Molecular cloning and characterization of Plasmodium falciparum transportin

A cDNA encoding transportin, a protein involved in the nuclear import of M9 nuclear localization signal-bearing proteins, has been cloned from the malaria parasite Plasmodium falciparum. The complete cDNA consists of 3,667 bp encoding 1,136 amino acid residues. Amino acid sequence analysis revealed that Ran-GTP and M9 binding domains are highly conserved in P. falciparum, suggesting that the transportin-mediated nuclear transport pathway exists in this protozoan parasite. Southern blot analysis revealed that the transportin gene exists as a single copy in the malarial genome.     

Molecular cloning, expression, characterization and mutation of Plasmodium falciparum guanylate kinase

The present work describes cloning, expression, purification, characterization, and mutation of Plasmodium falciparum guanylate kinase (PlasmoDB ID PFI1420w). Amino-acid sequence alignment revealed important differences especially in K42-V51, Y73-A77, and F100-L110, which include residues important for kinase activity, and at helix 3, which is important for domain movements. The catalytic efficiency for dGMP was 22-fold lower than that for GMP, whose value is the lowest among known guanylate kinases. dGMP was found to a competitive inhibitor for GMP with K_i = 0.148 mM and a mixed-type inhibitor with regard to ATP with measured K_i = 0.4 mM. The specificity constant (K_cat/K_m) of the four examined mutants varied for natural substrate GMP/dGMP, indicating the involvement of different mechanisms in substrate recognition and subsequent loop-domain movement. These results show that P. falciparum guanylate kinase is structurally and biochemically distinct from other guanylate kinases and could be a possible target in drug development.     

Molecular characterization of bifunctional hydroxymethyldihydropterin pyrophosphokinase-dihydropteroate synthase from Plasmodium falciparum

A 2118-base pair gene encoding the bifunctional hydroxymethyldihydropterin pyrophosphokinase-dihydropteroate syntheses of Plasmodium falciparum (pfPPPK-DHPS) was expressed under the control of the T5 promoter in a DHPS-deficient Escherichia coli strain. The enzyme was purified to near homogeneity using nickel affinity chromatography followed by gel filtration and migrates as an intense band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with apparent mass of approximately 83 kDa. Gel filtration suggested that the native pfPPPK-DHPS might exist as a tetramer of identical subunits. The enzyme was found to be Mg2+ - and ATP-dependent and had optimal temperature ranging from 37 to 45 degrees C with peak activity at pH 10. Sodium chloride and potassium chloride at 0.2 and 0.4 M, respectively, activated the activity of the enzyme but higher salt concentrations were inhibitory. Guanidine-HCl and urea inhibited the enzyme activity by 50% at 0.25 and 0.9 M, respectively. Kinetic properties of the recombinant pfPPPK-DHPS were investigated. Sulfathiazole and dapsone were potent inhibitors of pfPPPK-DHPS, whilst sulfadoxine, sulfanilamide, sulfacetamide and p-aminosalicylic acid were less inhibitory. Our construct provides an abundant source of recombinant pfPPPK-DHPS for crystallization and drug screening.     

Molecular analysis of Plasmodium falciparum hexokinase.

Hexokinase, a key glycolytic enzyme, is involved in the initial phosphorylation reaction of imported glucose and specific blocking of this activity may therefore arrest the development of malaria parasites. We describe here the cloning of a single copy hexokinase gene of Plasmodium falciparum (PfHK) from cDNA or genomic DNA libraries. The deduced amino acid sequence of PfHK has 26% identity with human hexokinase I and its predicted molecular mass assigns it as an invertebrate type isoenzyme of hexokinase. A single 1.5-kb exon is translated from a 3-kb mRNA in asexual stages of the parasite. In contrast to aldolase and GPI, the gene for this glycolytic enzyme is located on chromosome 8. Poly- and monoclonal antibodies against recombinant PfHK support our cloning results at the protein level as they detect a protein of the predicted size and isoelectric point by Western blotting in parasite protein samples. Moreover, polyclonal rabbit IgG against recombinant PfHK partially inhibits the hexokinase activity of a P. falciparum lysate which provides direct proof that the gene cloned encodes hexokinase of the parasite.     

Purification and characterization of Plasmodium falciparum succinate dehydrogenase

Succinate dehydrogenase (SDH), a Krebs cycle enzyme and complex II of the mitochondrial electron transport system was purified to near homogeneity from the human malarial parasite Plasmodium falciparum cultivated in vitro by FPLC on Mono Q, Mono S and Superose 6 gel filtration columns. The malarial SDH activity was found to be extremely labile. Based on Superose 6 FPLC, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and nondenaturing-PAGE analyses, it was demonstrated that the malarial enzyme had an apparent native molecular mass of 90 +/- 8 kDa and contained two major subunits with molecular masses of 55 +/- 6 and 35 +/- 4 kDa (n = 8). The enzymatic reaction required both succinate and coenzyme Q (CoQ) for its maximal catalysis with Km values of 3 and 0.2 microM, and k(cat) values of 0.11 and 0.06 min(-1), respectively. Catalytic efficiency of the malarial SDH for both substrates were found to be relatively low (approximately 600-5000 M(-1) s(-1)). Fumarate, malonate and oxaloacetate were found to inhibit the malarial enzyme with Ki values of 81, 13 and 12 microM, respectively. The malarial enzyme activity was also inhibited by substrate analog of CoQ, 5-hydroxy-2-methyl-1,4-naphthoquinone, with a 50% inhibitory concentration of 5 microM. The quinone had antimalarial activity against the in vitro growth of P. falciparum with a 50% inhibitory concentration of 0.27 microM and was found to completely inhibit oxygen uptake of the parasite at a concentration of 0.88 microM. A known inhibitor of mammalian mitochondrial SDH, 2-thenoyltrifluoroacetone. had no inhibitory effect on both the malarial SDH activity and the oxygen uptake of the parasite at a concentration of 50 microM. Many properties observed in the malarial SDH were found to be different from the host mammalian enzyme.     

Molecular aspects of Plasmodium falciparum Infection during pregnancy

Cytoadherence of Plasmodium-falciparum-parasitized red blood cells (PRBCs) to host receptors is the key phenomenon in the pathological process of the malaria disease. Some of these interactions can originate poor outcomes responsible for 1 to 3 million annual deaths mostly occurring among children in sub-Saharan Africa. Pregnancy-associated malaria (PAM) represents an important exception of the disease occurring at adulthood in malaria endemic settings. Consequences of this are shared between the mother (maternal anemia) and the baby (low birth weight and infant mortality). Demonstrating that parasites causing PAM express specific variant surface antigens (VSA(PAM)), including the P. falciparum erythrocyte membrane protein 1 (P f EMP1) variant VAR2CSA, that are targets for protective immunity has strengthened the possibility for the development of PAM-specific vaccine. In this paper, we review the molecular basis of malaria pathogenesis attributable to the erythrocyte stages of the parasites, and findings supporting potential anti-PAM vaccine components evidenced in PAM.     

Purification and characterization of culture-derived exoantigens of Plasmodium falciparum

An 83 kDa glycoprotein and a 100 kDa glycoprotein have been purified from the supernatant fluid of in vitro cultures of Plasmodium falciparum by conventional cation-exchange liquid chromatography, size exclusion high performance liquid chromatography, and anion-exchange high performance liquid chromatography. Both proteins exist as dimers in the native state and have been identified as parasite antigens by Western immunoblotting and by their specific reactivity in the indirect enzyme-linked immunosorbent assay. The N-terminal amino acid sequence of these two proteins has been determined and they are at least 90% homologous. The use of monospecific rabbit antisera raised against the individual pure proteins confirm their cross-reactivity. We postulate that the 83 kDa protein is a specific processing product of the larger 100 kDa protein. The presence of these proteins in the culture supernatant suggests they could both be derived from the merozoite surface coat and are potential protective antigens.     

Isolation and characterization of rhoptries of Plasmodium falciparum.

Rhoptries have been isolated from Plasmodium falciparum schizont-infected erythrocytes by isopycnic density centrifugation. Gradient fractions were analyzed by immunoblotting with antibodies against two polypeptides of 140 and 110 kDa, known to be components of the rhoptry. The proteins were present primarily in fractions with a density of 1.16 g ml-1. Electron microscopy of these fractions indicated they were enriched in rhoptries. For the most part, the isolated organelle retained in situ morphology, although some rhoptries were distorted, indicating the structure of some of the organelles is not rigid. Electrophoretic analysis of the rhoptry fractions indicated the presence of a number of proteins, many of which have not been identified to date. Properties of proteins in the isolated rhoptry were examined using the 140 and 110 kDa proteins as representative markers. Both proteins are present in a complex with a 130-kDa protein, as all three co-immunoprecipitate. At the late schizont stage, the rhoptry proteins are present in two distinct forms; a soluble form with an Mr of 480 000 which would correspond to a single copy of the 140/130/110 kDa complex and a form that can be sedimented at 130 000 x g. Properties of the sedimentable form suggest that the proteins are included in structures that resemble membranes. Ionic detergents were required to solubilize the proteins while high concentrations of NaCl and Na2CO3 resulted in only partial solubilization. Furthermore, treatment of disrupted rhoptries with phospholipase A and C resulted in the release of proteins into the soluble form.     

Purification and characterization of an aminopeptidase from Plasmodium falciparum

A soluble aminopeptidase from Plasmodium falciparum was purified by high performance liquid chromatography. The enzyme has a molecular weight of 100 000 and pI 6.8. Activity can be monitored conveniently with L-alanine-p-nitroanilide or L-leucine-p-nitroanilide at 405 nm or with L-leucine-7-amido-4-methylcoumarin in a fluorescence assay. The enzyme is inhibited by bestatin and phosphoramidone but not by leupeptin, chymostatin, antipain or pepstatin. pH-rate studies indicated the presence of a group on the free enzyme, pKa = 6.6, which must be in the conjugate base form for activity. The aminopeptidase has an essential sulfhydryl group at the active site which is rapidly modified by Hg2+ or Zn2+, is slowly modified by p-hydroxymercuribenzoate, but is not accessible to iodoacetamide or N-ethylmaleimide. The aminopeptidase is inhibited noncompetitively by chloroquine, mefloquine and quinacrine (Ki = 410, 280 and 20 microM, respectively) but is not inhibited by quinine or primaquine. Hemin does not inhibit. Complexation of hemin with quinacrine prevents inhibition by quinacrine.     

Molecular characterization of a 2-Cys peroxiredoxin from the human malaria parasite Plasmodium falciparum.

We have identified the 2-Cys peroxiredoxin (PfPrx-1) from the human malaria parasite Plasmodium falciparum. The PfPrx-1 showed the highest identity at amino acid level to the type II Prx among the currently known six subfamilies of mammalian Prx. The sequence identity between the PfPrx-1 and the previously reported 1-Cys Prx of P. falciparum (PfPrx-2), which corresponded to mammalian type VI Prx, was 25%. This suggests that the parasite possesses two Prx subfamilies. The PfPrx-1 showed significant sequence similarities with those of 2-Cys peroxiredoxins of plants in the BLASTX search. This may reflect the consequences of a genetic transfer from an algal endosymbiont to the parasite nucleus during evolution. The recombinant PfPrx-1 protein (rPfPrx-1) was expressed as a histidine fusion protein in Escherichia coli and purified with Ni chromatography. The rPfPrx-1 existed as dimers under non-reducing conditions and dissociated into monomers in the presence of dithiothreitol. The PfPrx-1 protein also exists as a dimer in the parasites themselves. The reduction of the oxidized enzyme by the donation of electrons from E. coli thioredoxin (Trx)/Trx reductase system was demonstrated in its reaction with H(2)O(2), using the rPfPrx-1 protein. These results suggested that the PfPrx-1 can act as a terminal peroxidase of the parasite Trx system. An elevated expression of the PfPrx-1 protein seen in the trophozoite, the stage with active metabolism, suggests an association of the parasite Trx system with its intracellular redox control.     

Identification and characterization of the Plasmodium falciparum RhopH2 ortholog in Plasmodium vivax

Plasmodium vivax is one of the most important human malaria species that is geographically widely endemic and potentially affects a larger number of people than its more notorious cousin, Plasmodium falciparum. During invasion of red blood cells, the parasite requires the intervention of high molecular weight complex rhoptry proteins (RhopH) that are also essential for cytoadherence. PfRhopH2, a member of the RhopH multigene family, has been characterized as being crucial during P. falciparum infection. This study describes identifying and characterizing the pfrhoph2 orthologous gene in P. vivax (hereinafter named pvrhoph2). The PvRhopH2 is a 1,369-amino acid polypeptide encoded by PVX_099930 gene, for which orthologous genes have been identified in other Plasmodium species by bioinformatic approaches. Both P. falciparum and P. vivax genes contain nine introns, and there is a high degree of similarity between the deduced amino acid sequences of the two proteins. Moreover, PvRhopH2 contains a signal peptide at its N-terminus and 12 cysteines predominantly in its C-terminal half. PvRhopH2 is localized in one of the apical organelles of the merozoite, the rhoptry, and the localization pattern is similar to that of PfRhopH2 in P. falciparum. The recombinant PvRhopH2 protein is recognized by serum antibodies of patients naturally exposed to P. vivax, suggesting that PvRhopH2 is immunogenic in humans.     

Molecular analysis of Plasmodium falciparum infections in man]

Plasmodium falciparum diversity has been analysed in two Senegalese villages with different transmission conditions and distinct kinetics of immunity acquisition. A very large allelic polymorphism was observed in both villages, with a similar number of alleles but quite distinct allelic frequencies, indicating a substantial micro-geographical heterogeneity of malaria parasite populations. In addition, the molecular characteristics of the infections differed in both villages. As in most endemic areas, many infected subjects carry multiple parasite clones. In Dielmo, the number of distinct clones hosted decreases at the age of acquisition of an efficient immunity. There was no influence of age on the number of clones hosted in Ndiop where adults experience clinical attacks. This indicates that complexity reflects acquired immunity. The precise longitudinal follow-up of parasitaemia, clinical signs and parasite genetic characteristics showed a rapid turn over of parasite populations in the peripheral blood during the transmission season, suggesting that immunity does not prevent infection but restricts multiplication of numerous genotypes at the erythrocytic stage. Clinical malaria occurs after a rapid, apparently unrestricted growth of recently inoculated parasites. The successive clinical attacks experienced by children are associated with genotypes different for each attack and different from those that the child carried during preceding asymptomatic phases. These data indicate that parasite diversity contributes to the pathology of infection and that control of parasite density, which is at least in part strain-specific, is an essential element of protection against malaria clinical attacks.     

Biochemical and structural characterization of Plasmodium falciparum glutamate dehydrogenase 2

Glutamate dehydrogenases (GDHs) play key roles in cellular redox, amino acid, and energy metabolism, thus representing potential targets for pharmacological interventions. Here we studied the functional network provided by the three known glutamate dehydrogenases of the malaria parasite Plasmodium falciparum. The recombinant production of the previously described PfGDH1 as hexahistidyl-tagged proteins was optimized. Additionally, PfGDH2 was cloned, recombinantly produced, and characterized. Like PfGDH1, PfGDH2 is an NADP(H)-dependent enzyme with a specific activity comparable to PfGDH1 but with slightly higher K(m) values for its substrates. The three-dimensional structure of hexameric PfGDH2 was solved to 3.1 ? resolution. The overall structure shows high similarity with PfGDH1 but with significant differences occurring at the subunit interface. As in mammalian GDH1, in PfGDH2 the subunit-subunit interactions are mainly assisted by hydrogen bonds and hydrophobic interactions, whereas in PfGDH1 these contacts are mediated by networks of salt bridges and hydrogen bonds. In accordance with this, the known bovine GDH inhibitors hexachlorophene, GW5074, and bithionol were more effective on PfGDH2 than on PfGDH1. Subcellular localization was determined for all three plasmodial GDHs by fusion with the green fluorescent protein. Based on our data, PfGDH1 and PfGDH3 are cytosolic proteins whereas PfGDH2 clearly localizes to the apicoplast, a plastid-like organelle specific for apicomplexan parasites. This study provides new insights into the structure and function of GDH isoenzymes of P. falciparum, which represent potential targets for the development of novel antimalarial drugs.     

Expression and biochemical characterization of Plasmodium falciparum DNA ligase I

We report that Plasmodium falciparum (Pf) encodes a 912 amino acid ATP-dependent DNA ligase. Protein sequence analysis of Pf DNA ligase I indicates a strong sequence similarity, particularly in the C-terminal region, to DNA ligase I homologues. The activity of recombinant Pf DNA ligase I (PfLigI) was investigated using protein expressed in HEK293 cells. The PfLigI gene product is approximately 94kDa and catalyzes phosphodiester bond formation on a singly nicked DNA substrate. The enzyme is most active at alkaline pH (8.5) and with Mg(2+) or Mn(2+) and ATP as cofactors. Kinetic studies of PfLigI revealed that the enzyme has similar substrate affinity (K(m) 2.6nM) as compared to human DNA ligase I and k(cat) (2.3x10(-3)s(-1)) and k(cat)/K(m) (8.8x10(5)M(-1)s(-1)) which are similar to other ATP-dependent DNA ligases. PfLigI was able to join RNA-DNA substrates only when the RNA sequence was upstream of the nick, confirming that it is DNA ligase I and has no associated DNA ligase III like activity.     

Molecular cloning, genomic structure and localization in a blood stage antigen of Plasmodium falciparum characterized by a serine stretch

Two short DNA segments were isolated by screening of a lambda gt11 library from Plasmodium falciparum schizont cDNA with an antiserum against the 140 kDa protein, which confers protective immunity to monkeys. The segments were used to identify a genomic fragment which carries the entire coding sequence for a protein of 113 kDa characterized by a stretch of serine residues (SERP I). We present the complete nucleotide and deduced amino acid sequence as well as the structure of the SERP I gene. The gene consists of four exons interrupted by three short introns located at the amino-terminal half. Exon 1 and the first part of exon 2 code for hydrophobic amino acids of a putative signal sequence. Exon 2 contains two repetitive segments, the first encoding six glycine rich octapeptides and a second region coding for 37 consecutive serine residues. Southern blot analysis demonstrated the conservation of the SERP I gene in four different parasite strains. SERP I could be localized in the parasitophorous vacuole and in the surrounding membranes. We discuss the relationship of this protein to the recently described P126 polypeptide and the possible role of this antigen as a vaccine candidate.     

Expression and characterization of glucose-6-phosphate dehydrogenase of Plasmodium falciparum

Glucose-6-phosphate dehydrogenase (G6PD) from Plasmodium falciparum has been detected previously in cultures of parasites grown in G6PD-deficient red blood cells. Using polyacrylamide gel electrophoresis, a semi-quantitative assay has been developed to compare the level of the parasite enzyme activity in G6PD normal and in G6PD-deficient host cells. The results do not support the previous contention that the host cell G6PD-deficiency necessarily affects the level of expression of the parasite enzyme. The plasmodial enzyme was partially purified from extracts of parasites prepared by digitonin lysis of infected red blood cells, and its distinctive biochemical properties are described. P. falciparum G6PD has a KmG6P of 27 microM, a KmNADP of 4.5 microM, and KiNADPH of 4.5 microM, indicating an affinity for all its main ligands much higher than that of normal human red cell G6PD.