A naturally occurring gene encoding the major surface antigen precursor p190 of Plasmodium falciparum lacks tripeptide repeats.

Plasmodium falciparum merozoites have variable surface proteins that are processed from a 190-kd precursor protein (p190). The gene encoding p190 exists in two allelic forms and cross-over events occurring mainly near the 5' end, combined with isolate-specific tripeptide repeats, contribute to its antigen diversity. We have sequenced a large portion of the p190 gene from the parasite isolate RO-33 (Ghana). Remarkably, the typical N-terminal tripeptide repeat structure is lacking. Apart from mutations in the variable parts, the gene appears identical to the MAD-20 allele (Papua, New Guinea). Southern blot analysis detects p190 genes similar to RO-33 in other parasite isolates independent of their geographical origin. The lack of p190 repeats in RO-33 eliminates the possibility that they are involved in host cell recognition or integration and restricts their function to immune escape.     

Allelic dimorphism in a surface antigen gene of the malaria parasite Plasmodium falciparum

Merozoites of the malaria parasite Plasmodium falciparum carry surface proteins processed from a precursor termed p190 or p195. Polymorphism has been reported in this protein. Since the protein is a candidate for a malaria vaccine, it is important to understand the nature of this polymorphism. We have determined the complete nucleotide sequence of the p190 gene from the MAD20 strain (a Papua New Guinea isolate). Comparisons of the gene with that from other strains of P. falciparum allowed us to study the genetic basis of the antigen's polymorphism. The gene consists of sequences distributed in variable blocks, which are separated by conserved or semi-conserved sequences. Variable sequences occur both in regions that code for tripeptide repeats and in regions with no apparent repeats. Interestingly, according to the present data, variable sequences are not widely polymorphic but fall into two distinct types. We argue that the p190 protein is encoded by dimorphic alleles capable of limited genetic exchange and present evidence at the nucleotide level documenting intragenic recombination in Plasmodium.     

Third form of the precursor to the major merozoite surface antigens of Plasmodium falciparum.

The precursor to the major merozoite surface antigens of Plasmodium falciparum appears to be encoded by two distinctly different (dimorphic) alleles able to undergo limited recombination. We analyzed 18 previously uncharacterized P. falciparum isolates to test the dimorphic model. All but one, a Thailand isolate, conformed to the dimorphic model, and this isolate conformed to the dimorphic model in all but variable block 2. Sequence analysis revealed that block 2 of isolate CSL2 was a third form. Hence, the dimorphic model is not strictly correct. Recombination between alleles was found only within two conserved blocks near the 5' end of the gene.     

A principal target of human immunity to malaria identified by molecular population genetic and immunological analyses

New strategies are required to identify the most important targets of protective immunity in complex eukaryotic pathogens. Natural selection maintains allelic variation in some antigens of the malaria parasite Plasmodium falciparum. Analysis of allele frequency distributions could identify the loci under most intense selection. The merozoite surface protein 1 (Msp1) is the most-abundant surface component on the erythrocyte-invading stage of P. falciparum. Immunization with whole Msp1 has protected monkeys completely against homologous and partially against non-homologous parasite strains. The single-copy msp1 gene, of about 5 kilobases, has highly divergent alleles with stable frequencies in endemic populations. To identify the region of msp1 under strongest selection to maintain alleles within populations, we studied multiple intragenic sequence loci in populations in different regions of Africa and Southeast Asia. On both continents, the locus with the lowest inter-population variance in allele frequencies was block 2, indicating selection in this part of the gene. To test the hypothesis of immune selection, we undertook a large prospective longitudinal cohort study. This demonstrated that serum IgG antibodies against each of the two most frequent allelic types of block 2 of the protein were strongly associated with protection from P. falciparum malaria.     

Positive selection and interallelic recombination at the merozoite surface antigen-1 (MSA-1) locus of Plasmodium falciparum.

DNA sequences of alleles at the merozoite surface antigen-1 (MSA-1) gene locus of the malaria parasite Plasmodium falciparum show evidence of repeated past recombination events between alleles. These include both (1) nonreciprocal recombination events that have homogenized certain gene regions among alleles and (2) reciprocal recombination events that have combined allelic segments with divergent evolutionary histories, thereby enhancing allelic diversity. In three different gene regions, the rate of nonsynonymous nucleotide substitution significantly exceeds that of synonymous nucleotide substitution, implying that positive Darwinian selection has acted to diversify alleles at the amino acid level. The MSA-1 polymorphism seems to be quite ancient; the two major allelic types have been maintained for approximately 35 Myr.     

Structural and antigenic polymorphism of the 35- to 48-kilodalton merozoite surface antigen (MSA-2) of the malaria parasite Plasmodium falciparum.

Merozoite surface antigen MSA-2 of the human parasite Plasmodium falciparum is being considered for the development of a malaria vaccine. The antigen is polymorphic, and specific monoclonal antibodies differentiate five serological variants of MSA-2 among 25 parasite isolates. The variants are grouped into two major serogroups, A and B. Genes encoding two different variants from serogroup A have been sequenced, and their DNA together with deduced amino acid sequences were compared with sequences encoded by other alleles. The comparison shows that the serological classification reflects differences in DNA sequences and deduced primary structure of MSA-2 variants and serogroups. Thus, the overall homologies of DNA and amino acid sequences are over 95% among variants in the same serogroup. In contrast, similarities between the group A variants and a group B variant are only 70 and 64% for DNA and amino acid sequences, respectively. We propose that the MSA-2 protein is encoded by two highly divergent groups of alleles, with limited additional polymorphism displayed within each group.     

The expression of Plasmodium falciparum bloodstage antigens in Escherichia coli

A library of cDNA clones expressing proteins of the asexual blood stages of a Papua New Guinean isolate of Plasmodium falciparum (isolate FCQ27/PNG (FC27] was constructed in the bacteriophage vector lambda gt11-Amp3. In an in situ colony immunoassay, human serum was used to identify colonies producing natural immunogens. Sera from donors of defined clinical status, or reactive to a defined subset of natural immunogens were used to identify clones of particular interest (for example, clones reacting with convalescent but not with acute serum or clones expressing the isolate specific S-antigen of FC27). Antisera raised by immunizing mice and rabbits with cloned antigens were used to characterize the P. falciparum proteins corresponding to the antigen-positive clones. Nucleotide sequence analysis of an antigen found on the surface of cells infected with ring stage parasites revealed an unusual sequence coding for eight, four and three amino acid repeats rich in acidic amino acids. The discussion centres on the use of cloned antigens as tools for the analysis of the host-protective immune response and selection of candidate vaccine molecules.     

Genetic diversity in merozoite surface protein (MSP)-1 and MSP-2 genes of Plasmodium falciparum in a major endemic region of Iran

Merozoite surface protein-1 (MSP-1) and merozoite surface protein-2 (MSP-2) were used to develop vaccines and to investigate the genetic diversity in Plasmodium falciparum malaria in Iran. Nested polymerase chain reaction amplification was used to determine polymorphisms of block 2 of the MSP-1 and the central domain of MSP-2 genes. A total of 67 microscopically positive P. falciparum infected individuals from a major endemic region, southeast Iran, were included in this trial. Nine alleles of MSP-1 and 11 alleles of MSP-2 were identified. The results showed that amplified product from these surface antigen genes varied in size and there was specific pattern for each isolate. Besides, regarding this pattern, 23 multiple infections with at least 2 alleles were observed. While the endemic regions of malaria in Iran is classified in low to moderate group, but extensive polymorphism was observed for each marker and the MSP-2 central repeat was the most diverse that could be considered in designing malaria vaccine.     

A conserved region of the MSP-1 surface protein of Plasmodium falciparum contains a recognition sequence for erythrocyte spectrin.

The major surface protein MSP-1 of Plasmodium falciparum blood-stage malaria parasites contains notably conserved sequence blocks with unknown function. The recombinant protein 190L, which represents such a block, exhibits a high affinity for red blood cell membranes. We demonstrate that both 190L and native MSP-1 protein bind to the inner red blood cell membrane skeleton protein spectrin. By using overlapping peptides covering the 190L molecule, we show that the spectrin contact site of 190L is included in a linear sequence of 30 amino acid residues. Association of 190L with naturally occurring spectrin deficient red blood cells is drastically reduced. In the same cells parasite invasion is normal, but the intracellular parasite development arrests late in the trophozoite stage. A similar situation arises when synthetic peptides covering the spectrin recognition sequence of 190L are added to P.falciparum cultures. These data and the cellular localization of MSP-1 suggest the possibility that MSP-1 associates with spectrin under natural conditions.     

Differential selective pressures on the merozoite surface protein 2 locus of Plasmodium falciparum in a low endemic area

195 Plasmodium falciparum merozoite surface protein-2 alleles collected in Tak Province, Thailand, in 1996 and 2006 revealed extremely limited sequence polymorphism except in the variable (V) region, which defines the two allelic families 3D7 and FC27. This pattern is most easily explained by repeated inter-allelic gene conversion events homogenizing alleles outside the V region. Comparison of synonymous and nonsynonymous differences in V regions within allelic families supported the hypothesis that amino acid sequence polymorphism in this region is selectively favored. The pattern of sequence differentiation supported the hypothesis that repeats in the V region have evolved by concerted evolution in the 3D7 family but not in the FC27 family. In the FC27 family two alleles of relatively high frequency were the most common V-region alleles in both 1996 and 2006, while 3D7 alleles constituted a significantly greater proportion of the sequences collected in 2006 (56.1%) than of those collected in 1996 (28.9%). These changes in the frequencies of 3D7 alleles may reflect increased intensity of selection on the P. falciparum population in Thailand as a result of effective control measures that have sharply reduced the incidence of malaria infection.     

Highly reiterated non-coding sequence in the genome of Plasmodium falciparum is composed of 21 base-pair tandem repeats

Clones containing highly reiterated DNA sequences were isolated from a Plasmodium falciparum genomic library. One clone, Rep2, was selected for further characterization by nucleotide sequence analysis. The results revealed that the insert of this clone is composed of tandemly arranged 21 base-pair imperfect repeats. These repeats are estimated to comprise approximately 1% of the P. falciparum genome and there are 10(4) to 2 X 10(5) copies, depending on the genome size estimate used for calculation. Moreover, the repeats are organized in clusters and do not appear to be transcribed in non-synchronized P. falciparum cultures.     

Conservation and antigenicity of N-terminal sequences of GP185 from different Plasmodium falciparum isolates

Complementary DNA (cDNA) clones for GP185, a major antigenically diverse glycoprotein of Plasmodium falciparum, were isolated from a cDNA library of the Honduras I/CDC (Honduras I) isolate, and 1052 bp were sequenced. The expression of cDNA fragments in Escherichia coli using the vector pCQV2 allowed verification of the reading frame. This GP185 cDNA sequence, like the cDNA sequence for a homologous gene of the K1 isolate [Hall et al., Nature 311 (1984) 379-382], codes for a polypeptide which is truncated due to multiple, in-frame stop codons. This polypeptide corresponds to the N-terminal 15% of the proposed coding region of the GP185 gene [Holder et al., Nature 317 (1985) 270-273]. Comparison of the nucleotide sequences for the GP185 gene of Honduras I and five other isolates indicated that there are two areas of conserved DNA sequence, one of 310 bp (beginning 181 bp upstream from the proposed initiation codon) and the other of greater than or equal to 360 bp (located entirely within the coding region), separated by a region encoding isolate-specific tandem amino acid repeats. Rat antiserum was raised to a fusion protein derived from the conserved regions and the intervening repeat region of this Honduras I protein. This antiserum bound GP185 on immunoblots of the homologous Honduras I isolate and the heterologous K1 isolate, which has different tandem repeats. Serum from owl monkeys and humans previously infected with P. falciparum reacted with the fusion protein on immunoblots demonstrating that determinants in the N-terminal 15% of GP185 were immunogenic in infected individuals and suggesting that some of these sites are conserved among isolates.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification of a rare point mutation at C-terminus of merozoite surface antigen-1 gene of Plasmodium falciparum in eastern Indian isolates

Merozoite surface antigen-1 (MSA-1) of Plasmodium falciparum is highly immunogenic in human. Several studies suggest that MSA-1 protein is an effective target for a protective immune response. Attempt has been made to find new point mutations by analyzing 244 bp [codon 1655(R) to 1735 (I)] relatively conserved C-terminus region of MSA-1 gene in 125 isolates. This region contains two EGF like domains, which are involved in generating protective immune response in human. Point mutations in this region are very much important in view of vaccine development. Searching of mutational hot spots in MSA-1 protein by sequencing method in a representative number of isolates is quite critical and expensive. Therefore, in this study slot blot and PCR-SSCP method have been used to find out new mutations in the individual isolates showing alterations in the mobility of DNA fragment. Sequencing of the altered bands from the SSCP gel shows a rare non-synonymous point mutation in 7 (5.6%) of the 125 isolates at amino acid position 1704 of MSA-1 gene where isoleucine is replaced by valine.     

Molecular Characterization of a Plasmodium chabaudi Erythrocyte Membrane-Associated Protein with Glutamate-Rich Tandem Repeats

The malarial parasite dramatically affects the structure and function of the erythrocyte membrane by exporting proteins that specifically interact with the host membrane. This report describes the complete sequence and some biochemical properties of a 93-kDa Plasmodium chabaudi chabaudi protein that interacts with the host erythrocyte membrane. Approximately 40% of the deduced protein sequence consists of tandem repeats of 14 amino acids that are rich in glutamic acid residues. Comparison of the repeat sequences from two different P. c. chabaudi strains derived from the same initial isolate revealed an exact duplication of 294 nucleotides suggesting a recent gel electrophoresis and gel filtration chromatography suggest that the protein is a long rod-shaped or fibrillar. protein. Attributes shared between the 93-kDa protein, some P. falciparum proteins with glutamate-rich tandem repeats, and cytoskeletal proteins suggest that these parasite proteins function as cytoskeletal proteins that possibly stabilize the erythrocyte membrane.     

Polymorphism of the precursor for the major surface antigens of Plasmodium falciparum merozoites: studies at the genetic level.

The gene for the precursor of Plasmodium falciparum merozoite surface antigens has been cloned. The entire sequence of the gene from a Thai isolate of the parasite is reported. It provides evidence for a signal peptide, a region containing short repeating peptides and an anchor sequence. In addition, the 5' end of a Papua New Guinea isolate has been sequenced. Comparison of these and other sequences defines, at the genetic level, a polymorphic region in the protein, and suggests that other parts of the protein are less susceptible to variation. Furthermore it appears that several signal peptides of P. falciparum exhibit extensive sequence homologies.     

The 230-kDa gamete surface protein of Plasmodium falciparum is also a target for transmission-blocking antibodies

Immunization with extracellular sexual stages of the malaria parasites can induce the production of antibodies which block the development of the parasites in the midgut of a mosquito after a blood meal. We have generated a number of monoclonal antibodies against gametes and zygotes of the human malaria Plasmodium falciparum. Two monoclonal antibodies (mAb) reacting with a 230-kDa gamete surface protein (mAb 1B3 and 2B4 both isotype IgG2a) were found to block transmission of P. falciparum to mosquitoes. Blocking was complement dependent and this was verified in vitro by the rapid lysis of newly formed gametes and zygotes in the presence of the mAb and active complement. Both mAb reacted by immunofluorescence with the surface of gametes and zygotes from isolates of P. falciparum from various geographical areas. Each mAb immunoprecipitated a 230-kDa protein from 125I-labeled surface proteins of newly formed gametes and zygotes and immunoblotted a protein doublet of about molecular mass 260 and 230 kDa from gametocytes and gametes of P. falciparum. Only the 230-kDa protein is expressed on the surface of newly formed macrogametes and zygotes. The 230-kDa gamete surface protein forms a molecular complex with two proteins of 48 and 45 kDa. The 48- and 45-kDa gamete surface proteins have previously been shown to be targets of mAb which block infectivity of P. falciparum to mosquitoes. The present study now demonstrates that antibodies against the 230-kDa gamete surface protein block transmission of P. falciparum to mosquitoes. The 230-kDa gamete protein is thus a potential candidate for a gamete vaccine.     

Plasmodium falciparum: a simple polymerase chain reaction method for differentiating strains.

Laboratory studies of the protozoan parasite Plasmodium falciparum have been hampered by difficulties in defining differences between isolates. We have developed a method based on the polymerase chain reaction (PCR) that allowed us to identify quickly the various strains with which we routinely work. We also adapted methods for easily purifying enough DNA to produce a PCR product from a small volume of culture: 100 microliters of an in vitro culture infected at 1% parasitemia. The primers were chosen from conserved regions flanking the variable repeats in four cloned genes, RESA, MSA-1, MSA-2, and CSP. The PCR products amplified from three of these genes differed in size and allowed us to identify particular isolates on this basis alone. The variation was between strains, and not a reflection of genetic instability during in vitro culture of one isolate. The method is sufficiently sensitive to detect a 1% contamination of one strain with another, an advantage for monitoring the integrity of strains when different isolates are grown in the same laboratory. The technical ease and speed of this assay and the small amount of culture required make it ideal for monitoring strains in the laboratory.     

Interaction of the 140/130/110 kDa rhoptry protein complex of Plasmodium falciparum with the erythrocyte membrane and liposomes

During Plasmodium falciparum merozoite invasion into human and mouse erythrocytes, a 110-kDa rhoptry protein is secreted from the organelle into the erythrocyte membrane. In the present study our interest was to examine the interaction of rhoptry proteins of P. falciparum with the erythrocyte membrane. It was observed that the complex of rhoptry proteins of 140/130/110 kDa bind directly to a trypsin sensitive site on intact mouse erythrocytes, and not human, saimiri, or other erythrocytes. However, when erythrocytes were disrupted by hypotonic lysis, rhoptry proteins of 140/130/110 kDa were found to bind to membranes and inside-out vesicles prepared from human, mouse, saimiri, rhesus, rat, and rabbit erythrocytes. A binding site on the cytoplasmic face of the erythrocyte membrane suggests that the rhoptry proteins may be translocated across the lipid bilayer during merozoite invasion. Furthermore, pretreatment of human erythrocytes with a specific peptide derived from MSA-1, the major P. falciparum merozoite surface antigen of MW 190,000-200,000, induced binding of the 140/130/110-kDa complex. The rhoptry proteins bound equally to normal human erythrocytes and erythrocytes treated with neuraminidase, trypsin, and chymotrypsin indicating the binding site was independent of glycophorin and other major surface proteins. The rhoptry protein complex also bound specifically to liposomes prepared from different types of phospholipids. Liposomes containing PE effectively block binding of the rhoptry proteins to mouse cells, suggesting that there are two binding sites on the mouse membrane for the 140/130/110-kDa complex, one protein and a second, possibly lipid in nature. The results of this study suggest that the 140/130/110 kDa protein complex may interact directly with sites in the lipid bilayer of the erythrocyte membrane.