RESUMEN
Like many other apicomplexan parasites, Toxoplasma gondii contains a plastid harboring key metabolic pathways, including the sulfur utilization factor (SUF) pathway that is involved in the biosynthesis of iron-sulfur clusters. These cofactors are crucial for a variety of proteins involved in important metabolic reactions, potentially including plastidic pathways for the synthesis of isoprenoid and fatty acids. It was shown previously that impairing the NFS2 cysteine desulfurase, involved in the first step of the SUF pathway, leads to an irreversible killing of intracellular parasites. However, the metabolic impact of disrupting the pathway remained unexplored. Here, we generated another mutant of this pathway, deficient in the SUFC ATPase, and investigated in details the phenotypic consequences of TgNFS2 and TgSUFC depletion on the parasites. Our analysis confirms that Toxoplasma SUF mutants are severely and irreversibly impacted in division and membrane homeostasis, and suggests a defect in apicoplast-generated fatty acids. However, we show that increased scavenging from the host or supplementation with exogenous fatty acids do not fully restore parasite growth, suggesting that this is not the primary cause for the demise of the parasites and that other important cellular functions were affected. For instance, we also show that the SUF pathway is key for generating the isoprenoid-derived precursors necessary for the proper targeting of GPI-anchored proteins and for parasite motility. Thus, we conclude plastid-generated iron-sulfur clusters support the functions of proteins involved in several vital downstream cellular pathways, which implies the SUF machinery may be explored for new potential anti-Toxoplasma targets.
Asunto(s)
Apicoplastos , Proteínas Hierro-Azufre , Proteínas Protozoarias , Toxoplasma , Apicoplastos/genética , Apicoplastos/metabolismo , Ácidos Grasos/metabolismo , Proteínas Hierro-Azufre/genética , Proteínas Hierro-Azufre/metabolismo , Plastidios/genética , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Terpenos/metabolismo , Toxoplasma/genética , Toxoplasma/metabolismoRESUMEN
Mutations within the central region of prion protein (PrP) have been shown to be associated with severe neurotoxic activity similar to that observed with Dpl, a PrP-like protein. To further investigate this neurotoxic effect, we generated lines of transgenic (Tg) mice expressing three different chimeric PrP-Dpl proteins. Chi1 (amino acids 1-57 of Dpl replaced by amino acids 1-125 of PrP) and Chi2 (amino acids 1-66 of Dpl replaced by amino acids 1-134 of PrP) abrogated the pathogenicity of Dpl indicating that the presence of a N-terminal domain of PrP (23-134) reduced the toxicity of Dpl, as reported. However, when the amino acids 1-24 of Dpl were replaced by amino acids 1-124 of PrP, Chi3 Tg mice, which express the chimeric protein at a very low level, start developing ataxia at the age of 5-7 weeks. This phenotype was not counteracted by a single copy of full-length-PrP(c) but rather by its overexpression, indicating the strong toxicity of the chimeric protein Chi3. Chi3 Tg mice exhibit severe cerebellar atrophy with a significant loss of granule cells. We concluded that aa25 to aa57 of Dpl, which are not present in Chi1 and Chi2 constructs, confer toxicity to the protein. We tested this possibility by using the 25-57 Dpl peptide in primary culture of mouse embryo cortical neurons and found a significant neurotoxic effect. This finding identifies a protein domain that plays a role in mediating Dpl-related toxicity.
Asunto(s)
Ataxia/genética , Ataxia/patología , Cerebelo/patología , Priones/genética , Animales , Ataxia/metabolismo , Western Blotting , Proteínas Ligadas a GPI/química , Proteínas Ligadas a GPI/genética , Proteínas Ligadas a GPI/metabolismo , Inmunohistoquímica , Ratones , Ratones Transgénicos , Microscopía Electrónica de Transmisión , Priones/química , Priones/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Quimera por TrasplanteRESUMEN
A growing number of studies have investigated the interaction between C1q and PrP, but the oligomeric form of PrP involved in this interaction remains to be determined. Aggregation of recombinant full-length murine PrP in the presence of 100 mm NaCl allowed us to isolate three different types of oligomers by size-exclusion chromatography. In contrast to PrP monomers and fibrils, these oligomers activate the classical complement pathway, the smallest species containing 8-15 PrP protomers being the most efficient. We used Thioflavine T fluorescence to monitor PrP aggregation and showed that, when added to the reaction, C1q has a cooperative effect on PrP aggregation and leads to the formation of C1q-PrP complexes. In these complexes, C1q interacts through its globular domains preferentially with the smallest oligomers, as shown by electron microscopy, and retains the ability to activate the classical complement pathway. Using two cell lines, we also provide evidence that C1q inhibits the cytotoxicity induced by the smallest PrP oligomers. The cooperative interaction between C1q and PrP could represent an early step in the disease, where it prevents elimination of the prion seed, leading to further aggregation.
Asunto(s)
Complemento C1q/metabolismo , Priones/química , Amiloide/química , Animales , Benzotiazoles , Cromatografía/métodos , Complemento C4/química , Proteínas del Sistema Complemento , Humanos , Inmunidad Innata , Ratones , Microscopía Electrónica/métodos , Neuronas/metabolismo , Unión Proteica , Tiazoles/químicaRESUMEN
Sex effect on the incubation period of variant Creutzfeldt-Jakob disease (vCJD) disease in human and ME-7 murine models was investigated. In the 167 vCJD cases reported in the United Kingdom as of January 2009, age at onset was significantly lower in female patients (by 2 years) than in male patients after stratification on birth cohort. In C57/Bl6N mice infected with ME-7 scrapie strain, incubation was shorter in female than in male mice. The incubation period increased in castrated male mice after intraperitoneal infection but not after intracerebral inoculation. In the absence of androgen receptors, the incubation period for prion disease increased after intraperitoneal inoculation. In ovariectomized or estrogen receptor alpha-defective female mice, no effect was observed on the incubation period of mouse prion disease. These results show that androgens influence the prion diseases incubation period after inoculation at a peripheral site.
Asunto(s)
Periodo de Incubación de Enfermedades Infecciosas , Enfermedades por Prión/fisiopatología , Adulto , Factores de Edad , Andrógenos/fisiología , Animales , Castración , Modelos Animales de Enfermedad , Femenino , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Proteínas Priónicas , Priones/administración & dosificación , Factores SexualesRESUMEN
Innate immunity is the major host defense against invasive aspergillosis. To determine whether the collectin mannan-binding lectin (MBL) is involved in the initial protective immunity through complement activation against opportunistic fungal infections caused by Aspergillus, we performed in vitro studies on 29 different strains of Aspergillus conidia from five different species. Incubation of Aspergillus conidia in human normal serum leads to activation of the alternative pathway, whereas neither the classical nor the lectin pathways through C4 and C2 cleavage are activated. Complement response to conidia was investigated using a MBL-deficient serum and reconstitution experiments were conducted with MBL/MASPs complexes. We found that MBL can directly support C3 activation by a C2 bypass mechanism. Finally, a stronger activation of the alternative pathway was observed for the clinical strains isolated from patients with invasive aspergillosis, compared with the environmental strains.
Asunto(s)
Aspergillus/inmunología , Lectina de Unión a Manosa de la Vía del Complemento/fisiología , Esporas Fúngicas/inmunología , HumanosRESUMEN
Transmissible spongiform encephalopathies are fatal neurodegenerative disorders thought to be transmitted by self-perpetuating conformational conversion of a neuronal membrane glycoprotein (PrP(C), for "cellular prion protein") into an abnormal state (PrP(Sc), for "scrapie prion protein"). Doppel (Dpl) is a protein that shares significant biochemical and structural homology with PrP(C). In contrast to its homologue PrP(C), Dpl is unable to participate in prion disease progression or to achieve an abnormal PrP(Sc)-like state. We have constructed a chimeric mouse protein, composed of the N-terminal domain of PrP(C) (residues 23-125) and the C-terminal part of Dpl (residues 58-157). This chimeric protein displays PrP-like biochemical and structural features; when incubated in presence of NaCl, the alpha-helical monomer forms soluble beta-sheet-rich oligomers which acquire partial resistance to pepsin proteolysis in vitro, as do PrP oligomers. Moreover, the presence of aggregates akin to protofibrils is observed in soluble oligomeric species by electron microscopy.
Asunto(s)
Fragmentos de Péptidos/química , Proteínas PrPC/química , Priones/química , Animales , Proteínas Ligadas a GPI , Ratones , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Proteínas PrPC/genética , Proteínas PrPC/metabolismo , Priones/genética , Priones/metabolismo , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Cloruro de Sodio/química , SolucionesRESUMEN
Expression of the cellular prion protein (PrP(c)) by host cells is required for prion replication and neuroinvasion in transmissible spongiform encephalopathies. As a consequence, identification of the cell types expressing PrP(c) is necessary to determine the target cells involved in the cerebral propagation of prion diseases. To identify the cells expressing PrP(c) in the mouse brain, the immunocytochemical localization of PrP(c) was investigated at the cellular and ultrastructural levels in several brain regions. In addition, we analyzed the expression pattern of a green fluorescent protein reporter gene under the control of regulatory sequences of the bovine prion protein gene in the brain of transgenic mice. By using a preembedding immunogold technique, neuronal PrP(c) was observed mainly bound to the cell surface and presynaptic sites. Dictyosomes and recycling organelles in most of the major neuron types also exhibited PrP(c) antigen. In the olfactory bulb, neocortex, putamen, hippocampus, thalamus, and cerebellum, the distribution pattern of both green fluorescent protein and PrP(c) immunoreactivity suggested that the transgenic regulatory sequences of the bovine PrP gene were sufficient to promote expression of the reporter gene in neurons that express immunodetectable endogenous PrP(c). Transgenic mice expressing PrP-GFP may thus provide attractive murine models for analyzing the transcriptional activity of the Prnp gene during prion infections as well as the anatomopathological kinetics of prion diseases.
Asunto(s)
Encéfalo/metabolismo , Regulación de la Expresión Génica/fisiología , Genes Reporteros/fisiología , Proteínas Luminiscentes/biosíntesis , Proteínas Luminiscentes/genética , Proteínas PrPC/biosíntesis , Animales , Encéfalo/ultraestructura , Bovinos , Proteínas Fluorescentes Verdes , Inmunohistoquímica , Proteínas Luminiscentes/análisis , Ratones , Ratones Transgénicos , Proteínas PrPC/análisis , Proteínas PrPC/genéticaRESUMEN
Fatal neurodegenerative prion diseases are caused by the transmissible PrP(Sc) prion agent whose initial replication after peripheral inoculation takes place in follicular dendritic cells present in germinal centers of lymphoid organs. However, prion replication also occurs in lymphoid cells. To assess the role of the hematopoietic compartment in neuroinvasion and prion replication, we generated chimeric mice, on a uniform congenic C57/BL6J background, by bone marrow replacement with hematopoietic cells expressing different levels of PrP protein. Nine different types of chimeric mice were inoculated intraperitoneally either with the lymphotropic Rocky Mountain Laboratory (RML) strain or the non lymphotropic ME-7 scrapie strain, at different doses. Here, we clearly demonstrate that overexpression of PrP by the hematopoietic system, or the lack of PrP expression by the bone marrow derived cells, does not change the incubation time period of the disease, even when the mice are infected at limiting doses. We conclude that the hematopoietic compartment is more or less permissive to prion replication, both for RML and ME-7, but does not play a role in neuroinvasion.
Asunto(s)
Médula Ósea/fisiopatología , Priones/fisiología , Scrapie/fisiopatología , Animales , Ratones , Ratones Endogámicos C57BL , Proteínas PrPC/metabolismoRESUMEN
Mice defective for C1q complement factor show enhanced resistance to peripheral prion inoculation, and previous work demonstrated a direct interaction between C1q and conformationally modified PrP. However, the nature and physiological consequences of this interaction remain uncharacterized. PrP amino acids 141-159 has been identified as a potential C1q binding site; we show, by both surface plasmon resonance (SPR) spectroscopy and ELISA, that C1q and its globular region bind to PrP mutagenized in the region of interest with comparable efficiency to that of wild-type protein. To test PrP's ability to activate complement, soluble oligomers of the PrP constructs were made. Only PrP and mutagenized PrP oligomers activate the classical complement cascade while PrP monomer and the C-terminal domain, both in oligomeric and in monomeric form, failed to induce activation. This suggests that a conformational change in PrP, which occurs both when PrP is bound to an SPR sensor chip and when it undergoes oligomerization, is requisite for PrP/C1q interaction and activation of the complement cascade. We propose that C1q may act as a natural sensor for prions, leading to activation of the classical complement cascade, which could result in local inflammation and subsequent recruitment of the immune cells that prions initially infect.
Asunto(s)
Complemento C1q/metabolismo , Vía Clásica del Complemento/inmunología , Priones/inmunología , Animales , Ensayo de Inmunoadsorción Enzimática , Ratones , Mutagénesis , Priones/genética , Unión Proteica , Conformación Proteica , Mapeo de Interacción de Proteínas , Resonancia por Plasmón de SuperficieRESUMEN
Prion infection relies on a continuous chain of PrP(c)-expressing tissues to spread from peripheral sites to the central nervous system (CNS). Direct neuroinvasion via peripheral nerves has long been considered likely. However, the speed of axonal flow is incompatible with the lengthy delay prior to the detection of PrP(Sc) in the brain. We hypothesized that Schwann cells could be the candidate implicated in this mechanism; for that, it has to express PrP(c) and to allow PrP(Sc) conversion. We investigated in vivo localization of PrP(c) in sciatic nerve samples from different strains of mice. We demonstrated that PrP(c) is mainly localized at the cell membrane of the Schwann cell. We also studied in vitro expression of PrP(c) in the Schwann cell line MSC-80 and demonstrated that it expresses PrP(c) at the same location. More specifically, we demonstrated that this glial cell line, when infected in vitro with the mouse Chandler prion strain, both produces the PrP(Sc) till after 18 passages and is able to transmit disease to mice, which then develop the typical signs of prion diseases. It is the first time that infection and replication of PrP(Sc) are shown in a peripheral glial cell line.