Widespread functional specialization of Plasmodium falciparum erythrocyte membrane protein 1 family members to bind CD36 analysed across a parasite genome

Plasmodium falciparum-infected erythrocytes sequester from blood circulation by binding host endothelium. A large family of variant proteins mediates cytoadherence and their binding specificity determines parasite sequestration patterns and potential for disease. The aim of the present study was to understand how binding properties are encoded into family members and to develop sequence algorithms for predicting binding. To accomplish these goals computational approaches and a binding assay were used to characterize adhesion across Plasmodium falciparum erythrocyte membrane 1 (PfEMP1) proteins in the 3D7 parasite genome. We report that most family members encode the capacity to bind CD36 in the protein's semi-conserved head structure and describe the sequence characteristics of a group of PfEMP1 proteins that do not. Structural and functional grouping of PfEMP1 proteins based upon head structure and additional domain architectural properties provide new insights into the protein family. These can be used to investigate the role of proteins in malaria pathogenesis and potentially tailor vaccines to recognize particular binding variants.     

Evidence for the importance of genetic structuring to the structural and functional specialization of the Plasmodium falciparum var gene family

The var gene family encodes Plasmodium falciparum erythrocyte membrane 1 (PfEMP1) proteins that act as virulence factors responsible for both antigenic variation and cytoadherence of infected erythrocytes. These proteins orchestrate infected erythrocyte sequestration from blood circulation and contribute to adhesion-based complications of P. falciparum malaria infections. For this study, we analysed the genetic organization and strain structure of var genes and present evidence for three separately evolving groups that have, in part, functionally diverged and differ between subtelomeric and central chromosomal locations. Our analyses suggest that a recombination hierarchy limits reassortment between groups and may explain why some var genes are unusually conserved between parasite strains. This recombination hierarchy, coupled with binding and immune selection, shapes the variant antigen repertoire and has structural, functional and evolutionary consequences for the PfEMP1 protein family that are directly relevant to malaria pathogenesis.     

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.     

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.     

Characterization of the pathway for transport of the cytoadherence-mediating protein, PfEMP1, to the host cell surface in malaria parasite-infected erythrocytes

The Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) family of antigenically diverse proteins is expressed on the surface of human erythrocytes infected with the malaria parasite P. falciparum, and mediates cytoadherence to the host vascular endothelium. In this report, we show that export of PfEMP1 is slow and inefficient as it takes several hours to traffic newly synthesized proteins to the erythrocyte membrane. Upon removal by trypsin treatment, the surface-exposed population of PfEMP1 is not replenished during subsequent culture indicating that there is no cycling of PfEMP1 between the erythrocyte surface and an intracellular compartment. The role of Maurer's clefts as an intermediate sorting compartment in trafficking of PfEMP1 was investigated using immunoelectron microscopy and proteolytic digestion of streptolysin O-permeabilized parasitized erythrocytes. We show that PfEMP1 is inserted into the Maurer's cleft membrane with the C-terminal domain exposed to the erythrocyte cytoplasm, whereas the N-terminal domain is buried inside the cleft. Transfer of PfEMP1 to the erythrocyte surface appears to involve electron-lucent extensions of the Maurer's clefts. Thus, we have delineated some important aspects of the unusual trafficking mechanism for delivery of this critical parasite virulence factor to the erythrocyte surface.     

The Maurer's cleft protein MAHRP1 is essential for trafficking of PfEMP1 to the surface of Plasmodium falciparum-infected erythrocytes

During the intra-erythrocytic development of Plasmodium falciparum, the parasite modifies the host cell surface by exporting proteins that interact with or insert into the erythrocyte membrane. These proteins include the principal mediator of cytoadherence, P. falciparum erythrocyte membrane protein 1 (PfEMP1). To implement these changes, the parasite establishes a protein-trafficking system beyond its confines. Membrane-bound structures called Maurer's clefts are intermediate trafficking compartments for proteins destined for the host cell membrane. We disrupted the gene for the membrane-associated histidine-rich protein 1 (MAHRP1). MAHRP1 is not essential for parasite viability or Maurer's cleft formation; however, in its absence, these organelles become disorganized in permeabilized cells. Maurer's cleft-resident proteins and transit cargo are exported normally in the absence of MAHRP1; however, the virulence determinant, PfEMP1, accumulates within the parasite, is depleted from the Maurer's clefts and is not presented at the red blood cell surface. Complementation of the mutant parasites with mahrp1 led to the reappearance of PfEMP1 on the infected red blood cell surface, and binding studies show that PfEMP1-mediated binding to CD36 is restored. These data suggest an important role of MAHRP1 in the translocation of PfEMP1 from the parasite to the host cell membrane.     

Mapping a common interaction site used by Plasmodium falciparum Duffy binding-like domains to bind diverse host receptors

The Duffy binding-like (DBL) domain is a key adhesive module in Plasmodium falciparum, present in both erythrocyte invasion ligands (EBLs) and the large and diverse P. falciparum erythrocyte membrane protein 1 (PfEMP1) family of cytoadherence receptors. DBL domains bind a variety of different host receptors, including intercellular adhesion molecule 1 (ICAM-1), a receptor interaction that may have a role in infected erythrocyte binding to cerebral blood vessels and cerebral malaria. In this study, we expressed the nearly full complement of DBLbeta-C2 domains from the IT4/25/5 (IT4) parasite isolate and showed that ICAM-1-binding domains (DBLbeta-C2(ICAM-1)) were confined to group B and group C PfEMP1 proteins and were not present in group A, suggesting that ICAM-1 selection pressure differs between PfEMP1 groups. To further dissect the molecular determinants of binding, we modelled a DBLbeta-C2(ICAM-1) domain on a solved DBL structure and created alanine substitution mutants in two DBLbeta-C2(ICAM-1) domains. This analysis indicates that the DBLbeta-C2::ICAM-1 interaction maps to the equivalent glycan binding region of EBLs, and suggests a general model for how DBL domains evolve under dual selection for host receptor binding and immune evasion.     

Mass spectrometric analysis of Plasmodium falciparum erythrocyte membrane protein-1 variants expressed by placental malaria parasites

Surface proteins from Plasmodium falciparum are important malaria vaccine targets. However, the surface proteins previously identified are highly variant and difficult to study. We used tandem mass spectrometry to characterize the variant antigens (Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1)) expressed on the surface of malaria-infected erythrocytes that bind to chondroitin sulfate A (CSA) in the placenta. Whereas PfEMP1 variants previously implicated as CSA ligands were detected, in unselected parasites four novel variants were detected in CSA-binding or placental parasites but not in unselected parasites. These novel PfEMP1 variants require further study to confirm whether they play a role in placental malaria.     

Delivery of the malaria virulence protein PfEMP1 to the erythrocyte surface requires cholesterol-rich domains

The particular virulence of the human malaria parasite Plasmodium falciparum derives from export of parasite-encoded proteins to the surface of the mature erythrocytes in which it resides. The mechanisms and machinery for the export of proteins to the erythrocyte membrane are largely unknown. In other eukaryotic cells, cholesterol-rich membrane microdomains or "rafts" have been shown to play an important role in the export of proteins to the cell surface. Our data suggest that depletion of cholesterol from the erythrocyte membrane with methyl-beta-cyclodextrin significantly inhibits the delivery of the major virulence factor P. falciparum erythrocyte membrane protein 1 (PfEMP1). The trafficking defect appears to lie at the level of transfer of PfEMP1 from parasite-derived membranous structures within the infected erythrocyte cytoplasm, known as the Maurer's clefts, to the erythrocyte membrane. Thus our data suggest that delivery of this key cytoadherence-mediating protein to the host erythrocyte membrane involves insertion of PfEMP1 at cholesterol-rich microdomains. GTP-dependent vesicle budding and fusion events are also involved in many trafficking processes. To determine whether GTP-dependent events are involved in PfEMP1 trafficking, we have incorporated non-membrane-permeating GTP analogs inside resealed erythrocytes. Although these nonhydrolyzable GTP analogs reduced erythrocyte invasion efficiency and partially retarded growth of the intracellular parasite, they appeared to have little direct effect on PfEMP1 trafficking.     

Malaria's deadly grip: cytoadhesion of Plasmodium falciparum‐infected erythrocytes

Cytoadhesion of Plasmodium falciparum‐infected erythrocytes to host microvasculature is a key virulence determinant. Parasite binding is mediated by a large family of clonally variant adhesion proteins, termed P. falciparum erythrocyte membrane protein 1 (PfEMP1), encoded by var genes and expressed at the infected erythrocyte surface. Although PfEMP1 proteins have extensively diverged under opposing selection pressure to maintain ligand binding while avoiding antibody‐mediated detection, recent work has revealed they can be classified into different groups based on chromosome location and domain composition. This grouping reflects functional specialization of PfEMP1 proteins for different human host and microvascular binding niches and appears to be maintained by gene recombination hierarchies. Inone extreme, a specific PfEMP1 variant is associated with placental binding and malaria during pregnancy, while other PfEMP1 subtypes appear to be specialized for infection of malaria naïve hosts. Here, we discuss recent findings on the origins and evolution of the var gene family, the structure–function of PfEMP1 proteins, and a distinct subset of PfEMP1 variants that have been associated with severe childhood malaria.     

A family affair: var genes, PfEMP1 binding, and malaria disease

An immunovariant adhesion protein family in Plasmodium falciparum named erythrocyte membrane protein 1 (PfEMP1), encoded by var genes, is responsible for both antigenic variation and cytoadhesion of infected erythrocytes at blood microvasculature sites throughout the body. Elucidation of the genome sequence of P. falciparum has revealed that var genes can be classified into different groups, each with distinct 5' flanking sequences, chromosomal locations and gene orientations. Recent binding and serological comparisons suggest that this genomic organization might cause var genes to diversify into separately recombining adhesion groups that have different roles in infection and disease. Detailed understanding of PfEMP1 expression and receptor binding mechanisms during infection and of the antigenic relatedness of disease variants might lead to new approaches in prevention of malaria disease.     

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.     

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: 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.     

Targeted mutagenesis of Plasmodium falciparum erythrocyte membrane protein 3 (PfEMP3) disrupts cytoadherence of malaria-infected red blood cells

Adhesion of parasite-infected red blood cells to the vascular endothelium is a critical event in the pathogenesis of malaria caused by Plasmodium falciparum. Adherence is mediated by the variant erythrocyte membrane protein 1 (PfEMP1). Another protein, erythrocyte membrane protein-3 (PfEMP3), is deposited under the membrane of the parasite-infected erythrocyte but its function is unknown. Here we show that mutation of PfEMP3 disrupts transfer of PfEMP1 to the outside of the P.FALCIPARUM:-infected cell. Truncation of the C-terminal end of PfEMP3 by transfection prevents distribution of this large (>300 kDa) protein around the membrane but does not disrupt trafficking of the protein from the parasite to the cytoplasmic face of the erythrocyte membrane. The truncated PfEMP3 accumulates in structures that appear to be associated with the erythrocyte membrane. We show that accumulation of mutated PfEMP3 blocks the transfer of PfEMP1 onto the outside of the parasitized cell surface and suggest that these proteins traffic through an erythrocyte membrane-associated compartment that is involved in the transfer of PfEMP1 to the surface of the parasite-infected red blood cell.     

A Maurer's cleft-associated protein is essential for expression of the major malaria virulence antigen on the surface of infected red blood cells

The high mortality of Plasmodium falciparum malaria is the result of a parasite ligand, PfEMP1 (P. falciparum) erythrocyte membrane protein 1), on the surface of infected red blood cells (IRBCs), which adheres to the vascular endothelium and causes the sequestration of IRBCs in the microvasculature. PfEMP1 transport to the IRBC surface involves Maurer's clefts, which are parasite-derived membranous structures in the IRBC cytoplasm. Targeted gene disruption of a Maurer's cleft protein, SBP1 (skeleton-binding protein 1), prevented IRBC adhesion because of the loss of PfEMP1 expression on the IRBC surface. PfEMP1 was still present in Maurer's clefts, and the transport and localization of several other Maurer's cleft proteins were unchanged. Maurer's clefts were altered in appearance and were no longer found as close to the periphery of the IRBC. Complementation of mutant parasites with sbp1 led to the reappearance of PfEMP1 on the IRBC surface and the restoration of adhesion. Our results demonstrate that SBP1 is essential for the translocation of PfEMP1 onto the surface of IRBCs and is likely to play a pivotal role in the pathogenesis of P. falciparum malaria.     

Molecular mechanisms of Plasmodium falciparum placental adhesion

In natural Plasmodium falciparum infections, parasitized erythrocytes (PEs) circulate in the peripheral blood for a period corresponding roughly to the first part of the erythrocytic life cycle (ring stage). Later, in blood-stage development, parasite-encoded adhesion molecules are inserted into the erythrocyte membrane, preventing the circulation of the PEs. The principal molecule mediating PE adhesion is P. falciparum erythrocyte membrane protein 1 (PfEMP1), encoded by the polymorphic var gene family. The population of parasites is subject to clonal antigenic variation through changes in var expression, and a single PfEMP1 variant is expressed at the PE surface in a mutually exclusive manner. In addition to its role in immune evasion, switches in PfEMP1 expression may be associated with fundamental changes in parasite tissue tropism in malaria patients. A switch from CD36 binding to chondroitin sulphate A (CSA) binding may lead to extensive sequestration of PEs in placenta syncytiotrophoblasts. This is probably a key event in malaria pathogenesis during pregnancy. The CSA-binding phenotype of mature PEs is linked to another distinct adhesive phenotype: the recently described CSA-independent cytoadhesion of ring-stage PEs. Thus, a subpopulation of PEs that sequentially displays these two different phenotypes may bind to an individual endothelial cell or syncytiotrophoblast throughout the asexual blood-stage cycle. This suggests that non-circulating (cryptic) parasite subpopulations are present in malaria patients.     

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.