Diversity and evolution of the rhoph1/clag multigene family of Plasmodium falciparum

A complex of high-molecular-mass proteins (PfRhopH) of the human malaria parasite Plasmodium falciparum induces host protective immunity and therefore is a candidate for vaccine development. Understanding the level of polymorphism and the evolutionary processes is important for advancements in both vaccine design and knowledge of the evolution of cell invasion in this parasite. In the present study, we sequenced the entire open reading frames of seven genes encoding the proteins of the PfRhopH complex (rhoph2, rhoph3, and five rhoph1/clag gene paralogs). We found that four rhoph1/clag genes (clag2, 3.1, 3.2, and 8) were highly polymorphic. Amino acid substitutions and indels are predominantly clustered around amino acid positions 1000-1200 of these four rhoph1/clag genes. An excess of nonsynonymous substitutions over synonymous substitutions was detected for clag8 and 9, indicating positive selection. The McDonald-Kreitman test with a Plasmodium reichenowi orthologous sequence also supports positive selection on clag8. Based on the ratio of interspecific genetic distance to intraspecific distance, the time to the most recent common ancestor of the clag2 and 8 polymorphisms was estimated to be 1.89 and 0.87 million years ago, respectively, assuming divergence of P. falciparum and P. reichenowi 6 million years ago. In addition to a copy number polymorphism, gene conversion events were detected for the rhoph1/clag genes on chromosome 3, which likely play a role in increasing the diversity of each locus. Our results indicate that a high diversity of the PfRhopH1/Clag multigene family is maintained by diversifying selection forces over a considerably long period.     

Properties of the Plasmodium falciparum homologue of a protective vaccine candidate of Plasmodium yoelii.

We describe an unusual tryptophan-rich protein of Plasmodium falciparum that contains threonine-rich repeats. The protein is encoded by a 2.5 kb gene with a two-exon structure including a short AT-rich intron that is spliced out of the mature message. The 5' end of the gene encodes a hydrophobic region, which is assumed to be a signal peptide. The peptide sequence is characterised by a tryptophan-rich region and a block of degenerate threonine repeats. The protein is synthesised throughout the asexual life cycle and has an apparent molecular weight of approximately 94 kDa. It has a variable molecular weight in different strains of P. falciparum. Length polymorphisms can be found in the intron region and the second exon. Four single nucleotide mutations are localised in the tryptophan-rich region and two were found in the threonine-repeat block. Homology searches based on gene structure and amino acid sequence revealed a relationship with a P. yoelii antigen that has been used successfully in vaccine studies. Thus, this P. falciparum antigen should be considered an additional candidate for assessment in vaccination against the asexual blood-stages of P. falciparum.     

Characterisation of exogenous folate transport in Plasmodium falciparum

Folate salvage by Plasmodium falciparum is an important source of key cofactors, but little is known about the underlying mechanism. Using synchronised parasite cultures, we observed that uptake of this dianionic species against the negative-inward electrochemical gradient is highly dependent upon cell-cycle stage, temperature and pH, but not on mono- or divalent metal ions. Energy dependence was tested with different sugars; glucose was necessary for folate import, although fructose was also able to function in this role, unlike sugars that cannot be processed through the glycolytic pathway. Import into both infected erythrocytes and free parasites was strongly inhibited by the anion-channel blockers probenecid and furosemide, which are likely to be acting predominantly on specific folate transporters in both cases. Import was not affected by high concentrations of the antifolate drugs pyrimethamine and sulfadoxine, but was inhibited by the close folate analogue methotrexate. The pH optimum for folate uptake into infected erythrocytes was 6.5-7.0. Dinitrophenol and nigericin, which strongly facilitate the equilibration of H(+) ions across biological membranes and thus abolish or substantially reduce the proton gradient, inhibited folate uptake profoundly. The ATPase inhibitor concanamycin A also greatly reduced folate uptake, further demonstrating a link to ATP-powered proton transport. These data strongly suggest that the principal folate uptake pathway in P. falciparum is specific, highly regulated, dependent upon the proton gradient across the parasite plasma membrane, and is likely to be mediated by one or more proton symporters.     

DNA immunization by Plasmodium falciparum liver-stage antigen 3 induces protection against Plasmodium yoelii sporozoite challenge

DNA-based immunization of mice by Plasmodium falciparum liver-stage antigen 3 (PfLSA3), a novel highly conserved P. falciparum preerythrocytic antigen, was evaluated. Animals developed a dominant Th1 immune response (high gamma interferon T-cell responses and predominance of immunoglobulin G2a) to each of three recombinant proteins spanning the molecule. We have exploited the immunological cross-reactivity of PfLSA3 with its putative homologue on sporozoites of the rodent parasite Plasmodium yoelii, and we show for the first time that responses induced by PfLSA3 in mice significantly protect against a heterologous challenge by P. yoelii sporozoites. These results support a significant effect of DNA-induced immune responses on preerythrocytic stages.     

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.     

Antibodies against ribosomal phosphoprotein P0 of Plasmodium falciparum protect mice against challenge with Plasmodium yoelii

Antibodies against the Plasmodium falciparum P0 ribosomal phosphoprotein (PfP0) have been detected exclusively but extensively in malaria-immune persons. Polyclonal rabbit and mice sera were raised against two recombinant polypeptides of P. falciparum P0 protein, PfP0N and PfP0C, covering amino acids 17 to 61 and the remaining amino acids 61 to 316, respectively. Sera against both these domains detected a 35-kDa protein from Plasmodium yoelii subsp. yoelii, a rodent malarial parasite, and stained the surface of merozoites in immunofluorescence assays. Total immunoglobulin G (IgG) purified from rabbit and mouse anti-PfP0 sera by ammonium sulfate and DEAE-cellulose chromatography was used for passive transfer experiments in mice. Mice passively immunized with both anti-PfP0N and anti-PfP0C showed distinctly lower levels of parasitemia than control mice. With immunizations on days -1, 0, 1, 3, and 5, about 50% of both sets of mice receiving anti-PfP0N and anti-PfP0C cleared the lethal 17XL strain of P. yoelii and revived by day 25. All the control mice died by day 10. By extending the immunization schedule, the survival period of the mice could be extended for every mouse that received anti-PfP0 IgG. These data demonstrate the cross-protection of the anti-PfP0 IgG and establish parasite P0 protein as a target for invasion-blocking antibodies.     

Serological cross-reaction between high molecular weight proteins synthesized in blood schizonts of Plasmodium yoelii, Plasmodium chabaudi and Plasmodium falciparum

A 230 000 molecular weight (MW) Plasmodium yoelii protein, a 250 000 MW P. chabaudi protein and a 195 000 MW P. falciparum protein, identified using monoclonal antibodies, have similar characteristics, and have been implicated as protective antigens. In this study the serological relationship between these proteins was investigated by Western transfer analysis. The monoclonal antibodies specific for each of the high molecular weight proteins did not cross-react with antigens of the other two parasites, but a polyvalent mouse serum raised against the purified 230 000 MW P. yoelii protein cross-reacted with the high molecular weight proteins of P. chabaudi and P. falciparum and also with the fragments derived from these proteins. This result indicates that these proteins belong to the same class of malaria parasite antigen.     

Characterisation and sequence of a protective rhoptry antigen from Plasmodium falciparum

We have recently demonstrated that a non-polymorphic rhoptry antigen, RAP-1 (rhoptry associated protein-1), which is recognised by human immune serum, can successfully protect Saimiri monkeys from a lethal infection of Plasmodium falciparum malaria. In this report we further characterise the antigen, which consists of four major proteins of 80, 65, 42 and 40 kDa and two minor proteins of 77 and 70 kDa, and present the antigen's gene sequence. Monoclonal antibody evidence, autocatalytic processing and immunological cross-reactivity suggest that all components of this antigen are derived from the same precursor protein. The antigen is lipophilic, and disulphide bonding plays an important role in its structure. We discuss the structure and function of RAP-1 in the light of its deduced amino acid sequence and consider the relationship of this antigen to other rhoptry antigens of similar subunit size and composition.     

Characterisation of a delta-COP homologue in the malaria parasite,Plasmodium falciparum

The mature human erythrocyte is a simple haemoglobin-containing cell with no internal organelles and no protein synthesis machinery. The malaria parasite invades this cell and develops inside a parasitophorous vacuole (PV). The parasite exports proteins into the erythrocyte to bring about extensive remodelling of its adopted cellular home. Plasmodial homologues of two COPII proteins, PfSar1p and PfSec31p, are exported to the erythrocyte cytosol where they appear to play a role in the trafficking of proteins across the erythrocyte cytoplasm [Eur. J. Cell Biol. 78 (1999) 453; J. Cell Sci. 114 (2001) 3377]. We have now characterised a homologue of the COPI protein, delta-COP. A recombinant protein corresponding to 90% of the Pfdelta-COP sequence was used to raise antibodies. The affinity-purified antiserum recognised a protein with an apparent M(r) of 58 x 10(3) on Western blots of malaria parasite-infected erythrocytes but not on blots of uninfected erythrocytes. Pfdelta-COP was shown to be largely insoluble in non-ionic detergent, possibly suggesting cytoskeletal attachment. Confocal immunofluorescence microscopy of parasitised erythrocytes was used to show that, in contrast to the COPII proteins, Pfdelta-COP is located entirely within the parasite. The location of Pfdelta-COP partly overlaps that of the endoplasmic reticulum (ER)-located protein, PfERC, and partly that of the trans-Golgi-associated protein, PfRab6. Treatment of ring-stage plasmodium-infected erythrocytes with brefeldin A (BFA) inhibited development of the ER structure within the parasite cytosol and prevented the trafficking of the P. falciparum erythrocyte membrane protein-1, PfEMP1, to the erythrocyte cytosol. The Pfdelta-COP and PfSec31p populations each appear to be associated with the restricted ER structure in brefeldin-treated rings. When more mature stage parasites were treated with BFA, erythrocyte cytosol-located populations of parasite proteins were not reorganised, however, the overlap between Pfdelta-COP and PfERC in parasite cytosol was more complete suggesting a possible redistribution of the Golgi compartment into the ER. These data support the suggestion that both COPI and COPII proteins are involved in the trafficking of proteins within the parasite cytoplasm. However, only COPII proteins are exported to the erythrocyte cytosol to establish a vesicle-mediated protein trafficking pathway to the erythrocyte membrane.     

Characterization of the merozoite surface protein 4/5 gene of Plasmodium berghei and Plasmodium yoelii

The genes encoding merozoite surface protein 4/5 (MSP4/5) from Plasmodium berghei and Plasmodium yoelii have been cloned and completely sequenced. Comparisons of the predicted protein sequences with those of Plasmodium chabaudi MSP4/5 and Plasmodium falciparum MSP4 and MSP5 show general structural similarities. All predicted proteins contain hydrophobic signal sequences, potential GPI attachment sequences and a single epidermal growth factor (EGF)-like domain at the C-terminus. The amino acid sequence of the EGF-like motif is highly conserved in rodent malaria species and also shows a considerable degree of similarity with the EGF-like domains found in the P. falciparum proteins. Both the P. yoelii and P. berghei genes show evidence of both spliced and unspliced mRNA at steady state. This phenomenon is similar to that seen for the P. chabaudi MSP4/5 gene, and is believed to be involved in regulation of protein expression. We describe here the construction of clones expressing full length recombinant protein. Antibodies directed against recombinant MSP4/5 proteins recognize a single polypeptide on parasite material and show crossreactivity between MSP4/5 from different murine malaria species, but do not crossreact with either MSP4 or MSP5 from P. falciparum. The various antisera show reactivity against reduction sensitive epitopes as well as reduction insensitive epitopes.     

Structure and expression of the Plasmodium falciparum SERA gene

Plasmodium falciparum, strain FCR3, genomic DNA that encodes the SERA gene of P. falciparum was isolated and sequenced. The SERA gene coding region was interrupted by 3 introns, the largest number observed, so far, in any Plasmodium gene. Two SERA gene alleles, allele I and allele II, were identified in the FCR3 strain, while only allele I was found in the Honduras-1 strain. Allele I mRNA was abundant in vivo during the late trophozoite and schizont stages. Allele II mRNA was either not expressed, or it was labile.     

Characterisation of the phosphatidylinositol synthase gene of Plasmodium species

Phosphatidylinositol (PI) is a versatile lipid that not only serves as a structural component of cellular membranes, but also plays important roles in membrane anchorage of proteins and in signal transduction through distinct phosphorylated derivatives of the inositol head group. PI is synthesised by PI synthase from CDP-diacylglycerol and myo-inositol. The enzymatic activity in Plasmodium falciparum and P. knowlesi has previously been characterised at the biochemical level. Here we characterise the PI synthase gene of P. falciparum and P. knowlesi. The cDNA sequence identified a highly spliced gene consisting of nine exons and encoding a protein of 209 and 207 amino acids, respectively. High sequence conservation enabled the prediction of the PI synthase genes of P. berghei, P. chabaudi and P. vivax. All Plasmodium PI synthase proteins appear to be highly hydrophobic, although no consensus for the number and location of distinct transmembrane domains could be detected. The P. falciparum PI synthase (PfPIS) gene successfully complemented a Saccharomyces cerevisiae PIS1 deletion mutant, demonstrating its enzymatic function. Complementation efficiency was dramatically improved when hybrid constructs between N-terminal S. cerevisiae and C-terminal P. falciparum sequences were used. Determination of in vitro PIS activities of complemented yeast strains confirmed the enzymatic function of the Plasmodium protein.     

Characterisation of a repetitive DNA sequence from the malaria parasite,Plasmodium falciparum

A repetitive DNA fragment cloned from the malaria parasite, Plasmodium falciparum, has been analysed. It contains a 21 base pair sequence which occurs in multiple tandem repeats. Two clusters of the same repeat are found in opposite orientations on the same DNA fragment. The repetitive DNA provides an additional way to distinguish between different strains of parasite by hybridisation to genomic blots and may serve as a species-specific probe for diagnosis.     

Reduced antibody response to the repetitive sequence of the Plasmodium falciparum circumsporozoite protein in mice infected with Plasmodium yoelii blood forms

The immunogenicity of the carrier-free synthetic peptide, (NANP)₄₀, from the repetitive region of the Plasmodium falciparum circumsporozoite (CS) protein was investigated in genetically responder mice (C57BL/6, H-2^b) acutely infected with blood forms of the non-lethal murine malaria parasite, P. yoelii. As compared to non-infected mice, P. yoelii-infected C57BL/6 mice produced significantly lower titers of anti-(NANP)₄₀ IgG antibodies. This decrease in the anti-(NANP)₄₀ antibody response peaked with the peak of parasitemia, and involved all the IgG subclasses. Interestingly, this P. yoelii-mediated effect was evident both on the development of the antibody response to the (NANP)₄₀ peptide, and on an already established anti-(NANP)₄₀ antibody titer, as seen in mice immunized with the peptide 1 month before the infection. Since (NANP)_n-based constructs are strongly envisaged as potential vaccines against falciparum malaria, these results might be important in the evaluation of the efficacy of these vaccine candidates, when they will be used in individuals living in endemic areas.     

Limited genetic diversity of the Plasmodium falciparum aquaglyceroporin gene

In Plasmodium falciparum small solutes like water, ammonium, glycerol and others are transported by a parasite-encoded channel into the parasite. The gene encoding this channel is termed P. falciparum aquaglyceroporin (PfAQP) and is a single-copy gene and highly homologous to other aquaporins from other protozoa. Aquaporins are considered to be attractive targets for drug treatment and more so since the human and parasite aquaporins show considerable sequence differences. To investigate whether PfAQP may be suitable as a conserved target for potential aquaporin blocking agents we determined the DNA sequences of PfAQP from 65 parasite strains, either from in vitro cultured laboratory strains or from parasites obtained in an malaria-endemic region of Gabon. Only two non-synonymous mutations were found and functionally tested by a methylamine efflux assay. The efflux activity of all variants tested was similar. The lack of functionally variability suggests an invariable protein core, which may restrict parasite populations from evading therapeutic pressure if PfAQP inhibitors will be found.     

Plasmodium falciparum aldolase: gene structure and localization

A genomic clone was isolated which codes for the fructose bisphosphate aldolase of Plasmodium falciparum. The aldolase gene is interrupted by one intron which divides the coding region into two exons. The first one codes for one amino acid only, the initiation methionine, while the second one encodes the residual 368 amino acids of the protein. The gene, which is represented only once in the genome, is transcribed at high rates as a 2.4-kb mRNA in the P. falciparum blood stage. The aldolase gene encodes a protein of 40,105 Da, which is 61-68% homologous to known eukaryotic aldolases. The protein was expressed in Escherichia coli cells in an unfused and enzymatically active form. Antisera raised against amino acids 9-96 recognize a 41-kDa protein band previously shown to protect monkeys against a P. falciparum infection. These antisera cross-react with aldolases of different species, which confirms the strong conservation of this enzyme during evolution. The aldolase could be localized in the cytoplasm of the parasite as an active and soluble form. An inactive form was found to be associated with the membrane fraction. Digestion data with phospholipase C suggest a membrane association of this polypeptide via a glycosylphosphatidylinositol anchor.