Erythrocyte-binding antigen 175 mediates invasion in Plasmodium falciparum utilizing sialic acid-dependent and -independent pathways

The Plasmodium falciparum erythrocyte-binding antigen 175 (EBA-175) is a ligand for merozoite invasion into human erythrocytes that binds to glycophorin A in a sialic acid-dependent manner. P. falciparum strain W2mef depends on sialic acid for invasion of erythrocytes, whereas 3D7 is sialic acid-independent. We generated parasites that lack expression or express truncated forms of EBA-175 in W2mef and 3D7. Lack of EBA-175 expression in W2mef parasites was associated with a switch to sialic acid-independent invasion. 3D7 parasites lacking expression of EBA-175 showed no alteration in their ability to utilize sialic acid-independent pathways. Strikingly, both W2mef and 3D7 parasites lacking EBA-175 expression invaded chymotrypsin-treated erythrocytes inefficiently compared with the parental lines. This loss of function suggests that the EBA-175/glycophorin A ligand-receptor interaction is the major chymotrypsin-resistant invasion pathway. Parasite lines with truncated EBA-175 had invasion phenotypes equivalent to parasites lacking expression of EBA-175. The EBA-175 ligand is functional in erythrocyte invasion by merozoites that utilize either sialic acid-dependent or -independent invasion pathways. This finding suggests a model where a minimal affinity supplied by multiple ligand-receptor interactions is required for successful invasion and has implications for EBA-175 as a malaria vaccine candidate.     

Polymorphism in the Plasmodium falciparum erythrocyte-binding ligand JESEBL/EBA-181 alters its receptor specificity

The malaria parasite lives within erythrocytes and depends on the binding of parasite ligands to host cell surface receptors for invasion. The most virulent human malaria parasite, Plasmodium falciparum, uses multiple ligands, including EBA-175, BAEBL, and JESEBL of the Duffy-binding-like (DBL) family of erythrocyte-binding proteins, for invasion of human erythrocytes. Region II of these parasite ligands is the erythrocyte-binding domain. Previously, we had shown that polymorphism in region II of BAEBL leads to different erythrocyte-binding specificities. We have now identified and characterized the binding specificity of six JESEBL variants. We sequenced region II of JESEBL from 20 P. falciparum clones collected from various parts of the world where malaria is endemic. We observed eight JESEBL variants that contained amino acid polymorphisms at five positions among all clones. Seven of the eight variants could be connected by a single base change that led to an amino acid change. We investigated the functional significance of these polymorphisms by transiently expressing region II from six of JESEBL variants on the surface of Chinese hamster ovary cells. We observed four erythrocyte-binding patterns to enzyme-treated erythrocytes. Thus, P. falciparum DBL ligands JESEBL and BAEBL can recognize multiple receptors on the erythrocyte surface. In contrast to Plasmodium vivax, which has disappeared from West Africa because of the Duffy-negative blood group, P. falciparum may have been successful in endemic areas because it has mutated the ligands of the DBL family to create multiple pathways of invasion, thus making selection of refractory erythrocytes unlikely.     

Targeted disruption of an erythrocyte binding antigen in Plasmodium falciparum is associated with a switch toward a sialic acid-independent pathway of invasion

Erythrocyte invasion by Plasmodium requires molecules present both on the merozoite surface and within the specialized organelles of the apical complex. The Plasmodium erythrocyte binding protein family includes the Plasmodium falciparum sialic acid-binding protein, EBA-175 (erythrocyte binding antigen-175), which binds sialic acid present on glycophorin A of human erythrocytes. We address the role of the conserved 3'-cysteine rich region, the transmembrane, and cytoplasmic domains through targeted gene disruption. Truncation of EBA-175 had no measurable effect on either the level of EBA-175 protein expression or its subcellular localization. Similarly, there appears to be no impairment in the ability of soluble EBA-175 to be released into the culture supernatant after schizont rupture. Additionally, the 3'-cys rich region, transmembrane, and cytoplasmic domains of EBA-175 are apparently non-essential for merozoite invasion. In contrast, erythrocyte invasion via the EBA-175/glycophorin A route appears to have been disrupted to such a degree that the mutant lines have undergone a stable switch in invasion phenotype. As such, EBA-175 appears to have been functionally inactivated within the truncation mutants. The sialic acid-independent invasion pathway within the mutant parasites accounts for approximately 85% of invasion into normal erythrocytes. These data demonstrate the ability of P. falciparum to utilize alternate pathways for invasion of red blood cells, a property that most likely provides a substantial survival advantage in terms of overcoming host receptor heterogeneity and/or immune pressure.     

Recombinant Plasmodium falciparum reticulocyte homology protein 4 binds to erythrocytes and blocks invasion

Plasmodium falciparum invasion of human erythrocytes involves several parasite and erythrocyte receptors that enable parasite invasion by multiple redundant pathways. A key challenge to the development of effective vaccines that block parasite infection of erythrocytes is identifying the players in these pathways and determining their function. Invasion by the parasite clone, Dd2, requires sialic acid on the erythrocyte surface; Dd2/NM is a variant selected for its ability to invade neuraminidase-treated erythrocytes that lack sialic acid. The P. falciparum protein, reticulocyte homology 4 (PfRH4), is uniquely up-regulated in Dd2/NM compared with Dd2, suggesting that it may be a parasite receptor involved in invasion. The aim of the present study was to determine the role of PfRH4 in invasion of erythrocytes and to determine whether it is a target of antibody-mediated blockade and thus a vaccine candidate. We show that both native PfRH4 and a recombinant 30-kDa protein to a conserved region of PfRH4 (rRH4(30)) bind strongly to neuraminidase-treated erythrocytes. rRH4(30) blocks both the erythrocyte binding of the native PfRH4 and invasion of neuraminidase-treated erythrocytes by Dd2/NM. Taken together, these results indicate that PfRH4 is a parasite receptor involved in sialic acid-independent invasion of erythrocytes. Although antibodies to rRH4(30) block binding of the native protein to erythrocytes, these antibodies failed to block invasion. These findings suggest that, although PfRH4 is required for invasion of neuraminidase-treated erythrocytes by Dd2/NM, it is inaccessible for antibody-mediated inhibition of the invasion process.     

Characterization of a Plasmodium falciparum erythrocyte-binding protein paralogous to EBA-175

A member of a Plasmodium receptor family for erythrocyte invasion was identified on chromosome 13 from the Plasmodium falciparum genome sequence of the Sanger Centre (Cambridge, U.K.). The protein (named BAEBL) has homology to EBA-175, a P. falciparum receptor that binds specifically to sialic acid and the peptide backbone of glycophorin A on erythrocytes. Both EBA-175 and BAEBL localize to the micronemes, organelles at the invasive ends of the parasites that contain other members of the family. Like EBA-175, the erythrocyte receptor for BAEBL is destroyed by neuraminidase and trypsin, indicating that the erythrocyte receptor is a sialoglycoprotein. Its specificity, however, differs from that of EBA-175 in that BAEBL can bind to erythrocytes that lack glycophorin A, the receptor for EBA-175. It has reduced binding to erythrocytes with the Gerbich mutation found in another erythrocyte, sialoglycoprotein (glycophorin C/D). The interest in BAEBL's reduced binding to Gerbich erythrocytes derives from the high frequency of the Gerbich phenotype in some regions of Papua New Guinea where P. falciparum is hyperendemic.     

Receptor-binding residues lie in central regions of Duffy-binding-like domains involved in red cell invasion and cytoadherence by malaria parasites

Erythrocyte invasion by malaria parasites and cytoadherence of Plasmodium falciparum-infected erythrocytes to host capillaries are 2 key pathogenic mechanisms in malaria. The receptor-binding domains of erythrocyte-binding proteins (EBPs) such as Plasmodium falciparum EBA-175, which mediate invasion, and P falciparum erythrocyte membrane protein 1 (PfEMP-1) family members, which are encoded by var genes and mediate cytoadherence, have been mapped to conserved cysteine-rich domains referred to as Duffy-binding-like (DBL) domains. Here, we have mapped regions within DBL domains from EBPs and PfEMP-1 that contain receptor-binding residues. Using biochemical and molecular methods we demonstrate that the receptor-binding residues of parasite ligands that bind sialic acid on glycophorin A for invasion as well as complement receptor-1 and chondroitin sulfate A for cytoadherence map to central regions of DBL domains. In contrast, binding to intercellular adhesion molecule 1 (ICAM-1) requires both the central and terminal regions of DBLbetaC2 domains. Determination of functional regions within DBL domains is the first step toward understanding the structure-function bases for their interaction with diverse host receptors.     

Mapping regions containing binding residues within functional domains of Plasmodium vivax and Plasmodium knowlesi erythrocyte-binding proteins

Invasion of erythrocytes by malaria parasites is mediated by specific molecular interactions. Whereas Plasmodium vivax and Plasmodium knowlesi use the Duffy blood group antigen, Plasmodium falciparum uses sialic acid residues of glycophorin A as receptors to invade human erythrocytes. P. knowlesi uses the Duffy antigen as well as other receptors to invade rhesus erythrocytes by multiple pathways. Parasite ligands that bind these receptors belong to a family of erythrocyte-binding proteins (EBP). The EBP family includes the P. vivax and P. knowlesi Duffy-binding proteins, P. knowlesi beta and gamma proteins, which bind alternate receptors on rhesus erythrocytes, and P. falciparum erythrocyte-binding antigen (EBA-175), which binds sialic acid residues of human glycophorin A. Binding domains of each EBP lie in a conserved N-terminal cysteine-rich region, region II, which contains around 330 amino acids with 12 to 14 conserved cysteines. Regions containing binding residues have now been mapped within P. vivax and P. knowlesi beta region II. Chimeric domains containing P. vivax region II sequences fused to P. knowlesi beta region II sequences were expressed on the surface of COS cells and tested for binding to erythrocytes. Binding residues of P. vivax region II lie in a 170-aa stretch between cysteines 4 and 7, and binding residues of P. knowlesi beta region II lie in a 53-aa stretch between cysteines 4 and 5. Mapping regions responsible for receptor recognition is an important step toward understanding the structural basis for the interaction of these parasite ligands with host receptors.     

Complement receptor 1 is the host erythrocyte receptor for Plasmodium falciparum PfRh4 invasion ligand

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 K_d 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.     

Invasion of erythrocytes by Plasmodium falciparum malaria parasites: evidence for receptor heterogeneity and two receptors

Plasmodium falciparum malaria parasites with different capabilities of invading sialic acid-deficient erythrocytes were identified. Thai-2 parasites cultured in Tn erythrocytes invaded neuraminidase-treated and Tn erythrocytes twice as efficiently as Thai-2 parasites cultured in normal erythrocytes and seven to ten times more efficiently than a cloned line of Camp parasites cultured in normal erythrocytes. All three parasite lines required sialic acid for optimal invasion, but Thai-2 parasites cultured in Tn erythrocytes invaded neuraminidase- treated erythrocytes with 45% efficiency whereas Camp parasites invaded neuraminidase-treated erythrocytes with less than 10% efficiency. P falciparum malaria parasites probably possess two receptors: one that binds to a sialic acid-dependent ligand and another that binds to a sialic acid-independent ligand. Parasites may differ in the quantity or affinity of their receptors for the sialic acid-independent ligand.     

Differential antibody responses to Plasmodium falciparum invasion ligand proteins in individuals living in malaria-endemic areas in Brazil and Cameroon

Antibody responses to malaria invasion ligands and proteins on the merozoite surface have been shown to interfere with red cell invasion and correlate with immunity to malaria. The current study is the first to characterize the antibody responses to EBA-140 and EBA-181, Plasmodium falciparum invasion ligands implicated in the alternative pathways of invasion, in age-matched populations of individuals living in endemic areas in both Brazil and Cameroon. Antibody responses to the proteins screened were different between populations. The African individuals reacted strongly with most fragments of these two EBAs, while the majority of the individuals from Mato Grosso, Brazil, reacted weakly and those from the Amazon had elevated responses to these EBA proteins. When compared with the responses against MSP-1(19) and EBA-175, it appeared that the Brazilian population has a variable ability to recognize P. falciparum invasion ligand proteins and that these responses are distinct from the African population.     

Polyethylene glycol-coated red blood cells fail to bind glycophorin A-specific antibodies and are impervious to invasion by the Plasmodium falciparum malaria parasite

This study was designed to assess the binding of glycophorin A-specific antibodies to polyethylene glycol (PEG)-modified red blood cells (RBCs) and evaluate their resistance to invasion by Plasmodium falciparum malaria parasites. RBCs were conjugated with a range of concentrations (0.05 to 7.5 mM) of activated PEG derivatives of either 3.35 or 18.5 kd molecular mass. The binding of glycophorin A-specific antibodies was assessed by hemagglutination and flow cytometry. PEG-modified RBCs were assessed for their ability to form rosettes around Chinese hamster ovary (CHO) cells transiently expressing the glycophorin A binding domain of EBA-175, a P falciparum ligand crucial to RBC invasion. PEG-RBCs were also tested for their ability to be invaded by the malaria parasite. RBCs coated with 3.35 and 18.5 kd PEG demonstrated a dose-dependent inhibition of glycophorin A-specific antibody binding, CHO cell rosetting, and P falciparum invasion. These results indicate that glycophorin A epitopes responsible for antibody and parasite binding are concealed by PEG coating, rendering these cells resistant to P falciparum invasion. These studies confirm the effectiveness of PEG modification for masking RBC-surface glycoproteins. This may provide a means to prevent alloimmunization in the setting of RBC transfusion and suggests a novel method to enhance the effectiveness of exchange transfusion for the treatment of cerebral malaria.     

The glycophorin C N-linked glycan is a critical component of the ligand for the Plasmodium falciparum erythrocyte receptor BAEBL

Plasmodium vivax uses a single member of the Duffy binding-like (DBL) receptor family to invade erythrocytes and is not found in West Africa where its erythrocyte ligand, the Duffy blood group antigen, is missing. In contrast, Plasmodium falciparum expresses four members of the DBL family, and remarkably, single-point mutations of two of these receptors (BAEBL and JESEBL) bind to entirely different erythrocyte ligands, greatly expanding the range of erythrocytes that P. falciparum can invade. In this article, we describe the molecular basis of the binding specificity for one BAEBL variant (VSTK) that binds to glycophorin C. We demonstrate that soluble glycophorin C completely blocks the binding of BAEBL (VSTK) to human erythrocytes, requiring 0.7 microM for 50% inhibition, a concentration similar to that required by glycophorin A to block the binding of erythrocyte-binding antigen 175 to erythrocytes. BAEBL (VSTK) does not bind to Gerbich-negative erythrocytes that express a truncated form of glycophorin C because it lacks exon 3. The N-linked oligosaccharide of Gerbich-negative glycophorin C has a markedly different composition than the wild-type glycophorin C. Moreover, removal of the N-linked oligosaccharide from the wild-type glycophorin C eliminates its ability to inhibit binding of BAEBL (VSTK) to erythrocytes. These findings are consistent with the ligand for BAEBL (VSTK) being, in part, the N-linked oligosaccharide and suggest that single-point mutations in BAEBL allow P. falciparum to recognize oligosaccharides on different erythrocyte surface glycoproteins or glycolipids, greatly increasing its invasion range.     

Search for the sialic acid-independent receptor on red blood cells for invasion by Plasmodium falciparum

BACKGROUND AND OBJECTIVES: Plasmodium falciparum uses multiple red blood cell (RBC) receptors and parasite ligands to invade RBCs. One pathway uses a sialic acid-independent protein or carbohydrate for invasion. The present study searches for this RBC receptor. MATERIALS AND METHODS: We determined whether antigen-negative and null RBCs (including PNH cells that lack all glycosylphosphatidyl inositol-linked proteins) could be invaded after neuraminidase treatment. We used two P. falciparum clones for the study: one that requires sialic acid for invasion and was an indication of removal of sialic acid and a second clone that can invade neuraminidase-treated RBCs. RESULTS: All neuraminidase-treated variant RBCs in this study were invaded. CONCLUSION: This study indicates that some molecule other than those studied (e.g., a carbohydrate) is the receptor for the sialic acid-independent pathway. This powerful tool for the identification of receptors for microorganisms should be used more extensively.     

恶性疟原FCC-1 HN株EBA-175和HRP-II基因片段在HeLa细胞中的表达及鉴定

目的　在 Ｈｅ Ｌａ 细胞中表达恶性疟原虫红细胞结合蛋白 Ｅ Ｂ Ａ－ １７５ 和富组氨酸蛋白 Ｈ Ｒ Ｐ－ Ｉ Ｉ, 进一步研究其生物学功能和免疫学特性。方法　将 Ｅ Ｂ Ａ－ １７５ 和 Ｈ Ｒ Ｐ－ Ｉ Ｉ 部分基因串接插入 Ｐ Ｃ Ｄ Ｎ Ａ３ 真核表达载体, 获得重组表达质粒 Ｐ Ｃ Ｄ Ｎ Ａ３ Ｅ Ｂ Ａ１７５ － Ｈ Ｒ Ｐ Ｉ Ｉ。采用磷酸钙－ Ｄ Ｎ Ａ 共沉淀法将重组质粒转染 Ｈｅ Ｌａ 细胞进行体外表达, 对表达产物进行 Ｓ Ｄ Ｓ－ Ｐ Ａ Ｇ Ｅ、 Ｄｏｔ－ Ｅ Ｌ Ｉ Ｓ Ａ 和 Ｗｅｓｔｅｒｎ － ｂｌｏｔ 鉴定。结果　表达产物占表达蛋白总量的１６４ ％ , 相对分子量与预设计的４８ｋ Ｄａ 基本相符。表达产物与鼠抗恶性疟原虫血清及构建的重组质粒免疫鼠血清产生特异免疫反应。结论　表达载体 Ｐ Ｃ Ｄ Ｎ Ａ３ 在 Ｈｅ Ｌａ 细胞内可表达出具有一定免疫学活性的恶性疟原虫重组抗原蛋白。     

Human antibodies to a Mr 155,000 Plasmodium falciparum antigen efficiently inhibit merozoite invasion.

IgG from a donor clinically immune to Plasmodium falciparum malaria strongly inhibited reinvasion in vitro of human erythrocytes by the parasite. When added to monolayers of glutaraldehyde-fixed and air-dried erythrocytes infected with the parasite, this IgG also displayed a characteristic immunofluorescence restricted to the surface of infected erythrocytes. Elution of the IgG adsorbed to such monolayers gave an antibody fraction that was 40 times more efficient in the reinvasion inhibition assay (50% inhibition titer, less than 1 microgram/ml) than the original IgG preparation. The major antibody in this eluate was directed against a parasite-derived antigen of Mr 155,000 (Pf 155) deposited by the parasite in the erythrocyte membrane in the course of invasion. A detailed study of IgG fractions from 11 donors with acute P. falciparum malaria or clinical immunity revealed the existence of an excellent correlation between their capacities to stain the surface of infected erythrocytes, their titers in reinvasion inhibition, and the presence of antibodies to Pf 155 as detected by immunoblotting. No such correlations were seen when the IgG fractions were analyzed for immunofluorescence of intracellular parasites or for the presence of antibodies to other parasite antigens as detected by immunoprecipitation of [35S]methionine-labeled and NaDodSO4/PAGE-separated parasite extracts. The results suggest that Pf 155 has an important role in the process of erythrocyte infection and that host antibodies to this antigen may efficiently interfere with this process.     

P195蛋白及其抗体抑制恶性疟原虫裂殖子入侵人红细胞的研究

目的寻找恶性疟原虫裂殖子表面主要蛋白Ｐ１９５中的红细胞结合位点,为设计疫苗阻断裂殖子入侵红细胞提供实验依据。方法在大肠杆菌中分８段表达Ｐ１９５蛋白。各段蛋白用镍亲和层析柱分离,然后复性。将得到的各段蛋白免疫家兔,制备抗血清。在体外培养疟原虫至成熟裂殖体期,将各段蛋白及其相应的抗血清分别加入到培养基上清中,继续培养２４小时,检查红细胞感染率。通过感染率了解各段蛋白及其抗体对裂殖子入侵红细胞的影响。结果Ｐ１９５蛋白中氨基酸序列为３８３～５９５（Ｍ６）,５９５～８９７（Ｍ７）,１３９７～１６６３（Ｍ１１）的三段蛋白的抗血清具有抑制裂殖子入侵红细胞的作用,而其中Ｍ６蛋白片段也具有抑制裂殖子入侵红细胞的作用。结论Ｐ１９５蛋白中氨基酸序列为３８３～５９５的一段序列,Ｍ６可能含恶性疟原虫识别人红细胞的位点,该位点可以作为疟疾疫苗的候选抗原。     

A lectin-like receptor is involved in invasion of erythrocytes by Plasmodium falciparum.

Glycophorin both in solution and inserted into liposomes blocks invasion of erythrocytes by the malaria parasite Plasmodium falciparum. Furthermore, one sugar, N-acetyl-D-glucosamine (GlcNAc), completely blocks invasion of the erythrocyte by this parasite. GlcNAc coupled to bovine serum albumin to prevent the sugar entering infected erythrocytes was at least 100,000 times more effective than GlcNAc alone. Bovine serum albumin coupled to lactose or bovine serum albumin alone had no effect on invasion. These results suggest that the binding of P. falciparum to erythrocytes is lectin-like and is determined by carbohydrates on glycophorin.     

Antigenic diversity amongst ten geographic isolates of Plasmodium falciparum defined by merozoite invasion inhibition assay.

The extent to which human antibodies involved in functional immunity react with antigenic determinants varying between different isolates or strains of human malaria parasite Plasmodium falciparum will influence the design of vaccine against malaria. In this study, in vitro inhibition of merozoite invasion in erythrocytes by an immune human serum was used to define the antigenic differences in 10 isolates of P. falciparum from three endemic areas, i.e. Africa, South America and Southeast Asia. The serum inhibited the invasion of merozoites of all the strains but the extent of inhibition varied from low to moderate to high degree indicating antigenic differences amongst isolates of P. falciparum. The antigenic differences could not be correlated to the geographic origin of the parasite isolate.