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.     

Plasmodium falciparum: characterization of a 33-kDa soluble antigen

A 33-kDa soluble antigen identified in the culture supernatant by patient serum and monoclonal antibodies was present in rings, trophozoites, schizonts, and merozoites of Plasmodium falciparum. The antigen which is released into the culture supernatant by growing parasites was also observed in the host cells of trophozoites and schizonts and could be localized on the host cell surface. Its specificity for the surface of trophozoites and schizonts was observed to decrease with increased duration without subculture. The antigen could then be detected on the surface of noninfected erythrocytes. The antigenicity of the 33-kDa antigen was destroyed by heating at 65 degrees C. Monoclonal and polyclonal specific antibodies weakly inhibited parasite growth in vitro. The antigen was present in both knob positive and knob negative parasites in all the P. falciparum isolates tested.     

Variation in the gene encoding a major merozoite surface antigen of the human malaria parasite Plasmodium falciparum.

Plasmodium falciparum merozoites have a variable surface protein of about 195,000 molecular weight which may be involved in strain-specific immunity. We have cloned and sequenced a major portion of the gene encoding this antigen from the CAMP strain and have located sites of preferred mung bean nuclease cleavage around the gene. These sites depend on reaction conditions, but at 40% formamide and 2 units of mung bean nuclease per microgram DNA, the intact gene was excised from the chromosome. Comparison of the CAMP strain gene with the same gene from other strains of P. falciparum by matching available DNA sequences and by DNA hybridization revealed five regions of homology separated by divergent segments. Two of the variable regions encoded three amino acid repeats, predominantly Ser-Gly-Thr and Thr-Glu-Glu. Implications of these findings on the function of the antigen, and possible mechanisms for generation of variants are discussed.     

Plasmodium falciparum: isolation and characterisation of a gene encoding protozoan GMP synthase

The final step in guanylate nucleotide biosynthesis is catalysed by GMP synthase. This paper presents the first isolation of a gene encoding a protozoan GMP synthase. The deduced amino acid sequence from Plasmodium falciparum shares 40% identity with yeast GMP synthase and contains motifs conserved in catalysis. Expression of the gene is regulated through the parasite's development in human red blood cells with maximal expression during the point of DNA replication. Psicofuranine, which inhibits GMP synthase, interrupts parasite growth, supporting the role of this enzyme. These findings will aid development of inhibitors of purine salvage in malaria parasites.     

Plasmodium falciparum and Plasmodium chabaudi: characterization of glycosylphosphatidylinositol-degrading activities.

Merozoites of malaria parasites have a membrane-bound serine protease whose solubilization and subsequent activity depend on a parasite-derived glycosylphosphatidylinositol-phospholipase C (GPI-PLC). The GPI-degrading activities from both Plasmodium falciparum and Plasmodium chabaudi have been characterized and partially purified by phenylboronate chromatography. They are membrane-bound, developmentally regulated, calcium-independent enzymes and as such they resemble GPI-PLC of Trypanosoma brucei. Furthermore, a T. brucei GPI-PLC-specific monoclonal antibody (mAT3) immunoprecipitates the plasmodial GPI-degrading activity. Thin-layer chromatography is suggestive of two activities: a GPI-PLC and a phospholipase A.     

Isolation and functional characterization of a dynamin-like gene from Plasmodium falciparum

A novel dynamin-like GTPase gene, Pfdyn1, was cloned from an asexual stage cDNA library of Plasmodium falciparum Dd2 strain. Pfdyn1 contains a highly conserved N-terminal tripartite GTPase domain, a coiled-coil region, and a C-terminal 129 aa unknown function domain. Like yeast Vps1p, it lacks pleckstrin homology domain and proline-rich region. Western blot analysis showed that Pfdyn1 is a Triton X-100 insoluble protein expressed only in the mature sub-stage. Morphological studies indicated that Pfdyn1 is partly co-localized with PfGRP, a known ER-resident protein, and localizes diffusely with several membrane structures and a 60-100 nm vesicle both inside and on surface of the parasites and also in the cytoplasm of infected erythrocytes. The dsRNA originated by C-terminus fragment of Pfdyn1 inhibits markedly the growth of P. falciparum parasite at the erythrocyte stage. Those data showed that Pfdyn1 is a conservative, membrane related protein and plays an essential role for the survival of Plasmodium parasite.     

The 11-1 gene of Plasmodium falciparum codes for distinct fast evolving repeats.

The 11-1 gene of Plasmodium falciparum has been investigated by DNA sequence analysis. It begins at the 5' end with a putative miniexon coding for a polypeptide which has the characteristics of a signal sequence. The miniexon is followed by a small intron. This again is followed by a large exon consisting of 9-, 18- and 27-bp repeats embedded in unique DNA. Specific antibodies isolated by affinity chromatography on a purified recombinant fusion protein expressing the three- and six-amino acid repeats were used to identify the product of the 11-1 gene. In exhibits size variations from 260 to 350 kd in different strains. Southern blot analysis with synthetic DNA as probe demonstrates that the 18-bp repeat is absent or drastically altered in two strains whereas the other repeats are present in all seven strains investigated. The unusual preference for G in the third position of some codons of the repeats but not in the unique sequences indicates rapid evolution of the repeats. Slippage during replication, unequal crossing over and selection are discussed as possible mechanisms leading rapidly to extreme diversity.     

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.     

Plasmodium falciparum: differentiation of isolates with DNA hybridization using antigen gene probes

Chromosomal DNA was prepared from seven Plasmodium falciparum isolates that had been cultured in vitro and from a cloned P. falciparum line. The DNA was cleaved with restriction endonucleases, fractionated by agarose gel electrophoresis, blotted to nitrocellulose, and hybridized with a series of radioactively labeled DNA probes. The probes had been derived from cDNA clones encoding portions of P. falciparum antigens. Simple, reproducible band patterns that differed for many of the isolates were obtained. Parasite isolates collected from different continents could be readily distinguished, as could some but not all isolates collected from one restricted region of Papua New Guinea. Application of this technique for the identification and differentiation of parasite strains was explored. The patterns of hybridization observed were consistent with the proposition that blood stages of P. falciparum have a haploid genome.     

Plasmodium falciparum: characterization of a late asexual stage golgi protein containing both ankyrin and DHHC domains

Proteins containing the DHHC motif have been shown to function as palmitoyl transferases. The palmitoylation of proteins has been shown to play an important role in the trafficking of proteins to the proper subcellular location. Herein, we describe a protein containing both ankyrin domains and a DHHC domain that is present in the Golgi of late schizonts of P. falciparum. The timing of expression as well as the location of this protein suggests that it may play an important role in the sorting of proteins to the apical organelles during the development of the asexual stage of the parasite.     

Plasmodium falciparum: chymotryptic-like proteolysis associated with a 101-kDa acidic-basic repeat antigen.

Malaria proteinases appear to function in the release of merozoites from infected erythrocytes and the invasion of merozoites into erythrocytes. Chymostatin, an inhibitor of chymotrypsin-like proteinases, inhibits malaria invasion and also inhibits apparent autoproteolysis of a 101-kDa acidic-basic repeat antigen (p101-ABRA) that is found in the vacuolar space surrounding merozoites in Plasmodium falciparum-infected erythrocytes. After purification by a monoclonal antibody (MAb 3D5), p101-ABRA degrades into smaller fragments in the absence of chymostatin. In this study fluorogenic proteinase substrates of the type peptidyl-7-amino-4-trifluoromethyl coumarin with phenylalanine or tyrosine linked to AFC were used to characterize chymotryptic-like activity associated with p101-ABRA. When p101-ABRA from the cell extract of P. falciparum-schizont-infected erythrocytes was affinity purified on MAb 3D5 beads, chymotryptic-like activity bound to the beads. Seventy-four percent to 96% of the activity detected using MeOSuc-KLF-AFC, Suc-LLVY-AFC, or SY-AFC at a pH optimum of 7.0 was removed from the extract and 6 to 33% was detected on the washed beads. Attempts to recover active enzyme eluted from the beads were not successful. Enzymes cleaving two other substrates (MeOSuc-AAPM-AFC and F-AFC) did not significantly bind to mAB 3D5 beads. Chymotryptic-like activity was also associated with p101-ABRA in fractions from sequential DEAE-Sephacel chromatography, Sephacryl S-200 chromatography, and nondenaturing polyacrylamide gel electrophoresis.     

Molecular characterization and inhibition of a Plasmodium falciparum aspartic hemoglobinase.

Intraerythrocytic malaria parasites rapidly degrade virtually all of the host cell hemoglobin. We have cloned the gene for an aspartic hemoglobinase that initiates the hemoglobin degradation pathway in Plasmodium falciparum. It encodes a protein with 35% homology to human renin and cathepsin D, but has an unusually long pro-piece that includes a putative membrane spanning anchor. Immunolocalization studies place the enzyme in the digestive vacuole and throughout the hemoglobin ingestion pathway, suggesting an unusual protein targeting route. A peptidomimetic inhibitor selectively blocks the aspartic hemoglobinase, prevents hemoglobin degradation and kills the organism. We conclude that Plasmodium hemoglobin catabolism is a prime target for antimalarial chemotherapy and have identified a lead compound towards this goal.