Joint effects of one year of marine omega-3 fatty acid supplementation and participant dietary fish intake upon circulating lipid mediators of inflammation resolution in a randomized controlled trial.

OBJECTIVES: We assessed the joint effects of omega (n)-3 fatty acid supplementation and dietary fish intake on systemic lipid mediators of inflammation among adults. METHODS: Within VITAL, a double-blind randomized controlled trial, adults were randomized to ω-3 fatty acids (460 mg EPA + 380 mg DHA/d) or placebo. We selected participants who reported low (<1 serving/mo) baseline dietary fish intake and matched them by age, sex, race, and trial arm to participants with self-reported highest fish intake (≥3.9 servings/wk). Baseline and 1-y plasma samples were tested for 9 ω-3 fatty acid-derived lipid mediators. Multivariable linear models assessed lipid mediator changes and joint effects of ω-3 fatty acid supplementation and dietary fish intake. RESULTS: Forty-eight participants with low baseline fish intake were matched to 48 with high fish intake. Mean age was 64.6 (±7.26), 50% were female, and 85% non-Hispanic white. One-year lipid mediator changes in expected directions were observed in those receiving ω-3 fatty acids versus placebo: reductions in proinflammatory mediators, PGD2, 5-HETE, and 12-HETE; increases in proresolving mediators, EPA and DHA. Larger 1-y lipid biomarker changes were seen in those with low baseline fish intake randomized to active ω-3 fatty acids for DHA, EPA, PGD2, Resolvin D1, and Resolvin D4 were observed, although no significant multiplicative interactions were detected. DISCUSSION: Beneficial changes in circulating proresolving and proinflammatory mediators were found with 1-y of ω-3 fatty acid supplementation versus placebo for all participants, with a trend toward larger effects among those with low baseline fish intake, although interactions were not significant.

Over-the-counter fish oil supplementation and pro-resolving and pro-inflammatory lipid mediators in rheumatoid arthritis.

OBJECTIVE: Little is known about the effects of over-the-counter fish oil (FO) supplements on circulating omega-3 polyunsaturated fatty acid (n-3 PUFA)-derived specialized pro-resolving mediators (SPMs), nor about whether having a chronic inflammatory disease such as rheumatoid arthritis (RA) influences SPM levels. We investigated associations between over-the-counter n-3 PUFA FO supplementation and circulating SPMs among patients with vs. without RA. METHODS: We studied 104 participants: 26 with RA taking FO matched by age and sex to 26 with RA not taking FO, 26 without RA taking FO, and 26 without RA not taking FO. Targeted-liquid chromatography-tandem mass spectroscopy was performed on patient plasma to identify and quantify 27 lipid mediators (including eicosanoids and SPMs). We performed t-tests and then multivariable linear regression analyses to assess whether having RA or taking FO supplements was associated with circulating lipid mediator concentrations, adjusting for age, race, sex, smoking, body mass index, and current medication use (statins, prednisone and immunomodulators among RA cases only). We tested for interactions between FO supplementation and RA status. We also conducted Spearman's correlations between EPA, DHA, and ARA and their downstream metabolites. RESULTS: Among patients who were taking FO compared to those who were not, in multivariable- adjusted analyses, SPM substrates EPA and DHA were both elevated as were several of their pro-resolving bioactive products, including 15- and 18-HEPE from EPA, and 14- and 17-HDHA from DHA, which are substrates for specific SPMs. While E-series and D-series resolvins were present and identified, we did not find statistical elevations of other SPMs. Results were similar among patients with RA and patients without RA, taking vs. not taking FO supplementation (no formal statistical interaction observed). There was a strong positive correlation between EPA and DHA and their immediate downstream SPM precursors (18-HEPE and15-HEPE from EPA; 17-HDHA and 14-HDHA from DHA) among all patients. CONCLUSION: Patients taking FO supplements, regardless of RA status, not only had higher blood levels of EPA and DHA, but also of their enzymatic products 18-HEPE (E-series resolvin precursors), 15-HEPE and 17-HDHA (D-series resolvin and protectin precursors). Patients with RA, an inflammatory autoimmune disease, may be able to augment some SPM precursor reserves, similarly to matched controls without RA, by taking oral FO supplements.

Structural basis for acyl chain control over glycosphingolipid sorting and vesicular trafficking.

The complex sphingolipids exhibit a diversity of ceramide acyl chain structures that influence their trafficking and intracellular distributions, but it remains unclear how the cell discerns among the different ceramides to affect such sorting. To address the mechanism, we synthesize a library of GM1 glycosphingolipids with naturally varied acyl chains and quantitatively assess their sorting among different endocytic pathways. We find that a stretch of at least 14 saturated carbons extending from C1 at the water-bilayer interface dictate lysosomal sorting by exclusion from endosome sorting tubules. Sorting to the lysosome by the C14∗ motif is cholesterol dependent. Perturbations of the C14∗ motif by unsaturation enable GM1 entry into endosomal sorting tubules of the recycling and retrograde pathways independent of cholesterol. Unsaturation occurring beyond the C14∗ motif in very long acyl chains rescues lysosomal sorting. These results define a structural motif underlying the membrane organization of sphingolipids and implicate cholesterol-sphingolipid nanodomain formation in sorting mechanisms.

Characterization of the Uncommon Lipid Families in Corynebacterium glutamicum by Mass Spectrometry.

This book chapter provides readers the step-by-step instruction for cell growth, lipid isolation, and lipid analysis to obtain the lipidome of Corynebacterium glutamicum (C. glutamicum) in the genus Corynebacterium, a biotechnologically important bacterium. We separate the lipid families by preparative HPLC with an analytical C-8 column, followed by linear ion-trap multiple stage mass spectrometry (LIT MSn) with high-resolution mass measurement to define the structures of cytidine diphosphate diacylglycerol (CDP-DAG), glucuronosyl diacylglycerol (GlcA-DAG), α-D-mannopyranosyl-(1 â†’ 4)-α-D-glucuronyl diacylglycerol (Man-GlcA-DAG), 1-mycolyl-2-acyl-phosphatidylglycerol (MA-PG), and acyl trehalose monomycolate (acyl-TMM) whose structures have been previously mis-assigned or not defined by mass spectrometric means. We also define the structures of mycolic acid, phosphatidylglycerol, phosphatidylinositol, cardiolipin, trehalose dimycolate lipids in the cell wall. The similarity of the lipidome to that in the Mycobacterium genera is consistent with the notion that Corynebacterium and Mycobacterium are gram-positive bacteria belonging to the suborder Corynebacterineae.

Unveiling the biodiversity of lipid species in Corynebacteria- characterization of the uncommon lipid families in C. glutamicum and pathogen C. striatum by mass spectrometry.

Uncommon lipids in biotechnologically important Corynebacterium glutamicum and pathogen Corynebacterium striatum in genus Corynebacterium are isolated and identified by linear ion-trap multiple stage mass spectrometry (LIT MSn) with high resolution mass measurement. We redefined several lipid structures that were previously mis-assigned or not defined, including cytidine diphosphate diacylglycerol (CDP-DAG), glucuronosyl diacylglycerol (GlcA-DAG), (α-d-mannopyranosyl)-(1 â†’ 4)-(α-D-glucuronyl diacyglycerol (Man-GlcA-DAG), 1-mycolyl-2-acyl-phosphatidylglycerol (MA-PG), acyl trehalose monomycolate (acyl-TMM). We also report the structures of mycolic acid, phosphatidylglycerol, phosphatidylinositol, cardiolipin, trehalose dimycolate lipids in which many isomeric structures are present. The LIT MSn approaches afford identification of the functional group, the fatty acid substituents and their regiospecificity in the molecules, revealing the biodiversities of the lipid species in two Corynebacterium strains that have played very different and important roles in human nutrition and health.

Palmitic Acid-Rich High-Fat Diet Exacerbates Experimental Pulmonary Fibrosis by Modulating Endoplasmic Reticulum Stress.

The impact of lipotoxicity on the development of lung fibrosis is unclear. Saturated fatty acids, such as palmitic acid (PA), activate endoplasmic reticulum (ER) stress, a cellular stress response associated with the development of idiopathic pulmonary fibrosis (IPF). We tested the hypothesis that PA increases susceptibility to lung epithelial cell death and experimental fibrosis by modulating ER stress. Total liquid chromatography and mass spectrometry were used to measure fatty acid content in IPF lungs. Wild-type mice were fed a high-fat diet (HFD) rich in PA or a standard diet and subjected to bleomycin-induced lung injury. Lung fibrosis was determined by hydroxyproline content. Mouse lung epithelial cells were treated with PA. ER stress and cell death were assessed by Western blotting, TUNEL staining, and cell viability assays. IPF lungs had a higher level of PA compared with controls. Bleomycin-exposed mice fed an HFD had significantly increased pulmonary fibrosis associated with increased cell death and ER stress compared with those fed a standard diet. PA increased apoptosis and activation of the unfolded protein response in lung epithelial cells. This was attenuated by genetic deletion and chemical inhibition of CD36, a fatty acid transporter. In conclusion, consumption of an HFD rich in saturated fat increases susceptibility to lung fibrosis and ER stress, and PA mediates lung epithelial cell death and ER stress via CD36. These findings demonstrate that lipotoxicity may have a significant impact on the development of lung injury and fibrosis by enhancing pro-death ER stress pathways.

Pathogenic mycobacteria achieve cellular persistence by inhibiting the Niemann-Pick Type C disease cellular pathway.

BACKGROUND: Tuberculosis remains a major global health concern. The ability to prevent phagosome-lysosome fusion is a key mechanism by which intracellular mycobacteria, including Mycobacterium tuberculosis, achieve long-term persistence within host cells. The mechanisms underpinning this key intracellular pro-survival strategy remain incompletely understood. Host macrophages infected with persistent mycobacteria share phenotypic similarities with cells taken from patients suffering from Niemann-Pick Disease Type C (NPC), a rare lysosomal storage disease in which endocytic trafficking defects and lipid accumulation within the lysosome lead to cell dysfunction and cell death. We investigated whether these shared phenotypes reflected an underlying mechanistic connection between mycobacterial intracellular persistence and the host cell pathway dysfunctional in NPC. METHODS: The induction of NPC phenotypes in macrophages from wild-type mice or obtained from healthy human donors was assessed via infection with mycobacteria and subsequent measurement of lipid levels and intracellular calcium homeostasis. The effect of NPC therapeutics on intracellular mycobacterial load was also assessed. RESULTS: Macrophages infected with persistent intracellular mycobacteria phenocopied NPC cells, exhibiting accumulation of multiple lipid types, reduced lysosomal Ca2+ levels, and defects in intracellular trafficking. These NPC phenotypes could also be induced using only lipids/glycomycolates from the mycobacterial cell wall. These data suggest that persistent intracellular mycobacteria inhibit the NPC pathway, likely via inhibition of the NPC1 protein, and subsequently induce altered acidic store Ca2+ homeostasis. Reduced lysosomal calcium levels may provide a mechanistic explanation for the reduced levels of phagosome-lysosome fusion in mycobacterial infection. Treatments capable of correcting defects in NPC mutant cells via modulation of host cell calcium were of benefit in promoting clearance of mycobacteria from infected host cells. CONCLUSION: These findings provide a novel mechanistic explanation for mycobacterial intracellular persistence, and suggest that targeting interactions between the mycobacteria and host cell pathways may provide a novel avenue for development of anti-TB therapies.

Human autoreactive T cells recognize CD1b and phospholipids.

In contrast with the common detection of T cells that recognize MHC, CD1a, CD1c, or CD1d proteins, CD1b autoreactive T cells have been difficult to isolate in humans. Here we report the development of polyvalent complexes of CD1b proteins and carbohydrate backbones (dextramers) and their use in identifying CD1b autoreactive T cells from human donors. Activation is mediated by αß T-cell receptors (TCRs) binding to CD1b-phospholipid complexes, which is sufficient to activate autoreactive responses to CD1b-expressing cells. Using mass spectrometry and T-cell responses to scan through the major classes of phospholipids, we identified phosphatidylglycerol (PG) as the immunodominant lipid antigen. T cells did not discriminate the chemical differences that distinguish mammalian PG from bacterial PG. Whereas most models of T-cell recognition emphasize TCR discrimination of differing self and foreign structures, CD1b autoreactive T cells recognize lipids with dual self and foreign origin. PG is rare in the cellular membranes that carry CD1b proteins. However, bacteria and mitochondria are rich in PG, so these data point to a more general mechanism of immune detection of infection- or stress-associated lipids.

Identification of a Potent Microbial Lipid Antigen for Diverse NKT Cells.

Semi-invariant/type I NKT cells are a well-characterized CD1d-restricted T cell subset. The availability of potent Ags and tetramers for semi-invariant/type I NKT cells allowed this population to be extensively studied and revealed their central roles in infection, autoimmunity, and tumor immunity. In contrast, diverse/type II NKT (dNKT) cells are poorly understood because the lipid Ags that they recognize are largely unknown. We sought to identify dNKT cell lipid Ag(s) by interrogating a panel of dNKT mouse cell hybridomas with lipid extracts from the pathogen Listeria monocytogenes. We identified Listeria phosphatidylglycerol as a microbial Ag that was significantly more potent than a previously characterized dNKT cell Ag, mammalian phosphatidylglycerol. Further, although mammalian phosphatidylglycerol-loaded CD1d tetramers did not stain dNKT cells, the Listeria-derived phosphatidylglycerol-loaded tetramers did. The structure of Listeria phosphatidylglycerol was distinct from mammalian phosphatidylglycerol because it contained shorter, fully-saturated anteiso fatty acid lipid tails. CD1d-binding lipid-displacement studies revealed that the microbial phosphatidylglycerol Ag binds significantly better to CD1d than do counterparts with the same headgroup. These data reveal a highly potent microbial lipid Ag for a subset of dNKT cells and provide an explanation for its increased Ag potency compared with the mammalian counterpart.

Characterization of polar lipids of Listeria monocytogenes by HCD and low-energy CAD linear ion-trap mass spectrometry with electrospray ionization.

Listeria monocytogenes (L. monocytogenes) is a facultative, Gram-positive, food-borne bacterium, which causes serious infections. Although it is known that lipids play important roles in the survival of Listeria, the detailed structures of these lipids have not been established. In this contribution, we described linear ion-trap multiple-stage mass spectrometric approaches with high-resolution mass spectrometry toward complete structural analysis including the identities of the fatty acid substituents and their position on the glycerol backbone of the polar lipids, mainly phosphatidylglycerol, cardiolipin (CL), and lysyl-CL from L. monocytogenes. The location of the methyl side group along the fatty acid chain in each lipid family was characterized by a charge-switch strategy. This is achieved by first alkaline hydrolysis to release the fatty acid substituents, followed by tandem mass spectrometry on their N-(4-aminomethylphenyl) pyridinium (AMPP) derivatives as the M+ ions. Several findings in this study are unique: (1) we confirm the presence of a plasmalogen PG family that has not been previous reported; (2) an ion arising from a rare internal loss of lysylglycerol residue was observed in the MS(2) spectrum of lysyl-CL, permitting its distinction from other CL subfamilies.

Activation of iNKT cells by a distinct constituent of the endogenous glucosylceramide fraction.

Invariant natural killer T (iNKT) cells are a specialized T-cell subset that recognizes lipids as antigens, contributing to immune responses in diverse disease processes. Experimental data suggests that iNKT cells can recognize both microbial and endogenous lipid antigens. Several candidate endogenous lipid antigens have been proposed, although the contextual role of specific antigens during immune responses remains largely unknown. We have previously reported that mammalian glucosylceramides (GlcCers) activate iNKT cells. GlcCers are found in most mammalian tissues, and exist in variable molecular forms that differ mainly in N-acyl fatty acid chain use. In this report, we purified, characterized, and tested the GlcCer fractions from multiple animal species. Although activity was broadly identified in these GlcCer fractions from mammalian sources, we also found activity properties that could not be reconciled by differences in fatty acid chain use. Enzymatic digestion of ß-GlcCer and a chromatographic separation method demonstrated that the activity in the GlcCer fraction was limited to a rare component of this fraction, and was not contained within the bulk of ß-GlcCer molecular species. Our data suggest that a minor lipid species that copurifies with ß-GlcCer in mammals functions as a lipid self antigen for iNKT cells.

Recognition of microbial and mammalian phospholipid antigens by NKT cells with diverse TCRs.

CD1d-restricted natural killer T (NKT) cells include two major subgroups. The most widely studied are Vα14Jα18(+) invariant NKT (iNKT) cells that recognize the prototypical α-galactosylceramide antigen, whereas the other major group uses diverse T-cell receptor (TCR) α-and ß-chains, does not recognize α-galactosylceramide, and is referred to as diverse NKT (dNKT) cells. dNKT cells play important roles during infection and autoimmunity, but the antigens they recognize remain poorly understood. Here, we identified phosphatidylglycerol (PG), diphosphatidylglycerol (DPG, or cardiolipin), and phosphatidylinositol from Mycobacterium tuberculosis or Corynebacterium glutamicum as microbial antigens that stimulated various dNKT, but not iNKT, hybridomas. dNKT hybridomas showed distinct reactivities for diverse antigens. Stimulation of dNKT hybridomas by microbial PG was independent of Toll-like receptor-mediated signaling by antigen-presenting cells and required lipid uptake and/or processing. Furthermore, microbial PG bound to CD1d molecules and plate-bound PG/CD1d complexes stimulated dNKT hybridomas, indicating direct recognition by the dNKT cell TCR. Interestingly, despite structural differences in acyl chain composition between microbial and mammalian PG and DPG, lipids from both sources stimulated dNKT hybridomas, suggesting that presentation of microbial lipids and enhanced availability of stimulatory self-lipids may both contribute to dNKT cell activation during infection.

Saposins utilize two strategies for lipid transfer and CD1 antigen presentation.

Transferring lipid antigens from membranes into CD1 antigen-presenting proteins represents a major molecular hurdle necessary for T-cell recognition. Saposins facilitate this process, but the mechanisms used are not well understood. We found that saposin B forms soluble saposin protein-lipid complexes detected by native gel electrophoresis that can directly load CD1 proteins. Because saposin B must bind lipids directly to function, we found it could not accommodate long acyl chain containing lipids. In contrast, saposin C facilitates CD1 lipid loading in a different way. It uses a stable, membrane-associated topology and was capable of loading lipid antigens without forming soluble saposin-lipid antigen complexes. These findings reveal how saposins use different strategies to facilitate transfer of structurally diverse lipid antigens.

Structural elucidation of diglycosyl diacylglycerol and monoglycosyl diacylglycerol from Streptococcus pneumoniae by multiple-stage linear ion-trap mass spectrometry with electrospray ionization.

The cell wall of the pathogenic bacterium Streptococcus pneumoniae contains glucopyranosyl diacylglycerol (GlcDAG) and galactoglucopyranosyldiacylglycerol (GalGlcDAG). The specific GlcDAG consisting of vaccenic acid substituent at sn-2 was recently identified as another glycolipid antigen family recognized by invariant natural killer T-cells. Here, we describe a linear ion-trap multiple-stage (MS(n) ) mass spectrometric approach towards structural analysis of GalGlcDAG and GlcDAG. Structural information derived from MS(n) (n = 2, 3) on the [M + Li](+) adduct ions desorbed by electrospray ionization affords identification of the fatty acid substituents, assignment of the fatty acyl groups on the glycerol backbone, as well as the location of double bond along the fatty acyl chain. The identification of the fatty acyl groups and determination of their regio-specificity were confirmed by MS(n) (n = 2, 3) on the [M + NH(4) ](+) ions. We establish the structures of GalGlcDAG and GlcDAG isolated from S. pneumoniae, in which the major species consists of a 16:1- or 18:1-fatty acid substituent mainly at sn-2, and the double bond of the fatty acid is located at ω-7 (n-7). More than one isomers were found for each mass in the family. This mass spectrometric approach provides a simple method to achieve structure identification of this important lipid family that would be very difficult to define using the traditional method.

Invariant natural killer T cells recognize lipid self antigen induced by microbial danger signals.

Invariant natural killer T cells (iNKT cells) have a prominent role during infection and other inflammatory processes, and these cells can be activated through their T cell antigen receptors by microbial lipid antigens. However, increasing evidence shows that they are also activated in situations in which foreign lipid antigens would not be present, which suggests a role for lipid self antigen. We found that an abundant endogenous lipid, ß-D-glucopyranosylceramide (ß-GlcCer), was a potent iNKT cell self antigen in mouse and human and that its activity depended on the composition of the N-acyl chain. Furthermore, ß-GlcCer accumulated during infection and in response to Toll-like receptor agonists, contributing to iNKT cell activation. Thus, we propose that recognition of ß-GlcCer by the invariant T cell antigen receptor translates innate danger signals into iNKT cell activation.

Innate recognition of cell wall ß-glucans drives invariant natural killer T cell responses against fungi.

iNKT cells are innate T lymphocytes recognizing endogenous and foreign lipid antigens presented in the MHC-like molecule CD1d. The semi-invariant iNKT cell TCR can detect certain bacterial and parasitic lipids and drive iNKT cell responses. How iNKT cells respond to fungi, however, is unknown. We found that CD1d-deficient mice, which lack iNKT cells, poorly control infection with the fungal pathogen Aspergillus fumigatus. Furthermore, A. fumigatus rapidly activates iNKT cells in vivo and in vitro in the presence of APCs. Surprisingly, despite a requirement for CD1d recognition, the antifungal iNKT cell response does not require fungal lipids. Instead, Dectin-1- and MyD88-mediated responses to ß-1,3 glucans, major fungal cell-wall polysaccharides, trigger IL-12 production by APCs that drives self-reactive iNKT cells to secrete IFN-γ. Innate recognition of ß-1,3 glucans also drives iNKT cell responses against Candida, Histoplasma, and Alternaria, suggesting that this mechanism may broadly define the basis for antifungal iNKT cell responses.

Innate and cytokine-driven signals, rather than microbial antigens, dominate in natural killer T cell activation during microbial infection.

Invariant natural killer T cells (iNKT cells) are critical for host defense against a variety of microbial pathogens. However, the central question of how iNKT cells are activated by microbes has not been fully explained. The example of adaptive MHC-restricted T cells, studies using synthetic pharmacological α-galactosylceramides, and the recent discovery of microbial iNKT cell ligands have all suggested that recognition of foreign lipid antigens is the main driver for iNKT cell activation during infection. However, when we compared the role of microbial antigens versus innate cytokine-driven mechanisms, we found that iNKT cell interferon-γ production after in vitro stimulation or infection with diverse bacteria overwhelmingly depended on toll-like receptor-driven IL-12. Importantly, activation of iNKT cells in vivo during infection with Sphingomonas yanoikuyae or Streptococcus pneumoniae, pathogens which are known to express iNKT cell antigens and which require iNKT cells for effective protection, also predominantly depended on IL-12. Constitutive expression of high levels of IL-12 receptor by iNKT cells enabled instant IL-12-induced STAT4 activation, demonstrating that among T cells, iNKT cells are uniquely equipped for immediate, cytokine-driven activation. These findings reveal that innate and cytokine-driven signals, rather than cognate microbial antigen, dominate in iNKT cell activation during microbial infections.

Regulation of CD1 antigen-presenting complex stability.

For major histocompatibility complex class I and II molecules, the binding of specific peptide antigens is essential for assembly and trafficking and is at the center of their quality control mechanism. However, the role of lipid antigen binding in stabilization and quality control of CD1 heavy chain (HC).beta(2)-microglobulin (beta(2)m) complexes is unclear. Furthermore, the distinct trafficking and loading routes of CD1 proteins take them from mildly acidic pH in early endososmal compartments (pH 6.0) to markedly acidic pH in lysosomes (pH 5.0) and back to neutral pH of the cell surface (pH 7.4). Here, we present evidence that the stability of each CD1 HC.beta(2)m complex is determined by the distinct pH optima identical to that of the intracellular compartments in which each CD1 isoform resides. Although stable at acidic endosomal pH, complexes are only stable at cell surface pH 7.4 when bound to specific lipid antigens. The proposed model outlines a quality control program that allows lipid exchange at low endosomal pH without dissociation of the CD1 HC.beta(2)m complex and then stabilizes the antigen-loaded complex at neutral pH at the cell surface.

Identification of an alpha(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 alpha(1-->6) and alpha(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 beta-D-mannopyranosyl-1-monophosphoryl-decaprenol together with the neoglycolipid acceptor alpha-D-Manp-(1-->6)-alpha-D-Manp-O-C8 as a substrate, confirmed NCgl2093 and Rv2174 as an alpha(1-->6) mannopyranosyltransferase (MptA), involved in the latter stages of the biosynthesis of the alpha(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.