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.     

A novel ligand from Plasmodium falciparum that binds to a sialic acid-containing receptor on the surface of human erythrocytes

Invasion of the merozoite form of Plasmodium falciparum into human erythrocytes involves multiple receptor-ligand interactions. The EBA175 protein of P. falciparum has been shown to be the ligand that binds to a sialic acid-dependent site on glycophorin A. We have identified a novel P. falciparum ligand, termed erythrocyte-binding antigen 140 (EBA140), that shares structural features and homology with EBA175. Subcellular localization of EBA140 suggests that it is located in the micronemes, the same localization as EBA175. EBA140 binds to a sialic acid-dependent receptor on the surface of human erythrocytes. Binding of EBA140 to this erythrocyte receptor is sensitive to neuraminidase and resistant to trypsin, proteinase K and pronase. The protease-resistant properties of the erythrocyte receptor suggests that it is not glycophorin A or C. Additionally, analysis of mutant erythrocytes from humans has shown that EBA140 does not bind glycophorin B. Interestingly, we have identified a parasite line that lacks the eba140 gene, suggesting that this protein is not essential for in vitro invasion. These results suggest that EBA140 may be involved in merozoite invasion using a sialic acid-dependent receptor on human erythrocytes.     

Inhibitory effects of erythrocyte membrane proteins on the in vitro invasion of the human malarial parasite (Plasmodium falciparum) into its host cell

The intracellular development of the erythrocytic stage of the malarial parasite (merozoite) is initiated by the attachment of the parasite to the erythrocyte surface. This paper describes an assay system to investigate Plasmodium falciparum merozoite entry into the host cell and reports on three observations regarding this interaction. (a) Merozoites do not invade human erythrocytes treated with either trypsin or neuraminidase, and both enzymes partially cleave glycophorin A, the major erythrocyte surface sialoglycoprotein. (b) A membrane protein fraction containing glycophorin A will, at low concentrations, inhibit the invasion of isolated merozoites into erythrocytes; no other fractions of membrane proteins have appreciable effects on the reinvasion. (c) Merozoites do not reinvade erythrocytes preincubated with F ab' fragments of antibody prepared against glycophorin A. Together, these three observations imply a role for glycophorin A in the attachment of the malarial parasite to the erythrocyte surface.     

Secretion of Plasmodium falciparum rhoptry protein into the plasma membrane of host erythrocytes

The rhoptry is an organelle of the malarial merozoite which has been suggested to play a role in parasite invasion of its host cell, the erythrocyte. A monoclonal antibody selected for reactivity with this organelle identifies a parasite synthesized protein of 110 kD. From biosynthetic labeling experiments it was demonstrated that the protein is synthesized midway through the erythrocytic cycle (the trophozoite stage) but immunofluorescence indicates the protein is not localized in the organelle until the final stage (segmenter stage) of intraerythrocytic development. Immunoelectron microscopy shows that the protein is localized in the matrix of the rhoptry organelle and on membranous whorls secreted from the merozoite. mAb recognition of the protein is dithiothreitol (DTT) labile, indicating that the conformation of the epitope is dependent on a disulfide linkage. During erythrocyte reinvasion by the extracellular merozoite, immunofluorescence shows the rhoptry protein discharging from the merozoite and spreading around the surface of the erythrocyte. The protein is located in the plasma membrane of the newly invaded erythrocyte. These studies suggest that the 110-kD rhoptry protein is inserted into the membrane of the host erythrocyte during merozoite invasion.     

The cytoplasmic domain of the Plasmodium falciparum ligand EBA-175 is essential for invasion but not protein trafficking

The invasion of host cells by the malaria parasite Plasmodium falciparum requires specific protein-protein interactions between parasite and host receptors and an intracellular translocation machinery to power the process. The transmembrane erythrocyte binding protein-175 (EBA-175) and thrombospondin-related anonymous protein (TRAP) play central roles in this process. EBA-175 binds to glycophorin A on human erythrocytes during the invasion process, linking the parasite to the surface of the host cell. In this report, we show that the cytoplasmic domain of EBA-175 encodes crucial information for its role in merozoite invasion, and that trafficking of this protein is independent of this domain. Further, we show that the cytoplasmic domain of TRAP, a protein that is not expressed in merozoites but is essential for invasion of liver cells by the sporozoite stage, can substitute for the cytoplasmic domain of EBA-175. These results show that the parasite uses the same components of its cellular machinery for invasion regardless of the host cell type and invasive form.     

Characterization with monoclonal antibodies of a surface antigen of Plasmodium falciparum merozoites

The merozoite, the extracellular form of the erythrocyte stage of the malarial parasite, invades the erythrocyte and develops intracellularly. Cloned hybridoma cell lines secreting monoclonal antibodies directed against the merozoite surface were selected by indirect immunofluorescent assay by using intact isolated merozoites. Monoclonal antibodies to a 200,000 m.w. merozoite surface antigen were selected and were used to characterize this protein and its role in erythrocyte invasion. Immunoelectron microscopy demonstrated that the antigen was located exclusively on the merozoite surface coat, distributed evenly over the entire surface. The 200,000 m.w. protein incorporated [3H]glucosamine, suggesting that it is a glycoprotein and could be purified to homogeneity by using immuno-affinity chromatography. Freshly isolated, invasive merozoites retained the 200,000 m.w. antigen, but the protein was rapidly cleaved to proteins of 90,000 and 50,000 m.w. when the merozoite was extracellular. The 50,000 m.w. fragment was retained the epitope binding to monoclonal antibody 5B1 and were labeled with [3H]glucosamine. Monoclonal antibodies against the 200,000 m.w. antigen partially inhibited merozoite invasion into erythrocytes.     

A role of falcipain-2, principal cysteine proteases of Plasmodium falciparum in merozoite egression

The process of merozoite release in Plasmodium falciparum involves rupture of the parasitophorous vacuole membrane and erythrocyte plasma membrane. Through the use of protease inhibitors that halt the merozoite release, a number of parasite proteases, especially serine, aspartic, and cysteine proteases, have been implicated in the schizont rupture. To understand the precise role of cysteine proteases in the merozoite release, in the present study, we treated P. falciparum cultures with siRNAs corresponding to falcipain-1, falcipain-2, and falcipain-3, the three papain-family proteases of the parasite. Treatment of malaria parasites with either of the falcipain siRNAs considerably reduced parasite growth. Morphological examination of the siRNA treated parasite cultures revealed that most of the parasites in falcipain-2 siRNA treated cultures were arrested at schizont stage. Analysis of a transgenic P. falciparum line expressing chimeric-GFP upon treatment with falcipain-2 siRNA revealed block in the rupture of erythrocyte membrane at the time of merozoite egression. These results suggest that falcipain-2 is an important parasitic protease that participates in hemoglobin degradation and in the merozoite release.     

A tandemly repeated sequence determines the binding domain for an erythrocyte receptor binding protein of P. falciparum

Erythrocyte invasion by the malarial merozoite is a receptor-mediated process, an obligatory step in the development of the parasite. The Plasmodium falciparum protein GBP-130, which binds to the erythrocyte receptor glycophorin, is shown here to encode the binding site in a domain composed of a tandemly repeated 50 amino acid sequence. The amino acid sequence of GBP-130, deduced from the cloned and sequenced gene, reveals that the protein contains 11 highly conserved 50 amino acid repeats and a charged N-terminal region of 225 amino acids. Binding studies on recombinant proteins expressing different numbers of repeats suggest that a correlation exists between glycophorin binding and repeat number. Thus, a repeat domain, a common feature of plasmodial antigens, has been shown to have a function independent of the immune system. This conclusion is further supported by the ability of antibodies directed against the repeat sequence to inhibit the in vitro invasion of erythrocytes by merozoites.     

Receptors for the Malarial Parasite

Abstract

During the erythrocytic cycle of Plasmodium, the parasite develops within an enclosed space, the parasitophorous vacuole, formed by endocytosis of an invasive stage, the merozoite. Among the erythrocyte membrane proteins possibly acting as a receptor for the attachment of P. falciparum merozoites to human erythrocytes is glycophorin A Isolated glycophorin inhibits merozoite entry in a competitive manner, perhaps via association with a 155 kDa surface protein. Another protein that competitively inhibits merozoite invasion, is band 3, the erythrocyte anion transport protein. The protein bearing Duffy blood group antigens may act to modulate invasion, but does not behave as a receptor.     

Plasmodium falciparum-infected erythrocytes: qualitative and quantitative analyses of parasite-induced knobs by atomic force microscopy

We used the combination of an atomic force microscope and a light microscope equipped with epifluorescence to serially image Plasmodium falciparum-infected erythrocytes. This procedure allowed us to determine unambiguously the presence and developmental stage of the malaria parasite as well as the number and size of knobs in singly, doubly, and triply infected erythrocytes. Knobs are not present during the ring stage of a malaria infection but a lesion resulting from invasion by a merozoite is clearly visible on the erythrocyte surface. This lesion is visible into the late trophozoite stage of infection. Knobs begin to form during the early trophozoite stage of infection and have a single-unit structure. Our data suggest the possibility that a two-unit structure of knobs, which was reported by Aikawa et al. (1996, Exp. Parasitol. 84, 339-343) using atomic force microscopy, appears to be a double-tipped image. The number of knobs per unit of host cell surface area is directly proportional to parasite number in both early and late trophozoite stages. These results indicate that knob formation by one parasite does not influence knob formation by other parasites in a multiply infected erythrocyte. In addition, knob volume is not influenced by either parasite stage or number at the late trophozoite stage, indicating that the number of component molecules per knob is constant throughout the parasite maturation process.     

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.     

Skeleton binding protein 1 (SBP1) of Plasmodium falciparum accumulates in electron-dense material before passing through the parasitophorous vacuole membrane

Plasmodium falciparum proteins involved in vascular endothelial cell adherence are transported to the surface of infected erythrocytes. These proteins are exported through parasite-derived membrane structures within the erythrocyte cytoplasm called Maurer's clefts. Skeleton binding protein 1 (SBP1) is localized in the Maurer's clefts and plays an important role in transporting molecules to the surface of infected erythrocytes. Details of the translocation pathway are unclear and in this study we focused on the subcellular localization of SBP1 at an early intraerythrocytic stage. We performed immunoelectron microscopy using specific anti-SBP1 antibodies generated by immunization with recombinant SBP1 of P. falciparum. At the early trophozoite (ring form) stage, SBP1 was detected within an electron dense material (EDM) found in the parasite cytoplasm and in the parasitophorous vacuolar (PV) space. These findings demonstrate that SBP1 accumulates in EDM in the early trophozoite cytoplasm and is transported to the PV space before translocation to the Maurer's clefts formed in the erythrocyte cytoplasm.     

The structure of the Plasmodium falciparum EBA175 ligand domain and the molecular basis of host specificity

Erythrocyte-binding antigen 175 (EBA175) is one of the best-characterized Plasmodium falciparum merozoite ligands; the recently solved crystal structure of EBA175 reveals that terminal sialic acids on the erythrocyte glycoprotein glycophorin A are a crucial factor for erythrocyte recognition by EBA175 because they lock into pockets on its surface. Comparison with Plasmodium reichenowi EBA175 indicates that these interactions have a pivotal role in the host-specific adaptations of parasite ligands.     

Unraveling the ubiquitome of the human malaria parasite

Malaria is one of the deadliest infectious diseases worldwide. The most severe form is caused by the eukaryotic protozoan parasite Plasmodium falciparum. Recent studies have highlighted the importance of post-translational regulations for the parasite's progression throughout its life cycle, protein ubiquitylation being certainly one of the most abundant. The specificity of its components and the wide range of biological processes in which it is involved make the ubiquitylation pathway a promising source of suitable targets for anti-malarial drug development. Here, we combined immunofluorescent microscopy, biochemical assays, in silico prediction, and mass spectrometry analysis using the multidimensional protein identification technology, or MudPIT, to describe the P. falciparum ubiquitome. We found that ubiquitin conjugates are detected at every morphological stage of the parasite erythrocytic cycle. Furthermore, we detected that more than half of the parasite's proteome represents possible targets for ubiquitylation, especially proteins found to be present at the most replicative stage of the asexual cycle, the trophozoite stage. A large proportion of ubiquitin conjugates were also detected at the schizont stage, consistent with a cell activity slowdown to prepare for merozoite differentiation and invasion. Finally, for the first time in the human malaria parasite, our results strongly indicate the presence of heterologous mixed conjugations, SUMO/UB. This discovery suggests that sumoylated proteins may be regulated by ubiquitylation in P. falciparum. Altogether, our results present the first stepping stone toward a better understanding of ubiquitylation and its role(s) in the biology of the human malaria parasite.     

Intramembrane proteolysis mediates shedding of a key adhesin during erythrocyte invasion by the malaria parasite

Apicomplexan pathogens are obligate intracellular parasites. To enter cells, they must bind with high affinity to host cell receptors and then uncouple these interactions to complete invasion. Merozoites of Plasmodium falciparum, the parasite responsible for the most dangerous form of malaria, invade erythrocytes using a family of adhesins called Duffy binding ligand-erythrocyte binding proteins (DBL-EBPs). The best-characterized P. falciparum DBL-EBP is erythrocyte binding antigen 175 (EBA-175), which binds erythrocyte surface glycophorin A. We report that EBA-175 is shed from the merozoite at around the point of invasion. Shedding occurs by proteolytic cleavage within the transmembrane domain (TMD) at a site that is conserved across the DBL-EBP family. We show that EBA-175 is cleaved by PfROM4, a rhomboid protease that localizes to the merozoite plasma membrane, but not by other rhomboids tested. Mutations within the EBA-175 TMD that abolish cleavage by PfROM4 prevent parasite growth. Our results identify a crucial role for intramembrane proteolysis in the life cycle of this pathogen.     

Crystal structure of a Fab complex formed with PfMSP1-19, the C-terminal fragment of merozoite surface protein 1 from Plasmodium falciparum: a malaria vaccine candidate

Merozoite surface protein 1 (MSP1) is the major protein component on the surface of the merozoite, the erythrocyte-invasive form of the malaria parasite Plasmodium. Present in all species of Plasmodium, it undergoes two distinct proteolytic maturation steps during the course of merozoite development that are essential for invasion of the erythrocyte. Antibodies specific for the C-terminal maturation product, MSP1-19, can inhibit erythrocyte invasion and parasite growth. This polypeptide is therefore considered to be one of the more promising malaria vaccine candidates. We describe here the crystal structure of recombinant MSP1-19 from P.falciparum (PfMSP1-19), the most virulent species of the parasite in humans, as a complex with the Fab fragment of the monoclonal antibody G17.12. This antibody recognises a discontinuous epitope comprising 13 residues on the first epidermal growth factor (EGF)-like domain of PfMSP1-19. Although G17.12 was raised against the recombinant antigen expressed in an insect cell/baculovirus system, it binds uniformly to the surface of merozoites from the late schizont stage, showing that the cognate epitope is exposed on the naturally occurring MSP1 polypeptide complex. Although the epitope includes residues that have been mapped to regions recognised by invasion-inhibiting antibodies studied by other workers, G17.12 does not inhibit erythrocyte invasion or MSP1 processing.     

Plasmodial ortholog of Toxoplasma gondii rhoptry neck protein 3 is localized to the rhoptry body

The proteins in apical organelles of Plasmodium falciparum merozoite play an important role in invasion into erythrocytes. Several rhoptry neck (RON) proteins have been identified in rhoptry proteome of the closely-related apicomplexan parasite, Toxoplasma gondii. Recently, three of P. falciparum proteins orthologous to TgRON proteins, PfRON2, 4 and 5, were found to be located in the rhoptry neck and interact with the micronemal protein apical membrane antigen 1 (PfAMA1) to form a moving junction complex that helps the invasion of merozoite into erythrocyte. However, the other P. falciparum RON proteins have yet to be characterized. Here, we determined that "PFL2505c" (hereafter referred to as pfron3) is the ortholog of the tgron3 in P. falciparum and characterized its protein expression profile, subcellular localization, and complex formation. Protein expression analysis revealed that PfRON3 was expressed primarily in late schizont stage parasites. Immunofluorescence microscopy (IFA) showed that PfRON3 localizes in the apical region of P. falciparum merozoites. Results from immunoelectron microscopy, along with IFA, clarified that PfRON3 localizes in the rhoptry body and not in the rhoptry neck. Even after erythrocyte invasion, PfRON3 was still detectable at the parasite ring stage in the parasitophorous vacuole. Moreover, co-immunoprecipitation studies indicated that PfRON3 interacts with PfRON2 and PfRON4, but not with PfAMA1. These results suggest that PfRON3 partakes in the novel PfRON complex formation (PfRON2, 3, and 4), but not in the moving junction complex (PfRON2, 4, 5, and PfAMA1). The novel PfRON complex, as well as the moving junction complex, might play a fundamental role in erythrocyte invasion by merozoite stage parasites.     

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

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