Plasmodium falciparum merozoites: isolation by density gradient centrifugation using Percoll and antigenic analysis

Merozoites of Plasmodium falciparum were isolated and immunocytochemically analyzed. Mature parasites from knobby (K+) and knobless (K-) strains were incubated for 4 to 5 hr in RPMI 1640 with 10% serum and 10% RBC extract. About 12 to 14% of the merozoites released were recovered by density gradient centrifugation using Percoll. From 1 to 3 X 10(9) merozoites were obtained per collection. The merozoite preparations were contaminated with 10% residual bodies, about 0.1% infected and uninfected erythrocytes, about 0.1% RBC-free trophozoites and schizonts, and numerous small (less than 0.5 microns) membrane vesicles. Merozoites from the K+ and K- strains were morphologically and, by an indirect, ferritin-labeled antibody assay using serum from immune Aotus, antigenically indistinguishable. Although the residual body coats reacted with the immune Aotus serum, the membrane vesicles, some of which were seen to be blebbing from merozoites, did not react with this serum or a serum against erythrocytes. This paper describes a procedure that can be used to obtain large numbers of merozoites with little contamination by host erythrocytes.     

Babesia argentina, Plasmodium vivax and P. falciparum: antigenic cross-reactions

An indirect fluorescent antibody test was used to analyze the antigenic relationships between Babesia argentina, a parasite of cattle, and two human malaria parasites, Plasmodium falciparum and Plasmodium vivax. Elevated antibody titers to P. falciparum were found in cattle infected with B. argentina. Some persons infected with P. falciparum or P. vivax were found to produce antibodies to B. argentina. Explanations for the occurrence of these cross reactions are considered.     

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.     

Cloning, over-expression, purification and characterization of Plasmodium falciparum enolase.

We have cloned, over-expressed and purified enolase from Plasmodium falciparum strain NF54 in Escherichia coli in active form, as an N-terminal His6-tagged protein. The sequence of the cloned enolase from the NF54 strain is identical to that of strain 3D7 used in full genome sequencing. The recombinant enolase (r-Pfen) could be obtained in large quantities (approximately 50 mg per litre of culture) in a highly purified form (> 95%). The purified protein gave a single band at approximately 50 kDa on SDS/PAGE. MALDI-TOF analysis gave a mean +/- SD mass of 51396 +/- 16 Da, which is in good agreement with the mass calculated from the sequence. The molecular mass of r-Pfen determined in gel-filtration experiments was approximately 100 kDa, indicating that P. falciparum enolase is a homodimer. Kinetic measurements using 2-phosphoglycerate as substrate gave a specific activity of approximately 30 U.mg(-1) and K(m2PGA) = 0.041 +/- 0.004 mm. The Michaelis constant for the reverse reaction (K(mPEP)) is 0.25 +/- 0.03 mm. pH-dependent activity measurements gave a maximum at pH 7.4-7.6 irrespective of the direction of catalysis. The activity of this enzyme is inhibited by Na+, whereas K+ has a slight activating effect. The cofactor Mg2+ has an apparent activation constant of 0.18 +/-0.02 mm. However, at higher concentrations, it has an inhibitory effect. Polyclonal antibody raised against pure recombinant P. falciparum enolase in rabbit showed high specificity towards recombinant protein and is also able to recognize enolase from the murine malarial parasite, Plasmodium yoelii, which shares 90% identity with the P. falciparum protein.     

The origin of antigenic diversity in Plasmodium falciparum

Most studies of genetic variability of Plasmodium falciparum have focused on protein antigens and the genes that encode them. The consensus is that populations exhibit high levels of genetic polymorphism, most notably the genes encoding surface proteins of the merozoite (Msp1, Msp2) and the sporozoite (Csp). The age and derivation of this variation is a subject that warrants further careful consideration, as discussed here by Stephen Rich, Marcelo Ferreira and Francisco Ayala.     

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.     

Characterization of an eukaryotic peptide deformylase from Plasmodium falciparum.

Ribosomal protein synthesis in eubacteria and eukaryotic organelles initiates with an N-formylmethionyl-tRNA(i), resulting in N-terminal formylation of all nascent polypeptides. Peptide deformylase (PDF) catalyzes the subsequent removal of the N-terminal formyl group from the majority of bacterial proteins. Until recently, PDF has been thought as an enzyme unique to the bacterial kingdom. Searches of the genomic DNA databases identified several genes that encode proteins of high sequence homology to bacterial PDF from eukaryotic organisms. The cDNA encoding Plasmodium falciparum PDF (PfPDF) has been cloned and overexpressed in Escherichia coli. The recombinant protein is catalytically active in deformylating N-formylated peptides, shares many of the properties of bacterial PDF, and is inhibited by specific PDF inhibitors. Western blot analysis indicated expression of mature PfPDF in trophozoite, schizont, and segmenter stages of intraerythrocytic development. These results provide strong evidence that a functional PDF is present in P. falciparum. In addition, PDF inhibitors inhibited the growth of P. falciparum in the intraerythrocytic culture.     

Production, characterization and crystallization of the Plasmodium falciparum aquaporin

The causative agent of malaria, Plasmodium falciparum posses a single aquaglyceroporin (PfAQP) which represents a potential drug target for treatment of the disease. PfAQP is localized to the parasite membrane to transport water, glycerol, ammonia and possibly glycolytic intermediates. In order to enable design of inhibitors we set out to determine the 3D structure of PfAQP, where the first bottleneck to overcome is achieving high enough yield of recombinant protein. The wild type PfAQP gene was expressed to low or undetectable levels in the expression hosts, Escherichia coli and Pichia pastoris, which was assumed to be due to different genomic A+T content and different codon usage. Thus, two codon-optimized PfAQP genes were generated. The Opt-PfAQP for E. coli still did not result in high production yields, possibly due to folding problems. However, PfAQP optimized for P. pastoris was successfully expressed in P. pastoris for production and in Saccharomyces cerevisiae for functional studies. In S. cerevisiae, PfAQP mediated glycerol transport but unexpectedly water transport could not be confirmed. Following high-level membrane-localized expression in P. pastoris (estimated to 64mg PfAQP per liter cell culture) PfAQP was purified to homogeneity (18mg/L) and initial attempts at crystallization of the protein yielded several different forms.     

Fine-scale genetic characterization of Plasmodium falciparum chromosome 7 encompassing the antigenic var and the drug-resistant pfcrt genes

The fact that malaria is still an uncontrolled disease is reflected by the genetic organization of the parasite genome. Efforts to curb malaria should begin with proper understanding of the mechanism by which the parasites evade human immune system and evolve resistance to different antimalarial drugs. We have initiated such a study and presented herewith the results from the in silico understanding of a seventh chromosomal region of the malarial parasite Plasmodium falciparum encompassing the antigenic var genes (coding pfemp1) and the drug-resistant gene pfcrt located at a specified region of the chromosome 7. We found 60 genes of various functions and lengths, majority (61.67%) of them were performing known functions. Almost all the genes have orthologs in other four species of Plasmodium, of which P. chabaudi seems to be the closest to P. falciparum. However, only two genes were found to be paralogous. Interestingly, the drug-resistant gene, pfcrt was found to be surrounded by seven genes coding for several CG proteins out of which six were reported to be responsible for providing drug resistance to P. vivax. The intergenic regions, in this specified region were generally large in size, majority (73%) of them were of more than 500 nucleotide bp length. We also designed primers for amplification of 21 noncoding DNA fragments in the whole region for estimating genetic diversity and inferring the evolutionary history of this region of P. falciparum genome.     

Kinetic and thermodynamic characterization of dUTP hydrolysis by Plasmodium falciparum dUTPase

Deoxyuridine 5'-triphosphate nucleotidohydrolase (dUTPase) catalyzes the hydrolysis of dUTP to dUMP and pyrophosphate and plays an important role in nucleotide metabolism and DNA replication controlling relative cellular levels of dTTP/dUTP, both of which can be incorporated into DNA. Isothermal titration calorimetry has been applied to the determination of the kinetic and thermodynamic parameters of the trimeric Plasmodium falciparum dUTPase, a potential drug target against malaria. The role of divalent ions in binding, and inhibition by different uridine derivatives has been assessed. When dUTP hydrolysis in the presence of EDTA was evaluated, a 105-fold decrease and a 12-fold increase of the k(cat) and K(m) values, respectively, were observed when compared with the dUTP.Mg(2+) complex. Calculation of the activation energy, E(a), and the thermodynamic activation parameters showed that the energetic barrier was ~4-fold higher when Mg(2+) was depleted. Other divalent ions such as Co(2+) or Mn(2+) can substitute the physiological cofactor, however the k(cat) was significantly reduced compared to dUTP.Mg(2+). Binding and inhibition by dU, dUMP, dUDP, and alpha,beta-imido-dUTP were analysed by ITC and compared with data obtained by spectrophotometric methods and binding equilibrium studies. Product inhibition (K(ip) dUMP: 99.34 muM) was insignificant yet K(i) values for dUDP and alpha,beta-imido-dUTP were in the low micromolar range. The effect of ionic strength on protein stability was also monitored. DSC analysis evidenced a slight increase in the unfolding temperature, T(m), with increasing salt concentrations. Moreover, the thermal unfolding pathway in the presence of salt fits adequately to an irreversible two-state model (N(3)-->3D).     

Immunological and biological characterization of Plasmodium falciparum exoantigens

Exoantigens (E-antigens) of P. falciparum prepared by cationic exchange chromatography from asynchronous culture supernatant were evaluated by High Performance Liquid Chromatography (HPLC), Chromatofocusing, Enzyme Linked ImmunoSorbent Assay (ELISA), Gel Electrophoresis of Derivated ELISA (GEDELISA) and Immunoblotting. Their mol. wt after denaturation and immunoblotting were: 170,000,135,000 and 100,000. Their isoelectric points (pI) in their native forms were: ⩾ 6.3, 4.1 and ⩽ 3.7. Thermostability tests showed that 65% of their antigenic activity remained after 5 min at 100°C. Metabolic labelling with ³H glucosamine, periodate oxidation and borohydride reduction showed that they were in part glycoproteins. These antigens are excreted or secreted during merozoite release and invasion of erythrocytes. Metabolically labelled (³Ft leucine) culture fluid from synchronous P. falciparum cultures was analysed by Inhibition ELISA (ELISA-I) and CounterImmunoElectrophoresis (CIE) to determine the presence of E-antigens. These antigens inhibited the in vitro growth of Plasmodium falciparum.     

Mitogenic and antigenic activity of Plasmodium falciparum in primate and rodent lymphocytes

Goldenser J., Marva E., Spira D. T., Gabrielsen A. A. and Jensen J. B. 1985. Mitogenic and antigenic activity of Plasmodium falciparum in primate and rodent lymphocytes. International Journal for Parasitology15: 435–440. Considerable reaction of human leucocytes to a wide range of concentrations of plasmodial preparations derived from in vitro cultures of Plasmodium falciparum was observed. Highest responses were recorded after 6 days in culture. This differed from the response to PHA or CON-A which peak with a narrow range of concentrations after 3 days in culture. Parasitized erythrocytes (PE) or parasites released from PE as well as soluble antigens obtained from the particulate preparations had a pronounced mitogenic activity which was unaffected by heating to 56°C for 1 h. Peripheral lymphocytes from man and monkey but not from rats reacted to P. falciparum preparations. Spleen cells obtained from normal rats did not react towards any P. falciparum preparation. Spleen cells of rats immune to P. berghei, responded to normal human erythrocytes but the response against P. falciparum antigens was much higher, indicating cross-reactivity with genus specific antigens. The combination of experimental procedures using human peripheral and rat spleen lymphocytes is suggested for differentiation between mitogenic and antigenic activity. Heat inactivation of some proteases present in the plasmodial preparations, while retaining mitogenic activity, may enable further purification of the mitogenic factors.     

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.     

Plasmodium falciparum: induction, selection, and characterization of pyrimethamine-resistant mutants

We have selected eight pyrimethamine resistant mutants of a cloned, drug sensitive, Plasmodium falciparum malaria parasite, strain FCR3. The mutants exhibited resistance to between 10 and 200 times higher concentrations of drug than the wild type parasite. The mutants were selected from cultured parasites that were either unmutagenized or N-methyl-N'-nitro-N-nitrosoguanidine mutagenized. One mutant was shown to contain a mutant dihydrofolate reductase enzyme in parasite extracts that exhibited (1) a five- to ninefold reduction in its binding of methotrexate, (2) an undetectable enzyme activity based on the spectrophotometric conversion of dihydrofolate to tetrahydrofolate, and (3) essentially normal amounts of the parasite's bifunctional thymidylate synthetase-dihydrofolate reductase enzyme. Other mutants exhibited both normal dihydrofolate reductase specific activity and normal enzyme sensitivity to the inhibitory activity of the drug.     

Plasmodium falciparum: isolation and purification of spontaneously released merozoites by nylon membrane sieves

A new procedure for isolating spontaneously released merozoites from in vitro cultures of Plasmodium falciparum (FVO and FCB strains) is described. The mature forms of relatively synchronous cultures containing predominantly trophozoites and few schizonts were concentrated with Plasmagel and then incubated at 37 C, without adding fresh red blood cells, until trophozoites matured into schizonts. Merozoites which were subsequently released were harvested and freed from host red blood cell material by low-speed centrifugations and nylon membrane sieves (3- and 1.2-μm pore size). From a culture containing about 5.2 × 10⁹ mature-form parasites, a total of about 10.7 × 10⁹ merozoites were released during three consecutive harvests and about 69% of these merozoites were recovered after the isolation and purification procedures. As demonstrated by both light and electron microscopy, most merozoites were morphologically intact and the merozoite preparations were free of host cell constituents. SDS-acrylamide gel electrophoresis confirmed the absence of host cell material and also showed that merozoites had a complex protein pattern of apparent molecular weights between 225 and 15 kdaltons. Such purified merozoite preparations will be invaluable for malaria immunization studies, for identification of protective antigens of P. falciparum, and for other immunological and biochemical studies.     

Molecular characterization and localization of Plasmodium falciparum choline kinase

Generation of phosphocholine by choline kinase is important for phosphatidylcholine biosynthesis via Kennedy pathway and phosphatidylcholine biosynthesis is essential for intraerythrocytic growth of malaria parasite. A putative gene (Gene ID PF14_0020) in chromosome 14, having highest sequence homology with choline kinase, has been identified by BLAST searches from P. falciparum genome sequence database. This gene has been PCR amplified, cloned, over-expressed and characterized. Choline kinase activity of the recombinant protein (PfCK) was validated as it catalyzed the formation of phosphocholine from choline in presence of ATP. The K(m) values for choline and ATP are found to be 145+/-20 microM and 2.5+/-0.3 mM, respectively. PfCK can phosphorylate choline efficiently but not ethanolamine. Southern blotting indicates that PfCK is a single copy gene and it is a cytosolic protein as evidenced by Western immunoblotting and confocal microscopy. A model structure of PfCK was constructed based on the crystal structure of choline kinase of C. elegans to search the structural homology. Consistent with the homology modeling predictions, CD analysis indicates that the alpha and beta content of PfCK are 33% and 14%, respectively. Since choline kinase plays a vital role for growth and multiplication of P. falciparum during intraerythrocytic stages, we can suggest that this well characterized PfCK may be exploited in the screening of new choline kinase inhibitors to evaluate their antimalarial activity.     

Kinetic and biochemical characterization of Plasmodium falciparum GMP synthetase

Plasmodium falciparum, the causative agent of the fatal form of malaria, synthesizes GMP primarily from IMP and, hence, needs active GMPS (GMP synthetase) for its survival. GMPS, a G-type amidotransferase, catalyses the amination of XMP to GMP with the reaction occurring in two domains, the GAT (glutamine amidotransferase) and ATPPase (ATP pyrophosphatase). The GAT domain hydrolyses glutamine to glutamate and ammonia, while the ATPPase domain catalyses the formation of the intermediate AMP-XMP from ATP and XMP. Co-ordination of activity across the two domains, achieved through channelling of ammonia from GAT to the effector domain, is the hallmark of amidotransferases. Our studies aimed at understanding the kinetic mechanism of PfGMPS (Plasmodium falciparum GMPS) indicated steady-state ordered binding of ATP followed by XMP to the ATPPase domain with glutamine binding in a random manner to the GAT domain. We attribute the irreversible, Ping Pong step seen in initial velocity kinetics to the release of glutamate before the attack of the adenyl-XMP intermediate by ammonia. Specific aspects of the overall kinetic mechanism of PfGMPS are different from that reported for the human and Escherichia coli enzymes. Unlike human GMPS, absence of tight co-ordination of activity across the two domains was evident in the parasite enzyme. Variations seen in the inhibition by nucleosides and nucleotide analogues between human GMPS and PfGMPS highlighted differences in ligand specificity that could serve as a basis for the design of specific inhibitors. The present study represents the first report on recombinant His-tagged GMPS from parasitic protozoa.