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PubMed1984

Clonal diversity in a single isolate of the malaria parasite Plasmodium falciparum.

Clones of an isolate of Plasmodium falciparum from Mae Sod (Thailand) were prepared by a dilution procedure. Some of the parasite cultures thus obtained have been typed for the following characters: (i) electrophoretic variants of three enzymes; (ii) susceptibility to chloroquine and pyrimethamine; (iii) antigen diversities recognized by ten strain-specific monoclonal antibodies; (iv) presence or absence of knobs on infected erythrocytes and (v) two-dimensional PAGE variants of seven proteins. Amongst the clones there was variation involving each of these five characters. At least seven different types of clones were found in ten cultures produced by dilution. The amount of phenotypic variation within a single isolate has thus been shown to be surprisingly great. Variations in drug susceptibility and antigens are considered to be particularly important in view of their relevance to anti-malarial treatments.

Animals↗

PubMed1987

Identification and characterisation of proteins associated with the rhoptry organelles of Plasmodium falciparum merozoites.

We describe antigens of Plasmodium falciparum recognised by murine monoclonal antibodies which by immunofluorescence react with the rhoptry organelles of the extracellular merozoite stage. Immunoblotting shows that the antibodies recognise two major parasite antigens of Mr 82 and 65 kilodaltons (kDa). Immunoprecipitations from detergent extracts of [35S]-methionine-labelled parasites show that the 82-kDa and 65-kDa antigens are parasite proteins. Pulse-chase experiments on synchronous parasite cultures show that the 82-kDa protein is synthesised during early schizogony and is later processed into the 65-kDa antigen in segmenting schizonts. In Nonidet P-40, these antigens are non-covalently associated with two other proteins of 40 kDa and 42 kDa. The 40/42-kDa doublet is synthesised in parallel with the 82 kDa antigen and persists, apparently unchanged, till the end of the cell cycle.

Animals↗

PubMed1982

Comparison of various serological test results using antigens from different strains of Plasmodium falciparum.

Suitability of different strains of Plasmodium falciparum grown continuously in vitro was compared using the indirect haemagglutination (IHA) the indirect immunofluorescent (IFA) tests and ELISA. In the tests employing soluble antigens (IHA and ELISA), there was a significant higher mean log titer of the same sera tested against different strains. Ranking of the strains in term of sensitivity for the detection of malaria antibody in people in the endemic area were G-112 = SO = CC greater than SU greater than PS in the IHA test and G-112 = SO greater than CC greater than SU greater than PS in the ELISA. The difference in the mean log titers appear to relate neither to the geographical location not the isoenzyme markers tested. There was also an apparent correlation between the results of the IHA and the IFA test but not between these two tests and ELISA.

Enzyme-Linked Immunosorbent Assay↗

PubMed1984

Isoenzyme characterization of chloroquine-resistant isolates of Plasmodium falciparum from Burma.

Sixty isolates of Plasmodium falciparum collected from hospital in-patients in Rangoon and out-patients from villages near Rangoon were tested in vitro for chloroquine sensitivity and then cultured to carry out starch gel electrophoresis of the following parasite enzyme: glucose phosphate isomerase (GPI) (EC.5.3.1.9.), NADP-dependent glutamate dehydrogenase (GDH) EC.1.4.1.4), lactate dehydrogenase (LDH) (EC.1.1.1.27) and 6-phosphogluconate dehydrogenase (6PGD) (E.C.1.1.1.44). Variations were observed in three (GPI, GDH and LDH) of the four enzymes examined. Three forms of parasite GPI, two forms of parasite GDH, two forms of parasite LDH and only one form of parasite 6PGD were identified. Enzyme polymorphism was more common in isolates with higher resistance to chloroquine. One isolate with parasite GDH-2 and six isolates with parasite LDH-2 were recorded from chloroquine-resistant cases.

Animals↗

PubMed1979

Characterization of a protein correlated with the production of knob-like protrusions on membranes of erythrocytes infected with Plasmodium falciparum.

Membranes of erythrocytes infected with Plasmodium falciparum develop protrusions called "knobs." These protrusions are not apparent on erythrocytes infected with young parasites (rings) but develop with the growth of parasites to the trophozoite and schizont stages. The nature and origin of knobs were characterized by comparing the stage-specific proteins of two culture lines of P. falciparum, K+ and K-. K+ parasites produce knobs; K- parasites do not. Erythrocytes infected with both types of parasites were labeled metabolically and samples were analyzed by electrophoresis in sodium dodecyl sulfate/polyacrylamide gels. There were no apparent differences in Coomassie blue-stained or radioactive components of rings of K+ and K- parasites. However, erythrocytes infected with K+ trophozoites or schizonts showed a major labeled protein, with an apparent molecular weight of approximately 80,000, that was not present in any developmental stage of K- parasites or in K+ rings. A fraction enriched in membranes from erythrocytes infected with K+ trophozoites showed enrichment of this protein. The results indicate that this protein, synthesized by the parasites, is correlated with the formation of knobs on the host cell membrane. Two additional labeled components were identified. These appeared with the development of schizonts in both K+ and K- parasites and are therefore stage-dependent and not correlated with knobs.

Antigens, Surface↗

PubMed1994

Transcriptional and nucleosomal characterization of a subtelomeric gene cluster flanking a site of chromosomal rearrangements in Plasmodium falciparum.

We have recently demonstrated that Plasmodium falciparum chromosomes are compartmentalized into different domains: conserved and polymorphic domains; transcriptionally active and silent domains. Here we have analyzed the transition between these domains at the structural and nucleosomal level. This study was conducted with the end of chromosome 2 that is associated with cytoadherence. At this end of the chromosome, the first set of erythrocytic genes has been mapped 85 kb from the telomere. These genes are monocistronically transcribed as revealed by nuclear run-on analysis. Two of these genes, PfEMP3 and KAHRP, are deleted in cytoadherent negative mutants, whereas the third gene, GLARP, is expressed in all the strains investigated. The data indicate that the polymorphic domain at this end of chromosome 2 extends into the transcribed region. Analysis of the chromatin structure revealed that both the transcribed domain and the subtelomeric region are organized as nucleosomes with a periodicity of 155 +/- 5 bp.

Animals↗

PubMed2025

Identification of novel PfEMP1 variants containing domain cassettes 11, 15 and 8 that mediate the Plasmodium falciparum virulence-associated rosetting phenotype.

Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) is a diverse family of variant surface antigens, encoded by var genes, that mediates binding of infected erythrocytes to human cells and plays a key role in parasite immune evasion and malaria pathology. The increased availability of parasite genome sequence data has revolutionised the study of PfEMP1 diversity across multiple P. falciparum isolates. However, making functional sense of genomic data relies on the ability to infer binding phenotype from var gene sequence. For P. falciparum rosetting, the binding of infected erythrocytes to uninfected erythrocytes, the analysis of var gene/PfEMP1 sequences encoding the phenotype is limited, with only eight rosette-mediating PfEMP1 variants described to date. These known rosetting PfEMP1 variants fall into two types, characterised by N-terminal domains known as "domain cassette" 11 (DC11) and DC16. Here we test the hypothesis that DC11 and DC16 are the only PfEMP1 types in the P. falciparum genome that mediate rosetting, by examining a set of thirteen recent culture-adapted Kenyan parasite lines. We first analysed the var gene/PfEMP1 repertoires of the Kenyan lines and identified an average of three DC11 or DC16 PfEMP1 variants per genotype. In vitro rosette selection of the parasite lines yielded four with a high rosette frequency, and analysis of their var gene transcription, infected erythrocyte PfEMP1 surface expression, rosette disruption and erythrocyte binding function identified four novel rosette-mediating PfEMP1 variants. Two of these were of the predicted DC11 type (one showing the dual rosetting/IgM-Fc-binding phenotype), whereas two contained DC15 (DBLα1.2-CIDRα1.5b) a PfEMP1 type not previously associated with rosetting. We also showed that a Thai parasite line expressing a DC8-like PfEMP1 binds to erythrocytes to form rosettes. Hence, these data expand current knowledge of rosetting mechanisms and emphasize that the PfEMP1 types mediating rosetting are more diverse than previously recognised.

Plasmodium falciparum↗

PubMed2005

Dictyostelium discoideum expresses a malaria chloroquine resistance mechanism upon transfection with mutant, but not wild-type, Plasmodium falciparum transporter PfCRT.

Chloroquine resistance in Plasmodium falciparum malaria results from mutations in PfCRT, a member of a unique family of transporters present in apicomplexan parasites and Dictyostelium discoideum. Mechanisms that have been proposed to explain chloroquine resistance are difficult to evaluate within malaria parasites. Here we report on the targeted expression of wild-type and mutant forms of PfCRT to acidic vesicles in D. discoideum. We show that wild-type PfCRT has minimal effect on the accumulation of chloroquine by D. discoideum, whereas forms of PfCRT carrying a key charge-loss mutation of lysine 76 (e.g. K76T) enable D. discoideum to expel chloroquine. As in P. falciparum, the chloroquine resistance phenotype conferred on transformed D. discoideum can be reversed by the channel-blocking agent verapamil. Although intravesicular pH levels in D. discoideum show small acidic changes with the expression of different forms of PfCRT, these changes would tend to promote intravesicular trapping of chloroquine (a weak base) and do not account for reduced drug accumulation in transformed D. discoideum. Our results instead support outward-directed chloroquine efflux for the mechanism of chloroquine resistance by mutant PfCRT. This mechanism shows structural specificity as D. discoideum transformants that expel chloroquine do not expel piperaquine, a bisquinoline analog of chloroquine used frequently against chloroquine-resistant parasites in Southeast Asia. PfCRT, nevertheless, may have some ability to act on quinine and quinidine. Transformed D. discoideum will be useful for further studies of the chloroquine resistance mechanism and may assist in the development and evaluation of new antimalarial drugs.

Acids↗

PubMed1993

Two-color flow cytometric analysis of intraerythrocytic malaria parasite DNA and surface membrane-associated antigen in erythrocytes infected with Plasmodium falciparum.

A method for fixation of Plasmodium falciparum infected erythrocytes and solubilization of the erythrocyte membrane with detergent was developed. This method was applied to two-color flow cytometric analysis of both intraerythrocytic (IE) malaria DNA and parasite-derived antigen on the erythrocyte surface membrane. Infected erythrocytes were fixed with 0.025% glutaraldehyde followed by treatment with 1% saponin to gain access to intramembranous components and allow antibody to interact with antigen. DNA of IE parasite was subsequently stained with propidium iodide. Using this procedure cell morphology was well preserved with excellent parasite DNA staining. Using anti-malaria antibodies which recognize ring-infected erythrocyte surface antigen (Pf155/RESA), we observed that glutaraldehyde-fixed saponin treated infected erythrocytes exhibited a variable immunofluorescence intensity as assessed by both flow cytometry and fluorescence microscopy. Ring-infected cells displayed strong immunofluorescence staining, whereas a weak signal was detected on cells containing schizonts. Simultaneous measurement of parasite DNA and antigen in the infected erythrocyte membrane can facilitate the study of antigen expression in the cell membrane in association with development of IE parasites.

Animals↗

PubMed1985

Interspersed blocks of repetitive and charged amino acids in a dominant immunogen of Plasmodium falciparum.

We describe an antigen of Plasmodium falciparum that is a dominant immunogen in man. The corresponding cDNA clone, Ag231, expressing this antigen in Escherichia coli reacted in an in situ colony assay with sera from up to approximately equal to 93% of 65 people living in an area in which P. falciparum is endemic. Human antibodies affinity purified on immobilized Ag231 lysates identified the corresponding parasite antigen as a polypeptide of Mr approximately equal to 300,000. It was present in schizonts and also in ring-stage trophozoites, where a speckled immunofluorescence pattern suggested an association with the erythrocyte. Its mRNA was enriched in merozoites relative to other blood stages, a distinctive property shared by a recently described antigen located on the surface of ring-infected erythrocytes, and it is encoded by a single gene having a number of allelic variants. The complete nucleotide sequence of Ag231 revealed a structural unit composed of 13 hexapeptide repeats flanked by a highly charged region containing both acidic and basic amino acids. This structural unit is itself repeated, so that blocks of repeats and charged units are interspersed along the molecule. The sequences within the repeats vary much more extensively than those in the charged units.

Amino Acid Sequence↗

PubMed2001

A single, bi-functional aquaglyceroporin in blood-stage Plasmodium falciparum malaria parasites.

The malaria parasite Plasmodium falciparum faces drastic osmotic changes during kidney passages and is engaged in the massive biosynthesis of glycerolipids during its development in the blood-stage. We identified a single aquaglyceroporin (PfAQP) in the nearly finished genome of P. falciparum with highest similarity to the Escherichia coli glycerol facilitator (50.4%), but both canonical Asn-Pro-Ala (NPA) motifs in the pore region are changed to Asn-Leu-Ala (NLA) and Asn-Pro-Ser (NPS), respectively. Expression in Xenopus oocytes renders them highly permeable for both water and glycerol. Sugar alcohols up to five carbons and urea pass the pore. Mutation analyses of the NLA/NPS motifs showed their structural importance, but the symmetrical pore properties were maintained. PfAQP is expressed in blood-stage parasites throughout the development from rings via trophozoites to schizonts and is localized to the parasite but not to the erythrocyte cytoplasm or membrane. Its unique bi-functionality indicates functions in the protection from osmotic stress and efficiently provides access to the serum glycerol pool for the use in ATP generation and primarily in the phospholipid synthesis.

Adenosine Triphosphate↗

PubMed2006

Programmed transcription of the var gene family, but not of stevor, in Plasmodium falciparum gametocytes.

The var genes encode Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) proteins, a set of highly diverse surface-expressed proteins that mediate adhesion of erythrocytes infected with asexual blood-stage parasites to host endothelium. Switching among expressed PfEMP1 variants in the course of a blood-stage infection is a key component of antigenic variation, and thus immune evasion, by the parasite. The majority of var loci are found in the subtelomeric regions of P. falciparum chromosomes associated with members of other multigene families, including stevor. Both PfEMP1 and STEVOR are expressed in gametocytes, the transmissible parasite stage, but the role of these proteins in the biology of sexual-stage parasites remains unknown. PfEMP1 may continue to mediate antigenic variation in gametocytes, which need to persist in the host for many days before reaching maturity. Using quantitative reverse transcription-PCR and Northern hybridization, we demonstrate that transcription of a defined subset of type C var loci occurs during gametocyte development in vitro. This transcriptional program occurs in gametocytes regardless of the var expression phenotype of their asexual progenitors and therefore is subject to regulatory processes distinct from those that manage antigenic variation in the asexual parasite. In contrast, the same stevor variants are transcribed in both gametocytes and their asexual progenitors. We also provide evidence that for both asexual parasites and gametocytes, var and stevor transcription patterns are not linked to each other.

Animals↗

PubMed2001

The H89 cAMP-dependent protein kinase inhibitor blocks Plasmodium falciparum development in infected erythrocytes.

In Plasmodium falciparum, the causative agent of human malaria, the catalytic subunit gene of cAMP-dependent protein kinase (Pfpka-c) exists as a single copy. Interestingly, its expression appears developmentally regulated, being at higher levels in the pathogenic asexual stages than in the sexual forms of parasite that are responsible for transmission to the mosquito vector. Within asexual parasites, PfPKA activity can be readily detected in schizonts. Similar to endogenous PKA activity of noninfected red blood cells, the parasite enzyme can be stimulated by cAMP and inhibited by protein kinase inhibitor.Importantly, ex vivo treatment of infected erythrocytes with the classical PKA-C inhibitor H89 leads to a block in parasite growth. This suggests that the PKA activities of infected red blood cells are essential for parasite multiplication. Finally, structural considerations suggest that drugs targeting the parasite, rather than the erythrocyte enzyme, might be developed that could help in the fight against malaria.

Amino Acid Sequence↗

PubMed2004

Distinct trafficking and localization of STEVOR proteins in three stages of the Plasmodium falciparum life cycle.

The genome of Plasmodium falciparum harbors three extensive multigene families, var, rif, and stevor (for subtelomeric variable open reading frame), located mainly in the subtelomeric regions of the parasite's 14 chromosomes. STEVOR variants are known to be expressed in asexual parasites, but no function has as yet been ascribed to this protein family. We have examined the expression of STEVOR proteins in intraerythrocytic sexual stages, gametocytes, and extracellular sporozoites isolated from infected Anopheles mosquitoes. In gametocytes, stevor transcripts appear transiently early in development but STEVOR proteins persist for several days and are transported out of the parasite, travel through the host cell cytoplasm, and localize to the erythrocyte plasma membrane. In contrast to asexual parasites, gametocytes move STEVOR to the periphery via a trafficking pathway independent of Maurer's clefts. In sporozoites, STEVOR appear dispersed throughout the cytoplasm in vesicle-like structures. The pattern of STEVOR localization we have observed in gametocytes and sporozoites differs significantly from that in asexual parasite stages. STEVOR variants are therefore likely to perform different functions in each stage of the parasites life cycle in which they occur.

Animals↗

PubMed2002

Characterization of proteases involved in the processing of Plasmodium falciparum serine repeat antigen (SERA).

The Plasmodium falciparum serine repeat antigen (SERA), a malaria vaccine candidate, is processed into several fragments (P73, P47, P56, P50, and P18) at the late schizont stage prior to schizont rupture in the erythrocytic cycle of the parasite. We have established an in vitro cell-free system using a baculovirus-expressed recombinant SERA (bvSERA) that mimics the SERA processing that occurs in parasitized erythrocytes. SERA processing was mediated by parasite-derived trans-acting proteases, but not an autocatalytic event. The processing activities appeared at late schizont stage. The proteases are membrane associated, correlating with the secretion and accumulation of SERA within the parasitophorous vacuole membrane (PVM). The activity responsible for the primary processing step of SERA to P47 and P73 was inhibited by serine protease inhibitor DFP. In contrast, the activity responsible for the conversion of P56 into P50 was inhibited by each of the cysteine protease inhibitors E-64, leupeptin and iodoacetoamide. Moreover, addition of DFP, E-64 or leupeptin to the cultures of schizont-stage parasites blocked schizont rupture and release of merozoites from PVM. These results indicate that SERA processing correlates to schizont rupture and the processing is mediated by at least three distinct proteases.

Animals↗

PubMed2001

Systematic optimization of expression and refolding of the Plasmodium falciparum cysteine protease falcipain-2.

The Plasmodium falciparum cysteine protease falcipain-2 is a potential new target for antimalarial chemotherapy. In order to obtain large quantities of active falcipain-2 for biochemical and structural analysis, a systematic assessment of optimal parameters for the expression and refolding of the protease was carried out. High-yield expression was achieved using M15(pREP4) Escherichia coli transformed with the pQE-30 plasmid containing a truncated profalcipain-2 construct. Recombinant falcipain-2 was expressed as inclusion bodies, solubilized, and purified by nickel affinity chromatography. A systematic approach was then used to optimize refolding parameters. This approach utilized 100-fold dilutions of reduced and denatured falcipain-2 into 203 different buffers in a microtiter plate format. Refolding efficiency varied markedly. Optimal refolding was obtained in an alkaline buffer containing glycerol or sucrose and equal concentrations of reduced and oxidized glutathione. After optimization of the expression and refolding protocols and additional purification with anion-exchange chromatography, 12 mg of falcipain-2 was obtained from 5 liters of E. coli, and crystals of the protease were grown. The systematic approach described here allowed the rapid evaluation of a large number of expression and refolding conditions and provided milligram quantities of recombinant falcipain-2.

Animals↗

PubMed1997

Sequence diversity and molecular evolution of the merozoite surface antigen 2 of Plasmodium falciparum.

Eleven new alleles of the Plasmodium falciparum merozoite surface antigen 2 (MSA2) from Papua New Guinea were analyzed by direct sequencing of polymerase chain reaction (PCR) products. We have used the sequence information to trace the molecular evolution of MSA2. The repeats of ten alleles belonging to the 3D7 allelic family differed considerably in size, nucleotide sequence, and repeat copy number. In the repeat region of these new alleles, codon usage was extremely biased with an exclusive use of NNT codons. Another new allele sequenced belonged to the FC27 family and confirmed the family-specific conserved structure of 96 and 36 bp repeats. In order to assess sequence microheterogeneity within samples defined as the same genotype by restriction fragment length polymorphism (RFLP), we have analyzed single-strand conformation polymorphism (SSCP) of different samples of the most frequent allele (D10 of the FC27 family) in the study population. No sequence heterogeneity could be detected within the repeat region. Based on analysis of the repeat regions in both allelic families, we discuss the hypothesis of a different evolutionary strategy being represented by each of the allelic families. Kew words: Merozoite surface antigen 2 - Nucleotide sequence comparisons - Molecular evolution

Amino Acid Sequence↗

PubMed2003

Neutralization of toxic heme by Plasmodium falciparum histidine-rich protein 2.

Plasmodium falciparum histidine-rich protein 2 (PfHRP2) has been suggested to be an initiator of the polymerization of heme, which is produced as by-product on the digestion of hemoglobin, and a promoter of the H(2)O(2)-induced degradation of heme in food vacuoles of the malarial parasite. In this work, we have designed PfHRP2 model peptides, R18 and R27 (18 and 27 residues, respectively), and used them for optical and electron spin resonance spectroscopic measurements to confirm that the axial ligands of the heme-PfHRP2 complex are the nitrogenous donors derived from the imidazole moieties of histidine residues of PfHRP2. In addition, we revealed that the affinities of R18 and R27 for heme (K(d) = 2.21 x 10(-6) M and 0.71 x 10(-6) M, respectively) might be as high as that of PfHRP2 (K(d) = 0.94 x 10(-6) M). The R27 peptide can remove heme from membrane-intercalated heme and inhibit heme-induced hemolysis. Therefore, we suggest another function of PfHRP2: it may play an important role in the neutralization of toxic heme in the parasite cytoplasm and infected erythrocytes by removing heme from heme-bound membranes or reducing heme-induced hemolysis.

Amino Acid Sequence↗