Targeting malaria parasite proteins to the erythrocyte

The intraerythrocytic stages of the protozoan parasite Plasmodium falciparum reside within a parasitophorous vacuole (PV) and set up unique "extraparasite, intraerythrocyte" protein-trafficking pathways that target parasite-encoded proteins to the erythrocyte cytoplasm and cell surface. Two recent articles report the identification of trafficking motifs that regulate the transport of parasite-encoded proteins across the PV. These articles greatly aid the annotation of the parasite "secretome" catalog of proteins that are targeted to the erythrocyte cytoplasm or cell membrane.     

Malaria parasite transmission stages: an update

The Molecular Approaches to Malaria 2004 meeting provided an opportunity to see the impressive progress in all research fields and in the four years since the previous Molecular Approaches to Malaria meeting, when much of the Plasmodium falciparum genome sequence was already available. Study of the part of the Plasmodium life cycle associated with transmission through the vector, which begins with the commitment of blood-stage forms to sexual development, has been especially fruitful. This success is a result of several reasons including: (i) the availability of the genome sequence; (ii) the availability of good animal models that allow parasite culture and facile in vivo studies of many of the life cycle stages involved in transmission; (iii) the availability of genetic manipulation technologies for the animal models of malaria, as well as P. falciparum; and (iv) the ability to study lethal gene knockouts at this stage of the life cycle.     

The malaria parasite Plasmodium falciparum has only one pyruvate dehydrogenase complex, which is located in the apicoplast

The relict plastid (apicoplast) of apicomplexan parasites synthesizes fatty acids and is a promising drug target. In plant plastids, a pyruvate dehydrogenase complex (PDH) converts pyruvate into acetyl-CoA, the major fatty acid precursor, whereas a second, distinct PDH fuels the tricarboxylic acid cycle in the mitochondria. In contrast, the presence of genes encoding PDH and related enzyme complexes in the genomes of five Plasmodium species and of Toxoplasma gondii indicate that these parasites contain only one single PDH. PDH complexes are comprised of four subunits (E1alpha, E1beta, E2, E3), and we confirmed four genes encoding a complete PDH in Plasmodium falciparum through sequencing of cDNA clones. In apicomplexan parasites, many nuclear-encoded proteins are targeted to the apicoplast courtesy of two-part N-terminal leader sequences, and the presence of such N-terminal sequences on all four PDH subunits as well as phylogenetic analyses strongly suggest that the P. falciparum PDH is located in the apicoplast. Fusion of the two-part leader sequences from the E1alpha and E2 genes to green fluorescent protein experimentally confirmed apicoplast targeting. Western blot analysis provided evidence for the expression of the E1alpha and E1beta PDH subunits in blood-stage malaria parasites. The recombinantly expressed catalytic domain of the PDH subunit E2 showed high enzymatic activity in vitro indicating that pyruvate is converted to acetyl-CoA in the apicoplast, possibly for use in fatty acid biosynthesis.     

Protein kinases of malaria parasites: an update

Protein kinases (PKs) play crucial roles in the control of proliferation and differentiation in eukaryotic cells. Research on protein phosphorylation has expanded tremendously in the past few years, in part as a consequence of the realization that PKs represent attractive drug targets in a variety of diseases. Activity in Plasmodium PK research has followed this trend, and several reports on various aspects of this subject were delivered at the Molecular Approaches to Malaria 2008 meeting (MAM2008), a sharp increase from the previous meeting. Here, the authors of most of these communications join to propose an integrated update of the development of the rapidly expanding field of Plasmodium kinomics.     

The biochemistry and fidelity of synthesis by the apicoplast genome replication DNA polymerase Pfprex from the malaria parasite Plasmodium falciparum

Plasmodium falciparum, the major causative agent of human malaria, contains three separate genomes. The apicoplast (an intracellular organelle) contains an ∼ 35-kb circular DNA genome of unusually high A/T content (> 86%) that is replicated by the nuclear-encoded replication complex Pfprex. Herein, we have expressed and purified the DNA polymerase domain of Pfprex [KPom1 (Klenow-like polymerase of malaria 1)] and measured its fidelity using a LacZ-based forward mutation assay. In addition, we analyzed the kinetic parameters for the incorporation of both complementary and noncomplementary nucleotides using Kpom1 lacking 3′ → 5′ exonucleolytic activity. KPom1 exhibits a strongly biased mutational spectrum in which T → C is the most frequent single-base substitution and differs significantly from the closely related Escherichia coli DNA polymerase I. Using E. coli harboring a temperature-sensitive polymerase I allele, we established that KPom1 can complement the growth-defective phenotype at an elevated temperature. We propose that the error bias of KPom1 may be exploited in the complementation assay to identify nucleoside analogs that mimic this base-mispairing and preferentially inhibit apicoplast DNA replication.     

Protein export in malaria parasites: an update

Symptomatic malaria is caused by the infection of human red blood cells (RBCs) with Plasmodium parasites. The RBC is a peculiar environment for parasites to thrive in as they lack many of the normal cellular processes and resources present in other cells. Because of this, Plasmodium spp. have adapted to extensively remodel the host cell through the export of hundreds of proteins that have a range of functions, the best known of which are virulence‐associated. Many exported parasite proteins are themselves involved in generating a novel trafficking system in the RBC that further promotes export. In this review we provide an overview of the parasite synthesized export machinery as well as recent developments in how different classes of exported proteins are recognized by this machinery.     

Vaccines against malaria - an update

Malaria vaccine discovery and development follow two principal strategies. Most subunit vaccines are designed to mimic naturally acquired immunity that develops over years upon continuous exposure to Plasmodium transmission. Experimental model vaccines, such as attenuated live parasites and transmission-blocking antigens, induce immune responses superior to naturally acquired immunity. The promises and hurdles of the different tracks towards an effective and affordable vaccine against malaria are discussed.     

Chemical rescue of malaria parasites lacking an apicoplast defines organelle function in blood-stage Plasmodium falciparum

Plasmodium spp parasites harbor an unusual plastid organelle called the apicoplast. Due to its prokaryotic origin and essential function, the apicoplast is a key target for development of new anti-malarials. Over 500 proteins are predicted to localize to this organelle and several prokaryotic biochemical pathways have been annotated, yet the essential role of the apicoplast during human infection remains a mystery. Previous work showed that treatment with fosmidomycin, an inhibitor of non-mevalonate isoprenoid precursor biosynthesis in the apicoplast, inhibits the growth of blood-stage P. falciparum. Herein, we demonstrate that fosmidomycin inhibition can be chemically rescued by supplementation with isopentenyl pyrophosphate (IPP), the pathway product. Surprisingly, IPP supplementation also completely reverses death following treatment with antibiotics that cause loss of the apicoplast. We show that antibiotic-treated parasites rescued with IPP over multiple cycles specifically lose their apicoplast genome and fail to process or localize organelle proteins, rendering them functionally apicoplast-minus. Despite the loss of this essential organelle, these apicoplast-minus auxotrophs can be grown indefinitely in asexual blood stage culture but are entirely dependent on exogenous IPP for survival. These findings indicate that isoprenoid precursor biosynthesis is the only essential function of the apicoplast during blood-stage growth. Moreover, apicoplast-minus P. falciparum strains will be a powerful tool for further investigation of apicoplast biology as well as drug and vaccine development.     

The genome of the malaria parasite

The genome of the human malaria parasite Plasmodium falciparum is being sequenced by an international consortium. Two of the parasite's 14 chromosomes have been completed and several other chromosomes are nearly finished. Even at this early stage of the project, analysis of the genome sequence has provided promising new leads for drug and vaccine development.     

The transportome of the malaria parasite

Membrane transport proteins, also known as transporters, control the movement of ions, nutrients, metabolites, and waste products across the membranes of a cell and are central to its biology. Proteins of this type also serve as drug targets and are key players in the phenomenon of drug resistance. The malaria parasite has a relatively reduced transportome, with only approximately 2.5% of its genes encoding transporters. Even so, assigning functions and physiological roles to these proteins, and ascertaining their contributions to drug action and drug resistance, has been very challenging. This review presents a detailed critique and synthesis of the disruption phenotypes, protein subcellular localisations, protein functions (observed or predicted), and links to antimalarial drug resistance for each of the parasite's transporter genes. The breadth and depth of the gene disruption data are particularly impressive, with at least one phenotype determined in the parasite's asexual blood stage for each transporter gene, and multiple phenotypes available for 76% of the genes. Analysis of the curated data set revealed there to be relatively little redundancy in the Plasmodium transportome; almost two‐thirds of the parasite's transporter genes are essential or required for normal growth in the asexual blood stage of the parasite, and this proportion increased to 78% when the disruption phenotypes available for the other parasite life stages were included in the analysis. These observations, together with the finding that 22% of the transportome is implicated in the parasite's resistance to existing antimalarials and/or drugs within the development pipeline, indicate that transporters are likely to serve, or are already serving, as drug targets. Integration of the different biological and bioinformatic data sets also enabled the selection of candidates for transport processes known to be essential for parasite survival, but for which the underlying proteins have thus far remained undiscovered. These include potential transporters of pantothenate, isoleucine, or isopentenyl diphosphate, as well as putative anion‐selective channels that may serve as the pore component of the parasite's ‘new permeation pathways’. Other novel insights into the parasite's biology included the identification of transporters for the potential development of antimalarial treatments, transmission‐blocking drugs, prophylactics, and genetically attenuated vaccines. The syntheses presented herein set a foundation for elucidating the functions and physiological roles of key members of the Plasmodium transportome and, ultimately, to explore and realise their potential as therapeutic targets.     

Adhesive proteins of the malaria parasite

Malaria infection of the host cells requires host-parasite recognition events mediated by adhesion and signaling molecules. Recent development of systems for stable transformation and targeted integration of exogenous DNA in malaria parasites provides a powerful tool to study the structure and function of Plasmodium attachment motifs, and their role in infection and disease.     

Subtilisin-like proteases of the malaria parasite

Proteases play critical roles in the life cycle of the malaria parasite, Plasmodium spp. Within the asexual erythrocytic cycle, responsible for the clinical manifestations of malaria, substantial interest has focused on the role of parasite serine proteases as a result of indications that they are involved in red blood cell invasion. Over the past 6 years, three Plasmodium genes encoding serine proteases of the subtilisin-like clan, or subtilases, have been identified. All are expressed in the asexual blood stages and, in at least two cases, the gene products localize to secretory organelles of the invasive merozoite. They may have potential as novel drug targets. Here, we review progress in our understanding of the maturation, specificity, structure and function of these Plasmodium subtilases.     

Severe malaria in children and pregnancy: an update and perspective

This review summarizes progress in preventing and treating severe malaria, which has been accompanied by advances in our understanding of the pathogenesis of severe malaria complications. New drugs such as intravenous artesunate and oral artemisinin combinations, with increased access to insecticide-treated bed nets, are improving outcomes and decreasing malaria deaths. Several groups are beginning to identify characteristics of parasite var genes associated with cerebral malaria. Understanding of the interactions between malaria and other diseases in causing severe anaemia and cerebral malaria has increased substantially, and at the cellular level, the disturbances leading to coma or other complications are becoming clearer.