RESUMEN
SAG1 (P30) is the major surface protein of the Toxoplasma gondii tachyzoite, the life cycle stage associated with the acute phase of infection. The protein is inserted into the parasite's plasma membrane by a glycosyl-phosphatidylinositol anchor, a modification that is present on all T. gondii surface proteins characterized so far. Here we describe a detailed structural analysis of this anchor. GPI anchor peptides were isolated from [3H]glucosamine labeled purified P30 by protease digestion and phase partitioning. Neutral glycans were prepared from this material by dephosphorylation and deamination. Two glycoforms were characterized by gel filtration and high performance ion exchange chromatography in combination with exoglycosidase treatment. Both forms were shown to carry an N-acetylgalactosamine bound to the first mannose of the conserved three-mannosyl core. Glycan B carries an additional terminal hexose linked to GalNAc. To identify the nature of this hexose, bulk anchor peptide was prepared and glycans were purified by aminopropyl-HPLC. Highly purified glycans were subjected to MALDI-TOF-MS and, after derivatization, to FAB-MS and methylation linkage analysis. The structures of the two anchors found on SAG1 were determined to be: Man-alpha1,2-Man-alpha1,6-Man-[GalNAc-beta1,4-]-alpha1,4-GlcN-PI and Man-alpha1,2-Man-alpha1,6-Man [Glc-alpha1,4-GalNAc-beta1,4-]-alpha1,4-GlcN-PI. Comparison of these structures with free GPI glycolipid precursors characterized in T. gondii suggests that core modification of the anchor takes place prior to transfer to the protein.
Asunto(s)
Antígenos de Protozoos , Glicosilfosfatidilinositoles/química , Polisacáridos/análisis , Proteínas Protozoarias/química , Toxoplasma/inmunología , Animales , Cromatografía en Gel , Cromatografía Líquida de Alta Presión , Endopeptidasas , Glicósido Hidrolasas , Modelos MolecularesRESUMEN
Tachyzoite endodyogeny is characterized by a three phase cell cycle comprised of major G1 and S phases with mitosis following immediately upon the conclusion of DNA replication. Cytokinesis, which begins with the formation of daughter apical complexes, initiates in late S phase and overlaps mitosis. There is no evidence to support an extended G2 period in these parasites. In all strains, parasites with a 2 N DNA content are a relatively small subpopulation and when tachyzoites expressing a fluorescent nuclear marker (green-fluorescent-protein fused to proliferating-cell-nuclear-antigen) were observed by time-lapse microscopy, there appeared to be little delay between S phase and mitosis. Measurements of the DNA content of RH parasites by flow cytometry demonstrated that the G1 and S periods were approximately 60 and approximately 30% of a single division cycle, although these phases were longer in strains that display a slower growth rate. The overall length of S phase was determined by [3H]-thymidine autoradiography using transgenic parasites expressing herpes simplex thymidine kinase and validated by Northern analysis of S phase specific genes during synchronous growth. The fraction of S phase parasites by flow cytometry paralleled autoradiography, however, within S phase, the distribution of parasites was bimodal in all strains examined. Parasites containing a 1-1.7 N DNA complement were a small fraction when compared to the major S phase population which contained a near-diploid ( approximately 1.8 N) complement, suggesting parasites in late S phase have a slower rate of DNA replication. In lieu of a short or missing G2, where checkpoints are thought to operate in other eukaryotes, the bimodal replication of tachyzoite chromosomes may represent a distinct premitotic checkpoint associated with endodyogeny.
Asunto(s)
Ciclo Celular/fisiología , Toxoplasma/crecimiento & desarrollo , Animales , División Celular , ADN Protozoario/análisis , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Fase G1 , Fase G2 , Expresión Génica , Mitosis , Antígeno Nuclear de Célula en Proliferación/análisis , ARN Mensajero/genética , ARN Mensajero/metabolismo , Fase S , Toxoplasma/citología , Toxoplasma/genéticaRESUMEN
Interleukin (IL)-10 is an inhibitor of cell mediated immunity and an antagonist of the development of protective immune responses associated with resistance to T. gondii. These observations led to the hypothesis that the production of IL-10 could contribute to the ability of T. gondii to replicate and survive in an immune competent host. To determine whether the production of IL-10 affects the ability of the RH strain of T. gondii to cause a lethal infection in mice, we compared the immune response to RH in IL-10+/+ and IL-10-/- BALB/c mice. Both groups of mice produced comparable amounts of IL-12 and interferon (IFN)-gamma and had similar mortality curves and parasite burdens. The use of green fluorescent protein-labelled parasites allowed us to infect IL-10+/+ and IL-10-/- mice and use a fluorescence-activated cell sorter to distinguish infected and uninfected populations of macrophages and compare their expression of CD80, CD86 and major histocompatibility complex (MHC) class II. Although infected cells expressed higher overall levels of these molecules than uninfected cells, there were no differences between cells isolated from IL-10+/+ and IL-10-/- mice. Taken together, these results indicate that IL-10 is not required for the virulence of the RH strain of T. gondii, nor is it involved in the regulation of the CD80, CD86 and MHC class II molecules during RH-infection.
Asunto(s)
Interleucina-10/fisiología , Toxoplasma/patogenicidad , Toxoplasmosis/inmunología , Animales , Antígenos CD/biosíntesis , Antígeno B7-1/biosíntesis , Antígeno B7-2 , Antígenos de Histocompatibilidad Clase II/biosíntesis , Interferón gamma/biosíntesis , Interleucina-10/deficiencia , Interleucina-12/biosíntesis , Glicoproteínas de Membrana/biosíntesis , Ratones , Ratones Endogámicos BALB C , Ratones NoqueadosRESUMEN
The apicoplast is a distinctive organelle associated with apicomplexan parasites, including Plasmodium sp. (which cause malaria) and Toxoplasma gondii (the causative agent of toxoplasmosis). This unusual structure (acquired by the engulfment of an ancestral alga and retention of the algal plastid) is essential for long-term parasite survival. Similar to other endosymbiotic organelles (mitochondria, chloroplasts), the apicoplast contains proteins that are encoded in the nucleus and post-translationally imported. Translocation across the four membranes surrounding the apicoplast is mediated by an N-terminal bipartite targeting sequence. Previous studies have described a recombinant "poison" that blocks plastid segregation during mitosis, producing parasites that lack an apicoplast and siblings containing a gigantic, nonsegregating plastid. To learn more about this remarkable phenomenon, we examined the localization and processing of the protein produced by this construct. Taking advantage of the ability to isolate apicoplast segregation mutants, we also demonstrated that processing of the transit peptide of nuclear-encoded apicoplast proteins requires plastid-associated activity.
Asunto(s)
Núcleo Celular/metabolismo , Citometría de Flujo/métodos , Plastidios/genética , Toxoplasma/genética , Animales , Membrana Celular/metabolismo , Separación Celular , Cloranfenicol O-Acetiltransferasa/metabolismo , Colorantes Fluorescentes/metabolismo , Proteínas Fluorescentes Verdes , Proteínas Luminiscentes/metabolismo , Plásmidos/metabolismo , Plastidios/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Fracciones Subcelulares , Proteína Fluorescente RojaRESUMEN
Rhoptry and microneme organelles of the protozoan parasite Toxoplasma gondii are closely associated with host cell adhesion/invasion and establishment of the intracellular parasitophorous vacuole. In order to study the targeting of proteins to these specialized secretory organelles, we have engineered green fluorescent protein (GFP) fusions to the rhoptry protein ROP1 and the microneme protein MIC3. Both chimeras are correctly targeted to the appropriate organelles, permitting deletion analysis to map protein subdomains critical for targeting. The propeptide and a central 146 amino acid region of ROP1 are sufficient to target GFP to the rhoptries. More extensive deletions result in a loss of rhoptry targeting; the GFP reporter is diverted into the parasitophorous vacuole via dense granules. Certain MIC3 deletion mutants were also secreted into the parasitophorous vacuole via dense granules, supporting the view that this route constitutes the default pathway in T. gondii, and that specific signals are required for sorting to rhoptries and micronemes. Deletions within the cysteine-rich central region of MIC3 cause this protein to be arrested at various locations within the secretory pathway, presumably due to improper folding. Although correctly targeted to the appropriate organelles in living parasites, ROP1-GFP and MIC3-GFP fusion proteins were not secreted during invasion. GFP fusion proteins were readily secreted from dense granules, however, suggesting that protein secretion from rhoptries and micronemes might involve more than a simple release of organellar contents.
Asunto(s)
Adhesinas Bacterianas , Proteínas Protozoarias/metabolismo , Toxoplasma/metabolismo , Animales , Proteínas Portadoras/análisis , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Adhesión Celular , Células Cultivadas , Eliminación de Gen , Proteínas Fluorescentes Verdes , Humanos , Immunoblotting , Indicadores y Reactivos , Proteínas Luminiscentes , Proteínas de la Membrana/análisis , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Microscopía Fluorescente , Mutación , Orgánulos/metabolismo , Proteínas Protozoarias/análisis , Proteínas Protozoarias/genética , Toxoplasma/genética , Transfección , Proteína Fluorescente RojaRESUMEN
Apicomplexan parasites--including the causative agents of malaria (Plasmodium sp.) and toxoplasmosis (Toxoplasma gondii)--harbor a secondary endosymbiotic plastid, acquired by lateral genetic transfer from a eukaryotic alga. The apicoplast has attracted considerable attention, both as an evolutionary novelty and as a potential target for chemotherapy. We report a recombinant fusion (between a nuclear-encoded apicoplast protein, the green fluorescent protein and a rhoptry protein) that targets to the apicoplast but grossly alters its morphology, preventing organellar segregation during parasite division. Apicoplast-deficient parasites replicate normally in the first infectious cycle and can be isolated by fluorescence-activated cell sorting, but die in the subsequent host cell, confirming the 'delayed death' phenotype previously described pharmacologically, and validating the apicoplast as essential for parasite viability.
Asunto(s)
Plastidios/genética , Toxoplasma/genética , Animales , Eucariontes/genética , Genes Protozoarios , Proteínas Fluorescentes Verdes , Proteínas Luminiscentes/genética , Proteínas de la Membrana/genética , Microscopía Electrónica , Mutación , Fenotipo , Proteínas Protozoarias/genética , Proteínas Recombinantes de Fusión/genética , Simbiosis/genética , Toxoplasma/crecimiento & desarrollo , Toxoplasma/microbiologíaRESUMEN
Apicomplexan parasites harbor a single nonphotosynthetic plastid, the apicoplast, which is essential for parasite survival. Exploiting Toxoplasma gondii as an accessible system for cell biological analysis and molecular genetic manipulation, we have studied how these parasites ensure that the plastid and its 35-kb circular genome are faithfully segregated during cell division. Parasite organelles were labeled by recombinant expression of fluorescent proteins targeted to the plastid and the nucleus, and time-lapse video microscopy was used to image labeled organelles throughout the cell cycle. Apicoplast division is tightly associated with nuclear and cell division and is characterized by an elongated, dumbbell-shaped intermediate. The plastid genome is divided early in this process, associating with the ends of the elongated organelle. A centrin-specific antibody demonstrates that the ends of dividing apicoplast are closely linked to the centrosomes. Treatment with dinitroaniline herbicides (which disrupt microtubule organization) leads to the formation of multiple spindles and large reticulate plastids studded with centrosomes. The mitotic spindle and the pellicle of the forming daughter cells appear to generate the force required for apicoplast division in Toxoplasma gondii. These observations are discussed in the context of autonomous and FtsZ-dependent division of plastids in plants and algae.
Asunto(s)
Centrosoma/metabolismo , Segregación Cromosómica , Plastidios/genética , Plastidios/metabolismo , Toxoplasma/citología , Compuestos de Anilina/farmacología , Animales , División Celular/efectos de los fármacos , Membrana Celular/metabolismo , Membrana Celular/ultraestructura , Núcleo Celular/efectos de los fármacos , Núcleo Celular/genética , Núcleo Celular/metabolismo , Centriolos/efectos de los fármacos , Centriolos/metabolismo , Centriolos/ultraestructura , Centrosoma/efectos de los fármacos , Centrosoma/ultraestructura , Segregación Cromosómica/efectos de los fármacos , Replicación del ADN , Genoma , Microscopía Electrónica , Microscopía por Video , Modelos Biológicos , Plastidios/efectos de los fármacos , Antígeno Nuclear de Célula en Proliferación/metabolismo , Huso Acromático/efectos de los fármacos , Huso Acromático/metabolismo , Huso Acromático/ultraestructura , Toxoplasma/efectos de los fármacos , Toxoplasma/genéticaRESUMEN
The discovery of a plastid in Plasmodium, Toxoplasma and related protozoan parasites provides a satisfying resolution to several long-standing mysteries: the mechanism of action for various surprisingly effective antibiotics; the subcellular location of an enigmatic 35 kb episomal DNA; and the nature of an unusual intracellular structure containing multiple membranes. The apicomplexan plastid highlights the importance of lateral genetic transfer in evolution and provides an accessible system for the investigation of protein targeting to secondary endosymbiotic organelles. Combining molecular genetic identification of targeting signals with whole genome analysis promises to yield a complete picture of organellar metabolic pathways and new targets for drug design.
Asunto(s)
Apicomplexa/fisiología , Plastidios/fisiología , Animales , Apicomplexa/genética , Apicomplexa/metabolismo , Evolución Biológica , Transporte Biológico , Plastidios/genética , Plastidios/metabolismo , Proteínas Protozoarias/metabolismoRESUMEN
Morphological examination of the highly polarized protozoan parasite Toxoplasma gondii suggests that secretory traffic in this organism progresses from the endoplasmic reticulum to the Golgi apparatus using the nuclear envelope as an intermediate compartment. While the endoplasmic reticulum is predominantly located near the basal end of the parasite, the Golgi is invariably adjacent to the apical end of the nucleus, and the space between the Golgi and nuclear envelope is filled with numerous coatomer-coated vesicles. Staining with antiserum raised against recombinant T. gondii beta-COP confirms its association with the apical juxtanuclear region. Perturbation of protein secretion using brefeldin A, microtubule inhibitors or dithiothreitol disrupts the Golgi, causing swelling of the nuclear envelope, particularly at its basal end. Prolonged drug treatment leads to gross distention of the endoplasmic reticulum, filling the basal end of the parasite. Cloning and sequencing of the T. gondii homolog of the chaperonin protein BiP identifies the carboxy-terminal amino acid sequence HDEL as this organism's endoplasmic reticulum-retention signal. Appending the HDEL motif to a recombinant secretory protein (a chimera between the parasite's major surface protein fusion, P30, and the Green Fluorescent Protein) causes this secretory reporter to be retained intracellularly. P30-GFP-HDEL fluorescence was most intense within the nuclear envelope, particularly at the apical end. These data support a model of secretion in which protein traffic from the endoplasmic reticulum to Golgi occurs via the apical end of the nuclear envelope.
Asunto(s)
Retículo Endoplásmico/metabolismo , Aparato de Golgi/metabolismo , Proteínas de Choque Térmico , Membrana Nuclear/metabolismo , Toxoplasma/metabolismo , Animales , Transporte Biológico/efectos de los fármacos , Transporte Biológico/fisiología , Brefeldino A/farmacología , Proteínas Portadoras/genética , Núcleo Celular/química , Núcleo Celular/metabolismo , Núcleo Celular/ultraestructura , Proteína Coatómero/análisis , Proteína Coatómero/metabolismo , Gránulos Citoplasmáticos/química , Gránulos Citoplasmáticos/metabolismo , Gránulos Citoplasmáticos/ultraestructura , Ditiotreitol/farmacología , Retículo Endoplásmico/química , Retículo Endoplásmico/ultraestructura , Chaperón BiP del Retículo Endoplásmico , Células Epiteliales/parasitología , Técnica del Anticuerpo Fluorescente , Proteínas Fúngicas/genética , Aparato de Golgi/química , Aparato de Golgi/ultraestructura , Proteínas Fluorescentes Verdes , Proteínas HSP70 de Choque Térmico/genética , Humanos , Indicadores y Reactivos/farmacocinética , Proteínas Luminiscentes/farmacocinética , Microscopía Electrónica , Chaperonas Moleculares/genética , Membrana Nuclear/química , Membrana Nuclear/ultraestructura , Señales de Clasificación de Proteína/metabolismo , Inhibidores de la Síntesis de la Proteína/farmacología , Proteínas Protozoarias/genética , Piel/citología , Especificidad de la Especie , Reactivos de Sulfhidrilo/farmacología , Toxoplasma/química , Toxoplasma/ultraestructuraRESUMEN
Toxoplasma gondii is an obligate intracellular parasite of the phylum apicomplexa and a common and often life-threatening opportunistic infection associated with AIDS. A family of parasite-specific glycosylphosphatidylinositols containing a novel glucosylated side chain has been shown to be highly immunogenic in humans (Striepen et al. (1997) J. Mol. Biol. 266, 797-813). In contrast to trypanosomes in T. gondii side chain modification takes place before addition to protein in the endoplasmic reticulum. The biosynthesis of these modifications was studied in an in vitro system prepared from hypotonically lysed T. gondii parasites. Radiolabeled glucose-containing glycosylphosphatidylinositol precursors were synthesized by T. gondii membrane preparations upon incubation with uridine diphosphate-[3H]glucose. Synthesis of glucosylated glycolipids took place only in the presence of exogenous uridine diphosphate-glucose and was stimulated by unlabeled uridine diphosphate-glucose in a dose-dependent manner. In contrast to glycosylphosphatidylinositol mannosylation, glucosylation was shown to be insensitive to amphomycin treatment. In addition, the glucose analogue 2-deoxy-D-glucose was used to trace the glycosylphosphatidylinositol glucosylation pathway. Detailed analysis of glycolipids synthesized in vitro in the presence of UDP and GDP derivatives of D-glucose and 2-deoxy-D-glucose ruled out an involvement of dolichol phosphate-glucose and demonstrates direct transfer of glucose from uridine diphosphate-glucose.
Asunto(s)
Glicosilfosfatidilinositoles/metabolismo , Azúcares de Guanosina Difosfato/metabolismo , Uridina Difosfato Glucosa/análogos & derivados , Uridina Difosfato Glucosa/metabolismo , Animales , Antibacterianos/farmacología , Bovinos , Sistema Libre de Células , Glucosa/metabolismo , Glucolípidos/química , Glucolípidos/metabolismo , Glicosilfosfatidilinositoles/biosíntesis , Glicosilfosfatidilinositoles/química , Azúcares de Guanosina Difosfato/química , Humanos , Lipopéptidos , Oligopéptidos/farmacología , Polisacáridos/química , Polisacáridos/metabolismo , Toxoplasma , Uridina Difosfato Glucosa/química , Uridina Difosfato N-Acetilglucosamina/metabolismo , Uridina Difosfato N-Acetilglucosamina/farmacologíaRESUMEN
The signals and the molecular machinery mediating release of dense matrix granules from pathogenic protozoan parasites are unknown. We compared the secretion of the endogenous dense granule marker GRA3 in Toxoplasma gondii with the release of a stably transfected foreign reporter, beta-lactamase, that localizes to parasite dense granules. Both proteins were released constitutively in a calcium-independent fashion, as shown using both intact and streptolysin O-permeabilized parasites. N-Ethylmaleimide and recombinant bovine Rab-guanine dissociation inhibitor inhibited beta-lactamase secretion in permeabilized parasites, whereas recombinant hamster N-ethylmaleimide-sensitive fusion protein and bovine alpha-SNAP augmented release. Guanosine 5'-3-O-(thio)triphosphate, but not cAMP, augmented secretion in the presence but not in the absence of ATP. The T. gondii NSF/SNAP/SNARE/Rab machinery participates in dense granule release using parasite protein components that can interact functionally with their mammalian homologues.
Asunto(s)
Calcio/metabolismo , Proteínas Portadoras/metabolismo , Proteínas de Unión al GTP/metabolismo , Proteínas de la Membrana/metabolismo , Penicilamina/análogos & derivados , Toxoplasma/metabolismo , Proteínas de Transporte Vesicular , Animales , Bovinos , Cricetinae , AMP Cíclico/metabolismo , Exocitosis , Proteínas Sensibles a N-Etilmaleimida , Penicilamina/metabolismo , Proteínas SNARE , TemperaturaRESUMEN
Like Plasmodium, the protozoan parasite Toxoplasma gondii is a member of the phylum Apicomplexa, and an obligate intracellular pathogen. Unlike Plasmodium, however, Toxoplasma is highly amenable to experimental manipulation in the laboratory. The development of molecular transformation protocols for T. gondii has provided both scientific precedent and practical selectable markers for Plasmodium. Beyond the feasibility of molecular biological experimentation now possible in both systems, the high frequency of stable transformation in Toxoplasma allows this parasite to be used for molecular genetic analysis. The ability to control homologous vs. non-homologous recombination in T. gondii permits gene knockouts/allelic replacements at previously cloned loci, and saturation insertional mutagenesis of the entire parasite genome (and cloning of the tagged loci). T. gondii also exhibits unusual ultrastructural clarity, facilitating cell biological analysis. The accessibility of Toxoplasma as an experimental system allows this parasite to be used as a surrogate for asking many questions that cannot easily be addressed in Plasmodium itself. T. gondii also serves as a model system for genetic exploration of parasite biology and host-parasite interactions. Success stories include: biochemical analysis of antifolate resistance mechanisms; pharmacological studies on the mechanisms of macrolide activity; genetic identification of nucleobase/nucleoside transporters and metabolic pathways; and cell biological characterization of the apicomplexan plastid. As with any model system, not all questions of interest to malariologists can be addressed in Toxoplasma; differentiating between sensible and foolish questions requires familiarity with the biological similarities and differences of these systems.
Asunto(s)
Plasmodium/metabolismo , Toxoplasma/metabolismo , Animales , Transporte Biológico Activo , Modelos Biológicos , Proteínas Protozoarias/metabolismo , ReproducciónRESUMEN
A vestigial, nonphotosynthetic plastid has been identified recently in protozoan parasites of the phylum Apicomplexa. The apicomplexan plastid, or "apicoplast," is indispensable, but the complete sequence of both the Plasmodium falciparum and Toxoplasma gondii apicoplast genomes has offered no clue as to what essential metabolic function(s) this organelle might perform in parasites. To investigate possible functions of the apicoplast, we sought to identify nuclear-encoded genes whose products are targeted to the apicoplast in Plasmodium and Toxoplasma. We describe here nuclear genes encoding ribosomal proteins S9 and L28 and the fatty acid biosynthetic enzymes acyl carrier protein (ACP), beta-ketoacyl-ACP synthase III (FabH), and beta-hydroxyacyl-ACP dehydratase (FabZ). These genes show high similarity to plastid homologues, and immunolocalization of S9 and ACP verifies that the proteins accumulate in the plastid. All the putatively apicoplast-targeted proteins bear N-terminal presequences consistent with plastid targeting, and the ACP presequence is shown to be sufficient to target a recombinant green fluorescent protein reporter to the apicoplast in transgenic T. gondii. Localization of ACP, and very probably FabH and FabZ, in the apicoplast implicates fatty acid biosynthesis as a likely function of the apicoplast. Moreover, inhibition of P. falciparum growth by thiolactomycin, an inhibitor of FabH, indicates a vital role for apicoplast fatty acid biosynthesis. Because the fatty acid biosynthesis genes identified here are of a plastid/bacterial type, and distinct from those of the equivalent pathway in animals, fatty acid biosynthesis is potentially an excellent target for therapeutics directed against malaria, toxoplasmosis, and other apicomplexan-mediated diseases.
Asunto(s)
Proteínas Nucleares/metabolismo , Plasmodium falciparum/metabolismo , Plastidios/metabolismo , Toxoplasma/metabolismo , Animales , Secuencia de Bases , Clonación Molecular , ADN Complementario , Ácidos Grasos/biosíntesis , Proteínas Fluorescentes Verdes , Proteínas Luminiscentes/genética , Datos de Secuencia Molecular , Proteínas Nucleares/genética , Filogenia , Plasmodium falciparum/genética , Toxoplasma/genéticaRESUMEN
We have engineered a mutant version of the green fluorescent protein GFP (Cormack et al. Selected for bright fluorescence in E. coli. Gene 1996;173:33-38) for expression in the protozoan parasite Toxoplasma gondii. Although intact GFP was not expressed at any detectable level, GFP fusion proteins could be detected by fluorescence microscopy, flow cytometry (FACS), and immunoblotting. Both extracellular tachyzoites and T. gondii-infected host cells could readily be sorted by FACS, which should facilitate a variety of selection strategies. Several selectable markers were tested for their ability to produce stable green transgenic parasites. Fluorescence intensity was directly correlated with gene copy number and protein expression level. Weak selectable markers such as chloramphenicol acetyl transferase (CAT) driven by the SAG1 promoter, which yield multicopy insertions, are therefore most effective for selecting green fluorescent parasites-particularly when coupled to constructs which employ a strong promoter to drive GFP expression. Transformation vectors developed in the course of this work should be of general utility for the overexpression of heterologous transgenes in Toxoplasma. CAT-GFP fusion proteins were expressed in the parasite cytoplasm. GFP fusions to the P30 major surface antigen (linked on the same plasmid to a CAT selectable marker under control of various promoters) could be detected in dense granules within living cells, and were efficiently secreted into the parasitophorous vacuole. GFP fusions to the rhoptry protein ROP1 were targeted to rhoptries (specialized secretory organelles at the apical end of the parasite).
Asunto(s)
Regulación de la Expresión Génica , Proteínas Luminiscentes/genética , Orgánulos/metabolismo , Toxoplasma/genética , Animales , Animales Modificados Genéticamente , Transporte Biológico , Biomarcadores , Clonación Molecular , Dosificación de Gen , Proteínas Fluorescentes Verdes , Proteínas Luminiscentes/metabolismo , Microscopía Fluorescente , Proteínas Protozoarias/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Toxoplasma/citología , Toxoplasma/metabolismoAsunto(s)
Antígenos de Protozoos/análisis , Oligosacáridos/análisis , Proteínas Protozoarias/análisis , Toxoplasma/inmunología , Animales , Chlorocebus aethiops , Cromatografía de Afinidad , Gránulos Citoplasmáticos/química , Gránulos Citoplasmáticos/inmunología , Femenino , Glicoproteínas/análisis , Glicoproteínas/metabolismo , Oligosacáridos/metabolismo , Embarazo , Proteínas Protozoarias/metabolismo , Toxoplasma/química , Células VeroRESUMEN
Toxoplasma gondii is a ubiquitous parasitic protozoan causing congenital infection and severe encephalitis in the course of the acquired immunodeficiency syndrome. Glycosyl-phosphatidylinositols of T. gondii have been shown to be identical with the low molecular weight antigen which elicits an early immunoglobulin M immune response in humans. A detailed study of the structures of these glycolipid antigens was performed. Radiolabelled glycolipids were extensively analysed by chemical and exoglycosidase treatments in combination with high pH anion-exchange chromatography, gel-filtration and lectin affinity chromatography. In addition, carbohydrate fragments prepared and purified from bulk preparations of unlabelled glycolipids by high performance liquid chromatography were subjected to two-dimensional 1H nuclear magnetic resonance spectroscopy, fast-atom bombardment-mass spectrometry, and methylation linkage analysis in order to elucidate the structure of T. gondii GPIs. The following structures were identified: (ethanolamine-PO4)-Man alpha 1-2Man alpha 1-6(GalNAc beta 1-4)Man alpha 1-4GlcN alpha-inositol-PO4-lipid and the novel structure (ethanolamine-PO4)-Man alpha 1-2Man alpha 1-6(Glc alpha 1-4GalNAc beta 1-4)Man alpha 1-4 GlcN alpha-inositol-PO4-lipid both with and without terminal ethanolamine phosphate. Evidence is provided, that only T. gondii GPIs bearing the unique glucose-N-acetylgalactosamine side branch are immunogenic in humans and that this structure is widely distributed among T. gondii isolates. Monoclonal antibodies have been characterized to recognize structures with different degrees of side-chain modification. We suggest that these reagents in combination with recently devised techniques for insertional mutagenesis in T. gondii should greatly facilitate the cloning of genes essential for GPI side-chain modification.
Asunto(s)
Antígenos de Protozoos/química , Glicosilfosfatidilinositoles/inmunología , Polisacáridos/química , Toxoplasma/inmunología , Acetilgalactosamina/análogos & derivados , Acetilgalactosamina/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Antígenos de Protozoos/inmunología , Western Blotting , Conformación de Carbohidratos , Secuencia de Carbohidratos , Cromatografía de Afinidad , Cromatografía Líquida de Alta Presión , Epítopos/inmunología , Etanolamina , Etanolaminas/análisis , Glucósidos/química , Glucósidos/inmunología , Glicosilfosfatidilinositoles/química , Humanos , Espectroscopía de Resonancia Magnética , Metilación , Datos de Secuencia Molecular , Estructura Molecular , Monosacáridos/análisis , Oligosacáridos/química , Oligosacáridos/inmunología , Polisacáridos/inmunología , Polisacáridos/aislamiento & purificación , Espectrometría de Masa Bombardeada por Átomos Veloces , Toxoplasma/química , Toxoplasmosis/inmunologíaRESUMEN
We are investigating the structure and biosynthesis of glycosyl-phosphatidylinositols (GPI) in the protozoa Toxoplasma gondii, Plasmodium falciparum, Plasmodium yoelii and Paramecium primaurelia. This comparison of structural and biosynthesis data should lead us to common and individual features of the GPI-biosynthesis and transport in different organisms.
Asunto(s)
Eucariontes/metabolismo , Glicosilfosfatidilinositoles/metabolismo , Animales , Glicosilfosfatidilinositoles/biosíntesis , Glicosilfosfatidilinositoles/química , Histocitoquímica , Estructura Molecular , Paramecium/metabolismo , Plasmodium falciparum/metabolismo , Plasmodium yoelii/metabolismo , Toxoplasma/metabolismoRESUMEN
Plasmid vectors that incorporate sequence elements from the dehydrofolate reductase-thymidylate synthase (DHFR-TS) locus of Toxoplasma gondii integrate into the parasite genome with remarkably high frequency (>1% of transfected parasites). These vectors may-but need not-include mutant DHFR-TS alleles that confer pyrimethamine resistance to transgenic parasites. Large genomic constructs integrate at the endogenous locus by homologous recombination, but cDNA-derived sequences lacking long stretches of contiguous genomic DNA (due to intron excision) typically integrate into chromosomal DNA by nonhomologous recombination. Nonhomologous integration occurs effectively at random; and coupled with the high frequency of transformation, this allows a large fraction of the parasite genome to be tagged in a single electroporation cuvette. Genomic tagging permits insertional mutagenesis studies conceptually analogous to transposon mutagenesis in bacteria, yeast, Drosophila, etc. In theory (and, thus far, in practice), this allows identification of any gene whose inactivation is not lethal to the haploid tachyzoite form of T. gondii and for which a suitable selection or screen is available. Transformation vectors can be engineered to facilitate rescue of the tagged locus and to include a variety of reporters or selectable markers. Genetic strategies are also possible, using reporters whose function can be assayed by metabolic, visual, or immunological screens to "trap" genes that are activated (or inactivated) under various conditions of interest.
Asunto(s)
Mutagénesis Insercional/métodos , Regiones Promotoras Genéticas , Toxoplasma/genética , Transfección/métodos , Animales , Cloranfenicol O-Acetiltransferasa/biosíntesis , ADN Complementario , Electroporación , Genes Reporteros , Vectores Genéticos , Proteínas Fluorescentes Verdes , Hipoxantina Fosforribosiltransferasa/biosíntesis , Intrones , Proteínas Luminiscentes/biosíntesis , Ácido Micofenólico/metabolismo , Plásmidos , Reacción en Cadena de la Polimerasa , Pirimetamina/farmacología , Proteínas Recombinantes/biosíntesis , Tetrahidrofolato Deshidrogenasa/biosíntesis , Timidilato Sintasa/biosíntesis , Toxoplasma/efectos de los fármacosRESUMEN
The parasitic protozoan Trypanosoma congolense exhibits a dense surface coat which is pivotal for immunoevasion of the parasite. This dense surface coat is made of a single protein species, the variant surface glycoprotein, which is present in a high copy number. The protein is anchored to the plasma membrane by a glycosyl-phosphatidylinositol membrane anchor. A detailed study of the structure of T. congolense strain 423 (clone BENat 1.3) variant surface glycoprotein glycosyl-phosphatidylinositol membrane anchor was performed. Radioactively labelled core-glycan prepared by dephosphorylation, deamination and reduction was analysed by high-pH anion-exchange chromatography, size-exclusion and lectin affinity chromatography. Additionally the glycosyl-phosphatidylinositol membrane anchor core-glycan was purified from a bulk preparation of variant surface glycoprotein and subjected to mass spectrometry and methylation analysis. Using these methods we could identify a novel galactose-beta 1,6-N-acetyl-glucosamine-beta 1,4-branch modifying the mannose adjacent to the glucosamine of the mannose-alpha 1,2-mannose-alpha 1,6-mannose-alpha 1,4-glucosamine core-glycan of the variant surface glycoprotein glycosyl-phosphatidylinositol membrane anchor. Furthermore the biosynthetic pathway leading to this novel structure was investigated. Two putative glycosyl-phosphatidylinositol anchor precursors were identified having structures identical to the previously characterized Trypanosoma brucei brucei glycolipids P2 and P3 (also designated glycolipid A and C) consistent with a trimannosyl core and a dimyristoyl-glycerol. Both glycosyl-phosphatidylinositol anchor precursors of T. congolense do not possess the side-branch modification found on the mature protein membrane anchor, implying that the sugar side-chain is added to the anchor during its passage through the Golgi-apparatus.
Asunto(s)
Glicosilfosfatidilinositoles/química , Trypanosoma congolense/química , Glicoproteínas Variantes de Superficie de Trypanosoma/química , Animales , Secuencia de Carbohidratos , Glicosilfosfatidilinositoles/biosíntesis , Hexosaminas/análisis , Manosa/química , Datos de Secuencia Molecular , Polisacáridos/análisis , Polisacáridos/aislamiento & purificación , Glicoproteínas Variantes de Superficie de Trypanosoma/biosíntesisRESUMEN
Glycolipids synthesized in a cell-free system prepared from the free-living protozoan Paramecium primaurelia and labelled with [3H]mannose and [3H]glucosamine using GDP-[3H]mannose and UDP-[3H]N-acetyl glucosamine, respectively, were identified and structurally characterized as glycosylinositol-phosphoceramides (GIP-ceramides). The ceramide-based lipid was also found in the GIP membrane anchor of the G surface antigen of P.primaurelia, strain 156. Using a combination of in vitro labelling with GDP-[3H]mannose and in vivo labelling with 33P, we found that the core glycans of the P.primaurelia GIP-ceramides were substituted with an acid-labile modification identified as mannosyl phosphate. The modification of the glycosylinositol-phospholipid core glycan by mannosyl phosphate has not been described to date in other organisms. The biosynthesis of GIP-ceramide intermediates in P.primaurelia was studied by a pulse-chase analysis. Their structural characterization is reported. We propose the following structure for the putative GIP-ceramide membrane anchor precursor of P.primaurelia surface proteins: ethanolamine phosphate-6Man-alpha 1-2Man-alpha 1-6Man-(mannosyl phosphate)-alpha 1-4glucosamine-inositol-phosphoceramide.