RESUMO
PFGCN20 is a member of the ATP-binding cassette family of proteins that is closely related to the yeast translational regulator Gcn20p. We have generated a polyclonal antibody against the N-terminal region of PFGCN20 and studied the cellular localization of PFGCN20 throughout the erythrocytic life cycle of Plasmodium falciparum. PFGCN20 was found to be present at all stages and a pronounced export of PFGCN20 into the erythrocyte was observed in the trophozoite and schizont stages. In the indirect immunofluorescence assay, PFGCN20 was found to display significant colocalization with antigens detected by the monoclonal antibody 41E11. In contrast, there was only a minimal overlap of PFGCN20 localization with EMP2 and HRP2. Immunoelectron microscopy demonstrated a pronounced accumulation of PFGCN20 in the lumen of the parasitophorous vacuole and deconvolution fluorescence microscopy showed membrane association with selective regions of a tubovesicular network in the red cell. We also observed a concentration of PFGCN20 in electron-dense plaques just underneath the parasite's plasma membrane and an association of PFGCN20 with cytoplasmic vesicular structures within the parasite. The observed export of PFGCN20 and its association with the tubovesicular network in host red cells, may be indicative of the fact that PFGCN20 functions as ATP-binding subunit of an unknown multimeric ABC-transporter. The cytoplasmic localization of PFGCN20 in the parasite, however, suggests that the involvement of PFGCN20 in translational regulation or other cytoplasmic biological functions cannot be ruled out.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Proteínas de Bactérias/metabolismo , Membrana Eritrocítica/metabolismo , Plasmodium falciparum/química , Plasmodium falciparum/metabolismo , Proteínas de Protozoários/metabolismo , Animais , Membrana Eritrocítica/parasitologia , Técnica Indireta de Fluorescência para Anticorpo , Humanos , Immunoblotting , Estágios do Ciclo de Vida/fisiologia , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Eletrônica , Plasmodium falciparum/crescimento & desenvolvimento , Plasmodium falciparum/ultraestrutura , Proteínas/metabolismoRESUMO
There is great need to identify and characterize drug targets and chemotherapeutic strategies against malaria. Here we show that a vacuolar-network induced by the human malaria parasite Plasmodium falciparum, is a major import pathway for artemisinin, a leading, new anti-malarial that is known to be effective against drug resistant strains. We also show that artemisinin-treatment induces aberrant, budding of a vacuolar-network membrane protein and its antimalarial activity is additive with toxic sphingolipid analogues that block the network. The data suggest that artemisinin alters membrane protein export from the vacuolar-network and combinations with anti-network reagents have the potential to provide powerful new chemotherapy for drug resistant malaria.
Assuntos
Antimaláricos/metabolismo , Artemisininas , Plasmodium falciparum/metabolismo , Sesquiterpenos/metabolismo , Esfingolipídeos/farmacologia , Vacúolos/metabolismo , Animais , Antimaláricos/farmacologia , Transporte Biológico , Cloroquina/farmacologia , Resistência a Medicamentos , Sinergismo Farmacológico , Eritrócitos/metabolismo , Eritrócitos/parasitologia , Técnica Indireta de Fluorescência para Anticorpo , Humanos , Malária Falciparum/parasitologia , Proteínas de Membrana/metabolismo , Microscopia Eletrônica , Plasmodium falciparum/efeitos dos fármacos , Proteínas de Protozoários/metabolismo , Sesquiterpenos/farmacologia , Vacúolos/efeitos dos fármacosAssuntos
Proteínas Luminescentes/genética , Plasmodium falciparum/genética , Animais , Expressão Gênica , Genes Reporter , Proteínas de Fluorescência Verde , Proteínas Luminescentes/metabolismo , Microscopia de Fluorescência , Plasmídeos/genética , Plasmodium falciparum/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , TransfecçãoAssuntos
Proteínas de Transporte/genética , Genes de Protozoários , Complexo de Golgi/química , Plasmodium berghei/genética , Proteínas rab de Ligação ao GTP , Proteínas ras/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Proteínas de Transporte/análise , Proteínas de Transporte/química , DNA Complementar , Dados de Sequência Molecular , Plasmodium/genética , Análise de Sequência de DNA , Proteínas ras/análise , Proteínas ras/químicaRESUMO
Nucleotide sequences in the GenEMBL database were analyzed using strategies designed to reveal species-specific patterns of DNA bending and DNA sequence. The results uncovered striking species-dependent patterns of bending with more variations among individual organisms than between prokaryotes and eukaryotes. The frequency of bent sites in sequences from different bacteria was related to genomic A + T content and this relationship was confirmed by electrophoretic analysis of genomic DNA. However, base composition was not an accurate predictor for DNA bending in eukaryotes. Sequences from C. elegans exhibited the highest frequency of bent sites in the database and the RNA polymerase II locus from the nematode was the most bent gene in GenEMBL. Bent DNA extended throughout most introns and gene flanking segments from C.elegans while exon regions lacked A-tract bending characteristics. Independent evidence for the strong bending character of this genome was provided by electrophoretic studies which revealed that a large number of the fragments from C.elegans DNA exhibited anomalous gel mobilities when compared to genomic fragments from over 20 other organisms. The prevalence of bent sites in this genome enabled us to detect selectively C.elegans sequences in a computer search of the database using as probes C.elegans introns, bending elements, and a 20 nucleotide consensus sequence for bent DNA. This approach was also used to provide additional examples of species-specific sequence patterns in eukaryotes where it was shown that (A) greater than or equal to 10 and (A.T) greater than or equal to 5 tracts are prevalent throughout the untranslated DNA of D.discodium and P.falciparum, respectively. These results provide new insight into the organization of eukaryotic DNA because they show that species-specific patterns of simple sequences are found in introns and in other untranslated regions of the genome.
Assuntos
DNA/química , Conformação de Ácido Nucleico , Algoritmos , Animais , Composição de Bases , Sequência de Bases , Caenorhabditis/genética , DNA Bacteriano/química , Bases de Dados Factuais , Dictyostelium/genética , Eletroforese em Gel Bidimensional , Biblioteca Gênica , Íntrons/genética , Dados de Sequência Molecular , Plasmodium falciparum/genética , RNA Polimerase II/genética , Sequências Repetitivas de Ácido Nucleico , Especificidade da Espécie , Zea mays/genéticaRESUMO
Erythrocytes, which are incapable of endocytosis or phagocytosis, can be infected by the malaria parasite Plasmodium falciparum. We find that a transmembrane protein (Duffy), glycosylphosphatidylinositol (GPI)-anchored and cytoplasmic proteins, associated with detergent-resistant membranes (DRMs) that are characteristic of microdomains in host cell membranes, are internalized by vacuolar parasites, while the major integral membrane and cytoskeletal proteins are not. The internalized host proteins and a plasmodial transmembrane resident parasitophorous vacuolar membrane (PVM) protein are detected in DRMs associated with vacuolar parasites. This is the first report of a host transmembrane protein being recruited into an apicomplexan vacuole and of the presence of vacuolar DRMs; it establishes that integral association does not preclude protein internalization into the P.FALCIPARUM: vacuole. Rather, as shown for Duffy, intracellular accumulation occurs at the same rate as that seen for a DRM-associated GPI-anchored protein. Furthermore, novel mechanisms regulated by the DRM lipids, sphingomyelin and cholesterol, mediate (i) the uptake of host DRM proteins and (ii) maintenance of the intracellular vacuole in the non-endocytic red cell, which may have implications for intracellular parasitism and pathogenesis.