A truncated lipoglycan from mycobacteria with altered immunological properties

Maintenance of cell-wall integrity in Mycobacterium tuberculosis is essential and is the target of several antitubercular drugs. For example, ethambutol targets arabinogalactan and lipoarabinomannan (LAM) biosynthesis through the inhibition of several arabinofuranosyltransferases. Apart from their role in cell-wall integrity, mycobacterial LAMs also exhibit important immunomodulatory activities. Here we report the isolation and detailed structural characterization of a unique LAM molecule derived from Mycobacterium smegmatis deficient in the arabinofuranosyltransferase AftC (AftC-LAM). This mutant LAM expresses a severely truncated arabinan domain completely devoid of 3,5-Araf–branching residues, revealing an intrinsic involvement of AftC in the biosynthesis of LAM. Furthermore, we found that ethambutol efficiently inhibits biosynthesis of the AftC-LAM arabinan core, unambiguously demonstrating the involvement of the arabinofuranosyltransferase EmbC in early stages of LAM-arabinan biosynthesis. Finally, we demonstrate that AftC-LAM exhibits an enhanced proinflammatory activity, which is due to its ability to activate Toll-like receptor 2 (TLR2). Overall, our efforts further describe the mechanism of action of an important antitubercular drug, ethambutol, and demonstrate a role for specific arabinofuranosyltransferases in LAM biosynthesis. In addition, the availability of sufficient amounts of chemically defined wild-type and isogenic truncated LAMs paves the way for further investigations of the structure–function relationship of TLR2 activation by mycobacterial lipoglycans

Identification and structural characterisation of a partially arabinosylated lipoarabinomannan variant isolated from a Corynebacterium glutamicum ubiAmutant

Arabinan polysaccharide side-chains are present in both Mycobacterium tuberculosis and Corynebacterium glutamicum in the heteropolysaccharide arabinogalactan (AG), and in M. tuberculosis in the lipoglycan, lipoarabinomannan (LAM). Herein, we show by quantitative sugar and glycosyl linkage analysis that C. glutamicum possesses a much smaller LAM version, Cg-LAM, characterised by single t-Araf residues linked to th

Dimerization of inositol monophosphatase Mycobacterium tuberculosis SuhB is not constitutive, but induced by binding of the activator Mg2+

Background The cell wall of Mycobacterium tuberculosis contains a wide range of phosphatidyl inositol-based glycolipids that play critical structural roles and, in part, govern pathogen-host interactions. Synthesis of phosphatidyl inositol is dependent on free myo-inositol, generated through dephosphorylation of myo-inositol-1-phosphate by inositol monophosphatase (IMPase). Human IMPase, the putative target of lithium therapy, has been studied extensively, but the function of four IMPase-like genes in M. tuberculosis is unclear. Results We determined the crystal structure, to 2.6 Å resolution, of the IMPase M. tuberculosis SuhB in the apo form, and analysed self-assembly by analytical ultracentrifugation. Contrary to the paradigm of constitutive dimerization of IMPases, SuhB is predominantly monomeric in the absence of the physiological activator Mg2+, in spite of a conserved fold and apparent dimerization in the crystal. However, Mg2+ concentrations that result in enzymatic activation of SuhB decisively promote dimerization, with the inhibitor Li+ amplifying the effect of Mg2+, but failing to induce dimerization on its own. Conclusion The correlation of Mg2+-driven enzymatic activity with dimerization suggests that catalytic activity is linked to the dimer form. Current models of lithium inhibition of IMPases posit that Li+ competes for one of three catalytic Mg2+ sites in the active site, stabilized by a mobile loop at the dimer interface. Our data suggest that Mg2+/Li+-induced ordering of this loop may promote dimerization by expanding the dimer interface of SuhB. The dynamic nature of the monomer-dimer equilibrium may also explain the extended concentration range over which Mg2+ maintains SuhB activity

Mycobacterium marinum MMAR_2380, a predicted transmembrane acyltransferase, is essential for the presence of the mannose cap on lipoarabinomannan

Lipoarabinomannan (LAM) is a major glycolipid in the mycobacterial cell envelope. LAM consists of a mannosylphosphatidylinositol (MPI) anchor, a mannan core and a branched arabinan domain. The termini of the arabinan branches can become substituted with one to three α(1→2)-linked mannosyl residues, the mannose cap, producing ManLAM. ManLAM has been associated with a range of different immunomodulatory properties of Mycobacterium tuberculosis during infection of the host. In some of these effects, the presence of the mannose cap on ManLAM appears to be crucial for its activity. So far, in the biosynthesis of the mannose cap on ManLAM, two enzymes have been reported to be involved: a mannosyltransferase that adds the first mannosyl residue of the mannose caps to the arabinan domain of LAM, and another mannosyltransferase that elongates the mannose cap up to three mannosyl residues. Here, we report that a third gene is involved, MMAR_2380, which is the Mycobacterium marinum orthologue of Rv1565c. MMAR_2380 encodes a predicted transmembrane acyltransferase. In M. marinum ΔMMAR_2380, the LAM arabinan domain is still intact, but the mutant LAM lacks the mannose cap. Additional effects of mutation of MMAR_2380 on LAM were observed: a higher degree of branching of both the arabinan domain and the mannan core, and a decreased incorporation of [1,2-14C]acetate into the acyl chains in mutant LAM as compared with the wild-type form. This latter effect was also observed for related lipoglycans, i.e. lipomannan (LM) and phosphatidylinositol mannosides (PIMs). Furthermore, the mutant strain showed increased aggregation in liquid cultures as compared with the wild-type strain. All phenotypic traits of M. marinum ΔMMAR_2380, the deficiency in the mannose cap on LAM and changes at the cell surface, could be reversed by complementing the mutant strain with MMAR_2380. Strikingly, membrane preparations of the mutant strain still showed enzymic activity for the arabinan mannose-capping mannosyltransferase similar to that of the wild-type strain. Although the exact function of MMAR_2380 remains unknown, we show that the protein is essential for the presence of a mannose cap on LAM

Lipoglycans Contribute to Innate Immune Detection of Mycobacteria

Innate immune recognition is based on the detection, by pattern recognition receptors (PRRs), of molecular structures that are unique to microorganisms. Lipoglycans are macromolecules specific to the cell envelope of mycobacteria and related genera. They have been described to be ligands, as purified molecules, of several PRRs, including the C-type lectins Mannose Receptor and DC-SIGN, as well as TLR2. However, whether they are really sensed by these receptors in the context of a bacterium infection remains unclear. To address this question, we used the model organism Mycobacterium smegmatis to generate mutants altered for the production of lipoglycans. Since their biosynthesis cannot be fully abrogated, we manipulated the biosynthesis pathway of GDP-Mannose to obtain some strains with either augmented (∼1.7 fold) or reduced (∼2 fold) production of lipoglycans. Interestingly, infection experiments demonstrated a direct correlation between the amount of lipoglycans in the bacterial cell envelope on one hand and the magnitude of innate immune signaling in TLR2 reporter cells, monocyte/macrophage THP-1 cell line and human dendritic cells, as revealed by NF-κB activation and IL-8 production, on the other hand. These data establish that lipoglycans are bona fide Microbe-Associated Molecular Patterns contributing to innate immune detection of mycobacteria, via TLR2 among other PRRs

The C-Terminal Domain of the Arabinosyltransferase Mycobacterium tuberculosis EmbC Is a Lectin-Like Carbohydrate Binding Module

The D-arabinan-containing polymers arabinogalactan (AG) and lipoarabinomannan (LAM) are essential components of the unique cell envelope of the pathogen Mycobacterium tuberculosis. Biosynthesis of AG and LAM involves a series of membrane-embedded arabinofuranosyl (Araf) transferases whose structures are largely uncharacterised, despite the fact that several of them are pharmacological targets of ethambutol, a frontline drug in tuberculosis therapy. Herein, we present the crystal structure of the C-terminal hydrophilic domain of the ethambutol-sensitive Araf transferase M. tuberculosis EmbC, which is essential for LAM synthesis. The structure of the C-terminal domain of EmbC (EmbCCT) encompasses two sub-domains of different folds, of which subdomain II shows distinct similarity to lectin-like carbohydrate-binding modules (CBM). Co-crystallisation with a cell wall-derived di-arabinoside acceptor analogue and structural comparison with ligand-bound CBMs suggest that EmbCCT contains two separate carbohydrate binding sites, associated with subdomains I and II, respectively. Single-residue substitution of conserved tryptophan residues (Trp868, Trp985) at these respective sites inhibited EmbC-catalysed extension of LAM. The same substitutions differentially abrogated binding of di- and penta-arabinofuranoside acceptor analogues to EmbCCT, linking the loss of activity to compromised acceptor substrate binding, indicating the presence of two separate carbohydrate binding sites, and demonstrating that subdomain II indeed functions as a carbohydrate-binding module. This work provides the first step towards unravelling the structure and function of a GT-C-type glycosyltransferase that is essential in M. tuberculosis. Author Summary Top Tuberculosis (TB), an infectious disease caused by the bacillus Mycobacterium tuberculosis, burdens large swaths of the world population. Treatment of active TB typically requires administration of an antibiotic cocktail over several months that includes the drug ethambutol. This front line compound inhibits a set of arabinosyltransferase enzymes, called EmbA, EmbB and EmbC, which are critical for the synthesis of arabinan, a vital polysaccharide in the pathogen's unique cell envelope. How precisely ethambutol inhibits arabinosyltransferase activity is not clear, in part because structural information of its pharmacological targets has been elusive. Here, we report the high-resolution structure of the C-terminal domain of the ethambutol-target EmbC, a 390-amino acid fragment responsible for acceptor substrate recognition. Combining the X-ray crystallographic analysis with structural comparisons, site-directed mutagenesis, activity and ligand binding assays, we identified two regions in the C-terminal domain of EmbC that are capable of binding acceptor substrate mimics and are critical for activity of the full-length enzyme. Our results begin to define structure-function relationships in a family of structurally uncharacterised membrane-embedded glycosyltransferases, which are an important target for tuberculosis therapy

Structural characterization of a partially arabinosylated lipoarabinomannan variant isolated from a Corynebacterium glutamicum ubiA mutant

Arabinan polysaccharide side-chains are present in both Mycobacterium tuberculosis and Corynebacterium glutamicum in the heteropolysaccharide arabinogalactan (AG), and in M. tuberculosis in the lipoglycan lipoarabinomannan (LAM). This study shows by quantitative sugar and glycosyl linkage analysis that C. glutamicum possesses a much smaller LAM version, Cg-LAM, characterized by single t-Araf residues linked to the α(1→6)-linked mannan backbone. MALDI-TOF MS showed an average molecular mass of 13 800–15 400 Da for Cg-LAM. The biosynthetic origin of Araf residues found in the extracytoplasmic arabinan domain of AG and LAM is well known to be provided by decaprenyl-monophosphoryl-d-arabinose (DPA). However, the characterization of LAM in a C. glutamicum : : ubiA mutant devoid of prenyltransferase activity and devoid of DPA-dependent arabinan deposition into AG revealed partial formation of LAM, albeit with a slightly altered molecular mass. These data suggest that in addition to DPA utilization as an Araf donor, alternative pathways exist in Corynebacterianeae for Araf delivery, possibly via an unknown sugar nucleotide

Identification of an α(1→6) mannopyranosyltransferase (MptA), involved in Corynebacterium glutamicum lipomanann biosynthesis, and identification of its orthologue in Mycobacterium tuberculosis

Corynebacterium glutamicum and Mycobacterium tuberculosis share a similar cell wall architecture, and the availability of their genome sequences has enabled the utilization of C. glutamicum as a model for the identification and study of, otherwise essential, mycobacterial genes involved in lipomannan (LM) and lipoarabinomannan (LAM) biosynthesis. We selected the putative glycosyltransferase-Rv2174 from M. tuberculosis and deleted its orthologue NCgl2093 from C. glutamicum. This resulted in the formation of a novel truncated lipomannan (Cg-t-LM) and a complete ablation of LM/LAM biosynthesis. Purification and characterization of Cg-t-LM revealed an overall decrease in molecular mass, a reduction of α(1→6) and α(1→2) glycosidic linkages illustrating a reduced degree of branching compared with wild-type LM. The deletion mutant's biochemical phenotype was fully complemented by either NCgl2093 or Rv2174. Furthermore, the use of a synthetic neoglycolipid acceptor in an in vitro cell-free assay utilizing the sugar donor β-d-mannopyranosyl-1-monophosphoryl-decaprenol together with the neoglycolipid acceptor α-d-Manp-(1→6)-α-d-Manp-O-C8 as a substrate, confirmed NCgl2093 and Rv2174 as an α(1→6) mannopyranosyltransferase (MptA), involved in the latter stages of the biosynthesis of the α(1→6) mannan core of LM. Altogether, these studies have identified a new mannosyltransferase, MptA, and they shed further light on the biosynthesis of LM/LAM in Corynebacterianeae

Ultralong C100 Mycolic Acids Support the Assignment of Segniliparus as a New Bacterial Genus

Mycolic acid-producing bacteria isolated from the respiratory tract of human and non-human mammals were recently assigned as a distinct genus, Segniliparus, because they diverge from rhodococci and mycobacteria in genetic and chemical features. Using high accuracy mass spectrometry, we determined the chemical composition of 65 homologous mycolic acids in two Segniliparus species and separately analyzed the three subclasses to measure relative chain length, number and stereochemistry of unsaturations and cyclopropyl groups within each class. Whereas mycobacterial mycolate subclasses are distinguished from one another by R groups on the meromycolate chain, Segniliparus species synthesize solely non-oxygenated α-mycolates with high levels of cis unsaturation. Unexpectedly Segniliparus α-mycolates diverge into three subclasses based on large differences in carbon chain length with one bacterial culture producing mycolates that range from C58 to C100. Both the overall chain length (C100) and the chain length diversity (C42) are larger than previously seen for mycolic acid-producing organisms and provide direct chemical evidence for assignment of Segniliparus as a distinct genus. Yet, electron microscopy shows that the long and diverse mycolates pack into a typical appearing membrane. Therefore, these new and unexpected extremes of mycolic acid chemical structure raise questions about the modes of mycolic acid packing and folding into a membrane

REUL Is a Novel E3 Ubiquitin Ligase and Stimulator of Retinoic-Acid-Inducible Gene-I

RIG-I and MDA5 are cytoplasmic sensors that recognize different species of viral RNAs, leads to activation of the transcription factors IRF3 and NF-κB, which collaborate to induce type I interferons. In this study, we identified REUL, a RING-finger protein, as a specific RIG-I-interacting protein. REUL was associated with RIG-I, but not MDA5, through its PRY and SPRY domains. Overexpression of REUL potently potentiated RIG-I-, but not MDA5-mediated downstream signalling and antiviral activity. In contrast, the RING domain deletion mutant of REUL suppressed Sendai virus (SV)-induced, but not cytoplasmic polyI:C-induced activation of IFN-β promoter. Knockdown of endogenous REUL by RNAi inhibited SV-triggered IFN-β expression, and also increased VSV replication. Full-length RIG-I, but not the CARD domain deletion mutant of RIG-I, underwent ubiquitination induced by REUL. The Lys 154, 164, and 172 residues of the RIG-I CARD domain were critical for efficient REUL-mediated ubiquitination, as well as the ability of RIG-I to induce activation of IFN-β promoter. These findings suggest that REUL is an E3 ubiquitin ligase of RIG-I and specifically stimulates RIG-I-mediated innate antiviral activity