Structural and functional studies of interaction between Plasmodium falciparum knob-associated histidine-rich protein (KAHRP) and erythrocyte spectrin

Plasmodium falciparum dramatically modifies the structure and function of the membrane of the parasitized host erythrocyte. Altered membrane properties are the consequence of the interaction of a group of exported malaria proteins with host cell membrane proteins. KAHRP (the knob-associated histidine-rich protein), a member of this group, has been shown to interact with erythrocyte membrane skeletal protein spectrin. However, the molecular basis for this interaction has yet to be defined. In the present study, we defined the binding motifs in both KAHRP and spectrin and identified a functional role for this interaction. We showed that spectrin bound to a 72-amino-acid KAHRP fragment (residues 370-441). Among nine-spectrin fragments, which encompass the entire alpha and beta spectrin molecules (four alpha spectrin and five beta spectrin fragments), KAHRP bound only to one, the alpha N-5 fragment. The KAHRP-binding site within the alpha N-5 fragment was localized uniquely to repeat 4. The interaction of full-length spectrin dimer to KAHRP was inhibited by repeat 4 of alpha spectrin. Importantly, resealing of this repeat peptide into erythrocytes mislocalized KAHRP in the parasitized cells. We concluded that the interaction of KAHRP with spectrin is critical for appropriate membrane localization of KAHRP in parasitized erythrocytes. As the presence of KAHRP at the erythrocyte membrane is necessary for cytoadherence in vivo, our findings have implications for the development of new therapies for mitigating the severity of malaria infection.     

Plasmodium falciparum erythrocyte membrane protein 3 (PfEMP3) destabilizes erythrocyte membrane skeleton

Plasmodium falciparum erythrocyte membrane protein 3 (PfEMP3) is a parasite-derived protein that appears on the cytoplasmic surface of the host cell membrane in the later stages of the parasite's development where it associates with membrane skeleton. We have recently demonstrated that a 60-residue fragment (FIa1, residues 38-97) of PfEMP3 bound to spectrin. Here we show that this polypeptide binds specifically to a site near the C terminus of alpha-spectrin at the point that spectrin attaches to actin and protein 4.1R in forming the junctions of the membrane skeletal network. We further show that this polypeptide disrupts formation of the ternary spectrin-actin-4.1R complex in solution. Importantly, when incorporated into the cell, the PfEMP3 fragment causes extensive reduction in shear resistance of the cell. We conjecture that the loss of mechanical cohesion of the membrane may facilitate the exit of the mature merozoites from the cell.     

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.     

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.     

Plasmodium falciparum Plasmodium helical interspersed subtelomeric proteins contribute to cytoadherence and anchor P. falciparum erythrocyte membrane protein 1 to the host cell cytoskeleton

Adherence of Plasmodium falciparum‐infected erythrocytes to host endothelium is conferred through the parasite‐derived virulence factor P. falciparum erythrocyte membrane protein 1 (PfEMP1), the major contributor to malaria severity. PfEMP1 located at knob structures on the erythrocyte surface is anchored to the cytoskeleton, and the Plasmodium helical interspersed subtelomeric (PHIST) gene family plays a role in many host cell modifications including binding the intracellular domain of PfEMP1. Here, we show that conditional reduction of the PHIST protein PFE1605w strongly reduces adhesion of infected erythrocytes to the endothelial receptor CD36. Adhesion to other endothelial receptors was less affected or even unaltered by PFE1605w depletion, suggesting that PHIST proteins might be optimized for subsets of PfEMP1 variants. PFE1605w does not play a role in PfEMP1 transport, but it directly interacts with both the intracellular segment of PfEMP1 and with cytoskeletal components. This is the first report of a PHIST protein interacting with key molecules of the cytoadherence complex and the host cytoskeleton, and this functional role seems to play an essential role in the pathology of P. falciparum.     

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.     

The complete sequence of the gene for the knob-associated histidine-rich protein from Plasmodium falciparum.

'Knobs' at the surface of erythrocytes infected with mature stages of Plasmodium falciparum are believed to be important in adherence of these cells to capillary walls. They contain at least one parasite protein, designated the knob-associated histidine-rich protein (KAHRP). We present here the sequences of a cDNA and chromosomal clone that predict the complete sequence of KAHRP. The gene contains a single intervening sequence, located at the 3' boundary of the hydrophobic core of a putative signal sequence. Exon two encodes a short region that is rich in histidine as well as two separate regions of repetitive sequence, the 5' repeats (five copies related to SKKHKDNEDAESVK) and the 3' repeats (seven copies related to SKGATKEAST). These repeat blocks were both shown to bear epitopes recognized by the human immune system during natural infection by expressing them separately in Escherichia coli, and reacting human antibodies affinity-purified on lysates of the resulting clones with the corresponding synthetic oligopeptides. The 3' end of the molecule, presumably the repetitive region, is a site of size variation in KAHRP from different isolates.     

Optimized expression of Plasmodium falciparum erythrocyte membrane protein 1 domains in Escherichia coli

BACKGROUND: Removal of exhaled air from total body emanations or artificially standardising carbon dioxide (CO2) outputs has previously been shown to eliminate differential attractiveness of humans to certain blackfly (Simuliidae) and mosquito (Culicidae) species. Whether or not breath contributes to between-person differences in relative attractiveness to the highly anthropophilic malaria vector Anopheles gambiae sensu stricto remains unknown and was the focus of the present study. METHODS: The contribution to and possible interaction of breath (BR) and body odours (BO) in the attraction of An. gambiae s.s. to humans was investigated by conducting dual choice tests using a recently developed olfactometer. Either one or two human subjects were used as bait. The single person experiments compared the attractiveness of a person's BR versus that person's BO or a control (empty tent with no odour). His BO and total emanations (TE = BR+BO) were also compared with a control. The two-person experiments compared the relative attractiveness of their TE, BO or BR, and the TE of each person against the BO of the other. RESULTS: Experiments with one human subject (P1) as bait found that his BO and TE collected more mosquitoes than the control (P = 0.005 and P < 0.001, respectively), as did his BO and the control versus his BR (P < 0.001 and P = 0.034, respectively). The TE of P1 attracted more mosquitoes than that of another person designated P8 (P < 0.021), whereas the BR of P8 attracted more mosquitoes than the BR of P1 (P = 0.001). The attractiveness of the BO of P1 versus the BO of P8 did not differ (P = 0.346). The BO from either individual was consistently more attractive than the TE from the other (P < 0.001). CONCLUSIONS: We demonstrated for the first time that human breath, although known to contain semiochemicals that elicit behavioural and/or electrophysiological responses (CO2, ammonia, fatty acids) in An. gambiae also contains one or more constituents with allomonal (~repellent) properties, which inhibit attraction and may serve as an important contributor to between-person differences in the relative attractiveness of humans to this important malaria vector.     

Specific erythrocyte binding capacity and biological activity of Plasmodium falciparum erythrocyte binding ligand 1 (EBL-1)-derived peptides

Erythrocyte binding ligand 1 (EBL-1) is a member of the ebl multigene family involved in Plasmodium falciparum invasion of erythrocytes. We found that five EBL-1 high-activity binding peptides (HABPs) bound specifically to erythrocytes: 29895 ((41)HKKKSGELNNNKSGILRSTY(60)), 29903 ((201)LYECGK-KIKEMKWICTDNQF(220)), 29923 ((601)CNAILGSYADIGDIVRGLDV(620)), 29924((621)WRDINTNKLSEK-FQKIFMGGY(640)), and 30018 ((2481)LEDIINLSKKKKKSINDTSFY(2500)). We also show that binding was saturable, not sialic acid-dependent, and that all peptides specifically bound to a 36-kDa protein on the erythrocyte membrane. The five HABPs inhibited in vitro merozoite invasion depending on the peptide concentration used, suggesting their possible role in the invasion process.     

Viruslike particles containing knob-associated histidine-rich protein are secreted into the culture medium of Plasmodium falciparum in vitro cultures

This report describes the isolation of a viruslike particle from in vitro cultures of the human malaria parasite P. falciparum. Electronmicroscopic observations suggest that the particles are liberated into the culture medium by budding from the erythrocyte membrane. The density of the free particles is 1.16, they contain nucleic acid and two distinct molecular species of the knob-associated Histidine-rich protein. Proteins of the particles are recognized by sera from malaria patients. The previously described knobs may correspond to viral coats inserted in the membrane.     

Plasmodium falciparum histidine-rich protein 1 associates with the band 3 binding domain of ankyrin in the infected red cell membrane

Infection of erythrocytes by the malaria parasite Plasmodium falciparum results in the export of several parasite proteins into the erythrocyte cytoplasm. Changes occur in the infected erythrocyte due to altered phosphorylation of proteins and to novel interactions between host and parasite proteins, particularly at the membrane skeleton. In erythrocytes, the spectrin based red cell membrane skeleton is linked to the erythrocyte plasma membrane through interactions of ankyrin with spectrin and band 3. Here we report an association between the P. falciparum histidine-rich protein (PfHRP1) and phosphorylated proteolytic fragments of red cell ankyrin. Immunochemical, biochemical and biophysical studies indicate that the 89 kDa band 3 binding domain and the 62 kDa spectrin-binding domain of ankyrin are co-precipitated by mAb 89 against PfHRP1, and that native and recombinant ankyrin fragments bind to the 5' repeat region of PfHRP1. PfHRP1 is responsible for anchoring the parasite cytoadherence ligand to the erythrocyte membrane skeleton, and this additional interaction with ankyrin would strengthen the ability of PfEMP1 to resist shear stress.     

Mapping the membrane topology and extracellular ligand binding domains of the retinol binding protein receptor

STRA6 is a multitransmembrane domain protein not homologous to any other proteins with known function. It functions as the high-affinity receptor for plasma retinol binding protein (RBP) and mediates cellular uptake of vitamin A from the vitamin A-RBP complex. Consistent with the diverse roles of vitamin A and the wide tissue expression pattern of STRA6, mutations in STRA6 are associated with severe pathological phenotypes in humans. The structural basis for STRA6's biochemical function is unknown. Although computer programs predict 11 transmembrane domains for STRA6, its topology has never been studied experimentally. Elucidating the transmembrane topology of STRA6 is critical for understanding its structure and function. By inserting an epitope tag into all possible extracellular and intracellular domains of STRA6, we systematically analyzed the accessibility of each tag on the surface of live cells, the accessibility of each tag in permeabilized cells, and the effect of each tag on RBP binding and STRA6-mediated vitamin A uptake from the vitamin A-RBP complex. In addition, we used a new lysine accessibility technique combining cell-surface biotinylation and tandem-affinity purification to study a region of the protein not revealed by the epitope tagging method. These studies not only revealed STRA6's extracellular, transmembrane, and intracellular domains but also implicated extracellular regions of STRA6 in RBP binding.     

Investigating the function of Fc‐specific binding of IgM to Plasmodium falciparum erythrocyte membrane protein 1 mediating erythrocyte rosetting

Acquired protection from Plasmodium falciparum malaria takes years to develop, probably reflecting the ability of the parasites to evade immunity. A recent example of this is the binding of the F_c region of IgM to VAR2CSA‐type PfEMP1. This interferes with specific IgG recognition and phagocytosis of opsonized infected erythrocytes (IEs) without compromising the placental IE adhesion mediated by this PfEMP1 type. IgM also binds via F_c to several other PfEMP1 proteins, where it has been proposed to facilitate rosetting (binding of uninfected erythrocytes to a central IE). To further dissect the functional role of F_c‐mediated IgM binding to PfEMP1, we studied the PfEMP1 protein HB3VAR06, which mediates rosetting and binds IgM. Binding of IgM to this PfEMP1 involved the F_c domains Cμ3‐Cμ4 in IgM and the penultimate DBL domain (DBLζ2) at the C‐terminus of HB3VAR06. However, IgM binding did not inhibit specific IgG labelling of HB3VAR06 or shield IgG‐opsonized IEs from phagocytosis. Instead, IgM was required for rosetting, and each pentameric IgM molecule could bind two HB3VAR06 molecules. Together, our data indicate that the primary function of F_c‐mediated IgM binding in rosetting is not to shield IE from specific IgG recognition and phagocytosis as in VAR2CSA‐type PfEMP1. Rather, the function appears to be strengthening of IE–erythrocyte interactions. In conclusion, our study provides new evidence on the molecular details and functional significance of rosetting, a long‐recognized marker of parasites that cause severe P. falciparum malaria.     

Comparison of cellular and humoral responses to recombinant protein and synthetic peptides of exon2 region of Plasmodium falciparum erythrocyte membrane protein1 (PfEMP1) among malaria patients from an endemic region

Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) expressed on the surface of parasitized red blood cells (PRBCs) mediate adhesion of PRBCs to host vascular endothelial receptors and is considered responsible for pathogenesis of severe P. falciparum malaria. The present study was undertaken to measure cellular immune responses and serum antibody responses against recombinant exon2 protein, the most conserved region of PfEMP1, and its synthetic peptides. T cell recognizing this domain could provide universal help to B cells in recognizing variant epitopes located in the extracellular region of PfEMP1. Human peripheral blood mononuclear cells from malaria-exposed immune adults (IA), malaria patients with varying severity, and malaria unexposed healthy donors were stimulated with recombinant exon2 protein and six synthetic peptides from its sequence to estimate the proliferative, IFN-gamma, and IL-4 responses. Antibody responses against these synthetic peptides and exon2 protein were also studied. Positive proliferative, IFN-gamma, and IL-4 responses in IA group each were 60% with recombinant exon2 protein and 27-47% with different synthetic peptides. Antibody recognition was observed in 67% with exon2 and between 40 and 53% with different peptides. In malaria patients, frequency and magnitude of proliferative response, IL-4 concentration, and antibody recognition were far less than immune adults but IFN-gamma response was almost similar. Proportion of positive responders and the magnitude of response to synthetic peptides were low. Also, there was no consistency in response of different peptides towards proliferative, cytokine, and antibody responses in IA and malaria patient groups except for peptide 1. We presume peptide 1 is a potential vaccine candidate and different cocktails containing peptide 1 are being evaluated for their T cell immunogenicity.     

Allelic diversity of the Plasmodium falciparum erythrocyte membrane protein 1 entails variant-specific red cell surface epitopes

The clonally variant Plasmodium falciparum PfEMP1 adhesin is a virulence factor and a prime target of humoral immunity. It is encoded by a repertoire of functionally differentiated var genes, which display architectural diversity and allelic polymorphism. Their serological relationship is key to understanding the evolutionary constraints on this gene family and rational vaccine design. Here, we investigated the Palo Alto/VarO and IT4/R29 and 3D7/PF13_003 parasites lines. VarO and R29 form rosettes with uninfected erythrocytes, a phenotype associated with severe malaria. They express an allelic Cys2/group A NTS-DBL1α(1) PfEMP1 domain implicated in rosetting, whose 3D7 ortholog is encoded by PF13_0003. Using these three recombinant NTS-DBL1α(1) domains, we elicited antibodies in mice that were used to develop monovariant cultures by panning selection. The 3D7/PF13_0003 parasites formed rosettes, revealing a correlation between sequence identity and virulence phenotype. The antibodies cross-reacted with the allelic domains in ELISA but only minimally with the Cys4/group B/C PFL1955w NTS-DBL1α. By contrast, they were variant-specific in surface seroreactivity of the monovariant-infected red cells by FACS analysis and in rosette-disruption assays. Thus, while ELISA can differentiate serogroups, surface reactivity assays define the more restrictive serotypes. Irrespective of cumulated exposure to infection, antibodies acquired by humans living in a malaria-endemic area also displayed a variant-specific surface reactivity. Although seroprevalence exceeded 90% for each rosetting line, the kinetics of acquisition of surface-reactive antibodies differed in the younger age groups. These data indicate that humans acquire an antibody repertoire to non-overlapping serotypes within a serogroup, consistent with an antibody-driven diversification pressure at the population level. In addition, the data provide important information for vaccine design, as production of a vaccine targeting rosetting PfEMP1 adhesins will require engineering to induce variant-transcending responses or combining multiple serotypes to elicit a broad spectrum of immunity.     

Structural basis for binding of Plasmodium falciparum erythrocyte membrane protein 1 to chondroitin sulfate and placental tissue and the influence of protein polymorphisms on binding specificity

Chondroitin sulfate (CS) A is a key receptor for adhesion of Plasmodium falciparum-infected erythrocytes (IEs) in the placenta and can also mediate adhesion to microvascular endothelial cells. IEs that adhere to CSA express var2csa-type genes, which encode specific variants of the IE surface antigen P. falciparum erythrocyte membrane protein 1 (PfEMP1). We report direct binding of native PfEMP1, isolated from IEs and encoded by var2csa, to immobilized CSA. Binding of PfEMP1 was dependent on 4-O-sulfated disaccharides and glucuronic acid rather than iduronic acid, consistent with the specificity of intact IEs. Using immobilized CS oligosaccharides as neoglycolipid probes, the minimum chain length for direct binding of PfEMP1 was eight monosaccharide units. Similarly for IE adhesion to placental tissue there was a requirement for 4-O-sulfated GalNAc and glucuronic acid mixed with non-sulfated disaccharides; 6-O-sulfation interfered with the interaction between placental CSA and IEs. The minimum chain length for maximal inhibition of adhesion was 10 monosaccharide residues. Partially 4-O-sulfated CS oligosaccharides (45-55% sulfation) were highly effective inhibitors of placental adhesion (IC(50), 0.15 microg/ml) and may have potential for therapeutic development. We used defined P. falciparum isolates expressing different variants of var2csa in adhesion assays and found that there were isolate-specific differences in the preferred structural motifs for adhesion to CSA that correlated with polymorphisms in PfEMP1 encoded by var2csa-type genes. This may influence sites of IE sequestration or parasite virulence. These findings have significant implications for understanding the pathogenesis and biology of malaria, particularly during pregnancy, and the development of targeted interventions.