipoarabinomannan (LAM) are structural and immunomodulatory components of this cell envelope. LM/LAM biosynthesis involves a number of mannosyltransferases and acyltransferases, and MptA is an α1,6-mannosyltransferase involved in the final extension of the mannan chain. Recently, we reported the periplasmic protein LmeA being involved in the maturation of the mannan backbone in Mycobacterium smegmatis. Here, we examined the role of LmeA under stress conditions. We found that lmeA transcription was upregulated under two stress conditions: stationary growth phase and nutrient starvation. Under both conditions, LAM was decreased, but LM was relatively stable, suggesting that maintaining the cellular level of LM under stress is important. Surprisingly, the protein levels of MptA were decreased in an lmeA deletion (ΔlmeA) mutant under both stress conditions. The transcript levels of mptA in the ΔlmeA mutant were similar to or even higher than those in the wild type, indicating that the decrease of MptA protein was a posttranscriptional event. The ΔlmeA mutant was unable to maintain the cellular level of LM under stress, consistent with the decrease in MptA. Even during active growth, overexpression of LmeA led the cells to produce more LM and become more resistant to several antibiotics. Altogether, our study reveals the roles of LmeA in the homeostasis of the MptA mannosyltransferase, particularly under stress conditions, ensuring the stable expression of LM and the maintenance of cell envelope integrity. IMPORTANCE Mycobacteria differentially regulate the cellular amounts of lipoglycans in response to environmental changes, but the molecular mechanisms of this regulation remain unknown. Here, we demonstrate that cellular lipoarabinomannan (LAM) levels rapidly decline under two stress conditions, stationary growth phase and nutrient starvation, while the levels of another related lipoglycan, lipomannan (LM), stay relatively constant. The persistence of LM under stress correlated with the maintenance of two key mannosyltransferases, MptA and MptC, in the LM biosynthetic pathway. We further showed that the stress exposures lead to the upregulation of lmeA gene expression and that the periplasmic protein LmeA plays a key role in maintaining the enzyme MptA and its product LM under stress conditions. These findings reveal new aspects of how lipoglycan biosynthesis is regulated under stress conditions in mycobacteria.
- Published
- 2020
14. The role of LmeA, a mycobacterial periplasmic protein, in stabilizing the mannosyltransferase MptA and its product lipomannan under stress
- Author
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Kathryn C. Rahlwes, Yasu S. Morita, and Sarah H Osman
- Subjects
Mannosyltransferase ,Lipomannan ,Lipoarabinomannan ,biology ,Chemistry ,Mycobacterium smegmatis ,Inner membrane ,Periplasmic space ,Cell envelope ,biology.organism_classification ,Mannan ,Cell biology - Abstract
The mycobacterial cell envelope has a diderm structure, composed of an outer mycomembrane, an arabinogalactan-peptidoglycan cell wall, periplasm and an inner membrane. Lipomannan (LM) and lipoarabinomannan (LAM) are structural and immunomodulatory components of this cell envelope. LM/LAM biosynthesis involves a number of mannosyltransferases and acyltransferases, and MptA is an α1,6-mannosyltransferase involved in the final extension of the mannan backbones. Recently, we reported the periplasmic protein LmeA being involved in the maturation of the mannan backbone in Mycobacterium smegmatis. Here, we examined the role of LmeA under stress conditions. We found that the lmeA transcription was upregulated under two stress conditions: stationary growth phase and nutrient starvation. Under both conditions, LAM was decreased, but LM was relatively stable, suggesting that maintaining the cellular level of LM under stress is important. Surprisingly, the protein levels of MptA were decreased in lmeA deletion mutant (ΔlmeA) in both stress conditions. The transcript levels of mptA in ΔlmeA were similar to or even higher than those in the wildtype, indicating that the decrease of MptA protein was a post-transcriptional event. Consistent with the decrease in MptA, ΔlmeA was unable to maintain the cellular level of LM under stress. Even during active growth, overexpression of LmeA led the cells to produce more LM and become more resistant to several antibiotics. Altogether, our study reveals the roles of LmeA in the homeostasis of the MptA mannosyltransferase particularly under stress conditions, ensuring the stable expression of LM and the maintenance of cell envelope integrity.
- Published
- 2020
15. Improving the biotransformation of phytosterols to 9α-hydroxy-4-androstene-3,17-dione by deleting embC associated with the assembly of cell envelope in Mycobacterium neoaurum
- Author
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Feng-Qing Wang, Liang-Bin Xiong, Hao-Hao Liu, Yu-Qing Ji, Xin-Wei Song, Xian-Zhou Liu, Xiang-Guo Meng, and Dongzhi Wei
- Subjects
0106 biological sciences ,0301 basic medicine ,Lipopolysaccharides ,Bioengineering ,01 natural sciences ,Applied Microbiology and Biotechnology ,Permeability ,03 medical and health sciences ,Biotransformation ,Bacterial Proteins ,Mycobacterium neoaurum ,Cell Wall ,010608 biotechnology ,Mycobacteriaceae ,chemistry.chemical_classification ,Lipoarabinomannan ,Lipomannan ,biology ,Cell Membrane ,Androstenedione ,Phytosterols ,Biological Transport ,General Medicine ,biology.organism_classification ,Sterol ,Sterols ,030104 developmental biology ,Enzyme ,chemistry ,Biochemistry ,Metabolic Engineering ,Genes, Bacterial ,Cell envelope ,Gene Deletion ,Biotechnology ,Transformation efficiency - Abstract
The conversion of low value-added phytosterols into 9α-hydroxy-4-androstene-3,17-dione (9-OHAD) by mycobacteria is an important step in the steroid pharmaceutical industry. However, the highly dense cell envelope with extremely low permeability largely affects the overall transformation efficiency. Here, we preliminarily located the key gene embC required for the synthesis of lipoarabinomannan from lipomannan in Mycobacterium neoaurum. The genetic manipulation of embC indicated that it might be the only functional enzyme catalyzing the above synthesis process. The deficiency of lipoarabinomannan led to a significantly increased cell permeability, which in turn caused the enhanced uptake capacity of cells. The sterol substrate conversion efficiency of mycobacterial cells was increased by about 52.4 % after 72-h conversion. Ultimately, the absence of embC increased the productivity from 0.0927 g/L/h to 0.1031 g/L/h, as confirmed by a resting cell system. This study verified the feasibility of improving the efficiency of the microbial conversion system through the cell envelope engineering strategy.
- Published
- 2020
16. Architecture and Biogenesis of the Cell Envelope of Corynebacterium glutamicum
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Nicolas Bayan, Christine Houssin, Cécile Labarre, Florence Constantinesco, Célia de Sousa d’Auria, and Christiane Dietrich
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chemistry.chemical_compound ,Lipoarabinomannan ,Lipomannan ,Chemistry ,Arabinogalactan ,Peptidoglycan ,Computational biology ,Cell envelope ,Biogenesis ,Function (biology) ,Corynebacterium glutamicum - Abstract
Corynebacteriales cells are surrounded by a multilayered cell envelope with unique architecture and composition. Corynebacterium glutamicum has become an increasingly used model to understand both biosynthesis and assembly of this complex compartment of the cell. The specific core of this envelope consists of a huge mycoloyl-arabinogalactan-peptidoglycan (mAGP) complex that constitutes the building support for an outer hydrophobic barrier mainly composed of mycolates containing glycolipids. Besides this basic specific core, the envelope contains other polymers such as lipomannan (LM) and lipoarabinomannan (LAM) but their localization and their functional role is still elusive. Finally, the more external layers encompass some strains of C. glutamicum: the outer layer and the S-layer which may have important roles in specific environments. The biosynthetic pathways involved in the synthesis of peptidoglycan, arabinogalactan and mycolates have been quite well characterized and are exhaustively described. In the frame of this review, we focused exclusively on what has been clearly demonstrated in Corynebaterium glutamicum and, when relevant, we compare to what is known in Mycobacterium species. The precise assembly of the whole envelope as a function of time and space is still to be discovered and represent an exciting challenge for the next decade.
- Published
- 2020
17. Controlled rapid synthesis and in vivo immunomodulatory effects of LM α(1,6)mannan with an amine linker
- Author
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Somsak Ruchirawat, Siwarutt Boonyarattanakalin, Jiraporn Paha, Warunda Bunthitsakda, Runglawan Chawengkirttikul, Marisa Ponpuak, Niwat Kangwanrangsan, and Haris Leelayuwapan
- Subjects
Lipopolysaccharides ,0301 basic medicine ,Glycan ,Polymers and Plastics ,chemical and pharmacologic phenomena ,Proinflammatory cytokine ,Mice ,03 medical and health sciences ,0302 clinical medicine ,Adjuvants, Immunologic ,In vivo ,Tetanus Toxoid ,Materials Chemistry ,Animals ,Amines ,Mannan ,Vaccines, Conjugate ,Lipomannan ,biology ,Chemistry ,Organic Chemistry ,Toxoid ,Mice, Inbred C57BL ,carbohydrates (lipids) ,Cross-Linking Reagents ,030104 developmental biology ,Biochemistry ,biology.protein ,Amine gas treating ,Linker ,030215 immunology - Abstract
The synthetic lipomannan (LM) α(1,6)mannans, already equipped with an amine linker on the reducing end, are rapidly synthesized in a size-, regio-, and stereocontrolled reaction. The size of the mannans is regulated through the concentration of the linker, applied during the controlled ring-opening polymerization reaction. The versatile amine linker enables a variety of glycan conjugations. The synthetic α(1,6)mannans exert adjuvant activities for a real vaccine antigen, tetanus toxoid (TT) in vitro, as demonstrated by the increased secretion of proinflammatory cytokines TNF-α and IL-6 from the treated macrophages. A conjugation of synthetic α(1,6)mannan with TT can also enhance immune response to TT in vivo after immunization as shown by an increase in TNF-α, IFN-γ, and IL-2 production in splenocytes.
- Published
- 2018
18. Cryo-EM Structures and Regulation of Arabinofuranosyltransferase AftD from Mycobacteria
- Author
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Ana Lúcia Rosário, Clinton S. Potter, Maria João Catalão, Margarida Archer, Yong Zi Tan, Sabrina I. Giacometti, Richard Brunton, Lei Zhang, Bridget Carragher, Madalena Pimentel, Hui Wei, Todd L. Lowary, Michael Niederweis, José Rodrigues, Ruixiang Blake Zheng, Brian Kloss, Ashleigh M. Raczkowski, Diogo Athayde, Venkata P. Dandey, Filippo Mancia, and Oliver B. Clarke
- Subjects
Lipopolysaccharides ,Conformational change ,Glycan ,Protein Conformation ,Mutant ,Mycobacterium smegmatis ,Galactans ,Article ,Substrate Specificity ,Mycobacterium tuberculosis ,03 medical and health sciences ,0302 clinical medicine ,Glycolipid ,Bacterial Proteins ,Arabinogalactan ,Cell Wall ,Catalytic Domain ,Glycosyltransferase ,Acyl Carrier Protein ,Molecular Biology ,Phylogeny ,030304 developmental biology ,0303 health sciences ,Lipomannan ,Lipoarabinomannan ,biology ,030306 microbiology ,Chemistry ,Cell Membrane ,Cryoelectron Microscopy ,Glycosyltransferases ,Cell Biology ,biology.organism_classification ,3. Good health ,Acyl carrier protein ,Biochemistry ,Mutation ,biology.protein ,lipids (amino acids, peptides, and proteins) ,030217 neurology & neurosurgery - Abstract
SUMMARYMycobacterium tuberculosiscauses tuberculosis, a disease that kills over one million people each year. Its cell envelope is a common antibiotic target and has a unique structure due, in part, to two lipidated polysaccharides – arabinogalactan and lipoarabinomannan. Arabinofuranosyltransferase D (AftD) is an essential enzyme involved in assembling these glycolipids. We present the 2.9 Å resolution structure ofM. abscessusAftD determined by single particle cryo-electron microscopy. AftD has a conserved GT-C glycosyltransferase fold and three carbohydrate binding modules. Glycan array analysis shows that AftD binds complex arabinose glycans. Additionally, AftD is non-covalently complexed with an acyl carrier protein (ACP). 3.4 and 3.5 Å structures of a mutant with impaired ACP binding reveal a conformational change that suggests the ACP may regulate AftD function. Using a conditional knock-out constructed inM. smegmatis, mutagenesis experiments confirm the essentiality of the putative active site and the ACP binding for AftD function.
- Published
- 2019
19. Cloning and Partial Characterization of an Endo-α-(1→6)-d-Mannanase Gene from
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Shiva Kumar, Angala, Wei, Li, Zuzana, Palčeková, Lu, Zou, Todd L, Lowary, Michael R, McNeil, and Mary, Jackson
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Lipopolysaccharides ,Mycobacterium smegmatis ,Bacillus ,lipomannan ,Mannosyltransferases ,Article ,Substrate Specificity ,Mycobacterium ,Protein Domains ,Genes, Bacterial ,endo-α-(1→6)-d-mannase ,mannoside ,Mannosides ,phosphatidylinositol mannosides ,lipoarabinomannan ,Cloning, Molecular - Abstract
Mycobacteria produce two major lipoglycans, lipomannan (LM) and lipoarabinomannan (LAM), whose broad array of biological activities are tightly related to the fine details of their structure. However, the heterogeneity of these molecules in terms of internal and terminal covalent modifications and complex internal branching patterns represent significant obstacles to their structural characterization. Previously, an endo-α-(1→6)-D-mannanase from Bacillus circulans proved useful in cleaving the mannan backbone of LM and LAM, allowing the reducing end of these molecules to be identified as Manp-(1→6) [Manp-(1→2)]-Ino. Although first reported 45 years ago, no easily accessible form of this enzyme was available to the research community, a fact that may in part be explained by a lack of knowledge of its complete gene sequence. Here, we report on the successful cloning of the complete endo-α-(1→6)-D-mannanase gene from Bacillus circulans TN-31, herein referred to as emn. We further report on the successful production and purification of the glycosyl hydrolase domain of this enzyme and its use to gain further insight into its substrate specificity using synthetic mannoside acceptors as well as LM and phosphatidyl-myo-inositol mannoside precursors purified from mycobacteria.
- Published
- 2019
20. Inositol lipid metabolism in mycobacteria: Biosynthesis and regulatory mechanisms
- Author
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Morita, Yasu S., Fukuda, Takeshi, Sena, Chubert B.C., Yamaryo-Botte, Yoshiki, McConville, Malcolm J., and Kinoshita, Taroh
- Subjects
- *
INOSITOL , *LIPID metabolism , *LIPID synthesis , *METABOLIC regulation , *MYCOBACTERIA , *BIOSYNTHESIS , *PHOSPHOINOSITIDES , *BIOCHEMISTRY - Abstract
Abstract: Background: The genus Mycobacterium includes a number of medically important pathogens. The cell walls of these bacteria have many unique features, including the abundance of various inositol lipids, such as phosphatidylinositol mannosides (PIMs), lipomannan (LM), and lipoarabinomannan (LAM). The biosynthesis of these lipids is believed to be prime drug targets, and has been clarified in detail over the past several years. Scope of review: Here we summarize our current understanding of the inositol lipid metabolism in mycobacteria. We will highlight unsolved issues and future directions especially in the context of metabolic regulation. Major conclusions: Inositol is a building block of phosphatidylinositol (PI), which is further elaborated to become PIMs, LM and LAM. d-myo-inositol 3-phosphate is an intermediate of the de novo inositol synthesis, but it is also the starting substrate for mycothiol synthesis. Controlling the level of d-myo-inositol 3-phosphate appears to be important for maintaining the steady state levels of mycothiol and inositol lipids. Several additional control mechanisms must exist to control the complex biosynthetic pathways of PI, PIMs, LM and LAM. These may include regulatory proteins such as a lipoprotein LpqW, and spatial separation of enzymes, such as the amphipathic PimA mannosyltransferase and later enzymes in the PIMs/LM biosynthetic pathway. Finally, we discuss mechanisms that underlie control of LM/LAM glycan polymer elongation. General significance: Mycobacteria have evolved a complex network of inositol metabolism. Clarifying its metabolism will not only provide better understanding of bacterial pathogenesis, but also understanding of the evolution and general functions of inositol lipids in nature. [Copyright &y& Elsevier]
- Published
- 2011
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21. Polymerization of mannosyl tricyclic orthoesters for the synthesis of α(1–6) mannopyranan—the backbone of lipomannan
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Yongyat, Chanokpon, Ruchirawat, Somsak, and Boonyarattanakalin, Siwarutt
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- *
POLYMERIZATION , *ESTERS , *MYCOBACTERIUM tuberculosis , *IMMUNE response , *CARBOHYDRATES , *MONOMERS , *GLYCOSIDES , *STEREOCHEMISTRY - Abstract
Abstract: Tuberculosis (TB) remains a major health problem worldwide. Understanding the interactions between the surface components of Mycobacterium tuberculosis (Mtb), the main causative agent of TB, with host immune response will be critical for developments of effective treatments and prevention of TB. Chemically defined mimics of the bacterial envelope components serve as important tools for biological studies of the bacterial interactions with mammalian hosts. We report here a rapid synthetic approach utilizing mannosyl tricyclic orthoesters as monomers for regio- and stereo-controlled polymerizations to generate α(1–6) mannopyranan—the backbone of lipomannan. The polymerizations generated multiple glycosidic bonds in a single chemical transformation in regio- and stereo-selective manners. TMSOTf is the optimum catalyst to promote the selective and high yielding polymerization when compared with other Lewis acids. In addition, the monomers 3,4-O-benzyl-β-d-mannopyranose 1,2,6-orthobenzoate (1) and 3,4-O-benzyl-β-d-mannopyranose 1,2,6-orthopivalate (2) can be synthesized in multiple-gram scale and in a rapid fashion. Characterizations by GPC and NMR indicate the identity of α(1–6) mannopyranan with DPn (degree of polymerization)=20. [Copyright &y& Elsevier]
- Published
- 2010
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22. Detection of Lipomannan in Cattle Infected with Bovine Tuberculosis
- Author
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Vu, Dung M., Sakamuri, Rama M., Waters, W. Ray, Swanson, Basil I., and Mukundan, Harshini
- Published
- 2017
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23. Structural characterization and functional properties of a novel lipomannan variant isolated from a Corynebacterium glutamicum pimB' mutant.
- Author
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Mishra, Arun K., Klein, Christina, Gurcha, Sudagar S., Alderwick, Luke J., Babu, Ponnusamy, Hitchen, Paul G., Morris, Howard R., Dell, Anne, Besra, Gurdyal S., and Eggeling, Lothar
- Abstract
The genus Corynebacterium is part of the phylogenetic group nocardioform actinomycetes, which also includes the genus Mycobacterium. Members of this phylogenetic group have a characteristic cell envelope structure, which is dominated by complex lipids and amongst these, lipoglycans are of particular interest. The disruption of NCgl2106 in C. glutamicum resulted in a mutant devoid of monoacylated phosphatidyl-myo-inositol dimannoside (Ac
1 PIM2 ) resulting in the accumulation of Ac1 PIM1 and cessation of phosphatidyl-myo-inositol (PI) based lipomannan (Cg-LM, now also termed ‘Cg-LM-A’) and lipoarabinomannan (Cg-LAM) biosynthesis. Interestingly, SDS-analysis of the lipoglycan fraction from the mutant revealed the synthesis of a single novel lipoglycan, now termed ‘Cg-LM-B’. Further chemical analyses established the lipoglycan possessed an a-D-glucopyranosyluronic acid-(1 → 3)-glycerol (GlcAGroAc2 ) based anchor which was then further glycosylated by 8–22 mannose residues, with Man12–20 GlcAGroAC2molecular species being the most abundant, to form a novel lipomannan structure (Cg-LM-B). The deletion of NCgl2106 in C. glutamicum has now provided a useful strain, in addition with a deletion mutant of NCgl0452 in C. glutamicum for the purification of Cg-LM-A and Cg-LM-B. Interestingly, both Cg-LM species induced a similar production of TNF-a by a human macrophage cell line suggesting that the phospho-myo-inositol residue of the PI-anchor does not play a key role in lipoglycan pro-inflammatory activity. [ABSTRACT FROM AUTHOR]- Published
- 2008
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24. Mycobacterial lipomannan induces MAP kinase phosphatase-1 expression in macrophages
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Elass, Elisabeth, Coddeville, Bernadette, Kremer, Laurent, Mortuaire, Marlène, Mazurier, Joël, and Guérardel, Yann
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- *
MYCOBACTERIUM , *KILLER cells , *PROTEIN kinases , *LYMPHOID tissue - Abstract
Abstract: Mycobacterial lipomannan (LM) and lipoarabinomannan (LAM) regulate macrophage activation by interacting with Toll-like receptors (TLRs). The intracellular signalling pathways elicited by these complex molecules are poorly defined. We have demonstrated that LM purified from various mycobacterial species, but not LAM from Mycobacterium kansasii or Mycobacterium bovis BCG, induced expression of the MAP kinase phosphatase 1 (MKP-1) in macrophages. Anti-TLR2 antibodies, as well as specific ERK and p38 MAPK inhibitors, decreased MKP-1 transcription in LM-stimulated cells. These findings suggest that the binding of LM to TLR2 triggers MAPK activation, followed by an up-regulation of MKP-1 expression, which in turn may act as a negative regulator of MAPK activation. [Copyright &y& Elsevier]
- Published
- 2008
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25. Identification by surface plasmon resonance of the mycobacterial lipomannan and lipoarabinomannan domains involved in binding to CD14 and LPS-binding protein
- Author
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Elass, Elisabeth, Coddeville, Bernadette, Guérardel, Yann, Kremer, Laurent, Maes, Emmanuel, Mazurier, Joël, and Legrand, Dominique
- Subjects
- *
DEFENSE reaction (Physiology) , *ANIMAL defenses , *PATTERN perception , *RESONANCE - Abstract
Abstract: The mycobacterial lipoglycans, lipomannan (LM) and lipoarabinomannan (LAM), regulate host defence mechanisms through their interaction with pattern recognition receptors such as Toll-like receptors (TLRs). We have developed a surface plasmon resonance assay to analyse the molecular basis for the recognition of Mycobacterium kansasii LM or LAM, by immobilized CD14 and LPS-binding protein (LBP) both being capable to promote presentation of bacterial glycolipids to TLRs. The affinity of either LM/LAM was higher to CD14 than to LBP. Kinetic and Scatchard analyses were consistent with a model involving a single class of binding sites. These interactions required the lipidic anchor, but not the carbohydrate domains, of LM or LAM. We also provide evidence that addition of recombinant LBP enhanced the stimulatory effect of LM or LAM on matrix metalloproteinase-9 expression and secretion in macrophages, through a TLR1/TLR2-dependent mechanism. [Copyright &y& Elsevier]
- Published
- 2007
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26. Anti-tumor immunity via the superoxide-eosinophil axis induced by a lipophilic component of Mycobacterium lipomannan
- Author
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Atsushi Onodera, Toshinori Nakayama, Toshihiro Ito, Ryo Koyama-Nasu, Kiyoshi Hirahara, and Ikuya Yano
- Subjects
Cytotoxicity, Immunologic ,Lipopolysaccharides ,0301 basic medicine ,Carcinogenesis ,Immunology ,Immunotherapy, Adoptive ,complex mixtures ,CCL5 ,Cell therapy ,Mice ,03 medical and health sciences ,chemistry.chemical_compound ,Th2 Cells ,0302 clinical medicine ,Immune system ,Cell Movement ,Cell Wall ,Superoxides ,Cell Line, Tumor ,Tumor Microenvironment ,Animals ,Immunology and Allergy ,Chemokine CCL5 ,Mice, Inbred BALB C ,Tumor microenvironment ,Lipomannan ,Lipoarabinomannan ,Chemistry ,Superoxide ,Dendritic Cells ,Neoplasms, Experimental ,General Medicine ,Mycobacterium bovis ,Tumor Burden ,Cell biology ,Eosinophils ,030104 developmental biology ,Tumor progression ,Female ,Transcriptome ,Immunologic Memory ,030215 immunology - Abstract
Mycobacterium bovis Bacille Calmette–Guérin (BCG) has been shown to possess potent anti-tumor activity particularly in various animal models, while the cellular and molecular mechanisms underlying its activity are not well understood. We found that lipomannan (BCG-LM), a lipophilic component of the mycobacterial cell envelope, specifically inhibits tumor growth and induces the infiltration of eosinophils at local tumor invasion sites. In contrast, neither lipoarabinomannan (BCG-LAM) nor the cell wall of Mycobacterium bovis BCG (BCG-CW) exerted anti-tumor immunity. BCG-LM enhances cytotoxic activity of eosinophils via the increased production of superoxide. Global transcriptomic analyses of BCG-LM-pulsed dendritic cells identified C-C motif ligand (CCL) 5 as a crucial chemokine for the anti-tumor immunity induced by BCG-LM, indicating that CCL5 plays an important role for the accumulation of eosinophils in the tumor microenvironment. Furthermore, BCG-LM and memory Th2 cells exerted a synergetic effect on tumor progression by cooperatively enhancing the eosinophil function. Thus, this study revealed an un-identified BCG-LM-mediated anti-tumor mechanism via superoxide produced by infiltrated eosinophils in the tumor microenvironment. Since BCG-LM activates this unique pathway, it may have potent therapeutic potential as immune cell therapy for cancer patients.
- Published
- 2017
27. Synthesis and Immunological Studies of the Lipomannan Backbone Glycans Found on the Surface of Mycobacterium tuberculosis
- Author
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Marisa Ponpuak, Runglawan Chawengkirttikul, Somsak Ruchirawat, Ratthaphol Charlermroj, Siwarutt Boonyarattanakalin, Kanokthip Boonyarattanakalin, Niwat Kangwanrangsan, and Harin Leelayuwapan
- Subjects
chemistry.chemical_classification ,Glycan ,Lipomannan ,biology ,010405 organic chemistry ,Stereochemistry ,Chemistry ,Organic Chemistry ,Glycosidic bond ,010402 general chemistry ,biology.organism_classification ,01 natural sciences ,0104 chemical sciences ,Mycobacterium tuberculosis ,Biochemistry ,Polymerization ,In vivo ,biology.protein ,Surface plasmon resonance ,Mannan - Abstract
Investigations into novel bacterial drug targets and vaccines are necessary to overcome tuberculosis. Lipomannan (LM), found on the surface of Mycobacterium tuberculosis (Mtb), is actively involved in the pathogenesis and survival of Mtb. Here, we report for the first time a rapid synthesis and biological activities of an LM glycan backbone, α(1-6)mannans. The rapid synthesis is achieved via a regio- and stereoselective ring opening polymerization to generate multiple glycosidic bonds in one simple chemical step, allowing us to finish assembling the defined polysaccharides of 5-20 units within days rather than years. Within the same pot, the polymerization is terminated by a thiol-linker to serve as a conjugation point to carrier proteins and surfaces for immunological experiments. The synthetic glycans are found to have adjuvant activities in vivo. The interactions with DC-SIGN demonstrated the significance of α(1-6)mannan motif present in LM structure. Moreover, surface plasmon resonance (SPR) showed that longer chain of synthetic α(1-6)mannans gain better lectin's binding affinity. The chemically defined components of the bacterial envelope serve as important tools to reveal the interactions of Mtb with mammalian hosts and facilitate the determination of the immunologically active molecular components.
- Published
- 2017
28. CORYNEBACTERIUM: FEATURES OF THE STRUCTURE OF THE BACTERIAL CELL
- Author
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N A Voronina and G G Kharseeva
- Subjects
0301 basic medicine ,nucleoid ,030106 microbiology ,surface proteins ,Corynebacterium ,Medicine (miscellaneous) ,peptidoglycan ,Microbiology ,Bacterial cell structure ,cord-factor ,Cell wall ,03 medical and health sciences ,chemistry.chemical_compound ,Arabinogalactan ,Nucleoid ,pili (fimbriae) ,Lipomannan ,biology ,General Medicine ,biology.organism_classification ,QR1-502 ,Cell biology ,chemistry ,Cytoplasm ,corynebacterium ,bacteria ,Peptidoglycan - Abstract
In a review of the features of the bacterial cells are Corynebacterium structure: characterized by an upper layer, highly organized cell wall, cytoplasmic membrane, cytoplasm, nucleoid. Described in detail the structure of the upper layer containing pili (fimbriae), microcapsule surface proteins - PS-2, DIP1281, 67-72r protein (hemagglutinin), porins, sialidase (neuraminidase). These components are the ability to initiate a serial of Corynebacterium work with the host cell, followed by colonization. It submitted a detailed description of the structure and functions of cell wall structures - cord factor, which is a second barrier permeability; arabinogalactan, peptidoglycan, lipomannan and lipoarabinomannan. The structure and function of the cytoplasmic membrane as the main diffusion barrier cell cytoplasm and the genome of Corynebacterium. Presented different molecular genetic methods for the identification and differentiation of closely related species of Corynebacterium.
- Published
- 2017
29. Recognition of Mycobacterial Lipids by Immune Receptors
- Author
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Daiki Mori, Shou Yamasaki, and Eri Ishikawa
- Subjects
0301 basic medicine ,Tuberculosis ,Immunology ,Population ,Biology ,Microbiology ,Mycobacterium tuberculosis ,03 medical and health sciences ,0302 clinical medicine ,Immune system ,Adjuvants, Immunologic ,medicine ,Animals ,Humans ,Immunologic Factors ,Immunology and Allergy ,Lectins, C-Type ,Receptor ,education ,education.field_of_study ,Innate immune system ,Lipoarabinomannan ,Lipomannan ,medicine.disease ,biology.organism_classification ,Immunity, Innate ,030104 developmental biology ,Host-Pathogen Interactions ,lipids (amino acids, peptides, and proteins) ,Glycolipids ,030215 immunology - Abstract
Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), infects one-third of the world's population and causes 1.5 million deaths each year. The cell envelopes of mycobacteria comprise a wealth of unique glycolipids, including trehalose-6,6'-dimycolate (TDM), lipoarabinomannan (LAM), lipomannan (LM), and phosphatidylinositol (PI) mannosides (PIMs). These lipids are important modulators of the host immune responses during infection and in some cases have been used as adjuvants [e.g., complete Freund's adjuvant (CFA)]. Despite this abundant basic knowledge, the identities of the host immune receptors for mycobacterial lipids have long been elusive. Here we review and summarize our current state of knowledge regarding innate immune receptors for mycobacteria, focusing particularly on immunoreceptor tyrosine-based activation motif (ITAM)-coupled C-type lectin receptors (CLRs), which have been shown to recognize mycobacteria-derived glycolipids.
- Published
- 2017
30. Disruption of the SucT acyltransferase in Mycobacterium smegmatis abrogates succinylation of cell envelope polysaccharides
- Author
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Zuzana Palčeková, Haig A. Eskandarian, Richard Brunton, Victoria Jones, Michael R. McNeil, Jérôme Nigou, Maju Joe, Mary Jackson, Shiva K. Angala, Todd L. Lowary, Juan Manuel Belardinelli, Christopher D. Rithner, Martine Gilleron, Mycobacteria Research Laboratories, Department of Microbiology, Immunology, and Pathology, Colorado State University [Fort Collins] (CSU), Institut de pharmacologie et de biologie structurale (IPBS), Centre National de la Recherche Scientifique (CNRS)-Université Toulouse III - Paul Sabatier (UT3), and Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées
- Subjects
0301 basic medicine ,Lipopolysaccharides ,mycobacteria ,[SDV]Life Sciences [q-bio] ,galactosamine substituent ,Mutant ,Mycobacterium smegmatis ,Glycobiology and Extracellular Matrices ,Biochemistry ,Galactans ,03 medical and health sciences ,Succinylation ,Bacterial Proteins ,Arabinogalactan ,Cell Wall ,[SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM] ,Molecular Biology ,Mycobacterium marinum ,acetylation ,Lipoarabinomannan ,030102 biochemistry & molecular biology ,biology ,Chemistry ,apoptosis ,Succinates ,Cell Biology ,lipomannan ,biology.organism_classification ,bacterial infections and mycoses ,arabinogalactan ,cell surface ,succinylation ,030104 developmental biology ,tuberculosis ,polysaccharide ,transport ,Mutation ,impact ,identification ,bacteria ,lipoarabinomannan ,lipids (amino acids, peptides, and proteins) ,biosynthesis ,Cell envelope ,Acyltransferases ,Mycobacterium - Abstract
Similar to other prokaryotes, mycobacteria decorate their major cell envelope glycans with minor covalent substituents whose biological significance remains largely unknown. We report on the discovery of a mycobacterial enzyme, named here SucT, that adds succinyl groups to the arabinan domains of both arabinogalactan (AG) and lipoarabinomannan (LAM). Disruption of the SucT-encoding gene in Mycobacterium smegmatis abolished AG and LAM succinylation and altered the hydrophobicity and rigidity of the cell envelope of the bacilli without significantly altering AG and LAM biosynthesis. The changes in the cell surface properties of the mutant were consistent with earlier reports of transposon mutants of the closely related species Mycobacterium marinum and Mycobacterium avium harboring insertions in the orthologous gene whose ability to microaggregate and form biofilms were altered. Our findings point to an important role of SucT-mediated AG and LAM succinylation in modulating the cell surface properties of mycobacteria.
- Published
- 2019
31. An essential periplasmic protein coordinates lipid trafficking and is required for asymmetric polar growth in mycobacteria
- Author
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Kuldeepkumar R. Gupta, Celena M. Gwin, Kathryn C. Rahlwes, Kyle J. Biegas, Chunyan Wang, Jin Ho Park, Jun Liu, Benjamin M. Swarts, Yasu S. Morita, and E. Hesper Rego
- Subjects
Lipoarabinomannan ,Lipomannan ,General Immunology and Microbiology ,biology ,Cell growth ,Chemistry ,General Neuroscience ,Cell Membrane ,Mycobacterium tuberculosis ,General Medicine ,biology.organism_classification ,General Biochemistry, Genetics and Molecular Biology ,Bacterial cell structure ,Cell biology ,Glycolipid ,Mycolic Acids ,Periplasm ,Humans ,Periplasmic Proteins ,Cell envelope ,Bacterial outer membrane ,Bacteria - Abstract
Mycobacteria grow and divide differently compared to well-studied model bacteria. They insert new cell envelope at their poles instead of along their side walls; and, they lack obvious homologs of many well-conserved cell growth and division proteins. Furthermore, while mycobacteria share several similarities to Gram-positive bacteria, unlike these organisms they possess an outer membrane, which is abundant in long-chain fatty acids and several glycolipids and lipoglycans. A better understanding of the unique factors that make this unusual structure could lead to therapeutic targets for pathogenic mycobacteria. Here, we study a gene of previously unknown function – msmeg_0317 - predicted to be essential and associated with mycobacterial cell growth and division proteins. We find that transcriptional depletion of msmeg_0317 leads to loss of polar growth, disruption of the mycobacterial outer membrane, and cell death. Surprisingly, we also observe that depletion results in the accumulation of cell-associated lipoglycans lipomannan and lipoarabinomannan (LM/LAM), while overexpression of CwdA leads to increased shedding of LM/LAM. LM/LAM have been extensively studied in relation to infection but their role in bacterial physiology is less clear. Altogether, our data suggest that MSMEG_0317, renamed CwdA, is involved in the transport of LM/LAM to the mycobacterial outer membrane, and reveal unexpected connections between the correct localization of LM/LAM, polar growth, and the structural integrity of the mycobacterial cell envelope. IMPORTANCE Some of the most successful antibiotics target bacterial cell envelope synthesis. However, the cell envelope of mycobacteria is significantly different from that of other, more well-studied, bacteria. Its core structure consists of a covalently-linked network of peptides, carbohydrates, and fatty acids. Additionally, there are several lipids non-covalently interspersed throughout. Many of the enzymes which synthesize these lipids are known, but how they are transported remains largely unclear. Here, we discover that an essential protein, CwdA, is involved in the transport of LM/LAM, abundant lipoglycans in the mycobacterial cell envelope. Depletion of CwdA leads to the loss of polar growth and the disintegration of the mycobacterial outer membrane. These results suggest that the proteins which transport molecules across the mycobacterial cell envelope may represent an abundant source of novel drug targets for the treatment of mycobacterial infections, like tuberculosis.
- Published
- 2019
32. Purification and Analysis of Mycobacterial Phosphatidylinositol Mannosides, Lipomannan, and Lipoarabinomannan
- Author
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Yasu S. Morita, Julia Puffal, and Kathryn C. Rahlwes
- Subjects
0301 basic medicine ,Mannosides ,Glycan ,Lipomannan ,Chromatography ,Lipoarabinomannan ,biology ,Chemistry ,030106 microbiology ,Phospholipid ,bacterial infections and mycoses ,Thin-layer chromatography ,03 medical and health sciences ,chemistry.chemical_compound ,030104 developmental biology ,Glycolipid ,hemic and lymphatic diseases ,biology.protein ,lipids (amino acids, peptides, and proteins) ,Phosphatidylinositol - Abstract
Mycobacteria and related bacteria in the Actinobacteria phylum are unusual in that they produce phosphatidylinositol (PI) as a major phospholipid species. PI can be further modified by glycan polymers, leading to the synthesis of PI mannosides (PIMs), lipomannan (LM), and lipoarabinomannan (LAM). Small lipids such as PI and PIMs are extracted with a mixture of chloroform, methanol, and water and analyzed by thin layer chromatography. For larger glycolipids, such as LM and LAM, more hydrophilic solvent is needed for the extraction, and SDS-PAGE is better suited for the analysis. For LM, further structural characterization can be performed by MALDI-TOF mass spectrometry. Precise quantification of PIMs, LM, and LAM can be performed by quantification of glycan staining using analytical software. The metabolic radiolabeling protocol is also described.
- Published
- 2019
33. Stomatococcus mucilaginosus produces a mannose-containing lipoglycan rather than lipoteichoic acid.
- Author
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Sutcliffe, Iain and Old, Lesley
- Abstract
Five strains of Stomatococcus mucilaginosus were investigated to determine whether the organism produces a lipoteichoic acid or a lipoglycan. Crude phenol extracts were purified by hydrophobic interaction chromatography and shown to contain lipoglycan. The major carbohydrate component present was mannose, indicating that the macroamphiphile is a lipomannan. The fatty acid composition of the lipoglycan was similar to that of stomatococcal whole cells. These data provide additional chemotaxonomic evidence supporting the suprageneric classification of the genus Stomatococcus within a group of actinomycete genera that also includes the genus Micrococcus. [ABSTRACT FROM AUTHOR]
- Published
- 1995
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34. Structural definition of acylated phosphatidylinositol mannosides from Mycobacterium tuberculosis: definition of a common anchor for lipomannan and lipoarabinomannan.
- Author
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Khoo, Kay-Hooi, Dell, Anne, Morris, Howard R., Breman, Patrick J., and Chatterjee, Delphi
- Abstract
Based on chemical analysis, we have previously concluded that the biologically important lipoarabinomannan (LAM) and lipomannan (LM) from are multiglycosylated forms of the phosphatidylinositol mannosides (PIMs), the characteristic cell envelope mannophosphoinositides of mycobacteria Using definitive analytical techniques, we have now re-examined the reported multiacylated nature of PIMs in order to gain a better insight into their possible roles as biosynthethic precursors of LM and LAM. High-sensitivity fast atom bombardment-mass spectrometry analyses of the perdeuteroacetyl and permethyl derivatives of PIMs from and enabled us to define the exact fatty acyl compositions of the multiacylated, heterogeneous PIM families, notably the dimannoside (PIM) and the hexamannoside (PIM). Specifically, in conjunction with other chemical and gas chromatography-mass spectrometry (GCMS) analyses, the additional C16 fatty acyl substituent on PIM and its lyso form were defined as attached to the C6 position of mannose. We also present evidence for triacylated mannophosphoinositide as a common lipid anchor for both LM and LAM, and further postulate that acylation of PIM may constitute a key regulatory step in their biosynthesis. [ABSTRACT FROM PUBLISHER]
- Published
- 1995
35. A paucity of knowledge regarding nontuberculous mycobacterial lipids compared to the tubercle bacillus
- Author
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Tru Tran, Jennifer R. Honda, Edward D. Chan, and Andrew J. Bonham
- Subjects
0301 basic medicine ,Microbiology (medical) ,Tuberculosis ,030106 microbiology ,Immunology ,Virulence ,Bacillus ,Microbiology ,Mycobacterium tuberculosis ,03 medical and health sciences ,Glycolipid ,Cell Wall ,medicine ,Immunity, Cellular ,Lipomannan ,Lipoarabinomannan ,biology ,Mycobacterium smegmatis ,Nontuberculous Mycobacteria ,bacterial infections and mycoses ,biology.organism_classification ,medicine.disease ,Lipids ,030104 developmental biology ,Infectious Diseases ,lipids (amino acids, peptides, and proteins) ,Nontuberculous mycobacteria - Abstract
All mycobacteria, including nontuberculous mycobacteria (NTM), synthesize an array of lipids including phosphatidylinositol mannosides (PIM), lipomannan (LM), and lipoarabinomannan (LAM). While absent from Mycobacterium tuberculosis (M. tb), glycopeptidolipids (GPL) are critical to the biology of NTM. M. tb and some NTM also synthesize trehalose-containing glycolipids and phenolic glycolipids (PGL), key membrane constituents with essential roles in metabolism. While lipids facilitate immune evasion, they also induce host immunity against tuberculosis. However, much less is known about the significance of NTM-derived PIM, LM, LAM, GPL, trehalose-containing glycolipids, and PGL as virulence factors, warranting further investigation. While culling the scientific literature on NTM lipids, it's evident that such studies were relatively few in number with the overwhelming majority of prior work dedicated to understanding lipids from the saprophyte Mycobacterium smegmatis. The identification and functional analysis of immune reactive NTM-derived lipids remain challenging, but such work is likely to yield a greater understanding of the pathogenesis of NTM lung disease. In this review, we juxtapose the vast literature of what is currently known regarding M. tb lipids to the lesser number of studies for comparable NTM lipids. But because GPL is the most widely recognized NTM lipid, we highlight its role in disease pathogenesis.
- Published
- 2018
36. Mycobacterium tuberculosis Lipoprotein and Lipoglycan Binding to Toll-Like Receptor 2 Correlates with Agonist Activity and Functional Outcomes
- Author
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Clifford V. Harding, Supriya Shukla, Edward T. Richardson, W. Henry Boom, and Michael G. Drage
- Subjects
0301 basic medicine ,Agonist ,Lipopolysaccharides ,medicine.drug_class ,Lipoproteins ,Immunology ,Biology ,Microbiology ,Mycobacterium tuberculosis ,03 medical and health sciences ,Mice ,0302 clinical medicine ,Bacterial Proteins ,medicine ,Animals ,Humans ,Tuberculosis ,Functional ability ,Receptor ,Toll-like receptor ,Host Response and Inflammation ,Lipomannan ,Lipoarabinomannan ,biology.organism_classification ,Molecular biology ,Toll-Like Receptor 1 ,Toll-Like Receptor 2 ,Mice, Inbred C57BL ,TLR2 ,030104 developmental biology ,Infectious Diseases ,Parasitology ,Female ,030215 immunology ,Protein Binding ,Signal Transduction - Abstract
Mycobacterium tuberculosis causes persistent infection due to its ability to evade host immune responses. M. tuberculosis induces Toll-like receptor 2 (TLR2) signaling, which influences immune responses to M. tuberculosis. TLR2 agonists expressed by M. tuberculosis include lipoproteins (e.g., LprG), the glycolipid phosphatidylinositol mannoside 6 (PIM6), and the lipoglycan lipomannan (LM). Another M. tuberculosis lipoglycan, mannose-capped lipoarabinomannan (ManLAM), lacks TLR2 agonist activity. In contrast, PILAM, from Mycobacterum smegmatis, does have TLR2 agonist activity. Our understanding of how M. tuberculosis lipoproteins and lipoglycans interact with TLR2 is limited, and binding of these molecules to TLR2 has not been measured directly. Here, we directly measured M. tuberculosis lipoprotein and lipoglycan binding to TLR2 and its partner receptor, TLR1. LprG, LAM, and LM were all found to bind to TLR2 in the absence of TLR1, but not to TLR1 in the absence of TLR2. Trimolecular interactions were revealed by binding of TLR2-LprG or TLR2-PIM6 complexes to TLR1, whereas binding of TLR2 to TLR1 was not detected in the absence of the lipoprotein or glycolipid. ManLAM exhibited low affinity for TLR2 in comparison to PILAM, LM, and LprG, which correlated with reduced ability of ManLAM to induce TLR2-mediated extracellular-signal-regulated kinase (ERK) activation and tumor necrosis factor alpha (TNF-α) secretion in macrophages. We provide the first direct affinity measurement and kinetic analysis of M. tuberculosis lipoprotein and lipoglycan binding to TLR2. Our results demonstrate that binding affinity correlates with the functional ability of agonists to induce TLR2 signaling.
- Published
- 2018
37. Cloning and Partial Characterization of an Endo-α-(1→6)-d-Mannanase Gene from Bacillus circulans
- Author
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Todd L. Lowary, Michael R. McNeil, Wei Li, Shiva K. Angala, Lu Zou, Mary Jackson, and Zuzana Palčeková
- Subjects
0301 basic medicine ,education ,Catalysis ,d<%2Fspan>-mannase%22">endo-α-(1→6)-d-mannase ,Inorganic Chemistry ,03 medical and health sciences ,chemistry.chemical_compound ,mannoside ,Hydrolase ,Glycosyl ,Mycobacterium ,Physical and Theoretical Chemistry ,Molecular Biology ,Spectroscopy ,Mannan ,chemistry.chemical_classification ,Lipomannan ,Lipoarabinomannan ,030102 biochemistry & molecular biology ,biology ,Chemistry ,Organic Chemistry ,General Medicine ,bacterial infections and mycoses ,lipomannan ,biology.organism_classification ,3. Good health ,Computer Science Applications ,030104 developmental biology ,Enzyme ,Biochemistry ,Bacillus circulans ,phosphatidylinositol mannosides ,lipoarabinomannan - Abstract
Mycobacteria produce two major lipoglycans, lipomannan (LM) and lipoarabinomannan (LAM), whose broad array of biological activities are tightly related to the fine details of their structure. However, the heterogeneity of these molecules in terms of internal and terminal covalent modifications and complex internal branching patterns represent significant obstacles to their structural characterization. Previously, an endo-&alpha, (1&rarr, 6)-D-mannanase from Bacillus circulans proved useful in cleaving the mannan backbone of LM and LAM, allowing the reducing end of these molecules to be identified as Manp-(1&rarr, 6) [Manp-(1&rarr, 2)]-Ino. Although first reported 45 years ago, no easily accessible form of this enzyme was available to the research community, a fact that may in part be explained by a lack of knowledge of its complete gene sequence. Here, we report on the successful cloning of the complete endo-&alpha, 6)-D-mannanase gene from Bacillus circulans TN-31, herein referred to as emn. We further report on the successful production and purification of the glycosyl hydrolase domain of this enzyme and its use to gain further insight into its substrate specificity using synthetic mannoside acceptors as well as LM and phosphatidyl-myo-inositol mannoside precursors purified from mycobacteria.
- Published
- 2019
38. Deletion of PimE mannosyltransferase results in increased copper sensitivity in Mycobacterium smegmatis
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Kathryn C. Rahlwes, Lisa R. Baumoel, Sarah H Osman, Yasu S. Morita, and William J Eagen
- Subjects
0301 basic medicine ,Mannosyltransferase ,Mycobacterium smegmatis ,030106 microbiology ,Microbial Sensitivity Tests ,Phosphatidylinositols ,Mannosyltransferases ,Microbiology ,03 medical and health sciences ,chemistry.chemical_compound ,Drug Resistance, Bacterial ,Genetics ,Phosphatidylinositol ,Molecular Biology ,Sequence Deletion ,Lipoarabinomannan ,Lipomannan ,biology ,biology.organism_classification ,Anti-Bacterial Agents ,Biosynthetic Pathways ,Phenotype ,030104 developmental biology ,chemistry ,Middlebrook 7H10 Agar ,Cell envelope ,Copper ,Mycobacterium - Abstract
The unique cell envelope structure of Mycobacterium tuberculosis is fundamental to its pathogenesis. Phosphatidylinositol (PI)-anchored glycolipids, such as phosphatidylinositol mannosides (PIMs), lipomannan and lipoarabinomannan, are essential components of the cell envelope widely conserved among mycobacteria, but their roles in the cell envelope integrity are not fully understood. We previously identified PimE in Mycobacterium smegmatis, a nonpathogenic model organism, as a mannosyltransferase that catalyzes the fifth mannose transfer for the biosynthesis of hexamannosyl PIMs. Our analyses, reported here, further demonstrate that the growth of the pimE deletion mutant (ΔpimE) is defective in the presence of copper. We first found that the small colony phenotype of ΔpimE on a solid Middlebrook 7H10 agar surface was alleviated when grown on M63 agar. Comparative analysis of the two media led us to identify copper in Middlebrook 7H10 as the cause of growth retardation seen in ΔpimE. We further demonstrated that ΔpimE is sensitized to several antibiotics, but the increased sensitivities were independent of the presence of copper. We conclude that the deletion of the pimE gene does not cause growth defects under optimal growth conditions, but makes the cell envelope vulnerable to toxic compounds such as copper and antibiotics.
- Published
- 2018
39. Controlled ring-opening polymerization of 1,2,6-tricyclic orthoesters of mannose toward size–controlled α-d-mannopyranan
- Author
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Chanokpon Yongyat, Siwarutt Boonyarattanakalin, and Somsak Ruchirawat
- Subjects
chemistry.chemical_classification ,chemistry.chemical_compound ,Materials science ,Lipomannan ,Polymers and Plastics ,chemistry ,Polymerization ,General Chemical Engineering ,Polymer chemistry ,Mannose ,Physical and Theoretical Chemistry ,Ring-opening polymerization ,Tricyclic - Abstract
Lipomannan (LM), one of the major components of glycans found on the cell wall of Mycobacterium tuberculosis, plays a critical role in interacting with host cells and moderating immune response. The backbone of LM is composed of α(1–6) mannopyranan, consisting of approximately 10–15 repeating mannose residues. Rapid synthesis of α(1–6) mannopyranan has been accomplished by ring-opening polymerization using 3,4-O-benzyl-β-d-mannopyranose 1,2,6-orthobenzoate (1) as a starting monomer. Here, we report the progress on attempts to control the size of α(1–6) mannopyranan polymer products. It is possible to prepare α-d-mannopyranan polymer products in different sizes by varying the monomer concentration.
- Published
- 2014
40. The cell envelope–associated phospholipid-binding protein LmeA is required for mannan polymerization in mycobacteria
- Author
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Jacob A. Mayfield, Shota Nakamura, Stephanie A. Ha, Kathryn C. Rahlwes, Ana P. Torres-Ocampo, Yasu S. Morita, Justin N. Strickland, Daisuke Motooka, and Lisa R. Baumoel
- Subjects
0301 basic medicine ,Lipopolysaccharides ,Mannosyltransferase ,030106 microbiology ,Mutant ,Mycobacterium smegmatis ,Glycobiology and Extracellular Matrices ,Biochemistry ,Mannosyltransferases ,Mannans ,03 medical and health sciences ,chemistry.chemical_compound ,Bacterial Proteins ,Phosphatidylinositol ,Molecular Biology ,Phospholipids ,Mannan ,Lipomannan ,Lipoarabinomannan ,biology ,Cell Membrane ,Cell Biology ,biology.organism_classification ,030104 developmental biology ,chemistry ,Cell envelope - Abstract
The integrity of the distinguishing, multilaminate cell envelope surrounding mycobacteria is critical to their survival and pathogenesis. The prevalence of phosphatidylinositol mannosides in the cell envelope suggests an important role in the mycobacterial life cycle. Indeed, deletion of the pimE gene (ΔpimE) encoding the first committed step in phosphatidylinositol hexamannoside biosynthesis in Mycobacterium smegmatis results in the formation of smaller colonies than wild-type colonies on Middlebrook 7H10 agar. To further investigate potential contributors to cell-envelope mannan biosynthesis while taking advantage of this colony morphology defect, we isolated spontaneous suppressor mutants of ΔpimE that reverted to wild-type colony size. Of 22 suppressor mutants, 6 accumulated significantly shorter lipomannan or lipoarabinomannan. Genome sequencing of these mutants revealed mutations in genes involved in the lipomannan/lipoarabinomannan biosynthesis, such as those encoding the arabinosyltransferase EmbC and the mannosyltransferase MptA. Furthermore, we identified three mutants carrying a mutation in a previously uncharacterized gene, MSMEG_5785, that we designated lmeA. Complementation of these suppressor mutants with lmeA restored the original ΔpimE phenotypes and deletion of lmeA in wild-type M. smegmatis resulted in smaller lipomannan, as observed in the suppressor mutants. LmeA carries a predicted N-terminal signal peptide, and density gradient fractionation and detergent extractability experiments indicated that LmeA localizes to the cell envelope. Using a lipid ELISA, we found that LmeA binds to plasma membrane phospholipids, such as phosphatidylethanolamine and phosphatidylinositol. LmeA is widespread throughout the Corynebacteriales; therefore, we concluded that LmeA is an evolutionarily conserved cell-envelope protein critical for controlling the mannan chain length of lipomannan/lipoarabinomannan.
- Published
- 2017
41. Signaling C-type lectin receptors in antimycobacterial immunity
- Author
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Hlumani Ndlovu and Mohlopheni J. Marakalala
- Subjects
0301 basic medicine ,Physiology ,Immune receptor ,Biochemistry ,Immune Receptors ,Pearls ,Cell-Mediated Immunity ,White Blood Cells ,chemistry.chemical_compound ,Cell Signaling ,Animal Cells ,C-type lectin ,Lectins ,Immune Physiology ,Medicine and Health Sciences ,Membrane Receptor Signaling ,lcsh:QH301-705.5 ,Innate Immune System ,Immune System Proteins ,T Cells ,Chemistry ,Pattern recognition receptor ,Immune Receptor Signaling ,Actinobacteria ,Trehalose dimycolate ,Cytokines ,Cellular Types ,Signal Transduction ,lcsh:Immunologic diseases. Allergy ,Immune Cells ,Immunology ,Microbiology ,Mycobacterium ,03 medical and health sciences ,Virology ,Genetics ,Animals ,Humans ,Lectins, C-Type ,Molecular Biology ,Blood Cells ,Innate immune system ,Lipoarabinomannan ,Lipomannan ,Bacteria ,Macrophages ,Organisms ,Immunity ,Biology and Life Sciences ,Proteins ,Cell Biology ,Dendritic cell ,Molecular Development ,Immunity, Innate ,030104 developmental biology ,lcsh:Biology (General) ,Immune System ,Parasitology ,lcsh:RC581-607 ,Mycobacterium Tuberculosis ,Developmental Biology - Abstract
The mammalian innate immune system is composed of phagocytes such as macrophages and dendritic cells that serve as the first line of defense against microbial infections. These cells express various pattern recognition receptors (PRRs) that recognize specific pathogen-associated molecular patterns (PAMPs) on the surface of or inside microorganisms [1]. PRRs such as Toll-like receptors (TLRs), C-type lectin receptors (CLRs), and Nucleotide-binding Oligomerization Domain (NOD)-like receptors (NLRs) have been widely studied in antimicrobial immunity and homeostasis. These PRRs have also been implicated in antimycobacterial immunity, with CLRs recently receiving considerable attention. CLRs are a large family of proteins containing at least 1 carbohydrate-recognition domain (CRD) that in most cases binds a range of carbohydrate-based PAMPs, including trehalose 6,6’ dimycolate (TDM), lipoarabinomannan (LAM), lipomannan (LM), and phosphatidylinositol mannosides (PIMs) [2–4]. Interactions of CLRs with mycobacterial PAMPs induce intracellular signaling that triggers responses ranging from cytokine production to induction of adaptive immunity (Table 1). Here, we discuss signaling CLRs that recognize mycobacterial PAMPs and contribute to antimycobacterial immunity. We focus on the receptors that signal through the Spleen tyrosine kinase (Syk)/Caspase recruitment domain family member 9 (CARD9) pathway, including Dectin-1, Dectin-2, macrophage-inducible C-type lectin (Mincle), C-type lectin superfamily member 8 (Clecsf8) also called macrophage C-type lectin (MCL), and dendritic cell immunoactivating receptor (DCAR) (Fig 1). Table 1 C-type lectin receptors, mycobacterial ligands, and their effects on pro-inflammatory cytokine production and contributions in host resistance to mycobacterial infections in vivo. C-type lectin receptor Mtb ligand Cellular expression Effects on pro-inflammatory cytokine production Role in host resistance to mycobacterial infection References Dectin-1 unknown DCs, monocytes, macrophages, neutrophils, eosinophils, mast cells, and lung epithelium ↑IL-6, IL-23, IL-1β, TNF-α, IL-12p40, and IL-17 Dispensable for host resistance to Mycobacterium tuberculosis H37Rv infection in mice [5, 7, 9–11, 35] Dectin-2 ManLAM DCs, monocytes, tissue macrophages, CD8+ T cells, and CD19+ B cells ↑TNF-α, IL-6, and IL-17 Survival studies not performed. Required to control lung damage during M. avium infection. [4, 12, 14, 36] Mincle TDM Monocytes, macrophages, neutrophils, myeloid DCs, and B cells. ↑IL-8, IL-6 andIL-1β Required for bacterial clearance. Inconsistent results on essentiality. [4, 17, 19, 21–23] ClecSF8 (MCL) TDM Neutrophils, monocytes, and DCs ↑IL-6, TNF-α and IL-1β Required for resistance to M. bovis BCG and M. tuberculosis H37Rv infection in mice [25, 28, 29] Mannose receptor ManLAM, DIM, mannosylated proteins Macrophages and MDCs ↑IFN-γ Survival studies not performed [10, 34, 37, 38] DC-SIGN ManLAM, PIMs, mannosylated glycoproteins Myeloid DCs and macrophages ↑IFN-γ hSIGN transgenic mice resistant to high-dose M. tuberculosis H37Rv infection. SIGNR3 KO mice have elevated CFUs. [10, 32, 33, 39] DCAR PIMs Peritoneal macrophages, monocyte-derived inflammatory cells in lung and spleen ↑IFN-γ and IL-12 Survival studies not performed. High CFU in DCAR KO mice infected with BCG or H37Rv. [4, 31] Open in a separate window Abbreviations: CFU, colony-forming unit; ClecSF8, C-type lectin superfamily member 8; DC, dendritic cells; DC-SIGN, Dendritic Cell-Specific Intercellular adhesion molecule-3-Grabbing Non-integrin; DCAR, dendritic cell immunoactivating receptor; DIM, Phthiol Dimycocerosates; KO, knock-out; ManLAM, Mannose-caped Lipoarabinomannan; Mincle, macrophage-inducible C-type lectin; MCL, macrophage C-type lectin; Mtb, Mycobacterium tuberculosis; PIM, Phosphatidyinositol Mannosides; TDM, Trehalose Dimycolate.
- Published
- 2017
42. AB0101 New mutated peptidylarginine deiminase from porphyromonas gingivalis a target in early ra citrullinates major ra-autoantigens
- Author
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M Jenning, G.-R. Burmester, Karl Skriner, J Yetterberg, and B Marklein
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Lipomannan ,biology ,medicine.drug_class ,business.industry ,Citrullination ,biology.organism_classification ,Monoclonal antibody ,Molecular biology ,Protein citrullination ,law.invention ,chemistry.chemical_compound ,chemistry ,law ,Immunology ,Recombinant DNA ,Citrulline ,biology.protein ,medicine ,Antibody ,business ,Porphyromonas gingivalis - Abstract
Objectives Previous reports showed that peptidylarginine deiminase (PPAD) form Porphyromonas gingivalis (P.g.) is not able to citrullinate proteins internaly. New mutated PPAD (mPPAD) from P.g. involved in periodontal disease (PD) cloned out of P.g. strain was characterized and analyzed for its reactivity in sera from patients with systemic autoimmune diseases Methods We cloned a new enzymatically active recombinant mutated PPAD from P.g. mPPAD mutations and citrullination sites were analyzed by DNA sequencing and/or protein mass spectrometry. Autocitrullination activity it9s enzymatic-activity and human autoantigen protein citrullination was investigated by 2D-Elektrophoresis, MS, immunoblot analysis and ELISA. Furthermore we tested anti-mPPAD/cit-mPPAD with human sera (n=93) from early RA before and after onset of RA (n=30), established RA (n=32), SLE (n=16) and healthy blood donors (n=15) in ELISA assays. To study a potential impact on the RA mouse model (CAIA), mPPAD-containing vesicles from P.g. were injected by intraperitoneal injection (IP). Results Recombinant mPPAD lacks 43 amino acids at the N-terminus and exhibits so far two new amino acid mutations (amino acid position 73 (F>L) and 447 (E>V). We were able to demonstrate, mPPAD is enzymatically active over a huge pH-range (3–10) and autocitrullinates at amino acid position 63 the arginine to citrulline. Moreover mPPAd citrullinates major autoantigens in RA (Fibrinogen, Vimentin and hnRNP-A2/B1) which are detectable by RA patient sera and specific anti-citrulline monoclonal antibodies. mPPAD citrullinates HeLa-protein extracts and these specific citrullinated proteins are recognized by RA patient sera. Anti-citrullinated mPPAD antibodies were detected in 41% (n=32) of patients with RA but not in SLE (n=16) and control sera (n=15). In a RA follow-up study (n=30), we detected nearly similar antibody-sensitivities for citrullinated mPPAD before and after onset of RA (13/20%). Only a minority (7%) of RA patients show higher mPPAD antibody levels after RA diagnosis. In the Collagen antibody-induced arthritis (CAIA) RA mouse model mPPAD containing P.g. vesicles when injected IP showed a TLR2-dependent protective anti- inflammatory effect like P.g. LPS and Lipomannan. Conclusions Pg. infection and RA disease diagnosis occurs on different timepoints and Pg. infection induces a TLR2-dependent protective anti-inflammatory effect.We show the first time that mPPAD can citrullinate major human autoantigens internally and their immunologically and diagnostic relevance. Disclosure of Interest None declared
- Published
- 2017
43. Detection of Lipomannan in Cattle Infected with Bovine Tuberculosis
- Author
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Rama Murthy Sakamuri, Basil I. Swanson, Dung M. Vu, Harshini Mukundan, and W. Ray Waters
- Subjects
0301 basic medicine ,Immunoassay ,Lipopolysaccharides ,Lipomannan ,medicine.diagnostic_test ,Chemistry ,02 engineering and technology ,Optical biosensor ,021001 nanoscience & nanotechnology ,Rapid detection ,Virology ,Analytical Chemistry ,03 medical and health sciences ,030104 developmental biology ,Bovine tuberculosis ,medicine ,Biomarker (medicine) ,Animals ,Cattle ,0210 nano-technology ,Tuberculosis, Bovine ,Blood Chemical Analysis ,Lipoprotein - Abstract
Early and rapid detection of bovine tuberculosis (bTB) is critical to controlling the spread of this disease in cattle and other animals. In this study, we demonstrate the development of an immunoassay for the direct detection of the bovine bTB biomarker, lipomannan (LM) in serum using a waveguide-based optical biosensor. We apply an ultra-sensitive detection strategy developed by our team, termed lipoprotein capture, that exploits the pull-down of high-density lipoprotein (HDL) nanodiscs from cattle blood that allows for the recovery and detection of associated LM. We also profile the change in the expression of these TB biomarkers as a function of time from a small set of samples collected from studies of bovine TB-infected cattle. We demonstrate for the first time the direct detection of bovine LM in serum, and clearly show that the biomarker is expressed in detectable concentrations during the entire course of the infection.
- Published
- 2017
44. Synthesis of synthetic mannan backbone polysaccharides found on the surface of Mycobacterium tuberculosis as a vaccine adjuvant and their immunological properties
- Author
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Siwarutt Boonyarattanakalin, Chakree Wattanasiri, Marisa Ponpuak, Somsak Ruchirawat, and Jiraporn Paha
- Subjects
0301 basic medicine ,Lipopolysaccharides ,Glycan ,Polymers and Plastics ,medicine.medical_treatment ,Mannose ,Biology ,Chemical synthesis ,Microbiology ,Mycobacterium tuberculosis ,03 medical and health sciences ,chemistry.chemical_compound ,Mice ,0302 clinical medicine ,Adjuvants, Immunologic ,Adjuvanticity ,Materials Chemistry ,medicine ,Animals ,Mannan ,Vaccines ,Lipomannan ,Organic Chemistry ,Polysaccharides, Bacterial ,biology.organism_classification ,030104 developmental biology ,RAW 264.7 Cells ,Biochemistry ,chemistry ,biology.protein ,Cytokines ,Adjuvant ,030215 immunology - Abstract
Surface components of Mycobacterium tuberculosis (Mtb) play crucial roles in modulating host immune responses. Thorough understandings of immunological properties of the Mtb’s surface components are essential for the development of tuberculosis treatment and prevention. Unfortunately, the accessibility to the molecules on the surface of Mtb is limited by the structural complexity due to their various macromolecular nature and the hazard of culturing Mtb. In this study, we reveal a practical synthesis of lipomannan (LM) backbone polysaccharides – the core glycans found on Mtb’s surface. A rapid synthetic approach based on a controlled polymerization was developed for the chemical synthesis of mannopyranans, the core structure of LM. The size of the LM glycans can be controlled by using specific monomer concentrations in addition to stereo- and regioselectivity derived from the versatile tricyclic orthoester mannose monomer. The immunological properties of the synthesized mannopyranans were investigated and their adjuvant potential was revealed. The adjuvanticity mechanism of the synthetic mannopyranans appears to involve the NF-κB and inflammasome pathways.
- Published
- 2017
45. Identification of a Membrane Protein Required for Lipomannan Maturation and Lipoarabinomannan Synthesis in Corynebacterineae*
- Author
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Rajini Brammananth, Ross L. Coppel, Paul K. Crellin, Malcolm J. McConville, Yoshiki Yamaryo-Botté, Arek K. Rainczuk, Tamaryn J. Cashmore, and Stephan Klatt
- Subjects
0301 basic medicine ,Lipopolysaccharides ,Mutant ,Biochemistry ,Corynebacterium glutamicum ,03 medical and health sciences ,Bacterial Proteins ,Cell Wall ,Corynebacterineae ,Molecular Biology ,Lipoarabinomannan ,Lipomannan ,030102 biochemistry & molecular biology ,biology ,Chemistry ,Mycobacterium smegmatis ,Cell Biology ,biology.organism_classification ,Lipids ,Biosynthetic Pathways ,030104 developmental biology ,Membrane protein ,Essential gene ,Gene Deletion - Abstract
Mycobacterium tuberculosis and related Corynebacterineae synthesize a family of lipomannans (LM) and lipoarabinomannans (LAM) that are abundant components of the multilaminate cell wall and essential virulence factors in pathogenic species. Here we describe a new membrane protein, highly conserved in all Corynebacterineae, that is required for synthesis of full-length LM and LAM. Deletion of the Corynebacterium glutamicum NCgl2760 gene resulted in a complete loss of mature LM/LAM and the appearance of a truncated LM (t-LM). Complementation of the mutant with the NCgl2760 gene fully restored LM/LAM synthesis. Structural studies, including monosaccharide analysis, methylation linkage analysis, and mass spectrometry of native LM species, indicated that the ΔNCgl2760 t-LM comprised a series of short LM species (8–27 residues long) containing an α1–6-linked mannose backbone with greatly reduced α1–2-mannose side chains and no arabinose caps. The structure of the ΔNCgl2760 t-LM was similar to that of the t-LM produced by a C. glutamicum mutant lacking the mptA gene, encoding a membrane α1–6-mannosyltransferase involved in extending the α1–6-mannan backbone of LM intermediates. Interestingly, NCgl2760 lacks any motifs or homology to other proteins of known function. Attempts to delete the NCgl2760 orthologue in Mycobacterium smegmatis were unsuccessful, consistent with previous studies indicating that the M. tuberculosis orthologue, Rv0227c, is an essential gene. Together, these data suggest that NCgl2760/Rv0227c plays a critical role in the elongation of the mannan backbone of mycobacterial and corynebacterial LM, further highlighting the complexity of lipoglycan pathways of Corynebacterineae.
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- 2017
46. In Situ Preactivation Strategies for the Expeditious Synthesis of Oligosaccharides: A Review
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Steven J. Sucheck and Samantha K. Bouhall
- Subjects
chemistry.chemical_compound ,Glycosylation ,Lipomannan ,Biochemistry ,Chemistry ,Carbohydrate chemistry ,Organic Chemistry ,Article - Abstract
Carbohydrates have gained increasing appreciation over the last few decades for their fundamental roles in all essential areas of life. As a result, there has been a surge of activity in synthetic glycosylation strategies to construct useful oligosaccharides. This review evaluates the advances in synthetic carbohydrate chemistry, specifically preactivation methodologies, stereoselective β-mannosylations, and an automated, electrochemical preactivation method. Also discussed is the use of preactivation as a tool to study reactive intermediates, and applications of preactivation protocols in the one pot-synthesis of a hyaluronic acid decasaccharide and one-pot synthesis of a tristearoyl lipomannan containing a pseudotrisaccharide.
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- 2014
47. Cryo-EM Structures and Regulation of Arabinofuranosyltransferase AftD from Mycobacteria.
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Tan, Yong Zi, Zhang, Lei, Rodrigues, José, Zheng, Ruixiang Blake, Giacometti, Sabrina I., Rosário, Ana L., Kloss, Brian, Dandey, Venkata P., Wei, Hui, Brunton, Richard, Raczkowski, Ashleigh M., Athayde, Diogo, Catalão, Maria João, Pimentel, Madalena, Clarke, Oliver B., Lowary, Todd L., Archer, Margarida, Niederweis, Michael, Potter, Clinton S., and Carragher, Bridget
- Subjects
- *
GLYCANS , *ACYL carrier protein , *MYCOBACTERIA , *MYCOBACTERIUM tuberculosis - Abstract
Mycobacterium tuberculosis causes tuberculosis, a disease that kills over 1 million people each year. Its cell envelope is a common antibiotic target and has a unique structure due, in part, to two lipidated polysaccharides—arabinogalactan and lipoarabinomannan. Arabinofuranosyltransferase D (AftD) is an essential enzyme involved in assembling these glycolipids. We present the 2.9-Å resolution structure of M. abscessus AftD, determined by single-particle cryo-electron microscopy. AftD has a conserved GT-C glycosyltransferase fold and three carbohydrate-binding modules. Glycan array analysis shows that AftD binds complex arabinose glycans. Additionally, AftD is non-covalently complexed with an acyl carrier protein (ACP). 3.4- and 3.5-Å structures of a mutant with impaired ACP binding reveal a conformational change, suggesting that ACP may regulate AftD function. Mutagenesis experiments using a conditional knockout constructed in M. smegmatis confirm the essentiality of the putative active site and the ACP binding for AftD function. • Cryo-EM structures of mycobacterial arabinofuranosyltransferase D (AftD) were solved • AftD has a conserved GT-C glycosyltransferase fold and binds complex arabinose glycans • Acyl carrier protein (ACP) is complexed to AftD, also endogenously • Impairment of ACP binding alters conformation, suggesting ACP plays a regulatory role Tan et al. present the cryo-EM structures of essential wild-type and mutant mycobacterial arabinofuranosyltransferase D (AftD), revealing the putative active site geometry and carbohydrate-binding modules. Acyl carrier protein (ACP) was tightly associated with AftD. Impairing ACP binding blocks AftD's active site, suggesting that ACP regulates enzyme function. [ABSTRACT FROM AUTHOR]
- Published
- 2020
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48. Synthesis of Phosphatidylinositol Mannosides
- Author
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Shang-Cheng Hung, Medel Manuel L. Zulueta, and Pratap S. Patil
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Glycan ,Mannosides ,Lipoarabinomannan ,Glycosylation ,Lipomannan ,biology ,General Chemistry ,Chemical synthesis ,carbohydrates (lipids) ,chemistry.chemical_compound ,Biochemistry ,chemistry ,biology.protein ,Phosphatidylinositol - Abstract
The cell envelope of mycobacterial species, which include the deadly Mycobacterium tuberculosis, carries unique glycan structures that are crucial for infection and bacterial survival. Notable among these glycans are the structurally-related phosphatidylinositol mannosides (PIMs), lipomannan, and lipoarabinomannan. The immunoactivities of these structures attracted considerable attention from synthetic chemists and biologists alike, who are keen on evaluating vaccine or adjuvant properties. Moreover, PIMs are interesting synthetic targets because of the regio- and stereoselective considerations in generating the inositol and mannosyl cores. In this paper, we summarize the recent efforts in the chemical synthesis of PIMs as well as their analogues. Particular emphasis is given on the challenges associated with the preparation of the chiral myo-inositol derivatives, the regioselective installation of mannosyl residues, and phosphatidylation strategies. The results of the bioevaluation of selected compounds are also briefly described.
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- 2013
49. Cloning and Partial Characterization of an Endo-α-(1→6)-d-Mannanase Gene from Bacillus circulans.
- Author
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Angala, Shiva kumar, Li, Wei, Palčeková, Zuzana, Zou, Lu, Lowary, Todd L., McNeil, Michael R., and Jackson, Mary
- Subjects
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BACILLUS (Bacteria) , *LIPOPOLYSACCHARIDES , *GENES , *SCIENTIFIC community - Abstract
Mycobacteria produce two major lipoglycans, lipomannan (LM) and lipoarabinomannan (LAM), whose broad array of biological activities are tightly related to the fine details of their structure. However, the heterogeneity of these molecules in terms of internal and terminal covalent modifications and complex internal branching patterns represent significant obstacles to their structural characterization. Previously, an endo-α-(1→6)-D-mannanase from Bacillus circulans proved useful in cleaving the mannan backbone of LM and LAM, allowing the reducing end of these molecules to be identified as Manp-(1→6) [Manp-(1→2)]-Ino. Although first reported 45 years ago, no easily accessible form of this enzyme was available to the research community, a fact that may in part be explained by a lack of knowledge of its complete gene sequence. Here, we report on the successful cloning of the complete endo-α-(1→6)-D-mannanase gene from Bacillus circulans TN-31, herein referred to as emn. We further report on the successful production and purification of the glycosyl hydrolase domain of this enzyme and its use to gain further insight into its substrate specificity using synthetic mannoside acceptors as well as LM and phosphatidyl-myo-inositol mannoside precursors purified from mycobacteria. [ABSTRACT FROM AUTHOR]
- Published
- 2019
- Full Text
- View/download PDF
50. Mannan core branching of lipo(arabino)mannan is required for mycobacterial virulence in the context of innate immunity
- Author
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Theo Verboom, Gurdyal S. Besra, Astrid M. van der Sar, Arun K. Mishra, Marion Sparrius, Wilbert Bitter, Esther J. M. Stoop, Gunny van Stempvoort, Nicole N. van der Wel, Susanne A. Raadsen, Ben J. Appelmelk, Maaike van Zon, Nicole N. Driessen, Lisanne M. van Leeuwen, Pascale Bouchier, and Jeroen Geurtsen
- Subjects
0303 health sciences ,Mannosyltransferase ,Lipomannan ,Lipoarabinomannan ,Mycobacterium smegmatis ,030302 biochemistry & molecular biology ,Immunology ,Virulence ,Biology ,bacterial infections and mycoses ,biology.organism_classification ,Microbiology ,3. Good health ,Mycobacterium tuberculosis ,03 medical and health sciences ,Virology ,Mycobacterium marinum ,030304 developmental biology ,Mannan - Abstract
The causative agent of tuberculosis (TB), Mycobacterium tuberculosis, remains an important worldwide health threat. Although TB is one of the oldest infectious diseases of man, a detailed understanding of the mycobacterial mechanisms underlying pathogenesis remains elusive. Here, we studied the role of the α(1→2) mannosyltransferase MptC in mycobacterial virulence, using the Mycobacterium marinum zebrafish infection model. Like its M. tuberculosis orthologue, disruption of M. marinum mptC (mmar_3225) results in defective elongation of mannose caps of lipoarabinomannan (LAM) and absence of α(1→2)mannose branches on the lipomannan (LM) and LAM mannan core, as determined by biochemical analysis (NMR and GC-MS) and immunoblotting. We found that the M. marinum mptC mutant is strongly attenuated in embryonic zebrafish, which rely solely on innate immunity, whereas minor virulence defects were observed in adult zebrafish. Strikingly, complementation with the Mycobacterium smegmatis mptC orthologue, which restored mannan core branching but not cap elongation, was sufficient to fully complement the virulence defect of the mptC mutant in embryos. Altogether our data demonstrate that not LAM capping, but mannan core branching of LM/LAM plays an important role in mycobacterial pathogenesis in the context of innate immunity.
- Published
- 2013
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