Genome sequence and comparative analysis of the model rodent malaria parasite Plasmodium yoelii yoelii

Species of malaria parasite that infect rodents have long been used as models for malaria disease research. Here we report the whole-genome shotgun sequence of one species, Plasmodium yoelii yoelii, and comparative studies with the genome of the human malaria parasite Plasmodium falciparum clone 3D7. A synteny map of 2,212 P. y. yoelii contiguous DNA sequences (contigs) aligned to 14 P. falciparum chromosomes reveals marked conservation of gene synteny within the body of each chromosome. Of about 5,300 P. falciparum genes, more than 3,300 P. y. yoelii orthologues of predominantly metabolic function were identified. Over 800 copies of a variant antigen gene located in subtelomeric regions were found. This is the first genome sequence of a model eukaryotic parasite, and it provides insight into the use of such systems in the modelling of Plasmodium biology and disease.     

Malaria research in the post-genomic era

For many pathogens the availability of genome sequence, permitting genome-dependent methods of research, can partially substitute for powerful forward genetic methods (genome-independent) that have advanced model organism research for decades. In 2002 the genome sequence of Plasmodium falciparum, the parasite causing the most severe type of human malaria, was completed, eliminating many of the barriers to performing state-of-the-art molecular biological research on malaria parasites. Although new, licensed therapies may not yet have resulted from genome-dependent experiments, they have produced a wealth of new observations about the basic biology of malaria parasites, and it is likely that these will eventually lead to new therapeutic approaches. This review will focus on the basic research discoveries that have depended, in part, on the availability of the Plasmodium genome sequences.     

Climate change and the resurgence of malaria in the East African highlands

The public health and economic consequences of Plasmodium falciparum malaria are once again regarded as priorities for global development. There has been much speculation on whether anthropogenic climate change is exacerbating the malaria problem, especially in areas of high altitude where P. falciparum transmission is limited by low temperature. The International Panel on Climate Change has concluded that there is likely to be a net extension in the distribution of malaria and an increase in incidence within this range. We investigated long-term meteorological trends in four high-altitude sites in East Africa, where increases in malaria have been reported in the past two decades. Here we show that temperature, rainfall, vapour pressure and the number of months suitable for P. falciparum transmission have not changed significantly during the past century or during the period of reported malaria resurgence. A high degree of temporal and spatial variation in the climate of East Africa suggests further that claimed associations between local malaria resurgences and regional changes in climate are overly simplistic.     

The global distribution of clinical episodes of Plasmodium falciparum malaria

Interest in mapping the global distribution of malaria is motivated by a need to define populations at risk for appropriate resource allocation and to provide a robust framework for evaluating its global economic impact. Comparison of older and more recent malaria maps shows how the disease has been geographically restricted, but it remains entrenched in poor areas of the world with climates suitable for transmission. Here we provide an empirical approach to estimating the number of clinical events caused by Plasmodium falciparum worldwide, by using a combination of epidemiological, geographical and demographic data. We estimate that there were 515 (range 300-660) million episodes of clinical P. falciparum malaria in 2002. These global estimates are up to 50% higher than those reported by the World Health Organization (WHO) and 200% higher for areas outside Africa, reflecting the WHO's reliance upon passive national reporting for these countries. Without an informed understanding of the cartography of malaria risk, the global extent of clinical disease caused by P. falciparum will continue to be underestimated.     

硫酸软骨素A介导的恶性疟原虫感染的红细胞粘附

对硫酸软骨素的粘附机制进行了初步探讨。研究表明,恶性疟原虫感染的红细胞可粘附于各种器官的微血管内皮细胞,这种粘附被认为是红细胞膜表面分子与内皮细胞表面分子间配体－受体相互作用的结果,ＣＳＡ为内皮细胞表面感染的红细胞的粘附受体和恶性疟原虫红细胞膜蛋白１的配体。     

Major surface antigen gene of a human malaria parasite cloned and expressed in bacteria

The late blood stages of the human malaria parasite, Plasmodium falciparum, carry a major surface antigen, p190, of molecular weight (Mr) 190,000. This antigenically variable protein is actively processed, first as the parasite matures and again when it is released into the blood stream and invades a new erythrocyte to initiate a cycle of growth. It elicits a strong immune response in man; all tested adult sera from endemic areas have antibodies against this protein. Our evidence indicates that purified p190 can alter the course of parasitaemia in monkeys with falciparum malaria. We have also succeeded in cloning part of the gene for p190 and expressing it in Escherichia coli. To this end we have developed a new technique, antibody select, which greatly simplifies final identification of expressing clones.     

Size variation in chromosomes from independent cultured isolates of Plasmodium falciparum

The complexity of the life cycle of the protozoan malaria parasite Plasmodium falciparum has hindered genetic analysis; even the number of chromosomes in P. falciparum is uncertain. The blood stages of rodent malaria parasites are haploid and hybridization with cloned complementary DNAs similarly suggests a haploid genome in P. falciparum blood stages (ref. 4 and our unpublished results). A novel approach to karyoptic and linkage analysis in P. falciparum has been provided recently by the technique of pulsed-field gradient (PFG) gel electrophoresis, which allows the fractionation of DNA molecules of 30-3,000 kilobases (kb), a range including the sizes of intact chromosomal DNA molecules from eukaryotes such as yeast and trypanosomatids. We describe here the fractionation by PFG electrophoresis of chromosomal DNA molecules from P. falciparum into at least seven discrete species which vary in size by up to 20% between different isolates. Several genes for P. faciparum antigens which contain repetitive sequences are located on different chromosomes. Surprisingly, two of the chromosomes seem to contain the same sequences.     

Malaria risk: estimating clinical episodes of malaria

Estimates of the disease burden caused by malaria are crucial for informing malaria control programmes. Snow and colleagues claim that their estimate of 515 million cases of malaria caused by Plasmodium falciparum globally is up to 50% higher than that reported by the World Health Organization (WHO), and 200% higher for areas outside Africa. However, this comparison refers to the WHO's estimates from 1990 and 1998, and not to the range of 300 million to 500 million that the WHO has used since 2000 (ref. 2). Both groups agree that the burden of malaria disease outside Africa, especially in South Asia, is greater than was estimated in the 1990s.     

Malaria risk: estimation of the malaria burden

Accurate estimates of the global burden of malaria are important for planning, monitoring and advocacy. Snow et al. attempt to address the shortcomings of previous estimates of the incidence of malaria caused by Plasmodium falciparum by combining current and historical data. However, we believe that the design of their model and its inputs have led to a significant overestimate of the malaria burden outside Africa--as in the example of the World Health Organization (WHO) western Pacific region (WPR), for which their model predicts 60 times the 2002 incidence reported by national malaria-control programmes.     

Human antisera detect a Plasmodium falciparum genomic clone encoding a nonapeptide repeat

Plasmodium falciparum causes malaria infections in its human host. Its wide distribution in tropical countries is a major world health problem. Before a vaccine can be produced, the identification and characterization of parasite antigens is necessary. This can be achieved by the cloning and subsequent analysis of genes coding for parasite antigens. Recently established cDNA banks allow the expression of cDNA derived from the simian parasite Plasmodium knowlesi and P. falciparum in Escherichia coli. Recombinants encoding parasite antigens have been identified by immunodetection in both banks. Two of them contain repetitive units of 11 (ref. 7) or 12 (ref. 5) amino acids. We describe here the construction of an expression bank made directly from randomly generated fragments of P. falciparum genomic DNA. We detect several clones which react strongly with human African immune sera. One clone expresses an antigenic determinant composed of occasionally degenerated repeats of a peptide nonamer.     

Synthetic GPI as a candidate anti-toxic vaccine in a model of malaria

The malaria parasite Plasmodium falciparum infects 5-10% of the world's population and kills two million people annually. Fatalities are thought to result in part from pathological reactions initiated by a malarial toxin. Glycosylphosphatidylinositol (GPI) originating from the parasite has the properties predicted of a toxin; however, a requirement for toxins in general and GPI in particular in malarial pathogenesis and fatality remains unproven. As anti-toxic vaccines can be highly effective public health tools, we sought to determine whether anti-GPI vaccination could prevent pathology and fatalities in the Plasmodium berghei/rodent model of severe malaria. The P. falciparum GPI glycan of the sequence NH(2)-CH(2)-CH(2)-PO(4)-(Man alpha 1-2)6Man alpha 1-2Man alpha 1-6Man alpha 1-4GlcNH(2)alpha 1-6myo-inositol-1,2-cyclic-phosphate was chemically synthesized, conjugated to carriers, and used to immunize mice. Recipients were substantially protected against malarial acidosis, pulmonary oedema, cerebral syndrome and fatality. Anti-GPI antibodies neutralized pro-inflammatory activity by P. falciparum in vitro. Thus, we show that GPI is a significant pro-inflammatory endotoxin of parasitic origin, and that several disease parameters in malarious mice are toxin-dependent. GPI may contribute to pathogenesis and fatalities in humans. Synthetic GPI is therefore a prototype carbohydrate anti-toxic vaccine against malaria.     

Cytoadherence of knobless Plasmodium falciparum-infected erythrocytes and its inhibition by a human monoclonal antibody

Red blood cells infected with mature stages of the malaria parasite Plasmodium falciparum bind to the endothelial lining of capillaries and venules. This sequestration is important for the survival of the parasite but may have severe consequences for the host. For example, it is involved in the causation of cerebral malaria which carries 25% mortality. Knob-like protrusions present on the surface of infected erythrocytes have been considered necessary but not sufficient for this cytoadherence. Here we describe the adhesion to endothelial cells of infected erythrocytes which do not have knobs. A human monoclonal antibody (33G2) which was specific for an epitope containing regularly spaced dimers of glutamic acid present in the repeated amino-acid sequences of some defined P. falciparum antigens was found to inhibit cyto-adherence and may therefore be an important reagent for elucidating the molecular basis of parasite sequestration.     

Adaptation of Plasmodium falciparum to glucose 6-phosphate dehydrogenase-deficient host red cells by production of parasite-encoded enzyme

There is impressive evidence from geographical data, studies in the field and in vitro culture work that genetically determined deficiency of glucose 6-phosphate dehydrogenase (G6PD) confers relative protection against the human malaria parasite, Plasmodium falciparum. G6PD is encoded by an X-chromosome-linked gene, and protection phenomenon is manifested in heterozygous females who are genetic mosaics but, surprisingly, not in hemizygous males with complete deficiency. We have shown previously that the parasite, when passaged serially through G6PD-deficient red cells, undergoes adaptive changes that gradually improve its ability to multiply in these deficient cells. To explain the above paradox, we now show that this adaptive process is associated with, and may consist in, the induction of synthesis of a novel G6PD coded by Plasmodium falciparum.     

Chloroquine resistance not linked to mdr-like genes in a Plasmodium falciparum cross

Chloroquine is thought to act against falciparum malaria by accumulating in the acid vesicles of the parasite and interfering with their function. Parasites resistant to chloroquine expel the drug rapidly in an unaltered form, thereby reducing levels of accumulation in the vesicles. The discovery that verapamil partially reverses chloroquine resistance in vitro led to the proposal that efflux may involve an ATP-driven P-glycoprotein pump similar to that in mammalian multidrug-resistant (mdr) tumor cell lines. Indeed, Plasmodium falciparum contains at least two mdr-like genes, one of which has been suggested to confer the chloroquine resistant (CQR) phenotype. To determine if either of these genes is linked to chloroquine resistance, we performed a genetic cross between CQR and chloroquine-susceptible (CQS) clones of P. falciparum. Examination of 16 independent recombinant progeny indicated that the rapid efflux phenotype is controlled by a single gene or a closely linked group of genes. But, there was no linkage between the rapid efflux, CQR phenotype and either of the mdr-like P. falciparum genes or amplification of those genes. These data indicate that the genetic locus governing chloroquine efflux and resistance is independent of the known mdr-like genes.     

Thrombospondin binds falciparum malaria parasitized erythrocytes and may mediate cytoadherence

Plasmodium falciparum infected erythrocytes containing mature trophozoites and schizonts sequester along venular endothelium and are not in the peripheral circulation of patients with malaria. Knobs appear on infected erythrocytes and are the points of attachment to endothelium. Sequestration may protect the parasite from splenic destruction and may play a role in the pathogenesis of cerebral malaria. Correlates of sequestration have been developed in vitro using cultured human endothelium and an amelanotic melanoma cell line. Knobless strains (K-) of P. falciparum fail to sequester in vivo and to bind to cells in vitro. We now present evidence that the receptor for cytoadherence is the glycoprotein, thrombospondin. Aotus monkey or human erythrocytes containing knobby (K+) but not Aotus erythrocytes containing knobless strains of P. falciparum bind to immobilized thrombospondin. Neither binds to the adhesive proteins laminin, fibronectin, factor VIII/von Willebrand factor or vitronectin. Both soluble thrombospondin and anti-thrombospondin antibodies inhibit binding of parasitized Aotus erythrocytes to immobilize thrombospondin and to melanoma cells which secrete thrombospondin.     

Induction of protective immunity against experimental infection with malaria using synthetic peptides

Synthetic peptides are potential vaccine candidates because they may be able to induce high antibody titres and specific cellular immune responses against native proteins and thus the whole invading organism. In a previous study we showed that immunization with molecules of relative molecular mass (Mr) 155,000 (155K) 83K, 55K and 35K, specific for the late schizont and merozoite stages of Plasmodium falciparum, could elicit either partial or total protection in Aotus trivirgatus monkeys experimentally infected with P. falciparum. Here we have chemically synthesized 18 peptides corresponding to different fragments of these proteins to immunize Aotus trivirgatus monkeys. Some peptides gave partial protection from challenge with P. falciparum parasites, but none provided complete protection individually. A combination of three partially protective peptides gave complete or almost complete protection, however, suggesting that this particular combination of peptides is a good candidate for a malaria vaccine.     

Primary structure of the precursor to the three major surface antigens of Plasmodium falciparum merozoites

Recently, a class of protein antigens of high relative molecular mass (Mt) which can induce protective immunity against blood-stage malaria has been identified. In Plasmodium falciparum the protein has a Mr of approximately 195,000 (P195). It is the precursor of three proteins of Mr 83,000 (83K), 42K and 19K which are the major surface antigens of merozoites; thus it may also be useful for immunization against P. falciparum. Three studies describing the isolation of single short complementary DNA clones for part of the P195 gene sequence have been reported. Here we describe the complete structure of the P195 gene determined from further DNA clones, its organization within genomic DNA and the location of the specific processing fragments within the primary amino-acid sequence.     

Chromosome-wide SNPs reveal an ancient origin for Plasmodium falciparum

The Malaria's Eve hypothesis, proposing a severe recent population bottleneck (about 3,000-5,000 years ago) of the human malaria parasite Plasmodium falciparum, has prompted a debate about the origin and evolution of the parasite. The hypothesis implies that the parasite population is relatively homogeneous, favouring malaria control measures. Other studies, however, suggested an ancient origin and large effective population size. To test the hypothesis, we analysed single nucleotide polymorphisms (SNPs) from 204 genes on chromosome 3 of P. falciparum. We have identified 403 polymorphic sites, including 238 SNPs and 165 microsatellites, from five parasite clones, establishing chromosome-wide haplotypes and a dense map with one polymorphic marker per approximately 2.3 kilobases. On the basis of synonymous SNPs and non-coding SNPs, we estimate the time to the most recent common ancestor to be approximately 100,000-180,000 years, significantly older than the proposed bottleneck. Our estimated divergence time coincides approximately with the start of human population expansion, and is consistent with a genetically complex organism able to evade host immunity and other antimalarial efforts.