Membranes of erythrocytes infected with Plasmodium falciparum develop protrusions called "knobs." These protrusions are not apparent on erythrocytes infected with young parasites (rings) but develop with the growth of parasites to the trophozoite and schizont stages. The nature and origin of knobs were characterized by comparing the stagespecific proteins of two culture lines of P. falciparum, K+ and K-. K+ parasites produce knobs; K-parasites do not. Erythrocytes infected with both types of parasites were labeled metabolically and samples were analyzed by electrophoresis in sodium dodecyl sulfate/polyacrylamide gels. There were no apparent differences in Coomassie blue-stained or radioactive components of rings of K+ Membranes of erythrocytes infected with Plasmodium falciparum develop morphological alterations that were first described as "knob-like protrusions" (1). These protrusions are not apparent on erythrocytes that are infected with ring stages; they develop with the growth of parasites. In infected hosts, erythrocytes parasitized with multinucleate stages become sequestered and are not seen in the peripheral circulation. The knobs have been shown to be the sites of adhesion between sequestered erythrocytes and venous endothelial cells (2).A previous study (3) demonstrated that these protrusions were antigenically different from adjacent areas devoid of them. In an attempt to determine whether knob constituents were antigens of parasite origin that were incorporated into the host cell membrane, membrane fractions enriched in knobs were isolated from metabolically labeled infected erythrocytes and compared with fractions devoid of knobs. An additional labeled protein, with an apparent molecular weight of 70,000-80,000, was identified in samples enriched with knobs (4). From these results it could not be concluded with certainty whether this protein was a knob constituent or some other protein associated with membranes. Recently, it was noted that in cultures of an African strain of P. falciparum (FCR-3/Gambia) that had been maintained in flow vials (5) for >2 years by W. Trager some parasites failed to produce knobs (6). We were able to select for these parasites and obtain a culture line that showed mainly (about 99%) parasitized erythrocytes with smooth membranes (A. Kilejian and W. Trager, unpublished data). For purposes of discussion, the parent strain will be referred to as K+ and the derived culture line that does not produce knobs, as K-.This communication is a report on the comparison of stagespecific proteins of K+ and K-parasites. The results show that a metabolically labeled protein, of -80,000 molecular weight, that had been noted in a previous study (4) is correlated with the production of knobs. Two additional components that are synthesized during the development of merozoites have been identified.MATERIALS AND METHODS Parasites. P. falciparum (FCR-3/Gambia) was cultured in 100mm petri dishes in a candle jar (7). Cultures of K+ and Kparasites were synchronized by treatment with s...
Establishment of highly synchronized cultures of Plasmodium falciparum enabled identification of stagespecific proteins, glycoproteins, and antigens. Comparison of metabolically labeled constituents of rings, trophozoites, mature schizonts, and merozoites indicated the absence of major proteins or glycoproteins unique to rings or trophozoites. A burst of new synthetic activity occurred during schizogony when several schizont-and merozoite-specific proteins and glycoproteins became apparent. In addition to the knob protein, which was previously shown to be associated with protrusions on the host erythrocyte membrane, a major glycoprotein of parasite origin was identified on the surface membrane of schizonts. Analysis of antigens solubilized from different developmental stages indicated that immune sera, which inhibit growth of parasites in vitro, react mainly with merozoite-and schizont-associated antigens.Successful experimental vaccinations with erythrocytic stages of malaria parasites have indicated that merozoites and schizonts are the best sources of antigens that elicit a protective immune response (1-3). Identification of these stage-specific functional antigens would provide a rational basis for developing a malaria vaccine suitable for humans. Although an in vitro method for the cultivation of Plasmodium falciparum, the most virulent species of human malaria, has been available for over 3 years (4), there is no published information on stage-specific constituents or antigens. The major obstacle has been the asynchronous development of parasites in culture which has created difficulties in collection of sufficient numbers of nondegraded free merozoites. After emergence from erythrocytes, merozoites lose infectivity within about half an hour.In this study, a combination of two published methods was used to establish highly synchronized cultures. These cultures enabled the identification of stage-specific proteins and glycoproteins. In addition, by using sera from infected humans and experimentally immunized rabbits, antigenic constituents of merozoites and schizonts were determined.MATERIALS AND METHODS Synchronized Cultures. P. falciparum (FCR-3/Gambia) was cultured in 100-mm petri dishes in a candle jar (4). To obtain highly synchronized cultures, the infection of erythrocytes by merozoites was limited to 3-4 hr as follows. Starting with asynchronous cultures, multinucleate parasites were concentrated by flotation in gelatin (5) and subcultured with fresh erythrocytes. After 7-8 hr, cultures were collected and all multinucleate parasites that had failed to develop to mature schizonts and reinfect erythrocytes were eliminated with sorbitol treatment (6). On subsequent subcultures the identical The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "ad- For collection of merozoites and mature schizonts, cultures (to which labeled substrate had been added at 32 hr) were collected at 40 hr and multinucleate parasites were conce...
Intraerythrocytic stages of Plasmodium lophurae contain prominent cytoplasmic granules. The main chemical constituent of isolated granules was found to be a rather peculiar protein with five major amino acids: 73% histidine, 7.5% proline, 7% alanine, 6% glutamic acid, and 2.1% aspartic acid (1, 2). Indirect experimental evidence indicated that polar organelles of merozoites may also contain a histidine-rich protein (3). Because the contents of polar organelles have been implicated to participate in infection of erythrocytes by merozoites, it seemed reasonable to test the purified protein as a vaccine. This antigen protected ducklings against infection with P. lophurae (4).Although erythrocytic stages of mammalian species of malaria do not have cytoplasmic granules similar to those of P. lophurae, polar organelles are a common feature of all malaria merozoites. Therefore, the presence of a histidine-rich protein in P.falciparum was investigated. A dual-labeling experiment with histidine (which comprises 70% of the protein) and serine (which is lacking) indicated the presence of a 55,000-mol wt protein that had a greater histidine:serine ratio than all other labeled proteins (5). This was considered as evidence for the presence of a histidine-rich protein in P. falciparum. In the course of recent experiments on metabolically labeled constituents of P. falciparum, a striking difference in extent of incorporation of various labeled amino acids was observed in an 80,000-tool wt protein. This protein had been previously shown to be correlated with the knob-like protrusions that appear on membranes of erythrocytes infected with P, falciparum (6).This communication is a report on the similarity of the amino acid composition of the histidine-rich protein (HRP) of P. lophurae and the knob protein (KP) of P. falctparum. Materials and MethodsP. falclparum (FCR-3/Gambia) was cultured in 100-mm Petri dishes in a candle jar (7).
Resealed erythrocyte ghosts were prepared under different experimental conditions and were tested in vitro for susceptibility to infection with the human malarial parasite, Plasmodium falciparum. Resealed ghosts, prepared by dialyzing erythrocytes in narrow membrane tubing against low ionic strength buffer that was supplemented with magnesium ATP, were as susceptible to parasite infection as were normal erythrocytes. There was a direct correlation between intraerythrocytic ATP content and susceptibility to parasite infection. Neither MgCI2 nor sodium ATP could be substituted for magnesium ATP in maintaining high intraerythrocytic ATP concentration. When resealed ghosts were loaded with antispectrin IgG, malaria merozoite invasion was inhibited. At an average intracellular antispectrin IgG concentration of 3.5/~g/108 cells, there was a 35% inhibition of parasite invasion. This inhibition was due to spectrin crosslinking within the resealed ghosts, since the monovalent, Fab' fragments of antispectrin IgG had no inhibitory effect on invasion. These results indicate that the cytoskeleton plays a role in the complex process of merozoite entry into the host erythrocyte.There have been several recent reports on the mechanism of entry of malaria merozoites into erythrocytes (1-6). This process of infection has been shown to involve attachment of the apical end of the merozoite to the host erythrocyte membrane, most likely by specific receptors (4-7). At the point of attachment, an electron-dense junction forms between the merozoite and the red cell membrane and, subsequently, the host membrane invaginates around the entering merozoite (8). As the merozoite invades, the junction moves along the orifice of the invaginating membrane by what has been proposed as a "modified zippering" type of endocytosis (8). Freeze-fracture studies of merozoites in the process of invasion have shown changes in the organization of transmembrane proteins within the invaginating host cell membrane (2, 9); its P face becomes depleted of intramembrane particles (IMPs) and clusters of IMPs appear at the moving junction.Although the red cell cytoskeleton is known to play a major role in erythrocyte shape, membrane deformability, and receptor distribution (10--12), the role of cytoskeletal components in merozoite invasion has not been examined. Resealed ghosts offer a good experimental system for perturbation of intraery-
Experiments on the primary structure of a histidine-rich polypeptide isolated from the malarial parasite Plasmodium Jophurae indicate that the smaller quantities of amino acids other than histidine form an integral part of the polypeptide and do not arise from a protein contaminating a histidine homopolymer. In culture, the parasites incorporate over 50% of exogenously supplied histidine into the histidine-rich polypeptide and this incorporation is inhibited by puromycin.The intraerythrocytic stages of the avian malarial parasite Plasmodium lophurae contain numerous cytoplasmic granules (1). The main chemical constituent of isolated granules was shown to migrate as a single polypeptide on polyacrylamide gels, and analysis of hydrolysates of the polypeptide showed five major constituent amino acids: 73% histidine, 7.5% proline, 7% alanine, 6% glutamic acid, and 2% aspartic acid (2). The unusually high content of histidine raised two questions: (a) could the smaller quantities of other amino acids come from a typical protein isolated as a contaminant with the histidine homopolymer rather than being integral parts of the polypeptide, and (b) could the polypeptide possibly be synthesized by a non-ribosomal enzymatic system (3-5). Even though non-ribosomal synthesis of peptides has been shown only in prokaryotes, the possibility that it might also exist in eukaryotes cannot be excluded. The unusually high content of one amino acid, histidine, and some parallelism with the polypeptide of the cyanophycin granules of blue-green algae made the malaria polypeptide a plausible candidate for testing. The algal polypeptide was shown to be a high-molecular-weight copolymer of argine and aspartic acid, and its synthesis was not inhibited by chloramphenicol (5, 6).To answer the above questions, we undertook experiments on the primary structure of the peptide and studied the effect of puromycin on the biosynthesis of the peptide.MATERIALS AND METHODS Materials. The histidine-rich polypeptide was isolated as previously described (2). Pepsin, trypsin, and chymotrypsin were purchased from Worthington; Nagarse was from En- 900), pH 6.5. The sample was applied at tUie middle of the strip. For preparative purposes, guide strips were cut and stained with ninhydrin-cadmium reagent (7) and peptides were eluted with 30% acetic acid.Amino-Acid Analysis. Peptides were hydrolyzed in 200Ml of 6 N HC1 containing 0.2% (w/v) phenol and 0.1% (v/v) mercaptoacetic acid in evacuated sealed tubes, at 1100 for 22 hr (8). Hydrolysates were analyzed with a modified amino-acid analyzer (9). Polyacrylamide Gel Electrophoresis. Electrophoresis was according to the method of Panyim and Chalkley (10).Incorporation of [3HlHistidine In Vitro. Uninucleate trophozoites of P. lophurae were removed from their host duck erythrocytes and cultured extracellularly under conditions described previously (11). To facilitate quantitative collection of parasites at the end of the incubation period, the plasma clot lining of the culture flask was omitted. To each...
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