RESUMEN
Glycosylphosphatidylinositol (GPI) is a complex glycolipid structure that acts as a membrane anchor for many cell-surface proteins of eukaryotes. GPI-anchored proteins are particularly abundant in protozoa and represent the major carbohydrate modification of many cell-surface parasite proteins. A minimal GPI-anchor precursor consists of core glycan (ethanolamine-PO4-Manα1-2Manα1-6Manα1-4GlcNH2) linked to the 6-position of the D-myo-inositol ring of phosphatidylinositol. Although the GPI core glycan is conserved in all organisms, many differences in additional modifications to GPI structures and biosynthetic pathways have been reported. The preassembled GPI-anchor precursor is post-translationally transferred to a variety of membrane proteins in the lumen of the endoplasmic reticulum in a transamidase-like reaction during which a C-terminal GPI attachment signal is released. Increasing evidence shows that a significant proportion of the synthesized GPIs are not used for protein anchoring, particularly in protozoa in which a large amount of free GPIs are being displayed at the cell surface. The characteristics of GPI biosynthesis are currently being explored for the development of parasite-specific inhibitors. Especially this pathway, at least for Trypanosoma brucei, has been validated as a drug target. Furthermore, thanks to an increase of new innovative strategies to produce pure synthetic carbohydrates, a novel era in the use of GPIs in diagnostic, anti-GPI antibody production, as well as parasitic protozoa GPI-based vaccine approach is developing fast.
Asunto(s)
Glicosilfosfatidilinositoles/química , Parásitos/química , Animales , Glucolípidos/química , Glicosilfosfatidilinositoles/metabolismo , Interacciones Hidrofóbicas e Hidrofílicas , Redes y Vías Metabólicas , Parásitos/metabolismo , Fosforilación , Plasmodium falciparum/química , Polisacáridos , Coloración y Etiquetado , Relación Estructura-ActividadRESUMEN
Glycosylphosphatidylinositol (GPI) anchor of Plasmodium falciparum origin is considered an important toxin leading to severe malaria pathology through stimulation of pro-inflammatory responses from innate immune cells. Even though the GPI-induced immune response is widely described to be mediated by pattern recognition receptors such as TLR2 and TLR4, previous studies have revealed that these two receptors are dispensable for the development of severe malaria pathology. Therefore, this study aimed at the identification of potential alternative Plasmodium GPI receptors. Herein, we have identified the host protein moesin as an interaction partner of Plasmodium GPI in vitro. Given previous reports indicating the relevance of moesin especially in the LPS-mediated induction of pro-inflammatory responses, we have conducted a series of in vitro and in vivo experiments to address the physiological relevance of the moesin-Plasmodium GPI interaction in the context of malaria pathology. We report here that although moesin and Plasmodium GPI interact in vitro, moesin is not critically involved in processes leading to Plasmodium-induced pro-inflammatory immune responses or malaria-associated cerebral pathology.
Asunto(s)
Glicosilfosfatidilinositoles/metabolismo , Interacciones Huésped-Parásitos/fisiología , Proteínas de Microfilamentos/metabolismo , Plasmodium/metabolismo , Plasmodium/patogenicidad , Animales , Células de la Médula Ósea , Quimiocinas/metabolismo , Citocinas/metabolismo , Femenino , Glicosilfosfatidilinositoles/química , Glicosilfosfatidilinositoles/genética , Glicosilfosfatidilinositoles/inmunología , Interacciones Huésped-Parásitos/inmunología , Humanos , Inmunidad Innata , Malaria/genética , Malaria/parasitología , Malaria/patología , Malaria Cerebral , Ratones , Ratones Endogámicos C57BL , Proteínas de Microfilamentos/genética , Fagocitosis , Plasmodium berghei/metabolismo , Plasmodium berghei/patogenicidad , Plasmodium falciparum , Transducción de Señal , Células THP-1RESUMEN
Vaccination against the ubiquitous parasite Toxoplasma gondii would provide the most efficient prevention against toxoplasmosis-related congenital, brain and eye diseases in humans. We investigated the immune response elicited by pathogen-specific glycosylphosphatidylinositol (GPI) glycoconjugates using carbohydrate microarrays in a BALB/c mouse model. We further examined the protective properties of the glycoconjugates in a lethal challenge model using the virulent T. gondii RH strain. Upon immunization, mice raised antibodies that bind to the respective GPIs on carbohydrate microarrays, but were mainly directed against an unspecific GPI epitope including the linker. The observed immune response, though robust, was unable to provide protection in mice when challenged with a lethal dose of viable tachyzoites. We demonstrate that anti-GPI antibodies raised against the here described semi-synthetic glycoconjugates do not confer protective immunity against T. gondii in BALB/c mice.
Asunto(s)
Anticuerpos Antiprotozoarios/inmunología , Glicosilfosfatidilinositoles/inmunología , Vacunas Antiprotozoos/inmunología , Toxoplasma/inmunología , Animales , Epítopos/inmunología , Femenino , Ratones , Ratones Endogámicos BALB CRESUMEN
Around 2 billion people worldwide are infected with the apicomplexan parasite Toxoplasma gondii which induces a variety of medical conditions. For example, primary infection during pregnancy can result in fetal death or mental retardation of the child. Diagnosis of acute infections in pregnant women is challenging but crucially important as the drugs used to treat T.â gondii infections are potentially harmful to the unborn child. Better, faster, more reliable, and cheaper means of diagnosis by using defined antigens for accurate serological tests are highly desirable. Synthetic pathogen-specific glycosylphosphatidylinositol (GPI) glycan antigens are diagnostic markers and have been used to distinguish between toxoplasmosis disease states using human sera.
Asunto(s)
Glicosilfosfatidilinositoles , Polisacáridos/química , Toxoplasmosis/diagnóstico , Secuencia de Carbohidratos , Glicosilfosfatidilinositoles/química , Humanos , Datos de Secuencia MolecularRESUMEN
We introduce a novel class of membrane active peptidomimetics, the amphiphilic cationic ß(3R3)-peptides, and evaluate their potential as antimicrobial agents. The design criteria, the building block and oligomer synthesis as well as a detailed structure-activity relationship (SAR) study are reported. Specifically, infrared reflection absorption spectroscopy (IRRAS) was employed to investigate structural features of amphiphilic cationic ß(3R3)-peptide sequences at the hydrophobic/hydrophilic air/liquid interface. Furthermore, Langmuir monolayers of anionic and zwitterionic phospholipids have been used to model the interactions of amphiphilic cationic ß(3R3)-peptides with prokaryotic and eukaryotic cellular membranes in order to predict their membrane selectivity and elucidate their mechanism of action. Lastly, antimicrobial activity was tested against Gram-positive M. luteus and S. aureus as well as against Gram-negative E. coli and P. aeruginosa bacteria along with testing hemolytic activity and cytotoxicity. We found that amphiphilic cationic ß(3R3)-peptide sequences combine high and selective antimicrobial activity with exceptionally low cytotoxicity in comparison to values reported in the literature. Overall, this study provides further insights into the SAR of antimicrobial peptides and peptidomimetics and indicates that amphiphilic cationic ß(3R3)-peptides are strong candidates for further development as antimicrobial agents with high therapeutic index.
Asunto(s)
Antibacterianos/farmacología , Membrana Celular/efectos de los fármacos , Péptidos/farmacología , Peptidomiméticos , Tensoactivos/farmacología , Antibacterianos/síntesis química , Antibacterianos/química , Cationes/síntesis química , Cationes/química , Cationes/farmacología , Membrana Celular/metabolismo , Relación Dosis-Respuesta a Droga , Escherichia coli/efectos de los fármacos , Pruebas de Sensibilidad Microbiana , Micrococcus luteus/efectos de los fármacos , Estructura Molecular , Péptidos/síntesis química , Péptidos/química , Pseudomonas aeruginosa/efectos de los fármacos , Staphylococcus aureus/efectos de los fármacos , Relación Estructura-Actividad , Tensoactivos/síntesis química , Tensoactivos/químicaRESUMEN
Glycosylphosphatidylinositols (GPIs) from several protozoan parasites are thought to elicit a detrimental stimulation of the host innate immune system aside their main function to anchor surface proteins. Here we analyzed the GPI biosynthesis of an avirulent Toxoplasma gondii type 2 strain (PTG) by metabolic radioactive labeling. We determined the biological function of individual GPI species in the PTG strain in comparison with previously characterized GPI-anchors of a virulent strain (RH). The GPI intermediates of both strains were structurally similar, however the abundance of two of six GPI intermediates was significantly reduced in the PTG strain. The side-by-side comparison of GPI-anchor content revealed that the PTG strain had only â¼ 34% of the protein-free GPIs as well as â¼ 70% of the GPI-anchored proteins with significantly lower rates of protein N-glycosylation compared to the RH strain. All mature GPIs from both strains induced comparable secretion levels of TNF-α and IL-12p40, and initiated TLR4/MyD88-dependent NF-κBp65 activation in macrophages. Taken together, these results demonstrate that PTG and RH strains differ in their GPI biosynthesis and possess significantly different GPI-anchor content, while individual GPI species of both strains induce similar biological functions in macrophages.
Asunto(s)
Glicosilfosfatidilinositoles/metabolismo , Macrófagos/parasitología , Toxoplasma/metabolismo , Toxoplasma/patogenicidad , Animales , Línea Celular , Chlorocebus aethiops , Subunidad p40 de la Interleucina-12/metabolismo , Ratones , Factor de Necrosis Tumoral alfa/metabolismo , Células VeroRESUMEN
Here, we present a new microparticle system for the selective detection and magnetic removal of bacteria from contaminated solutions. The novelty of this system lies in the combination of a biocompatible scaffold reducing unspecific interactions with high capacity for bacteria binding. We apply highly porous poly(ethylene glycol) (PEG) microparticles and functionalize them, introducing both sugar ligands for specific bacteria targeting and cationic moieties for electrostatic loading of superparamagnetic iron oxide nanoparticles. The resulting magnetic, porous, sugar-functionalized (MaPoS) PEG microgels are able to selectively bind and discriminate between different strains of bacteria Escherichia coli . Furthermore, they allow for a highly efficient removal of bacteria from solution as their increased surface area can bind three times more bacteria than nonporous particles. All in all, MaPoS particles represent a novel generation of magnetic beads introducing for the first time a porous, biocompatible and easy to functionalize scaffold and show great potential for various biotechnological applications.
Asunto(s)
Bacterias/aislamiento & purificación , Carbohidratos/química , Geles , Magnetismo , Polietilenglicoles/metabolismo , Microscopía Electrónica de Transmisión , Nanopartículas , SolucionesRESUMEN
Toxoplasma gondii is the causative agent of toxoplasmosis, one of the most widespread infections in humans and animals, and is a major opportunistic pathogen in immunocompromised patients. Toxoplasma gondii is unique as it can invade virtually any nucleated cell, although the mechanisms are not completely understood. Parasite attachment to the host cell is a prerequisite for reorientation and penetration and likely requires the recognition of molecules at the host cell surface. It has been reported that the affinity of tachyzoites, the invasive form of T. gondii, for host cells can be inhibited by a variety of soluble-sulfated glycosaminoglycans (GAGs), such as heparan sulfate. Using heparin-functionalized zeolites in the absence of host cells, we visualized heparin-binding sites on the surface of tachyzoites by confocal and atomic force microscopy. Furthermore, we report that protein components of the parasite rhoptry, dense granule and surface bind GAGs. In particular, the proteins ROP2 and ROP4 from the rhoptry, GRA2 from the dense granules and the surface protein SAG1 were found to bind heparin. The binding specificities and affinities of individual parasite proteins for natural heparin and heparin oligosaccharides were determined by a combination of heparin oligosaccharide microarrays and surface plasmon resonance. Our results suggest that interactions between sulfated GAGs and parasite surface antigens contribute to T. gondii attachment to host cell surfaces as well as initiating the invasion process, while rhoptries and dense granule organelles may play an important role during the establishment of the infection and during the life of the parasite inside the parasitophorous vacuole.
Asunto(s)
Heparina/química , Proteínas Protozoarias/metabolismo , Toxoplasma/metabolismo , Animales , Antígenos de Protozoos/química , Antígenos de Protozoos/metabolismo , Membrana Celular/metabolismo , Glicosaminoglicanos/metabolismo , Heparina/metabolismo , Interacciones Huésped-Parásitos , Proteínas Protozoarias/química , Resonancia por Plasmón de Superficie , ZeolitasRESUMEN
Building blocks: a new, general synthetic strategy, which allows the construction of branched glycosylphosphatidylinositols (GPIs), enables the synthesis of parasitic glycolipid 1 from Toxoplasma gondii. In addition, the structure is further confirmed by recognition of monoclonal antibodies.
Asunto(s)
Antígenos/química , Glicosilfosfatidilinositoles/síntesis química , Toxoplasma/química , Conformación de Carbohidratos , Glicosilfosfatidilinositoles/química , Datos de Secuencia Molecular , Peso MolecularRESUMEN
Biocompatible glyco-dendronized poly-l-lysine (PLL) polymers carry either three or nine mannose- or galactose-bearing dendrons that selectively bind, and thus can be used to detect, bacteria. Central to the synthesis of glyco-dendronized polymers was the development of a continuous flow [2 + 2] photocycloaddition reaction to connect the dendrons and PLL. Glycodendronized polymers cluster bacteria by binding to cell-surface carbohydrate receptors and thereby result in an easy read-out using microscopic analyses.
Asunto(s)
Técnicas Biosensibles/métodos , Dendrímeros/química , Escherichia coli/aislamiento & purificación , Manosa/química , Polilisina/química , Dendrímeros/metabolismo , Escherichia coli/metabolismo , Galactosa/química , Galactosa/metabolismo , Manosa/metabolismo , Microscopía de Fuerza Atómica , Microscopía Fluorescente , Nanotubos/química , Nanotubos/ultraestructura , Fotoquímica , Sensibilidad y EspecificidadRESUMEN
Carbohydrate-protein interactions are involved in various intracellular functions and play an essential role in biological system, particularly at the level of cell-cell recognition, cell adhesion, and cell signaling processes. The importance of carbohydrate-protein binding is now recognized as a major mode of interaction between microbial pathogens and animal cells. Using innovative synthetic methods for oligosaccharide assembly an increasing number of synthetic carbohydrates of biomedical importance is available. Here, we illustrate using some case studies that show the design and the use of a glycosylphosphatidylinositol oligosaccharide library. We discuss the importance of parasite glycosylphosphatidylinositol-protein interactions including receptors, enzymes, and antibodies. Furthermore, glycosylphosphatidylinositol epitope mapping studies are of interest in the field of parasitic diseases, and provide a promising platform to understand structure-function relationships of glycosylphosphatidylinositols.
Asunto(s)
Bases de Datos Factuales , Glicosilfosfatidilinositoles/síntesis química , Parásitos/química , Polisacáridos/química , Animales , Conformación de Carbohidratos , Secuencia de Carbohidratos , Glicosilfosfatidilinositoles/química , Glicosilfosfatidilinositoles/metabolismo , Humanos , Análisis por Micromatrices/instrumentación , Análisis por Micromatrices/métodos , Datos de Secuencia MolecularRESUMEN
Glycosylphosphatidylinositol (GPI) is a complex glycolipid structure that acts as a membrane anchor for many cell-surface proteins of eukaryotes. GPI-anchored proteins are particularly abundant in protozoa and represent the major carbohydrate modification of many cell-surface parasite proteins. A minimal GPI-anchor precursor consists of core glycan (ethanolamine-P-Manalpha1-2Manalpha1-6Manalpha1-4GlcNH2) linked to the 6-position of the D-myo-inositol ring of phos-phatidylinositol. Although the GPI core glycan is conserved in all organisms, many differences in additional modifications to GPI structures and biosynthetic pathways have been reported. The preassembled GPI-anchor precursor is post-translationally transferred to a variety of membrane proteins in the lumen of the endoplasmic reticulum in a transamidase-like reaction during which a C-terminal GPI attachment signal is released. Increasing evidence show that a significant proportion of the synthesized GPIs are not used for protein anchoring, particularly in protozoa in which a large amount of free GPIs are being displayed at the cell surface. The characteristics of GPI biosynthesis are currently being explored for the development of parasite-specific inhibitors. Especially as this pathway, at least for Trypanosoma brucei, has been validated as a drug target.
Asunto(s)
Glicosilfosfatidilinositoles/metabolismo , Plasmodium falciparum/metabolismo , Animales , Conformación de Carbohidratos , Cromatografía Líquida de Alta Presión , Cromatografía por Intercambio Iónico , Glicosilfosfatidilinositoles/químicaRESUMEN
Dolichol phosphate mannose synthase (DPM) catalyzes the reaction between dolichol phosphate (Dol-P) and guanosine diphosphate mannose (GDP-Man) to form dolichol-phosphate-mannose (Dol-P-Man). This molecule acts as mannose donor for N-glycosylation and glycosylphosphatidylinositol (GPI) biosynthesis. The Plasmodium falciparum DPM1 (Pfdpm1) possesses a single predicted transmembrane region near the N-, but not the C-terminus. Here we show that the cloned Pfdpm1 gene failed to complement a Saccharomyces cerevisiae mutant indicating that the parasite gene does not belong to the baker's yeast group, as was previously assumed. Furthermore, Pfdpm1 was unable to complement a mouse mutant deficient in DPM but efficiently complements the Schizosaccharomyces pombe fission yeast mutant, indicating a difference between fission yeast and mammalian DPM genes. Therefore, we reanalyzed the hydrophobicity scales of all known DPMs and consequently reclassify the DPM clade into six major novel subgroups. Furthermore, we show that Pfdpm1 represents a unique enzyme among these subgroups.
Asunto(s)
Manosiltransferasas/clasificación , Manosiltransferasas/genética , Plasmodium falciparum/enzimología , Proteínas Protozoarias/clasificación , Proteínas Protozoarias/genética , Animales , Clonación Molecular , Prueba de Complementación Genética , Humanos , Manosiltransferasas/metabolismo , Ratones , Proteínas Protozoarias/metabolismo , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , Schizosaccharomyces/enzimología , Schizosaccharomyces/genéticaRESUMEN
Glycosylphosphatidylinositols (GPIs) constitute a class of glycolipids that have various functions, the most basic being to attach proteins to the surface of eukaryotic cells. GPIs have to be taken into account, when expressing surface antigens from parasitic protozoa in heterologous systems. The synthesis of the GPI-anchors was previously reported to be drastically decreased to almost background level following baculovirus infection. Here we describe a new method to express GPI-anchor proteins in insect cells relying on using of a supplementary baculovirus construct that overexpresses the N-acetylglucosaminyl phosphatidylinositol de-N-acetylase, the enzyme catalyzing the second step in the GPI biosynthetic pathway.
Asunto(s)
Amidohidrolasas/metabolismo , Baculoviridae/genética , Glicosilfosfatidilinositoles/metabolismo , Ingeniería de Proteínas/métodos , Spodoptera/metabolismo , Amidohidrolasas/genética , Animales , Línea Celular , Vectores Genéticos/genética , Glicosilfosfatidilinositoles/genética , Proteínas Recombinantes/metabolismo , Spodoptera/genética , Transfección/métodosRESUMEN
Toxoplasma gondii is a ubiquitous parasitic protozoan that invades nucleated cells in a process thought to be in part due to several surface glycosylphosphatidylinositol (GPI)-anchored proteins, like the major surface antigen SAG1 (P30), which dominates the plasma membrane. The serine protease inhibitors phenylmethylsulfonyl fluoride and diisopropyl fluoride were found to have a profound effect on the T. gondii GPI biosynthetic pathway, leading to the observation and characterization of novel inositol-acylated mannosylated GPI intermediates. This inositol acylation is acyl-CoA-dependent and takes place before mannosylation, but uniquely for this class of inositol-acyltransferase, it is inhibited by phenylmethylsulfonyl fluoride. The subsequent inositol deacylation of fully mannosylated GPI intermediates is inhibited by both phenylmethylsulfonyl fluoride and diisopropyl fluoride. The use of these serine protease inhibitors allows observations as to the timing of inositol acylation and subsequent inositol deacylation of the GPI intermediates. Inositol acylation of the non-mannosylated GPI intermediate D-GlcNalpha1-6-D-myo-inositol-1-HPO4-sn-lipid precedes mannosylation. Inositol deacylation of the fully mannosylated GPI intermediate allows further processing, i.e. addition of GalNAc side chain to the first mannose. Characterization of the phosphatidylinositol moieties present on both free GPIs and GPI-anchored proteins shows the presence of a diacylglycerol lipid, whose sn-2 position contains almost exclusively an C18:1 acyl chain. The data presented here identify key novel inositol-acylated mannosylated intermediates, allowing the formulation of an updated T. gondii GPI biosynthetic pathway along with identification of the putative genes involved.
Asunto(s)
Glicosilfosfatidilinositoles/metabolismo , Inositol/metabolismo , Toxoplasma/metabolismo , Acilación/efectos de los fármacos , Animales , Vías Biosintéticas/efectos de los fármacos , Extractos Celulares , Chlorocebus aethiops , Isoflurofato/farmacología , Manosa/metabolismo , Fluoruro de Fenilmetilsulfonilo/farmacología , Inhibidores de Serina Proteinasa/farmacología , Células VeroRESUMEN
GPIs isolated from Toxoplasma gondii, as well as a chemically synthesized GPI lacking the lipid moiety, activated a reporter gene in Chinese hamster ovary cells expressing TLR4, while the core glycan and lipid moieties cleaved from the GPIs activated both TLR4- and TLR2-expressing cells. MyD88, but not TLR2, TLR4, or CD14, is absolutely needed to trigger TNF-alpha production by macrophages exposed to T. gondii GPIs. Importantly, TNF-alpha response to GPIs was completely abrogated in macrophages from TLR2/4-double-deficient mice. MyD88(-/-) mice were more susceptible to death than wild-type (WT), TLR2(-/-), TLR4(-/-), TLR2/4(-/-), and CD14(-/-) mice infected with the ME-49 strain of T. gondii. The cyst number was higher in the brain of TLR2/4(-/-), but not TLR2(-/-), TLR4(-/-), and CD14(-/-), mice, as compared with WT mice. Upon infection with the ME-49 strain of T. gondii, we observed no decrease of IL-12 and IFN-gamma production in TLR2-, TLR4-, or CD14-deficient mice. Indeed, splenocytes from T. gondii-infected TLR2(-/-) and TLR2/4(-/-) mice produced more IFN-gamma than cells from WT mice in response to in vitro stimulation with parasite extracts enriched in GPI-linked surface proteins. Together, our results suggest that both TLR2 and TLR4 receptors may participate in the host defense against T. gondii infection through their activation by the GPIs and could work together with other MyD88-dependent receptors, like other TLRs or even IL-18R or IL-1R, to obtain an effective host response against T. gondii infection.
Asunto(s)
Glicosilfosfatidilinositoles/metabolismo , Receptor Toll-Like 2/metabolismo , Receptor Toll-Like 4/metabolismo , Toxoplasmosis Animal/inmunología , Animales , Células CHO , Cricetinae , Cricetulus , Citometría de Flujo , Glicosilfosfatidilinositoles/química , Glicosilfosfatidilinositoles/inmunología , Receptores de Lipopolisacáridos/inmunología , Receptores de Lipopolisacáridos/metabolismo , Macrófagos/inmunología , Macrófagos/metabolismo , Masculino , Ratones , Ratones Noqueados , Factor 88 de Diferenciación Mieloide/inmunología , Factor 88 de Diferenciación Mieloide/metabolismo , Receptor Toll-Like 2/inmunología , Receptor Toll-Like 4/inmunología , Toxoplasma/inmunologíaRESUMEN
Plasmodium falciparum malaria kills roughly 2.5 million people, mainly children, annually. Much of this mortality is thought to arise from the actions of a malarial toxin. This toxin, identified as glycosylphosphatidylinositol (GPI), is a major pathogenicity determinant in malaria. A malarial molecule, Pfj, labeled by [3H]glucosamine like the GPIs, was identified as a non-GPI molecule. Here we show that Pfj is able to down-regulate tumor necrosis factor alpha (TNF-alpha) production induced by the GPI of P. falciparum. Mass spectrometry analysis showed that Pfj was not a single molecule but represented a number of molecules. Separation methods, such as cation-exchange chromatography and thin-layer chromatography, were used to isolate and identify the following four main fatty acids responsible for the inhibitory effect on TNF-alpha production: myristic, pentadecanoic, palmitic, and palmitoleic acids. This regulatory effect on cytokine production suggests that there is balanced bioactivity for the different categories of malarial lipids.
Asunto(s)
Ácidos Grasos/farmacología , Malaria Falciparum/inmunología , Plasmodium falciparum/metabolismo , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Factores de Virulencia/antagonistas & inhibidores , Animales , Regulación hacia Abajo , Ácidos Grasos/aislamiento & purificación , Ácidos Grasos Monoinsaturados/aislamiento & purificación , Ácidos Grasos Monoinsaturados/farmacología , Glicosilfosfatidilinositoles/toxicidad , Espectrometría de Masas , Ratones , Microscopía Electrónica , Ácido Mirístico/aislamiento & purificación , Ácido Mirístico/farmacología , Ácido Palmítico/aislamiento & purificación , Ácido Palmítico/farmacología , Factor de Necrosis Tumoral alfa/agonistas , Factores de Virulencia/toxicidadRESUMEN
Using hypotonically permeabilized Toxoplasma gondii tachyzoites, we investigated the topology of the free glycosylphosphatidylinositols (GPIs) within the endoplasmic reticulum (ER) membrane. The morphology and permeability of parasites were checked by electron microscopy and release of a cytosolic protein. The membrane integrity of organelles (ER and rhoptries) was checked by protease protection assays. In initial experiments, GPI biosynthetic intermediates were labeled with UDP-[6-(3)H]GlcNAc in permeabilized parasites, and the transmembrane distribution of the radiolabeled lipids was probed with phosphatidylinositol-specific phospholipase C (PI-PLC). A new early intermediate with an acyl modification on the inositol was identified, indicating that inositol acylation also occurs in T. gondii. A significant portion of the early GPI intermediates (GlcN-PI and GlcNAc-PI) could be hydrolyzed following PI-PLC treatment, indicating that these glycolipids are predominantly present in the cytoplasmic leaflet of the ER. Permeabilized T. gondii parasites labeled with either GDP-[2-(3)H]mannose or UDP-[6-(3)H]glucose showed that the more mannosylated and side chain (Glc-GalNAc)-modified GPI intermediates are also preferentially localized in the cytoplasmic leaflet of the ER.
Asunto(s)
Retículo Endoplásmico/metabolismo , Retículo Endoplásmico/ultraestructura , Glicosilfosfatidilinositoles/metabolismo , Toxoplasma/metabolismo , Toxoplasma/ultraestructura , Acilación , Animales , Secuencia de Carbohidratos , Membrana Celular/química , Membrana Celular/metabolismo , Permeabilidad de la Membrana Celular , Retículo Endoplásmico/química , Glicosilfosfatidilinositoles/análisis , Datos de Secuencia MolecularRESUMEN
Toxoplasma gondii is a ubiquitous parasite that infects nearly all warm-blooded animals. Developmental switching in T. gondii, from the virulent tachyzoite to the relatively quiescent bradyzoite stage, is responsible for the disease propagation after alteration of the immune status of the carrier. The redifferentiation event is characterized by an over expression of a tachyzoite specific set of glycosylphosphatidylinositol anchored surface antigens and free GPIs. T. gondii grown in animal cells uses two glycosylphosphatidylinositol precursors to anchor the parasite surface proteins. The first form has an N-acetylgalactosamine residue bound to a conserved three-mannosyl core glycan, while the second structure contains an additional terminal glucose linked to the N-acetylgalactosamine side branch. Sera from persons infected with T. gondii reacted only with the glucose-N-acetylgalactosamine-containing structure. Here we report that T. gondii cultured in human cells uses predominantly the N-acetylgalactosamine-containing structure to anchor the parasite surface antigens. On the other hand, glycosylphosphatidylinositol structures having an additional terminal glucose are found exclusively on the parasite cell surface as free glycolipids participating in the production of cytokines that are implicated in the pathogenesis of T. gondii. We also provide evidence that such free glycosylphosphatidylinositols are restricted mainly to the lipid microdomains in the parasite cell surface membrane and mostly associated with proteins involved in the parasite motility as well as invasion of the host cell.
Asunto(s)
Antígenos de Superficie/metabolismo , Glicosilfosfatidilinositoles/metabolismo , Toxoplasma/metabolismo , Animales , Antígenos de Protozoos/inmunología , Antígenos de Protozoos/metabolismo , Antígenos de Superficie/inmunología , Línea Celular , Chlorocebus aethiops , Cromatografía en Capa Delgada , Glicosilfosfatidilinositoles/inmunología , Glicosilfosfatidilinositoles/farmacología , Humanos , Lípidos/análisis , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Macrófagos/metabolismo , Microscopía Fluorescente , Modelos Biológicos , Proteínas Protozoarias/análisis , Proteínas Protozoarias/inmunología , Proteínas Protozoarias/metabolismo , Toxoplasma/crecimiento & desarrollo , Toxoplasma/inmunología , Factor de Necrosis Tumoral alfa/metabolismo , Células VeroRESUMEN
Parasitic glycosylphosphatidylinositols (GPIs) are thought to be involved in induced cell signaling that leads to proinflammatory responses. Increasing interest in elucidation of the mechanisms involved in signaling pathways drives the finding of rapid and reliable methods to purify GPIs. GPIs are usually extracted using mixtures of chloroform/methanol/water, followed by a phase partition between water and water-saturated n-butanol. GPIs recovered in the butanol phase are separated by thin-layer chromatography, scraped, eluted from the silica, and used for studying the structure-function relationship. The presence of phospholipid contaminants or other hydrophobic components in the samples cannot be excluded. Furthermore, the standard procedures to purify GPIs harbor several drawbacks, including the need to handle large amounts of culture, poor yields, time-consuming, and interfering contaminants. Here we report on the development of a simple and reliable method to isolate and purify both free and bound GPIs from one cell pellet. We exploited the low solubility of GPIs in water-saturated n-butanol to remove the phospholipid contaminants completely. After delipidation, GPI proteins were solubilized from the pellet using a mixture of organic solvent containing ethanol and water.