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Original Article |
mats.wahlgren{at}smi.ki.se
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Key Words: P. • falciparum • surface antigen • rif gene family • antibodies • strain-specific
The morbidity and mortality associated with Plasmodium falciparum malaria infections occurs exclusively during the erythrocytic phase of the parasite life cycle. Strategies used by P. falciparum for maximizing survival and proliferative capacity in the bloodstream include the receptor-mediated sequestration of P. falciparum–infected erythrocytes (pRBCs) on the vascular endothelium and the constant variation of antigenic epitopes on the pRBC surface to evade antiparasite mechanisms of protection in the human host 1.
A polypeptide exported by the parasite to the outer face of the pRBC membrane, the 200–400-kD P. falciparum erythrocyte membrane protein 1 (PfEMP1),1 is a cytoadherence ligand and undergoes clonal switching 23. PfEMP1 polypeptides are encoded by the var gene family 4, and they are expressed on the infected cell surface as the parasite develops from the ring-shaped early forms into the pigmented trophozoite stage, simultaneous with the onset of adhesive capacity and antigenicity of the pRBC 5.
Studies of humoral immune responses in natural infections of P. falciparum highlight the extreme diversity of antigenic determinants on the infected erythrocytes 678. Although sera from individuals with a history of exposure to the disease may contain antibodies that react with epitopes shared by many parasite isolates, the bulk of the natural or experimentally induced immune response to surface determinants on the pRBC is strain/clone specific 910. The variant antigen PfEMP1 has been postulated to be the sole target for specific antibodies that agglutinate pRBCs and confer protection against clinical disease 1112.
To date, PfEMP1 is the only molecularly characterized protein of P. falciparum shown to be located on the surface of the infected erythrocyte 2. PfEMP1 mediates binding to vascular endothelial receptors such as CD36, intercellular adhesion molecule 1, and thrombospondin 13, as well as to uninfected erythrocytes in the adhesion phenomenon known as rosetting 1415. Polypeptides of low molecular mass have been radiolabeled on the pRBC surface and termed rosettins after their identification in rosetting strains 16. Based on this and additional observations suggesting that the parasite exports more than one set of polypeptides to the host cell surface (reference 17 and our unpublished data), we set out to systematically reanalyze the surfaces of pRBCs harboring P. falciparum recently isolated from malaria patients or strains and clones adapted to laboratory culture conditions. Here, we have focused the analysis and characterization to parasite-derived products with a molecular size <200 kD, a size distinct from the known PfEMP1 antigens.
Sera were collected from (a) adults living in Yekepa, Liberia, an area characterized by high perennial malaria transmission (denoted 022, 102, 119, 142, 163, 164, 169, 174, 179, 198, 241, and 368), (b) adults from Fajara, The Gambia, a region with seasonal malaria transmission (denoted 072, 100, and 136), and (c) children 1–15 yr old living in Saradidi, an area in western Kenya holoendemic for malaria (denoted 011, 039, 080, 118, and 209). All donors had had repeated malaria attacks; none had symptoms of clinical malaria at the time of sampling. In all cases, informed consent was obtained from the patients and/or their parents. The sera were stored at –70°C and heat inactivated at 56°C before the assays. Control sera were obtained from healthy Swedish blood donors.
Culture of Parasites.
Surface Analysis of pRBCs.
Immunoprecipitation.
Trypsin Treatment of the PRBC Surface.
Materials and Methods
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Parasites and Sera.
The P. falciparum lines FCR3S/b (K–) and FCR3S1/b (K–) were selected from parasites FCR3S (K–) and FCR3S1 (K–), respectively, for cytoadherence to the CD36 receptor on C32 melanoma cells. FCR3S/a (K–) was obtained from FCR3S by repeated selection for nonrosetting parasites as described elsewhere 18. FCR3S1 was cloned by limiting dilution from FCR3S, which originated from the FCR3 strain isolated in The Gambia, West Africa. The subclones FCR3S1.2 (K–) and FCR3S1.6 (K–) were obtained by micromanipulation from FCR3S1. Clones TM284S2 (K+), TM284S3 (K+), TM284S11 (K+), TM284S12 (K+), TM284S20 (K+), TM284S7 (K–), TM284S9 (K–), and TM284S19 (K–) were derived by micromanipulation from the strain TM284 (K+), which, along with strain TM180 (K–), were isolated from malaria patients in Thailand. The F32 strain was isolated in Tanzania. The parasite 3D7 was obtained by limiting dilution cloning of the isolate NF54, which was derived from a patient who acquired malaria in the airport area in Amsterdam, The Netherlands. R29 (K+) was cloned from ITOR, a parasite from the ITO strain selected for the rosetting phenotype. The parasite Dd2 was originally cloned from the W2-MEF line of the Indochina III isolate. The P. falciparum isolates 186, 199, 341, 347, 352, and 354 were part of a larger panel of field parasites collected from African children infected with malaria. Upon collection, the blood samples were immediately frozen according to standard techniques. For their analysis, the frozen blood samples were thawed and maintained in culture in their own blood for 24–30 h until parasites developed into the mature trophozoite stage, at which time they were harvested and further processed.
All of the laboratory-adapted parasites used in this study were cultured in human group O Rh+ erythrocytes at 5% hematocrit with 10% AB+ serum added to the malaria culture medium containing RPMI 1640, 25 mM Hepes, 25 mM sodium bicarbonate, and 50 mg/ml gentamycin, pH 7.4. In some cases, cultured parasites were enriched for the rosetting phenotype (R+) as previously described 18.
Surface iodination of pRBCs was performed by the lactoperoxidase/Na125I/H2O2 method under conditions of minimal intracellular labeling. In brief, 2 x 109 cells of a culture at 7–15% parasitemia with a majority of parasites in the trophozoite stage were gently washed in PBS and resuspended to 1 ml in PBS plus 1 mM KI. 1 mCi of Na125I (Amersham) and 100 µl of 2 mg/ml lactoperoxidase (Sigma Chemical Co.) was added, and the reaction was initiated by the addition of 25 µl of 0.03% H2O2. Four subsequent additions of 25 µl of 0.03% H2O2 were administered at 1-min intervals. Radioiodinated cells were washed four times with ice-cold PBS containing 5 mM KI and resuspended in 1 ml of RPMI 1640 plus 5% sorbitol. Labeling of intracellular hemoglobin accounted for <2% of total acid-precipitable incorporated radiolabel. To disrupt rosettes/agglutinates, 100 IU/ml of heparin (Løvens) was added to the cell suspension, and this was passed five times through a 23-gauge (0.6 mm internal diameter) needle using a 1-ml syringe. The cell suspension was overlaid on top of a four-step (40, 60, 70, and 80%) Percoll gradient in RPMI/5% sorbitol and centrifuged in a JA 20 rotor (Beckman Instruments, Inc.) at 10,000 rpm for 30 min at room temperature. Cells floating between the 40 and 60% Percoll layers (>95% mature parasite–containing erythrocytes) were recovered and gently washed with PBS. Enriched pRBCs were first extracted with 1% Triton X-100/PBS containing a cocktail of protease inhibitors (1 mM EDTA-N2, 20 µg/ml leupeptin, 0.7 µg/ml pepstatin, 0.2 mM PMSF, and 50 µg/ml aprotinin) and subsequently extracted in a solution of 2% SDS and protease inhibitors in PBS. The polypeptides in the Triton-insoluble fraction were solubilized in 2% SDS-PAGE sample buffer and separated by a gradient of 5–8.5 or 7.5–17.5% SDS-PAGE. The dried gels were scanned and analyzed using a PhosphorImager and ImageQuant analysis software (Molecular Dynamics).
SDS or Triton X-100 extracts of surface-radioiodinated pRBCs (25 µl) were mixed with 375 µl of NETT (50 mM Tris, pH 7.4, 150 mM NaCl, 5 mM disodium EDTA, 20 µg/ml leupeptin, 50 µg/ml aprotinin, 0.02% sodium azide, and 0.5% Triton X-100) buffer/10 mg/ml IgG-free BSA (Sigma Chemical Co.) and 100 µl of 10% Triton X-100/PBS. 15 µl of serum was added to 500 µl of reconstituted extract, and the samples were incubated for 12 h at 4°C with rotation. Protein A–Sepharose (100 µl of a 50% suspension in NETT buffer/10 mg/ml IgG-free BSA) was added, and the samples were further incubated for 1 h at room temperature with rotation. The adsorbent was then washed twice with 1 ml of NETT, twice with 1 ml of NETT/0.35 M NaCl, and twice with 1 ml of NETT. The Sepharose beads were boiled for 4 min in the presence of 5% SDS-PAGE sample buffer, and the solubilized polypeptides were separated and analyzed as described above.
Intact or radiolabeled pRBCs purified on Percoll gradients were digested with trypsin (Sigma Chemical Co.) at the indicated concentrations (see Fig. 5 and Table ) for 10 min at 37°C. The reaction was terminated by the addition of 1 ml soybean of trypsin inhibitor (Sigma Chemical Co.) at 1 mg/ml in RPMI 1640/10% AB+ serum. Cells were washed with PBS and either resuspended in RPMI–Hepes/10% AB+ serum for binding and agglutination assays or extracted with Triton X-100/SDS and analyzed by SDS-PAGE as described above.
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Rosetting, Autoagglutination, and Cytoadherence.
Rosetting of uninfected red cells and autoagglutination of parasitized erythrocytes was assessed by direct staining of cultures with acridine orange (Sigma Chemical Co.) and examination using an epifluorescence microscope. Rosetting rates were measured as previously described 20. Spontaneous autoagglutination of pRBCs in the P. falciparum cultures was measured as described elsewhere 18. Adherence of pRBCs to unfixed C32 melanoma cells, human umbilical vein endothelial cells (HUVECs), chinese hamster ovary (CHO) cells transfected with CD36 or intercellular adhesion molecule 1, or L cells transfected with platelet/endothelial cell adhesion molecule (PECAM)1/CD31 was performed as described 21. In some cytoadherence assays on HUVECs, C32, and L cells, rosettes were first disrupted by adding 100 IU/ml of heparin (Løvens) to the culture and passage (five times) through a 23-gauge (0.6 mm internal diameter) needle using a 1-ml syringe. The cell suspension was overlaid on a four-step (40, 60, 70, and 80%) Percoll gradient, and the trophozoite-bearing red cells were selected as described above, washed twice in RPMI, and used for the assays in serum-free medium.
Surface Immunofluorescence.
The assay of the binding of human nonimmune normal Swedish serum Igs to pRBCs was performed by direct labeling of the cells using FITC-conjugated sheep anti–human Ig antibodies (State Bacteriology Laboratory, Sweden) as described elsewhere 22.
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To investigate the stability of expression of the small surface antigens in parasite populations that were or were not exposed to selective pressures other than those inherent to in vitro growth, we 125I-labeled pRBCs periodically collected from year-long continuous cultures of FCR3S1, maintained with regular enrichment for the rosetting phenotype, and the Dd2 strain, cultured without selection but displaying rosetting rates consistently >65%. The results showed no change in the type of radioiodinated polypeptides of these two parasites grown during at least 150 generations, as assessed in at least 10 separate experiments of surface labeling of pRBCs (data not shown). Although similar stability in the small surface antigen types is a characteristic of other parasite strains undergoing long-term culture, a decrease in the expression of these radiolabeled bands has been sporadically observed in some cultures.
Molecular Heterogeneity of the Small Surface Polypeptides.
We noticed that some radiolabeled bands in the 30–45-kD cluster appeared characteristically dispersed (Fig. 2 B). This observation prompted us to investigate possible molecular heterogeneity not discerned in regular SDS-PAGE gels. A two-dimensional electrophoresis analysis of an SDS extract of FCR3S1.2 pRBCs (the one-dimensional SDS-PAGE pattern of this parasite is shown in Fig. 2 B) revealed the existence of charge and size microheterogeneity in the surface-radioiodinated polypeptides (Fig. 3a and Fig. b). The parasite-specific bands of 35 and 36 kD resolved into several molecular species or isoforms in the pI range of 5.5–6.5, displaying a charge composition distinct from the major proteins of the human erythrocyte surface, including the heavily glycosylated anion transporter Band 3 and the sialoglycoproteins, glycophorins A and B. Polyclonal sera to total erythrocytic antigens and mAbs to Band 3 and glycophorins A, B, and C were used in immunoprecipitation and Western blot for the identification of the normal RBC proteins undergoing radioiodination (results not shown). None of these antibodies recognized the parasite-specific surface-radioiodinatable antigens of FCR3S1.2 or any other strain/clone tested.
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Controlled proteolytic digestion of intact radioiodinated pRBCs was used to further explore possible associations between the expression of surface antigens and binding phenotypes. Upon incubation of surface-radioiodinated, trophozoite-infected erythrocytes with trypsin, complete deletion of the 35-kD rifin band of FCR3S1 occurred at concentrations of the protease of 100 µg/ml or higher (Fig. 5). The 39-kD polypeptide of this parasite showed a reduced sensitivity to the protease. In comparison, the 285-kD PfEMP1 polypeptide expressed by FCR3S1 was cleaved at concentrations of trypsin <1 µg/ml, in agreement with our and others' data on the high trypsin sensitivity of most PfEMP1 polypeptides. The proteolytic treatment left the bulk of Triton X-100– and SDS-soluble polypeptides unaltered, with the exception of the glycophorins. The sensitivity of the rosetting phenotype to trypsin digestion of intact pRBCs closely matched that of the PfEMP1 protein, but not the removal of the 35- or 39-kD rifins from the pRBC surface. Similarly, the trypsin sensitivity of other adhesive phenotypes of erythrocytes bearing FCR3S1 or its clone FCR3S1.2, such as autoagglutination of infected RBCs, binding to the blood group A trisaccharide, adherence to the CD36 receptor, and binding of normal human Igs paralleled the cleavage of PfEMP1 (data not shown). In contrast, an association was found between the deletion of the 35-kD rifin polypeptide and the abrogation of pRBC binding to PECAM1/CD31, expressed constitutively on HUVECs or L cells transfected with the corresponding gene (Fig. 5).
The lack of association between the expression of non-PfEMP1 antigens on the pRBC surface and adherent phenotypes of the parasite, with the exception of binding to PECAM1/CD31, was further evidenced when we panned the rosetting FCR3S and FCR3S1 parasites on C32 melanoma cells expressing the CD36 receptor. The resulting highly CD36-cytoadherent, nonrosetting parasite lines expressed different patterns of surface-radioiodinatable polypeptides, 36 and 39 kD in FCR3S/b and 39 kD in FCR3S1/b. Polypeptides of 39 kD were also detected in noncytoadherent, nonrosetting parasites (Fig. 2 B).
Immune Recognition of Variant Rifin Antigens.
To assess the natural antigenicity of rifin polypeptides, we initially tested two human hyperimmune sera in immunoprecipitation of SDS extracts of surface 125I-labeled pRBCs of the FCR3S1.2 clone. Antibodies in these two sera recognized radioiodinated rifins of 35, 36 (doublet), and 39 kD in addition to PfEMP1 (Fig. 6 A). Rabbit antisera raised against GST fusion proteins comprising the semiconserved DBL1 and the highly conserved ATS domains of the var/PfEMP1 expressed by FCR3S1.2 immunoprecipitated PfEMP1 polypeptides but did not react with rif gene products. Having obtained the first evidence that rifin polypeptides were naturally immunogenic, we next carried out an expanded analysis of the antigenicity of these parasite products in natural infections. For this purpose, we used a panel of 18 sera from malaria-experienced individuals living in different geographical regions of Africa (Kenya, Liberia, and The Gambia) in assays of immunoprecipitation and agglutination of FCR3S1.2 and TM284 parasites (Fig. 6 B). Most of the sera immunoprecipitated one or more radioiodinated rifin polypeptides in the range of 31–39 kD, both in FCR3S1.2 and TM284 parasites. Qualitative variation was observed in the antigenic pattern recognized by different sera in each parasite, a result consistent with the extent of the rif gene repertoire and the switching in expressed rifin types. At the same time, sharing of epitopes between parasite-specific variant forms of rifins was suggested by the corresponding reactivities of a majority of the individual sera to the 36-kD doublet of the FCR3S1.2 parasite and the 39-kD band of the TM284 strain, a result anticipated if conservation at the 5' and 3' ends is a widespread feature of rif products. The major radioiodinated 170-kD polypeptide of TM284 was not immunoprecipitated by immune sera. The PfEMP1 polypeptides of the two parasites studied were recognized by all of the sera except 72, 100, and 136 (data not shown). The agglutination of trophozoite-bearing pRBCs by the immune sera was generally, but not strictly, correlated with their capacity to immunoprecipitate the small surface antigens.
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A salient feature of rifin polypeptides is their variability, in size as well as in the number of distinct components expressed in different parasites. The capacity of P. falciparum to undergo clonal antigenic variation has been established 2728, and the switching in the expression of different var genes, or their product PfEMP1, is correlated with clonal changes in surface antigenic determinants of the pRBC 3. Rifin antigens are clonally variant, as the analysis of clones and subclones of TM284 and FCR3S1.2 parasites demonstrate. These variable products, which are partially solubilized by neutral detergents (e.g., Triton X-100) but require treatment with 1–2% SDS, conditions that disrupt the host erythrocyte cytoskeleton for complete dissolution, are transported to the plasma membrane of the infected RBC and exposed on the surface according to several criteria. They are (a) readily and consistently labeled by the lactoperoxidase/Na125I/H2O2 method under conditions that fail to label hemoglobin and (b) cleaved by trypsin treatment of intact infected erythrocytes under conditions that do not cleave any other RBC or malarial proteins, except glycophorin polypeptides and the PfEMP1 antigen.
It is unknown how rif gene expression is regulated. The clusters with varying numbers of radiolabeled bands detected in freshly isolated as well as in recently cloned parasites may reflect, besides several other possible explanations, (a) the nonclonal composition of the parasites 2930, (b) posttranscriptional processing of the product of a single gene, or (c) the simultaneous expression of several rif genes. Implicit in this last alternative would be a partial lack of allelic restrictive processes (e.g., allelic exclusion) controlling the expression of products from the rif loci. This is in contrast to the "one parasite–one gene" modality of surface protein expression in the var/PfEMP1 multigene family 31. The finding of microheterogeneity in the rifins could be explained by the concurrent expression of several rif genes encoding polypeptides of similar molecular mass. Alternatively, superimposed to the variation created at the pRBC surface by the display of proteins comprising semivariable and hypervariable regions, e.g., var/PfEMP1, additional layers of antigenic variation could be generated in asexual stages of P. falciparum by the addition of carbohydrates, phosphate groups, or perhaps unusual modifications of polypeptides targeted to the erythrocyte surface. Examples of posttranslational modifications modulating the surface architecture, and likely the survival/virulence of pathogenic microorganisms, are the glycosylation and glycerophosphorylation of antigenically variable pilins in the prokaryote Neisseria species 3233 or the glycosylation and palmitoylation of the variant surface proteins of the eukaryotic parasite Giardia species 34. Analysis of the rif gene amino acid sequences available to date reveals numerous and relatively conserved potential sites for O- and N-glycosylation.
Allowing for the limited number of clinical isolates included in this study, it is interesting to note that rif gene expression is detected as prominent bands in each of the wild parasites examined but absent or faint in some of the strains or parasite lines that were long-term cultured in the laboratory. Chromosomal truncations, resulting in gene deletions as well as gametocytogenesis and cytoadherence phenotype losses, commonly occur in P. falciparum propagated mitotically in vitro 35363738. The function(s) of the variant rifin antigens being unknown, it is likely to be essential for survival of the parasite confronted with the host's environment and defence mechanisms but not indispensable for in vitro growth. The variant nature of the rifin antigens further suggests that presence at the pRBC surface is a compelling need.
Our data do not support a direct role of rif products in rosetting binding. An accessory function in conjunction with the rosetting ligand PfEMP1 1415 cannot be, however, completely disregarded. We have previously shown that the trypsin sensitivity of other binding phenotypes of multiadhesive parasites, i.e., binding to blood group A, autoagglutination of infected erythrocytes, binding of normal Igs, and adhesion to the CD36 receptor correlate with the enzymatic digestion of PfEMP1 18. In contrast, here we show that cytoadherence to the endothelial receptor PECAM1/CD31 is a phenotype associated with the presence on the pRBC surface of at least one radioiodinatable polypeptide, the 35-kD rifin in FCR3S1, but apparently not to the radioiodinated domain(s) of the PfEMP1 in this parasite. It remains a possibility that a trypsin-resistant domain of PfEMP1 contains the CD31 binding specificity. The 35-kD rifin may have, in conjunction with PfEMP1 or not, either a direct or an accessory role in the binding of pRBCs to CD31 and other receptors. Although binding to PECAM1/CD31 is a feature of only some laboratory-adapted strains 39, the extent of this binding trait in natural P. falciparum populations and its relationship to severe forms of malaria remains to be investigated. The prominent labeling of a 170-kD polypeptide in some isolates and laboratory strains and its paucity in nonbinding clones has not escaped our attention. Its labeling pattern may indicate molecular abundance at the pRBC surface. In immunoprecipitation and Western blot assays, this band is neither recognized by antibodies to the highly conserved COOH-terminal segment of PfEMP1 nor by immune sera that immunoprecipitate PfEMP1 and components of the 30–45-kD rifin cluster from the same parasite. It remains to be determined whether the 170-kD polypeptide is too antigenically diverse to be detected by heterologous antibodies or is not naturally immunogenic. Regardless of the function of rifins as well as the origin and function of the other novel surface-exposed polypeptides described here, their diversity, stability of expression, and multiplicity of combinatorial patterns (19 distinct types in 23 different parasites) suggests a potential for use as an isolate typing tool, conceivably in assays where the expressed PfEMP1 variant is simultaneously assessed.
Antigens exposed on the pRBC surface undergo clonal variation at rates of up to 1–2% per generation 28. The variant adhesin PfEMP1, which mediates binding of infected erythrocytes to vascular endothelial cells and to uninfected erythrocytes, plays a major role in this antigenic variation 32731. Adults living in areas of high malaria endemicity have antibodies in their sera capable of reacting with antigenic determinants of many P. falciparum isolates from distinct geographic regions 819. These antibodies recognize conserved and polymorphic parasite antigens, including PfEMP1 940. Our data indicate that rifin proteins are located on the surface of the pRBC and, moreover, that these parasite-derived components are antigenic in the course of a P. falciparum infection, eliciting a substantial humoral immune response. Furthermore, we show here that human immune sera agglutinate infected erythrocytes under conditions such that most, if not all, PfEMP1 products have been removed from the pRBC surface. The presence in sera of agglutinating or opsonizing antibodies reactive with the infected erythrocyte surface has been shown to correlate with protection from lethal disease 841. This preliminary data opens a new avenue for studies conducive to the eventual elucidation of targets for immune protection against P. falciparum malaria. The relative contribution of the rifins, PfEMP1, and other surface antigens on the pRBC to the triggering of the antibody response, Ig classes, and immunity buildup have yet to be determined.
This represents the first report of natural immune responses to surface molecules of intraerythrocytic P. falciparum distinct from PfEMP1. The results indicate that more than one family of genes encoding variable surface-targeted proteins, perhaps with additional epigenetic generation of submolecular variation, accounts for the remarkable diversity generated by the parasite in its host cell.
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Acknowledgments |
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These studies were supported by grants from the United Nations Developing Program/World Bank/World Health Organization Special Program for Research and Training in Tropical Diseases (TDR) and the Swedish Medical Research Council. V. Fernandez was supported in part by the Karolinska Institutet and the Swedish Society for Medical Research.
Submitted: 26 February 1999
Revised: 3 August 1999
Accepted: 3 September 1999
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50 genes per genome;