Genome sequence of the human malaria parasite Plasmodium falciparum

The parasite Plasmodium falciparum is responsible for hundreds of millions of cases of malaria, and kills more than one million African children annually. Here we report an analysis of the genome sequence of P. falciparum clone 3D7. The 23-megabase nuclear genome consists of 14 chromosomes, encodes about 5,300 genes, and is the most (A + T)-rich genome sequenced to date. Genes involved in antigenic variation are concentrated in the subtelomeric regions of the chromosomes. Compared to the genomes of free-living eukaryotic microbes, the genome of this intracellular parasite encodes fewer enzymes and transporters, but a large proportion of genes are devoted to immune evasion and host-parasite interactions. Many nuclear-encoded proteins are targeted to the apicoplast, an organelle involved in fatty-acid and isoprenoid metabolism. The genome sequence provides the foundation for future studies of this organism, and is being exploited in the search for new drugs and vaccines to fight malaria.     

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.     

A synthetic vaccine protects humans against challenge with asexual blood stages of Plasmodium falciparum malaria

We have previously shown that a mixture of three synthetic peptides (83.1, 55.1, 35.1), corresponding to fragments of the relative molecular mass 83,000 (83K), 55K and 35K Plasmodium falciparum merozoite-specific proteins, induces protection in Aotus triviroatus monkeys experimentally infected with P. falciparum. Here we describe two polymeric synthetic hybrid proteins based on these peptides that delay or suppress the development of parasitaemia in immunized human volunteers.     

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.     

Safety and immunogenicity in man of a synthetic peptide malaria vaccine against Plasmodium falciparum sporozoites

A 12 amino-acid synthetic peptide (NANP)3 comprising the immunodominant epitope of Plasmodium falciparum circumsporozoite protein was conjugated to tetanus toxoid (TT), adjuvanted with aluminium hydroxide, and administered intramuscularly in three doses at monthly intervals to 35 healthy males as a malaria vaccine. No significant adverse reactions were noted, with mild soreness at the injection site the only common symptom. Seroconversions against NANP occurred in 53% and 71% of recipients of 100 or 160 micrograms, respectively, measured by enzyme-linked immunosorbent assay (ELISA). Most ELISA-positive sera reacted with sporozoites by indirect immunofluorescence (IFA). Three vaccinees with the highest ELISA and IFA titres and four unimmunized controls were challenged with P. falciparum sporozoites introduced via the bites of infective Anopheles mosquitoes. Blood stage parasites were detected in all controls by 10 days (mean 8.5 days, range 7-10). In contrast, the two vaccinees who became infected did not manifest parasitaemia until day 11 and the third vacinee showed neither parasites nor symptoms during the 29 day observation period. This first synthetic peptide parenteral vaccine against a communicable disease tested in man is safe and stimulates biologically active antibodies. These observations encourage the development of improved vaccine formulations which, by enhancing immunogenicity, may lead to practical vaccines to assist in the control of falciparum malaria.     

Frequent ectopic recombination of virulence factor genes in telomeric chromosome clusters of P. falciparum

Persistent and recurrent infections by Plasmodium falciparum malaria parasites result from the ability of the parasite to undergo antigenic variation and evade host immune attack. P. falciparum parasites generate high levels of variability in gene families that comprise virulence determinants of cytoadherence and antigenic variation, such as the var genes. These genes encode the major variable parasite protein (PfEMP-1), and are expressed in a mutually exclusive manner at the surface of the erythrocyte infected by P. falciparum. Here we identify a mechanism by which var gene sequences undergo recombination at frequencies much higher than those expected from homologous crossover events alone. These recombination events occur between subtelomeric regions of heterologous chromosomes, which associate in clusters near the nuclear periphery in asexual blood-stage parasites or in bouquet-like configurations near one pole of the elongated nuclei in sexual parasite forms. We propose that the alignment of var genes in heterologous chromosomes facilitates gene conversion and promotes the diversity of antigenic and adhesive phenotypes. The association of virulence factors with a specific nuclear subcompartment may also have implications for variation during mitotic recombination in asexual blood stages.     

A proteomic view of the Plasmodium falciparum life cycle

The completion of the Plasmodium falciparum clone 3D7 genome provides a basis on which to conduct comparative proteomics studies of this human pathogen. Here, we applied a high-throughput proteomics approach to identify new potential drug and vaccine targets and to better understand the biology of this complex protozoan parasite. We characterized four stages of the parasite life cycle (sporozoites, merozoites, trophozoites and gametocytes) by multidimensional protein identification technology. Functional profiling of over 2,400 proteins agreed with the physiology of each stage. Unexpectedly, the antigenically variant proteins of var and rif genes, defined as molecules on the surface of infected erythrocytes, were also largely expressed in sporozoites. The detection of chromosomal clusters encoding co-expressed proteins suggested a potential mechanism for controlling gene expression.     

Several alleles of the multidrug-resistance gene are closely linked to chloroquine resistance in Plasmodium falciparum

The lethal form of human malaria caused by Plasmodium falciparum is virtually uncontrollable in many areas because of the development of drug resistance, in particular chloroquine resistance (CQR). CQR is biologically similar to the multiple drug resistance phenotype (MDR) of mammalian tumour cells, as both involve expulsion of drug from the cell and both can be reversed by calcium channel antagonists. A homologue (pfmdr1) of the mammalian multidrug resistance gene has been implicated in CQR because it is amplified in some CQR isolates of P. falciparum as is an mdr gene in MDR tumour cells. We show here that the complete sequences of pfmdr1 genes from 2 CQ sensitive (CQS) P. falciparum isolates are identical. In 5 CQR isolates, 1-4 key nucleotide differences resulted in amino acid substitutions. On the basis of these substitutions, we have correctly predicted the CQS/CQR status of a further 34 out of 36 isolates. This is a paradox as CQR arises much less frequently than would be predicted if single point mutations were sufficient. We conclude that a mutated pfmdr1 gene is one of at least two mutated genes required for CQR.     

Specific role of mitochondrial electron transport in blood-stage Plasmodium falciparum

The origin of all mitochondria can be traced to the symbiotic arrangement that resulted in the emergence of eukaryotes in a world that was exclusively populated by prokaryotes. This arrangement, however, has been in continuous genetic flux: the varying degrees of gene loss and transfer from the mitochondrial genome in different eukaryotic lineages seem to signify an ongoing 'conflict' between the host and the symbiont. Eukaryotic parasites belonging to the phylum Apicomplexa provide an excellent example to support this view. These organisms contain the smallest mitochondrial genomes known, with an organization that differs among various genera; one genus, Cryptosporidium, seems to have lost the entire mitochondrial genome. Here we show that erythrocytic stages of the human malaria parasite Plasmodium falciparum seem to maintain an active mitochondrial electron transport chain to serve just one metabolic function: regeneration of ubiquinone required as the electron acceptor for dihydroorotate dehydrogenase, an essential enzyme for pyrimidine biosynthesis. Transgenic P. falciparum parasites expressing Saccharomyces cerevisiae dihydroorotate dehydrogenase, which does not require ubiquinone as an electron acceptor, were completely resistant to inhibitors of mitochondrial electron transport. Maintenance of mitochondrial membrane potential, however, was essential in these parasites, as indicated by their hypersensitivity to proguanil, a drug that collapsed the membrane potential in the presence of electron transport inhibitors. Thus, acquisition of just one enzyme can render mitochondrial electron transport nonessential in erythrocytic stages of P. falciparum.     

A trans-acting class II regulatory gene unlinked to the MHC controls expression of HLA class II genes

Class II (or Ia) antigens are highly polymorphic surface molecules which are essential for the cellular interactions involved in the immune response. In man, these antigens are encoded by a complex multigene family which is located in the major histocompatibility complex (MHC) and which comprises up to 12 distinct alpha- and beta-chain genes, coding for the HLA-DR, -DQ and -DP antigens. One form of congenital severe combined immunodeficiency (SCID) in man, which is generally lethal, is characterized by an absence of HLA-DR histocompatibility antigens on peripheral blood lymphocytes (HLA class II-deficient SCID). In these patients, as reported here, we have observed an absence of messenger RNA for the alpha- and beta-chains of HLA-DR, -DQ and -DP, indicating a global defect in the expression of all class II genes. Moreover, the lack of expression of HLA class II mRNAs could not be corrected by gamma-interferon, an inducer of class II gene expression in normal cells. Family studies have established that the genetic defect does not segregate with the MHC. We conclude, therefore, that the expression of the entire family of class II genes is normally controlled by a trans-acting class II regulatory gene which is unlinked to the MHC and which is affected in the patients. This gene controls a function or a product necessary for the action of gamma-interferon on class II genes.     

Analysis of Plasmodium falciparum diversity in natural infections by deep sequencing

Malaria elimination strategies require surveillance of the parasite population for genetic changes that demand a public health response, such as new forms of drug resistance. Here we describe methods for the large-scale analysis of genetic variation in Plasmodium falciparum by deep sequencing of parasite DNA obtained from the blood of patients with malaria, either directly or after short-term culture. Analysis of 86,158 exonic single nucleotide polymorphisms that passed genotyping quality control in 227 samples from Africa, Asia and Oceania provides genome-wide estimates of allele frequency distribution, population structure and linkage disequilibrium. By comparing the genetic diversity of individual infections with that of the local parasite population, we derive a metric of within-host diversity that is related to the level of inbreeding in the population. An open-access web application has been established for the exploration of regional differences in allele frequency and of highly differentiated loci in the P.?falciparum genome.     

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.     

A novel mechanism controls anther opening and closing in Paris polyphylla var. Yunnanensis

The phenomena of anther opening and closing in Paris polyphylla var. yunnanensis (Franch) Hand.-Mazz were described in detail, and the effects of ecological factors on those phenomena related to anther opening, closing and the fly-pollination mechanism were discussed. Anthers of P. polyphylla var. yunnanensis open in the morning and close in the evening every day during an over 20 days’ period of anthesis. Light was detected as the main factor controlling this daily anther opening and closing. Anther openin...     

A superfamily of variant genes encoded in the subtelomeric region of Plasmodium vivax

The malarial parasite Plasmodium vivax causes disease in humans, including chronic infections and recurrent relapses, but the course of infection is rarely fatal, unlike that caused by Plasmodium falciparum. To investigate differences in pathogenicity between P. vivax and P. falciparum, we have compared the subtelomeric domains in the DNA of these parasites. In P. falciparum, subtelomeric domains are conserved and contain ordered arrays of members of multigene families, such as var, rif and stevor, encoding virulence determinants of cytoadhesion and antigenic variation. Here we identify, through the analysis of a continuous 155,711-base-pair sequence of a P. vivax chromosome end, a multigene family called vir, which is specific to P. vivax. The vir genes are present at about 600-1,000 copies per haploid genome and encode proteins that are immunovariant in natural infections, indicating that they may have a functional role in establishing chronic infection through antigenic variation.