Cooperativity between Plasmodium falciparum adhesive proteins for invasion into erythrocytes

Plasmodium falciparum is the most virulent of the Plasmodium species infective to humans. Different P. falciparum strains vary in their dependence on erythrocyte receptors for invasion and their ability to switch in their utilization of different receptor repertoires. Members of the reticulocyte-binding protein-like (RBL) family of invasion ligands are postulated to play a central role in defining ligand–receptor interactions, known as invasion pathways. Here we report the targeted gene disruption of PfRh2b and PfRh2a in W2mef, a parasite strain that is heavily dependent on sialic-acid receptors for invasion, and show that the PfRh2b ligand is functional in this parasite background. Like the parental line, parasites lacking either PfRh2a or PfR2b can switch to a sialic acid-independent invasion pathway. However, both of the switched lines exhibit a reduced efficiency for invasion into sialic acid-depleted cells, suggesting a role for both PfRh2b and PfRh2a in invasion via sialic acid-independent receptors. We also find a strong selective pressure for the reconstitution of PfRh2b expression at the expense of PfRh2a. Our results reveal the importance of genetic background in ligand–receptor usage by P. falciparum parasites, and suggest that the co-ordinate expression of PfRh2a, PfRh2b together mediate efficient sialic acid-independent erythrocyte invasion.     

Plasmodium falciparum TryThrA antigen synthetic peptides block in vitro merozoite invasion to erythrocytes

Tryptophan-threonine-rich antigen (TryThrA) is a Plasmodium falciparum homologue of Plasmodium yoelii-infected erythrocyte membrane pypAg-1 antigen. pypAg-1 binds to the surface of uninfected mouse erythrocytes and has been used successfully in vaccine studies. The two antigens are characterized by an unusual tryptophan-rich domain, suggesting similar biological properties. Using synthetic peptides spanning the TryThrA sequence and human erythrocyte we have done binding assays to identify possible TryThrA functional regions. We describe four peptides outside the tryptophan-rich domain having high activity binding to normal human erythrocytes. The peptides termed HABPs (high activity binding peptides) are 30884 ((61)LKEKKKKVLEFFENLVLNKKY(80)) located at the N-terminal and 30901 ((401)RKSLEQQFGDNMDKMNKLKKY(420)), 30902 ((421)KKILKFFPLFNYKSDLESIM(440)) and 30913 ((641)DLESTAEQKAEKKGGKAKAKY(660)) located at the C-terminal. Studies with polyclonal goat antiserum against synthetic peptides chosen to represent the whole length of the protein showed that TryThrA has fluorescence pattern similar to PypAg-1 of P. yoelii. All HABPs inhibited merozoite in vitro invasion, suggesting that TryThrA protein may be participating in merozoite-erythrocyte interaction during invasion.     

N-Acetylglucosamine-binding proteins on Plasmodium falciparum merozoite surface

Plasmodium falciparum merozoite surface is specifically labelledwith a neoglycoprotein bearing N-acetylgluco-samine (GlcNAc)residues in a sugar-dependent manner, as shown by affinity cytochemistryin fluorescence and electron microscopy. To ascertain the natureof the sugar receptor, merozoite proteins were blotted and testedby a two-step method using biotinylated GlcNAc—bovineserum albumin (BSA) and streptavidin—peroxidase conjugate.Three parasite proteins were specifically revealed and designatedas Pf 120, Pf 83 and Pf 45 GlcNAc-binding proteins. These proteinsbind to a gel substituted with GlcNAc and are specifically elutedwith 300 mM GlcNAc. Using a rabbit antiserum raised againstPf 83, the Pf 120 GlcNAc-binding protein, in addition to Pf83, was labelled by Western blotting. Comparative analyses withan antibody against the Pf 83 MSP derived from the P.falciparummerozoite surface protein (Pf MSP) indicated that the Pf 83GlcNAc-binding protein is not related to the fragment of thePf MSP antigen. Similarly, the Pf 83 GlcNAc-binding proteinis not related to the apical membrane antigen 1 (AMA 1) whichalso has the same molecular mass. Therefore the Pf 120, Pf 83and Pf 45 GlcNAc-binding proteins which are located on the merozoitesurface and recognize GlcNAc residues could be involved in thebinding of merozoites to the glycoconjugates of the surfaceof the red blood cells. GlcNAc lectin neoglycoprotein Plasmodium falciparum red blood cell     

A GBP 130 derived peptide from Plasmodium falciparum binds to human erythrocytes and inhibits merozoite invasion in vitro

The malarial GBP 130 protein binds weakly to intact human erythrocytes; the binding sites seem to be located in the repeat region and this region's antibodies block the merozoite invasion. A peptide from this region (residues from 701 to 720) which binds to human erythrocytes was identified. This peptide named 2220 did not bind to sialic acid; the binding site on human erythrocyte was affected by treatment with trypsin but not by chymotrypsin. The peptide was able to inhibit Plasmodium falciparum merozoite invasion of erythrocytes. The residues F701, K703, L705, T706, E713 (FYKILTNTDPNDEVERDNAD) were found to be critical for peptide binding to erythrocytes.     

MAEBL Plasmodium falciparum protein peptides bind specifically to erythrocytes and inhibit in vitro merozoite invasion

MAEBL is an erythrocyte binding protein located in the rhoptries and on the surface of mature merozoites, being expressed at the beginning of schizogony. The structure of MAEBL originally isolated from rodent malaria parasites suggested a molecule likely to be involved in invasion. We thus became interested in identifying possible MAEBL functional regions. Synthetic peptides spanning the MAEBL sequence were tested in erythrocyte binding assays to identify such possible MAEBL functional regions. Nine high activity binding peptides (HABPs) were identified: two were found in the M1 domain, one was found between the M1 and M2 regions, five in the erythrocyte binding domain (M2), and one in the protein's repeat region. The results showed that peptide binding was saturable; some HABPs inhibited in vitro merozoite invasion and specifically bound to a 33kDa protein on red blood cell membrane. HABPs' possible function in merozoite invasion of erythrocytes is also discussed.     

Functional analysis of proteins involved in Plasmodium falciparum merozoite invasion of red blood cells

Plasmodium falciparum causes the most lethal form of malaria in humans and is responsible for over two million deaths per year. The development of a vaccine against this parasite is an urgent priority and potential protein targets include those on the surface of the asexual merozoite stage, the form that invades the host erythrocyte. The development of methods to transfect P. falciparum has enabled the construction of gain-of-function and loss-of-function mutants and provided new strategies to analyse the role of parasite proteins. In this review, we describe the use of this technology to examine the role of merozoite antigens in erythrocyte invasion and to address their potential as vaccine candidates.     

Plasmodium falciparum: assay of invasion of erythrocytes

A method for quantitatively assaying Plasmodium falciparum merozoite invasion of particular erythrocytes is described. Erythrocytes were labeled with fluorescein isothiocyanate which did not affect parasite entry or growth, to distinguish them from uninfected erythrocytes in the original parasitized cell population. Parasites were detectable after staining with ethidium bromide. The time course of infection of the labeled cells was followed over 26 hr. The technique was used to determine the effect of serum from a patient with P. falciparum malaria on merozoite invasion of the labeled erythrocytes.     

Identification of Plasmodium falciparum RhopH3 protein peptides that specifically bind to erythrocytes and inhibit merozoite invasion

The identification of sequences involved in binding to erythrocytes is an important step for understanding the molecular basis of merozoite-erythrocyte interactions that take place during invasion of the Plasmodium falciparum malaria parasite into host cells. Several molecules located in the apical organelles (micronemes, rhoptry, dense granules) of the invasive-stage parasite are essential for erythrocyte recognition, invasion, and establishment of the nascent parasitophorous vacuole. Particularly, it has been demonstrated that rhoptry proteins play an important role in binding to erythrocyte surface receptors, among which is the PfRhopH3 protein, which triggers important immune responses in patients from endemic regions. It has also been reported that anti-RhopH3 antibodies inhibit in vitro invasion of erythrocytes, further supporting its direct involvement in erythrocyte invasion processes. In this study, PfRhopH3 consecutive peptides were synthesized and tested in erythrocyte binding assays for identifying those regions mediating binding to erythrocytes. Fourteen PfRhopH3 peptides presenting high specific binding activity were found, whose bindings were saturable and presented nanomolar dissociation constants. These high-activity binding peptides (HABPs) were characterized by having alpha-helical structural elements, as determined by circular dichroism, and having receptors of a possible sialic acid-dependent and/or glycoprotein-dependent nature, as evidenced in enzyme-treated erythrocyte binding assays and further corroborated by cross-linking assay results. Furthermore, these HABPs inhibited merozoite in vitro invasion of normal erythrocytes at 200 microM by up to 60% and 90%, suggesting that some RhopH3 protein regions are involved in the P. falciparum erythrocyte invasion.     

Plasmodium falciparum PhIL1-associated complex plays an essential role in merozoite reorientation and invasion of host erythrocytes

The human malaria parasite, Plasmodium falciparum possesses unique gliding machinery referred to as the glideosome that powers its entry into the insect and vertebrate hosts. Several parasite proteins including Photosensitized INA-labelled protein 1 (PhIL1) have been shown to associate with glideosome machinery. Here we describe a novel PhIL1 associated protein complex that co-exists with the glideosome motor complex in the inner membrane complex of the merozoite. Using an experimental genetics approach, we characterized the role(s) of three proteins associated with PhIL1: a glideosome associated protein- PfGAPM2, an IMC structural protein- PfALV5, and an uncharacterized protein—referred here as PfPhIP (PhIL1 Interacting Protein). Parasites lacking PfPhIP or PfGAPM2 were unable to invade host RBCs. Additionally, the downregulation of PfPhIP resulted in significant defects in merozoite segmentation. Furthermore, the PfPhIP and PfGAPM2 depleted parasites showed abrogation of reorientation/gliding. However, initial attachment with host RBCs was not affected in these parasites. Together, the data presented here show that proteins of the PhIL1-associated complex play an important role in the orientation of P. falciparum merozoites following initial attachment, which is crucial for the formation of a tight junction and hence invasion of host erythrocytes.     

Identifying Plasmodium falciparum merozoite surface antigen 3 (MSP3) protein peptides that bind specifically to erythrocytes and inhibit merozoite invasion

Receptor-ligand interactions between synthetic peptides and normal human erythrocytes were studied to determine Plasmodium falciparum merozoite surface protein-3 (MSP-3) FC27 strain regions that specifically bind to membrane surface receptors on human erythrocytes. Three MSP-3 protein high activity binding peptides (HABPs) were identified; their binding to erythrocytes became saturable, had nanomolar affinity constants, and became sensitive on being treated with neuraminidase and trypsin but were resistant to chymotrypsin treatment. All of them specifically recognized 45-, 55-, and 72-kDa erythrocyte membrane proteins. They all presented alpha-helix structural elements. All HABPs inhibited in vitro P. falciparum merozoite invasion of erythrocytes by ~55%-85%, suggesting that MSP-3 protein's role in the invasion process probably functions by using mechanisms similar to those described for other MSP family antigens.     

Sialic acid-dependent binding of Plasmodium falciparum merozoite surface antigen, Pf200, to human erythrocytes

Plasmodium falciparum merozoites, the extracellular stage of the erythrocytic cycle of the human malarial parasite, specifically invade human E. The major determinant of that specificity is the sialic acid residues of E glycophorin. In the present study we show that the merozoite surface Ag, Pf200 (m.w. 195,000 to 205,000), of two different isolates of P. falciparum, binds to the surface of human E but not E from other species not invaded by P. falciparum. Pf200 does not bind to neuraminidase-treated E, indicating the interaction is dependent on sialic acid residues. Binding is inhibited by soluble glycophorin and selective mAb against the glycosylated domain of glycophorin, but not by a mAb against the peptide domain of glycophorin. mAb.5B1 previously identified as reacting with Pf 200, blocks binding of the protein to the E. Binding between Pf200 and the E is not high affinity, as Pf200 can be released from the surface by 0.25 M NaCl.     

Plasmodium falciparum merozoite surface protein 6 (MSP-6) derived peptides bind erythrocytes and partially inhibit parasite invasion

This work shows that Plasmodium falciparum merozoite surface protein-6 (MSP-6) peptides specifically bind to membrane surface receptor on human erythrocytes. Three high activity binding peptides (HABPs) were found: peptides 31175 (41MYNNDKILSKNEVDTNIESN60) and 31178 (101YDIQATYQFPSTSGGNNVIP120) in the amino terminal region and 31191 (361EIDSTINNLVQEMIHLFSNNY380) at the carboxy terminal. Their binding to erythrocytes was saturable. HABPs 31191 and 31178 recognized 56 and 26 kDa receptors on erythrocyte membrane and inhibited in vitro Plasmodium falciparum merozoite invasion of erythrocytes by between 27% and 46% at 200 microg ml(-1) concentration, suggesting that these MSP-6 protein peptides play a possible role in the invasion process.     

Activation of a Plasmodium falciparum protease correlated with merozoite maturation and erythrocyte invasion

Protease-dependent processes of the P. falciparum schizogonic cycle are briefly described. The P. falciparum p76 protease is the first example of a biochemically regulated protease, the activation of which is related to merozoite maturation and/or erythrocyte invasion. The main known properties of the p76 protease are reviewed and some original results concerning its biosynthesis and biological properties are described.     

Plasmodium falciparum: merozoite invasion in vitro in the presence of chloroquine.

The fine structure of invasion of human erythrocytes by merozoites of the malaria parasite Plasmodium falciparum was observed in vitro. The invasion process is similar to that described for P. knowlesi. Merozoites enter apical end first by invagination of the erythrocyte membrane. At the rim of the invagination, where merozoite and erythrocyte are in closest contact, the erythrocyte membrane is thickened. The brushy cell coat of the P. falciparum merozoite appears to be lost at this attachment zone. The part of the merozoite within the erythrocyte invagination has no visible coat. The coat on the portion outside is unaltered. Merozoites can successfully invade erythrocytes after 3 hr in the presence of a concentration of chloroquine harmful to feeding stages.     

Functional analysis of Plasmodium falciparum merozoite antigens: implications for erythrocyte invasion and vaccine development

Malaria is a major human health problem and is responsible for over 2 million deaths per year. It is caused by a number of species of the genus Plasmodium, and Plasmodium falciparum is the causative agent of the most lethal form. Consequently, the development of a vaccine against this parasite is a priority. There are a number of stages of the parasite life cycle that are being targeted for the development of vaccines. Important candidate antigens include proteins on the surface of the asexual merozoite stage, the form that invades the host erythrocyte. The development of methods to manipulate the genome of Plasmodium species has enabled the construction of gain-of-function and loss-of-function mutants and provided new strategies to analyse the role of parasite proteins. This has provided new information on the role of merozoite antigens in erythrocyte invasion and also allows new approaches to address their potential as vaccine candidates.     

Reticulocyte-binding protein homologue 1 is required for sialic acid-dependent invasion into human erythrocytes by Plasmodium falciparum

The Apicomplexan parasite responsible for the most virulent form of malaria, Plasmodium falciparum, invades human erythrocytes through multiple ligand-receptor interactions. Some strains of P. falciparum are sensitive to neuraminidase treatment of the host erythrocyte and these parasites have been termed sialic acid-dependent as they utilize receptors containing sialic acid. In contrast, other strains can efficiently invade neuraminidase-treated erythrocytes and hence are sialic acid-independent. The molecular interactions that allow P. falciparum to differentially utilize receptors for merozoite invasion are not understood. The P. falciparum reticulocyte-binding protein homologue (PfRh or PfRBL) family have been implicated in the invasion process but their exact role is unknown. PfRh1, a member of this protein family, appears to be expressed in all parasite lines analysed but there are marked differences in the level of expression between different strains. We have used targeted gene disruption of the PfRh1 gene in P. falciparum to show that the encoded protein is required for sialic acid-dependent invasion of human erythrocytes. The DeltaPfRh1 parasites are able to invade normally; however, they utilize a pattern of ligand-receptor interactions that are more neuraminidase-resistant. Current data suggest a strategy based on the differential function of specific PfRh proteins has evolved to allow P. falciparum parasites to utilize alternative receptors on the erythrocyte surface for evasion of receptor polymorphisms and the host immune system.     

Effect of calpain inhibitors on the invasion of human erythrocytes by the parasite Plasmodium falciparum

17 different proteinase inhibitors were screened for their effect on the erythrocyte invasion by the malaria parasite Plasmodium falciparum. The effect was tested when the inhibitors were present in the culture medium and when they were trapped into erythrocyte ghosts. A very strong inhibition of invasion was observed in the presence of calpain inhibitors, with IC50 in the order of 10(-7) M. Chymostatin, leupeptin, pepstatin A and bestatin also caused inhibition of the invasion, but with IC50 in the order of 10(-5) M. The results suggest that participation of various proteinases in the process and point to the possibility of a calpain-mediated proteolytic event. This study may explain previous observations on the role of calcium in the invasion of the human erythrocyte by Plasmodium falciparum.     

Plasmodium falciparum: carbohydrates as receptor sites of invasion

Monosaccharides, disaccharides, and trisaccharides were tested as inhibitors of the in vitro growth of Plasmodium falciparum (strain FCB). While certain monosaccharides (N-acetyl-D-glucosamine, D-mannose, and 3-O-methyl-D-glucose) proved to exhibit a toxic or reversibly retarding effect on the intraerythrocytic development of the parasite, the corresponding alpha- or beta-methylglycosides did not. Several methylglycosides, synthetic di- and tri-saccharides, and artificial blood group antigens were further tested for inhibitory effects on invasion of host red blood cells in vitro. The synthetic disaccharides beta DGlcNAc(1----4) alpha DManOMe and beta DGlcNAc(1----4) DGlcNAc (chitobiose) were good inhibitors of invasion at 10 mM concentration, whereas beta DGal(1----4)beta DGlcNAcOMe was negligibly inhibitory. The inhibition rate of N-acetyl-D-glucosamine, beta-glycosidically linked to bovine serum albumin (BSA) by an alipathic spacer, -(CH2)8CO-, was not enhanced, compared to the corresponding hapten, beta DGlcNAcO(CH2)8COOCH3. The inhibition rates of blood group A- and B-trisaccharide haptens, which were inhibitors of invasion, were also not significantly enhanced when coupled to BSA by way of the corresponding amide spacer, -(CH2)2NHCO(CH2)7CO-. A remarkable enhancement of the inhibition rate was, however, observed when beta DGal(1----3) alpha DGalNAcO(CH2)2NHCO(CH2)7COOCH3 (T-hapten) was coupled to BSA. A clear-cut decrease in the inhibition rates of different beta-glycosides of N-acetyl-D-glucosamine, beta DGlcNAcOR, was observed, depending on the nature of the aglycon R(p-nitrophenyl greater than -(CH2)8COOCH3 greater than -(CH2)2NHCO(CH2)2COOCH3 greater than -CH3). Also, p-nitrophenyl-alpha-D-glucopyranoside was a much better inhibitor of invasion than the corresponding methyl glycoside, alpha DGlcOMe, which was not inhibitory. The properties of the aglycon spacer, used for the covalent attachment of the carbohydrate to the carrier protein, may thus be crucial for the outcome of the inhibition rate.     

Amino terminal peptides from the Plasmodium falciparum EBA-181/JESEBL protein bind specifically to erythrocytes and inhibit in vitro merozoite invasion

Several EBA-175 paralogues (EBA-140, EBA-165, EBA-175, EBA-181, and EBL-1) have been described among the Plasmodium falciparum malaria parasite proteins, which are important in the red blood cell (RBC) invasion process. EBA-181/JESEBL is a 181 kDa protein expressed in the late schizont stage and located in the micronemes; it belongs to the Plasmodium Duffy binding-like family and is able to interact with the erythrocyte surface. Here, we describe the synthesis of 78, 20-mer synthetic peptides derived from the reported EBA-181/JESEBL sequence and their ability to bind RBCs in receptor-ligand assays. Five peptides (numbered 30030, 30031, 30045, 30051, and 30060) displayed high specific binding to erythrocytes; their equilibrium binding parameters were then determined. These peptides interacted with 53 and 33 kDa receptor proteins on the erythrocyte surface, this binding being altered when RBCs were pretreated with enzymes. They were able to inhibit P. falciparum merozoite invasion of RBCs when tested in in vitro assays. According to these results, these five EBA-181/JESEBL high specific erythrocyte binding peptides, as well as the entire protein, were seen to be involved in the molecular machinery used by the parasite for invading RBCs. They are thus suggested as potential candidates in designing a multi-sub-unit vaccine able to combat the P. falciparum malaria parasite.