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The development of amine‐functionalized graphene quantum dots (GQDs) linked to mycolic acids (MAs) as a potential fluorescent biosensor to detect tuberculosis (TB) biomarkers is described. GQDs have attractive properties: high fluorescence, excellent biocompatibility, good water solubility, and low toxicity. MAs are lipids that are found in the cell wall of Mycobacterium tuberculosis that are antigenic, however, they are soluble only in chloroform and hexane. Chloroform‐soluble MAs were covalently linked to synthesized water‐soluble GQDs using an amide connection to create a potential fluorescent water‐soluble TB biosensor: MA‐GQDs. Fluorescence results showed that GQDs had a narrow emission spectrum with the highest emission at 440 nm, while MA‐GQDs had a broader spectrum with the highest emission at 470 nm, after exciting at 360 nm. The appearance of the peptide bond (amide linkage) in the Fourier‐transform infrared spectrum of MA‐GQDs confirmed the successful linking of MAs to GQDs. Powder X‐ray diffraction exhibited an increase in the number of peaks for MA‐GQDs relative to GQDs, suggesting that linking MAs to GQDs changed the crystal structure thereof. The linked MA‐GQDs showed good solubility in water, high fluorescence, and visual flow through a nitrocellulose membrane. These properties are promising for biomedical fluorescence sensing applications. [ABSTRACT FROM AUTHOR]

The Mycobacterium tuberculosis cell envelope is a critical interface between the host and pathogen and provides a protective barrier against the immune response and antibiotics. Cell envelope lipids are also mycobacterial virulence factors that influence the host immune response. The mycobacterial membrane protein large (MmpL) proteins transport cell envelope lipids and siderophores that are important for the basic physiology and pathogenesis of M. tuberculosis. We recently identified MmpL11 as a conserved transporter of mycolic acid‐containing lipids including monomeromycolyl diacylglycerol (MMDAG), mycolate wax ester (MWE), and long‐chain triacylglycerols (LC‐TAGs). These lipids contribute to biofilm formation in M. tuberculosis and M. smegmatis, and non‐replicating persistence in M. tuberculosis. In this report, we identified domains and residues that are essential for MmpL11TB lipid transporter activity. Specifically, we show that the D1 periplasmic loop and a conserved tyrosine are essential for the MmpL11 function. Intriguingly, we found that MmpL11 levels are regulated by the phosphorylation of threonine in the cytoplasmic C‐terminal domain, providing the first direct evidence of the phospho‐regulation of MmpL11 transporter activity in M. tuberculosis and M. smegmatis. Our results offer further insight into the function of MmpL transporters and regulation of mycobacterial cell envelope biogenesis. [ABSTRACT FROM AUTHOR]

The genomes of Corynebacteriales contain several genes encoding mycoloyltransferases (Myt) that are specific cell envelope enzymes essential for the biogenesis of the outer membrane. MytA is a major mycoloyltransferase of Corynebacterium glutamicum, displaying an N‐terminal domain with esterase activity and a C‐terminal extension containing a conserved repeated Leu‐Gly‐Phe‐Pro (LGFP) sequence motif of unknown function. This motif is highly conserved in Corynebacteriales and found associated with cell wall hydrolases and with proteins of unknown function. In this study, we determined the crystal structure of MytA and found that its C‐terminal domain is composed of five LGFP motifs and forms a long stalk perpendicular to the N‐terminal catalytic α/β‐hydrolase domain. The LGFP motifs are composed of a 4‐stranded β‐fold and occupy alternating orientations along the axis of the stalk. Multiple acetate binding pockets were identified in the stalk, which could correspond to putative ligand‐binding sites. By using various MytA mutants and complementary in vitro and in vivo approaches, we provide evidence that the C‐terminal LGFP domain interacts with the cell wall peptidoglycan‐arabinogalactan polymer. We also show that the C‐terminal LGFP domain is not required for the activity of MytA but rather contributes to the overall integrity of the cell envelope. [ABSTRACT FROM AUTHOR]

In Mycobacterium tuberculosis, mycolic acids and their glycerol, glucose, and trehalose esters ("cord factor") form the main part of the mycomembrane. Despite their first isolation almost a century ago, full stereochemical evaluation is lacking, as is a scalable synthesis required for accurate immunological, including vaccination, studies. Herein, we report an efficient, convergent, gram‐scale synthesis of four stereo‐isomers of a mycolic acid and its glucose ester. Binding to the antigen presenting protein CD1b and T cell activation studies are used to confirm the antigenicity of the synthetic material. The absolute stereochemistry of the syn‐methoxy methyl moiety in natural material is evaluated by comparing its optical rotation with that of synthetic material. [ABSTRACT FROM AUTHOR]

The final step in mycolic acid biosynthesis in Mycobacterium tuberculosis is catalysed by mycolyl reductase encoded by the Rv2509 gene. Sequence analysis and homology modelling indicate that Rv2509 belongs to the short‐chain fatty acid dehydrogenase/reductase (SDR) family, but with some distinct features that warrant its classification as belonging to a novel family of short‐chain dehydrogenases. In particular, the predicted structure revealed a unique α‐helical C‐terminal region which we demonstrated to be essential for Rv2509 function, though this region did not seem to play any role in protein stabilisation or oligomerisation. We also show that unlike the M. smegmatis homologue which was not essential for growth, Rv2509 was an essential gene in slow‐growing mycobacteria. A knockdown strain of the BCG2529 gene, the Rv2509 homologue in Mycobacterium bovis BCG, was unable to grow following the conditional depletion of BCG2529. This conditional depletion also led to a reduction of mature mycolic acid production and accumulation of intermediates derived from 3‐oxo‐mycolate precursors. Our studies demonstrate novel features of the mycolyl reductase Rv2509 and outline its role in mycobacterial growth, highlighting its potential as a new target for therapies. [ABSTRACT FROM AUTHOR]

The family of non-classical major histocompatibility complex (MHC) class-I like CD1 molecules has an emerging role in human disease. Group 1 CD1 includes CD1a, CD1b and CD1c, which function to display lipids on the cell surface of antigen-presenting cells for direct recognition by T-cells. The recent advent of CD1 tetramers and the identification of novel lipid ligands has contributed towards the increasing number of CD1-restricted T-cell clones captured. These advances have helped to identify novel donor unrestricted and semi-invariant T-cell populations in humans and new mechanisms of T-cell recognition. However, although there is an opportunity to design broadly acting lipids and harness the therapeutic potential of conserved T-cells, knowledge of their role in health and disease is lacking. We briefly summarize the current evidence implicating group 1 CD1 molecules in infection, cancer and autoimmunity and show that although CD1 are not as diverse as MHC, recent discoveries highlight their versatility as they exhibit intricate mechanisms of antigen presentation. [ABSTRACT FROM AUTHOR]

Abstract: Mycolic acid (MA) plays important role in Corynebacterium glutamicum, but the key enzymes in the biosynthetic pathway of MA in C. glutamicum ATCC13869 have not been characterized. Since the locus BBD29_RS14045 in C. glutamicum ATCC13869 shows high similarity to the gene Cgl2871, which encodes Pks13, the key enzyme for synthesizing MA in C. glutamicum ATCC13032, it was deleted, resulting in the mutant WG001. Compared with the wild‐type ATCC13869, MA was not synthesized in WG001, but more phosphatidylglycerol and phosphatidylinositol containing longer unsaturated fatty acids were produced. WG001 cells also show hindered cell growth and defective cell separation when compared with ATCC13869 cells. Transcriptomic analysis shows that many genes relevant to the pathways of fatty acids, inositol, phospholipids, cell wall, and cell division were significantly regulated in WG001 cells when compared with ATCC13869 cells. This study demonstrates that the locus BBD29_RS14045 encodes a key enzyme that plays important role for synthesizing MA in C. glutamicum ATCC13869. [ABSTRACT FROM AUTHOR]

Mycobacterium tuberculosis synthesizes a thick cell wall comprised of mycolic acids (MA), which are foreign antigens for human T cells. T-cell clones from multiple donors were used to determine the fine specificity of MA recognition by human αβ T cells. Most CD1-presented lipid antigens contain large hydrophilic head groups comprised of carbohydrates or peptides that dominate patterns of T-cell specificity. MA diverges from the consensus antigen motif in that it lacks a head group. Using multiple forms of natural and synthetic MA and MA-specific T-cells with different T-cell receptors, we found that, unlike antigens with larger head groups, lipid length strongly controlled T-cell responses to MA. In addition, the three forms of MA that naturally occur in M. tuberculosis that differ in modifications on the lipid tail, differ in their potency for activating MA-specific T-cell clones. Thus, naturally occurring MA forms should be considered as separate, partly cross-reactive antigens. Two of the three forms of MA could be loaded onto human CD1b proteins, creating working CD1b-MA tetramers. The creation of CD1b-MA tetramers represents a new tool for future studies that track the effector functions and kinetics of MA-specific T-cells ex vivo. [ABSTRACT FROM AUTHOR]

Mycolic acids (MAs) are highly hydrophobic long-chain α-alkyl β-hydroxy fatty acids present in the cell wall of Mycobacterium tuberculosis ( Mtb) as a complex mixture of molecules with a common general structure but with variable functional groups in the meromycolate chain. In this study, we addressed the relationship between the MA molecular structure and their contribution to the development of T-cell immune responses. Hereto, we used the model antigen ovalbumin and single synthetic MAs, differing in oxygenation class and cis versus trans proximal cyclopropane configuration, as immune stimulatory agents. Subcutaneous delivery of liposome-formulated MAs with a proximal cis cyclopropane elicited antigen-specific Th1 and cytotoxic T-cell immune responses, whereas intratracheal immunization elicited pulmonary Th17 responses. These immune stimulatory activities depended not only on the cis versus trans proximal cyclopropane configuration but also on the MA oxygenation class. Our study thus shows that both the presence and nature of the functional groups in the meromycolate chain affect the immune stimulatory adjuvant activity of Mtb mycolates and suggests that Mtb bacilli may impact on the host protective immune response by modulating the cis versus trans stereochemistry of its mycolates as well as by altering the oxygenation class of the meromycolate functional group. [ABSTRACT FROM AUTHOR]

While a deep understanding of the fungal and mammalian multi-enzyme type I fatty-acid synthases (FAS I) has been achieved in recent years, the bacterial FAS I family, which is narrowly distributed within the Actinomycetales genera Mycobacterium, Corynebacterium and Nocardia, is still poorly understood. This is of particular relevance for two reasons: (i) although homologous to fungal FAS I, cryo-electron microscopic studies have shown that bacterial FAS I has unique structural and functional properties, and (ii) M. tuberculosis FAS I is a drug target for the therapeutic treatment of tuberculosis (TB) and therefore is of extraordinary importance as a drug target. Crystals of FAS I from C. efficiens, a homologue of M. tuberculosis FAS I, were produced and diffracted X-rays to about 4.5 Å resolution. [ABSTRACT FROM AUTHOR]

Highlights: [•] The PorA–PorH complex forms an ion channel in C. glutamicum outer membrane. [•] Assignment of 15N labelled PorH was obtained in DMSO. [•] A specific shift on PorH Ser56 Hβ resonances revealed its O-acylation. [•] The O-mycoloylation position was confirmed by mutagenesis and MALDI-TOF MS. [Copyright &y& Elsevier]

Decaprenyl-phospho-arabinose (β-d-arabinofuranosyl-1- O-monophosphodecaprenol), the only known donor ofd-arabinose in bacteria, and its precursor, decaprenyl-phospho-ribose (β-d-ribofuranosyl-1- O-monophosphodecaprenol), were first described in 1992. En route tod-arabinofuranose, the decaprenyl-phospho-ribose 2′-epimerase converts decaprenyl-phospho-ribose to decaprenyl-phospho-arabinose, which is a substrate for arabinosyltransferases in the synthesis of the cell-wall arabinogalactan and lipoarabinomannan polysaccharides of mycobacteria. The first step of the proposed decaprenyl-phospho-arabinose biosynthesis pathway in Mycobacterium tuberculosis and related actinobacteria is the formation ofd-ribose 5-phosphate from sedoheptulose 7-phosphate, catalysed by the Rv1449 transketolase, and/or the isomerization ofd-ribulose 5-phosphate, catalysed by the Rv2465d-ribose 5-phosphate isomerase.d-Ribose 5-phosphate is a substrate for the Rv1017 phosphoribosyl pyrophosphate synthetase which forms 5-phosphoribosyl 1-pyrophosphate (PRPP). The activated 5-phosphoribofuranosyl residue of PRPP is transferred by the Rv3806 5-phosphoribosyltransferase to decaprenyl phosphate, thus forming 5′-phosphoribosyl-monophospho-decaprenol. The dephosphorylation of 5′-phosphoribosyl-monophospho-decaprenol to decaprenyl-phospho-ribose by the putative Rv3807 phospholipid phosphatase is the committed step of the pathway. A subsequent 2′-epimerization of decaprenyl-phospho-ribose by the heteromeric Rv3790/Rv3791 2′-epimerase leads to the formation of the decaprenyl-phospho-arabinose precursor for the synthesis of the cell-wall arabinans in Actinomycetales. The mycobacterial 2′-epimerase Rv3790 subunit is similar to the fungald-arabinono-1,4-lactone oxidase, the last enzyme in the biosynthesis ofd-erythroascorbic acid, thus pointing to an evolutionary link between thed-arabinofuranose- andl-ascorbic acid-related pathways. Decaprenyl-phospho-arabinose has been a lead compound for the chemical synthesis of substrates for mycobacterial arabinosyltransferases and of new inhibitors and potential antituberculosis drugs. The peculiar (ω,mono- E,octa- Z) configuration of decaprenol has yielded insights into lipid biosynthesis, and has led to the identification of the novel Z-polyprenyl diphosphate synthases of mycobacteria. Mass spectrometric methods were developed for the analysis of anomeric linkages and of dolichol phosphate-related lipids. In the field of immunology, the renaissance in mycobacterial polyisoprenoid research has led to the identification of mimetic mannosyl-β-1-phosphomycoketides of pathogenic mycobacteria as potent lipid antigens presented by CD1c proteins to human T cells. [ABSTRACT FROM AUTHOR]

Five mycolic acids [2-alkyl-3-hydroxy FA: R1C*H(OH)C*HR2COOH] were synthesized using acyl chlorides with alkyl chains of different lengths (total carbon numbers of mycolic acids, 12, 16, 20, 24, 36). The relationship between the chemical structures of the mycolic acids and their surface-active properties was determined. The acids were synthesized in three steps: (i) dimerization of acyl chloride into alkyl ketene dimer, (ii) selective reduction of C=C to C-C by hydrogenation, and (iii) β-lactone ring cleavage under alkaline conditions. The yields of C12-, C16-, C20-, C24-, and C36-mycolic acid were 72, 73, 73, 73, and 73%, respectively. The critical micelle concentrations (CMC) of C12-, C16-, and C20-mycolic acid were 2.2 x 10-4, 1.36 x 10-4, and 7.4 x 10-5 M, respectively. As the carbon number increased, the surface tension at the CMC value was also lower; the values for C12-, C16-, and C20-mycolic acid were 46.54, 43.59, and 41.57 dyn/cm, respectively. The emulsifying activities of mycolic acids were determined for n-tetradecane, n-hexadecane, cyclohexane, and diesel oil. The results showed that C12-mycolic acid was the best emulsifier for diesel oil, C16-mycolic acid was the best emulsifier for n-tetradecane and n-hexadecane, and C20-mycolic acid was the best emulsifier for cyclohexane. This study showed that mycolic acids having surface-active properties can be chemically synthesized for potential applications in the detergent/cleaning material industries, for example, in oil spill cleanup, oil recovery, textiles, pharmaceuticals, and cosmetics. [ABSTRACT FROM AUTHOR]

Mycolic acids (MAs) are α-alkyl, β-hydroxy long-chain fatty acids found in abundance in the cell envelope of the Mycobacterium tuberculosis complex (MTBC). MAs form an efficient permeability barrier, modulate host innate immune responses, and are the targets of several anti-tuberculosis drugs. Using mass spectrometry, we measured the relative abundance of 80 MA species across 36 clinical isolates of MTBC covering four major phylogenetic lineages. We found significant variations in the MA patterns between different MTBC strains and lineages. MA patterns of "ancient" lineages contrasted those from "modern" lineages, with a lower representation of alpha-mycolates among Lineage 6 strains and an inversion of the methoxy: keto-mycolates ratio in Lineage 1 strains. By interrogating the whole genome sequences of these MTBC strains, we identified relevant single-nucleotide polymorphisms that may sustain the lineage-specific MA patterns. Our results show that the strain genetic background influences MA metabolism and suggests that strain diversity should be considered in the development of new anti-tuberculosis drugs that target MA synthesis., (© 2014 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.)