Molecular Architecture of a Complex between an Adhesion Protein from the Malaria Parasite and Intracellular Adhesion Molecule 1

The adhesion of Plasmodium falciparum-infected erythrocytes to human tissues or endothelium is central to the pathology caused by the parasite during malaria. It contributes to the avoidance of parasite clearance by the spleen and to the specific pathologies of cerebral and placental malaria. The PfEMP1 family of adhesive proteins is responsible for this sequestration by mediating interactions with diverse human ligands. In addition, as the primary targets of acquired, protective immunity, the PfEMP1s are potential vaccine candidates. PfEMP1s contain large extracellular ectodomains made from CIDR (cysteine-rich interdomain regions) and DBL (Duffy-binding-like) domains and show extensive variation in sequence, size, and domain organization. Here we use biophysical methods to characterize the entire ∼300-kDa ectodomain from IT4VAR13, a protein that interacts with the host receptor, intercellular adhesion molecule-1 (ICAM-1). We show through small angle x-ray scattering that IT4VAR13 is rigid, elongated, and monomeric. We also show that it interacts with ICAM-1 through the DBLβ domain alone, forming a 1:1 complex. These studies provide a first low resolution structural view of a PfEMP1 ectodomain in complex with its ligand. They show that it combines a modular domain arrangement consisting of individual ligand binding domains, with a defined higher order architecture that exposes the ICAM-1 binding surface to allow adhesion.     

The cysteine-rich interdomain region from the highly variable plasmodium falciparum erythrocyte membrane protein-1 exhibits a conserved structure

Plasmodium falciparum malaria parasites, living in red blood cells, express proteins of the erythrocyte membrane protein-1 (PfEMP1) family on the red blood cell surface. The binding of PfEMP1 molecules to human cell surface receptors mediates the adherence of infected red blood cells to human tissues. The sequences of the 60 PfEMP1 genes in each parasite genome vary greatly from parasite to parasite, yet the variant PfEMP1 proteins maintain receptor binding. Almost all parasites isolated directly from patients bind the human CD36 receptor. Of the several kinds of highly polymorphic cysteine-rich interdomain region (CIDR) domains classified by sequence, only the CIDR1alpha domains bind CD36. Here we describe the CD36-binding portion of a CIDR1alpha domain, MC179, as a bundle of three alpha-helices that are connected by a loop and three additional helices. The MC179 structure, containing seven conserved cysteines and 10 conserved hydrophobic residues, predicts similar structures for the hundreds of CIDR sequences from the many genome sequences now known. Comparison of MC179 with the CIDR domains in the genome of the P. falciparum 3D7 strain provides insights into CIDR domain structure. The CIDR1alpha three-helix bundle exhibits less than 20% sequence identity with the three-helix bundles of Duffy-binding like (DBL) domains, but the two kinds of bundles are almost identical. Despite the enormous diversity of PfEMP1 sequences, the CIDR1alpha and DBL protein structures, taken together, predict that a PfEMP1 molecule is a polymer of three-helix bundles elaborated by a variety of connecting helices and loops. From the structures also comes the insight that DBL1alpha domains are approximately 100 residues larger and that CIDR1alpha domains are approximately 100 residues smaller than sequence alignments predict. This new understanding of PfEMP1 structure will allow the use of better-defined PfEMP1 domains for functional studies, for the design of candidate vaccines, and for understanding the molecular basis of cytoadherence.     

Plasmodium falciparum Erythrocyte Membrane Protein 1 Diversity in Seven Genomes – Divide and Conquer

The var gene encoded hyper-variable Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) family mediates cytoadhesion of infected erythrocytes to human endothelium. Antibodies blocking cytoadhesion are important mediators of malaria immunity acquired by endemic populations. The development of a PfEMP1 based vaccine mimicking natural acquired immunity depends on a thorough understanding of the evolved PfEMP1 diversity, balancing antigenic variation against conserved receptor binding affinities. This study redefines and reclassifies the domains of PfEMP1 from seven genomes. Analysis of domains in 399 different PfEMP1 sequences allowed identification of several novel domain classes, and a high degree of PfEMP1 domain compositional order, including conserved domain cassettes not always associated with the established group A–E division of PfEMP1. A novel iterative homology block (HB) detection method was applied, allowing identification of 628 conserved minimal PfEMP1 building blocks, describing on average 83% of a PfEMP1 sequence. Using the HBs, similarities between domain classes were determined, and Duffy binding-like (DBL) domain subclasses were found in many cases to be hybrids of major domain classes. Related to this, a recombination hotspot was uncovered between DBL subdomains S2 and S3. The VarDom server is introduced, from which information on domain classes and homology blocks can be retrieved, and new sequences can be classified. Several conserved sequence elements were found, including: (1) residues conserved in all DBL domains predicted to interact and hold together the three DBL subdomains, (2) potential integrin binding sites in DBLα domains, (3) an acylation motif conserved in group A var genes suggesting N-terminal N-myristoylation, (4) PfEMP1 inter-domain regions proposed to be elastic disordered structures, and (5) several conserved predicted phosphorylation sites. Ideally, this comprehensive categorization of PfEMP1 will provide a platform for future studies on var/PfEMP1 expression and function.     

Plasmodium falciparum: cloned and expressed CIDR domains of PfEMP1 bind to chondroitin sulfate A

Adherence of erythrocytes infected with mature asexual Plasmodium falciparum parasites (iRBC) to microvascular endothelial cells contributes to the pathology of P. falciparum malaria. It has been shown that the variant P. falciparum erythrocyte membrane protein 1 (PfEMP1) confers adhesion to a wide range of cell surface receptors. Previously, the cysteine-rich interdomain region (CIDR) of PfEMP1 has been identified as binding site to CD36. We provide evidence that the same region can also mediate binding to chondroitin sulfate A (CSA). CIDR domains of two different parasite strains were expressed in Escherichia coli as a 6xHis-tagged protein. Purified recombinant protein bound to Chinese hamster ovary (CHO) cells which naturally express chondroitin sulfate A. Treatment of wild-type CHO cells with chondroitinase ABC reduced binding up to 94.4%. Competitive binding using soluble CSA inhibited binding to CHO cells by up to 100% at 2 mg/ml and by 62.4% at 0.5 mg/ml, whereas 1 mg/ml heparan sulfate had only a little effect (18.1%). In contrast, a recombinant 6xHis-tagged DBL1 domain showed no binding to wild-type CHO cells. Such an approach of analyzing various domains of PfEMP1 as recombinant proteins may elucidate their functions and may lead to novel anti-adherence therapeutics, especially for maternal malaria infections.     

Structure-Function-Immunogenicity Studies of PfEMP1 Domain DBL2βPF11_0521, a Malaria Parasite Ligand for ICAM-1

Plasmodium falciparum virulence has been ascribed to its ability to sequester in deep vascular beds, mediated by the variant surface antigen family PfEMP1 binding endothelial receptors like ICAM-1. We previously observed that naturally-acquired antibodies that block a PfEMP1 domain, DBL2β of PF11_0521 allele, from binding to the human ICAM1 receptor, reduce the risk of malaria hospitalization in children. Here, we find that DBL2β_{PF11_0521} binds ICAM-1 in the low nM range and relate the structure of this domain with its function and immunogenicity. We demonstrate that the interaction with ICAM-1 is not impaired by point mutations in the N-terminal subdomain or in the flexible Loop 4 of DBL2β_{PF11_0521}, although both substructures were previously implicated in binding ICAM-1. These data will help to refine the existing model of DBLβ::ICAM-1 interactions. Antibodies raised against full-length DBL2β_{PF11_0521}, but not truncated forms lacking the N terminal fragment, block its interaction with ICAM-1. Our data suggest that full length domain is optimal for displaying functional epitopes and has a broad surface of interaction with ICAM-1 that is not disrupted by individual amino acid substitutions at putative key residues. This information might be important for the future design of anti-malarial vaccines based on PfEMP1 antigens.     

Recovery of adhesion to chondroitin-4-sulphate in Plasmodium falciparum varCSA disruption mutants by antigenically similar PfEMP1 variants

Protection against maternal malaria has been associated with the acquisition of a specific antibody response that prevents adhesion of Plasmodium falciparum-infected erythrocytes to the glycosaminoglycan chondroitin-4-sulphate (CSA), which is present in the placental intervillous space. These antibodies are directed against variant forms of the P. falciparum erythrocyte membrane protein 1 (PfEMP1) that mediate binding to CSA. We have generated insertional disruption mutants of the gene encoding the CSA-binding phenotype in the P. falciparum clone FCR3 (varCSA) to test the hypothesis that strategies targeting the parasite's determinant for this adhesive phenotype may prevent sequestration of infected erythrocytes in the placenta and hence the development of maternal malaria. The varCSA-disruption mutants were initially unable to adhere to CSA; however, they could recover the phenotype after repeated selection over CSA. We show that recovery of CSA binding is varCSA independent and mediated by the activation of a novel var variant. Importantly, the corresponding PfEMP1 protein reacts with a monoclonal antibody recognizing the DBL3 gamma domain of the varCSA gene product, indicating that the DBL3 gamma CSA-binding domains are conserved between these PfEMP1-binding variants. Our data support strategies exploring these conserved epitopes as vaccine candidates against maternal malaria.     

A plasma survey using 38 PfEMP1 domains reveals frequent recognition of the Plasmodium falciparum antigen VAR2CSA among young Tanzanian children

PfEMP1 proteins comprise a family of variant antigens that appear on the surface of P. falciparum-infected erythrocytes and bind to multiple host receptors. Using a mammalian expression system and BioPlex technology, we developed an array of 24 protein constructs representing 38 PfEMP1 domains for high throughput analyses of receptor binding as well as total and functional antibody responses. We analyzed the reactivity of 561 plasma samples from 378 young Tanzanian children followed up to maximum 192 weeks of life in a longitudinal birth cohort. Surprisingly, reactivity to the DBL5 domain of VAR2CSA, a pregnancy malaria vaccine candidate, was most common, and the prevalence of reactivity was stable throughout early childhood. Reactivity to all other PfEMP1 constructs increased with age. Antibodies to the DBL2βC2(PF11_0521) domain, measured as plasma reactivity or plasma inhibition of ICAM1 binding, predicted reduced risk of hospitalization for severe or moderately severe malaria. These data suggest a role for VAR2CSA in childhood malaria and implicate DBL2βC2(PF11_0521) in protective immunity.     

Decoding the language of var genes and Plasmodium falciparum sequestration

Sequestration and rosetting are key determinants of Plasmodium falciparum pathogenesis. They are mediated by a large family of variant proteins called P. falciparum erythrocyte membrane protein 1 (PfEMP1). PfEMP1 proteins are multispecific binding receptors that are transported to parasite-induced, 'knob-like' binding structures at the erythrocyte surface. To evade immunity and extend infections, parasites clonally vary their expressed PfEMP1. Thus, PfEMP1 are functionally selected for binding while immune selection acts to diversify the family. Here, we describe a new way to analyse PfEMP1 sequence that provides insight into domain function and protein architecture with potential implications for malaria disease.     

Antibodies targeting the PfRH1 binding domain inhibit invasion of Plasmodium falciparum merozoites

Invasion by the malaria merozoite depends on recognition of specific erythrocyte surface receptors by parasite ligands. Plasmodium falciparum uses multiple ligands, including at least two gene families, reticulocyte binding protein homologues (RBLs) and erythrocyte binding proteins/ligands (EBLs). The combination of different RBLs and EBLs expressed in a merozoite defines the invasion pathway utilized and could also play a role in parasite virulence. The binding regions of EBLs lie in a conserved cysteine-rich domain while the binding domain of RBL is still not well characterized. Here, we identify the erythrocyte binding region of the P. falciparum reticulocyte binding protein homologue 1 (PfRH1) and show that antibodies raised against the functional binding region efficiently inhibit invasion. In addition, we directly demonstrate that changes in the expression of RBLs can constitute an immune evasion mechanism of the malaria merozoite.     

Analysis of structure and function of the giant protein Pf332 in Plasmodium falciparum

Virulence of Plasmodium falciparum , the most lethal parasitic disease in humans, results in part from adhesiveness and increased rigidity of infected erythrocytes. Pf332 is trafficked to the parasite-infected erythrocyte via Maurer's clefts, structures for protein sorting and export in the host erythrocyte. This protein has a domain similar to the Duffy-binding-like (DBL) domain, which functions by binding to receptors for adherence and invasion. To address structure of the Pf332 DBL domain, we expressed this region, and validated its fold on the basis of the disulphide bond pattern, which conformed to the generic pattern for DBL domains. The modelled structure for Pf332 DBL had differences compared with the erythrocyte-binding region of the αDBL domain of Plasmodium knowlesi Duffy-binding protein (Pkα-DBL). We addressed the function of Pf332 by constructing parasites that either lack expression of the protein or express an altered form. We found no evidence that Pf332 is involved in cytoadhesion or merozoite invasion. Truncation of Pf332 had a significant effect on deformability of the P. falciparum -infected erythrocyte, while loss of the full protein deletion did not. Our data suggest that Pf332 may contribute to the overall deformability of the P. falciparum -infected erythrocyte by anchoring and scaffolding.     

Plasmodium falciparum-infected erythrocytes bind ICAM-1 at a site distinct from LFA-1, Mac-1, and human rhinovirus.

The attachment of erythrocytes infected with P. falciparum to human venular endothelium is the primary step leading to complications from severe and cerebral malaria. Intercellular adhesion molecule-1 (ICAM-1, CD54) has been implicated as a cytoadhesion receptor for P. falciparum-infected erythrocytes. Characterization of domain deletion, human/murine chimeric ICAM-1 molecules, and amino acid substitution mutants localized the primary binding site for parasitized erythrocytes to the first amino-terminal immunoglobulin-like domain of ICAM-1. The ICAM-1 binding site is distinct from those recognized by LFA-1, Mac-1, and the human major-type rhinoviruses. Synthetic peptides encompassing the binding site on ICAM-1 inhibited malaria-infected erythrocyte adhesion to ICAM-1-coated surfaces with a Ki of 0.1-0.3 mM, whereas the Ki for soluble ICAM-1 is 0.15 microM. These findings have implications for the therapeutic reversal of malaria-infected erythrocyte sequestration in the host microvasculature.     

Insights into Duffy Binding-like Domains through the Crystal Structure and Function of the Merozoite Surface Protein MSPDBL2 from Plasmodium falciparum

Invasion of human red blood cells by Plasmodium falciparum involves interaction of the merozoite form through proteins on the surface coat. The erythrocyte binding-like protein family functions after initial merozoite interaction by binding via the Duffy binding-like (DBL) domain to receptors on the host red blood cell. The merozoite surface proteins DBL1 and -2 (PfMSPDBL1 and PfMSPDBL2) (PF10_0348 and PF10_0355) are extrinsically associated with the merozoite, and both have a DBL domain in each protein. We expressed and refolded recombinant DBL domains for PfMSPDBL1 and -2 and show they are functional. The red cell binding characteristics of these domains were shown to be similar to full-length forms of these proteins isolated from parasite cultures. Futhermore, metal cofactors were found to enhance the binding of both the DBL domains and the parasite-derived full-length proteins to erythrocytes, which has implications for receptor binding of other DBL-containing proteins in Plasmodium spp. We solved the structure of the erythrocyte-binding DBL domain of PfMSPDBL2 to 2.09 Å resolution and modeled that of PfMSPDBL1, revealing a canonical DBL fold consisting of a boomerang shaped α-helical core formed from three subdomains. PfMSPDBL2 is highly polymorphic, and mapping of these mutations shows they are on the surface, predominantly in the first two domains. For both PfMSPDBL proteins, polymorphic variation spares the cleft separating domains 1 and 2 from domain 3, and the groove between the two major helices of domain 3 extends beyond the cleft, indicating these regions are functionally important and are likely to be associated with the binding of a receptor on the red blood cell.     

T-cell responses to the DBLα-tag, a short semi-conserved region of the Plasmodium falciparum membrane erythrocyte protein 1

The Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) is a variant surface antigen expressed on mature forms of infected erythrocytes. It is considered an important target of naturally acquired immunity. Despite its extreme sequence heterogeneity, variants of PfEMP1 can be stratified into distinct groups. Group A PfEMP1 have been independently associated with low host immunity and severe disease in several studies and are now of potential interest as vaccine candidates. Although antigen-specific antibodies are considered the main effector mechanism in immunity to malaria, the induction of efficient and long-lasting antibody responses requires CD4+ T-cell help. To date, very little is known about CD4+ T-cell responses to PfEMP1 expressed on clinical isolates. The DBLα-tag is a small region from the DBLα-domain of PfEMP1 that can be amplified with universal primers and is accessible in clinical parasite isolates. We identified the dominant expressed PfEMP1 in 41 individual clinical parasite isolates and expressed the corresponding DBLα-tag as recombinant antigen. Individual DBLα-tags were then used to activate CD4+ T-cells from acute and convalescent blood samples in children who were infected with the respective clinical parasite isolate. Here we show that CD4+ T-cell responses to the homologous DBLα-tag were induced in almost all children during acute malaria and maintained in some for 4 months. Children infected with parasites that dominantly expressed group A-like PfEMP1 were more likely to maintain antigen-specific IFNγ-producing CD4+ T-cells than children infected with parasites dominantly expressing other PfEMP1. These results suggest that group A-like PfEMP1 may induce long-lasting effector memory T-cells that might be able to provide rapid help to variant-specific B cells. Furthermore, a number of children induced CD4+ T-cell responses to heterologous DBLα-tags, suggesting that CD4+ T-cells may recognise shared epitopes between several DBLα-tags.     

Identification of residues in the Cmu4 domain of polymeric IgM essential for interaction with Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1)

The binding of nonspecific human IgM to the surface of infected erythrocytes is important in rosetting, a major virulence factor in the pathogenesis of severe malaria due to Plasmodium falciparum, and IgM binding has also been implicated in placental malaria. Herein we have identified the IgM-binding parasite ligand from a virulent P. falciparum strain as PfEMP1 (TM284var1 variant), and localized the region within this PfEMP1 variant that binds IgM (DBL4beta domain). We have used this parasite IgM-binding protein to investigate the interaction with human IgM. Interaction studies with domain-swapped Abs, IgM mutants, and anti-IgM mAbs showed that PfEMP1 binds to the Fc portion of the human IgM H chain and requires the IgM Cmu4 domain. Polymerization of IgM was shown to be crucial for the interaction because PfEMP1 binding did not occur with mutant monomeric IgM molecules. These results with PfEMP1 protein have physiological relevance because infected erythrocytes from strain TM284 and four other IgM-binding P. falciparum strains showed analogous results to those seen with the DBL4beta domain. Detailed investigation of the PfEMP1 binding site on IgM showed that some of the critical amino acids in the IgM Cmu4 domain are equivalent to those regions of IgG and IgA recognized by Fc-binding proteins from bacteria, suggesting that this region of Ig molecules may be of major functional significance in host-microbe interactions. We have therefore shown that PfEMP1 is an Fc-binding protein of malaria parasites specific for polymeric human IgM, and that it shows functional similarities with Fc-binding proteins from pathogenic bacteria.     

Serum lipoproteins promote efficient presentation of the malaria virulence protein PfEMP1 at the erythrocyte surface

The virulence of the malaria parasite Plasmodium falciparum is related to its ability to express a family of adhesive proteins known as P. falciparum erythrocyte membrane protein 1 (PfEMP1) at the infected red blood cell surface. The mechanism for the transport and delivery of these adhesins to the erythrocyte membrane is only poorly understood. In this work, we have used specific immune reagents in a flow cytometric assay to monitor the effects of serum components on the surface presentation of PfEMP1. We show that efficient presentation of the A4 and VAR2CSA variants of PfEMP1 is dependent on the presence of serum in the bathing medium during parasite maturation. Lipid-loaded albumin supports parasite growth but allows much less efficient presentation of PfEMP1 at the red blood cell surface. Analysis of the serum components reveals that lipoproteins, especially those of the low-density lipoprotein fraction, promote PfEMP1 presentation. Cytoadhesion of infected erythrocytes to the host cell receptors CD36 and ICAM-1 is also decreased in infected erythrocytes cultured in the absence of serum. The defect appears to be in the transfer of PfEMP1 from parasite-derived structures known as the Maurer's clefts to the erythrocyte membrane or in surface conformation rather than a down-regulation or switching of particular PfEMP1 variants.     

Genetic ablation of a Maurer's cleft protein prevents assembly of the Plasmodium falciparum virulence complex

The malaria parasite Plasmodium falciparum assembles knob structures underneath the erythrocyte membrane that help present the major virulence protein, P. falciparum erythrocyte membrane protein-1 (PfEMP1). Membranous structures called Maurer's clefts are established in the erythrocyte cytoplasm and function as sorting compartments for proteins en route to the RBC membrane, including the knob-associated histidine-rich protein (KAHRP), and PfEMP1. We have generated mutants in which the Maurer's cleft protein, the ring exported protein-1 (REX1) is truncated or deleted. Removal of the C-terminal domain of REX1 compromises Maurer's cleft architecture and PfEMP1-mediated cytoadherance but permits some trafficking of PfEMP1 to the erythrocyte surface. Deletion of the coiled-coil region of REX1 ablates PfEMP1 surface display, trapping PfEMP1 at the Maurer's clefts. Complementation of mutants with REX1 partly restores PfEMP1-mediated binding to the endothelial cell ligand, CD36. Deletion of the coiled-coil region or complete deletion of REX1 is tightly associated with the loss of a subtelomeric region of chromosome 2, encoding KAHRP and other proteins. A KAHRP-green fluorescent protein (GFP) fusion expressed in the REX1-deletion parasites shows defective trafficking. Thus, loss of functional REX1 directly or indirectly ablates the assembly of the P. falciparum virulence complex at the surface of host erythrocytes.     

Trafficking determinants for PfEMP3 export and assembly under the Plasmodium falciparum-infected red blood cell membrane

During the maturation of intracellular asexual stages of Plasmodium falciparum parasite-encoded proteins are exported into the erythrocyte cytosol. A number of these parasite proteins attach to the host cell cytoskeleton and facilitate transformation of a disk-shaped erythrocyte into a rounded and more rigid infected erythrocyte able to cytoadhere to the vasculature. Knob formation on the surface of infected erythrocytes is critical for this cytoadherence to the host endothelium. P. falciparum proteins have been identified that localize to the parasite-infected erythrocyte membrane: the variant cytoadherence ligand erythrocyte membrane protein 1 (PfEMP1), the knob-associated histidine-rich protein (KAHRP) and the erythrocyte membrane protein 3 (PfEMP3). In this study, we have generated parasites expressing PfEMP3-green fluorescent protein chimeras and identified domains involved in entry to the secretory pathway, export across the parasitophorous vacuolar membrane and attachment to Maurer's clefts and the erythrocyte membrane. Solubility assays, fluorescence photobleaching experiments and immunogold electron microscopy suggest that the exported chimeric proteins are trafficked in a complex rather than in vesicles. This study characterizes elements involved in the tight but transient binding of PfEMP3 to Maurer's clefts and shows that the same elements are necessary for correct assembly under the erythrocyte membrane.     

An Analysis of the Binding Characteristics of a Panel of Recently Selected ICAM-1 Binding Plasmodium falciparum Patient Isolates

The basis of severe malaria pathogenesis in part includes sequestration of Plasmodium falciparum-infected erythrocytes (IE) from the peripheral circulation. This phenomenon is mediated by the interaction between several endothelial receptors and one of the main parasite-derived variant antigens (PfEMP1) expressed on the surface of the infected erythrocyte membrane. One of the commonly used host receptors is ICAM-1, and it has been suggested that ICAM-1 has a role in cerebral malaria pathology, although the evidence to support this is not conclusive. The current study examined the cytoadherence patterns of lab-adapted patient isolates after selecting on ICAM-1. We investigated the binding phenotypes using variant ICAM-1 proteins including ICAM-1^Ref, ICAM-1^Kilifi, ICAM-1^S22/A, ICAM-1^L42/A and ICAM-1^L44/A using static assays. The study also examined ICAM-1 blocking by four anti-ICAM-1 monoclonal antibodies (mAb) under static conditions. We also characterised the binding phenotypes using Human Dermal Microvascular Endothelial Cells (HDMEC) under flow conditions. The results show that different isolates have variant-specific binding phenotypes under both static and flow conditions, extending our previous observations that this variation might be due to variable contact residues on ICAM-1 being used by different parasite PfEMP1 variants.