RESUMEN
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.
Asunto(s)
Autoantígenos/inmunología , Infecciones Bacterianas/inmunología , Glicoesfingolípidos/inmunología , Células T Asesinas Naturales/inmunología , Animales , Autoinmunidad/inmunología , Línea Celular , Glicoesfingolípidos/metabolismo , Humanos , Activación de Linfocitos/inmunología , Tejido Linfoide/inmunología , Tejido Linfoide/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Receptores de Antígenos de Linfocitos T/inmunología , Receptores de Antígenos de Linfocitos T/metabolismoRESUMEN
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.
Asunto(s)
Dieta Alta en Grasa/efectos adversos , Estrés del Retículo Endoplásmico/efectos de los fármacos , Ácido Palmítico/toxicidad , Fibrosis Pulmonar/inducido químicamente , Animales , Apoptosis/efectos de los fármacos , Antígenos CD36/deficiencia , Antígenos CD36/fisiología , Células Epiteliales/efectos de los fármacos , Pulmón/metabolismo , Pulmón/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ácido Palmítico/administración & dosificación , Ácido Palmítico/farmacocinética , Fibrosis Pulmonar/metabolismo , Fibrosis Pulmonar/patologíaRESUMEN
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.
Asunto(s)
Antígenos CD1/metabolismo , Fosfolípidos/metabolismo , Células Presentadoras de Antígenos/inmunología , Células HEK293 , Humanos , Células K562 , Activación de Linfocitos/inmunología , Espectrometría de Masas , Fosfatidilgliceroles/química , Linfocitos T/inmunología , TransfecciónRESUMEN
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.
Asunto(s)
Antígenos/inmunología , Listeria monocytogenes/inmunología , Lípidos de la Membrana/inmunología , Células T Asesinas Naturales/inmunología , Fosfatidilgliceroles/inmunología , Animales , Antígenos CD1d/inmunología , Línea Celular , Hibridomas/inmunología , Ratones , Subgrupos de Linfocitos T/inmunologíaRESUMEN
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.
Asunto(s)
Glucosilceramidas/farmacología , Activación de Linfocitos/efectos de los fármacos , Células T Asesinas Naturales/inmunología , Animales , Bovinos , Cromatografía en Capa Delgada , Glucosilceramidasa/metabolismo , Glucosilceramidas/química , Humanos , Activación de Linfocitos/inmunología , Espectrometría de Masas , Ratones , Leche/química , Células T Asesinas Naturales/efectos de los fármacosRESUMEN
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.
Asunto(s)
Antígenos Bacterianos/inmunología , Células T Asesinas Naturales/inmunología , Fosfolípidos/inmunología , Receptores de Antígenos de Linfocitos T alfa-beta/inmunología , Animales , Células Presentadoras de Antígenos/inmunología , Células Presentadoras de Antígenos/metabolismo , Antígenos Bacterianos/metabolismo , Antígenos CD1d/genética , Antígenos CD1d/inmunología , Antígenos CD1d/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/inmunología , Proteínas Bacterianas/metabolismo , Cardiolipinas/inmunología , Cardiolipinas/metabolismo , Línea Celular , Células Cultivadas , Corynebacterium glutamicum/genética , Corynebacterium glutamicum/inmunología , Corynebacterium glutamicum/metabolismo , Galactosilceramidas/inmunología , Galactosilceramidas/metabolismo , Hibridomas/inmunología , Hibridomas/metabolismo , Macrófagos/inmunología , Macrófagos/metabolismo , Macrófagos/microbiología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mycobacterium tuberculosis/inmunología , Mycobacterium tuberculosis/metabolismo , Factor 88 de Diferenciación Mieloide/genética , Factor 88 de Diferenciación Mieloide/inmunología , Factor 88 de Diferenciación Mieloide/metabolismo , Células T Asesinas Naturales/metabolismo , Fosfatidilgliceroles/inmunología , Fosfatidilgliceroles/metabolismo , Fosfolípidos/metabolismo , Receptores de Antígenos de Linfocitos T alfa-beta/metabolismo , Receptores Toll-Like/inmunología , Receptores Toll-Like/metabolismo , Transferasas (Grupos de Otros Fosfatos Sustitutos)/genética , Transferasas (Grupos de Otros Fosfatos Sustitutos)/inmunología , Transferasas (Grupos de Otros Fosfatos Sustitutos)/metabolismoRESUMEN
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.
Asunto(s)
Lípidos/química , Listeria monocytogenes/química , Espectrometría de Masas en Tándem/métodos , Metabolismo de los Lípidos , Listeria monocytogenes/metabolismo , Estructura Molecular , Espectrometría de Masa por Ionización de Electrospray/instrumentación , Espectrometría de Masa por Ionización de Electrospray/métodos , Espectrometría de Masas en Tándem/instrumentaciónRESUMEN
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.
Asunto(s)
Antígenos CD1/metabolismo , Saposinas/química , Saposinas/fisiología , Linfocitos T/citología , Animales , Células CHO , Cricetinae , Electroforesis en Gel de Agar , Glucolípidos/química , Humanos , Concentración de Iones de Hidrógeno , Células Asesinas Naturales/citología , Membrana Dobles de Lípidos/metabolismo , Lípidos/química , Liposomas/química , Modelos Biológicos , Proteínas Recombinantes/químicaRESUMEN
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.
Asunto(s)
Presentación de Antígeno , Antígenos CD1/metabolismo , Animales , Antígenos CD1/química , Antígenos CD1/genética , Línea Celular , Membrana Celular/inmunología , Membrana Celular/metabolismo , Endosomas/inmunología , Endosomas/metabolismo , Células HeLa , Antígenos de Histocompatibilidad Clase I/química , Antígenos de Histocompatibilidad Clase I/metabolismo , Antígenos de Histocompatibilidad Clase II/química , Antígenos de Histocompatibilidad Clase II/metabolismo , Humanos , Concentración de Iones de Hidrógeno , Lisosomas/inmunología , Lisosomas/metabolismo , Ratones , Complejos Multiproteicos , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Estabilidad Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transfección , Microglobulina beta-2/química , Microglobulina beta-2/metabolismoRESUMEN
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.
Asunto(s)
Corynebacterium glutamicum/crecimiento & desarrollo , Lipidómica/métodos , Lípidos/análisis , Técnicas Bacteriológicas , Cromatografía Líquida de Alta Presión , Corynebacterium glutamicum/química , Estructura Molecular , Espectrometría de Masa por Ionización de ElectrosprayRESUMEN
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.
Asunto(s)
Corynebacterium glutamicum/química , Corynebacterium/química , Lípidos/química , Lípidos/aislamiento & purificación , Factores Cordón/química , Diglicéridos/química , Humanos , Metabolismo de los Lípidos , Lípidos/clasificación , Fosfatidilgliceroles/química , Espectrometría de Masa por Ionización de ElectrosprayRESUMEN
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.
Asunto(s)
Aspergilosis/inmunología , Aspergillus fumigatus/inmunología , Pared Celular/inmunología , Células T Asesinas Naturales/inmunología , Células T Asesinas Naturales/microbiología , beta-Glucanos/inmunología , Animales , Células Presentadoras de Antígenos/inmunología , Antígenos CD1d/inmunología , Aspergilosis/microbiología , Ratones , Ratones Endogámicos C57BL , Ratones NoqueadosRESUMEN
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.
Asunto(s)
Antígenos Bacterianos/metabolismo , Citocinas/metabolismo , Células T Asesinas Naturales/microbiología , Animales , Femenino , Galactosilceramidas/química , Interferón gamma/metabolismo , Interleucina-12/metabolismo , Ligandos , Activación de Linfocitos , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Células T Asesinas Naturales/metabolismo , Fosforilación , Factor de Transcripción STAT4/metabolismo , Transducción de Señal , Espectrometría de Masa por Ionización de Electrospray/métodos , Sphingomonas/metabolismo , Streptococcus pneumoniae/metabolismoRESUMEN
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.
Asunto(s)
Proteínas Bacterianas/metabolismo , Corynebacterium glutamicum/enzimología , Lipopolisacáridos/biosíntesis , Manosiltransferasas/metabolismo , Mycobacterium tuberculosis/enzimología , Homología de Secuencia de Aminoácido , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Membrana Celular/enzimología , Corynebacterium glutamicum/genética , Corynebacterium glutamicum/crecimiento & desarrollo , Genoma Bacteriano , Glucolípidos/biosíntesis , Glucolípidos/química , Lipopolisacáridos/química , Lipopolisacáridos/aislamiento & purificación , Manosiltransferasas/química , Datos de Secuencia Molecular , Mutación/genética , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Especificidad por SustratoRESUMEN
Mycobacterium tuberculosis PimB has been demonstrated to catalyze the addition of a mannose residue from GDP-mannose to a monoacylated phosphatidyl-myo-inositol mannoside (Ac(1)PIM(1)) to generate Ac(1)PIM(2). Herein, we describe the disruption of its probable orthologue Cg-pimB and the chemical analysis of glycolipids and lipoglycans isolated from wild type Corynebacterium glutamicum and the C. glutamicum::pimB mutant. Following a careful analysis, two related glycolipids, Gl-A and Gl-X, were found in the parent strain, but Gl-X was absent from the mutant. The biosynthesis of Gl-X was restored in the mutant by complementation with either Cg-pimB or Mt-pimB. Subsequent chemical analyses established Gl-X as 1,2-di-O-C(16)/C(18:1)-(alpha-d-mannopyranosyl)-(1-->4)-(alpha-d-glucopyranosyluronic acid)-(1-->3)-glycerol (ManGlcAGroAc(2)) and Gl-A as the precursor, GlcAGroAc(2). In addition, C. glutamicum::pimB was still able to produce Ac(1)PIM(2), suggesting that Cg-PimB catalyzes the synthesis of ManGlcAGroAc(2) from GlcAGroAc(2). Isolation of lipoglycans from C. glutamicum led to the identification of two related lipoglycans. The larger lipoglycan possessed a lipoarabinomannan-like structure, whereas the smaller lipoglycan was similar to lipomannan (LM). The absence of ManGlcA-GroAc(2) in C. glutamicum::pimB led to a severe reduction in LM. These results suggested that ManGlcAGroAc(2) was further extended to an LM-like molecule. Complementation of C. glutamicum::pimB with Cg-pimB and Mt-pimB led to the restoration of LM biosynthesis. As a result, Cg-PimB, which we have assigned as MgtA, is now clearly defined as a GDP-mannose-dependent alpha-mannosyltransferase from our in vitro analyses and is involved in the biosynthesis of ManGlcAGroAc(2).