RESUMEN
The human malaria parasite Plasmodium falciparum harbors a relict, nonphotosynthetic plastid of algal origin termed the apicoplast. Although considerable progress has been made in defining the metabolic functions of the apicoplast, information on the composition and biogenesis of the four delimiting membranes of this organelle is limited. Here, we report an efficient method for preparing highly purified apicoplasts from red blood cell parasite stages and the comprehensive lipidomic analysis of this organelle. Apicoplasts were prepared from transgenic parasites expressing an epitope-tagged triosephosphate transporter and immunopurified on magnetic beads. Gas and liquid chromatography MS analyses of isolated apicoplast lipids indicated significant differences compared with total parasite lipids. In particular, apicoplasts were highly enriched in phosphatidylinositol, consistent with a suggested role for phosphoinositides in targeting membrane vesicles to apicoplasts. Apicoplast phosphatidylinositol and other phospholipids were also enriched in saturated fatty acids, which could reflect limited acyl exchange with other membrane phospholipids and/or a requirement for specific physical properties. Lipids atypical for plastids (sphingomyelins, ceramides, and cholesterol) were detected in apicoplasts. The presence of cholesterol in apicoplast membranes was supported by filipin staining of isolated apicoplasts. Galactoglycerolipids, dominant in plant and algal plastids, were not detected in P. falciparum apicoplasts, suggesting that these glycolipids are a hallmark of photosynthetic plastids and were lost when these organisms assumed a parasitic lifestyle. Apicoplasts thus contain an atypical melange of lipids scavenged from the human host alongside lipids remodeled by the parasite cytoplasm, and stable isotope labeling shows some apicoplast lipids are generated de novo by the organelle itself.
Asunto(s)
Lípidos/química , Malaria Falciparum/parasitología , Plasmodium falciparum/metabolismo , Plastidios/química , Colesterol/metabolismo , Cromatografía Liquida , Ácidos Grasos/metabolismo , Cromatografía de Gases y Espectrometría de Masas , Humanos , Metabolismo de los Lípidos , Plasmodium falciparum/ultraestructura , Plastidios/ultraestructuraRESUMEN
Live cell imaging of human malaria parasites Plasmodium falciparum during gametocytogenesis revealed that the apicoplast does not grow, whereas the mitochondrion undergoes remarkable morphological development. A close connection of the two organelles is consistently maintained. The apicoplast and mitochondrion are not components of the male gametes, suggesting maternal inheritance.
Asunto(s)
Células Germinativas/fisiología , Malaria Falciparum/parasitología , Mitocondrias/metabolismo , Plasmodium falciparum/fisiología , Plastidios/metabolismo , Animales , Femenino , Gametogénesis , Células Germinativas/citología , Humanos , Masculino , Mitocondrias/genética , Plasmodium falciparum/citología , Plasmodium falciparum/genética , Plastidios/genética , Especificidad de la EspecieRESUMEN
In eukaryotes, the enzyme GDP-mannose pyrophosphorylase (GDP-MP) is essential for the formation of GDP-mannose, the donor of activated mannose for all glycosylation reactions. Unlike other eukaryotes, where deletion of GDP-mannose pyrophosphorylase is lethal, deletion of this gene in Leishmania mexicana has no effect on viability, but leads to the generation of avirulent parasites. In this study, we show that the null mutants have a perturbed morphology and cytokinesis, retarded growth and increased adherence to the substratum where they form large colonies. The null mutants attach avidly to mouse macrophages, but unlike the wild type organisms, they do not bind to the complement receptor 3 and are slow to induce phagocytosis. Once internalised, they localise to the phagolysosome, but in contrast to wild type organisms which transform into the intracellular amastigote and establish in the macrophage, they are cleared by 24 h in culture and by 5 h in vivo. The null mutants are hypersensitive to human but not mouse complement and to temperature and acidic pH. Surprisingly, in view of the lack of several known host-protective antigens, injection of the mutant parasites into BALB/c mice confers significant and long lasting protection against infection, suggesting that these temperature sensitive mutants are an attractive candidate for a live attenuated vaccine.
Asunto(s)
Leishmania mexicana/fisiología , Animales , Anticuerpos/inmunología , Adhesión Celular/fisiología , Línea Celular , Citocinesis/fisiología , Femenino , Guanosina Difosfato Manosa/genética , Interacciones Huésped-Parásitos , Humanos , Concentración de Iones de Hidrógeno , Leishmania mexicana/genética , Leishmania mexicana/crecimiento & desarrollo , Antígeno de Macrófago-1/inmunología , Macrófagos/fisiología , Ratones , Ratones Endogámicos BALB C , Mutación , Fenotipo , Temperatura , Vacunación/métodos , VirulenciaRESUMEN
The apicoplast and mitochondrion of the malaria parasite Plasmodium falciparum are important intracellular organelles and targets of several anti-malarial drugs. In recent years, our group and others have begun to piece together the metabolic pathways of these organelles, with a view to understanding their functions and identifying further anti-malarial targets. This has involved localization of putative organellar proteins using fluorescent reporter proteins such as green fluorescent protein (GFP). A major limitation to such an approach is the difficulties associated with using existing plasmids to genetically modify P. falciparum. In this paper, we present a novel series of P. falciparum transfection vectors based around the Gateway recombinatorial cloning system. Our system makes it considerably easier to construct fluorescent reporter fusion proteins, as well as allowing the use of two selectable markers. Using this approach, we localize proteins involved in isoprenoid biosynthesis and the posttranslational processing of apicoplast-encoded proteins to the apicoplast, and a protein putatively involved in the citric acid cycle to the mitochondrion. To confirm the localization of these proteins, we have developed a new immunofluorescence assay (IFA) protocol using antibodies specific to the apicoplast and mitochondrion. In comparison with published IFA methods, we find that ours maintains considerably better structural preservation, while still allowing sufficient antibody binding as well as preserving reporter protein fluorescence. In summary, we present two important new tools that have enabled us to characterize some of the functions of the apicoplast and mitochondrion, and which will be of use to the wider malaria research community in elucidating the localization of other P. falciparum proteins.
Asunto(s)
Técnica del Anticuerpo Fluorescente/métodos , Vectores Genéticos , Mitocondrias/química , Orgánulos/química , Plasmodium falciparum/química , Proteínas Protozoarias/análisis , Animales , Fusión Artificial Génica , Ciclo del Ácido Cítrico , Clonación Molecular/métodos , Genes Reporteros , Orgánulos/enzimología , Plasmodium falciparum/enzimología , Plasmodium falciparum/genética , Procesamiento Proteico-Postraduccional , Transporte de Proteínas , Proteínas Recombinantes de Fusión , Selección Genética , Terpenos/metabolismo , TransfecciónRESUMEN
Red blood cells infected with Plasmodium falciparum (iRBCs) have been shown to modulate maturation of human monocyte-derived dendritic cells (DCs), interfering with their ability to activate T cells. Interaction between Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) and CD36 expressed by DCs is the proposed mechanism, but we show here that DC modulation does not require CD36 binding, PfEMP1, or contact between DCs and infected RBCs and depends on the iRBC dose. iRBCs expressing a PfEMP1 variant that binds chondroitin sulfate A (CSA) but not CD36 were phagocytosed, inhibited lipopolysaccharide (LPS)-induced phenotypic maturation and cytokine secretion, and abrogated the ability of DCs to stimulate allogeneic T-cell proliferation. CD36- and CSA-binding iRBCs showed comparable inhibition. P. falciparum lines rendered deficient in PfEMP1 expression by targeted gene knockout or knockdown also inhibited LPS-induced phenotypic maturation, and separation of DCs and iRBCs in transwells showed that inhibition was not contact dependent. Inhibition was observed at an iRBC:DC ratio of 100:1 but not at a ratio of 10:1. High doses of iRBCs were associated with apoptosis of DCs, which was not activation induced. Lower doses of iRBCs stimulated DC maturation sufficient to activate autologous T-cell proliferation. In conclusion, modulation of DC maturation by P. falciparum is dose dependent and does not require interaction between PfEMP1 and CD36. Inhibition and apoptosis of DCs by high-dose iRBCs may or may not be physiological. However, our observation that low-dose iRBCs initiate functional DC maturation warrants reevaluation and further investigation of DC interactions with blood-stage P. falciparum.
Asunto(s)
Antígenos CD36/metabolismo , Diferenciación Celular/inmunología , Células Dendríticas/citología , Células Dendríticas/inmunología , Eritrocitos/parasitología , Malaria Falciparum/inmunología , Plasmodium falciparum/patogenicidad , Proteínas Protozoarias/metabolismo , Animales , Células CHO , Adhesión Celular , Células Cultivadas , Sulfatos de Condroitina/metabolismo , Técnicas de Cocultivo , Cricetinae , Cricetulus , Células Dendríticas/fisiología , Eritrocitos/fisiología , Humanos , Activación de Linfocitos , Malaria Falciparum/parasitología , Fagocitosis , Plasmodium falciparum/crecimiento & desarrollo , Plasmodium falciparum/metabolismo , Linfocitos T/inmunologíaRESUMEN
Malaria parasites contain a nonphotosynthetic plastid homologous to chloroplasts of plants. The parasite plastid synthesizes fatty acids, heme, iron sulfur clusters and isoprenoid precursors and is indispensable, making it an attractive target for antiparasite drugs. How parasite plastid biosynthetic pathways are fuelled in the absence of photosynthetic capture of energy and carbon was not clear. Here, we describe a pair of parasite transporter proteins, PfiTPT and PfoTPT, that are homologues of plant chloroplast innermost membrane transporters responsible for moving phosphorylated C3, C5, and C6 compounds across the plant chloroplast envelope. PfiTPT is shown to be localized in the innermost membrane of the parasite plastid courtesy of a cleavable N-terminal targeting sequence. PfoTPT lacks such a targeting sequence, but is shown to localize in the outermost parasite plastid membrane with its termini projecting into the cytosol. We have identified these membrane proteins in the parasite plastid and determined membrane orientation for PfoTPT. PfiTPT and PfoTPT are proposed to act in tandem to transport phosphorylated C3 compounds from the parasite cytosol into the plastid. Thus, the transporters could shunt glycolytic derivatives of glucose scavenged from the host into the plastid providing carbon, reducing equivalents and ATP to power the organelle.