Identification of a role for the PfEMP1 semi‐conserved head structure in protein trafficking to the surface of Plasmodium falciparum infected red blood cells

Transport of Plasmodium falciparum Erythrocyte Membrane Protein 1 (PfEMP1) variants to the red blood cell (RBC) surface enables malarial parasite evasion of host immunity by modifying the antigenic and adhesive properties of infected RBCs. In this study, we applied the Bxb1 integrase system to integrate transgenes encoding truncated PfEMP1‐GFP fusions into cytoadherent A4 parasites and characterize their surface transport requirements. Our studies revealed that the semi‐conserved head structure of PfEMP1 proteins, in combination with the predicted transmembrane region and cytoplasmic tail, encodes sufficient information for RBC surface display. In contrast, miniPfEMP1 proteins with truncated head structures were exported to the RBC cytoplasm but were not detected at the RBC surface by flow cytometry or immuno‐electron microscopy. We demonstrated the absence of a mechanistic barrier to having native and miniPfEMP1 proteins displayed simultaneously at the RBC surface. However, surface‐exposed miniPfEMP1 proteins did not convey cytoadherence properties to their host cells, implicating potential steric considerations in host‐receptor interactions or the need for multiple domains to mediate cell binding. This study establishes a new system to investigate PfEMP1 transport and demonstrates that the PfEMP1 semi‐conserved head structure is under selection for protein transport, in addition to its known roles in 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.     

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.     

Clonal Variants of Plasmodium falciparum Exhibit a Narrow Range of Rolling Velocities to Host Receptor CD36 under Dynamic Flow Conditions

Cytoadhesion of Plasmodium falciparum parasitized red blood cells (pRBCs) has been implicated in the virulence of malaria infection. Cytoadhesive interactions are mediated by the protein family of Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1). The PfEMP1 family is under strong antibody and binding selection, resulting in extensive sequence and size variation of the extracellular domains. Here, we investigated cytoadhesion of pRBCs to CD36, a common receptor of P. falciparum field isolates, under dynamic flow conditions. Isogeneic parasites, predominantly expressing single PfEMP1 variants, were evaluated for binding to recombinant CD36 under dynamic flow conditions using microfluidic devices. We tested if PfEMP1 size (number of extracellular domains) or sequence variation affected the pRBC-CD36 interaction. Our analysis showed that clonal parasite variants varied ∼5-fold in CD36 rolling velocity despite extensive PfEMP1 sequence polymorphism. In addition, adherent pRBCs exhibited a characteristic hysteresis in rolling velocity at microvascular flow rates, which was accompanied by changes in pRBC shape and may represent important adaptations that favor stable binding.     

Structural analysis of the Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) intracellular domain reveals a conserved interaction epitope

Plasmodium falciparum-infected red blood cells adhere to endothelial cells, thereby obstructing the microvasculature. Erythrocyte adherence is directly associated with severe malaria and increased disease lethality, and it is mediated by the PfEMP1 family. PfEMP1 clustering in knob-like protrusions on the erythrocyte membrane is critical for cytoadherence, however the molecular mechanisms behind this system remain elusive. Here, we show that the intracellular domains of the PfEMP1 family (ATS) share a unique molecular architecture, which comprises a minimal folded core and extensive flexible elements. A conserved flexible segment at the ATS center is minimally restrained by the folded core. Yeast-two-hybrid data and a novel sequence analysis method suggest that this central segment contains a conserved protein interaction epitope. Interestingly, ATS in solution fails to bind the parasite knob-associated histidine-rich protein (KAHRP), an essential cytoadherence component. Instead, we demonstrate that ATS associates with PFI1780w, a member of the Plasmodium helical interspersed sub-telomeric (PHIST) family. PHIST domains are widespread in exported parasite proteins, however this is the first specific molecular function assigned to any variant of this family. We propose that PHIST domains facilitate protein interactions, and that the conserved ATS epitope may be targeted to disrupt the parasite cytoadherence system.     

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.     

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.     

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.     

Plasmodium falciparum antigenic variation. Mapping mosaic var gene sequences onto a network of shared, highly polymorphic sequence blocks

Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) is a potentially important family of immune targets, encoded by an extremely diverse gene family called var . Understanding of the genetic organization of var genes is hampered by sequence mosaicism that results from a long history of non-homologous recombination. Here we have used software designed to analyse social networks to visualize the relationships between large collections of short var sequences tags sampled from clinical parasite isolates. In this approach, two sequences are connected if they share one or more highly polymorphic sequence blocks. The results show that the majority of analysed sequences including several var -like sequences from the chimpanzee parasite Plasmodium reichenowi can be either directly or indirectly linked together in a single unbroken network. However, the network is highly structured and contains putative subgroups of recombining sequences. The major subgroup contains the previously described group A var genes, previously proposed to be genetically distinct. Another subgroup contains sequences found to be associated with rosetting, a parasite virulence phenotype. The mosaic structure of the sequences and their division into subgroups may reflect the conflicting problems of maximizing antigenic diversity and minimizing epitope sharing between variants while maintaining their host cell binding functions.     

Interactions of Plasmodium falciparum erythrocyte membrane protein 3 with the red blood cell membrane skeleton

Plasmodium falciparum parasites express and traffick numerous proteins into the red blood cell (RBC), where some associate specifically with the membrane skeleton. Importantly, these interactions underlie the major alterations to the modified structural and functional properties of the parasite-infected RBC. P. falciparum Erythrocyte Membrane Protein 3 (PfEMP3) is one such parasite protein that is found in association with the membrane skeleton. Using recombinant PfEMP3 proteins in vitro, we have identified the region of PfEMP3 that binds to the RBC membrane skeleton, specifically to spectrin and actin. Kinetic studies revealed that residues 38-97 of PfEMP3 bound to purified spectrin with moderately high affinity (K(D(kin))=8.5 x 10(-8) M). Subsequent deletion mapping analysis further defined the binding domain to a 14-residue sequence (IFEIRLKRSLAQVL; K(D(kin))=3.8 x 10(-7) M). Interestingly, this same domain also bound to F-actin in a specific and saturable manner. These interactions are of physiological relevance as evidenced by the binding of this region to the membrane skeleton of inside-out RBCs and when introduced into resealed RBCs. Identification of a 14-residue region of PfEMP3 that binds to both spectrin and actin provides insight into the potential function of PfEMP3 in P. falciparum-infected RBCs.     

Plasmodium falciparum Expressing Domain Cassette 5 Type PfEMP1 (DC5-PfEMP1) Bind PECAM1

Members of the Plasmodium falciparum Erythrocyte Membrane protein 1 (PfEMP1) family expressed on the surface of malaria-infected erythrocytes mediate binding of the parasite to different receptors on the vascular lining. This process drives pathologies, and severe childhood malaria has been associated with the expression of particular subsets of PfEMP1 molecules. PfEMP1 are grouped into subtypes based on upstream sequences and the presence of semi-conserved PfEMP1 domain compositions named domain cassettes (DCs). Earlier studies have indicated that DC5-containing PfEMP1 (DC5-PfEMP1) are more likely to be expressed in children with severe malaria disease than in children with uncomplicated malaria, but these PfEMP1 subtypes only dominate in a relatively small proportion of the children with severe disease. In this study, we have characterised the genomic sequence characteristic for DC5, and show that two genetically different parasite lines expressing DC5-PfEMP1 bind PECAM1, and that anti-DC5-specific antibodies inhibit binding of DC5-PfEMP1-expressing parasites to transformed human bone marrow endothelial cells (TrHBMEC). We also show that antibodies against each of the four domains characteristic for DC5 react with native PfEMP1 expressed on the surface of infected erythrocytes, and that some of these antibodies are cross-reactive between the two DC5-containing PfEMP1 molecules tested. Finally, we confirm that anti-DC5 antibodies are acquired early in life by individuals living in malaria endemic areas, that individuals having high levels of these antibodies are less likely to develop febrile malaria episodes and that the antibody levels correlate positively with hemoglobin levels.     

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.     

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.     

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.     

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.     

The malaria parasite Plasmodium falciparum encodes members of the Puf RNA-binding protein family with conserved RNA binding activity

A novel class of RNA-binding proteins, Puf, regulates translation and RNA stability by binding to specific sequences in the 3'-untranslated region of target mRNAs. Members of this protein family share a conserved Puf domain consisting of eight 36 amino acid imperfect repeats. Here we report two Puf family member genes, PfPuf1 and PfPuf2, from the human malaria parasite Plasmodium falciparum. Both genes are spliced with four and three introns clustered within or near the Puf domains, respectively. Northern and RT-PCR analysis indicated that both genes were differentially expressed in gametocytes during erythrocytic development of the parasite. Except for similarities in the Puf domain and expression profile, the deduced PfPuf1 and PfPuf2 proteins differ considerably in size and structure. PfPuf1 has 1894 amino acids and a central Puf domain, whereas PfPuf2 is much smaller with a C-terminal Puf domain. The presence of at least two Puf members in other Plasmodium species suggests that these proteins play evolutionarily similar roles during parasite development. Both in vivo studies using the yeast three-hybrid system and in vitro binding assays using the recombinant Puf domain of PfPuf1 expressed in bacteria demonstrated intrinsic binding activity of the PfPuf1 Puf domain to the NRE sequences in the hunchback RNA, the target sequence for Drosophila Pumilio protein. Altogether, these results suggest that PfPufs might function during sexual differentiation and development in Plasmodium through a conserved mechanism of translational regulation of their target mRNAs.     

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.