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Small, clonally variant antigens expressed on the surface of the Plasmodium falciparum-infected erythrocyte are encoded by the rif gene family and are the target of human immune responses.

Fernandez V, Hommel M, Chen Q, Hagblom P, Wahlgren M - J. Exp. Med. (1999)

Bottom Line: We have found these antigens to be identical to the rifins, predicted polypeptides encoded by the rif multigene family.When analyzed by two-dimensional electrophoresis, the rifins resolved into several isoforms in the pI range of 5.5-6.5, indicating molecular microheterogeneity, an additional potential novel source of antigenic diversity in P. falciparum.Prominent polypeptides of 20, 22, 76-80, 140, and 170 kD were also detected on the surfaces of pRBCs bearing in vitro-propagated or field-isolated parasites.

View Article: PubMed Central - PubMed

Affiliation: Microbiology and Tumor Biology Center, Karolinska Institutet, and the Swedish Institute for Infectious Disease Control, S-17177 Stockholm, Sweden.

ABSTRACT
Disease severity in Plasmodium falciparum infections is a direct consequence of the parasite's efficient evasion of the defense mechanisms of the human host. To date, one parasite-derived molecule, the antigenically variant adhesin P. falciparum erythrocyte membrane protein 1 (PfEMP1), is known to be transported to the infected erythrocyte (pRBC) surface, where it mediates binding to different host receptors. Here we report that multiple additional proteins are expressed by the parasite at the pRBC surface, including a large cluster of clonally variant antigens of 30-45 kD. We have found these antigens to be identical to the rifins, predicted polypeptides encoded by the rif multigene family. These parasite products, formerly called rosettins after their identification in rosetting parasites, are prominently expressed by fresh isolates of P. falciparum. Rifins are immunogenic in natural infections and strain-specifically recognized by human immune sera in immunoprecipitation of surface-labeled pRBC extracts. Furthermore, human immune sera agglutinate pRBCs digested with trypsin at conditions such that radioiodinated PfEMP1 polypeptides are not detected but rifins are detected, suggesting the presence of epitopes in rifins targeted by agglutinating antibodies. When analyzed by two-dimensional electrophoresis, the rifins resolved into several isoforms in the pI range of 5.5-6.5, indicating molecular microheterogeneity, an additional potential novel source of antigenic diversity in P. falciparum. Prominent polypeptides of 20, 22, 76-80, 140, and 170 kD were also detected on the surfaces of pRBCs bearing in vitro-propagated or field-isolated parasites. In this report, we describe the rifins, the second family of clonally variant antigens known to be displayed by P. falciparum on the surface of the infected erythrocyte.

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Novel variable antigens expressed on the surfaces of erythrocytes harboring clinical isolates of P. falciparum. Frozen blood samples from malaria patients were thawed, maintained in culture until the development of trophozoite stages, and subsequently surface-radioiodinated as explained in Materials and Methods. Labeled erythrocytes bearing trophozoite and schizont stages were separated from ring-infected and noninfected cells (RBC) in Percoll/sorbitol gradients and detergent extracted, and the polypeptides were fractionated by SDS-PAGE. Molecular sizes are indicated (kD). Extracts from autologous RBCs were always run in adjoining lanes for identification of parasite-derived polypeptides, which are indicated by asterisks (<45 kD) or closed arrowheads (100–200 kD). PfEMP1 polypeptides are not indicated.
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Figure 1: Novel variable antigens expressed on the surfaces of erythrocytes harboring clinical isolates of P. falciparum. Frozen blood samples from malaria patients were thawed, maintained in culture until the development of trophozoite stages, and subsequently surface-radioiodinated as explained in Materials and Methods. Labeled erythrocytes bearing trophozoite and schizont stages were separated from ring-infected and noninfected cells (RBC) in Percoll/sorbitol gradients and detergent extracted, and the polypeptides were fractionated by SDS-PAGE. Molecular sizes are indicated (kD). Extracts from autologous RBCs were always run in adjoining lanes for identification of parasite-derived polypeptides, which are indicated by asterisks (<45 kD) or closed arrowheads (100–200 kD). PfEMP1 polypeptides are not indicated.

Mentions: Frozen blood samples from P. falciparum malaria patients were thawed, and the cells were maintained in culture for 30 h. The intact cells were mildly radioiodinated under conditions ensuring >98% isotope incorporation to surface-exposed molecular moieties, and the labeled polypeptides were analyzed by protein gel electrophoresis. In all six isolates that developed into the mature stages, we found, in addition to PfEMP1 bands (>200 kD), several polypeptides with an estimated molecular mass between 33 and 39 kD that were readily labeled on the surfaces of erythrocytes containing parasites at the trophozoite/schizont stages, but not on the ring-infected or uninfected RBCs from the same blood sample (Fig. 1). The number, size, and degree of radiolabel incorporation of these polypeptides was variable and essentially unique for each isolate. Typically, the largest of these bands (39 kD) comigrated with the heavily radioiodinated monomer chains of erythrocytic glycophorin A and was therefore often indistinguishable from the host protein. One to three additional labeled polypeptides 33–37 kD in size formed a characteristic cluster of bands with isolate-specific patterns. We then proceeded to study the pRBC surfaces of 17 strains, lines, and clones of P. falciparum and found that radioiodinatable polypeptides in the range of 30–45 kD were commonly exported and surface expressed by a majority of these in vitro–propagated parasites (Fig. 2A–C). The number of bands and their relative radiolabel intensities was specific for each parasite. As determined by radioiodination experiments with highly synchronized cultures, the expression of the 30–45-kD polypeptide cluster on the pRBC surface occurred from 16 to 18 h after invasion until the end of the erythrocytic cycle (data not shown), matching the time course of expression of antigenic and adhesive phenotypes and the onset of PfEMP1 on the pRBC surface. These polypeptides were partially solubilized in Triton X-100, in contrast to the extreme insolubility of PfEMP1 in nonionic detergents. Additional parasite-specific radiolabeled bands were detected on pRBCs infected with some of the patient isolates or laboratory-cultured parasites (Fig. 1 and Fig. 2A–C). A 20-kD band was observed in isolate 199, whereas a polypeptide of 22 kD was radiolabeled in isolate 347 and the strain Dd2. Faint bands of 76–80 kD were visible in several of the in vitro–propagated parasites, and a polypeptide of 140 kD was detected in TM180, a strain also expressing several smaller (33–39 kD) polypeptides. Most prominent was the strongly radioiodinated band of 170 kD found in two of the clinical isolates (199 and 341) and strain F32, as well as strain TM284 and several of its clones (only TM284S2 is shown). None of these polypeptides were detected on pRBCs bearing trophozoite/schizont stages when the erythrocytes were surface 125I-labeled before parasite invasion (data not shown).


Small, clonally variant antigens expressed on the surface of the Plasmodium falciparum-infected erythrocyte are encoded by the rif gene family and are the target of human immune responses.

Fernandez V, Hommel M, Chen Q, Hagblom P, Wahlgren M - J. Exp. Med. (1999)

Novel variable antigens expressed on the surfaces of erythrocytes harboring clinical isolates of P. falciparum. Frozen blood samples from malaria patients were thawed, maintained in culture until the development of trophozoite stages, and subsequently surface-radioiodinated as explained in Materials and Methods. Labeled erythrocytes bearing trophozoite and schizont stages were separated from ring-infected and noninfected cells (RBC) in Percoll/sorbitol gradients and detergent extracted, and the polypeptides were fractionated by SDS-PAGE. Molecular sizes are indicated (kD). Extracts from autologous RBCs were always run in adjoining lanes for identification of parasite-derived polypeptides, which are indicated by asterisks (<45 kD) or closed arrowheads (100–200 kD). PfEMP1 polypeptides are not indicated.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2195703&req=5

Figure 1: Novel variable antigens expressed on the surfaces of erythrocytes harboring clinical isolates of P. falciparum. Frozen blood samples from malaria patients were thawed, maintained in culture until the development of trophozoite stages, and subsequently surface-radioiodinated as explained in Materials and Methods. Labeled erythrocytes bearing trophozoite and schizont stages were separated from ring-infected and noninfected cells (RBC) in Percoll/sorbitol gradients and detergent extracted, and the polypeptides were fractionated by SDS-PAGE. Molecular sizes are indicated (kD). Extracts from autologous RBCs were always run in adjoining lanes for identification of parasite-derived polypeptides, which are indicated by asterisks (<45 kD) or closed arrowheads (100–200 kD). PfEMP1 polypeptides are not indicated.
Mentions: Frozen blood samples from P. falciparum malaria patients were thawed, and the cells were maintained in culture for 30 h. The intact cells were mildly radioiodinated under conditions ensuring >98% isotope incorporation to surface-exposed molecular moieties, and the labeled polypeptides were analyzed by protein gel electrophoresis. In all six isolates that developed into the mature stages, we found, in addition to PfEMP1 bands (>200 kD), several polypeptides with an estimated molecular mass between 33 and 39 kD that were readily labeled on the surfaces of erythrocytes containing parasites at the trophozoite/schizont stages, but not on the ring-infected or uninfected RBCs from the same blood sample (Fig. 1). The number, size, and degree of radiolabel incorporation of these polypeptides was variable and essentially unique for each isolate. Typically, the largest of these bands (39 kD) comigrated with the heavily radioiodinated monomer chains of erythrocytic glycophorin A and was therefore often indistinguishable from the host protein. One to three additional labeled polypeptides 33–37 kD in size formed a characteristic cluster of bands with isolate-specific patterns. We then proceeded to study the pRBC surfaces of 17 strains, lines, and clones of P. falciparum and found that radioiodinatable polypeptides in the range of 30–45 kD were commonly exported and surface expressed by a majority of these in vitro–propagated parasites (Fig. 2A–C). The number of bands and their relative radiolabel intensities was specific for each parasite. As determined by radioiodination experiments with highly synchronized cultures, the expression of the 30–45-kD polypeptide cluster on the pRBC surface occurred from 16 to 18 h after invasion until the end of the erythrocytic cycle (data not shown), matching the time course of expression of antigenic and adhesive phenotypes and the onset of PfEMP1 on the pRBC surface. These polypeptides were partially solubilized in Triton X-100, in contrast to the extreme insolubility of PfEMP1 in nonionic detergents. Additional parasite-specific radiolabeled bands were detected on pRBCs infected with some of the patient isolates or laboratory-cultured parasites (Fig. 1 and Fig. 2A–C). A 20-kD band was observed in isolate 199, whereas a polypeptide of 22 kD was radiolabeled in isolate 347 and the strain Dd2. Faint bands of 76–80 kD were visible in several of the in vitro–propagated parasites, and a polypeptide of 140 kD was detected in TM180, a strain also expressing several smaller (33–39 kD) polypeptides. Most prominent was the strongly radioiodinated band of 170 kD found in two of the clinical isolates (199 and 341) and strain F32, as well as strain TM284 and several of its clones (only TM284S2 is shown). None of these polypeptides were detected on pRBCs bearing trophozoite/schizont stages when the erythrocytes were surface 125I-labeled before parasite invasion (data not shown).

Bottom Line: We have found these antigens to be identical to the rifins, predicted polypeptides encoded by the rif multigene family.When analyzed by two-dimensional electrophoresis, the rifins resolved into several isoforms in the pI range of 5.5-6.5, indicating molecular microheterogeneity, an additional potential novel source of antigenic diversity in P. falciparum.Prominent polypeptides of 20, 22, 76-80, 140, and 170 kD were also detected on the surfaces of pRBCs bearing in vitro-propagated or field-isolated parasites.

View Article: PubMed Central - PubMed

Affiliation: Microbiology and Tumor Biology Center, Karolinska Institutet, and the Swedish Institute for Infectious Disease Control, S-17177 Stockholm, Sweden.

ABSTRACT
Disease severity in Plasmodium falciparum infections is a direct consequence of the parasite's efficient evasion of the defense mechanisms of the human host. To date, one parasite-derived molecule, the antigenically variant adhesin P. falciparum erythrocyte membrane protein 1 (PfEMP1), is known to be transported to the infected erythrocyte (pRBC) surface, where it mediates binding to different host receptors. Here we report that multiple additional proteins are expressed by the parasite at the pRBC surface, including a large cluster of clonally variant antigens of 30-45 kD. We have found these antigens to be identical to the rifins, predicted polypeptides encoded by the rif multigene family. These parasite products, formerly called rosettins after their identification in rosetting parasites, are prominently expressed by fresh isolates of P. falciparum. Rifins are immunogenic in natural infections and strain-specifically recognized by human immune sera in immunoprecipitation of surface-labeled pRBC extracts. Furthermore, human immune sera agglutinate pRBCs digested with trypsin at conditions such that radioiodinated PfEMP1 polypeptides are not detected but rifins are detected, suggesting the presence of epitopes in rifins targeted by agglutinating antibodies. When analyzed by two-dimensional electrophoresis, the rifins resolved into several isoforms in the pI range of 5.5-6.5, indicating molecular microheterogeneity, an additional potential novel source of antigenic diversity in P. falciparum. Prominent polypeptides of 20, 22, 76-80, 140, and 170 kD were also detected on the surfaces of pRBCs bearing in vitro-propagated or field-isolated parasites. In this report, we describe the rifins, the second family of clonally variant antigens known to be displayed by P. falciparum on the surface of the infected erythrocyte.

Show MeSH
Related in: MedlinePlus