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.     

Plasmodium falciparum: isolation and characterization of a 55-kDa protease with a cathepsin D-like activity from P. falciparum

Native electrophoresis followed by imprint digest method using hemoglobin as substrate allowed the detection of parasite hemoglobinase activity at acidic pH (3.9 to 5). This protease was inhibited specifically by pepstatin A and insensitive to other protease inhibitors. The molecular weight determination using modified SDS-PAGE followed by imprint digest method, demonstrated a single area of activity at 55-58 kDa, similar to cathepsin D characterized in eucaryotic cells. The parasitic origin has been shown by radiolabeling experiments with [35S]-methionine. The 55-kDa protein was immunoprecipitated by a rabbit anti-cathepsin D serum.     

Plasmodium falciparum: enhanced soluble expression, purification and biochemical characterization of lactate dehydrogenase

Plasmodium lactate dehydrogenase (pLDH), owing to unique structural and kinetic properties, is a well known target for antimalarial compounds. To explore a new approach for high level soluble expression of Plasmodium falciparum lactate dehydrogenase (PfLDH) in E. coli, PfLDH encoding sequence was cloned into pQE-30 Xa vector. When transformed E. coli SG13009 cells were induced at 37 °C with 0.5 mM isopropyl β-d-thiogalactoside (IPTG) concentration, the protein was found to be exclusively associated with inclusion bodies. By reducing cell growth temperature to 15 °C and IPTG concentration to 0.25 mM, it was possible to get approximately 82% of expressed protein in soluble form. Recombinant PfLDH (rPfLDH) was purified to homogeneity yielding 18 mg of protein/litre culture. rPfLDH was found to be biologically active with specific activity of 453.8 μmol/min/mg. The enzyme exhibited characteristic reduced substrate inhibition and enhanced k_cat [(3.2 ± 0.02) × 10⁴] with 3-acetylpyridine adenine dinucleotide (APAD⁺). The procedure described in this study may provide a reliable and simple method for production of large quantities of soluble and biologically active PfLDH.     

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.     

Recombinant expression and partial characterization of an active soluble histo-aspartic protease from Plasmodium falciparum

Malaria aspartic proteases are attractive drug targets for the treatment of malaria, however, recombinant expression of active histo-aspartic proteinase (HAP) to facilitate its characterization has proven elusive. The present study reports on the first recombinant expression of soluble, active histo-aspartic proteinase from Plasmodium falciparum as a thioredoxin fusion protein. A truncated form of HAP (77p-451) was fused to thioredoxin in the pET32b(+) vector and the fusion protein (Trx-tHAP) was expressed in Escherichia coli Rosetta-gami B (DE3)pLysS. The fusion protein was partially purified from the culture medium using a combination of anion exchange and Ni(2+) affinity chromatography. Soluble tHAP was subsequently purified by enterokinase treatment and removal, followed by gel filtration chromatography. Although truncated HAP was incapable of autocatalytic activation, enterokinase digestion of partially purified fusion protein released the truncated prosegment yielding a mature form of tHAP (mtHAP). N-terminal sequencing of mtHAP indicated that enterokinase cleavage took place at Lys119-Ser120, four residues upstream of the native cleavage site (Gly123-Ser124). Initial activity tests showed that mtHAP was capable of hydrolyzing acid-denatured globin as well as cleavage of the synthetic substrate EDANS-CO-CH(2)-CH(2)-CO-ALERMFLSFP-Dap(DABCYL)-OH. Inhibition studies showed that the activity of mtHAP was completely inhibited by pepstatin A and to a lesser degree, PMSF. Using the synthetic substrate, mtHAP showed a pH optimum of 5.2, and Km=3.4 microM and kcat=1.6 x 10(-3)s(-1). The successful expression of active recombinant HAP from E. coli will accelerate the investigation of the structure-function relationships of HAP and facilitate the development of specific inhibitors with antimalarial activities.     

Plasmodium falciparum: characterization of gene R45 encoding a trophozoite antigen containing a central block of six amino acid repeats.

We describe here an antigen, called R45, expressed by the young trophozoites of Plasmodium falciparum. This antigen contains a block of tandem repeats of six amino acids which are recognized by sera from humans living in endemic areas. The R45 gene is located on chromosome 3. It is present in all strains examined and shows limited size polymorphism. The C-terminal unique region of the protein shows a strong homology with the catalytic domain of the serine protein kinases. Interestingly, the central repeats contain a large number of putative phosphorylation sites. The implications of these features are discussed.     

Characterization with monoclonal antibodies of a surface antigen of Plasmodium falciparum merozoites

The merozoite, the extracellular form of the erythrocyte stage of the malarial parasite, invades the erythrocyte and develops intracellularly. Cloned hybridoma cell lines secreting monoclonal antibodies directed against the merozoite surface were selected by indirect immunofluorescent assay by using intact isolated merozoites. Monoclonal antibodies to a 200,000 m.w. merozoite surface antigen were selected and were used to characterize this protein and its role in erythrocyte invasion. Immunoelectron microscopy demonstrated that the antigen was located exclusively on the merozoite surface coat, distributed evenly over the entire surface. The 200,000 m.w. protein incorporated [3H]glucosamine, suggesting that it is a glycoprotein and could be purified to homogeneity by using immuno-affinity chromatography. Freshly isolated, invasive merozoites retained the 200,000 m.w. antigen, but the protein was rapidly cleaved to proteins of 90,000 and 50,000 m.w. when the merozoite was extracellular. The 50,000 m.w. fragment was retained the epitope binding to monoclonal antibody 5B1 and were labeled with [3H]glucosamine. Monoclonal antibodies against the 200,000 m.w. antigen partially inhibited merozoite invasion into erythrocytes.     

A novel 40-kDa membrane-associated EF-hand calcium-binding protein in Plasmodium falciparum.

A 40-kDa sexual stage radiolabeled surface protein of Plasmodium falciparum, Pfs40, was previously identified as a potential target antigen of transmission blocking immunity by an immunogenetic approach. Synthetic oligonucleotide "guessmers," based on microsequenced tryptic peptides of Pfs40 purified by two-dimensional gel electrophoresis, were used to clone the full length cDNA and genomic DNA encoding Pfs40. The deduced amino acid sequence predicted an integral membrane protein containing five EF-hand calcium-binding domains. The biological activity of one or more of these domains was confirmed by binding of 45Ca to both native and recombinant Pfs40. Antisera to recombinant Pfs40 immunoprecipitated the native radiolabeled 40-kDa surface protein. The predicted noncytosolic membrane-associated localization of Pfs40 is unique within the EF-hand calcium-binding protein superfamily.     

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.     

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.     

Fine specificity of neonatal lymphocytes to an abundant malaria blood-stage antigen: epitope mapping of Plasmodium falciparum MSP1(33)

Cord blood T cells have been reported to respond to a variety of exogenous Ags, including environmental allergens and various viruses and parasites, as demonstrated by enhanced proliferation and cytokine secretion. This finding is evidence that Ags in the maternal environment transplacentally prime and result in fetal development of memory T cells. Some studies suggest these neonatal T cell responses may arise by nonspecific activation of T cells that express TCRs with low binding affinity, thus lacking fine lymphocyte specificity. To address this question, we examined malaria Ag stimulation of human cord and adult blood mononuclear cells in samples from residents of a malaria endemic area in Kenya. We constructed overlapping 18-mer peptides derived from sequences contained in dimorphic alleles of the C-terminal 33-kDa fragment of Plasmodium falciparum merozoite protein 1. This study identified a dominant T cell epitope for one MSP1(33) allele (MAD20) and two T cell epitopes for the second allele (K1); these epitopes were nonoverlapping and allele specific. In a given donor, peptide-specific proliferation and IFN-gamma secretion were highly concordant. However, IL-10 and IL-13 secretion were not correlated. Importantly, the fine specificity of lymphocyte proliferation and cytokine secretion in cord and adult blood mononuclear cells was similar. Cord blood cells obtained from malaria-infected pregnant women were 4-fold more likely to acquire a peptide-specific immune response. We conclude that the fetal malaria response functions in a fully adaptive manner and that this response may serve to help protect the infant from severe malaria during infancy.     

Plasmodium falciparum variant surface antigen expression patterns during malaria

The variant surface antigens expressed on Plasmodium falciparum-infected erythrocytes are potentially important targets of immunity to malaria and are encoded, at least in part, by a family of var genes, about 60 of which are present within every parasite genome. Here we use semi-conserved regions within short var gene sequence "tags" to make direct comparisons of var gene expression in 12 clinical parasite isolates from Kenyan children. A total of 1,746 var clones were sequenced from genomic and cDNA and assigned to one of six sequence groups using specific sequence features. The results show the following. (1) The relative numbers of genomic clones falling in each of the sequence groups was similar between parasite isolates and corresponded well with the numbers of genes found in the genome of a single, fully sequenced parasite isolate. In contrast, the relative numbers of cDNA clones falling in each group varied considerably between isolates. (2) Expression of sequences belonging to a relatively conserved group was negatively associated with the repertoire of variant surface antigen antibodies carried by the infected child at the time of disease, whereas expression of sequences belonging to another group was associated with the parasite "rosetting" phenotype, a well established virulence determinant. Our results suggest that information on the state of the host-parasite relationship in vivo can be provided by measurements of the differential expression of different var groups, and need only be defined by short stretches of sequence data.     

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: in vitro characterization and human infectivity of a cloned line.

The culture-adapted NF54 isolate of Plasmodium falciparum was subjected in vitro to three sequential limiting dilution titrations and the resulting clone was given the designation CVD1. DNA sequence analysis of the gene encoding the circumsporozoite (CS) protein revealed differences between CVD1 and the published NF54 CS gene. CVD1 had 1191 bp, 397 amino acids, and 42 repeat units while NF54 had 1218 bp, 405 amino acids, and 44 repeat units. The CVD1 clone was more sensitive to chloroquine than was the parental line, in vitro. Anopheles stephensi mosquitoes were infected equally by the cloned and uncloned parasites. Volunteers were readily infected by NF54 and CVD1 following infectious mosquito bites. The availability of a well-characterized, chloroquine-sensitive clone which safety infects humans should facilitate performance of experimental challenge studies to assess vaccine efficacy.     

Acylation of a Plasmodium falciparum merozoite surface antigen via sn-1,2-diacyl glycerol

The 195-kDa merozoite protein synthesized in schizonts of Plasmodium falciparum (Holder, A. A., and Freeman, R. R. (1982) J. Exp. Med. 156, 1528-1538) contains ester-linked fatty acid. Enzymatic treatment of the purified acylated protein established that the lipid is present as sn-1,2-diacyl glycerol, most probably linked to a phosphodiester at the 3-position of glycerol. The phosphodiglyceride is not directly esterified to an amino acid residue on the polypeptide backbone. The 195-kDa protein is processed to three fragments (83, 42, and 19 kDa) on the surface of free merozoites (Holder, A. A., and Freeman, R. R. (1984) J. Exp. Med. 160, 624-629), of which only the 42-kDa polypeptide is acylated.     

Biosynthesis of the 25-kDa protein in the macrogametes/zygotes of Plasmodium falciparum

Synthesis of the 25-kDa protein in the early midgut stages of Plasmodium falciparum was studied, using metabolic inhibitors (colchicine and actinomycin D) and pulse-labeling experiments. Experiments with colchicine showed that, immediately after induction of macrogametogenesis, 25-kDa protein synthesis occurs in both fertilized and nonfertilized macrogametes. The amount of 25-kDa protein synthesized increased slowly during time. Experiments with actinomycin D revealed that the slow increase of synthesis may be dependent on de novo messenger RNA synthesis.     

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.