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.     

Plasmodium falciparum aldolase: gene structure and localization

A genomic clone was isolated which codes for the fructose bisphosphate aldolase of Plasmodium falciparum. The aldolase gene is interrupted by one intron which divides the coding region into two exons. The first one codes for one amino acid only, the initiation methionine, while the second one encodes the residual 368 amino acids of the protein. The gene, which is represented only once in the genome, is transcribed at high rates as a 2.4-kb mRNA in the P. falciparum blood stage. The aldolase gene encodes a protein of 40,105 Da, which is 61-68% homologous to known eukaryotic aldolases. The protein was expressed in Escherichia coli cells in an unfused and enzymatically active form. Antisera raised against amino acids 9-96 recognize a 41-kDa protein band previously shown to protect monkeys against a P. falciparum infection. These antisera cross-react with aldolases of different species, which confirms the strong conservation of this enzyme during evolution. The aldolase could be localized in the cytoplasm of the parasite as an active and soluble form. An inactive form was found to be associated with the membrane fraction. Digestion data with phospholipase C suggest a membrane association of this polypeptide via a glycosylphosphatidylinositol anchor.     

Expression and biochemical characterization of the Plasmodium falciparum DNA repair enzyme, flap endonuclease-1 (PfFEN-1)

Flap endonuclease-1 (FEN-1) is a structure-specific endonuclease that is critical for the resolution of single-stranded DNA flap intermediates that form during long patch DNA base excision repair (BER). This investigation reports that Plasmodium species encode FEN-1 homologs. Protein sequence analysis revealed the N and I domains of Plasmodium falciparum (PfFEN-1) and Plasmodium yoelii (PyFEN-1) to be homologous to FEN-1 from other species. However, each possessed an extended C domain which had limited homology to apicomplexan FEN-1s and no homology to eukaryotic FEN-1s. A conserved proliferating cell nuclear antigen (PCNA)-binding site was identified at an internal location rather than the extreme C-terminal location typically seen in FEN-1 from other organisms. The endonuclease and exonuclease activities of PfFEN-1 and PyFEN-1 were investigated using recombinant protein produced in Escherichia coli. Pf and PyFEN-1 possessed DNA structure-specific flap endonuclease and 5'-->3' exonuclease activities, similar to FEN-1s from other species. Endonuclease activity was stimulated by Mg(2+) or Mn(2+) and inhibited by monovalent ions (>20.0 mM). A PfFEN-1 C-terminal truncation mutant lacking the terminal 250 amino acids (PfFEN-1DeltaC) had endonuclease activity that was approximately 130-fold greater (k(cat)=1.2x10(-1)) than full-length PfFEN-1 (k(cat)=9.1x10(-4)) or approximately 240-fold greater than PyFEN-1 (k(cat)=4.9x10(-4)) in vitro. PfFEN-1 generated a nicked DNA substrate that was ligated by recombinant Pf DNA Ligase I (PfLigI) using an in vitro DNA repair assay. Plasmodium FEN-1s have enzymatic activities similar to other species but contain extended C-termini and a more internally located PCNA-binding site.     

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.     

Expression, partial purification and immunogenicity of fragments of the knob protein of Plasmodium falciparum

Three structural domains of the histidine-rich knob protein (KP) of Plasmodium falciparum were expressed in Escherichia coli. A single-step purification scheme was devised to obtain great enrichment of expressed polypeptides for use in subsequent experiments. Immune human sera from Africa, South-East Asia and South America were tested for reactivity with each of the expressed fragments. While the two fragments which represented the central and C-terminal regions of KP showed a strong reactivity with all the antisera which were tested, the N-terminal fragment which contains the repetitive histidine-rich sequences showed almost no reactivity.     

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.     

Expression and characterization of catalytic domain of Plasmodium falciparum subtilisin-like protease 3

PfSUB3 is the third subtilisin-like protease annotated in Plasmodium genome database "PlasmoDB". The other two members, PfSUB1 and PfSUB2 have been implicated in merozoite egress and invasion in asexual blood stages. In this study, we recombinantly expressed a region of PfSUB3 spanning from Asn(334) to Glu(769) (PfSUB3c) which encompassed the predicted catalytic domain with all the active site residues and predicted mature region spanning from Thr(516) to Glu(769) (PfSUB3m) in E. coli. PfSUB3m showed PMSF-sensitive proteolytic activity in in vitro assays. Replacement of active site serine with alanine in PfSUB3m resulted in inactive protein. We found that PfSUB3c and PfSUB3m undergo truncation to produce a 25-kDa species which was sufficient for proteolytic activity. Quantitative real-time PCR, immnufluorescence assay and Western blot analyses revealed that PfSUB3 is expressed at late asexual blood stages. Serine protease activity of PfSUB3 and its expression in the late stages of erythrocytic schizogony are indicative of some possible role of the protease in merozoite egress and/or invasion processes.     

TNF and inhibition of growth of Plasmodium falciparum

The mechanism of intra-erythrocyte death of Plasmodium chabaudi in vivo has not yet been elucidated. Here we summarise recent experiments in which serum from mice undergoing a successful immune response to this parasite did not inhibit Plasmodium falciparum in vivo unless the P. chabaudi infection and TNF levels were high enough to cause illness in the host. This was true for the 556KA and DS strains of P. chabaudi in intact mice, but not for 556KA in nude mice, which did not generate inhibitory activity at any parasitaemia. Tumour necrosis factor (TNF) inhibits malaria parasites via some undefined secondary mediator. 10 mg of r hu TNF generated this inhibitory activity, as measured against P. falciparum in vitro, in the serum of mice only if they were pretreated with Corynebacterium parvum, which activates macrophages and sensitises the mice to the toxic effects of TNF. This implies a role for activated macrophages downstream from TNF in the process involved in intra-erythrocytic death of parasites.     

Expression of a recombinant IRP-like Plasmodium falciparum protein that specifically binds putative plasmodial IREs

Plasmodium falciparum iron regulatory-like protein (PfIRPa, accession AJ012289) has homology to a family of iron-responsive element (IRE)-binding proteins (IRPs) found in different species. We have previously demonstrated that erythrocyte P. falciparum PfIRPa binds a mammalian consensus IRE and that the binding activity is regulated by iron status. In the work we now report, we have cloned a C-terminus histidine-tagged PfIRPa and overexpressed it in a bacterial expression system in soluble form capable of binding IREs. To overexpress PfIRPa, we used the T7 promoter-driven vector, pET28a(+), in conjunction with the Rosetta(DE3)pLysS strain of E. coli, which carries extra copies of tRNA genes usually found in organisms such as P. falciparum whose genome is (A+T)-rich. The histidine-tagged recombinant protein (rPfIRPa) in soluble form was partially purified using His-bind resin. We searched the plasmodial database, plasmoDB, to identify sequences capable of forming IRE loops using a specially developed algorithm, and found three plasmodial sequences matching the search criteria. In gel retardation assays, rPfIRPa bound three 32P-labeled putative plasmodial IREs with affinity exceeding the affinity for the mammalian consensus IRE. The binding was concentration-dependent and was not inhibited by heparin, an inhibitor of non-specific binding. Immunodepletion of rPfIRPa resulted in substantial inhibition of the signal intensity in the gel retardation assays and in Western blot-determinations of rPfIRPa protein levels. Endogenous PfIRPa retained all three putative 32P-IREs at the same position on the gel as the recombinant PfIRPa.     

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 cloning and characterization of Plasmodium falciparum transketolase

The pentose phosphate pathway (PPP) is an important metabolic pathway for yielding reducing power in the form of NADPH and production of pentose sugar needed for nucleic acid synthesis. Transketolase, the key enzyme of non-oxidative arm of PPP, plays a vital role in the survival/replication of the malarial parasite. This enzyme in Plasmodium falciparum is a novel drug target as it has least homology with the human host. In the present study, the P. falciparum transketolase (PfTk) was expressed, localized and biochemically characterized. The recombinant PfTk harboring transketolase activity catalyzed the oxidation of donor substrates, fructose-6-phosphate (F6P) and hydroxypyruvate (HP), with K(m)(app) values of 2.25 and 4.78 mM, respectively. p-Hydroxyphenylpyruvate (HPP) was a potent inhibitor of PfTk, when hydroxypyruvate was used as a substrate, exhibiting a K(i) value of 305 microM. At the same time, noncompetitive inhibition was observed with F6P. The native PfTk is a hexamer with subunit molecular weight of 70kDa, which on treatment with low concentrations of guanidine hydrochloride (GdmCl) dissociated into functionally active dimers. This protein was localized in the cytosol and nucleus of the parasite as studied by confocal microscopy. A model structure of PfTk was constructed based on the crystal structure of the transketolases of Saccharomyces cerevisae, Leishmania mexicana and Escherichia coli to assess the structural homology. Consistent with the homology modeling predictions, CD analysis indicated that PfTk is composed of 39% alpha-helices and 26% beta-sheets. The availability of a structural model of PfTk and the observed differences in its kinetic properties compared to the host enzyme may facilitate designing of novel inhibitors of PfTk with potential anti-malarial activity.     

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.     

Immunogenicity of recombinant Plasmodium falciparum SERA proteins in rodents

We have expressed defined regions of the serine-repeat antigen (SERA) of the Honduras-1 strain of Plasmodium falciparum in the yeast Saccharomyces cerevisiae. Amino-terminal domains of the natural SERA protein have been shown previously to be targets for parasite-inhibitory murine monoclonal antibodies. Two recombinant SERA antigens were selected for purification and immunological analysis. The first (SERA 1), corresponding to amino acids 24-285 of the natural SERA precursor, was expressed by the ubiquitin fusion method. This allowed for in vivo cleavage by endogenous yeast ubiquitin hydrolase, and subsequent isolation of the mature polypeptide. The second, larger protein (SERA N), encompassing amino acids 24-506, was expressed at only low levels using this system, but could be isolated in high yields when fused to human gamma-interferon (gamma-IFN). Each purified protein was used to immunize mice with either Freund's adjuvant or a muramyl tripeptide adjuvant that has been used in humans. Sera from immunized mice were shown to be capable of in vitro inhibition of invasion of erythrocytes by the Honduras-1 strain of P. falciparum. The results suggest that a recombinant SERA antigen may be an effective component of a candidate malaria vaccine.     

Antisense oligonucleotides targeting malarial aldolase inhibit the asexual erythrocytic stages of Plasmodium falciparum

A major obstacle in the global effort to control malaria is the paucity of anti-malarial drugs. This is compounded by the continuing emergence and spread of resistance to old and new anti-malarial drugs in the malarial parasites. Here we describe the anti-malarial effect of phosphorothioate antisense (AS) oligodeoxynucleotides (ODNs) targeting the aldolase enzyme of Plasmodium falciparum, using the asexual blood stages of the parasite grown in vitro. The blood stages of P. falciparum depend almost entirely on the energy produced by their own glycolysis. Aldolase, the fourth enzyme of the glycolytic pathway, is highly upregulated during the malarial 48-h life cycle. We found that the mRNA of this enzyme can be inhibited, in a sequence specific manner, using AS-ODN to the splice sites on the pre-mRNA of malarial aldolase. At the enzyme level, both specific AS-ODNs for the splice sites, as well as for the translation initiation site on mature mRNA, can inhibit aldolase enzyme activity within the trophozoites of P. falciparum. Furthermore, this downregulation of the malarial aldolase results in a reduction in the production of ATP within the parasite. Finally, the treatment reduces parasitemia. In summary, AS-ODNs targeting the aldolase gene of P. falciparum can interfere with the blood-stage life cycle of this parasite in vitro by inhibiting the expression of the enzyme aldolase which results in decreased malarial glycolysis and energy production. Thus, we conclude that blockade of the expression of malarial glycolytic enzymes using specific AS-ODNs has the potential of a new anti-malarial strategy.     

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.     

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.     

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.     

Expression, purification, and characterization of the immunological response to a 40-kilodalton Plasmodium vivax tryptophan-rich antigen

We describe here an approximately 40-kDa Plasmodium vivax tryptophan-rich antigen (PvTRAg40) which contains 321 amino acids and 11.4% tryptophan residues. This protein shows 65% homology (35% identity) with the previously described PvTRAg, besides sharing 23 of 27 positionally conserved tryptophan residues and similar genomic organization. The nucleotide sequence of the entire tryptophan-rich domain of PvTRAg40 was identical among 35 P. vivax clinical isolates. The protein is expressed by ring, trophozoite, and schizont stages of the parasite. The cDNA covering exon 2 of PvTRAg40 was cloned and expressed in the pPROEXHTa vector, and recombinant protein was purified. A high humoral immune response (90.7% seropositivity; n = 43) against this recombinant protein was detected in humans during the course of natural P. vivax infection. Eighty percent of the total of 20 P. vivax-exposed individuals exhibited lymphoproliferative responses against this antigen. The T cells of these individuals produced larger amounts of interleukin-12 (IL-12), IL-4, and IL-10 than gamma interferon and tumor necrosis factor alpha cytokines in response to the recombinant protein. Production of Th2-biased cytokines, conserved T- and B-cell epitopes, and an enhanced humoral immune response indicate that PvTRAg40 could possibly induce antibody-mediated immune protection against infection.     

Identification and purification of glucose phosphate isomerase of Plasmodium falciparum.

The multiplication of malaria parasites within red blood cells is energy dependent. Since these parasites lack a functional tricarboxylic acid cycle, the energy needs of the parasite are met by anaerobic glycolysis of exogenous glucose. High levels of glycolytic enzymes such as fructose-1,6-diphosphate aldolase, phosphoglycerate kinase and pyruvate kinase have been detected in infected erythrocytes. Here we report a 4-9 times increase in glucose phosphate isomerase (GPI) activity of infected erythrocytes over that of normal erythrocytes. This increase is of parasitic origin, as additional enzyme bands were observed in lysates of infected erythrocytes. The expression of GPI parallels parasite maturation and reaches a maximum at the trophozoite/schizont stage. Two distinct but closely related activity patterns consisting of 3-4 GPI isoenzymes (not shown in normal erythrocytes) with neutral to weakly acidic isoelectric points were observed in 6 P. falciparum isolates tested by isoelectric focusing. The purified P. falciparum GPI has an apparent size of 66 kDa. No size variation was observed in the 6 P. falciparum isolates studied. Furthermore, antiserum raised against this protein in BALB/c mice specifically inhibits parasite encoded GPI activity while no effect was observed on host enzyme activity.