RESUMEN
A short-term in vitro culture system that allows for significant re-invasion of target erythrocytes by Plasmodium vivax was used to study the role of the Duffy blood group antigen as a ligand for merozoite invasion by this human malaria species. Using human Duffy-positive and -negative erythrocytes, various primate erythrocytes, enzymatic modification of erythrocytes, and mAb that defines a new Duffy determinant (Fy6) we conclude that the erythrocyte glycoprotein carrying Duffy determinants is required as a ligand for the invasion of human erythrocytes by P. vivax merozoites. Blockade of invasion by Fab fragments of the anti-Fy6 mAb equal to that of the intact molecule and the correlation of P. vivax susceptibility with the presence of the Fy6 determinant suggests this epitope or a nearby domain may be an active site on the Duffy glycoprotein. However, as for P. knowlesi, there is evidence that an alternate pathway for P. vivax invasion of simian erythrocytes may exist.
Asunto(s)
Antígenos de Grupos Sanguíneos , Sistema del Grupo Sanguíneo Duffy , Eritrocitos/parasitología , Plasmodium vivax/fisiología , Animales , Anticuerpos Monoclonales , Antígenos de Grupos Sanguíneos/inmunología , Células Cultivadas , Quimotripsina/farmacología , Sistema del Grupo Sanguíneo Duffy/inmunología , Epítopos/inmunología , Eritrocitos/efectos de los fármacos , Humanos , Fragmentos Fab de Inmunoglobulinas , Macaca mulatta , Malaria/sangre , Neuraminidasa/farmacología , Plasmodium/fisiología , Saimiri , Tripsina/farmacologíaRESUMEN
Invasion of erythrocytes by Plasmodium merozoites is an intricate process involving multiple receptor-ligand interactions. The glycophorins and an unknown trypsin sensitive factor are all erythrocyte receptors used during invasion by the major human pathogen Plasmodium falciparum. However, only one erythrocyte receptor, Glycophorin A, has a well-established cognate parasite ligand, the merozoite protein erythrocyte binding antigen-175 (EBA-175). The involvement of several other parasite proteins during invasion have been proposed, but no direct evidence links them with a specific invasion pathway. Here we report the identification and characterization of P. falciparum normocyte binding protein 1 (PfNBP1), an ortholog of Plasmodium vivax reticulocyte binding protein-1. PfNBP1 binds to a sialic acid dependent trypsin-resistant receptor on the erythrocyte surface that appears to be distinct from known invasion receptors. Antibodies against PfNBP1 can inhibit invasion of trypsinized erythrocytes and two P. falciparum strains that express truncated PfNBP1 are unable to invade trypsinized erythrocytes. One of these strain, 7G8, also does not invade Glycophorin B-negative erythrocytes. PfNBP1 therefore defines a novel trypsin-resistant invasion pathway and adds a level of complexity to current models for P. falciparum erythrocyte invasion.
Asunto(s)
Eritrocitos/metabolismo , Proteínas de la Membrana/metabolismo , Plasmodium falciparum/metabolismo , Proteínas Protozoarias/metabolismo , Tripsina/metabolismo , Animales , Anticuerpos Antiprotozoarios/metabolismo , Secuencia de Bases , ADN Complementario , Eritrocitos/parasitología , Humanos , Proteínas de la Membrana/genética , Datos de Secuencia Molecular , Mutagénesis , Plasmodium falciparum/genética , Plasmodium falciparum/patogenicidad , Plasmodium vivax/genética , Plasmodium vivax/metabolismo , Proteínas Protozoarias/genéticaRESUMEN
We have identified strain-specific antigens with Camp and St. Lucia strains of P. falciparum of Mr approximately 285,000 and approximately 260,000, respectively. These strain-specific antigens were metabolically labeled with radioactive amino acids, indicating that they were of parasite origin rather than altered host components. These proteins had the properties of a molecule exposed on the surface of infected erythrocytes (IE). First, the proteins are accessible to lactoperoxidase-catalyzed radioiodination of IE. Second, the radioiodinated proteins were cleaved by low concentrations of trypsin (0.1 microgram/ml). Third, these antigens were immunoprecipitated after addition of immune sera to intact IE. Fourth, the strain-specific immuno-precipitation of these proteins correlated with the capacity of immune sera to block cytoadherence of IE in a strain-specific fashion. Fifth, the strain-specific antigen had detergent solubility properties (i.e., insolubility in 1% Triton X-100, solubility in 5% sodium dodecyl sulfate) similar to the variant antigen of P. knowlesi, which has been proven to be a malarial protein exposed on the erythrocyte surface.
Asunto(s)
Antígenos de Superficie/análisis , Eritrocitos/parasitología , Plasmodium falciparum/inmunología , Animales , Antígenos de Superficie/inmunología , Aotus trivirgatus , Adhesión Celular/efectos de los fármacos , Eritrocitos/inmunología , Sueros Inmunes/farmacología , Técnicas de Inmunoadsorción , Melanoma , Proteínas de la Membrana/sangre , Peso Molecular , Especificidad de la Especie , Tripsina/farmacologíaRESUMEN
Plasmodium falciparum-infected erythrocytes (RBC) develop surface protrusions (knobs) which consist of electron-dense submembrane cups and the overlying RBC plasma membrane. Knobs mediate cytoadherence to endothelial cells. Falciparum variants exist that lack knobs. Using knobby (K+) and knobless (K-) variants of two strains of P. falciparum, we confirmed Kilejian's original observation that a histidine-rich protein occurred in K+ parasites but not K- variants (Kilejian, A., 1979, Proc. Natl. Acad. Sci. USA, 76:4650-4653; and Kilejian, A., 1980, J. Exp. Med., 151:1534-1538). Two additional histidine-rich proteins of lower molecular weight were synthesized by K+ and K- variants of both strains. We used differential detergent extraction and thin-section electron microscopy to investigate the subcellular location of the histidine-rich protein unique to K+ parasites. Triton X-100, Zwittergent 314, cholic acid, CHAPS, and Triton X-100/0.6 M KCl failed to extract the unique histidine-rich protein. The residues insoluble in these detergents contained the unique histidine-rich protein and electron-dense cups. The protein was extracted by 1% SDS and by 1% Triton X-100/9 M urea. The electron-dense cups were missing from the insoluble residues of these detergents. The electron-dense cups and the unique histidine-rich protein appeared to be associated with the RBC skeleton, particularly RBC protein bands 1, 2, 4.1, and 5. We propose that the unique histidine-rich protein binds to the RBC skeleton to form the electron-dense cup. The electron-dense cup produces knobs by forming focal protrusions of the RBC membrane. These protrusions are the specific points of attachment between infected RBC and endothelium.
Asunto(s)
Proteínas Sanguíneas , Membrana Eritrocítica/ultraestructura , Glicoproteínas/sangre , Malaria/sangre , Plasmodium falciparum/patogenicidad , Animales , Cebidae , Electroforesis en Gel de Poliacrilamida , Hemólisis , Microscopía Electrónica , Proteínas/aislamiento & purificaciónRESUMEN
The protein CD36 is a membrane receptor for thrombospondin (TSP), malaria-infected erythrocytes, and collagen. Three functional sequences were identified within a single disulfide loop of CD36: one that mediates TSP binding (amino acids 87 to 99) and two that support malarial cytoadhesion (amino acids 8 to 21 and 97 to 110). One of these peptides (p87-99) is a consensus protein kinase C (PKC) phosphorylation site. Dephosphorylation of constitutively phosphorylated CD36 in resting platelets and a megakaryocytic cell line led to the loss of collagen adhesion and platelet reactivity to collagen, with a reciprocal increase in TSP binding. PKC-mediated phosphorylation of this ectodomain resulted in a loss of TSP binding and the reciprocal acquisition of collagen binding. In site-directed mutagenesis studies, when the threonine phosphorylation site was changed to alanine, CD36 was expressed in a dephosphorylated state and bound to TSP constitutively.
Asunto(s)
Antígenos CD/metabolismo , Plaquetas/metabolismo , Colágeno/metabolismo , Glicoproteínas de Membrana/metabolismo , Glicoproteínas de Membrana Plaquetaria/metabolismo , Secuencia de Aminoácidos , Animales , Antígenos CD/química , Antígenos CD/genética , Secuencia de Bases , Antígenos CD36 , Adhesión Celular , Línea Celular , Eritrocitos/citología , Eritrocitos/parasitología , Humanos , Megacariocitos/metabolismo , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Fosforilación , Plasmodium falciparum/fisiología , Adhesividad Plaquetaria , Agregación Plaquetaria , Glicoproteínas de Membrana Plaquetaria/química , Glicoproteínas de Membrana Plaquetaria/genética , Proteína Quinasa C/metabolismo , Receptores de Citoadhesina/metabolismo , TrombospondinasRESUMEN
The dynamics of multiple Plasmodium infections in asymptomatic children living under intense malaria transmission pressure provide evidence for a density-dependent regulation that transcends species as well as genotype. This regulation, in combination with species- and genotype-specific immune responses, results in nonindependent, sequential episodes of infection with each species.
Asunto(s)
Malaria/parasitología , Parasitemia/parasitología , Plasmodium/fisiología , Adolescente , Animales , Niño , Preescolar , Femenino , Genotipo , Humanos , Malaria/inmunología , Vacunas contra la Malaria , Masculino , Papúa Nueva Guinea , Plasmodium/genética , Plasmodium falciparum/fisiología , Plasmodium malariae/fisiología , Plasmodium vivax/fisiología , Especificidad de la EspecieRESUMEN
The gene encoding the circumsporozoite (CS) protein of the human malaria parasite Plasmodium vivax has been cloned. The deduced sequence of the protein consists of 373 amino acids with a central region of 19 tandem repeats of the nonapeptide Asp-Arg-Ala-Asp/Ala-Gly-Gln-Pro-Ala-Gly. A synthetic 18-amino acid peptide containing two tandem repeats binds to a monoclonal antibody directed to the CS protein of Plasmodium vivax and inhibits the interaction of this antibody with the native protein in sporozoite extracts. The portions of the CS gene that do not contain repeats are closely related to the corresponding regions of the CS genes of two simian malarias, Plasmodium cynomolgi and Plasmodium knowlesi. In contrast, the homology between the CS genes of Plasmodium vivax and Plasmodium falciparum, another malaria parasite of humans, is very limited.
Asunto(s)
Antígenos de Superficie/genética , Epítopos/genética , Plasmodium vivax/genética , Proteínas Protozoarias , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/inmunología , Antígenos de Superficie/inmunología , Clonación Molecular , Epítopos/inmunología , Haplorrinos/parasitología , Humanos , Malaria/parasitología , Hibridación de Ácido Nucleico , Plasmodium/inmunología , Plasmodium vivax/inmunología , Secuencias Repetitivas de Ácidos NucleicosRESUMEN
Plasmodium falciparum-infected erythrocytes (IE) specifically adhere to vascular endothelium in vivo and to human endothelial cells, some human melanoma cell lines, and human monocytes in vitro. The tissue cell receptor for a ligand on the surface of the infected erythrocytes is an Mr 88,000 glycoprotein (GP88) recognized by the MAb OKM5, which also blocks cytoadherence of IE. Isolated, affinity-purified GP88 (CD36) competitively blocks cytoadherence and when absorbed to plastic surfaces, specifically binds P. falciparum IE. Additionally, monoclonal and polyclonal antibodies to GP88 block cytoadherence to both target cells and immobilized GP88. Binding to GP88 by IE is unaffected by the absence of calcium or the absence of thrombospondin, a putative mediator for cytoadherence of P. falciparum IE. Thus, GP88 (CD36), which has been demonstrated to be the same as platelet glycoprotein IV, interacts directly with P. falciparum IE, presumably via a parasite-induced ligand exposed on the surface of the infected erythrocytes. CD36 is shown to be present on brain endothelium in both individuals without malaria and individuals with cerebral malaria. This would suggest that factors other than just cerebral sequestration of IE play an initiating role in the genesis of cerebral malaria.
Asunto(s)
Antígenos de Diferenciación/inmunología , Eritrocitos/metabolismo , Glicoproteínas de Membrana/inmunología , Plasmodium falciparum/metabolismo , Complejo GPIb-IX de Glicoproteína Plaquetaria , Glicoproteínas de Membrana Plaquetaria , Receptores Inmunológicos/fisiología , Formación de Roseta , Animales , Anticuerpos Monoclonales/fisiología , Antígenos de Diferenciación/aislamiento & purificación , Unión Competitiva , Encéfalo/irrigación sanguínea , Antígenos CD36 , Línea Celular , Endotelio Vascular/análisis , Eritrocitos/inmunología , Humanos , Melanoma/inmunología , Glicoproteínas de Membrana/aislamiento & purificación , Glicoproteínas de Membrana/farmacología , Peso Molecular , Plasmodium falciparum/inmunología , TrombospondinasRESUMEN
A malarial antigen, Pc96, in the plasma membrane of erythrocytes infected with Plasmodium chabaudi has been identified. It is synthesized by the parasite and present during most of the growth stages of the intra-erythrocytic cycle as demonstrated by immunofluorescence. The antigen has a molecular weight of approximately 96,000. Monoclonal antibodies raised against this antigen were used to isolate the protein by affinity chromatography. Mice immunized with affinity-purified Pc96 were partially protected against blood induced-P. chabaudi infection. This result indicates the existence of a protective antigen in the membranes of erythrocytes parasitized by a rodent malaria and encourages the search for analogous antigens in human malaria parasites as possible candidate molecules for malaria vaccination.
Asunto(s)
Antígenos de Protozoos/análisis , Membrana Eritrocítica/inmunología , Eritrocitos/parasitología , Plasmodium/inmunología , Animales , Anticuerpos Monoclonales , Anticuerpos Antiprotozoarios/biosíntesis , Antígenos de Protozoos/biosíntesis , Antígenos de Protozoos/inmunología , Antígenos de Protozoos/aislamiento & purificación , Eritrocitos/inmunología , Femenino , Sueros Inmunes/inmunología , Inmunoensayo , Malaria/prevención & control , Ratones , VacunaciónRESUMEN
A subcellular fraction enriched in erythrocyte membranes has been isolated from rhesus monkey erythrocytes infected with Plasmodium knowlesi. Infected cells were lysed by centrifugation through a zone of hypotonic buffer and membranes isolated by equilibrium density gradient centrifugation in the same tube. The purified membrane fraction was shown to include the erythrocyte surface membrane by several methods: electron microscopy, identification of Coomassie Blue stained erythrocyte membrane proteins, identification of band 3 with a monoclonal antibody, and identification of radioiodinated cell surface proteins. The resulting ghosts were shown to be specifically reactive with monkey sera against the variant surface antigens of P. knowlesi by indirect immunofluorescence and membrane agglutination. No reactivity was seen with a monoclonal antibody (13C11) against the intracellular schizont surface. A number of metabolically labelled parasite proteins were enriched in this membrane function, including peptides of 277, 208, 173, 153, 134, 109, 80, 60 and 48 kDa and the variant surface antigens of variable molecular mass (180-207 kDa). These proteins were distinct from the major parasite proteins of total infected erythrocytes and isolated merozoites. The major glucosamine labelled glycoprotein of the internal schizont (230 kDa) was not found in this fraction. Moreover, no fragment of this parasite glycoprotein was found in this membrane fraction, indicating that no part of this molecule is transported to the erythrocyte surface. In contrast, the variant antigen of P. knowlesi, known to be on the erythrocyte surface, could be readily identified as peptides unique to specific cloned parasite lines. We propose that the other nine parasite proteins found within this membrane fraction represent a starting point for the identification of other parasite proteins transported to the surface membrane of the infected erythrocyte.
Asunto(s)
Membrana Eritrocítica/análisis , Malaria/sangre , Proteínas de la Membrana/análisis , Plasmodium/análisis , Proteínas/análisis , Animales , Antígenos de Superficie/análisis , Electroforesis en Gel de Poliacrilamida , Membrana Eritrocítica/inmunología , Membrana Eritrocítica/ultraestructura , Eritrocitos/parasitología , Femenino , Técnica del Anticuerpo Fluorescente , Glicoproteínas/análisis , Macaca mulatta , Masculino , Microscopía Electrónica , Péptidos/análisis , Plasmodium/inmunologíaRESUMEN
This paper reports the identification of 14-3-3 in Plasmodium. 14-3-3 is an evolutionarily conserved protein that is most noted as a mediator in signal transduction events and cell cycle regulation. The complete cDNA (approximately 2.6 kb) and gDNA (approximately 3.4 kb) of a Plasmodium knowlesi 14-3-3 (Pk14-3-3) is reported. The gene has three introns; two near the beginning and one close to the end of the coding sequence. Also reported, is the gDNA of the Plasmodium falciparum homologue (Pf14-3-3). Unlike in many other organisms, where multiple gene copies and different functional isoforms exist, Plasmodium 14-3-3 is encoded as a single-copy gene. Northern blot analyses show that the Pk14-3-3 transcript in asexual blood stages begins to be expressed in the ring-stage, predominates in young trophozoites, and thereafter declines. An antiserum produced against recombinant Pk14-3-3 reacts via immunoblot and immunoprecipitation with the approximately 30 kDa and the approximately 32 kDa Pk14-3-3 and Pf14-3-3 proteins, respectively. Protein expression in P. knowlesi closely mimics the pattern of the transcript.
Asunto(s)
Malaria/veterinaria , Plasmodium falciparum/genética , Plasmodium knowlesi/genética , Proteínas/genética , Tirosina 3-Monooxigenasa , Proteínas 14-3-3 , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Northern Blotting , Southern Blotting , ADN Complementario/genética , ADN Protozoario/genética , Dosificación de Gen , Malaria/parasitología , Datos de Secuencia Molecular , Enfermedades de los Monos/parasitología , Plasmodium falciparum/crecimiento & desarrollo , Plasmodium falciparum/metabolismo , Plasmodium knowlesi/crecimiento & desarrollo , Plasmodium knowlesi/metabolismo , Pruebas de Precipitina , Biosíntesis de Proteínas , Análisis de Secuencia de ADNRESUMEN
The genes encoding two merozoite surface proteins of Plasmodium vivax that are related to PvMSP3 [1] are reported. One of these genes was identified within P. vivax lambdagt11 clone 5.4, which was selected by immunoscreening with a Saimiri monkey antiserum. The insert DNA of this clone was used as a probe to isolate the complete gene from a P. vivax lambdaDASH genomic (g) DNA library. Antibodies to recombinant 5.4 and subsequent fusion proteins produce a pattern of circumferential surface fluorescence by indirect immunofluorescence assays (IFA) on segmented schizonts and free intact merozoites, and recognize a 125 kDa protein via western immunoblots. The gene, however, encodes a protein with a calculated size of 75677 Da, and 3' and 5' RACE analyses were employed to confirm the size of the gene and its coding region. The second related P. vivax gene was isolated by hybridization of a fragment of an orthologous P. knowlesi gene. The encoded proteins of all three related P. vivax genes have putative signal peptides, large central domains that contain >20% alanine residues bound by charged regions, are predicted to form alpha-helices with heptad repeat coiled-coil structures, and do not have a hydrophobic region that could anchor them to the surface of the merozoite. Although the overall identity in amino acid alignment among the three encoded proteins is low (<40%), the shared predicted structural features and motifs indicate that they are members of an intra-species family, which we are designating as the PvMSP-3 family with the reported members being Pvmsp-3alpha, Pvmsp-3beta, and Pvmsp-3gamma. We further demonstrate that this family also includes related proteins from P. knowlesi and P. falciparum.
Asunto(s)
Antígenos de Protozoos/química , Antígenos de Protozoos/genética , Genes Protozoarios , Malaria Vivax/parasitología , Plasmodium vivax/genética , Proteínas Protozoarias/química , Proteínas Protozoarias/genética , Secuencia de Aminoácidos , Animales , Antígenos de Protozoos/inmunología , Antígenos de Protozoos/metabolismo , Secuencia de Bases , Western Blotting , ADN Protozoario/análisis , ADN Protozoario/genética , Escherichia coli/genética , Escherichia coli/inmunología , Escherichia coli/metabolismo , Técnica del Anticuerpo Fluorescente , Humanos , Datos de Secuencia Molecular , Plasmodium vivax/crecimiento & desarrollo , Plasmodium vivax/metabolismo , Proteínas Protozoarias/inmunología , Proteínas Protozoarias/metabolismo , Conejos , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes de Fusión/metabolismo , Saimiri , Análisis de Secuencia de ADNRESUMEN
The karyotype and chromosomes of the human malaria parasite Plasmodium falciparum have been well characterized in recent years. Here we present karyotype maps of the three other human malaria species, P. vivax, P. malariae and P. ovale. Chromosomes of these species were found to be of significantly higher molecular weight than those of P. falciparum. Some 14 P. vivax chromosomes were distinguishable, and 12-14 P. malariae and P. ovale chromosomes. The chromosome location of 15 genes, known to be present within five synteny groups between P. falciparum and the rodent malarias, were analyzed, and four of these synteny groups were found to be conserved between all of the human malaria species. In addition, a more detailed genome map of P. vivax was made using ten housekeeping and antigen genes. These data represent the first karyotype maps of all species of malaria which infect man.
Asunto(s)
Mapeo Cromosómico , Genes Protozoarios , Cariotipificación , Plasmodium/clasificación , Plasmodium/genética , Animales , Electroforesis en Gel de Campo Pulsado , Humanos , Malaria Vivax/parasitología , Plasmodium falciparum/genética , Plasmodium malariae/genética , Plasmodium vivax/genéticaRESUMEN
Plasmodium merozoites are covered with a palisade layer of proteins that are arranged as organized bundles or appear as protruding spikes by electron microscopy. Here we present a third Plasmodium vivax merozoite surface protein, PvMSP-3, which is associated with but not anchored in the merozoite membrane. Serum from a P. vivax immune squirrel monkey was used to screen a lambdagt11 P. vivax genomic DNA (gDNA) library. Plaque-selected antibodies from clone no. 6.1, and rabbit antisera against its encoded protein, produced a pattern in immunofluorescence assays (IFAs) that is consistent with a localization at the surface of mature schizonts and free merozoites. Specific antisera also agglutinated merozoites and recognized a protein of 150 000 Da by SDS-PAGE. The complete msp-3 gene and flanking sequences were cloned from a P. vivax lambda Dash II gDNA library and also partly characterized by RACE (rapid amplification of cDNA ends). The immediate upstream sequence contains non-coding repeats and a putative protein encoding open reading frame (ORF), which are also present on the msp-3 5'RACE gene product. Pvmsp-3 encodes a protein with a calculated mass of 89 573 Da, which has a potential signal peptide and a major central alanine-rich domain (31%) that exhibits largely alpha-helical secondary structure and is flanked by charged regions. The protein does not have a putative transmembrane domain or a consensus sequence for a glycosylphosphatidylinositol (GPI) anchor modification. However, the alanine-rich domain has heptad repeats that are predicted to form coiled-coil tertiary structures, which mediate protein-protein interactions. PvMSP-3 is structurally related to P. falciparum MSP-3 and the 140000 Da MSP of P. knowlesi. Characterization of PvMSP-3, thus, also begins to define a new interspecies family of evolutionarily related Plasmodium merozoite proteins.
Asunto(s)
Antígenos de Protozoos/química , Plasmodium vivax/química , Proteínas Protozoarias/química , Alanina/química , Secuencia de Aminoácidos , Animales , Antígenos de Protozoos/genética , Antígenos de Protozoos/inmunología , Antígenos de Protozoos/metabolismo , Secuencia de Bases , Western Blotting , Clonación Molecular , Técnica del Anticuerpo Fluorescente , Genes Protozoarios , Humanos , Datos de Secuencia Molecular , Plasmodium vivax/genética , Estructura Secundaria de Proteína , Proteínas Protozoarias/genética , Proteínas Protozoarias/inmunología , Proteínas Protozoarias/metabolismo , Conejos , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Mapeo Restrictivo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , SaimiriRESUMEN
Two very large Plasmodium falciparum proteins are identified as constituents of the infected erythrocyte membrane. Sera were obtained from Aotus monkeys that had been repeatedly infected with asexual P. falciparum from one of four strains. The capacity of these sera to block in vitro cytoadherence of infected erythrocytes and agglutinate intact infected cells was determined. The sera were also used to immunoprecipitate protein antigens from detergent extracts of 125I-surface labeled or biosynthetically radiolabeled infected erythrocytes. For each serum/antigen combination, precipitation of only one protein correlated with the ability of the serum to interfere with cytoadherence and agglutinate infected cells. This malarial protein, denoted Pf EMP 1 (P. falciparum-erythrocyte-membrane-protein 1) bore strain-specific epitope(s) on the cell surface and displayed size heterogeneity (Mr approximately 220,000-350,000). Pf EMP 1 was strongly labeled by cell-surface radioiodination but was a quantitatively very minor malarial protein. Pf EMP 1 was distinguished by its size, surface accessibility and antigenic properties from a more predominant malarial protein in the same size range (Pf EMP 2) that is under the infected erythrocyte membrane at knobs. Monoclonal antibodies and rabbit antisera raised against Pf EMP 2 were used to show that this size heterogeneous antigen was indistinguishable from the previously described MESA (mature parasite infected erythrocyte surface antigen), identified by precipitation with rabbit antisera raised against the MESA hexapeptide repeats. Antibodies raised against Pf EMP 2/MESA did not precipitate Pf EMP 1. We conclude that Pf EMP 1 is either directly responsible for the cytoadherence phenomenon, or is very closely associated with another as yet unidentified functional molecule. Pf EMP 2/MESA must have a structural property/function that is important under the host cell membrane.
Asunto(s)
Antígenos de Protozoos/inmunología , Antígenos de Superficie/inmunología , Membrana Eritrocítica/inmunología , Plasmodium falciparum/análisis , Animales , Anticuerpos Antiprotozoarios/inmunología , Aotus trivirgatus/sangre , Aotus trivirgatus/parasitología , Humanos , Peso Molecular , Plasmodium falciparum/inmunologíaRESUMEN
The subcellular location of the major malarial glycoprotein in erythrocytes infected with schizonts of Plasmodium falciparum has been studied by two methods. In the first, glycoproteins were labelled with [3H]glucosamine or [3H]isoleucine during in vitro culture. Trypsin treatment of intact infected erythrocytes caused no major qualitative or quantitative changes in [3H]glucosamine labelled glycoproteins or [3H]isoleucine labelled proteins separated by sodium dodecylsulfate polyacrylamide gel electrophoresis. However, in the presence of Triton X-100 the labelled glycoproteins and proteins were completely cleaved by trypsin. In the second method, two monoclonal antibodies which specifically immunoprecipitate the major 195 kDa glycoprotein failed to react on indirect immunofluorescence with intact non-fixed schizont-infected erythrocytes, but reacted strongly with saponin released schizonts indicating specificity for the surface of mature intracellular parasites. Immunoelectronmicroscopy using ferritin-conjugated secondary antibody confirmed the location of the epitope(s) recognized by these monoclonals on the surface of intracellular parasites. Ferritin particles were not associated with knob-bearing erythrocyte membranes. The results indicate that only a small proportion or none of the 195 kDa glycoprotein is on the surface of the infected erythrocyte and that the largest proportion is expressed on the surface of mature intraerythrocytic parasites.
Asunto(s)
Glicoproteínas/metabolismo , Malaria/sangre , Proteínas de la Membrana/metabolismo , Plasmodium falciparum/metabolismo , Animales , Anticuerpos Monoclonales , Aotus trivirgatus , Membrana Eritrocítica/metabolismo , Eritrocitos/parasitología , Malaria/parasitología , Plasmodium falciparum/crecimiento & desarrollo , Fracciones Subcelulares/metabolismoRESUMEN
We have undertaken the first comparative pilot gene discovery analysis of approximately 25,000 random genomic and expressed sequence tags (ESTs) from three species of Plasmodium, the infectious agent that causes malaria. A total of 5482 genome survey sequences (GSSs) and 5582 ESTs were generated from mung bean nuclease (MBN) and cDNA libraries, respectively, of the ANKA line of the rodent malaria parasite Plasmodium berghei, and 10,874 GSSs generated from MBN libraries of the Salvador I and Belem lines of Plasmodium vivax, the most geographically wide-spread human malaria pathogen. These tags, together with 2438 Plasmodium falciparum sequences present in GenBank, were used to perform first-pass assembly and transcript reconstruction, and non-redundant consensus sequence datasets created. The datasets were compared against public protein databases and more than 1000 putative new Plasmodium proteins identified based on sequence similarity. Homologs of previously characterized Plasmodium genes were also identified, increasing the number of P. vivax and P. berghei sequences in public databases at least 10-fold. Comparative studies with other species of Apicomplexa identified interesting homologs of possible therapeutic or diagnostic value. A gene prediction program, Phat, was used to predict probable open reading frames for proteins in all three datasets. Predicted and non-redundant BLAST-matched proteins were submitted to InterPro, an integrated database of protein domains, signatures and families, for functional classification. Thus a partial predicted proteome was created for each species. This first comparative analysis of Plasmodium protein coding sequences represents a valuable resource for further studies on the biology of this important pathogen.
Asunto(s)
Biología Computacional/métodos , Genoma de Protozoos , Genómica , Malaria/parasitología , Plasmodium/genética , Proteínas Protozoarias/genética , Animales , Apicomplexa/clasificación , Apicomplexa/genética , ADN Complementario/genética , Bases de Datos de Ácidos Nucleicos , Humanos , Datos de Secuencia Molecular , Plasmodium/clasificación , Plasmodium berghei/genética , Plasmodium falciparum/genética , Plasmodium vivax/genética , Proteoma , Proteínas Protozoarias/metabolismo , Análisis de Secuencia de ADNRESUMEN
Erythrocytes infected with Plasmodium vivax show unique ultrastructural changes which include membranous structures in the host cell cytosol, called clefts, and caveola-vesicle complexes (CVC) in the infected erythrocyte membrane. It has been suggested that the latter structures correspond with the Schuffner's dots observed on Giemsastained thin films. The subcellular localization of a 28 kDa and a 95 kDa antigen of the erythrocytic stages of P. vivax was determined by post-embedding immunoelectron microscopy. Four monoclonal antibodies (MAbs) (2H12.B4,2H8.E10, 1H4.B6, and 4C12.G4) against the 95 kDa protein reacted with the vesicles of CVC and vesicles scattered in the cytoplasm of the infected erythrocytes. Two other MAbs (4C12.B10 and 4D7.B1) against a 28 kDa protein reacted with the cytoplasmic clefts and were also reactive with the vesicles and electron dense materials in parasitophorous vacuole. These parasite-induced structures make a contribution to the movement of some malaria proteins from the parasite to the erythrocyte surface.
Asunto(s)
Antígenos de Protozoos/análisis , Eritrocitos/inmunología , Plasmodium vivax/inmunología , Animales , Anticuerpos Monoclonales , Citosol/inmunología , Citosol/ultraestructura , Membrana Eritrocítica/inmunología , Membrana Eritrocítica/ultraestructura , Eritrocitos/ultraestructura , Inmunohistoquímica , Microscopía Electrónica , SaimiriRESUMEN
Mice can be partially protected against Plasmodium chabaudi by immunization with the antigen Pc96, isolated from the erythrocyte membranes of infected mice. We used a Pc96 specific monoclonal antibody to identify antigens which cross-react with Pc96 in P. falciparum, P. vivax, and P. cynomologi. The cross-reactive molecules are antigens of Mr 155,000 in P. falciparum, Mr 220,000 in P. cynomologi. They are located in the surface membranes of infected erythrocytes. Pc96 is characterized by immunoelectron microscopy and epitope mapping.
Asunto(s)
Antígenos de Protozoos/inmunología , Antígenos de Superficie/inmunología , Membrana Eritrocítica/inmunología , Malaria/inmunología , Plasmodium/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Antígenos de Protozoos/aislamiento & purificación , Antígenos de Superficie/análisis , Reacciones Cruzadas , Epítopos/inmunología , Humanos , Malaria/prevención & control , Ratones , Ratones Endogámicos BALB C , Peso Molecular , Plasmodium falciparum/inmunología , Plasmodium vivax/inmunologíaRESUMEN
Blockage of the cerebral microvasculature by Plasmodium falciparum-infected erythrocytes appears to be the principal cause of human cerebral malaria. Knobs which appear on the membrane of the infected erythrocytes adhere to the endothelium, causing the obstruction of cerebral microvessels. Protein molecules such as CD36, thrombospondin, and intercellular adhesion molecule-1, which are present on the membrane of endothelial cells, may act as receptors for the attachment of knobs of P. falciparum-infected erythrocytes. Each of these candidate host molecules for infected-cell recognition and attachment are expressed in microvessels of the human brain. The presence of HRP1 and HRP2 in the cerebral microvessels of cerebral malaria patients may indicate the involvement of knob proteins in the pathogenesis of cerebral malaria. Owl monkeys infected with P. falciparum do not develop cerebral malaria. There is no blockage of cerebral microvessels by infected erythrocytes and knob proteins are absent. These findings support the contention that cerebral microvessel blockage and the presence of knob proteins are the probable causes of cerebral malaria.