Complement receptor 1 is the host erythrocyte receptor for Plasmodium falciparum PfRh4 invasion ligand
Edited* by Louis H. Miller, National Institutes of Health, Rockville, MD, and approved August 27, 2010 (received for review June 10, 2010)
Abstract
Plasmodium falciparum is responsible for the most severe form of malaria disease in humans, causing more than 1 million deaths each year. As an obligate intracellular parasite, P. falciparum’s ability to invade erythrocytes is essential for its survival within the human host. P. falciparum invades erythrocytes using multiple host receptor–parasite ligand interactions known as invasion pathways. Here we show that CR1 is the host erythrocyte receptor for PfRh4, a major P. falciparum ligand essential for sialic acid–independent invasion. PfRh4 and CR1 interact directly, with a Kd of 2.9 μM. PfRh4 binding is strongly correlated with the CR1 level on the erythrocyte surface. Parasite invasion via sialic acid–independent pathways is reduced in low-CR1 erythrocytes due to limited availability of this receptor on the surface. Furthermore, soluble CR1 can competitively block binding of PfRh4 to the erythrocyte surface and specifically inhibit sialic acid–independent parasite invasion. These results demonstrate that CR1 is an erythrocyte receptor used by the parasite ligand PfRh4 for P. falciparum invasion.
Sign up for PNAS alerts.
Get alerts for new articles, or get an alert when an article is cited.
Erythrocyte invasion is essential for the survival of Plasmodium falciparum within the human host. The merozoite forms of P. falciparum invade erythrocytes through a multistep process that involves initial contact with the erythrocyte, apical reorientation, and formation of a tight junction that moves progressively toward the posterior end of the parasite until host cell membrane fusion is complete (see ref. 1 for a review). These steps in invasion are dependent on specific interactions between multiple parasite invasion ligands and their respective human erythrocyte receptors, which have been defined as distinct invasion pathways.
In P. falciparum, two gene families encode important proteins used in invasion: the erythrocyte-binding–like antigens (EBAs: EBA-140/BAEBL, EBA-175, EBA-181/JESEBL, and EBL-1) and reticulocyte-binding–like homolog proteins (RBPs, or PfRhs: PfRh1, PfRh2a, PfRh2b, PfRh4, and PfRh5) (2–5). During invasion, these ligands are localized at the apical tip of the merozoite and are able to bind erythrocytes. Invasion pathways have been identified by examining the entry of merozoites into erythrocytes that have deficient or mutant host receptors or that have been treated with enzymes that modify the properties or presence of erythrocyte surface proteins. The most common enzyme treatments involve neuraminidase, which removes sialic acid residues, and trypsin or chymotrypsin, which differentially cleave the peptide backbones of proteins. At present, only a handful of erythrocyte receptors that bind to P. falciparum invasion ligands have been identified, including glycophorin A for EBA-175, glycophorin B for EBL-1, and glycophorin C for EBA-140 (6–8). All three of these interactions are sensitive to neuraminidase treatment of erythrocytes and thus are involved in sialic acid–dependent invasion pathways. Complement receptor 1 (CR1) was recently identified as a receptor for the sialic acid–independent invasion pathways in multiple laboratory strains and wild isolates, although the parasite ligand with which it interacts has not yet been identified (9). CR1 also mediates rosetting through its interaction with PfEMP-1, a parasite-derived variant erythrocyte membrane protein (10). On the erythrocyte surface, CR1 is present as a ∼190- to 280-kDa single-chain transmembrane glycoprotein bearing the Knops blood group (11).
PfRh4 is essential in the sialic acid-independent pathway as disruption of the gene in W2mef results in the inability of this strain to switch invasion pathways to allow invasion into neuraminidase-treated erythrocytes (12). Growth assays in the presence of anti-PfRh4 antibodies have shown that PfRh4 is the major ligand responsible for invasion via the sialic acid–independent pathways (50–80%, depending on the parasite strain used) (13). By activating PfRh4 expression, the parasite is able to switch receptor usage from sialic acid–dependent to sialic acid-independent pathways, thereby providing a mechanism for the parasite to invade via different pathways (12). Because parasite entry into erythrocytes is an essential component of the life cycle of P. falciparum, the use of multiple redundant invasion pathways and the ability to switch pathways through differential expression of parasite ligands provides the parasite with mechanisms to increase successful invasion in the face of host immune responses and erythrocyte receptor polymorphisms in malaria endemic regions (SI Text, Fig. S1).
Identifying the parasite ligand–host erythrocyte receptors used in invasion is a crucial step toward a complete understanding of the full repertoire of invasion pathways available to P. falciparum. Because PfRh4 is a key player in phenotypic variation of invasion and sialic acid–independent pathway, we sought to identify its host erythrocyte receptor. We observed that erythroid CR1 is susceptible to trypsin and chymotrypsin treatment, an enzyme profile consistent with that obtained for the abolishment of PfRh4 erythrocyte binding (13, 14). Here we show that PfRh4 binds to CR1 on the erythrocyte surface, and that this interaction mediates a functional sialic acid–independent pathway for P. falciparum invasion of human erythrocytes.
Results
PfRh4 Binds to CR1 on the Erythrocyte Surface.
To examine whether CR1 is a receptor for PfRh4, we incubated erythrocytes with anti-CR1 antibodies, then performed an erythrocyte-binding assay using culture supernatants containing P. falciparum invasion ligands released from merozoites (Fig. 1A). Using erythrocytes precoated with anti-CR1 antibodies, we found that PfRh4 erythrocyte binding was reduced, whereas EBA-175 binding was not perturbed (Fig. 1A). The addition of increasing amounts of anti-CR1 antibodies resulted in enhanced inhibition of PfRh4 erythrocyte binding, with >90% of binding blocked at an antibody dose of 0.0107 mg/mL (Fig. 1A). Preincubation of erythrocytes with glycophorin A/B or decay accelerating factor (DAF) antibodies did not affect PfRh4 binding, indicating that the inhibition observed with anti-CR1 antibodies is specific to PfRh4 (Fig. 1B).
Fig. 1.
We also tested the ability of soluble recombinant CR1 to inhibit binding of PfRh4 to the surface of human erythrocytes. Soluble CR1 (sCR1) contains the extracellular domain but lacks the transmembrane and cytoplasmic domains (15). Culture supernatants were preincubated with sCR1 before being added to erythrocyte-binding assays. Increasing amounts of sCR1 competitively blocked native PfRh4 binding to CR1 on erythrocytes, with >90% inhibition obtained at 0.04 mg/mL (Fig. 1C). Probing of the same binding eluates with EBA-175 antibodies resulted in no perturbation of EBA-175 erythrocyte binding, indicating that the inhibition is specific to PfRh4. Thus, PfRh4 binds to CR1 on the erythrocyte surface.
Recombinant PfRh4 and CR1 Interact Directly.
To provide additional evidence of a PfRh4–CR1 interaction, we performed immunoprecipitation experiments using sCR1 and recombinant PfRh4, which is functional, as demonstrated by its ability to bind erythrocytes (Figs. S2 and S3) (13, 15). Using anti-PfRh4 monoclonal antibody 10C9, a complex containing both recombinant PfRh4 and sCR1 was immunoprecipitated (Fig. 2A). Given CR1’s ability to bind immune complexes, we showed, as a control, that the 10C9 monoclonal antibody does not nonspecifically immunoprecipitate sCR1 (Fig. 2A). For an antibody control, we used anti-Rh4 monoclonal antibody 2E8, which does not recognize recombinant PfRh4; this did not immunoprecipitate the CR1–PfRh4 complex (Fig. S3 B and C). Immunoprecipitation with an anti-CR1 antibody also isolated a complex containing recombinant PfRh4 and sCR1, but not another hexaHis-tagged control protein, further supporting the specificity of the PfRh4–CR1 interaction (Fig. 2B).
Fig. 2.
We also measured the CR1–PfRh4 interaction using an ELISA-based assay, in which sCR1 bound to immobilized recombinant PfRh4 but not to the control protein, and this binding increased with increasing concentrations of sCR1 (Fig. 2C and Fig. S3). This result was reinforced by surface plasmon resonance (SPR)-based studies (Fig. 2D). Soluble CR1, but not BSA, binds to recombinant PfRh4 that has been immobilized via amine coupling to a sensor chip. Measuring the binding of an sCR1 concentration series (at two different loadings on the chip of recombinant PfRh4) yielded a Kd of 2.9 ± 0.2 μM for the sCR1–PfRh4 interaction (Fig. 2D).
In an attempt to delineate the region within CR1 involved in PfRh4 binding, we measured the CR1–PfRh4 interaction in the presence of anti-CR1 monoclonal antibodies mapped to the extracellular domain of CR1 (Fig. S3) (16). In the most common isoform of CR1, the extracellular domain comprises 30 short consensus repeat modules (SCRs). Based on a high degree of internal homology, all except the last two carboxy terminal SCRs form larger units called long homologous repeats (LHRs), each of which is composed of seven SCRs. The subclass for the mouse monoclonal antibodies was IgG1 with the exception of 7G9, which was IgG2a. The addition of anti-CR1 monoclonal antibodies or Ig2a mouse isotype resulted in three distinct phenotypes for CR1–PfRh4 interaction in the ELISA-based assay. First, the addition of 4D6 (which recognizes SCRs 3, 10, and 17) or Ig2a mouse isotype did not affect this interaction. Second, the CR1–PfRh4 interaction was enhanced in the presence of 1B4, 3D9 (which also recognize SCRs 3, 10, and 17), and HB8592. Third, the CR1–PfRh4 interaction was disrupted by 7G9 and E11 monoclonal antibodies (Fig. 2E). These monoclonal antibodies recognize similar epitopes present at SCRs 5–7, 12–14, and 19–21. Although HB8592 recognizes the same class of epitopes as E11 (SCRs 5–7, 12–14, 19–21, and 26–28), it did not inhibit the interaction between sCR1 and PfRh4. This may be because E11 is able to immunoprecipitate LHR A and B, whereas HB8592 shows reactivity only to recombinant LHR D (16). Although this finding implicates one or more of SCRs 5–7, 12–14, and 19–21 of CR1 as the region involved in PfRh4 binding, the binding of antibodies to CR1 SCRs adjacent to (rather than within) ligand-binding sites has been reported to perturb function (16). Consistent with results for the inhibition of CR1–PfRh4 interaction, the addition of E11 and 7G9 produced a reduction in PfRh4 binding in standard erythrocyte-binding assays (Fig. 2F).
Levels of PfRh4 Binding Were Strongly Correlated with CR1 Level on the Erythrocyte Surface.
CR1 levels on erythrocytes vary between individuals in the range of 50–1,200 molecules per cell. In Caucasian populations, single nucleotide polymorphism (SNP) within exon 22 in the CR1 gene is linked to high CR1 expression (H, high allele) or low CR1 expression (L, low allele) (17). Homozygous HH individuals have higher erythrocyte surface levels of CR1, homozygous LL individuals have <200 molecules per erythrocyte, and HL individuals having intermediate CR1 surface levels. To test whether differential expression of CR1 on erythrocytes correlated with PfRh4 binding, we analyzed the CR1 phenotype of blood samples from Australian residents. CR1 erythrocyte levels from these individuals showed an association with exon 22 genotyping (Fig. 3A). We observed markedly reduced native PfRh4 binding in erythrocytes from a LL individual compared with those from an HH individual in a standard erythrocyte-binding assay (Fig. 3B and Fig. S4D). We probed the same binding eluates with EBA-175 antibodies, but found no difference in EBA-175 binding.
Fig. 3.
To measure the level of PfRh4 erythrocyte binding in more blood samples, we developed a FACS-based assay for erythrocyte binding using recombinant PfRh4 (Fig. S2) (18). The features of recombinant PfRh4 erythrocyte binding in a FACS-based assay are similar to those of standard binding assays in terms of enzyme sensitivity (Fig. S2). Analysis of 100 individual blood samples revealed a strong correlation between the percentage of erythrocytes that bound recombinant PfRh4 and the CR1 level on the erythrocyte surface (r2 = 0.8236; Fig. 3C and Fig. S2). In contrast, there was no significant correlation between recombinant PfRh4 binding and the level of surface glycophorin C (r2 = 10−5; Fig. 3D). This indicates that the level of native or recombinant PfRh4 binding to erythrocytes is dependent on the amount of CR1 expressed on the erythrocyte.
Sialic Acid-Independent Invasion Was Reduced in Low-CR1 Erythrocytes and Inhibited in the Presence of Soluble CR1.
To examine the role of CR1 in P. falciparum invasion, we screened 400 blood samples from nonoverlapping individuals to identify additional low-CR1 erythrocytes. We selected the 10 samples with the highest CR1 expression and the 10 samples with the lowest CR1 expression on the erythrocyte surface; in most cases, CR1 phenotype was associated with exon 22 genotyping (Fig. S4). These erythrocyte samples were within normal ranges for blood indices and glucose-6-phosphate dehydrogenase (G6PD) activity and were wild type for other blood polymorphisms, including Gerbich, South Asian Ovalocytosis (SAO), and α-thalassemia (Fig. S5). Although low-CR1 erythrocytes had higher levels of G6PD activity, this phenotype should not negatively affect parasite growth. Furthermore, none of the blood samples were microcytic or macrocytic. The level of recombinant PfRh4 binding was again strongly associated with CR1 expression, whereas binding of recombinant PfRh4 and EBA-175 did not correlate with levels of glycophorin C and CR1 expression, respectively (Figs. S4 and S5).
To examine the importance of CR1 as a receptor for the PfRh4 invasion pathway, we evaluated the invasion of parasite strains W2mefΔ175 and 3D7 into neuraminidase-treated erythrocytes (Fig. 4A). These strains have been shown to efficiently invade neuraminidase-treated erythrocytes, and PfRh4 is known to be an essential invasion ligand for this sialic acid–independent pathway (12, 19). Thus, if CR1 is the receptor for PfRh4, then this invasion pathway will be less efficient in the presence of neuraminidase-treated low-CR1 erythrocytes compared with invasion into neuraminidase-treated high-CR1 erythrocytes, due to the reduced availability of its receptor. Because both high-CR1 and low-CR1 erythrocytes showed variation in parasite growth rate, the efficiency of sialic acid-independent invasion in each sample (i.e., the invasion ratio) was calculated as a ratio of the percentage of parasitemia in neuraminidase-treated erythrocytes divided by the percentage of parasitemia in untreated erythrocytes using the same strain. These results are shown for high-CR1 and low-CR1 erythrocytes in Fig. 4A. For both W2mefΔ175 and 3D7, invasion into neuraminidase-treated low-CR1 erythrocytes was reduced compared with invasion into neuraminidase-treated high-CR1 erythrocytes (P = 0.0009 and 0.003, respectively). This decreased efficiency of the PfRh4 invasion pathway results from the limited availability of CR1 on the erythrocyte surface.
Fig. 4.
We also examined parasite invasion rates in the presence of sCR1, which competitively inhibits binding of PfRh4 to erythroid CR1 (Fig. 1C). We tested parasite strains W2mef, W2mefΔRh4, W2mefΔ175, and 3D7 in invasion assays into untreated or neuraminidase-treated erythrocytes (Fig. 4B). Anti-PfRh4 antibodies were shown to inhibit invasion of strains 3D7 (untreated), W2mefΔ175 (neuraminidase-treated) and 3D7 (neuraminidase-treated) to 20%, 60%, and 80%, respectively, but no inhibition was observed for W2mefΔRh4, W2mef, and W2mefΔ175 into untreated erythrocytes (13). This indicates that invasion of W2mefΔRh4, W2mef, and W2mefΔ175 into untreated erythrocytes is not dependent on PfRh4, whereas invasion of 3D7 (untreated), W2mefΔ175 (neuraminidase-treated), and 3D7 (neuraminidase-treated) is increasingly dependent on PfRh4 (Fig. S1). Parasite invasion into neuraminidase-treated erythrocytes by both 3D7 and W2mefΔ175 was greatly reduced in the presence of sCR1 (87% inhibition, P = 0.001 and 81% inhibition, P = 0.010, respectively) (Fig. 4B and Fig. S4), but not in the presence of control protein. This inhibition of parasite invasion could be titrated, with decreasing amounts of sCR1 resulting in less invasion inhibition (Fig. 4C). Invasion of 3D7 into untreated erythrocytes also was slightly reduced, consistent with results obtained with anti-PfRh4 antibodies, although the difference was not statistically significant (20% inhibition; P = 0.09). As controls, we observed no significant difference in parasite invasion in the presence of sCR1 into untreated erythrocytes with W2mef, W2mefΔRh4, and W2mefΔ175, strains that do not rely on PfRh4 as an invasion ligand (P = 0.913, 0.206, and 0.352, respectively). These results demonstrate that CR1 is the host erythrocyte receptor used by PfRh4, and that this pathway functions directly in P. falciparum invasion.
Discussion
Invasion of P. falciparum into human erythrocytes requires specific ligand–receptor interactions. The PfRh family of proteins is a group of key ligands important to invasion by direct binding to the erythrocyte. Up to now, no receptors have been identified, however. Here we show that CR1 serves as a receptor for P. falciparum invasion via direct binding of the parasite ligand PfRh4. The level of PfRh4 binding to erythrocytes is strongly correlated with the amount of CR1 molecules on the erythrocyte surfaces. In the presence of sCR1, low inhibition of parasite invasion into untreated erythrocytes was observed in sialic acid–independent strains 3D7 and W2mefΔ175. This is related to the redundancy of EBA and PfRh proteins, so that inhibition of one pathway is compensated for by the function of others. Treatment of erythrocytes with neuraminidase resulted in a blockage of invasion by such ligands as EBA-175, EBA-181, EBA-140, and PfRh1, which require sialic acid-containing receptors. As a result, parasite invasion into neuraminidase-treated erythrocytes was increasingly reliant on the PfRh4–CR1 pathway, as demonstrated by the strong inhibition of invasion by the addition of sCR1. This result is consistent with data reported by Spadafora et al. (9) showing that the addition of sCR1 inhibited parasite invasion into neuraminidase-treated erythrocytes in sialic acid-independent strains, such as 7G8 and 3D7. sCR1 was not able to inhibit invasion in strains that either lack PfRh4 expression (W2mef) or possess a genetic knockout of the PfRh4 gene (W2mefΔRh4), providing convincing evidence that the reduction in invasion was due to the sCR1–PfRh4 interaction. We also show that binding of PfRh4 to erythrocytes, as well as PfRh4–CR1 interactions, can be blocked by the presence of sCR1 or anti-CR1 monoclonal antibodies, thus providing a molecular basis for the inhibition of invasion. These results indicate that the PfRh4–CR1 interaction is a functional sialic acid–independent invasion pathway for the entry of P. falciparum into human erythrocytes. Although neuraminidase-treated erythrocytes do not exist in the field, this in vitro situation mimics situations in which host immune responses selectively block the function of specific parasite invasion pathways or erythrocyte polymorphisms in surface proteins result in a limited receptor repertoire.
CR1 has been shown to mediate rosetting through its interaction with PfEMP-1, a parasite-derived variant erythrocyte membrane virulence protein (10). Rosetting occurs when infected erythrocytes adhere to uninfected erythrocytes, resulting in “clumps.” The formation of rosettes can be disrupted by the addition of sCR1 and is reduced in the presence of low-CR1 erythrocytes (10). The CR1 region required for rosetting is SCRs 10 and 17. Anti-CR1 monoclonal antibodies that map to those regions (1B4, 3D9, and 4D6) do not inhibit either PfRh4 erythrocyte binding or the CR1–PfRh4 interaction, suggesting that the regions of binding to PfEMP-1 and PfRh4 might be distinct sites within CR1 (20). However, anti-CR1 monoclonal antibody J3B11 has been shown to inhibit parasite invasion into neuraminidase-treated erythrocytes and also to interfere with PfEMP-1 binding (9, 20). A major caveat with all of these experiments is that anti-CR1 monoclonal antibodies that recognize the same epitopes show contrasting results in terms of inhibition of rosetting, PfRh4 erythrocyte binding, and invasion assays (9, 20). In addition, it has been shown that binding of anti-CR1 antibodies to SCRs adjacent to (rather than within) ligand-binding sites can perturb function (16). Finer epitope mapping of the anti-CR1 monoclonal antibodies and further studies using functional protein domains of CR1 will help elucidate the precise location within CR1 that binds PfRh4. Of note, P. falciparum uses the same host erythrocyte receptor for two distinct functions important for the virulence and survival of the parasite, which probably exploits the essential nature of CR1 on erythrocytes, given the absence of CR1 null erythrocytes in the human population.
Field isolates from India, Gambia, Brazil, Tanzania, and Kenya use many different invasion pathways, as demonstrated by the ability of these field parasites to invade a wide variety of enzyme-treated erythrocytes (21–25). Interestingly, a majority of Kenyan P. falciparum isolates invade via a neuraminidase-resistant, trypsin-sensitive, and chymotrypsin-sensitive pathway—an enzyme profile identical to that observed for PfRh4 erythrocyte binding (19 out of 31 isolates) (22). It is highly likely that PfRh4 is involved in this invasion pathway, given the fact that no other PfRh or EBA has a comparable enzyme profile. In support of this possibility, Spadafora et al. (9) reported the inhibition of invasion of three clinical isolates into both intact and neuraminidase-treated erythrocytes by the addition of sCR1. Determining the prevalence of the CR1–PfRh4 pathway in field isolates would require growth assays on more samples in the presence of inhibitory anti-PfRh4 antibodies or with the addition of sCR1 (13).
To prevent invasion, the human population relies on two protective mechanisms: a polymorphic erythrocyte surface and antibody responses to block parasite ligand function. Blood polymorphisms prevalent in malaria-endemic regions, such as Gerbich negativity in Papua New Guinea (PNG), could result in less-efficient invasion via EBA-140 (26). Human sera from malaria-exposed individuals contain antibodies to P. falciparum ligands and inhibit invasion in both sialic acid-dependent and –independent pathways (27). Thus, receptor polymorphisms and host immune responses against individual ligands might lead to inhibition of particular invasion pathways. The population of Madang, PNG, has the lowest CR1 levels reported to date (28). Our results indicate that the PfRh4–CR1 invasion pathway was less effective in the presence of low-CR1 erythrocytes, most likely due to limited receptor availability. However, studies in PNG have found no evidence for differences in parasite densities among HH, HL and LL genotypes for CR1, although the HL genotype is significantly protected from severe malaria (29). The lack of correlation between protection against severe malaria and reduced parasitemia has been observed for other blood polymorphisms, including α-thalassemia and SAO, and remains a confounding dilemma in the field (30, 31). In terms of CR1 polymorphisms, the lack of measurable differences in parasite densities is likely due to the fact that HH, HL, and LL erythrocytes from PNG individuals all have low CR1 levels (<400 molecules per cell) compared with Caucasian individuals. The importance of PfRh4 as an invasion ligand in the PNG population is indicated by the strong reactivity to PfRh4 of sera from immune individuals (13). To date, no study has investigated the invasion phenotypes of PNG field isolates, and further work is needed to determine the relevance of these protective mechanisms.
Here we show that CR1 is a receptor used for P. falciparum invasion into erythrocytes. CR1 belongs to the regulator of complement activation (RCA) protein family and has been characterized as a negative regulator of complement activation. Several pathogens are known to bind to RCA proteins as a means to facilitate entry into host cells or to down-regulate complement activation (reviewed in ref. 32). RCA proteins, such as DAF, are present on erythrocytes and also may be involved in P. falciparum invasion.
Materials and Methods
See SI Materials and Methods for more detailed information.
Erythrocyte-Binding Assays.
Erythrocyte-binding assays and enzymatic treatment of erythrocytes were performed as described previously (13). For the antibody inhibition of erythrocyte binding, anti-CR1, anti-glycophorin A/B monoclonal, and anti-DAF monoclonal antibodies were preincubated with erythrocytes before the addition of culture supernatants. Competitive binding assays with sCR1 were performed by incubating sCR1 with culture supernatants before proceeding with standard erythrocyte-binding assays. The FACS-based erythrocyte-binding assay for P. falciparum is a modification of the protocol used for Duffy antigen binding (18).
In Vitro Immunoprecipitation.
Recombinant proteins used in immunoprecipitations were incubated together in T-NET buffer [1% Triton X-100, 150 mM NaCl, 10 mM EDTA, and 50 mM Tris (pH 7.4)]. Immunoprecipitation was performed using anti-CR1 (HB8592) or anti-PfRh4 (10C9 and 2E8) monoclonal antibody coupled to Protein G/A Sepharose beads and then eluted in glycine elution buffer. Eluted proteins were resuspended in nonreducing sample buffer, run on SDS/PAGE gels, and evaluated by immunoblot analysis.
ELISA.
Microtiter plates were coated with recombinant fusion protein, blocked, and incubated with ligand. Protein interactions were detected using a specific monoclonal antibody and an HRP-conjugated secondary antibody. Azino-bis-3-ethylbenthiazoline-6-sulfonic acid was used to detect HRP activity, and optical density was measured at 405 nm.
SPR.
Binding of sCR1 for PfRh4 was monitored by SPR using the Biacore T100 system (GE Healthcare). The sensor chip surfaces were prepared by immobilizing recombinant PfRh4 at different loading densities on two of the four flow cells of a Biacore Series S carboxymethylated dextran (CM5) sensor chip, using standard amine coupling. Experiments were performed at 25 °C at and a flow rate of 30 μL/minute. Duplicate injections of sCR1 samples in 10 mM Hepes-buffered 150 mM saline with 3 mM EDTA and 0.005% (vol/vol) polysorbate 20 (pH 7.4) were performed at the concentrations indicated in Fig. 2. Data were processed using Biacore T100 evaluation software version 2.0. Dissociation constants were calculated by fitting steady-state binding levels derived from the background-subtracted traces to a one-to-one binding steady-state model.
Growth Assays.
Parasite growth assays were performed over two cycles of parasite growth as described previously (27).
Acknowledgments
We thank Dr. Henry C. Marsh (Celldex Therapeutics, Needham, MA) for providing sCR1, CR1 monoclonal antibodies 4D6 and 6B1, and comments on the manuscript. We thank Ronald Taylor (University of Virginia School of Medicine, Charlottesville, VA) and Thalachallour Mohanakumar (University of Virginia School of Medicine, Charlottesville, VA) for CR1 monoclonal antibodies 1B4, 3D9, 7G9, 9H3, and KuN241; the Australian Red Cross (Dr. Jenny Condon) for the blood samples; The Walter and Eliza Hall Institute's Monoclonal Facility (The Walter and Eliza Hall Institute of Medical Research, Melbourne) for antibody production; and Stuart Wyithe (Department of Physics, University of Melbourne, Melbourne) for statistical analysis. Infrastructure was supported by Victoria State Government Offer Information Statement and National Health and Medical Research Council Independent Research Institute Infrastructure Support Scheme grants. A.F.C. is a Howard Hughes International Scholar and an Australia Fellow of the National Health and Medical Research Council. J.G.B. is supported by a National Health and Medical Research Council Career Development award. This work was supported by the National Health and Medical Research Council, the Darwin Trust of Edinburgh (studentship to P.B.T-Q.), and the Wellcome Trust.
Supporting Information
Supporting Information (PDF)
Supporting Information
- Download
- 914.95 KB
References
1
AF Cowman, BS Crabb, Invasion of red blood cells by malaria parasites. Cell 124, 755–766 (2006).
2
DS Peterson, TE Wellems, EBL-1, a putative erythrocyte-binding protein of Plasmodium falciparum, maps within a favored linkage group in two genetic crosses. Mol Biochem Parasitol 105, 105–113 (2000).
3
JH Adams, et al., A family of erythrocyte-binding proteins of malaria parasites. Proc Natl Acad Sci USA 89, 7085–7089 (1992).
4
JC Rayner, MR Galinski, P Ingravallo, JW Barnwell, Two Plasmodium falciparum genes express merozoite proteins that are related to Plasmodium vivax and Plasmodium yoelii adhesive proteins involved in host cell selection and invasion. Proc Natl Acad Sci USA 97, 9648–9653 (2000).
5
JC Rayner, E Vargas-Serrato, CS Huber, MR Galinski, JW Barnwell, A Plasmodium falciparum homologue of Plasmodium vivax reticulocyte binding protein (PvRBP1) defines a trypsin-resistant erythrocyte invasion pathway. J Exp Med 194, 1571–1581 (2001).
6
AG Maier, et al., Plasmodium falciparum erythrocyte invasion through glycophorin C and selection for Gerbich negativity in human populations. Nat Med 9, 87–92 (2003).
7
DC Mayer, et al., Glycophorin B is the erythrocyte receptor of Plasmodium falciparum erythrocyte-binding ligand, EBL-1. Proc Natl Acad Sci USA 106, 5348–5352 (2009).
8
BKL Sim, CE Chitnis, K Wasniowska, TJ Hadley, LH Miller, Receptor and ligand domains for invasion of erythrocytes by Plasmodium falciparum. Science 264, 1941–1944 (1994).
9
C Spadafora, et al., Complement receptor 1 is a sialic acid–independent erythrocyte receptor of Plasmodium falciparum. PLoS Pathog 6, e1000968 (2010).
10
JA Rowe, JM Moulds, CI Newbold, LH Miller, P. falciparum rosetting mediated by a parasite-variant erythrocyte membrane protein and complement-receptor 1. Nature 388, 292–295 (1997).
11
M Krych-Goldberg, JP Atkinson, Structure–function relationships of complement receptor type 1. Immunol Rev 180, 112–122 (2001).
12
J Stubbs, et al., Molecular mechanism for switching of P. falciparum invasion pathways into human erythrocytes. Science 309, 1384–1387 (2005).
13
WH Tham, et al., Antibodies to reticulocyte-binding protein-like homologue 4 inhibit invasion of Plasmodium falciparum into human erythrocytes. Infect Immun 77, 2427–2435 (2009).
14
D Gaur, et al., Recombinant Plasmodium falciparum reticulocyte homology protein 4 binds to erythrocytes and blocks invasion. Proc Natl Acad Sci USA 104, 17789–17794 (2007).
15
HF Weisman, et al., Soluble human complement receptor type 1: In vivo inhibitor of complement suppressing post-ischemic myocardial inflammation and necrosis. Science 249, 146–151 (1990).
16
M Nickells, et al., Mapping epitopes for 20 monoclonal antibodies to CR1. Clin Exp Immunol 112, 27–33 (1998).
17
L Xiang, JR Rundles, DR Hamilton, JG Wilson, Quantitative alleles of CR1: Coding sequence analysis and comparison of haplotypes in two ethnic groups. J Immunol 163, 4939–4945 (1999).
18
TM Tran, et al., Detection of a Plasmodium vivax erythrocyte-binding protein by flow cytometry. Cytometry A 63, 59–66 (2005).
19
MT Duraisingh, AG Maier, T Triglia, AF Cowman, Erythrocyte-binding antigen 175 mediates invasion in Plasmodium falciparum utilizing sialic acid–dependent and –independent pathways. Proc Natl Acad Sci USA 100, 4796–4801 (2003).
20
JA Rowe, et al., Mapping of the region of complement receptor (CR) 1 required for Plasmodium falciparum rosetting and demonstration of the importance of CR1 in rosetting in field isolates. J Immunol 165, 6341–6346 (2000).
21
CA Lobo, et al., Invasion profiles of Brazilian field isolates of Plasmodium falciparum: Phenotypic and genotypic analyses. Infect Immun 72, 5886–5891 (2004).
22
AM Deans, et al., Invasion pathways and malaria severity in Kenyan Plasmodium falciparum clinical isolates. Infect Immun 75, 3014–3020 (2007).
23
N Gomez-Escobar, et al., Erythrocyte invasion and merozoite ligand gene expression in severe and mild Plasmodium falciparum malaria. J Infect Dis 201, 444–452 (2010).
24
AK Bei, et al., Variant merozoite protein expression is associated with erythrocyte invasion phenotypes in Plasmodium falciparum isolates from Tanzania. Mol Biochem Parasitol 153, 66–71 (2007).
25
JN Okoyeh, CR Pillai, CE Chitnis, Plasmodium falciparum field isolates commonly use erythrocyte invasion pathways that are independent of sialic acid residues of glycophorin A. Infect Immun 67, 5784–5791 (1999).
26
SW Serjeantson, BS White, K Bhatia, RJ Trent, A 3.5-kb deletion in the glycophorin C gene accounts for the Gerbich-negative blood group in Melanesians. Immunol Cell Biol 72, 23–27 (1994).
27
KE Persson, et al., Variation in use of erythrocyte invasion pathways by Plasmodium falciparum mediates evasion of human inhibitory antibodies. J Clin Invest 118, 342–351 (2008).
28
IA Cockburn, et al., A human complement receptor 1 polymorphism that reduces Plasmodium falciparum rosetting confers protection against severe malaria. Proc Natl Acad Sci USA 101, 272–277 (2004).
29
FJ Fowkes, et al., Host erythrocyte polymorphisms and exposure to Plasmodium falciparum in Papua New Guinea. Malar J 7, 1 (2008).
30
B Genton, et al., Ovalocytosis and cerebral malaria. Nature 378, 564–565 (1995).
31
SJ Allen, et al., α+-Thalassemia protects children against disease caused by other infections as well as malaria. Proc Natl Acad Sci USA 94, 14736–14741 (1997).
32
JD Lambris, D Ricklin, BV Geisbrecht, Complement evasion by human pathogens. Nat Rev Microbiol 6, 132–142 (2008).
Information & Authors
Information
Published in
Classifications
Copyright
Freely available online through the PNAS open access option.
Submission history
Published online: September 20, 2010
Published in issue: October 5, 2010
Keywords
Acknowledgments
We thank Dr. Henry C. Marsh (Celldex Therapeutics, Needham, MA) for providing sCR1, CR1 monoclonal antibodies 4D6 and 6B1, and comments on the manuscript. We thank Ronald Taylor (University of Virginia School of Medicine, Charlottesville, VA) and Thalachallour Mohanakumar (University of Virginia School of Medicine, Charlottesville, VA) for CR1 monoclonal antibodies 1B4, 3D9, 7G9, 9H3, and KuN241; the Australian Red Cross (Dr. Jenny Condon) for the blood samples; The Walter and Eliza Hall Institute's Monoclonal Facility (The Walter and Eliza Hall Institute of Medical Research, Melbourne) for antibody production; and Stuart Wyithe (Department of Physics, University of Melbourne, Melbourne) for statistical analysis. Infrastructure was supported by Victoria State Government Offer Information Statement and National Health and Medical Research Council Independent Research Institute Infrastructure Support Scheme grants. A.F.C. is a Howard Hughes International Scholar and an Australia Fellow of the National Health and Medical Research Council. J.G.B. is supported by a National Health and Medical Research Council Career Development award. This work was supported by the National Health and Medical Research Council, the Darwin Trust of Edinburgh (studentship to P.B.T-Q.), and the Wellcome Trust.
Notes
*This Direct Submission article had a prearranged editor.
Authors
Competing Interests
The authors declare no conflict of interest.
Metrics & Citations
Metrics
Citation statements
Altmetrics
Citations
If you have the appropriate software installed, you can download article citation data to the citation manager of your choice. Simply select your manager software from the list below and click Download.
Cited by
Loading...
View Options
View options
PDF format
Download this article as a PDF file
DOWNLOAD PDFGet Access
Login options
Check if you have access through your login credentials or your institution to get full access on this article.
Personal login Institutional LoginRecommend to a librarian
Recommend PNAS to a LibrarianPurchase options
Purchase this article to get full access to it.