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PubMed1991

Antimalarial effects of peptide inhibitors of a Plasmodium falciparum cysteine proteinase.

We previously identified a Plasmodium falciparum trophozoite cysteine proteinase (TCP) and hypothesized that it is required for the degradation of host hemoglobin by intraerythrocytic malaria parasites. To test this hypothesis and to evaluate TCP as a chemotherapeutic target, we examined the antimalarial effects of a panel of peptide fluoromethyl ketone proteinase inhibitors. For each inhibitor, effectiveness at inhibiting the activity of TCP correlated with effectiveness at both blocking hemoglobin degradation and killing cultured parasites. Benzyloxycarbonyl (Z)-Phe-Arg-CH2F, the most potent inhibitor, inhibited TCP at picomolar concentrations and blocked hemoglobin degradation and killed parasites at nanomolar concentrations. Micromolar concentrations of the inhibitor were nontoxic to cultured mammalian cells. These results support the hypothesis that TCP is a necessary hemoglobinase and suggest that it is a promising chemotherapeutic target.

Animals↗
PubMed1990

The distribution of circumsporozoite protein (CS) in Anopheles stephensi mosquitoes infected with Plasmodium falciparum malaria.

We monitored the distribution of Plasmodium falciparum circumsporozoite protein (CS) in Anopheles stephensi using an immunohistochemical method. An alkaline phosphatase-labeled monoclonal antibody, specific for the CS protein of P. falciparum, was incubated with tissue sections from infected and non-infected mosquitoes. Sections were stained for phosphatase activity using a new fuchsin/naphthol AS-BI phosphate capture system. Distribution of the CS protein in mosquitoes was dependent on the time after post-infective blood meal. CS protein was first detected in immature oocysts on the mosquito midgut. As oocysts differentiated to mature sporoblasts, detectable CS protein increased. Between 11-16 days post infective blood meal, CS protein was detected on the surface of sporozoites that were released into the hemolymph from oocysts. Although sporozoites were found throughout the hemocoel, they were most frequently associated with the salivary glands and flight muscle. Once in the salivary glands, sporozoites massed into bundles. The amount of CS protein associated with bundles of sporozoites was highly variable.

Animals↗
PubMed2002

Functional characterization of the acyl carrier protein (PfACP) and beta-ketoacyl ACP synthase III (PfKASIII) from Plasmodium falciparum.

The genome of the malaria parasite, Plasmodium falciparum, appears to contain the proteins necessary for a Type II dissociated fatty acid biosynthetic system. Here we report the functional characterization of two proteins from this system. Purified recombinant acyl carrier protein (ACP) and beta-ketoacyl-ACP synthase III (KASIII) from P. falciparum are soluble and active in a truncated form. Malarial ACP is activated by the addition of a 4'-phosphopantetheine prosthetic group derived from coenzyme A, generating holo-PfACP. Holo-PfACP is an effective substrate for the transacylase activity of PfKASIII, but substitution of a key active site cysteine in PfKASIII to alanine or serine abolishes enzymatic activity. During the schizont stage of parasite development, there is a significant up-regulation of the mRNAs corresponding to these proteins, indicating an important metabolic requirement for fatty acids during this stage.

3-Oxoacyl-(Acyl-Carrier-Protein) Synthase↗
PubMed1992

Extracellular (axenic) development in vitro of the erythrocytic cycle of Plasmodium falciparum.

Merozoites of the erythrocytic stage of Plasmodium falciparum were suspended in erythrocyte sonicate medium with ATP and pyruvate and mixed with Matrigel to form a soft gel. The gel was overlaid with complete medium; this was replaced with fresh medium at 12, 24, and 36 hr. At these times and also at 45 hr rhodamine 123 was added to some cultures and gels were sampled. Viable extracellular forms showing rhodamine fluorescence were seen: rings at 12 hr, trophozoites and early schizonts with pigment at 36 hr, and late schizonts with developing merozoites at 45 hr. These merozoites were shown to be infective to erythrocytes added to the cultures at 45 hr. Electron micrographs of 36-hr trophozoites show the organisms to have only their single plasma membrane; no parasitophorous membrane is evident. We conclude that the complex process of entry, the intactness of the host erythrocyte, and the parasitophorous membrane are not essential to the development of a merozoite through its complete asexual cycle.

Adenosine Triphosphate↗
PubMed1993

Actin in the merozoite of the malaria parasite, Plasmodium falciparum.

Merozoites of the human malaria parasite, Plasmodium falciparum, when treated with cytochalasin B, will attach irreversibly to red cells with formation of a vestigial internal (parasitophorous) vacuole, but they are inhibited from moving into the cell. The existence of an actin-based motile mechanism is implied. Immunoblotting, peptide mapping and the DNase inhibition assay have been used to show that the merozoite contains actin. It makes up an estimated 0.3% of the total parasite protein and is partitioned in the ratio of about 1:2 between the cytosolic and particulate protein fractions. In the former it is unpolymerised and in the latter filamentous. Most of the anti-actin-reactive protein in the soluble fraction and about 20% of that in the pellet has an apparent molecular weight of 55,000 and reacts with an anti-ubiquitin antibody; it is thus evidently ubiquitinyl actin, or arthrin, which has so far been detected only in insect flight muscle.

Actins↗
PubMedDate not supplied

Seasonal fluctuation of Plasmodium falciparum gametocytaemia.

Two numerically minor components of Plasmodium falciparum prevalence--gametocytaemia and trophozoite densities greater than 99/500 white blood cells--displayed an annual cycle that reflected the seasonal abundance of infective Anopheles dirus at a hyperendemic focus in Thailand, even though the gross monthly prevalence for combined ages remained stable. Gametocyte prevalence rose more than 300% within 30 d after the capture of the dry season's first infective mosquito, remained at about 8% until the beginning of the monsoon 7 months later, then fell within 60 d to about 2%. The number of cases with a high density of trophozoites behaved similarly. These periodic fluctuations represented changes in incidence, at least half of which appeared to be due to superinfection. Almost 49% of all gametocyte carriers were older than 14 years, but nearly all gametocyte densities greater than 20/500 white blood cells were in children. These observations, as well as the calculated efficiency of human infectivity, imply that superinfection of adults may contribute significantly to transmission in semi-immune populations.

Animals↗
PubMed2002

A proteomic view of the Plasmodium falciparum life cycle.

The completion of the Plasmodium falciparum clone 3D7 genome provides a basis on which to conduct comparative proteomics studies of this human pathogen. Here, we applied a high-throughput proteomics approach to identify new potential drug and vaccine targets and to better understand the biology of this complex protozoan parasite. We characterized four stages of the parasite life cycle (sporozoites, merozoites, trophozoites and gametocytes) by multidimensional protein identification technology. Functional profiling of over 2,400 proteins agreed with the physiology of each stage. Unexpectedly, the antigenically variant proteins of var and rif genes, defined as molecules on the surface of infected erythrocytes, were also largely expressed in sporozoites. The detection of chromosomal clusters encoding co-expressed proteins suggested a potential mechanism for controlling gene expression.

Animals↗
PubMed2006

A conserved region in the EBL proteins is implicated in microneme targeting of the malaria parasite Plasmodium falciparum.

The proliferation of the malaria parasite Plasmodium falciparum within the human host is dependent upon invasion of erythrocytes. This process is accomplished by the merozoite, a highly specialized form of the parasite. Secretory organelles including micronemes and rhoptries play a pivotal role in the invasion process by storing and releasing parasite proteins. The mechanism of protein sorting to these compartments is unclear. Using a transgenic approach we show that trafficking of the most abundant micronemal proteins (members of the EBL-family: EBA-175, EBA-140/BAEBL, and EBA-181/JSEBL) is independent of their cytoplasmic and transmembrane domains, respectively. To identify the minimal sequence requirements for microneme trafficking, we generated parasites expressing EBA-GFP chimeric proteins and analyzed their distribution within the infected erythrocyte. This revealed that: (i) a conserved cysteine-rich region in the ectodomain is necessary for protein trafficking to the micronemes and (ii) correct sorting is dependent on accurate timing of expression.

Animals↗
PubMed1996

A 22 kDa protein associated with the Plasmodium falciparum merozoite surface protein-1 complex.

The Plasmodium falciparum merozoite surface protein-1 (MSP-1) is synthesized as a precursor of approximately 195 kDa and is processed to form a complex of polypeptides on the surface of free merozoites. As a result of a second processing event, the entire MSP-1 complex is shed from the surface, apart from a C-terminal fragment that remains anchored to the merozoite membrane. We have identified a 22 kDa protein (p22) on the surface of merozoites by cell surface radioiodination and indirect immunofluorescence assay on unfixed free merozoites. p22 is also a component of the shed MSP-1 complex where it is present in part as a 19 kDa form (p22(19)) as shown by immunochemical and peptide mapping analyses. The soluble complex contains MSP-1-derived polypeptides and p22 in approximately stoichiometrically equal amounts. N-terminal amino acid sequence analyses of p22/p22(19) showed that the protein is not derived from the MSP-1 precursor.

Amino Acid Sequence↗
PubMed1996

Chemical synthesis of the Plasmodium falciparum dihydrofolate reductase-thymidylate synthase gene.

Plasmodium falciparum dihydrofolate reductase-thymidylate synthase (DHFR-TS) is a well-known target for pyrimethamine and cycloguanil. The low amounts of enzyme obtainable from parasites or the currently available heterologous expression systems have thus far hindered studies of this enzyme. The 1912-base pair P. falciparum DHFR-TS gene was designed based on E. coli codon preference with unique restriction sites evenly placed throughout the coding sequence. The gene was designed and synthesized as three separated domains: the DHFR domain, the junctional sequence, and the TS domain. Each of these domains contained numerous unique restriction sites to facilitate mutagenesis. The three domains were assembled into a complete DHFR-TS gene which contained 30 unique restriction sites in the coding sequence. The bifunctional DHFR-TS was expressed from the synthetic gene as soluble enzyme in E. coli about 10-fold more efficiently than from the wild-type sequence. The DHFR-TS from the synthetic gene had kinetic properties similar to those of the wild-type enzyme and represents a convenient source of protein for further study. The unique restriction sites in the coding sequence permits easy mutagenesis of the gene which should facilitate further understanding of the molecular basis of antifolate resistance in malaria.

Animals↗
PubMed1999

Development of antibodies against chondroitin sulfate A-adherent Plasmodium falciparum in pregnant women.

In areas where Plasmodium falciparum is endemic, pregnant women are at increased risk for malaria, and this risk is greatest during the first pregnancy. The placenta sequesters parasites that are able to cytoadhere to chondroitin sulfate A (CSA), a molecule expressed by the placental syncytiotrophoblast, while parasites from a nonpregnant host do not bind to CSA. Cytoadherence is mediated by the expression of variants of the P. falciparum-erythrocyte membrane protein 1 family. Each member of this molecule family induces antibodies that specifically agglutinate infected erythrocytes and inhibit their cytoadherence ability. We investigated whether the higher susceptibility of primigravidae was related to the lack of immune response towards CSA-binding parasites. In a cross-sectional study, primigravidae delivering with a noninfected placenta were less likely to have antibodies agglutinating CSA-binding parasites than multigravidae (P < 0.01). In contrast, parasites from nonpregnant hosts were as likely to be recognized by the sera from women of various parities. In a longitudinal study, at 6 months of pregnancy, antibodies against CSA-binding parasites were present in 31.8% of primigravidae and in 76.9% of secundigravidae (P = 0.02). The antibodies against CSA-binding parasites inhibited the cytoadherence of a CSA-adherent parasite strain to the human placental trophoblast. Our data support the idea that the higher susceptibility of primiparae is related to a lack of a specific immune response to placental parasites.

Adhesiveness↗
PubMed1987

Evaluation of an in vitro test method for the assessment of sensitivity of Plasmodium falciparum to pyrimethamine and sulfadoxine.

Twenty three Plasmodium falciparum isolates collected from two highly pyrimethamine/sulfadoxine-resistant areas of Thailand were evaluated for their in vitro responses to pyrimethamine, sulfadoxine and combinations of these two drugs in various conditions. The test procedure was based on inhibition of parasite multiplication and of schizont formation, using the recommended modified RPMI medium 1640 with PABA 0.5 mug per litre and folic acid 10 mug per litre (LPLF). The optimum blood/medium ratios and inoculum sizes for parasite multiplication and for schizont formation were 1:19, 100 mul/well and 1:9, 50 mul/well, respectively. The appropriate incubation period was 48 hours. It was found that inhibition of either parasite multiplication or schizont formation could be used as the endpoint for evaluating the antiplasmodial action of pyrimethamine and combined pyrimethamine/sulfadoxine in vitro for field investigations; however, inhibition of only parasite multiplication should be used for determination of sulfadoxine activity. The actions of pyrimethamine in the combination pyrimethamine/sulfadoxine in ratios of 1:80 and 1:200 were similar. In vitro testing using combined pyrimethamine/sulfadoxine should be more precise than pyrimethamine alone for monitoring parasite susceptibility to the combined drug (Fansidar).

Animals↗
PubMed1995

Effect of 7-day daily replacement of culture medium containing Eurycoma longifolia Jack constituents on the Malaysian Plasmodium falciparum isolates.

Six Malaysian chloroquine-resistant Plasmodium falciparum isolates were cultured in vitro following the candle-jar method. Antimalarial evaluations of daily replacement of culture medium containing chloroquine and a semi-purified extract of Eurycoma longifolia Jack (containing 13 beta, 18-dihydroeurycomanol (1), eurycomanol-2-O-beta-D-glucopyranoside (2), eurycomanol (3) and eurycomanone (4)) were performed on 6-well plates at 37 degrees C for a week. Presence or absence of the parasites was determined microscopically on thin-film Giemsa-stained preparations. Results showed that the antimalarial activity of Eurycoma longifolia Jack was dose-dependent and reached a maximum of < 50% at 0.07-5.00 micrograms ml-1 after 1 day post-treatment. However, complete inhibitions were observed at 1.25-5.00 micrograms ml-1 extract after 3 days post-treatment and 0.62 and 0.31 micrograms ml-1 after 4 and 6 days post-treatment, respectively. Further results indicated that chloroquine exhibited total inhibition at concentrations > 2.50 and 0.62 micrograms ml-1 after 1 and 2 days post-treatment, respectively and at all concentrations after 3 days post-treatment.

Animals↗
PubMed1983

Developmental modulation of protein synthetic patterns by the human malarial parasite Plasmodium falciparum.

Under conditions of in vitro culture, Plasmodium falciparum incorporated amino acids into particulate (membrane) and soluble proteins in a pattern which changed sequentially and which was dependent upon the stage of parasite maturation. Synchronized cultures pulse labeled with a mixture of 15 14C-labeled amino acids or [14C]histidine alone displayed stage-related patterns of polypeptide biosynthesis. Certain plasmodial proteins were associated with both particulate (membrane) and soluble fractions, whereas others appeared to be specific to a given fraction. Proteolysis of intact infected cells with pronase under conditions which removed 97 +/- 2.2% of the endogenous red cell acetylcholinesterase activity did not cause the apparent removal of any radiolabeled proteins; this suggests the absence of externally exposed, parasite-synthesized proteins in the infected red cell membrane. Such a result was consistent whether the radiolabel was [14C]histidine or the 14C-labeled amino acid mixture. These results indicate that specific modulation of parasite biosynthetic patterns occurs during the asexual reproductive cycle and is probably one mechanism whereby parasite differentiation occurs. Despite the formation of surface excrescences on infected red cells containing mature parasites, results of surface digestion experiments failed to demonstrate the presence of surface-exposed plasmodial proteins.

Amino Acids↗
PubMed2001

Two-step chromatographic purification of recombinant Plasmodium falciparum circumsporozoite protein from Escherichia coli.

The Plasmodium falciparum circumsporozoite (PfCS) protein (aa 19-405) has been cloned and expressed in E. coli. The protein was purified in a two-step process that was rapid and reproducible. E. coli cells were grown to a high density before induction for 1 h. Cells were disrupted by high pressure microfluidization and the total bacterial protein solubilized in 6 M Gu-HCl. The protein was refolded while bound to Ni-NTA agarose by exchange of 6 M Gu-HCl for 8 M urea and then slow removal of the urea. The eluted protein was further purified on Q Sepharose Fast Flow using conditions developed to remove E. coli proteins and reduce endotoxin (to 10 EU/50 microg). Yield was 20 mg of PfCS protein from 10 g of wet cell paste. The final protein product bound to HepG2 liver cells in culture and inhibited the invasion of those cells by sporozoites in an ISI assay greater than 80% over control cultures when used at 10 microg/ml.

Amino Acid Sequence↗
PubMed1982

Gametocytogenesis of Plasmodium falciparum in vitro: an electron microscopic study.

Plasmodium falciparum was grown in vitro in blood taken from naturally infected Gambian patients, and the development of the cultured sexual parasites was studied by light and electron microscopy. The young (Stage II and III) female gametocytes undergo a single cryptomitotic nuclear division. This division immediately follows the S phase which Sinden & Smalley (1979) have demonstrated in the Stage I and II gametocytes of both sexes. The male gametocytes, by contrast, do not undergo mitosis during their maturation period in the erythrocyte and thus remain polyploid. Hence the cell cycles of the male and female gametocytes differ significantly. The ultrastructural basis of the characteristic changes in shape of the developing gametocyte are shown to be due to the assembly and subsequent loss of components of the subpellicular membranous and microtubular cytoskeleton. Stage I-III gametocytes synthesize numerous ribosomes and endoplasmic reticulum. This correlates with the active synthesis of RNA and protein in these young parasites (Sinden & Smalley, 1979). The marked reduction in macromolecular synthesis in the mature parasites is paralleled by a reduction in cytoplasmic ribosome density in the male gametocyte only. In the female, however, this reduction in activity is correlated with the appearance of a nucleolus. These changes suggest that different mechanisms are being used to control RNA synthesis in the two sexes of gametocyte.

Animals↗
PubMed2004

Target guided synthesis of 5-benzyl-2,4-diamonopyrimidines: their antimalarial activities and binding affinities to wild type and mutant dihydrofolate reductases from Plasmodium falciparum.

The resistance to pyrimethamine (PYR) of Plasmodium falciparum arising from mutation at position 108 of dihydrofolate reductase (pfDHFR) from serine to asparagine (S108N) is due to steric interaction between the bulky side chain of N108 and Cl atom of the 5-p-Cl aryl group of PYR, which consequently resulted in the reduction in binding affinity between the enzyme and inhibitor. Molecular modeling suggested that the flexible antifolate, such as trimethoprim (TMP) derivatives, could avoid this steric constraint and should be considered as new, potentially effective compounds. The hydrophobic interaction between the side chain of inhibitor and the active site of the enzyme around position 108 was enhanced by the introduction of a longer and more hydrophobic side chain on TMP's 5-benzyl moiety. The prepared compounds, especially those bearing aromatic substituents, exhibited better binding affinities to both wild type and mutant enzymes than the parent compound. Binding affinities of these compounds correlated well with their antimalarial activities against both wild type and resistant parasites. Molecular modeling of the binding of such compounds with pfDHFR also supported the experimental data and clearly showed that aromatic substituents play an important role in enhancing binding affinity. In addition, some compounds with 6-alkyl substituents showed relatively less decrease in binding constants with the mutant enzymes and relatively good antimalarial activities against the parasites bearing the mutant enzymes.

Animals↗
PubMed1992

Involvement of Pf155/RESA and cross-reactive antigens in Plasmodium falciparum merozoite invasion in vitro.

Lines of Plasmodium falciparum FCR3 either expressing or not expressing the blood-stage antigen Pf155/RESA were used to analyze the possible involvement of this antigen in the merozoite invasion process in vitro. Antibodies from human sera, affinity purified on synthetic peptides corresponding to C-terminal repeated sequences in Pf155/RESA, were shown to inhibit merozoite invasion of both types of parasites with similar efficiency. Reversal of the invasion inhibition by fusion proteins containing repeated sequences of Pf155/RESA but not of the cross-reactive antigens Ag332 and Pf11.1 indicated that the inhibitory antibodies had similar target antigens in both Pf155/RESA+ and Pf155/RESA- parasites that involved cross-reacting epitopes present in Pf155/RESA. Rabbit antibodies specific for Pf155/RESA repeats inhibited merozoite invasion of Pf155/RESA expressing parasites efficiently but had no or very small effect on the invasion of Pf155/RESA-deficient parasites. In contrast, rabbit antibodies specific for Ag332 repeats as well as human antibodies affinity purified on synthetic Ag332 peptides inhibited merozoite invasion of both types of parasites with high efficiency. A similar inhibition pattern was seen with the human monoclonal antibody 33G2, which has specificity for Ag332 but also cross-reacts with Pf155/RESA and Pf11.1. Taken together, our data suggest that Pf155/RESA and related cross-reactive antigens as well as Ag332 are involved in the merozoite invasion process and may constitute targets for invasion inhibitory antibodies.

Animals↗