Subunit complementation of thymidylate synthase in Plasmodium falciparum bifunctional dihydrofolate reductase-thymidylate synthase

Thymidylate synthase of Plasmodium falciparum dihydrofolate reductase-thymidylate synthase (PfDHFR-TS) functions as a dimeric enzyme with extensive contact between the two TS domains. Structural data of PfDHFR-TS shows that the formation of the two TS active sites involves contribution of the amino acid residues from both TS domains. Arg-470 donated from the adjoining domain is shown to hydrogen-bond to dUMP, while Cys-490 is a key nucleophile for TS catalysis by attacking C-6 of dUMP. However, mutants of the two series could complement one another, giving rise to active enzyme. By means of subunit complementation assay using Arg-470 and Cys-490 mutants, it is shown that co-transformants of both TS-inactive Arg-470 and Cys-490 mutants can complement the growth of thymidine auxotroph chi2913RecA(DE3) by formation of a functional TS heterodimer contributing from both Arg-470 and Cys-490 mutant subunits. 6-[3H]-FdUMP thymidylate synthase activity assay further elaborate the essence of restoration of TS activity. The TS k(cat) value of the R470D+C490A heterodimer is decreased by half from that of the wild-type PfDHFR-TS. However, the Km values for dUMP and CH2H4folate of the R470D+C490A heterodimer are similar to those of wild-type enzyme, indicating that the catalytic efficiency of the functional TS from the R470D+C490A heterodimer is similar to the wild-type TS enzyme in P. falciparum DHFR-TS.     

A Plasmodium falciparum blood stage antigen highly homologous to the glycophorin binding protein GBP.

We have isolated a gene coding for a protein highly homologous to an antigen known as the glycophorin binding protein (GBP) which was therefore called GBPH. The gene consists of 2 exons interrupted by an intron located at a position corresponding to that of the GBP gene. The deduced amino acid sequence of GBPH comprises 427 residues and is characterized by a signal sequence and by an extended repeat region consisting of 8 units of 40 amino acid residues. The comparison of the amino acid sequences of GBPH and GBP reveals an identity of 69%. Antisera raised against a GBPH fragment that carries part of the repetitive region cross-react with GBP (105 kDa) and additionally detect some bands between 40 and 70 kDa, one of which may correspond to GBPH. The genes coding for GBP and GBPH are located on chromosomes 10 and 14, respectively. The GBP gene is transcribed as a highly abundant 6.5 kb mRNA in the blood-stage form, whereas Northern blot analysis using a GBPH specific probe detects 2 less abundant mRNAs of 2.3 kb and 2.7 kb. Southern blot analysis of P. falciparum DNA identifies a third member of the GBP gene family.     

Plasmodium falciparum erythrocyte invasion: a conserved myosin associated complex

Host cell invasion by apicomplexan parasites is powered by an actin/myosin motor complex that has been most thoroughly described in Toxoplasma gondii tachyzoites. In T. gondii, two inner membrane complex (IMC) proteins, the peripheral protein TgGAP45 and the transmembrane protein TgGAP50, form a complex with the myosin, TgMyoA, and its light chain, TgMLC1. This complex, referred to as the glideosome, anchors the invasion motor to the IMC. We have identified and characterized orthologues of TgMLC1, TgGAP45 and TgGAP50 in blood-stages of the major human pathogen Plasmodium falciparum, supporting the idea that the same basic complex drives host cell invasion across the apicomplexan phylum. The P. falciparum glideosome proteins are transcribed, expressed and localized in a manner consistent with a role in erythrocyte invasion. Furthermore, PfMyoA interacts with PfMTIP through broadly conserved mechanisms described in other eukaryotes, and forms a complex with PfGAP45 and PfGAP50 in late schizonts and merozoites. P. falciparum is known to use multiple alternative invasion pathways to enter erythrocytes, hampering vaccine development efforts targeting erythrocyte invasion. Our data suggests that the same invasion motor underpins all alternative invasion pathways, making it an attractive target for the development of novel intervention strategies.     

Plasmodium falciparum drug resistance and sulfadoxine-pyrimethamine in Africa

The sulfadoxine-pyrimethamine combination has not been recommended for the prophylaxis of malaria since 1985 following serious accidents in the USA. However, this drug is worth considering for treatment since it has the advantage over mefloquine of being cheaper, having fewer side effects and it avoids using mefloquine. A study of Plasmodium falciparum resistance to Fansidar should be carried out on cases imported to France to determine an adapted utilisation of this drug. This would be an appreciable advantage for tropical Africa.     

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.     

Mechanisms and dynamics of drug resistance in Plasmodium falciparum

The emergence of chloroquine resistance has been associated with a dramatic increase in malaria mortality in some human populations from endemic regions. Plasmodium falciparum drug resistant malaria originates from chromosomal mutations. Analysis using molecular, genetic and biochemical approaches has shown that 1) impaired intake of chloroquine by the parasite vacuole is a common characteristic of resistant strains, the chloroquine-resistance mechanism regulates the access of chloroquine to hematin, this phenotype correlates with Pfmdr1 and Pfcg2 gene mutations; 2) one to four point mutations of dihydrofolate reductase, the enzyme target of antifolinics (pyrimethamine and proguanil), give moderate to high levels of resistance to these drugs but there is a fitness cost to resistance; 3) the mechanism of resistance to sulfonamides and sulfones involves mutations of dihydropteroate synthase, their enzyme target; 4) treatment with sulphadoxine-pyrimethamine (SP) selected for the variants Ile(51), Arg(59) and Asn(108) of DHFR and for the variants Ser(436), Gly(437), and Glu(540) of DHPS; 5)clones that were resistant to some traditional antimalarial agents acquired resistance to new ones at high frequency (accelerated resistance to multiple drugs--ARMD). Amino-alcohol (quinine, mefloquine, halofantrine) mechanisms of resistance are still unclear. Population genetic studies have confirmed that selfing is more frequent in Plasmodium falciparum where the transmission rate is lower in some regions such as Papua-New Guinea, whereas isolates from individuals on the Thai-Burmese border, an area of hypoendemic transmission, revealed a higher number of genotypes per infected person. It has been suggested that intense intra-host competition between co-infecting clones, low numbers of genes required to encode resistance, and high drug usage all encourage the emergence of drug resistance. On the other hand, the greater effective recombination in high transmission areas may breakdown multiple drug resistance when it is coded for by two unlinked loci. Epidemiological studies have established that the frequency of chloroquine resistant mutants varies among parasite isolate populations while resistance to antifolinics is highly prevalent in most malarial endemic countries (more than 92% of Kenyan field isolates have undergone at least one point mutation). Established and strong drug pressure as well as low antiparasitic immunity probably explains the multidrug-resistance encountered in forests of Southeast Asia and South America. In Africa, frequent genetic recombinations in Plasmodium originate from a high level of malaria transmission, and falciparum chloroquine-resistant prevalence seems to stabilise at an equal level as chloroquine-sensitive malaria. Clinical studies demonstrated that control of clinical symptoms is better when chloroquine is used with sulphadoxine-pyrimethamine (SP) than when SP is used alone, and the cure rate also tends to be higher with the triple combination regimen.     

Characterization of the 175-kilodalton erythrocyte binding antigen of Plasmodium falciparum

EBA-175 is a soluble 175-kDa Plasmodium falciparum antigen that is released into culture supernatants during rupture of schizont-infected erythrocytes. EBA-175 binds to erythrocytes and binding is sialic acid-dependent. A clone expressing the gene encoding EBA-175 was obtained previously by screening a genomic DNA expression library with antibodies that had been affinity-purified from EBA-175. Antibodies were raised against a 43-amino-acid peptide (EBA-peptide 4) synthesized according to the deduced amino acid sequence. Antibodies to peptide 4 and affinity-purified antibodies specific for EBA-175 were used to characterize further EBA-175 giving the following results: (1) EBA-175 differs biochemically and immunologically from other reported malarial antigens; (2) the EBA-175s from six geographical isolates of P. falciparum are antigenically conserved; (3) EBA-175 is expressed during schizogony as a 190-kDa protein which is larger than the culture supernatant form of the antigen. The 190-kDa form of the protein is recovered from the cell pellet in schizont-infected erythrocytes and partitions to the soluble fraction when extracted with detergent; (4) release of soluble EBA-175 into the culture supernatant coincides with schizont rupture; (5) there was no observable change in pI (pI=6.86) by isoelectric focusing between the cellular and supernatant species of the protein; and (6) release of EBA-175 into the culture supernatant is inhibited by the addition of chymostatin and leupeptin. The continued research into the role of EBA-175 during erythrocyte invasion may aid in vaccine development for malaria.     

A rhoptry antigen of Plasmodium falciparum contains conserved and variable epitopes recognized by inhibitory monoclonal antibodies

Four monoclonal antibodies produced against Plasmodium falciparum recognize an antigen in merozoites that is localized in rhoptries, as judged by a punctate, double dot fluorescence pattern. All four antibodies bound to the same affinity purified antigen in a two site immunoradiometric assay. Immunoprecipitation of antigen by monoclonal antibody followed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis yielded protein bands of 80, 66 and 42 kDa. Western blotting gave bands of 80 and 66 kDa only with three of the antibodies: the fourth did not blot. Based on protease inhibitor data the 66 kDa band is considered to be a cleavage product of the 80 kDa band, but the 42 kDa band does not appear to derive from the latter and may be a coprecipitation product. This group of antigens labels with both [35S]methionine and [3H]histidine. Two of the monoclonal antibodies inhibited merozoite invasion of erythrocytes. One of these inhibitors recognizes a variable epitope, whereas the second recognizes a highly conserved epitope present in all 106 primary isolates of P. falciparum tested from Brazil, Thailand and Papua New Guinea.     

Differential binding of clonal variants of Plasmodium falciparum to allelic forms of intracellular adhesion molecule 1 determined by flow adhesion assay

Adhesion of Plasmodium falciparum-infected erythrocytes to the endothelial ligand intercellular adhesion molecule 1 (ICAM-1) has been implicated in the pathogenesis of cerebral malaria. Recently, a high-frequency coding polymorphism in the N-terminal domain of ICAM-1 (ICAM-1(Kilifi)) that is associated with susceptibility to cerebral disease in Kenya has been described. Preliminary static adhesion assays suggested that two different selected P. falciparum lines, ITO4-A4 (A4) and ItG-ICAM (ItG), have different properties of binding to the natural variant proteins ICAM-1 and ICAM-1(Kilifi). Using a flow adhesion assay system, we have confirmed differences between the two lines in binding of parasitized erythrocytes to the variant ICAM-1 proteins. Total adhesion of ItG-infected erythrocytes to ICAM-1 and ICAM-1(Kilifi) is greater than that of A4-infected erythrocytes, and erythrocytes infected by both parasite strains show reduced binding to ICAM-1(Kilifi). However, under these physiologically relevant flow conditions, we have shown differences between A4 and ItG strains in dynamic rolling behavior on ICAM-1(Kilifi). The percentage of erythrocytes infected with A4 that roll on both ICAM-1 and ICAM-1(Kilifi) is greater than that of those infected with ItG. Also, the rolling velocity of A4-infected erythrocytes on ICAM-1(Kilifi) is markedly increased compared to that on ICAM-1, in contrast to the rolling velocity of ItG-infected erythrocytes, which is similar on both proteins. These findings suggest that different parasite lines can vary in their avidity for the same host ligand, which may have important consequences for the pathophysiology of P. falciparum malaria.     

Optimal tumor necrosis factor induction by Plasmodium falciparum requires the highly localized release of parasite products

Overproduction of tumor necrosis factor (TNF) has been linked with the pathogenesis of Plasmodium falciparum malaria. Here, we examined why the high levels of TNF-inducing activity associated with P. falciparum-parasitized erythrocytes (PE) appear to be lost after cell lysis. Static coculture of PE and peripheral blood mononuclear cells (PBMC), with or without separation by porous membranes, demonstrated that rupture of live PE in the presence of responder cells was required for optimal TNF induction. Although the insoluble fraction of lysed PE was found to partially inhibit TNF responses, supernatants prepared from large numbers of lysed PE still contained only low levels of TNF-inducing activity, which showed no evidence of instability. A dramatic reduction in TNF levels resulted when noncytoadherent PE lines were maintained under low-cell-proximity conditions by suspension coculture. This reduction was much less marked with PE capable of adhering to PBMC, despite the fact that cytoadherent and noncytoadherent parasite lines induced comparable levels of TNF in high-cell-proximity, static coculture. These results suggest that rupture of PE in a highly localized setting, facilitated by either static coculture or the more biologically relevant phenomenon of cytoadherence to PBMC, can result in considerable enhancement of the P. falciparum-induced TNF response.     

Induction and selection of drug resistant mutants of Plasmodium falciparum

Conditions for mutagenizing Plasmodium falciparum in culture with the mutagen N-methyl-N'-nitro-N-nitrosoguanidine have been established. Parasite mutants resistant to aphidicolin, cycloheximide and sinefungin have been selected and subcloned.     

Mapping the binding site for gossypol-like inhibitors of Plasmodium falciparum lactate dehydrogenase

Gossypol is a di-sesquiterpene natural-product in the form of a functionalised binaphthyl and is isolated from cotton plants. The compound has long been known to exhibit anti-malarial and other biological activities. Previous studies have indicated that compounds of this type target Plasmodium falciparum lactate dehydrogenase (pfLDH), an essential enzyme for energy generation within the parasite. In this study, we report that simple naphthalene-based compounds, the core of the gossypol structure, exhibit weak inhibition of the parasite lactate dehydrogenase. Crystal structures of the complexes formed by binding of these naphthalene-based compounds to their target enzyme have been used to delineate the molecular features likely to form the gossypol binding site. Two modes of binding are observed: one overlapping the pyruvate but not the co-factor site, the other bridging the binding sites for the co-factor nicontinamide group and pyruvate substrate. This latter site encompasses molecular features unique to Plasmodium forms of LDH and is likely to represent the mode of binding for gossypol derivatives that show selectivity for the parasite enzymes. We also report a substrate analogue that unexpectedly binds within the adenine pocket of the co-factor groove. Although these core pharmacophore-like molecules only exhibit low levels of inhibitory activity, these molecular snapshots provide a rational basis for renewed structure-based development of naphthalene-based compounds as anti-malarial agents.