Erythrocyte surface glycosylphosphatidyl inositol anchored receptor for the malaria parasite

Parasitophorous vacuole formation is a critical step for the successful invasion of host erythrocytes by the malaria parasite. Rhoptry proteins are believed to have essential roles in vacuole formation, although their biological roles are poorly understood. To understand the molecular interactions between parasite rhoptry proteins and the erythrocyte during invasion, we have characterized the binding specificity of the high molecular mass rhoptry protein (RhopH) complex to erythrocytes using the rodent malaria parasite, Plasmodium yoelii. RhopH complex binding to erythrocytes was species-specific, observed with mouse but not rabbit or human erythrocytes. Binding is abolished following treatment of erythrocytes with trypsin or chymotrypsin. Because host cell cholesterol-rich membrane domains are recruited into the nascent parasitophorous vacuole, we evaluated a possible role of RhopH complex binding to the cholesterol-rich membrane domain-associated glycosylphosphatidyl inositol (GPI)-anchored protein. Using chimeric mice harboring GPI-deficient erythrocytes, RhopH complex binding to GPI-deficient mouse erythrocytes was undetectable, indicating involvement of GPI-anchored protein in PyRhopH complex binding. Furthermore, a significant reduction of P. yoelii parasite infection of GPI-deficient erythrocytes was observed in vivo, probably due to inefficient invasion. We conclude that the major erythrocyte receptor for PyRhopH complex is a protein attached to the erythrocyte surface via GPI-anchor and that GPI-deficient erythrocytes are resistant to P. yoelii invasion.     

Characterisation of the rhoph2 gene of Plasmodium falciparum and Plasmodium yoelii

The high molecular mass protein complex (RhopH) in the rhoptries of the malaria parasite consists of three distinct polypeptides with estimated sizes in Plasmodium falciparum of 155kDa (PfRhopH1), 140kDa (PfRhopH2) and 110kDa (PfRhopH3). Using a number of reagents, including a new mAb 4E10 that is specific for the PfRhopH complex, it was shown that the RhopH complex is synthesised during schizogony and transferred intact to the ring stage in newly invaded erythrocytes. The genes encoding RhopH1 and RhopH3 have already been identified and characterised in both P. falciparum and Plasmodium yoelii. In this report, we describe the identification of the gene for RhopH2 in both these parasite species. Peptide sequences were obtained from purified RhopH2 proteins and used to generate oligonucleotide primers and search malaria sequence databases. In a parallel approach, mAb 4E10 was used to identify a clone coding for RhopH2 from a P. falciparum cDNA library. The sequences of both P. falciparum and P. yoelii genes for RhopH2 were completed and compared. They both contain nine introns and there is a high degree of similarity between the deduced amino acid sequences of the two proteins. The P. falciparum gene is a single copy gene located on chromosome 9, and is transcribed in schizonts.     

The high molecular mass rhoptry protein, RhopH1, is encoded by members of the clag multigene family in Plasmodium falciparum and Plasmodium yoelii.

Malarial merozoite rhoptries contain a high molecular mass protein complex called RhopH. RhopH is composed of three polypeptides, RhopH1, RhopH2, and RhopH3, encoded by distinct genes. Using monoclonal antibody-purified protein complex from both Plasmodium falciparum and Plasmodium yoelii, peptides were obtained by digestion of RhopH1 and their sequence determined either by mass spectrometry or Edman degradation. In both species the genes encoding RhopH1 were identified as members of the cytoadherence linked asexual gene (clag) family. In P. falciparum the family members on chromosome 3 were identified as encoding RhopH1. In P. yoelii two related genes were identified and sequenced. One of the genes, pyrhoph1a, was positively identified as encoding RhopH1 by the peptide analysis and the other gene, pyrhoph1a-p, was at least transcribed. Genes in the clag family present in both parasite species have a number of conserved features. The size and location of the P. yoelii protein complex in the rhoptries was confirmed. The first clag gene identified on chromosome 9 was implicated in cytoadherence, the binding of infected erythrocytes to host endothelial cells; this study shows that other members of the family encode merozoite rhoptry proteins, proteins that may be involved in merozoite-erythrocyte interactions. We propose that the family should be renamed as rhoph1/clag.     

Primary structure of a Plasmodium falciparum rhoptry antigen.

The high-molecular-weight rhoptry complex of Plasmodium falciparum consists of 3 non-covalently associated polypeptides of 150, 135 and 105 kDa. We present the complete nucleotide sequence of the 105-kDa (RhopH3) component of this complex derived from analysis of genomic and cDNA clones. The genomic structure is unusually complex for P. falciparum, consisting of 7 exons including 2 mini-exons of 19 and 21 amino acids. The sequence lacks tandem repeats and is conserved among several parasite isolates. B cell epitopes that induce antibody responses during natural infection were mapped to five different regions of the polypeptide.     

Plasmodium falciparum MAEBL is a unique member of the ebl family

Malaria is one of the deadliest human diseases and efforts to control it have been difficult due to the protozoan parasites' complex biology. Malaria merozoite invasion of erythrocytes is an essential part of blood-stage infections. The invasion process is mediated by numerous parasite molecules, such as EBA-175, a member of the ebl family of erythrocyte binding proteins. We have identified maebl, an ebl paralogue, in Plasmodium falciparum and found it highly conserved with its orthologues in P. yoelii and P. berghei, but distinct from other Plasmodium ebl. Importantly, the putative MAEBL ligand domains are highly conserved and are similar to AMA-1, but not the consensus DBL ligand domains present in all other ebl. In mature merozoites, MAEBL localized with rhoptry proteins (RhopH2, RAP-1), including surface localization with RhopH2, but not microneme proteins (EBA-175, BAEBL). MAEBL appears as proteolytically processed fragments in P. falciparum parasites. The amino cysteine-rich ligand domains were present primarily in culture supernatants, while the carboxyl cysteine-rich domain adjacent to the transmembrane domain was preferentially isolated from Triton X-100 extracted fractions. These data indicate that the primary structure of maebl is highly conserved among Plasmodium species, while its characteristics demonstrate a function unique among the ebl proteins.     

Identification of protein complexes in detergent-resistant membranes of Plasmodium falciparum schizonts

Merozoite surface proteins of the human malaria parasite Plasmodium falciparum are involved in initial contact with target erythrocytes, a process that begins a cascade of events required for successful invasion of these cells. In order to identify complexes that may play a role in invasion we purified detergent-resistant membranes (DRMs), known to be enriched in merozoite surface proteins, and used blue native-polyacrylamide gel electrophoresis (BN-PAGE) to isolate high molecular weight complexes for identification by mass spectrometry. Sixty-two proteins were detected and these mostly belonged to expected DRM proteins classes including GPI-anchored, multi-membrane spanning and rhoptry proteins. Proteins from seven known complexes were identified including MSP-1/7, the low (RAP1/2 and RAP1/3), and high (RhopH1/H2/H3) molecular weight rhoptry complexes, and the invasion motor complex (GAP45/GAP50/myosinA). Remarkably, a large proportion of identified spectra were derived from only 4 proteins: the GPI-anchored proteins MSP-1 and Pf92, the putative GPI-anchored protein Pf113 and RAP-1, the core component of the two RAP complexes. Each of these proteins predominated in high molecular weight species suggesting their aggregation in much larger complexes than anticipated. To demonstrate that the procedure had isolated novel complexes we focussed on MSP-1, which predominated as a distinct species at approximately 500 kDa by BN-PAGE, approximately twice its expected size. Chemical cross-linking supports the existence of a stable MSP-1 oligomer of approximately 500 kDa, probably comprising a highly stable homodimeric species. Our observations also suggests that oligomerization of MSP-1 is likely to occur outside the C-terminal epidermal growth factor (EGF)-like domains. Confirmation of MSP-1 oligomerization, together with the isolation of a number of known complexes by BN-PAGE, makes it highly likely that novel interactions occur amongst members of this proteome.     

Identification and disruption of the gene encoding the third member of the low-molecular-mass rhoptry complex in Plasmodium falciparum

The low-molecular-mass rhoptry complex of Plasmodium falciparum consists of three proteins, rhoptry-associated protein 1 (RAP1), RAP2, and RAP3. The genes encoding RAP1 and RAP2 are known; however, the RAP3 gene has not been identified. In this study we identify the RAP3 gene from the P. falciparum genome database and show that this protein is part of the low-molecular-mass rhoptry complex. Disruption of RAP3 demonstrated that it is not essential for merozoite invasion, probably because RAP2 can complement the loss of RAP3. RAP3 has homology with RAP2, and the genes are encoded on chromosome 5 in a head-to-tail fashion. Analysis of the genome databases has identified homologous genes in all Plasmodium spp., suggesting that this protein plays a role in merozoite invasion. The region surrounding the RAP3 homologue in the Plasmodium yoelii genome is syntenic with the same region in P. falciparum; however, there is a single gene. Phylogenetic comparison of the RAP2/3 protein family from Plasmodium spp. suggests that the RAP2/3 duplication occurred after divergence of these parasite species.     

Identification and characterization of the Plasmodium falciparum RhopH2 ortholog in Plasmodium vivax

Plasmodium vivax is one of the most important human malaria species that is geographically widely endemic and potentially affects a larger number of people than its more notorious cousin, Plasmodium falciparum. During invasion of red blood cells, the parasite requires the intervention of high molecular weight complex rhoptry proteins (RhopH) that are also essential for cytoadherence. PfRhopH2, a member of the RhopH multigene family, has been characterized as being crucial during P. falciparum infection. This study describes identifying and characterizing the pfrhoph2 orthologous gene in P. vivax (hereinafter named pvrhoph2). The PvRhopH2 is a 1,369-amino acid polypeptide encoded by PVX_099930 gene, for which orthologous genes have been identified in other Plasmodium species by bioinformatic approaches. Both P. falciparum and P. vivax genes contain nine introns, and there is a high degree of similarity between the deduced amino acid sequences of the two proteins. Moreover, PvRhopH2 contains a signal peptide at its N-terminus and 12 cysteines predominantly in its C-terminal half. PvRhopH2 is localized in one of the apical organelles of the merozoite, the rhoptry, and the localization pattern is similar to that of PfRhopH2 in P. falciparum. The recombinant PvRhopH2 protein is recognized by serum antibodies of patients naturally exposed to P. vivax, suggesting that PvRhopH2 is immunogenic in humans.     

Preferential binding of Plasmodium falciparum SERA and rhoptry proteins to erythrocyte membrane inner leaflet phospholipids.

Proteins of an apical organelle, the rhoptry, of Plasmodium falciparum are secreted into the host erythrocyte membrane during merozoite invasion. To identify the membrane-binding site for rhoptry proteins, we examined the binding of parasite proteins to phospholipid vesicles. A specific interaction between the rhoptry proteins of 140, 130, and 110 kDa to vesicles containing phosphatidylserine and phosphatidylinositol was observed. Both phospholipids are preferentially localized on the inner leaflet of the bilayer. Binding to other phospholipids, including sphingomyelin, was considerably less. In addition, the 120-kDa serine repeat antigen known as SERA, which was determined to be present on the merozoite, bound to phosphatidylserine vesicles and much less to vesicles of other phospholipids. Both the rhoptry and SERA proteins exhibited a preference for phosphatidylserine with short acyl side chains. Specific binding of SERA and the rhoptry proteins to phospholipids of the inner leaflet of membranes suggests a possible mechanism by which the protein facilitate invasion into host cells.     

Binding of Plasmodium falciparum rhoptry proteins to mouse erythrocytes and their possible role in invasion

Rhoptry proteins of Plasmodium falciparum merozoites, of 140, 130, and 110 kDa, identified by co-precipitation with Mab.1B9, bind selectively to mouse erythrocytes and reticulocytes. The properties of binding are shown to correlate with invasion of P. falciparum into mouse erythrocytes. Invasion of two strains of P. falciparum 7G8 and FCR-3, into mouse erythrocytes was examined, and was found to differ significantly. The 7G8 strain invades mouse erythrocytes at a rate of 40-60% compared to invasion into human erythrocytes, whereas FCR-3 invades at a rate of 5-15%. Both strains of P. falciparum preferentially invade reticulocytes in the in vitro invasion assay. This correlated with an increase in the amount of rhoptry protein of the 7G8 strain bound to mouse erythrocytes, compared to the FCR-3 strain and an increased binding to reticulocytes compared to mature erythrocytes. Binding of the rhoptry proteins and merozoite invasion into the erythrocyte is blocked in erythrocytes treated with trypsin and chymotrypsin but not in neuraminidase-treated erythrocytes, suggesting that the putative receptor site is exposed and accessible on the erythrocyte surface. Rabbit antiserum against gp3, the major glycophorin of mouse erythrocytes, blocks binding of the rhoptry proteins to erythrocytes and reduces merozoite invasion into mouse erythrocytes by 50%. Binding of rhoptry proteins to mouse reticulocytes was not blocked by alpha gp3 indicating a receptor difference between reticulocytes and erythrocytes. Mab.1B9 reduces merozoite invasion but does not decrease binding of the rhoptry proteins to the mouse erythrocyte. The mouse erythrocyte serves as a useful model to study the receptor-ligand interaction of rhoptry proteins and host surface proteins and to define the role of the rhoptry proteins during the invasion process.     

The cysteine-rich regions of <Emphasis Type="Italic">Plasmodium falciparum</Emphasis> RON2 bind with host erythrocyte and AMA1 during merozoite invasion

Invasion of Plasmodium falciparum merozoites into host erythrocyte involves a series of highly specific and sequential interaction between merozoite and host erythrocyte surface protein. The key step in the invasion process is the formation of a tight protein–protein interaction between host and parasite called as moving junction. A number of parasite proteins secreted from two organelles, microneme and rhoptry, play a role in initial interaction and junction formation between merozoite with host red blood cells (RBCs) during the invasion process. In the present study, we investigated the role of different domains of a P. falciparum rhoptry neck protein PfRON2. Immunofluorescence assay revealed close association of PfAMA1 and PfRON2 in the merozoites during the invasion process. PfRON2 domains were expressed on COS-7 cell surface, and their interaction was analysed with host RBCs and PfAMA1 protein by rosetting assays. The rosetting assays suggest that the C-terminal cysteine-rich domain of PfRON2 plays a role in binding with host erythrocyte. The C-terminal as well as the central cysteine-rich domain of PfRON2 interact with PfAMA1; this binding can be inhibited by monoclonal antibody (mAb 4 G2) against PfAMA1, suggesting that the hydrophobic groove of PfAMA1 binds to PfRON2. These results suggest that PfRON2 plays a role in merozoite invasion and thus it can be an important vaccine candidate antigen.     

Associations between responses to the rhoptry-associated membrane antigen of Plasmodium falciparum and immunity to malaria infection

Rhoptry proteins participate in the invasion of red blood cells by merozoites during the malaria parasite's asexual-stage cycle. Interference with the rhoptry protein function has been shown to prevent invasion, and three rhoptry proteins have been suggested as potential components of a vaccine against malaria. Rhoptry-associated membrane antigen (RAMA) is a 170-kDa protein of Plasmodium falciparum which is processed to a 60-kDa mature form in the rhoptries. p60/RAMA is discharged from rhoptries of free merozoites and binds to the red-cell membrane before being internalized to form part of the parasitophorous vacuole of the newly developing ring. We examined the range of anti-RAMA responses in individuals living in an area of endemicity for malaria and determined its association with clinical immunity. RAMA is immunogenic during infections, and at least three epitopes within RAMA are recognized by hyperimmune sera in immunoblots. Sera from individuals living in a region of Vietnam where malaria is endemic possessed strong antibody responses toward two C-terminal regions of RAMA. Cytophilic antibody isotypes (immunoglobulin G1 [IgG1] and IgG3) predominated in humoral responses to both C-terminal epitopes. Acute episodes of P. falciparum infection result in significant boosting of levels of antibody to an epitope at the extreme C terminus of RAMA that harbors the red-cell-binding domain. Immunity to P. falciparum infection was linked to elevated levels of IgG3 responses to this functional domain of RAMA, suggesting that the region may contain a protective epitope useful for inclusion in a multiepitope vaccine against malaria.     

Molecular characterisation of a Cryptosporidium parvum rhoptry protein candidate related to the rhoptry neck proteins TgRON1 of Toxoplasma gondii and PfASP of Plasmodium falciparum

Given the lack of knowledge on the rhoptry proteins of Cryptosporidium parvum, we searched for putative members of this protein class in the CryptoDB database using as queries known Toxoplasma gondii rhoptry molecules. We cloned a C. parvum sporozoite cDNA of 4269bp encoding the sushi domain-containing protein cgd8_2530, which shared low amino acid sequence identity, yet a highly conserved domain architecture with the rhoptry neck proteins TgRON1 of T. gondii and PfASP of Plasmodium falciparum. On denaturing and native gels, cgd8_2530 migrated at approximately 150 and 1000 kDa, respectively, suggesting an involvement in a multi-subunit protein complex. Immunoflorescence localised cgd8_2530 to a single, elongated area anterior to sporozoite micronemes and showed protein relocation to the parasite-host cell interface in early epicellular stages. Our data strongly suggest a rhoptry localization for the newly characterised protein, which was therefore renamed C. parvum putative rhoptry protein-1 (CpPRP1).     

Plasmodium falciparum homologue of the genes for Plasmodium vivax and Plasmodium yoelii adhesive proteins, which is transcribed but not translated

The 235-kDa family of rhoptry proteins in Plasmodium yoelii and the two reticulocyte binding proteins of P. vivax comprise a family of proteins involved in host cell selection and erythrocyte invasion. Here we described a member of the gene family found in P. falciparum (PfRH3) that is transcribed in its entirety, under stage-specific control, with correct splicing of the intron, but appears not to be translated, probably due to two reading frameshifts at the 5' end of the gene.     

Targeting of soluble proteins to the rhoptries and micronemes in Toxoplasma gondii

Rhoptry and microneme organelles of the protozoan parasite Toxoplasma gondii are closely associated with host cell adhesion/invasion and establishment of the intracellular parasitophorous vacuole. In order to study the targeting of proteins to these specialized secretory organelles, we have engineered green fluorescent protein (GFP) fusions to the rhoptry protein ROP1 and the microneme protein MIC3. Both chimeras are correctly targeted to the appropriate organelles, permitting deletion analysis to map protein subdomains critical for targeting. The propeptide and a central 146 amino acid region of ROP1 are sufficient to target GFP to the rhoptries. More extensive deletions result in a loss of rhoptry targeting; the GFP reporter is diverted into the parasitophorous vacuole via dense granules. Certain MIC3 deletion mutants were also secreted into the parasitophorous vacuole via dense granules, supporting the view that this route constitutes the default pathway in T. gondii, and that specific signals are required for sorting to rhoptries and micronemes. Deletions within the cysteine-rich central region of MIC3 cause this protein to be arrested at various locations within the secretory pathway, presumably due to improper folding. Although correctly targeted to the appropriate organelles in living parasites, ROP1-GFP and MIC3-GFP fusion proteins were not secreted during invasion. GFP fusion proteins were readily secreted from dense granules, however, suggesting that protein secretion from rhoptries and micronemes might involve more than a simple release of organellar contents.     

Differences in erythrocyte receptor specificity of different parts of the Plasmodium falciparum reticulocyte binding protein homologue 2a

The Plasmodium falciparum reticulocyte-binding-like protein homologue (RH) and erythrocyte binding-like (EBL) protein families play important roles during invasion, though their exact roles are not clear. Both EBL and RH proteins are thought to directly bind different receptors on the surface of the erythrocyte, and the binding properties for a number of EBLs and RHs have been described. While P. falciparum RH1 (PfRH1) and PfRH4 have been shown to act directly in two alternative invasion pathways used by merozoites, the functions of PfRH2a and PfRH2b during invasion are less defined. Here, using monoclonal antibodies raised against a unique region of PfRH2a, we show that PfRH2a moves from the rhoptry neck to the moving junction during merozoite invasion. The movement of PfRH2a to the junction is independent of the invasion pathway used by the merozoite, suggesting an additional function of the protein that is independent of receptor binding. We further show that PfRH2a is processed both in the schizont and during invasion, resulting in proteins with different erythrocyte binding properties. Our findings suggest that PfRH2a and, most likely, the other members of the RH family, depending on their processing stage, can engage different receptors at different stages of the invasion process.     

A component of an antigenic rhoptry complex of Plasmodium falciparum is modified after merozoite invasion

Human antibodies affinity purified on an adsorbent prepared from a cDNA clone (Ag44) expressing a portion of a rhoptry antigen were used to characterize the synthesis and fate of the antigen in the asexual blood stages of Plasmodium falcipartum. The rhoptry antigen is synthesized in the mature trophozoite-stage parasites as a 103 kDa polypeptide, is present in the schizonts and merozoites as a 105 kDa polypeptide, is discharged from the rhoptries and found in the newly invaded red cells as a 110 kDa polypeptide. Anti-Ag44 antibodies immunoprecipitate the antigen and two additional polypeptides of 135 and 150 kDa from lysates of infected cells and from culture supernatants. The three polypeptides are associated in a non-covalent complex that persists in the newly invaded red cells. All the components of the high molecular weight rhoptry complex are antigenic and can be precipitated with immune human serum. The 135 kDa polypeptide is identical to a 140 kDa rhoptry antigen previously identified by a monoclonal antibody.