Evidence for immunological cross-reaction between sporozoites and blood stages of a human malaria parasite

Malaria parasites (Plasmodium spp.) show a complex pattern of development in the mammalian host and many studies support the view that the surface of the sporozoite, injected by the mosquito, has no antigens in common with the erythrocytic stage of development. For example, immunization with the erythrocytic parasites generates antisera with negligible titre by indirect immunofluorescence to the sporozoite surface. Although monoclonal antibodies prepared against erythrocytic stages were reported to show cross-reaction to the sporozoite stage, this appeared to be due to cytoplasmic antigens exposed by the method of sporozoite preparation, and in Plasmodium knowlesi, a cDNA clone coding for the circumsporozoite antigen, the major protein of the sporozoite surface, showed no hydridization to mRNA isolated from the erythrocytic stages. Here, however, we present evidence for an antigenic determinant shared by the sporozoite surface and the erythrocytic stages of the human malaria parasite, P. falciparum. Moreover, our studies show that the antigen(s) elicit a strong immune response in man.     

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with cloned complementary DNAs similarly suggests a haploid genome in P. falciparum blood stages (ref. 4 and our unpublished results).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.     

An anaerobic mitochondrion that produces hydrogen

Hydrogenosomes are organelles that produce ATP and hydrogen, and are found in various unrelated eukaryotes, such as anaerobic flagellates, chytridiomycete fungi and ciliates. Although all of these organelles generate hydrogen, the hydrogenosomes from these organisms are structurally and metabolically quite different, just like mitochondria where large differences also exist. These differences have led to a continuing debate about the evolutionary origin of hydrogenosomes. Here we show that the hydrogenosomes of the anaerobic ciliate Nyctotherus ovalis, which thrives in the hindgut of cockroaches, have retained a rudimentary genome encoding components of a mitochondrial electron transport chain. Phylogenetic analyses reveal that those proteins cluster with their homologues from aerobic ciliates. In addition, several nucleus-encoded components of the mitochondrial proteome, such as pyruvate dehydrogenase and complex II, were identified. The N. ovalis hydrogenosome is sensitive to inhibitors of mitochondrial complex I and produces succinate as a major metabolic end product--biochemical traits typical of anaerobic mitochondria. The production of hydrogen, together with the presence of a genome encoding respiratory chain components, and biochemical features characteristic of anaerobic mitochondria, identify the N. ovalis organelle as a missing link between mitochondria and hydrogenosomes.     

Trichomonas hydrogenosomes contain the NADH dehydrogenase module of mitochondrial complex I

Hydrogenosomes are double-membraned ATP-producing and hydrogen-producing organelles of diverse anaerobic eukaryotes. In some versions of endosymbiotic theory they are suggested to be homologues of mitochondria, but alternative views suggest they arose from an anaerobic bacterium that was distinct from the mitochondrial endosymbiont. Here we show that the 51-kDa and 24-kDa subunits of the NADH dehydrogenase module in complex I, the first step in the mitochondrial respiratory chain, are active in hydrogenosomes of Trichomonas vaginalis. Like mitochondrial NADH dehydrogenase, the purified Trichomonas enzyme can reduce a variety of electron carriers including ubiquinone, but unlike the mitochondrial enzyme it can also reduce ferredoxin, the electron carrier used for hydrogen production. The presence of NADH dehydrogenase solves the long-standing conundrum of how hydrogenosomes regenerate NAD+ after malate oxidation. Phylogenetic analyses show that the Trichomonas 51-kDa homologue shares common ancestry with the mitochondrial enzyme. Recruitment of complex I subunits into a H2-producing pathway provides evidence that mitochondria and hydrogenosomes are aerobic and anaerobic homologues of the same endosymbiotically derived organelle.     

酮替酚配伍周效磺胺对红内期约氏疟原虫超微结构的影响

单独使用酮替酚或周效磺胺，红内期约氏疟原虫的超微结构受到一定程度的损伤，但配伍使用酮替酚和周效碘胺后虫体的结构变化更为迅速，给药８小时后裂殖体的形成受阻，滋养体除细胞结构逐渐消失外胞质中还出现伪食泡和逐渐增多增大的单膜或多膜空泡，给药２４小时后未查见结构完整的滋养体。     

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.     

Sequence of Plasmodium falciparum chromosomes 2, 10, 11 and 14

The mosquito-borne malaria parasite Plasmodium falciparum kills an estimated 0.7-2.7 million people every year, primarily children in sub-Saharan Africa. Without effective interventions, a variety of factors-including the spread of parasites resistant to antimalarial drugs and the increasing insecticide resistance of mosquitoes-may cause the number of malaria cases to double over the next two decades. To stimulate basic research and facilitate the development of new drugs and vaccines, the genome of Plasmodium falciparum clone 3D7 has been sequenced using a chromosome-by-chromosome shotgun strategy. We report here the nucleotide sequences of chromosomes 10, 11 and 14, and a re-analysis of the chromosome 2 sequence. These chromosomes represent about 35% of the 23-megabase P. falciparum genome.     

Malaria. Cooperative silencing elements in var genes

Each Plasmodium falciparum malaria parasite carries about 50 var genes from a diverse family that encode variable adhesion proteins on the infected red blood cells of the host, but individual parasites single out just one var gene for expression and silence all the others. Here we show that this silencing is established during the DNA-synthesis phase (S phase) of the cell cycle and that it depends on the cooperative interaction between two elements in separate control regions of each var gene (the 5'-flanking region and the intron). This finding should help to clarify the mechanisms by which parasites coordinate the silencing and activation of var genes that are responsible for antigenic variation in malaria.     

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.     

Gametocytogenesis by malaria parasites in continuous culture

Asexual proliferation of malaria parasites proceeds by multiplication of the parasites within red cells. Following rupture of the host cells the released merozoites re-invade other red cells. On re-invasion, a proportion of merozoites become, not asexual parasites but gametocytes, the sexual stages infective to the mosquito vectors. Conversion of asexual parasites to gametocytes occurs not only during natural infections but also in continuous in vitro culture as reported first by Trager and Jensen and by others. We showed previously that the proportion of early intra-erythrocytic stages (ring stages) of Plasmodium falciparum which developed into gametocytes in culture was influenced by culture conditions. Gametocyte formation was rare in conditions supporting rapid proliferation but frequent when parasite densisites were static. We now show that nearly 100% of ring stages develop into gametocytes in response to 1mM cyclic AMP in static cultures whereas in rapidly growing cultures few rings become gametocytes in response to cyclic AMP.     

Detecting selection using a single genome sequence of M. tuberculosis and P. falciparum

Selective pressures on proteins are usually measured by comparing nucleotide sequences. Here we introduce a method to detect selection on the basis of a single genome sequence. We catalogue the relative strength of selection on each gene in the entire genomes of Mycobacterium tuberculosis and Plasmodium falciparum. Our analysis confirms that most antigens are under strong selection for amino-acid substitutions, particularly the PE/PPE family of putative surface proteins in M. tuberculosis and the EMP1 family of cytoadhering surface proteins in P. falciparum. We also identify many uncharacterized proteins that are under strong selection in each pathogen. We provide a genome-wide analysis of natural selection acting on different stages of an organism's life cycle: genes expressed in the ring stage of P. falciparum are under stronger positive selection than those expressed in other stages of the parasite's life cycle. Our method of estimating selective pressures requires far fewer data than comparative sequence analysis, and it measures selection across an entire genome; the method can readily be applied to a large range of sequenced organisms.     

Advances in the study of helminth mitochondrial genomes and their associated applications

Helminths, including flatworms and roundworms, are abundant organisms that have a variety of life histories. Of these, the genera Schistosoma, Echinococcus, Trichinella are notable parasites of veterinary and medical importance, and cause substantial socio- economic losses throughout China and the rest of the world. Genetic markers in the mitochondrial (mt) genome have proven use- ful for systematic, ecological, evolutionary and population studies, and the growth of mt genomic research has increased in the last two decades. Technological improvements, such as the long-polymerase chain reaction method and high-throughput se- quencing have allowed minute amounts of DNA from single worms, biopsy samples or microscopic organisms to be used for whole mt genome characterization. To facilitate the retrieval, annotation and analyses of mitochondrial features, multiple data- bases and specific software have also been designed and established. This review focuses on current progress, applications and perspectives regarding helminth mt genomics. To date, the complete mt genomes for 93 species of helminths have been sequenced and analyzed. Analyses of the mt genes, including gene content, arrangement, composition and variation have revealed unique features among the helminths when compared with other metazoans. This provides important data concerning their functional and comparative mitochondrial genomics, molecular taxonomy and characterization, population genetics and systematics, and evolu- tionary history. Moreover, mt genome data for parasitic helminths are important for diagnosis, epidemiology and ecology of in- fections. Mitochondrial genome data offer a rich source of markers for the systematics and population genetics of socioeconomi- cally important parasitic helminths of humans and other animals.     

A novel route for ATP acquisition by the remnant mitochondria of Encephalitozoon cuniculi

Mitochondria use transport proteins of the eukaryotic mitochondrial carrier family (MCF) to mediate the exchange of diverse substrates, including ATP, with the host cell cytosol. According to classical endosymbiosis theory, insertion of a host-nuclear-encoded MCF transporter into the protomitochondrion was the key step that allowed the host cell to harvest ATP from the enslaved endosymbiont. Notably the genome of the microsporidian Encephalitozoon cuniculi has lost all of its genes for MCF proteins. This raises the question of how the recently discovered microsporidian remnant mitochondrion, called a mitosome, acquires ATP to support protein import and other predicted ATP-dependent activities. The E. cuniculi genome does contain four genes for an unrelated type of nucleotide transporter used by plastids and bacterial intracellular parasites, such as Rickettsia and Chlamydia, to import ATP from the cytosol of their eukaryotic host cells. The inference is that E. cuniculi also uses these proteins to steal ATP from its eukaryotic host to sustain its lifestyle as an obligate intracellular parasite. Here we show that, consistent with this hypothesis, all four E. cuniculi transporters can transport ATP, and three of them are expressed on the surface of the parasite when it is living inside host cells. The fourth transporter co-locates with mitochondrial Hsp70 to the E. cuniculi mitosome. Thus, uniquely among eukaryotes, the traditional relationship between mitochondrion and host has been subverted in E. cuniculi, by reductive evolution and analogous gene replacement. Instead of the mitosome providing the parasite cytosol with ATP, the parasite cytosol now seems to provide ATP for the organelle.     

Reduction of cytochrome c oxidase by a second electron leads to proton translocation

Cytochrome c oxidase, the terminal enzyme of cellular respiration in mitochondria and many bacteria, reduces O(2) to water. This four-electron reduction process is coupled to translocation (pumping) of four protons across the mitochondrial or bacterial membrane; however, proton pumping is poorly understood. Proton pumping was thought to be linked exclusively to the oxidative phase, that is, to the transfer of the third and fourth electron. Upon re-evaluation of these data, however, this proposal has been questioned, and a transport mechanism including proton pumping in the reductive phase--that is, during the transfer of the first two electrons--was suggested. Subsequently, additional studies reported that proton pumping during the reductive phase can occur, but only when it is immediately preceded by an oxidative phase. To help clarify the issue we have measured the generation of the electric potential across the membrane, starting from a defined one-electron reduced state. Here we show that a second electron transfer into the enzyme leads to charge translocation corresponding to pumping of one proton without necessity for a preceding turnover.     

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.     

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.     

Artemisinins target the SERCA of Plasmodium falciparum

Artemisinins are extracted from sweet wormwood (Artemisia annua) and are the most potent antimalarials available, rapidly killing all asexual stages of Plasmodium falciparum. Artemisinins are sesquiterpene lactones widely used to treat multidrug-resistant malaria, a disease that annually claims 1 million lives. Despite extensive clinical and laboratory experience their molecular target is not yet identified. Activated artemisinins form adducts with a variety of biological macromolecules, including haem, translationally controlled tumour protein (TCTP) and other higher-molecular-weight proteins. Here we show that artemisinins, but not quinine or chloroquine, inhibit the SERCA orthologue (PfATP6) of Plasmodium falciparum in Xenopus oocytes with similar potency to thapsigargin (another sesquiterpene lactone and highly specific SERCA inhibitor). As predicted, thapsigargin also antagonizes the parasiticidal activity of artemisinin. Desoxyartemisinin lacks an endoperoxide bridge and is ineffective both as an inhibitor of PfATP6 and as an antimalarial. Chelation of iron by desferrioxamine abrogates the antiparasitic activity of artemisinins and correspondingly attenuates inhibition of PfATP6. Imaging of parasites with BODIPY-thapsigargin labels the cytosolic compartment and is competed by artemisinin. Fluorescent artemisinin labels parasites similarly and irreversibly in an Fe2+-dependent manner. These data provide compelling evidence that artemisinins act by inhibiting PfATP6 outside the food vacuole after activation by iron.     

A protein on Plasmodium falciparum-infected erythrocytes functions as a transferrin receptor

Several observations suggest that iron is essential for the development of malaria parasites but there is evidence that the parasites in erythrocytes do not obtain iron from haemoglobin. The total haemin level in parasitized erythrocytes does not vary during parasite development, indicating that the iron-containing moiety of haemoglobin is not detectably metabolized. Although parasite proteases can degrade the protein part of haemoglobin in red cells, no parasite enzymes that degrade haemin have been identified. In mammalian cells, haemin is degraded to carbon monoxide and bilirubin by the enzyme haeme oxygenase. This enzyme has not been found in malaria parasites. In fact haemin has been found to be toxic to parasite carbohydrate metabolism. Thus, iron apparently cannot be liberated from haemin and instead is sequestered in infected red cells as haemozoin, the characteristic pigment associated with malarial infection. If iron bound to transferrin is the source of ferric ions for malaria parasites within mature erythrocytes, then the parasite must synthesize its own transferrin receptor and localize it on the surface of the infected cell, because the receptors for transferrin are lost during erythrocyte maturation. Our results here suggest that Plasmodium falciparum synthesizes its own transferrin receptors enabling it to take up iron from transferrin by receptor-mediated endocytosis.     

Coenzyme Q is an obligatory cofactor for uncoupling protein function

Uncoupling proteins (UCPs) are thought to be intricately controlled uncouplers that are responsible for the futile dissipation of mitochondrial chemiosmotic gradients, producing heat rather than ATP. They occur in many animal and plant cells and form a subfamily of the mitochondrial carrier family. Physiological uncoupling of oxidative phosphorylation must be strongly regulated to avoid deterioration of the energy supply and cell death, which is caused by toxic uncouplers. However, an H+ transporting uncoupling function is well established only for UCP1 from brown adipose tissue, and the regulation of UCP1 by fatty acids, nucleotides and pH remains controversial. The failure of UCP1 expressed in Escherichia coli inclusion bodies to carry out fatty-acid-dependent H+ transport activity inclusion bodies made us seek a native UCP cofactor. Here we report the identification of coenzyme Q (ubiquinone) as such a cofactor. On addition of CoQ10 to reconstituted UCP1 from inclusion bodies, fatty-acid-dependent H+ transport reached the same rate as with native UCP1. The H+ transport was highly sensitive to purine nucleotides, and activated only by oxidized but not reduced CoQ. H+ transport of native UCP1 correlated with the endogenous CoQ content.