The human malaria parasite Plasmodium vivax is responsible for 25-40% of the ~515 million annual cases of malaria worldwide. Although seldom fatal, the parasite elicits severe and incapacitating clinical symptoms and often relapses months after a primary infection has cleared. Despite its importance as a major human pathogen, P. vivax is little studied because it cannot be propagated in the laboratory except in non-human primates. We determined the genome sequence of P. vivax in order to shed light on its distinctive biologic features, and as a means to drive development of new drugs and vaccines. Here we describe the synteny and isochore structure of P. vivax chromosomes, and show that the parasite resembles other malaria parasites in gene content and metabolic potential, but possesses novel gene families and potential alternate invasion pathways not recognized previously. Completion of the P. vivax genome provides the scientific community with a valuable resource that can be used to advance scientific investigation into this neglected species.

Several hundred malaria parasite proteins are exported beyond an encasing vacuole and into the cytosol of the host erythrocyte, a process that is key to the virulence and viability of the causative Plasmodium species. The trafficking machinery responsible for this export is unknown. Here, we identify a Plasmodium Translocon of EXported proteins (PTEX), which is located in the vacuole membrane. The PTEX complex is ATP-powered and comprises HSP101, which is a ClpA/B-like AAA+ ATPase of a type commonly associated with protein translocons, a novel protein termed PTEX150 and a known parasite protein EXP2. EXP2 is the potential channel as it is the membrane-associated component of the core PTEX complex. Two other proteins, a novel protein PTEX88 and a thioredoxin known as TRX2, were also identified as PTEX components. As a common portal for numerous crucial processes, this novel translocon offers an exciting new avenue for therapeutic intervention.

During blood stage Plasmodium falciparum infection, merozoites invade uninfected erythrocytes via a complex, multistep process involving a series of distinct receptor-ligand binding events. Understanding each element in this process increases the potential to block the parasite’s life cycle via drugs or vaccines. To investigate specific receptor-ligand interactions, they were systematically blocked using a combination of genetic deletion, enzymatic receptor cleavage and inhibition of binding via antibodies, peptides and small molecules, and the resulting temporal changes in invasion and morphological effects on erythrocytes were filmed using live cell imaging. Analysis of the videos have shown receptor-ligand interactions occur in the following sequence with the following cellular morphologies; 1) an early heparin-blockable interaction which weakly deforms the erythrocyte, 2) EBA and PfRh ligands which strongly deform the erythrocyte, a process dependant on the merozoite’s actin-myosin motor, 3) a PfRh5-basigin binding step which results in a pore or opening between parasite and host through which it appears small molecules and possibly invasion components can flow and 4) an AMA1–RON2 interaction that mediates tight junction formation, which acts as an anchor point for internalization. In addition to enhancing general knowledge of apicomplexan biology, this work provides a rational basis to combine sequentially acting merozoite vaccine candidates in a single multi-receptor-blocking vaccine.

Plasmodium falciparum causes the virulent form of malaria and disease manifestations are linked to growth inside infected erythrocytes. In order to survive and evade host responses the parasite remodels the erythrocyte by exporting several hundred effector proteins beyond the surrounding parasitophorous vacuole membrane. A feature of exported proteins is a pentameric motif (RxLxE/Q/D) that is a substrate for an unknown protease. Here, we show the protein responsible for cleavage of this motif is Plasmepsin V, an aspartic acid protease located in the endoplasmic reticulum. Plasmepsin V cleavage reveals the export signal (xE/Q/D) at the N-terminus of cargo proteins. Expression of an identical mature protein with xQ at the N-terminus generated by signal peptidase was not exported demonstrating Plasmepsin V activity is essential and linked with other key export events. Identification of the protease responsible for export into erythrocytes provides a novel target for therapeutic intervention against this devastating disease.

During the blood stages of malaria, several hundred parasite-encoded proteins are exported beyond the double-membrane barrier that separates the parasite from the host cell cytosol. These proteins have a variety of roles that are essential to virulence or parasite growth. There is keen interest in understanding how proteins are exported and whether common machineries are involved in trafficking the different classes of exported proteins. One potential trafficking machine is a protein complex known as the Plasmodium translocon of exported proteins (PTEX). Although PTEX has been linked to the export of one class of exported proteins, there has been no direct evidence for its role and scope in protein translocation. Here we show, through the generation of two parasite lines defective for essential PTEX components (HSP101 or PTEX150), and analysis of a line lacking the non-essential component TRX2 (ref. 12), greatly reduced trafficking of all classes of exported proteins beyond the double membrane barrier enveloping the parasite. This includes proteins containing the PEXEL motif (RxLxE/Q/D) and PEXEL-negative exported proteins (PNEPs). Moreover, the export of proteins destined for expression on the infected erythrocyte surface, including the major virulence factor PfEMP1 in Plasmodium falciparum, was significantly reduced in PTEX knockdown parasites. PTEX function was also essential for blood-stage growth, because even a modest knockdown of PTEX components had a strong effect on the parasite's capacity to complete the erythrocytic cycle both in vitro and in vivo. Hence, as the only known nexus for protein export in Plasmodium parasites, and an essential enzymic machine, PTEX is a prime drug target.

The introduction of plastids into different heterotrophic protists created lineages of algae that diversified explosively, proliferated in marine and freshwater environments, and radically altered the biosphere. The origins of these secondary plastids are usually inferred from the presence of additional plastid membranes. However, two examples provide unique snapshots of secondaryendosymbiosis-in-action, because they retain a vestige of the endosymbiont nucleus known as the nucleomorph. These are chlorarachniophytes and cryptomonads, which acquired their plastids from a green and red alga respectively. To allow comparisons between them, we have sequenced the nucleomorph genome from the chlorarachniophyte Bigelowiella natans: at a mere 373,000 bp and with only 331 genes, the smallest nuclear genome known and a model for extreme reduction. The genome is eukaryotic in nature, with three linear chromosomes containing densely packed genes with numerous overlaps. The genome is replete with 852 introns, but these are the smallest introns known, being only 18, 19, 20, or 21 nt in length. These pygmy introns are shown to be miniaturized versions of normal-sized introns present in the endosymbiont at the time of capture. Seventeen nucleomorph genes encode proteins that function in the plastid. The other nucleomorph genes are housekeeping entities, presumably underpinning maintenance and expression of these plastid proteins. Chlorarachniophyte plastids are thus serviced by three different genomes (plastid, nucleomorph, and host nucleus) requiring remarkable coordination and targeting. Although originating by two independent endosymbioses, chlorarachniophyte and cryptomonad nucleomorph genomes have converged upon remarkably similar architectures but differ in many molecular details that reflect two distinct trajectories to hypercompaction and reduction.plastid ͉ secondary endosymbiosis ͉ intron ͉ endosymbiosis T he origin of plastids through endosymbiosis of a cyanobacterium-like prokaryote transferred photosynthesis into eukaryotes and launched a massive wave of diversification that subsequently generated a tremendous range of algae and plants (1). This initial event is referred to as primary endosymbiosis ( Fig. 1) and created a plastid with two membranes such as those of green algae, plants, red algae, and glaucophyte algae (1). Transfer of genes from the endosymbiont to the nuclear genome of the host initially led to dependence of the endosymbiont on the host that was necessary to stabilize the partnership (2). Ongoing transfer has resulted in reduction of the prokaryotic genome, so that plastid DNA now represents probably Ͻ10% of its original gene content, and increasingly sophisticated regulation of the endosymbiont by the host has resulted in endosymbiont replication, gene expression, metabolic activity, and even death being managed by the eukaryotic host (3). Indeed, primary plastids seem to retain some autonomy only in the synthesis and deployment of redox proteins involved in photosynthetic electron transfer (4).A...

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Glycosylphosphatidylinositol (GPI)-anchored proteins coat the surface of extracellular Plasmodium falciparum merozoites, of which several are highly validated candidates for inclusion in a blood-stage malaria vaccine. Here we determined the proteome of gradient-purified detergent-resistant membranes of mature bloodstage parasites and found that these membranes are greatly enriched in GPI-anchored proteins and their putative interacting partners. Also prominent in detergent-resistant membranes are apical organelle (rhoptry), multimembrane-spanning, and proteins destined for export into the host erythrocyte cytosol. Four new GPIanchored proteins were identified, and a number of other novel proteins that are predicted to localize to the merozoite surface and/or apical organelles were detected. Three of the putative surface proteins possessed six-cysteine (Cys 6 ) motifs, a distinct fold found in adhesive surface proteins expressed in other life stages. All three Cys 6 proteins, termed Pf12, Pf38, and Pf41, were validated as merozoite surface antigens recognized strongly by antibodies present in naturally infected individuals. In addition to the merozoite surface, Pf38 was particularly prominent in the secretory apical organelles. A different cysteine-rich putative GPI-anchored protein, Pf92, was also localized to the merozoite surface. This insight into merozoite surfaces provides new opportunities for understanding both erythrocyte invasion and anti-parasite immunity.Developing a vaccine to control human malaria is a global health priority. The recent availability of the genome sequence of the protozoan parasite Plasmodium falciparum, the major cause of malaria, allows the use of genomic technologies such as microarray and proteomics to identify novel vaccine and drug targets (1-5). Most membrane proteins that coat the surface of the erythrocyte-invasive merozoite form of the parasite are attached to the plasma membrane via a C-terminal glycosylphosphatidylinositol (GPI) 5 anchor. To date, four GPI-anchored merozoite surface proteins (MSP-1, -2, -4, and -5) have been identified, and two others (MSP-10 and rhoptry-associated membrane antigen (RAMA)) appear to reside at least in part in organelles at the apical end of the parasite (6 -10). Another protein originally localized to the merozoite surface, MSP-8, appears to instead reside in the ring stage (11). Most other merozoite surface proteins (e.g. MSP-3/6 family members, MSP-7, and acidic basic repeat antigen (ABRA)) are not directly membrane-associated but are indirectly linked to the surface, probably in most cases via interactions with GPI-anchored proteins (12-15). In contrast to the apical and peripheral classes of bloodstage antigens, the GPI-anchored proteins appear to be essential to blood-stage growth with repeated attempts to genetically disrupt six GPI-anchored merozoite proteins resulting in only one "knock-out," that of the msp-5 gene (16). 6 This, together with considerable data highlighting their potential as targets of protective antibodies, place...