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PubMed2002

Topology and replication of a nuclear episomal plasmid in the rodent malaria Plasmodium berghei.

The rodent malaria Plasmodium berghei is one of a small number of species of Plasmodium that can currently be genetically transformed through experimentally controlled uptake of exogenous DNA by bloodstage parasites. Circular DNA containing a selectable marker replicates and is maintained under selection pressure in a randomly segregating episomal form during the first weeks after transformation. In this study, using pulsed field gel electrophoresis and ionising radiation, we show that in dividing asexual blood stage parasites the episomes are completely converted, within 2 weeks post-infection, into non-rearranged circular concatamers ranging in size between about 9 and 15 copies of the monomer. These occur as slow-moving aggregates held together by radiation-sensitive linkers consisting partly of single-stranded DNA. The process generating these complexes is not clear but 2D gel analysis showed that Cairns-type replication origins were absent and it seems most likely that the initial concatamerisation takes place using a rolling circle mechanism followed by circularisation through internal recombination. We propose a model in which continued rolling circle replication of the large circular concatamers and the recombinational activity of the tails of the rolling circles could lead to the formation of the large aggregates.

Animals↗
PubMed2006

Comparative whole genome transcriptome analysis of three Plasmodium falciparum strains.

Gene expression patterns have been demonstrated to be highly variable between similar cell types, for example lab strains and wild strains of Saccharomyces cerevisiae cultured under identical growth conditions exhibit a wide range of expression differences. We have used a genome-wide approach to characterize transcriptional differences between strains of Plasmodium falciparum by characterizing the transcriptome of the 48 h intraerythrocytic developmental cycle (IDC) for two strains, 3D7 and Dd2 and compared these results to our prior work using the HB3 strain. These three strains originate from geographically diverse locations and possess distinct drug sensitivity phenotypes. Our goal was to identify transcriptional differences related to phenotypic properties of these strains including immune evasion and drug sensitivity. We find that the highly streamlined transcriptome is remarkably well conserved among all three strains, and differences in gene expression occur mainly in genes coding for surface antigens involved in parasite-host interactions. Our analysis also detects several transcripts that are unique to individual strains as well as identifying large chromosomal deletions and highly polymorphic regions across strains. The majority of these genes are uncharacterized and have no homology to other species. These tractable transcriptional differences provide important phenotypes for these otherwise highly related strains of Plasmodium.

Animals↗
PubMed2006

Development and application of a positive-negative selectable marker system for use in reverse genetics in Plasmodium.

A limitation of transfection of malaria parasites is the availability of only a low number of positive selectable markers for selection of transformed mutants. This is exacerbated for the rodent parasite Plasmodium berghei as selection of mutants is performed in vivo in laboratory rodents. We here report the development and application of a negative selection system based upon transgenic expression of a bifunctional protein (yFCU) combining yeast cytosine deaminase and uridyl phosphoribosyl transferase (UPRT) activity in P.berghei followed by in vivo selection with the prodrug 5-fluorocytosine (5-FC). The combination of yfcu and a positive selectable marker was used to first achieve positive selection of mutant parasites with a disrupted gene in a conventional manner. Thereafter through negative selection using 5-FC, mutants were selected where the disrupted gene had been restored to its original configuration as a result of the excision of the selectable markers from the genome through homologous recombination. This procedure was carried out for a Plasmodium gene (p48/45) encoding a protein involved in fertilization, the function of which had been previously implied through gene disruption alone. Such reversible recombination can therefore be employed for both the rapid analysis of the phenotype by targeted disruption of a gene and further associate phenotype and function by genotype restoration through the use of a single plasmid and a single positive selectable marker. Furthermore the negative selection system may also be adapted to facilitate other procedures such as 'Hit and Run' and 'vector recycling' which in principle will allow unlimited manipulation of a single parasite clone. This is the first demonstration of the general use of yFCU in combination with a positive selectable marker in reverse genetics approaches and it should be possible to adapt its use to many other biological systems.

Animals↗
PubMed1995

Stage-specific expression of the calmodulin gene in Plasmodium falciparum.

We studied the synthesis of calmodulin mRNA and of calmodulin during the asexual cell cycle of Plasmodium falciparum. The expression of the gene was low during the early stages of the erythrocytic asexual cycle. When the parasites were 28 h old the level of mRNA increased steadily to reach a peak at the age of 40 h. During the last 8 h of the cycle a constant decrease in the amount of calmodulin mRNA was observed. The synthesis of the protein followed the expression pattern of the mRNA, but without the final decrease. The average amounts of calmodulin measured were 2.5 +/- 0.9 ng/10(8) 14-h-old rings, 4.38 +/- 0.35 ng/10(8) 36-h-old trophozoites, 11.8 +/- 1.45 ng/10(8) 44-h-old trophozoites, and 19.6 +/- 1.9 ng/10(8) 48-h-old schizonts. The in vivo stability of mRNA in different developmental stages was evaluated by pulse and chase experiments. The RNA of very mature trophozoites was quickly degraded, in contrast with the high stability shown by the RNA in other stages. The results here suggest that the calmodulin gene expression in Plasmodium does not follow a housekeeping pattern of expression such as in other eukaryotic cells, but that it is regulated at the level of transcription and RNA degradation.

Animals↗
PubMed1976

Beta-actinin-like protein from plasmodium.

A beta-actinin-like protein was isolated from plasmodia of the slime mold. The chain weight was the same as that of actin (43,000), but the amino acid composition was significantly different. The action of plasmodium beta-actinin on actin was the same as that of beta-actinin from rabbit skeletal muscle: inhibition of the recombination of F-actin fragments; formation of Mg polymer; inhibition of interfilamental interaction of F-actin and retardation of depolymerization of F-actin. The only difference observed was its sensitivity to trypsin: plasmodium actinin was less quickly digested by trypsin than rabbit beta-actinin.

Actinin↗
PubMed1997

Erythrocyte membrane alterations in Plasmodium falciparum malaria sequestration.

Plasmodium falciparum malaria, the most lethal form of human malaria, claims at least 2 million lives worldwide each year. Recently, there has been a significant advance in our understanding of the molecular basis of P. falciparum sequestration, a distinctive pathologic feature that often leads to fatal human cerebral malaria. Parasite-derived VAR proteins (Plasmodium falciparum-infected erythrocyte membrane protein 1) have been cloned and identified as antigenically diverse cytoadherent receptors localized to the knob protrusions that act as attachment points in parasite sequestration. Evidence now supports the hypothesis that cryptic regions of band 3 protein are parasite-induced, host-derived erythrocyte receptors mediating parasite sequestration. Knob structures have been localized to spectrin-actin-protein 4.1 junctions in intact spread membrane skeletons. A recombinant domain of knob-associated histidine-rich protein, a major protein found in both membrane-intact and isolated knobs, has been shown to associate with filamentous actin and spectrin. Parasite- and host-derived erythrocyte membrane proteins involved in P. falciparum sequestration are discussed in this review.

Animals↗
PubMed2006

Modulation of malaria virulence by determinants of Plasmodium falciparum erythrocyte membrane protein-1 display.

PURPOSE OF REVIEW: Plasmodium falciparum malaria parasites carry approximately 60 var genes that encode variable adhesins termed P. falciparum erythrocyte membrane protein-1. Clonal expression of a single P. falciparum erythrocyte membrane protein-1 variant on the surface of the parasitized host erythrocyte promotes binding of the cell to blood elements (including noninfected erythrocytes, leukocytes) and walls of microvessels. These binding events enable parasitized erythrocytes to sequester and avoid clearance by the spleen, and they also contribute to disease by causing microvascular inflammation and obstruction. RECENT FINDINGS: Steps by which P. falciparum erythrocyte membrane protein-1 is exported to the parasitized erythrocyte surface have recently been elucidated. The ability of parasites to cytoadhere and cause disease depends on the variant of P. falciparum erythrocyte membrane protein-1 as well as its amount and distribution at the erythrocyte surface. An example of a host polymorphism that affects P. falciparum erythrocyte membrane protein-1 display is hemoglobin C, which may protect against malaria by impairing the parasite's ability to adhere to microvessels and induce inflammation. Interference with P. falciparum erythrocyte membrane protein-1-mediated phenomena appears to diminish cytoadherence in vivo and to protect against disease in animal models. SUMMARY: Plasmodium falciparum erythrocyte membrane protein-1-mediated sequestration of parasitized erythrocytes plays a central role in malaria pathogenesis. Clinical interventions aimed at reducing cytoadherence and microvascular inflammation may improve disease outcome.

Animals↗
PubMed1980

A malaria parasite of the white-tailed deer (Odocoileus virginianus) and its relation with known species of Plasmodium in other ungulates.

With the exception of the human malaria parasites, the mammalian species of Plasmodium have a fairly restricted geographical distribution, while the incidence of the infection in the different orders of mammals is patchy. A few species have been described in ungulates in the Old World Tropics, but Plasmodium odocoilei sp.nov. of the white-tailed deer (Odocoileus virginianus) of Texas is the first example to be found in the New World. It was discovered in an adult specimen that had had its spleen removed. Only the blood stages of the parasite are known, but these show various remarkable features: the presence of large vacuoles in the cytoplasm, and the enormous distortion and discoloration of the infected red blood cell. The phylogeny of the parasite is discussed in relation to that of the vertebrate host and it is suggested that Cervid stock crossed the Bering Land Bridge in the Pliocene Age, carrying a plasmodial infection that was the common source of malaria in the Old World Tragulidae and in the New World Odocoileus.

Animals↗
PubMed2000

Life history of a malaria parasite (Plasmodium mexicanum): independent traits and basis for variation.

Plasmodium mexicanum, a malaria parasite of lizards, exhibits substantial variation among infections in the life-history traits which define its blood-dwelling stages. Such variation in life histories among infections is common in Plasmodium and may influence the ecology and evolution of the parasite's transmission success and virulence. Insight into these issues requires identification of independent traits (some traits may be bound by developmental trade-offs) and the importance of genetic versus host effects producing the variation. We studied 11 life-history traits in 120 induced infections of P. mexicanum in its natural lizard host (20 each from six donor infections). The traits varied among infections and fell into three clusters: rate/peak (rate of increase and peak parasitaemia of asexuals and gametocytes), time (duration of pre-patent period and the infection's growth) and maturity (timing of first gametocytes). Thus, few life-history traits define an infection in the lizard's blood. Donor effects were significant for ten traits and two trait clusters (maturity was the exception) suggesting genetic differences among infections may influence the rate of increase and peak parasitaemia, but not the timing of the first production of gametocytes.

Animals↗
PubMed2002

Mining the Plasmodium genome database to define organellar function: what does the apicoplast do?

Apicomplexan species constitute a diverse group of parasitic protozoa, which are responsible for a wide range of diseases in many organisms. Despite differences in the diseases they cause, these parasites share an underlying biology, from the genetic controls used to differentiate through the complex parasite life cycle, to the basic biochemical pathways employed for intracellular survival, to the distinctive cell biology necessary for host cell attachment and invasion. Different parasites lend themselves to the study of different aspects of parasite biology: Eimeria for biochemical studies, Toxoplasma for molecular genetic and cell biological investigation, etc. The Plasmodium falciparum Genome Project contributes the first large-scale genomic sequence for an apicomplexan parasite. The Plasmodium Genome Database (http://PlasmoDB.org) has been designed to permit individual investigators to ask their own questions, even prior to formal release of the reference P. falciparum genome sequence. As a case in point, PlasmoDB has been exploited to identify metabolic pathways associated with the apicomplexan plastid, or 'apicoplast' - an essential organelle derived by secondary endosymbiosis of an alga, and retention of the algal plastid.

Animals↗
PubMed2000

Targeted terminal deletions as a tool for functional genomics studies in Plasmodium.

We describe a transfection system that induces terminal deletions at specific chromosome ends in malaria parasites using a linear construct containing telomeric repeats at one end and plasmodial sequences able to drive homologous recombination at the other. A site-specific deletion was generated at one extremity of chromosome 5 of Plasmodium berghei, which was stably maintained in the parasite population selected after transfection. The telomeric repeat array introduced with the construct reached the average length observed in natural telomeres of Plasmodium, indicating that in vivo telomere addition occurred at the newly formed extremity. The expression of a mutant dhfr/ts gene conferring pyrimethamine resistance, used as a selectable marker, was not affected by the proximity to the telomeric sequences, either in the presence or absence of drug pressure. In addition, no transcriptional silencing was observed on insertion of the mutant dhfr/ts gene either in subtelomeric or internal positions that are transcriptionally silent in blood-stage parasites. This suggests that the activity of its promoter is not affected by the chromatin organization of the chromosomal context.

Animals↗
PubMed2004

Comparative genomics of transcriptional control in the human malaria parasite Plasmodium falciparum.

The life cycle of the parasite Plasmodium falciparum, responsible for the most deadly form of human malaria, requires specialized protein expression for survival in the mammalian host and insect vector. To identify components of processes controlling gene expression during its life cycle, the malarial genome--along with seven crown eukaryote group genomes--was queried with a reference set of transcription-associated proteins (TAPs). Following clustering on the basis of sequence similarity of the TAPs with their homologs, and together with hidden Markov model profile searches, 156 P. falciparum TAPs were identified. This represents about a third of the number of TAPs usually found in the genome of a free-living eukaryote. Furthermore, the P. falciparum genome appears to contain a low number of sequences, which are highly conserved and abundant within the kingdoms of free-living eukaryotes, that contribute to gene-specific transcriptional regulation. However, in comparison with these other eukaryotic genomes, the CCCH-type zinc finger (common in proteins modulating mRNA decay and translation rates) was found to be the most abundant in the P. falciparum genome. This observation, together with the paucity of malarial transcriptional regulators identified, suggests Plasmodium protein levels are primarily determined by posttranscriptional mechanisms.

Animals↗
PubMed2006

Computational modeling of the Plasmodium falciparum interactome reveals protein function on a genome-wide scale.

Many thousands of proteins encoded by the genome of Plasmodium falciparum, the causal organism of the deadliest form of human malaria, are of unknown function. It is of utmost importance that these proteins be characterized if we are to develop combative strategies against malaria based on the biology of the parasite. In an attempt to infer protein function on a genome-wide scale, we computationally modeled the P. falciparum interactome, elucidating local and global functional relationships between gene products. The resulting interaction network, reconstructed by integrating in silico and experimental functional genomics data within a Bayesian framework, covers approximately 68% of the parasite genome and provides functional inferences for more than 2000 uncharacterized proteins, based on their associations. Network reconstruction involved the use of a novel strategy, where we incorporated continuously updated, uniform reference priors in our Bayesian model. This method for generating interaction maps is thus also well suited for application to other genomes, where pre-existing interactome knowledge is sparse. Additionally, we superimposed this map on genomes of three apicomplexan pathogens--Plasmodium yoelii, Toxoplasma gondii, and Cryptosporidium parvum--describing relationships between these organisms based on retained functional linkages. This comparison provided a glimpse of the highly evolved nature of P. falciparum; for instance, a deficit of nearly 26% in terms of predicted interactions is observed against P. yoelii, because of missing ortholog partners in pairs of functionally linked proteins.

Animals↗
PubMed2001

Low-complexity regions in Plasmodium falciparum proteins.

Full-sequence data available for Plasmodium falciparum chromosomes 2 and 3 are exploited to perform a statistical analysis of the long tracts of biased amino acid composition that characterize the vast majority of P. falciparum proteins and to make a comparison with similarly defined tracts from other simple eukaryotes. When the relatively minor subset of prevalently hydrophobic segments is discarded from the set of low-complexity segments identified by current segmentation methods in P. falciparum proteins, a good correspondence is found between prevalently hydrophilic low-complexity segments and the species-specific, rapidly diverging insertions detected by multiple-alignment procedures when sequences of bona fide homologs are available. Amino acid preferences are fairly uniform in the set of hydrophilic low-complexity segments identified in the two P. falciparum chromosomes sequenced, as well as in sequenced genes from Plasmodium berghei, but differ from those observed in Saccharomyces cerevisiae and Dictyostelium discoideum. In the two plasmodial species, amino acid frequencies do not correlate with properties such as hydrophilicity, small volume, or flexibility, which might be expected to characterize residues involved in nonglobular domains but do correlate with A-richness in codons. An effect of phenotypic selection versus neutral drift, however, is suggested by the predominance of asparagine over lysine.

Amino Acids↗
PubMed2005

Structures of Plasmodium falciparum purine nucleoside phosphorylase complexed with sulfate and its natural substrate inosine.

Purine metabolism in the parasite Plasmodium has been identified as a promising target for antimalarial therapies. Purine nucleoside phosphorylase (PNP) is part of a salvage pathway for the biosynthesis of purines, which are essential for parasite survival. Two crystal structures of PNP from Plasmodium falciparum (PfPNP) in two space groups, each with a single subunit in the asymmetric unit, are described here. One structure, refined to 2.4 A, has an empty nucleoside-binding site and a sulfate ion bound in the phosphate-binding pocket. The second structure, refined to 2.0 A, has the substrate inosine bound to the active centre. Structure comparison reveals alterations in the active site upon ligand binding. The new structures presented here specifically highlight the likely roles of Asp206 and two loops flanking the active site: the beta7-alpha6 loop (residues approximately 161-169) and the beta9-alpha8 loop (residues approximately 208-223). Comparison with PNP in complex with transition-state inhibitors suggests that the purine substrate moves towards the phosphate substrate, rather than vice versa, upon forming the transition state. The single-substrate-containing PfPNP structures also appear to be more flexible than PfPNP bound to inhibitors. Together, these structures serve as a basis for better understanding of ligand binding and mechanism that can be further exploited to optimize the specificity of anti-PfPNP drugs.

Animals↗
PubMed2005

Crystallization and preliminary X-ray analysis of the aspartic protease plasmepsin 4 from the malarial parasite Plasmodium malariae.

Plasmepsin 4 from the malarial parasite Plasmodium malariae (PmPM4) is a member of the plasmepsins (Plasmodium pepsins), a subfamily of the pepsin-like aspartic proteases whose ortholog in the malarial parasite P. falciparum is involved in hemoglobin digestion in its digestive vacuole. Crystals of PmPM4 in complex with the small-molecule inhibitor AG1776 have been grown from a precipitant of 15% PEG 4000 and 200 mM ammonium sulfate in 100 mM sodium acetate pH 4.5. X-ray diffraction data were collected on a Rigaku rotating-anode generator from a single crystal under cryoconditions, with a maximal useful diffraction pattern to 3.3 A resolution. The crystals are shown to be orthorhombic and have been assigned to space group P2(1)2(1)2, with unit-cell parameters a = 95.88, b = 112.58, c = 90.40 A and a scaling Rsym of 0.104 for 14,334 unique reflections. Packing consideration and self-rotation function results indicate that there are two molecules per asymmetric unit. It is expected that in the near future the structure of PmPM4 will be obtained using molecular-replacement methods, obtaining phases from previously determined plasmepsin structures. Elucidation of the structure of PmPM4 in complex with inhibitors may be paramount to producing new antimalarial therapeutic agents.

Amino Acid Sequence↗
PubMed2005

The crystal structure of PfFabZ, the unique beta-hydroxyacyl-ACP dehydratase involved in fatty acid biosynthesis of Plasmodium falciparum.

The unique beta-hydroxyacyl-ACP dehydratase in Plasmodium falciparum, PfFabZ, is involved in fatty acid biosynthesis and catalyzes the dehydration of beta-hydroxy fatty acids linked to acyl carrier protein. The structure was solved by single anomalous dispersion (SAD) phasing using a quick-soaking experiment with potassium iodide and refined to a resolution of 2.1 A. The crystal structure represents the first structure of a Plasmodium beta-hydroxyacyl-ACP dehydratase with broad substrate specificity. The asymmetric unit contains a hexamer that appears as a trimer of dimers. Each dimer shows the known "hot dog" fold that has been observed in only a few other protein structures. Each of the two independent active sites in the dimer is formed by equal contributions from both subunits. The active site is mainly hydrophobic and looks like an L-shaped tunnel. The catalytically important amino acids His 133 and Glu 147' (from the other subunit), together with His98', form the only hydrophilic site in this tunnel. The inner end of the active site tunnel is closed by the phenyl ring of Phe 169, which is located in a flexible, partly visible loop. In order to explain the acceptance of substrates longer than ~C-7, the phenyl ring must move away to open the tunnel. The present structure supports an enzymatic mechanism consisting of an elimination reaction catalyzed by His 133 and Glu147'. 3-decynoyl-N-acetylcysteamine, an inhibitor known to interact with the E. coli dehydratase/isomerase, turned out to interact covalently with PfFabZ. A first model of PfFabZ with this potent inhibitor is presented.

Amino Acid Sequence↗
PubMed1988

Pharmacokinetics of chloroquine in Thais: plasma and red-cell concentrations following an intravenous infusion to healthy subjects and patients with Plasmodium vivax malaria.

1. Chloroquine diphosphate (15 mg base kg-1) was given by constant rate intravenous infusion to two groups of Thai subjects. Eleven were patients with malaria (10 with Plasmodium vivax and one case with Plasmodium malariae) and 10 were healthy normal volunteers. 2. Plasma and packed red-cell concentrations of chloroquine, electrocardiographic intervals, arterial blood pressure and pulse were measured at frequent intervals. 3. Peak plasma concentrations at the end of the infusion ranged from 979 to 2,900 ng ml-1 in the malaria patients. In the group of healthy subjects the range was 550-2,200 ng ml-1. Values for terminal elimination rate constant, (lambda z) plasma clearance (CL), initial volume of distribution (V1) and volume of distribution at steady state (Vss) were calculated. For the healthy subjects, mean estimates of these parameters were lambda z = 0.062 +/- 0.030 day-1, CL = 597 +/- 238 ml min-1, V1 = 0.66 +/- 0.71 l kg-1 and Vss = 132 +/- 50 l.kg-1 For the group of malaria patients, the corresponding values were lambda z = 0.055 +/- 0.032 day-1, CL = 535 +/- 246 ml min-1, V1 = 0.74 +/- 0.75 l kg-1 and Vss = 136 +/- 64 l kg-1 There was no statistically significant difference in the estimates for any parameter between groups (P less than or equal to 0.05). 4. Chloroquine concentrations in packed red blood cells consistently exceeded those in plasma and showed no consistent change with time throughout the period of study in either group. The median value for the red cell to plasma ratio was between 3 and 4 in each group.(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗