Extensive proteolytic processing of the malaria parasite merozoite surface protein 7 during biosynthesis and parasite release from erythrocytes

In Plasmodium falciparum, merozoite surface protein 7 (MSP7) was originally identified as a 22kDa protein on the merozoite surface and associated with the MSP1 complex shed during erythrocyte invasion. MSP7 is synthesised in schizonts as a 351-amino acid precursor that undergoes proteolytic processing. During biosynthesis the MSP1 and MSP7 precursors form a complex that is targeted to the surface of developing merozoites. In the sequential proteolytic processing of MSP7, N- and C-terminal 20 and 33kDa products of primary processing, MSP7(20) and MSP7(33) are formed and MSP7(33) remains bound to full length MSP1. Later in the mature schizont, MSP7(20) disappears from the merozoite surface and on merozoite release MSP7(33) undergoes a secondary cleavage yielding the 22kDa MSP7(22) associated with MSP1. In free merozoites, both MSP7(22) and a further cleaved product, MSP7(19) present only in some parasite lines, were detected; these two derivatives are shed as part of the protein complex with MSP1 fragments during erythrocyte invasion. Primary processing of MSP7 is brefeldin A-sensitive while secondary processing is resistant to both calcium chelators and serine protease inhibitors. Primary processing of MSP7 occurs prior to that of MSP1 in a post-Golgi compartment, whereas the secondary cleavage occurs on the surface of the developing merozoite, possibly at the time of MSP1 primary processing and well before the secondary processing of MSP1.     

The human immune response to Plasmodium falciparum includes both antibodies that inhibit merozoite surface protein 1 secondary processing and blocking antibodies

Malaria merozoite surface protein 1 (MSP1) is cleaved in an essential step during erythrocyte invasion. The responses of children to natural malaria infection included antibodies that inhibit this cleavage and others that block the binding of these inhibitory antibodies. There was no correlation between the titer of the antibody to the 19-kDa fragment of MSP1 and its inhibitory activity. These findings have implications for the design of MSP1-based vaccines.     

Plasmodium falciparum merozoite surface protein 6 is a dimorphic antigen

Merozoite surface protein 1 (MSP1) is a highly polymorphic Plasmodium falciparum merozoite surface protein implicated in the invasion of human erythrocytes during the asexual cycle. It forms a complex with MSP6 and MSP7 on the merozoite surface, and this complex is released from the parasite around the time of erythrocyte invasion. MSP1 and many other merozoite surface proteins contain dimorphic elements in their protein structures, and here we show that MSP6 is also dimorphic. The sequences of eight MSP6 genes indicate that the alleles of each dimorphic form of MSP6 are highly conserved. The smaller 3D7-type MSP6 alleles are detected in parasites from all malarious regions of the world, whereas K1-type MSP6 alleles have only been detected in parasites from mainland Southeast Asia. Cleavage of MSP6, which produces the p36 fragment in 3D7-type MSP6 and associates with MSP1, also occurs in K1-type MSP6 but at a different site in the protein. Anti-3D7 MSP6 antibodies weakly inhibited erythrocyte invasion by homologous 3D7 merozoites but did not inhibit a parasite line expressing the K1-type MSP6 allele. Antibodies from hyperimmune individuals affinity purified on an MSP3 peptide cross-reacted with MSP6; therefore, MSP6 may also be a target of antibody-dependent cellular inhibition.     

Surface proteins of schizont-infected erythrocytes and merozoites of Plasmodium falciparum

Schizont-infected erythrocytes and merozoites were isolated from in vitro cultures of the human parasite, Plasmodium falciparum labeled with various radioactive substrates. The isolated merozoites were viable since they were able to reinvade fresh erythrocytes. On the basis of sensitivity to specific enzymes, eleven proteins synthesised by the parasite, were localised on the surface of the schizont-infected erythrocyte. Eight of these were glycoproteins, six of which appeared to represent three doublets. Five merozoite surface proteins were identified on the basis of their sensitivity to trypsin and chymotrypsin, treatments which also rendered the merozoite incapable of erythrocyte invasion. Merozoites appeared not to contain any glycoproteins; all of the glycoproteins synthesised by the parasite were apparently transported to the surface of the schizont-infected erythrocyte.     

Comparison of Surface Proteins of Anaplasma marginale Grown in Tick Cell Culture, Tick Salivary Glands, and Cattle

Anaplasma marginale, a tick-borne rickettsial pathogen of cattle, infects bovine erythrocytes, resulting in mild to severe hemolytic disease that causes economic losses in domestic livestock worldwide. Recently, the Virginia isolate of A. marginale was propagated in a continuous tick cell line, IDE8, derived from embryonic Ixodes scapularis. Development of A. marginale in cell culture was morphologically similar to that described previously in ticks. In order to evaluate the potential of the cell culture-derived organisms for use in future research or as an antigen for serologic tests and vaccines, the extent of structural conservation of the major surface proteins (MSPs) between the cell culture-derived A. marginale and the bovine erythrocytic stage, currently the source of A. marginale antigen, was determined. Structural conservation on the tick salivary-gland stage was also examined. Monoclonal and monospecific antisera against MSPs 1 through 5, initially characterized against erythrocyte stages, also reacted with A. marginale from cell culture and tick salivary glands. MSP1a among geographic A. marginale isolates is variable in size because of different numbers of a tandemly repeated 28- or 29-amino-acid peptide. The cell culture-derived A. marginale maintained the same-size MSP1a as that found on the Virginia isolate of A. marginale in bovine erythrocytes and tick salivary glands. Although differences were observed in the polymorphic MSP2 antigen between culture and salivary-gland stages, MSP2 did not appear to vary, by two-dimensional gel electrophoresis, during continuous passage in culture. These data show that MSPs of erythrocyte-stage A. marginale are present on culture stages and may be structurally conserved during continuous culture. The presence of all current candidate diagnostic and vaccine antigens suggests that in vitro cultures are a valuable source of rickettsiae for basic research and for the development of improved diagnostic reagents and vaccines against anaplasmosis.     

RALP1 Is a Rhoptry Neck Erythrocyte-Binding Protein of Plasmodium falciparum Merozoites and a Potential Blood-Stage Vaccine Candidate Antigen

Erythrocyte invasion by merozoites is an obligatory stage of Plasmodium infection and is essential to disease progression. Proteins in the apical organelles of merozoites mediate the invasion of erythrocytes and are potential malaria vaccine candidates. Rhoptry-associated, leucine zipper-like protein 1 (RALP1) of Plasmodium falciparum was previously found to be specifically expressed in schizont stages and localized to the rhoptries of merozoites by immunofluorescence assay (IFA). Also, RALP1 has been refractory to gene knockout attempts, suggesting that it is essential for blood-stage parasite survival. These characteristics suggest that RALP1 can be a potential blood-stage vaccine candidate antigen, and here we assessed its potential in this regard. Antibodies were raised against recombinant RALP1 proteins synthesized by using the wheat germ cell-free system. Immunoelectron microscopy demonstrated for the first time that RALP1 is a rhoptry neck protein of merozoites. Moreover, our IFA data showed that RALP1 translocates from the rhoptry neck to the moving junction during merozoite invasion. Growth and invasion inhibition assays revealed that anti-RALP1 antibodies inhibit the invasion of erythrocytes by merozoites. The findings that RALP1 possesses an erythrocyte-binding epitope in the C-terminal region and that anti-RALP1 antibodies disrupt tight-junction formation, are evidence that RALP1 plays an important role during merozoite invasion of erythrocytes. In addition, human sera collected from areas in Thailand and Mali where malaria is endemic recognized this protein. Overall, our findings indicate that RALP1 is a rhoptry neck erythrocyte-binding protein and that it qualifies as a potential blood-stage vaccine candidate.     

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.     

Parasite polypeptides lost during schizogony and erythrocyte invasion by the malaria parasites, Plasmodium chabaudi and Plasmodium knowlesi

By biosynthetically labelling Plasmodium chabaudi and P. knowlesi stage-specific polypeptides and allowing continued development, schizogony and reinvasion in vivo or in vitro, we have identified parasite polypeptides not taken into the erythrocyte by the invading mezozoite. Three major and two minor parasite polypeptides synthesized by rings or mid-stage trophozoites of P. chabaudi were either degraded preferentially during further development, or lost during schizogony and reinvasion. For both P. chabaudi and P. knowlesi, a 250 000 mol. wt. polypeptide synthesized during maturation of trophozoites to schizonts and merozoites was not taken into the erythrocyte by the invading merozoite. The late stage synthesis of this polypeptide by P. chabaudi and its loss at schizogony and reinvasion was confirmed by immunofluorescence staining with a monoclonal antibody to this antigen. The importance of these antigens in the erythrocyte invasion process and in the induction and expression of immunity to malaria is discussed.     

Secondary processing of the Plasmodium falciparum merozoite surface protein-1 (MSP1) by a calcium-dependent membrane-bound serine protease: shedding of MSP133 as a noncovalently associated complex with other fragments of the MSP1.

Merozoites of the malaria parasite Plasmodium falciparum possess on their surface proteolytically processed fragments of the merozoite surface protein-1 (MSP1). Secondary processing of one of these fragments, MSP1₄₂, always occurs prior to, or at the point of successful erythrocyte reinvasion. It is shown that a product of this secondary processing, MSP1₃₃, is shed in the form of a noncovalently-associated complex with a number of other proteins, including the MSP1-derived species MSP1₃₈ and MSP1₈₃. Secondary processing of MSP1₄₂, is inhibited by the chelating agents ethylenediaminetetraacetic acid (EDTA) and ethyleneglycol-bis-(β-aminoethyl ether)-tetraacetic acid (EGTA), and this inhibition is reversible by addition of excess calcium. Secondary processing occurs in preparations of washed, disrupted merozoites, and is inhibited by the protease inhibitors phenylmethylsulphonyl fluoride (PMSF) and diisopropyl fluorophosphate (DFP), indicating that the protease responsible is a membrane-associated serine protease.     

Secondary processing of the Plasmodium falciparum merozoite surface protein-1 (MSP1) by a calcium-dependent membrane-bound serine protease: shedding of MSP133 as a noncovalently associated complex with other fragments of the MSP1

Merozoites of the malaria parasite Plasmodium falciparum possess on their surface proteolytically processed fragments of the merozoite surface protein-1 (MSP1). Secondary processing of one of these fragments, MSP1₄₂, always occurs prior to, or at the point of successful erythrocyte reinvasion. It is shown that a product of this secondary processing, MSP1₃₃, is shed in the form of a noncovalently-associated complex with a number of other proteins, including the MSP1-derived species MSP1₃₈ and MSP1₈₃. Secondary processing of MSP1₄₂, is inhibited by the chelating agents ethylenediaminetetraacetic acid (EDTA) and ethyleneglycol-bis-(β-aminoethyl ether)-tetraacetic acid (EGTA), and this inhibition is reversible by addition of excess calcium. Secondary processing occurs in preparations of washed, disrupted merozoites, and is inhibited by the protease inhibitors phenylmethylsulphonyl fluoride (PMSF) and diisopropyl fluorophosphate (DFP), indicating that the protease responsible is a membrane-associated serine protease.     

Babesia bovis merozoite surface antigen 2 proteins are expressed on the merozoite and sporozoite surface,and specific antibodies inhibit attachment and invasion of erythrocytes

The Babesia bovis merozoite surface antigen 2 (MSA-2) locus encodes four proteins, MSA-2a(1), -2a(2), -2b, and -2c. With the use of specific antibodies, each MSA-2 protein was shown to be expressed on the surface of live extracellular merozoites and coexpression on single merozoites was confirmed. Individual antisera against MSA-2a, MSA-2b, and MSA-2c significantly inhibited merozoite invasion of bovine erythrocytes. As tick-derived sporozoites also directly invade erythrocytes, expression of each MSA-2 protein on the sporozoite surface was examined and verified. Finally, statistically significant inhibition of sporozoite binding to the erythrocytes was demonstrated by using antisera specific for MSA-2a, MSA-2b, and MSA-2c. These results indicate an important role for MSA-2 proteins in the initial binding and invasion of host erythrocytes and support the hypothesis that sporozoites and merozoites use common surface molecules in erythrocyte invasion.     

Binding of Plasmodium falciparum 175-kilodalton erythrocyte binding antigen and invasion of murine erythrocytes requires N-acetylneuraminic acid but not its O-acetylated form.

Sialic acid on human erythrocytes is involved in invasion by the human malaria parasite, Plasmodium falciparum. Mouse erythrocytes were used as a reagent to explore the question of whether erythrocyte sialic acid functions as a nonspecific negative charge or whether the sialic acid is a necessary structural part of the receptor for merozoites. Human erythrocytes contain N-acetylneuraminic acid (Neu5Ac), whereas mouse erythrocytes, which are also invaded by P. falciparum merozoites, contain 9-O-acetyl-N-acetylneuraminic acid (Neu5,9Ac2) and N-glycoloylneuraminic acid (Neu5Gc), in addition to Neu5Ac. We compared the effects of sialidase and influenza C virus esterase treatments of mouse erythrocytes on invasion and the binding of a 175-kDa P. falciparum protein (EBA-175), a sialic acid-dependent malaria ligand implicated in the invasion process. Sialidase-treated mouse erythrocytes were refractory to invasion by P. falciparum merozoites and failed to bind EBA-175. Influenza C virus esterase, which converts Neu5,9Ac2 to Neu5Ac, increased both invasion efficiency and EBA-175 binding to mouse erythrocytes. Thus, the parasite and EBA-175 discriminate between Neu5Ac and Neu5,9Ac2, that is, the C-9 acetyl group interferes with EBA-175 binding and invasion by P. falciparum merozoites. This indicates that sialic acid is part of a receptor for invasion.     

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.     

Identification of a Potent Combination of Key Plasmodium falciparum Merozoite Antigens That Elicit Strain-Transcending Parasite-Neutralizing Antibodies

Blood-stage malaria vaccines that target single Plasmodium falciparum antigens involved in erythrocyte invasion have not induced optimal protection in field trials. Blood-stage malaria vaccine development has faced two major hurdles, antigenic polymorphisms and molecular redundancy, which have led to an inability to demonstrate potent, strain-transcending, invasion-inhibitory antibodies. Vaccines that target multiple invasion-related parasite proteins may inhibit erythrocyte invasion more efficiently. Our approach is to develop a receptor-blocking blood-stage vaccine against P. falciparum that targets the erythrocyte binding domains of multiple parasite adhesins, blocking their interaction with their receptors and thus inhibiting erythrocyte invasion. However, with numerous invasion ligands, the challenge is to identify combinations that elicit potent strain-transcending invasion inhibition. We evaluated the invasion-inhibitory activities of 20 different triple combinations of antibodies mixed in vitro against a diverse set of six key merozoite ligands, including the novel ligands P. falciparum apical asparagine-rich protein (PfAARP), EBA-175 (PfF2), P. falciparum reticulocyte binding-like homologous protein 1 (PfRH1), PfRH2, PfRH4, and Plasmodium thrombospondin apical merozoite protein (PTRAMP), which are localized in different apical organelles and are translocated to the merozoite surface at different time points during invasion. They bind erythrocytes with different specificities and are thus involved in distinct invasion pathways. The antibody combination of EBA-175 (PfF2), PfRH2, and PfAARP produced the most efficacious strain-transcending inhibition of erythrocyte invasion against diverse P. falciparum clones. This potent antigen combination was selected for coimmunization as a mixture that induced balanced antibody responses against each antigen and inhibited erythrocyte invasion efficiently. We have thus demonstrated a novel two-step screening approach to identify a potent antigen combination that elicits strong strain-transcending invasion inhibition, supporting its development as a receptor-blocking malaria vaccine.     

Glycoprotein recognition mediates attachment of Plasmodium chabaudi to mouse erythrocytes

The interaction between Plasmodium falciparum merozoites and human erythrocytes is mediated by specific parasite proteins and sialoglycoproteins (SGPs) on the surface of the host cell. To investigate whether a similar mechanism functions in rodent malaria, a series of experiments was performed to identify the proteins involved in the interaction of Plasmodium chabaudi parasites and mouse erythrocytes. Labeled parasite proteins incubated with purified mouse SGP bound specifically to glycoprotein 2.1. Two parasite proteins (72 and 126 kilodaltons [kDa]) were coprecipitated with antibody directed to mouse erythrocyte membrane proteins. The lower band (72 kDa) as well as a band of 105 kDa were also observed to bind to N-acetyl-D-galactosamine affinity columns, suggesting a carbohydrate component in the binding of these parasites to erythrocytes. These experiments indicate that P. chabaudi possesses specific proteins which recognized SGP on the surface of murine erythrocytes in a manner similar to that of the merozoites of P. falciparum. Thus P. chabaudi in mice may provide an in vivo model of the human parasite for testing ways to inhibit merozoite recognition and invasion of host cells.     

Processing of the Plasmodium falciparum major merozoite surface protein-1: identification of a 33-kilodalton secondary processing product which is shed prior to erythrocyte invasion.

We have previously shown that only a single 19-kDa fragment of the Plasmodium falciparum major merozoite surface protein (MSP1) is carried with an invading merozoite into the infected red cell. This fragment (MSP1(19] is derived from the C-terminal membrane-bound end of a major product, MSP1(42), of the primary stage of MSP1 proteolytic processing. Using a monoclonal antibody mapped to an epitope within the N-terminal region of MSP1(42), we have shown that a soluble 33-kDa polypeptide (MSP1(33) corresponding to the N-terminal region of MSP1(42) is shed into culture supernatants during merozoite release and erythrocyte invasion. These observations provide further evidence that the secondary processing of MSP1(42) involves a highly site-specific proteolytic activity.     

The 22 kDa component of the protein complex on the surface of Plasmodium falciparum merozoites is derived from a larger precursor, merozoite surface protein 7

The gene coding for merozoite surface protein 7 has been identified and sequenced in three lines of Plasmodium falciparum. The gene encodes a 351 amino acid polypeptide that is the precursor of a 22-kDa protein (MSP7(22)) on the merozoite surface and non-covalently associated with merozoite surface protein 1 (MSP1) complex shed from the surface at erythrocyte invasion. A second 19-kDa component of the complex (MSP7(19)) was shown to be derived from MSP7(22) and the complete primary structure of this polypeptide was confirmed by mass spectrometry. The protein sequence contains several predicted helical and two beta elements, but has no similarity with sequences outside the Plasmodium databases. Four sites of sequence variation were identified in MSP7, all within the MSP7(22) region. The MSP7 gene is expressed in mature schizonts, at the same time as other merozoite surface protein genes. It is proposed that MSP7(22) is the result of cleavage by a protease that may also cleave MSP1 and MSP6. A related gene was identified and cloned from the rodent malaria parasite, Plasmodium yoelii YM; at the amino acid level this sequence was 23% identical and 50% similar to that of P. falciparum MSP7.     

Truncation of Plasmodium berghei merozoite surface protein 8 does not affect in vivo blood-stage development

Merozoite surface protein 8 (MSP8) has shown promise as a vaccine candidate in the Plasmodium yoelii rodent malaria model and has a proposed role in merozoite invasion of erythrocytes. However, the temporal expression and localisation of MSP8 are unusual for a merozoite antigen. Moreover, in Plasmodium falciparum the MSP8 gene could be disrupted with no apparent effect on invitro growth. To address the invivo function of full-length MSP8, we truncated MSP8 in the rodent parasite Plasmodium berghei. PbDeltaMSP8 disruptant parasites displayed a normal blood-stage growth rate but no increase in reticulocyte preference, a phenomenon observed in P. yoelii MSP8 vaccinated mice. Expression levels of erythrocyte surface antigens were similar in P. berghei wild-type and PbDeltaMSP8-infected erythrocytes, suggesting that a parasitophorous vacuole function for MSP8 does not involve global trafficking of such antigens. These data demonstrate that a full-length membrane-associated form of PbMSP8 is not essential for blood-stage growth.     

Expression of Plasmodium falciparum genes involved in erythrocyte invasion varies among isolates cultured directly from patients

Plasmodium falciparum merozoites invade erythrocytes using a range of alternative ligands that includes erythrocyte binding antigenic proteins (EBAs) and reticulocyte binding protein homologues (Rh). Variation in the expression of some of these genes among culture-adapted parasite lines correlates with the use of different erythrocyte receptors. Here, expression profiles of four Rh genes and eba175 are analysed in a sample of 42 isolates cultured from malaria patients in Kenya. The profiles cluster into distinct groups, largely because of very strong negative correlations between the levels of expression of particular gene pairs (Rh1 versus Rh2b, eba175 versus Rh2b, and eba175 versus Rh4), previously associated with alternative invasion pathways in culture-adapted parasite lines. High levels of eba175 are seen in isolates in expression profile group I, and may be associated with sialic acid-dependent invasion. Groups II and III are, respectively, characterized by high levels of Rh2b and Rh4, and are more likely to be associated with sialic acid-independent invasion.