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PubMed2001

Stage-dependent localization of a novel gene product of the malaria parasite, Plasmodium falciparum.

A novel Plasmodium falciparum gene, MB2, was identified by screening a sporozoite cDNA library with the serum of a human volunteer protected experimentally by the bites of P. falciparum-infected and irradiated mosquitoes. The single-exon, single-copy MB2 gene is predicted to encode a protein with an M(r) of 187,000. The MB2 protein has an amino-terminal basic domain, a central acidic domain, and a carboxyl-terminal domain with similarity to the GTP-binding domain of the prokaryotic translation initiation factor 2. MB2 is expressed in sporozoites, the liver, and blood-stage parasites and gametocytes. The MB2 protein is distributed as a approximately 120-kDa moiety on the surface of sporozoites and is imported into the nucleus of blood-stage parasites as a approximately 66-kDa species. Proteolytic processing is favored as the mechanism regulating the distinct subcellular localization of the MB2 protein. This differential localization provides multiple opportunities to exploit the MB2 gene product as a vaccine or therapeutic target.

Amino Acid Sequence↗
PubMed1990

Initial extracellular development in vitro of erythrocytic stages of malaria parasites (Plasmodium falciparum).

Merozoites of Plasmodium falciparum placed in culture medium with a 50% erythrocyte extract and supplemented with ATP and pyruvate differentiated extracellularly into early trophic forms. Erythrocyte extract prepared by sonication was found superior to extract prepared by freezing and thawing. Under the best conditions, up to 30% of the merozoites showed some development after 16 hr of incubation and approximately 5% developed into larger forms, often ring-shaped and occasionally showing pigment. The small as well as the larger forms took up the fluorescent dye rhodamine 123. Under similar conditions, partial further development was also obtained of young rings freed from their host cells. Again, the sonicated erythrocyte extract gave better development than the frozen-thawed extract, and ATP with pyruvate had a marked favorable effect. These parasites had both a plasma membrane and a surrounding closely apposed parasitophorous membrane, whereas the forms derived by extracellular development of merozoites had only their plasma membrane. We conclude that initial development requires neither an intact erythrocyte nor a parasitophorous membrane.

Adenosine Diphosphate↗
PubMed1993

Monoclonal antibody epitope mapping of Plasmodium falciparum rhoptry proteins.

Plasmodium falciparum rhoptry proteins of the 140/130/110-kDa high molecular weight complex (HMWC) are secreted into the erythrocyte membrane during merozoite invasion. Epitopes of membrane-associated HMWC proteins can be detected using rhoptry-specific antibodies by immunofluorescence assays. Phospholipase treatment of ring-infected intact human erythrocytes, membrane ghosts, and inside-out vesicles results in the release of the HMWC as demonstrated by immunoblotting. We characterized the membrane-associating properties of the 110-kDa protein in more detail. PLA2 from three different sources; bee venom, Naja naja venom, and porcine pancreas, were examined and all were equally effective in releasing the 110-kDa protein. Furthermore, PLA2 activity was inhibited by o-phenanthroline, quinacrine, maleic anhydride, and partially by p-bromophenacyl bromide, indicating that the activity of PLA2 is specific. Using sequential protease and phospholipase digestion experiments to map the immunoreactive and functional epitopes of the 110-kDa protein, a 35-kDa protease-resistant protein associated with mouse and human erythrocyte membranes was identified. Limited proteolysis of the 110-kDa protein and analysis by immunoblotting demonstrated several immunoreactive cleavage products, including a highly protease-resistant peptide fragment of approximately 35-kDa which corresponds to the membrane-associated protein. Epitope mapping of the 130-kDa rhoptry protein resulted in a different pattern of cleavage products. Stage-specific metabolic labeling of P. falciparum with [3H] palmitate and [3H] myristate was performed to determine the lipophilic properties of the HMWC. Results showed the incorporation of label into proteins of approximate molecular weight 200 and 45-kDa, predominantly in the late schizont stage. Interestingly, proteins of 140 and 110/100-kDa, corresponding to [35S] methionine-labeled proteins were labeled with [3H]palmitate in ring-infected erythrocyte membranes. However, these proteins were not immunoprecipitated by a rhoptry protein-specific monoclonal antibody, 1B9. Similar label incorporation was not obtained with [3H]myristate. In Triton X-114 solubility studies, the HMWC proteins partitioned into the aqueous phase, suggesting that they are not integral membrane proteins. In addition, the proteins were extracted by 100 mM Na2CO3, pH 11.5, and immunoprecipitated by rhoptry-specific antibody. These results suggest that the HMWC proteins may exist in a soluble and membrane bound form. The latter may participate in membrane expansion and the formation of the parasitophorous vacuole during merozoite invasion.

Animals↗
PubMed1984

Mechanism of pyrimethamine resistance in recent isolates of Plasmodium falciparum.

Clones of Plasmodium falciparum prepared from recent isolates of infected blood were studied to determine the molecular mechanism of naturally occurring pyrimethamine resistance. Total DNA, as well as thymidylate synthetase and dihydrofolate reductase activities, were characterized from these lines. Restriction analysis of DNA from pyrimethamine-susceptible and -resistant lines of the parasite showed no obvious amplification of any DNA fragment. Further, analysis of DNA from resistant and susceptible lines by centrifugation in cesium chloride-ethidium bromide revealed no extrachromosomal amplification in the resistant line. Comparison of the dihydrofolate reductase enzyme activity in the two lines revealed similar KmS for substrate but a large difference in the inhibition constant for pyrimethamine. Additionally, the enzyme from the resistant line was considerably more stable in vitro than the corresponding enzyme from the susceptible line. The thymidylate synthetase activity in the two lines was similar and unaffected by pyrimethamine. The mechanism of drug resistance in this isolate involves altered properties of the dihydrofolate reductase conferring both a different affinity for the drug and increased stability.

Animals↗
PubMed2003

Native and inhibited structure of a Mu class-related glutathione S-transferase from Plasmodium falciparum.

The parasite Plasmodium falciparum causes malaria tropica, the most prevailing parasitic disease worldwide, with 300-500 million infections and 1.5-2.7 million deaths/year. The emergence of strains resistant to drugs used for prophylaxis and treatment and no vaccine available makes the structural analysis of potential drug targets essential. For that reason, we analyzed the three-dimensional structure of the glutathione S-transferase from P. falciparum (Pf-GST1) in the apoform and in complex with its inhibitor S-hexyl-glutathione. The structures have been analyzed to 2.6 and 2.2 A, respectively. Pf-GST1 shares several structural features with the Mu-type GSTs and is therefore closely related to this class, even though alignments with its members display low sequence identities in the range of 20-33%. Upon S-hexyl-glutathione binding, the overall structure and the glutathione-binding site (G-site) remain almost unchanged with the exception of the flexible C terminus. The detailed comparison of the parasitic enzyme with the human host Mu-class enzyme reveals that, although the overall structure is homologue, the shape of the hydrophobic binding pocket (H-site) differs substantially. In the human enzyme, it is shielded from one side by the large Mu-loop, whereas in Pf-GST1 the Mu-loop is truncated and the space to recognize and bind voluminous substrates is extended. This structural feature can be exploited to support the design of specific and parasite-selective inhibitors.

Amino Acid Sequence↗
PubMed2000

Evidence for vesicle-mediated trafficking of parasite proteins to the host cell cytosol and erythrocyte surface membrane in Plasmodium falciparum infected erythrocytes.

Plasmodium falciparum malaria parasites actively remodel the host cell cytosol and plasma membrane during the erythrocytic cycle. The focus of this investigation was to characterize intra-parasitic and -erythrocytic secretory pathways. Electron-dense vesicles, similar in appearance to mammalian secretory vesicles were detected in proximity to smooth tubo-vesicular elements at the periphery of the parasite cytoplasm in mature parasites by transmission electron microscopy. Vesicles (60-100 nm diameter), which appeared to be coated, were visualized on the erythrocytic side of the parasite vacuolar membrane and in the erythrocyte cytosol. The vesicles seemed to bind to and fuse with the erythrocyte membrane, giving rise to cup-shaped electron-dense structures, which might be intermediates in knob structure formation. Treatment of mature parasites with aluminum tetrafluoride, an activator of GTP-binding proteins, resulted in the accumulation of the vesicles with an electron-dense limiting membrane in the erythrocyte cytosol into multiple vesicle strings. These vesicle complexes were often associated with and closely abutted the erythrocyte membrane, but were apparently prevented from fusing by the aluminum fluoride treatment. The parasite proteins PfEMP1 and PfEMP3 were found by immunoelectron microscopy to be associated with these vesicles, suggesting they are responsible for transporting these proteins to the erythrocyte membrane.

Aluminum Compounds↗
PubMed1999

The complete nucleotide sequence of chromosome 3 of Plasmodium falciparum.

Analysis of Plasmodium falciparum chromosome 3, and comparison with chromosome 2, highlights novel features of chromosome organization and gene structure. The sub-telomeric regions of chromosome 3 show a conserved order of features, including repetitive DNA sequences, members of multigene families involved in pathogenesis and antigenic variation, a number of conserved pseudogenes, and several genes of unknown function. A putative centromere has been identified that has a core region of about 2 kilobases with an extremely high (adenine + thymidine) composition and arrays of tandem repeats. We have predicted 215 protein-coding genes and two transfer RNA genes in the 1,060,106-base-pair chromosome sequence. The predicted protein-coding genes can be divided into three main classes: 52.6% are not spliced, 45.1% have a large exon with short additional 5' or 3' exons, and 2.3% have a multiple exon structure more typical of higher eukaryotes.

Animals↗
PubMed2005

High-throughput screening for potent and selective inhibitors of Plasmodium falciparum dihydroorotate dehydrogenase.

Plasmodium falciparum is the causative agent of the most serious and fatal malarial infections, and it has developed resistance to commonly employed chemotherapeutics. The de novo pyrimidine biosynthesis enzymes offer potential as targets for drug design, because, unlike the host, the parasite does not have pyrimidine salvage pathways. Dihydroorotate dehydrogenase (DHODH) is a flavin-dependent mitochondrial enzyme that catalyzes the fourth reaction in this essential pathway. Coenzyme Q (CoQ) is utilized as the oxidant. Potent and species-selective inhibitors of malarial DHODH were identified by high-throughput screening of a chemical library, which contained 220,000 drug-like molecules. These novel inhibitors represent a diverse range of chemical scaffolds, including a series of halogenated phenyl benzamide/naphthamides and urea-based compounds containing napthyl or quinolinyl substituents. Inhibitors in these classes with IC(50) values below 600 nm were purified by high pressure liquid chromatography, characterized by mass spectroscopy, and subjected to kinetic analysis against the parasite and human enzymes. The most active compound is a competitive inhibitor of CoQ with an IC(50) against malarial DHODH of 16 nm, and it is 12,500-fold less active against the human enzyme. Site-directed mutagenesis of residues in the CoQ-binding site significantly reduced inhibitor potency. The structural basis for the species selective enzyme inhibition is explained by the variable amino acid sequence in this binding site, making DHODH a particularly strong candidate for the development of new anti-malarial compounds.

Animals↗
PubMed1999

A comparison of thick smears, QBC malaria, PCR and PATH falciparum malaria test trip in Plasmodium falciparum diagnosis.

Blood samples from 182 patients presenting at the out-patient clinic in Richard-Toll. Senegal were analysed by Thick smear microscopy, the QBC, PCR and the new dipstick PATH Malaria assay which detects the histidine rich protein II antigen of Plasmodium falciparum. Thick smear microscopy was used as the reference method. Sensitivity, specificity, predictive positive and negative values were 100%, 83.6%, 93.4% and 100% QBC respectively; 100%, 72.7%, 89.4% and 100% for PCR; 96%, 92.7%, 96.8% and 91% for the PATH assay. PATH assay failed to detect one positive sample with Plasmodium malariae. Assays were also compared with regard to the expense of equipment and reagents and speed and ease of use. The rapid PATH assay can be performed with minimal training and may be specially useful in areas where P. falciparum is the predominant malaria species, in epidemic malaria regions, and where skilled microscopy is not readily available.

Ambulatory Care Facilities↗
PubMed2006

Assessment of the drug susceptibility of Plasmodium falciparum clinical isolates from africa by using a Plasmodium lactate dehydrogenase immunodetection assay and an inhibitory maximum effect model for precise measurement of the 50-percent inhibitory concentration.

The extension of drug resistance among malaria-causing Plasmodium falciparum parasites in Africa necessitates implementation of new combined therapeutic strategies. Drug susceptibility phenotyping requires precise measurements. Until recently, schizont maturation and isotopic in vitro assays were the only methods available, but their use was limited by technical constraints. This explains the revived interest in the development of replacement methods, such as the Plasmodium lactate dehydrogenase (pLDH) immunodetection assay. We evaluated a commercially controlled pLDH enzyme-linked immunosorbent assay (ELISA; the ELISA-Malaria antigen test; DiaMed AG, Cressier s/Morat, Switzerland) to assess drug susceptibility in a standard in vitro assay using fairly basic laboratory equipment to study the in vitro resistance of malaria parasites to major antimalarials. Five Plasmodium falciparum clones and 121 clinical African isolates collected during 2003 and 2004 were studied by the pLDH ELISA and the [8-(3)H]hypoxanthine isotopic assay as a reference with four antimalarials. Nonlinear regression with a maximum effect model was used to estimate the 50% inhibitory concentration (IC(50)) and its confidence intervals. The two methods were observed to have similar reproducibilities, but the pLDH ELISA demonstrated a higher sensitivity. The high correlation (r = 0.98) and the high phenotypic agreement (kappa = 0.88) between the two methods allowed comparison by determination of the IC(50)s. Recently collected Plasmodium falciparum African isolates were tested by pLDH ELISA and showed drug resistance or decreased susceptibilities of 62% to chloroquine and 11.5% to the active metabolite of amodiaquine. No decreased susceptibility to lumefantrine or the active metabolite of artemisinin was detected. The availability of this simple and highly sensitive pLDH immunodetection assay will provide an easier method for drug susceptibility testing of malaria parasites.

Africa↗
PubMed1987

[Current aspects of imported drug-resistant Plasmodium falciparum].

24 cases of Plasmodium falciparum malaria in a group of 73 non-immune subjects coming mostly from central Africa have been tested in vitro for chloroquine sensitivity: 5 are sensitive, 19 are resistant among whom one is also quinine-resistant. The comparison of the major clinical and biological features in the two groups by methods of statistical calculation shows that the parasitological diagnosis is retarded and that hemolysis is more frequent in the strains of Plasmodium falciparum resistant to chloroquine. Thrombopenia is more frequent with chemosensitives strains. In any case, the delay between the first symptoms and the parasitological diagnosis is increased with an average of 17 days in comparison to what was observed in the 1970's. Under the influence of several complex factors including human ones, the clinical aspects are changed in comparison to forms usually observed. In this context of an extension of the emergence of the chemoresistance and in the aim of efficiency, only the quinine I.V., with an average dose of 16.5 mg/kg per day of quinine-base, has been chosen as a curative treatment during 5 days allowing a rapid recovery without after-effects for all the cases.

Adult↗
PubMed2000

Plasmodium falciparum: detection and strain identification of Indian isolates by polymerase chain reaction.

The polymerase chain reaction (PCR) was employed for detection and strain identification of P. falciparum in a comparative field study of Indian isolates. The primers were selected from highly conserved regions flanking the variable, tandemly repeated regions of highly polymorphic cell surface antigens, major merozoite surface antigen-1 (MSP-1), major surface antigen-2 (MSP-2), circumsporozoite surface antigen (CSP) and ring-infected erythrocyte surface antigen (RESA). Out of the 52 microscopically positive P. falciparum infected field samples, 47 samples were positive by PCR. Variation in the size of the amplified products was observed using MSP-1, MSP-2 specific primers respectively in different field isolates of P. falciparum, but CSP and RESA did not exhibit any variation in size of the amplified product. The multiplex PCR results demonstrated that amplified products from these surface antigens vary in size and there is a specific pattern for each strain and this could be utilized to identify a particular field isolate. One P. falciparum infected field sample detected by the above PCR method was found to be a mixed infection by two different strains. Five microscopically positive P. vivax infeced samples were also analyzed by PCR method using P. falciparum cell surface antigen (MSP-2) specific primers. PCR results showed one P. vivax infected sample was positive when P. falciparum specific primers were used, this could be due to inaccurate and reduced limit of detection of Plasmodial species by microscopic examination.

Animals↗
PubMed1992

Malaria mimicry with tumor necrosis factor. Contrasts between species of murine malaria and Plasmodium falciparum.

Because Plasmodium berghei ANKA induces cerebral malaria and P. vinckei does not, the former has often been studied as a model for human falciparum malaria. It lacks, however, many of the systemic changes seen in the human disease. Because both of these murine models and the human disease have now been defined in terms of excess tumor necrosis factor (TNF) production, the authors have more closely examined the two murine models in this light to see which provides the better overall model for falciparum malaria. Administering TNF to malaria-infected mice did not cause cerebral symptoms nor breakdown of the blood-brain barrier, which is the hallmark of P. berghei ANKA cerebral malaria and is generally absent in human cerebral malaria. Tumor necrosis factor did, however, induce hypoglycemia and liver injury, pathology that is seen in terminal P. vinckei and falciparum malaria, but is absent in terminal P. berghei ANKA malaria. Plasma TNF and interleukin-6 (IL-6) also were found to be consistently higher in infections caused by P. vinckei than in those caused by P. berghei ANKA. The pathology of P. vinckei malaria is thus consistent with raised systemic levels of TNF and other cytokines, as is falciparum malaria. The authors therefore conclude that P. vinckei malaria, although lacking a cerebral component, is the better model for the human disease.

Animals↗
PubMed1999

The Thai-Burmese border: drug studies of Plasmodium falciparum in pregnancy.

Plasmodium falciparum malaria is increasing world-wide, as is resistance to the available antimalarials. On the Thai-Burmese border this problem is most acute in pregnant women, as options for their treatment are even more restricted because of the unknown effects of antimalarials on the foetus. Presented here are the results of descriptive, clinical, drug studies on quinine, mefloquine and artemisinin derivatives for P. falciparum in pregnant women. Mefloquine and quinine have high failure rates for primary and recrudescent infections. Artemisinin-based treatments in pregnant women have proved safe, tolerable and efficacious. However, randomized drug studies with these drugs and other new antimalarials are required to define the true safety and efficacy of these drugs in pregnant women.

Antimalarials↗
PubMed1995

The use of the polymerase chain reaction for more sensitive detection of Plasmodium falciparum.

The prevalence of Plasmodium falciparum in children and adults living in a malaria-endemic area in Papua New Guinea was determined by microscopy and by polymerase chain reaction (PCR). The sensitivity of detecting P. falciparum infections increased two-fold with PCR. Undetected infections by microscopy were more frequent in adults (including adolescents) than in children. Detecting this subpatent parasitaemia by PCR resulted in an equal P. falciparum prevalence in children and adults; in children the parasitaemia rate increased from 32% to 48% and in adults from 23% to 47%. In more than 50% of all blood samples positive for P. vivax and P. malariae an underlying P. falciparum infection remained undetected by microscopy. The introduction of PCR has opened up new possibilities in malaria diagnosis and research.

Adolescent↗
PubMed1990

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-2b) 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.

Amino Acid Sequence↗
PubMed1992

Cytoadherence characteristics of rosette-forming Plasmodium falciparum.

Sequestration of Plasmodium falciparum-infected erythrocytes to the capillary endothelium can cause obstruction and localized tissue damage. Occlusion of vessels in falciparum malaria infection has been related to two properties of the parasite: adhesion to endothelial cells and rosette formation. Our study on P. falciparum isolates from Thailand producing variable numbers of rosettes suggests the involvement of rosettes in capillary blockage caused by direct adhesion of the rosette-forming infected erythrocytes to various target cells, e.g., live human umbilical vein endothelial cells, monocytes, and platelets. These rosettes did not bind Formalin-fixed target cells, nor did they bind to live or fixed C32 or G361 melanoma cells. Classification of the receptors involved in cytoadherence of endothelial cells and monocytes by specific antibody blocking and flow cytometry indicated that CD36 was involved in the adherence of monocytes but that other receptors besides CD36 may be involved in parasite adherence to endothelial cells. The cytoadherence of infected erythrocytes to monocytes was also associated with CD54 (ICAM-1). Further, differentiation of adherent monocytes resulted in an inversion of CD36 and CD54 levels on the cell surface which correlated with a decrease in surface binding of infected erythrocytes. This observation suggests that the state of cell activation and differentiation may also contribute to sequestration of parasites and to the pathogenesis of malaria.

Animals↗
PubMed2005

Combinatorial gene regulation in Plasmodium falciparum.

The malaria parasite Plasmodium falciparum has a complicated life cycle with large variations in its gene expression pattern, but it contains relatively few specific transcriptional regulators. To elucidate this paradox, we identified regulatory sequences, using an approach that integrates the sequence conservation among species and the correlation in mRNA expression within a species. Our analysis identified several DNA sequence motifs that are associated with mRNA expression, two of which were previously determined experimentally. We found more putative regulatory sequences per gene in P. falciparum than in other eukaryotes, such as yeast. We propose that Plasmodium uses the few regulatory proteins it has in a combinatorial approach for gene regulation, explaining the relative paucity in regulatory proteins.

Animals↗