RESUMEN
The protozoan parasite Giardia lamblia synthesizes a diverse and surprisingly abundant array of sterile transcripts unable to code for proteins. Random sampling of cDNAs from two evolutionarily divergent Giardia strains indicates that approximately 20% of cDNAs in the libraries represent polyadenylated sterile transcripts. RNase protection analysis and northern blot hybridization of three sterile transcript loci demonstrated that both the sterile transcript and a complementary mRNA were made in each case, further categorizing these sterile transcripts as antisense transcripts. Investigation of the genomic loci for these same three sterile antisense transcripts showed typical transcription units for the sense transcripts, but still failed to reveal a usable open reading frame for the sterile antisense transcripts. 5'-RACE mapped the transcription start site for one of the sterile antisense transcripts to an AT-rich region, as is typical for GIARDIA: It is unclear whether these sterile transcripts represent errors in transcription or whether they have regulatory functions within the cell, although preliminary investigations failed to reveal evidence for a role in developmental gene regulation. In either case, the presence of such a large pool of sterile antisense transcripts is dramatic evidence of the unusual molecular machinery of the early diverging protist G.lamblia.
Asunto(s)
Giardia lamblia/genética , ARN no Traducido/genética , Animales , Secuencia de Bases , Northern Blotting , Cisteína Endopeptidasas/genética , ADN Complementario/química , ADN Complementario/genética , ADN Protozoario/química , ADN Protozoario/genética , Regulación del Desarrollo de la Expresión Génica , Giardia lamblia/crecimiento & desarrollo , Datos de Secuencia Molecular , NADH NADPH Oxidorreductasas/genética , Sistemas de Lectura Abierta/genética , Biosíntesis de Proteínas , ARN Helicasas/genética , ARN Mensajero/genética , Análisis de Secuencia de ADN , Transcripción GenéticaRESUMEN
Giardia lamblia, one of the earliest diverging eukaryotes and a major cause of diarrhea world-wide, has unusually short intergenic regions, raising questions concerning its regulation of gene expression. We have approached this issue through examination of the alpha2-tubulin promoter and in particular investigated the function of an AT-rich element surrounding the transcription start site. Its placement and the ability of this sequence to direct transcription initiation in the absence of any other promoter elements is similar to the initiator element in higher eukaryotes. However, the sequence diversity of extremely short (8-10 bp) initiator elements is surprising, as is their ability to independently direct substantial levels of transcription. We also identified a large AT-rich element located between -64 and -29 bp upstream of the transcriptional start site and show using both deletions and site-specific mutations of this region that sequences between -60 and the start of transcription are important for promoter strength; interestingly this AT-rich sequence is not highly conserved among different Giardia promoters. These data suggest that while the overall structure of the core promoter has been conserved throughout eukaryotic evolution, significant variation and flexibility is allowed in element consensus sequences and roles in transcription. In particular, the short and diverse sequences that function in transcription initiation in Giardia suggest the potential for relaxed transcriptional regulation.
Asunto(s)
Genoma de Protozoos , Giardia lamblia/genética , Tubulina (Proteína)/genética , Animales , Secuencia de Bases , Eliminación de Gen , Giardia lamblia/química , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Regiones Promotoras Genéticas , Transcripción Genética/genéticaRESUMEN
Immune evasion is frequently cited as the main reason for antigenic variation in pathogenic microorganisms. To better understand the role of switching of variant-specific surface proteins (VSPs) in Giardia lamblia-host interactions, antigenic variation during infections of mice and gerbils was examined, using clones that predominantly expressed unique VSPs. As expected, VSPs were selected against during infections of immunocompetent hosts. In contrast, in immunodeficient hosts, some VSPs were selected for and others were selected against. These diverse patterns of selection demonstrate that there are host-VSP interactions that exert both positive and negative selective pressures on parasites, independent of the adaptive immune response. Furthermore, selection was dependent on both the particular VSP and the host. Thus, the large number of VSP genes in G. lamblia may allow the parasite to infect multiple different hosts, and antigenic variation could be a mechanism to expand the parasite's host range.
Asunto(s)
Antígenos de Protozoos/inmunología , Antígenos de Superficie/inmunología , Giardia lamblia/inmunología , Giardiasis/parasitología , Animales , Variación Antigénica , Femenino , Gerbillinae , Interacciones Huésped-Parásitos , Inmunocompetencia , Terapia de Inmunosupresión , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones SCIDAsunto(s)
Giardia lamblia/metabolismo , Proteínas Protozoarias/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Transporte Biológico Activo , Núcleo Celular/metabolismo , ADN Protozoario/genética , Giardia lamblia/genética , Proteínas Fluorescentes Verdes , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Datos de Secuencia Molecular , Señales de Localización Nuclear/genética , Plásmidos/genética , Proteínas Protozoarias/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
The rab6 gene product in mammalian cells and yeast is localized to and regulates protein transport in the medial and trans Golgi cisternae, as well as the trans Golgi network. We have identified a homologue in the malaria parasite Plasmodium falciparum which displays a rab-like sequence that is 62.4% identical to mammalian rab6. In addition the parasite gene (Pfrab6 gene) contains an N-terminal hydrophobic domain, unique to P. falciparum. Antibodies developed to Pfrab6 localize protein in 4-7 well-resolved sites in a ring-stage parasite, as detected by high resolution fluorescence microscopy. This suggests that there are multiple, distinct foci of medial/trans Golgi membranes in a ring. ERD2 is a cis Golgi marker in mammalian cells. The plasmodial homologue of ERD2 (PfERD2) is concentrated in a single perinuclear region in a ring-stage parasite. This site is distinct from the Pfrab6 membranes, indicating that early and late Golgi markers can be segregated in P. falciparum. Mammalian cells contain a single Golgi complex where cis medial and trans markers are tightly stacked in closely apposed cisternae. In P. falciparum-rings however, rab6-associated membranes are not invariably 'stacked' with an ERD2 structure. In immunoelectron microscopy studies, both the PfERD2- and Pfrab6-associated membranes appear tubulovesicular in nature, devoid of cisternal morphology. Hence the Golgi of ring stage parasites may comprise of multiple, 'unstacked' tubulovesicular clusters, suggesting a primitive organization of the organelle in Plasmodia.
Asunto(s)
Proteínas Portadoras/aislamiento & purificación , Aparato de Golgi/química , Proteínas de la Membrana/aislamiento & purificación , Plasmodium falciparum/química , Proteínas Protozoarias/aislamiento & purificación , Receptores de Péptidos , Proteínas de Saccharomyces cerevisiae , Proteínas de Unión al GTP rab , Proteínas ras/aislamiento & purificación , Secuencia de Aminoácidos , Animales , Biomarcadores , Proteínas Portadoras/genética , Proteínas Portadoras/inmunología , Clonación Molecular , Técnica del Anticuerpo Fluorescente Indirecta , Genes Protozoarios , Aparato de Golgi/ultraestructura , Microscopía Fluorescente/instrumentación , Microscopía Inmunoelectrónica , Modelos Estructurales , Datos de Secuencia Molecular , Plasmodium falciparum/genética , Plasmodium falciparum/crecimiento & desarrollo , Plasmodium falciparum/ultraestructura , Reacción en Cadena de la Polimerasa , Proteínas Protozoarias/genética , Proteínas Protozoarias/inmunología , Homología de Secuencia de Aminoácido , Proteínas ras/genética , Proteínas ras/inmunologíaRESUMEN
During its asexual life cycle, the human malaria parasite Plasmodium falciparum exports numerous proteins beyond its surface to its host erythrocyte. We have studied the biosynthesis, processing and export of a 45 kDa parasite protein resident in membrane clefts in the erythrocyte cytoplasm. Our results indicate that this cleft protein is made as a single tightly membrane-bound 45 kDa polypeptide in ring- and trophozoite-infected erythrocytes (0-36 h in the life cycle). Using ring/trophozoite parasites released from erythrocytes, the 45 kDa protein is shown to be efficiently transported to the cell surface. This export is specifically blocked by the drug brefeldin A, and at 15 and 20 degrees C. These results indicate that transport blocks seen in the Golgi of mammalian cells are conserved in P. falciparum. Further, the newly synthesized 45 kDa protein passes through parasite Golgi compartments before its export to clefts in the erythrocyte. In mid-to-late-ring-infected erythrocytes, a fraction of the newly synthesized 45 kDa protein is processed to a second membrane-bound phosphorylated 47 kDa protein. The t1/2 of this processing step is about 4 h, suggesting that it occurs subsequent to protein export from the parasite. Evidence is presented that, in later trophozoite stages (24-36 h), the exported 45 and 47 kDa proteins are partially converted into soluble molecules in the intraerythrocytic space. Taken together, the results indicate that the lower eukaryote P. falciparum modulates a classical secretory pathway to support membrane export beyond its plasma membrane to clefts in the erythrocyte. Subsequent to export, phosphorylation and/or conversion into a soluble form may regulate the interactions of the 45 kDa protein with the clefts during parasite development.
Asunto(s)
Membrana Eritrocítica/metabolismo , Eritrocitos/metabolismo , Plasmodium falciparum/metabolismo , Procesamiento Proteico-Postraduccional , Proteínas Protozoarias/metabolismo , Animales , Transporte Biológico , Medios de Cultivo , Eritrocitos/parasitología , Técnica del Anticuerpo Fluorescente , Aparato de Golgi/metabolismo , Humanos , Membranas Intracelulares/metabolismo , Peso Molecular , Fosforilación , Plasmodium falciparum/crecimiento & desarrollo , Pruebas de Precipitina , Proteínas Protozoarias/biosíntesisRESUMEN
This work describes two unusual features of membrane development in a eukaryotic cell. (a) The induction of an extensive network of tubovesicular membranes by the malaria parasite Plasmodium falciparum in the cytoplasm of the mature erythrocyte, and its visualization with two ceramide analogues C5-DMB-ceramide and C6-NBD-ceramide. "Sectioning" of the infected erythrocytes using laser confocal microscopy has allowed the reconstruction of detailed three-dimensional images of this novel membrane network. (b) The stage-specific export of sphingomyelin synthase, a biosynthetic activity concentrated in the Golgi of mammalian cells, to this tubovesicular network. Evidence is presented that in the extracellular merozoite stage the parasite retains sphingomyelin synthase within its plasma membrane. However, intracellular ring- and trophozoite-stage parasites export a substantial fraction (approximately 26%) of sphingomyelin synthase activity to membranes beyond their plasma membrane. Importantly we do not observe synthesis of new enzyme during these intracellular stages. Taken together these results strongly suggest that the export of this classic Golgi enzyme is developmentally regulated in Plasmodium. We discuss the significance of this export and the tubovesicular network with respect to membrane development and function in the erythrocyte cytosol.
Asunto(s)
Eritrocitos/parasitología , Aparato de Golgi/metabolismo , Plasmodium falciparum/metabolismo , Transferasas (Grupos de Otros Fosfatos Sustitutos)/metabolismo , Animales , Transporte Biológico , Biomarcadores , Citoplasma/metabolismo , Eritrocitos/metabolismo , Humanos , Immunoblotting , Técnicas In VitroRESUMEN
The ERD2 gene product in mammalian cells and yeast is a receptor required for protein retention in the endoplasmic reticulum (ER); immunolocalization studies indicate that the protein is concentrated in the cis Golgi. We have identified a homologue of ERD2 in the malaria parasite, Plasmodium falciparum (PfERD2). The deduced protein sequence is 42% identical to mammalian and yeast homologues and bears striking homology in its proposed tertiary structure. PfERD2 is tightly confined to a single focus of staining in the perinuclear region as seen by indirect immunofluorescence. This is redistributed by brefeldin A (BFA) to a diffuse pattern similar to that of parasite BiP, a marker for the ER; removal of the drug results in recovery of the single focus, consistent with the localization of PfERD2 to the parasite Golgi and its participation in a retrograde transport pathway to the ER. Sphingomyelin synthesis is a second resident activity of the cis Golgi whose organization is sensitive to BFA in mammalian cells. Within the parasite it again localizes to a perinuclear region but does not reorganize upon BFA treatment. The results strongly suggest that these two activities are in distinct compartments of the Golgi in the malaria parasite.
Asunto(s)
Aparato de Golgi/metabolismo , Proteínas de la Membrana/metabolismo , Plasmodium falciparum/metabolismo , Proteínas Protozoarias , Receptores de Péptidos , Proteínas de Saccharomyces cerevisiae , Esfingomielinas/biosíntesis , Secuencia de Aminoácidos , Animales , Anticuerpos Antiprotozoarios/inmunología , Secuencia de Bases , Brefeldino A , Clonación Molecular , Ciclopentanos/farmacología , Técnica del Anticuerpo Fluorescente , Proteínas de la Membrana/inmunología , Datos de Secuencia Molecular , Oligodesoxirribonucleótidos , Homología de Secuencia de AminoácidoRESUMEN
The asexual blood stage of the human malaria parasite Plasmodium falciparum resides within the mature erythrocyte - a cell that has no intracellular organelles and few biosynthetic activities. However, Plasmodium, as on actively growing and dividing cell, has numerous requirements for the uptake o f nutrients and expulsion of waste. Hence, the parasite must extensively remodel the erythrocyte to facilitate its survival, not only by exporting numerous proteins, but also by providing the requisite machinery for their .trafficking. In this review, Heidi Elmendorf and Kastun Haldar propose a model for secretion in P. falciparum.
RESUMEN
Controlled mechanical homogenization of Plasmodium falciparum-infected erythrocytes releases parasites of a quality sufficient for studying the export of newly synthesized plasmodial proteins. Protein synthesis occurs within intact released parasites as defined by resistance of acid-insoluble incorporation of radiolabel to high levels of exogenously added EDTA, hexokinase, and RNaseA. While exogenously added ATP and erythrocyte cytosol were not essential for biosynthetic activity at levels comparable to that seen in infected erythrocytes, the addition of an extracellular ATP regenerating system (ARS) stimulated the synthesis of parasite proteins. Conversely, parasite viability and biosynthetic activity are decreased by the addition of a non-hydrolyzable ATP analogue (ATP gamma S), ADP, or ATP in the absence of a regenerating system. These data suggest a metabolic interdependence between extracellular energy metabolism and biosynthetic functions within the parasite. The export of a predominant subset of proteins was retarded in the presence of Brefeldin A, indicating the existence of a classical secretory pathway characteristic of that seen in higher eukaryotic cells. Interestingly, a Brefeldin A-insensitive component of export was also consistently observed; this may suggest the existence of an additional alternative secretory mechanism in malaria.
Asunto(s)
Plasmodium falciparum/metabolismo , Proteínas Protozoarias/biosíntesis , Adenosina Trifosfato/metabolismo , Animales , Antibacterianos/farmacología , Brefeldino A , Ciclopentanos/farmacología , Citosol/metabolismo , Eritrocitos/parasitología , Hexoquinasa/metabolismo , Microscopía Electrónica , Plasmodium falciparum/efectos de los fármacos , Plasmodium falciparum/ultraestructura , Proteínas Protozoarias/metabolismo , Ribonucleasa Pancreática/metabolismoRESUMEN
We have examined the accumulation and metabolism of N-[7-(4-nitrobenzo-2-oxa-1,3-diazole)]aminocaproyl sphingosine (C6-NBD-cer) in Plasmodium falciparum FCR-3/A2-infected erythrocytes. C6-NBD-cer transferred to live infected erythrocytes at 2 degrees C to label the infected red cell surface and intracellular parasite membranes. Subsequent incubation for 30 min at 2 degrees C, resulted in a depletion of the ceramide label from the red cell membrane and an accumulation of fluorescence in parasite membranes, by an energy independent process. When the cells were subsequently warmed to 37 degrees C for 30 min, virtually all of the ceramide was converted to N-[7-(4-nitrobenzo-2-oxa-1,3- diazole)]aminocaproyl sphingosine-1-phosphocholine (C6-NBD-Sm). Uninfected erythrocytes were incapble of sphingomyelin synthesis. By fluorescence microscopy, sphingomyelin synthesis in infected erythrocytes occurred in compartments morphologically similar to those accumulating ceramide. To examine the intracellular sites of ceramide accumulation glutaraldehyde fixed cells were labeled with C6-NBD-ceramide and subsequently back extracted to remove excess probe. This resulted in a depletion of label at the red cell membrane but prominent fluorescence remained associated with the parasite. Photobleaching in the presence of diaminobenzidine resulted in precipitates in intraerythrocytic cisternae and the vacuolar membrane surrounding the parasite, rather than a perinuclear Golgi apparatus within the organism. The results support a novel organisation of plasmodial membranes regulating the accumulation and metabolism of C6-NBD-cer in infected erythrocytes.