An antigenic complex in the rhoptries of Plasmodium falciparum

A previously identified putative rhoptry antigen of Plasmodium falciparum is composed of two major components, one of 80 kDa and a doublet at 42/40 kDa. An inhibitory monoclonal antibody immunoprecipitated both the 80 kDa protein and the 42/40 kDa doublet, but immunoblotted only the 80 kDa component. A second monoclonal antibody, raised against the affinity purified complex, immunoblotted only the 42 kDa band under non-reducing conditions. Electron microscopic examination of thin sections of parasites immunolabeled with these monoclonal antibodies and colloidal gold anti-mouse conjugate has confirmed that this antigen is localised in the rhoptry organelles of mature schizonts and free merozoites. The antigen is associated with apparent membranous structures released from free merozoites. Immunoblotting and immunoprecipitation with two different monoclonal antibodies, and protease digestion experiments, have clearly demonstrated that this antigen is a complex composed of two separate and distinct proteins, and does not represent a monomer/dimer pair. The 80 kDa protein is synthesised as an 84 kDa precursor.     

Two Plasmodium falciparum merozoite surface polypeptides share epitopes with a single Mr 185 000 parasite glycoprotein

The malarial parasite Plasmodium falciparum synthesizes a major glycoprotein (gp) of Mr 185 000 during its asexual blood cycle. Immunoprecipitation of [35S]methionine- or [3H]glucosamine-labeled schizont antigens indicated that two groups of polypeptides were distinguished with anti-gp 185 mouse monoclonal antibodies: group A was composed of glycosylated molecules of Mr 185 000, 120 000, 90 000, 88 000, 46 000, and 40 000 while group B contained, in addition to gp 185, polypeptides of Mr 152 000, 106 000 and 83 000. The latter polypeptides lacked detectable amounts of radiolabeled saccharide. The smaller Mr polypeptides were specifically immunoprecipitated and not merely coprecipitated with gp 185. Our results suggest that gp 185 contains at least two structurally distinct domains which may be processed independently into either the group A or group B polypeptides. Although gp 185 may not be a merozoite surface protein, representative group A and group B-specific monoclonal antibodies bound to surface antigens of the merozoite as demonstrated by immunolabeling followed by electron microscopy. Therefore, at least one group A antigen and one group B antigen appeared to be on the extracellular surface of the merozoite. The proteins found in immunoprecipitates after both (1) sonication in aqueous medium and ultracentrifugation and (2) solubilization and phase separation of parasite molecules with Triton X-114 suggested that the group A and group B polypeptides and glycoproteins are either soluble or peripheral membrane proteins. Some of these, therefore, may be components of the surface coat of the merozoite.     

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.     

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.     

Structural and antigenic properties of merozoite surface protein 4 of Plasmodium falciparum

Merozoite surface protein 4 (MSP4) of Plasmodium falciparum is a glycosylphosphatidylinositol-anchored integral membrane protein of 272 residues that possesses a single epidermal growth factor (EGF)-like domain near the carboxyl terminus. We have expressed both full-length MSP4 and a number of fragments in Escherichia coli and have used these recombinant proteins to raise experimental antisera. All recombinant proteins elicited specific antibodies that reacted with parasite-derived MSP4 by immunoblotting. Antibody reactivity was highly dependent on the protein conformation. For example, reduction and alkylation of MSP4 almost completely abolished the reactivity of several antibody preparations, including specificities directed to regions of the protein that do not contain cysteine residues and are far removed from the cysteine-containing EGF-like domain. This indicated the presence of conformation-dependent epitopes in MSP4 and demonstrated that proper folding of the EGF-like domain influenced the antigenicity of the entire molecule. The recombinant proteins were used to map epitopes recognized by individuals living in areas where malaria is endemic, and at least four distinct regions are naturally antigenic during infection. Binding of human antibodies to the EGF-like domain was essentially abrogated after reduction of the recombinant protein, indicating the recognition of conformational epitopes by the human immune responses. This observation led us to examine the importance of conformation dependence in responses to other integral membrane proteins of asexual stages. We analyzed the natural immune responses to a subset of these antigens and demonstrated that there is diminished reactivity to several antigens after reduction. These studies demonstrate the importance of reduction-sensitive structures in the maintenance of the antigenicity of several asexual-stage antigens and in particular the importance of the EGF-like domain in the antigenicity of MSP4.     

Surface charge of Plasmodium falciparum merozoites as revealed by atomic force microscopy with surface potential spectroscopy

Electric charges on the surface of Plasmodium falciparum merozoites and erythrocytes were investigated by atomic force microscopy with surface potential spectroscopy. The apical end of merozoites was positively charged, while the entire erythrocyte surface was negatively charged. Transmission electron microscopy also demonstrated that negatively charged nanogold particles attached to the apical end of merozoites, and cationized ferritin particles attached to the entire surface of the erythrocyte. This indicates that the surface charge at the apical end of the merozoite may play an important role in invasion of the erythrocyte.     

Immunological fine structure of the variable and constant regions of a polymorphic malarial surface antigen from Plasmodium falciparum.

The 51-kDa merozoite surface antigen MSA2 of Plasmodium falciparum shows considerable strain-dependent polymorphism. Although marked sequence variation occurs in the central region of the molecule, the N and C-terminal sequences are highly conserved. A number of monoclonal antibodies directed against MSA2 have been described which inhibit parasite growth in vitro, but these are all directed against variable regions. In an attempt to raise strain independent antibodies we have prepared peptide-diphtheria toxoid (DT) constructs from 36 N-terminal octapeptides spanning the constant region and extending into the variable region of the FCQ/27 PNG variant staggered by one amino acid at either end. Similarly, we prepared 26 C-terminal octapeptides spanning the C-terminal constant region as well as 10 octapeptides from the variable region of the Indochina I variant MSA2. Most of the peptides elicited antipeptide titres in excess of 1/10(4) when administered to mice as peptide-DT adducts emulsified with Freund's complete adjuvant. Only 3 of the 43 N- and C-terminal constant region peptides elicited antibodies which reacted appropriately on immunofluorescence (IFA) or immunoblotting analysis with the intact MSA2 of both strains studied (FCQ/27 and Indochina I), whereas 3 other peptides from the variable region elicited antibodies reactive with the parent MSA2 only. Peptide constructs eliciting antibodies recognising the intact protein corresponded to elements in the cognate sequence of high antigenicity as predicted by the Jameson and Wolf algorithm.     

Identification and characterisation of proteins associated with the rhoptry organelles of Plasmodium falciparum merozoites

We describe antigens of Plasmodium falciparum recognised by murine monoclonal antibodies which by immunofluorescence react with the rhoptry organelles of the extracellular merozoite stage. Immunoblotting shows that the antibodies recognise two major parasite antigens of M_r 82 and 65 kilodaltons (kDa). Immunoprecipitations from detergent extracts of [³⁵S]-methioninelabelled parasites show that the 82-kDa and 65-kDa antigens are parasite proteins. Pulse-chase experiments on synchronous parasite cultures show that the 82-kDa protein is synthesised during early schizogony and is later processed into the 65-kDa antigen in segmenting schizonts. In Nonidet P-40, these antigens are non-covalently associated with two other proteins of 40 kDa and 42 kDa. The 40/42-kDa doublet is synthesised in parallel with the 82 kDa antigen and persists, apparently unchanged, till the end of the cell cycle.Abbreviations HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - EDTA ethylenediaminetetraacetic acid - EGTA ethyleneglycolbis-(aminoethylether) tetraacetic acid - PMSF phenylmethylsulphonylfluoride - TLCK tosyl-L-lysine chloromethyl ketone - SDS-PAGE sodium dodecyl sulphate polyacrylamide gel electrophoresis - M _r relative molecular mass     

Patterns of polymorphism in genomic regions flanking three highly polymorphic surface antigens in Plasmodium falciparum

Many surface antigens of the human malaria parasite Plasmodium falciparum show extraordinary diversity, with different alleles being so divergent as to be unalignable in some coding regions. To better understand the population history and modes of selection on such loci, we sequenced genomic regions flanking the highly polymorphic genes merozoite surface protein-1, merozoite surface protein-2, and circumsporozoite protein, from reference isolates of P. falciparum. Diversity was much lower in genomic flanking regions than in the coding sequences. Average pairwise nucleotide diversity for these regions was 0.00088, similar to other genomic regions not thought to be evolving under balancing selection, suggesting against balancing selection acting on promoter regions of these genes. Most observed polymorphisms were singletons. A higher ratio of SNPs to indels than previously reported for P. falciparum was observed. An 11 bp repeat upstream of msp2 showed an intriguing pattern of polymorphism possibly suggestive of purifying selection on total allele length.     

Membrane orientation and antigenic peptides of an immunoprotective 74 kDa Plasmodium knowlesi glycoprotein

Vaccination trials have shown that a purified, 74 kDa glycoprotein, GP74, isolated from the host cell membrane of Plasmodium knowlesi-infected rhesus erythrocytes, can provide protective immunity against P. knowlesi malaria. We have extended this work by a tryptic peptide analysis of the disposition of GP74 in the host cell membrane. Of the 18 peptides characterized by high-performance liquid chromatography only four were accessible to lactoperoxidase-catalyzed radioiodination of non-leaky, schizont-infected host cells from the extracellular space. Metabolic labeling with radioactive glucosamine indicates that two of the surface exposed peptides are glycopeptides, and one of these, peptide 12 appears to carry a dominant antigenic site, according to its reactivity with immunoglobulin from sera of monkeys protected against P. knowlesi malaria.     

Purification and characterization of an aminopeptidase from Plasmodium falciparum

A soluble aminopeptidase from Plasmodium falciparum was purified by high performance liquid chromatography. The enzyme has a molecular weight of 100 000 and pI 6.8. Activity can be monitored conveniently with L-alanine-p-nitroanilide or L-leucine-p-nitroanilide at 405 nm or with L-leucine-7-amido-4-methylcoumarin in a fluorescence assay. The enzyme is inhibited by bestatin and phosphoramidone but not by leupeptin, chymostatin, antipain or pepstatin. pH-rate studies indicated the presence of a group on the free enzyme, pKa = 6.6, which must be in the conjugate base form for activity. The aminopeptidase has an essential sulfhydryl group at the active site which is rapidly modified by Hg2+ or Zn2+, is slowly modified by p-hydroxymercuribenzoate, but is not accessible to iodoacetamide or N-ethylmaleimide. The aminopeptidase is inhibited noncompetitively by chloroquine, mefloquine and quinacrine (Ki = 410, 280 and 20 microM, respectively) but is not inhibited by quinine or primaquine. Hemin does not inhibit. Complexation of hemin with quinacrine prevents inhibition by quinacrine.     

Plasmodium falciparum: surface modifications of infected erythrocytes from clinical isolates

 Infections of human erythrocytes with the mature asexual blood stages of Plasmodium falciparum result in antigenic changes in the host cell membrane that, by virtue of their position, length of exposure, and close association with functional changes critical to pathogenesis, are a potential important target for host effector mechanisms. These parasite-induced antigens expressed on the surface of infected erythrocytes have been shown to exhibit considerable polymorphism. An antibody-mediated agglutination assay using malaria serum samples from different regions of Venezuela has been developed to examine the extent of antigenic diversity of infected red blood cells (IRBC) taken from subjects with naturally acquired P. falciparum infections. An important humoral immune recognition of surface molecules from red blood cells infected with a wide variety of clinical isolates of P. falciparum was observed even when sera from individuals experiencing a single episode of malaria were used. A process of in vivo antigenic variation of surface molecules is postulated, since agglutination of IRBC was observed with acute heterologous but not autologous sera. When sera obtained from Amerindians inhabiting the Venezuelan Amazon were assayed, a strong immune response to different parasite isolates, including those of another geographic region, was observed, suggesting the recognition of highly conserved immunogenic parasitic epitopes in people exposed to multiple malaria infections. Received: 20 June 1995 / Accepted: 3 November 1995     

Role of the carbohydrate domains of glycophorins as erythrocyte receptors for invasion by Plasmodium falciparum merozoites.

Solubilized preparations of purified glycophorins and specific domains of these molecules were assessed for their effects as inhibitors of Plasmodium falciparum invasion of human erythrocytes in vitro. The ability of newly invaded merozoites to continue developing and incorporating [3H]hypoxanthine during a 24-h period after their invasion was used as an assay for merozoite invasion. Glycophorins A, B, and C were found to be equally effective as inhibitors. Previous studies had shown N-acetylglucosamine, a sugar component of glycophorins A and C but not B, to be an effective inhibitor. Accordingly, molecular domains common to all of the glycophorins were further assessed. Sialic acid was shown to act almost as effectively as N-acetylglucosamine, presumably because of the structural similarities between these sugars. The inhibitory ability of sialic acid is considerably enhanced when presented to the parasite in a clustered form, as in an oligosaccharide. The acetyl group of these sugars does not appear to play an essential role in this inhibition. How the P. falciparum merozoite recognizes and interacts with the sugar domains of the glycophorin molecule remains to be determined.     

Identification of surface and internal antigens from spontaneously releasedPlasmodium falciparum merozoites by radio-iodination and metabolic labelling

Spontaneously released merozoites from synchronousPlasmodium falciparum cultures were isolated in the presence of protease blocker. 1–5×10¹⁰ merozoites were obtained in each experiment. The isolated merozoites possessed a thick surface coat and about 80% were invasive to human erythrocytes although they did not subsequently develop into ring stages. Tests using several analytical methods showed the merozoite preparations to be free of any erythrocyte contamination. Six labelled proteins were identified after surface radio-iodination, the largest with a molecular weight of 82000. All six proteins were precipitated with various immune sera. Four other proteins with molecular weights of 200000 and 160000–145000 (a triplet) were identified by precipitation with the same immune sera after metabolically labelling the merozoites. The six surface proteins were not prominent in the metabolically labelled preparations. Using these methods it is possible to identify and differentiate between surface and internal merozoite antigens.     

Studies on the role of red blood cell glycoproteins as receptors for invasion by Plasmodium falciparum merozoites

The mechanism of invasion of human red blood cells by Plasmodium falciparum merozoites has been studied by several indirect methods. Red blood cells of the S+s+U+ and S-s-U- blood group phenotypes were trypsin treated and their susceptibility to invasion measured. Trypsin-treated S+s+U+ cells lack the portion of glycophorin A which bears the MN blood group determinants but possess glycophorin B, whereas trypsin-treated S-s-U- cells lack both the glycophorin A MN determinants and the glycophorin B molecule. Since the treated S-s-U- cells showed an even greater loss in susceptibility to invasion that the treated S+s+U+ cells, we conclude that glycophorin B does have a role In merozoite recognition, although it appears less important than glycophorin A. Attempts to decrease invasion by pretreatment with glycosidases were unsuccessful, except for the previously reported effect of neuraminidase. N-acetyl-D-glucosamine decreases the appearance of ring-stage parasites after in vitro reinvasion of P. falciparum. However, the persistence of intact and lysed schizont-infected cells when N-acetyl-D-glucosamine was present, several hours after disappearance of these cells from control cultures, leads us to conclude that this sugar has a deleterious effect on terminal stages of parasite maturation. It is therefore not possible to conclude that N-acetyl-D-glucosamine inhibits merozoite attachment and reinvasion specifically by competition for the receptor.     

Complement activation by the surface of Plasmodium falciparum infected erythrocytes

The surface of trophozoite-stage Plasmodium falciparum infected erythrocytes will, in the presence of immune human or owl monkey serum, activate the classical complement pathway. This was demonstrated with a sensitive, enzyme-linked immunosorbent assay which detects the complex, C1s-C1 inhibitor, which is only generated when the classical pathway is activated. A second enzyme-linked immunosorbent assay, as well as Covaspheres coated with affinity-purified anti-C3, showed that immune activation of the classical pathway by infected erythrocytes resulted in the accumulation of significant amounts of C3b on the erythrocyte surface. During the development of the parasite to the trophozoite stage, the erythrocyte membrane is also transformed from a non-activator into a surface capable of activating complement by the alternative pathway. Erythrocytes infected with trophozoite-stage parasites directly activated the alternative complement pathway. This activation led to the specific binding of an average of 15,000 C3b molecules per infected cell. Alternative pathway activation was augmented by anti-parasite antibody. Such conditions mediated the accumulation of an average of 36,000 C3b molecules per infected erythrocyte. The amounts of C3b on the infected erythrocyte surface did not lead to cellular lysis. They are, however, likely to have a major impact on the total in vivo response to this parasite.