Plasmodium falciparum: detection of a novel asparagine-rich protein on the surface of sporozoite.

We had previously cloned and characterized a gene for a novel asparagine-rich protein from P. falciparum (PfARP), a target of natural human immune response. The antibodies to PfARP were localized to the surface of parasitized red blood cells and reacted with intracellular components in all erythrocytic asexual and sexual stages of the parasite. We here describe reactivity of antibodies against this novel PfARP on the surface of mosquito stage sporozoite of P. falciparum by indirect immunofluorescence assay and immunoelectron microscopy, the latter revealing a highly periodic punctate pattern of distribution of PfARP on the surface of sporozoite. These results suggest a possibility that PfARP might represent yet another sporozoite surface protein.     

Plasmodium vivax: cloning and expression of a major blood-stage surface antigen

Plasmodium vivax is a highly prevalent malaria pathogen of man; the following report is the first to describe the cloning and expression of a major asexual erythrocytic stage antigen of this species. The screening of a genomic DNA expression library with a monoclonal antibody directed against a 200-kDa surface component (Pv200) of the more mature schizonts of P. vivax led to the selection of a recombinant bacterial clone which produced a fusion protein. Mouse and rabbit immune sera raised against the purified fusion protein recognized the 200-kDa parasite antigen on Western blots and reacted with the surface of segmenters by immunofluorescence. Sequencing of the 1.9-kb P. vivax DNA insert coding for this fusion protein revealed a 45-47% homology at the nucleotide level with the P. falciparum gene of a parasite surface antigen, Pf195, which has been shown to be a promising candidate for a malaria vaccine in primates and in man.     

The Plasmodium falciparum heat shock protein 40, Pfj4, associates with heat shock protein 70 and shows similar heat induction and localisation patterns

Human cerebral malaria is caused by the protozoan parasite Plasmodium falciparum, which establishes itself within erythrocytes. The normal body temperature in the human host could constitute a possible source of heat stress to the parasite. Molecular chaperones belonging to the heat shock protein (Hsp) class are thought to be important for parasite subsistence in the host cell, as the expression of some members of this family has been reported to increase upon heat shock. In this paper we investigated the possible functions of the P. falciparum heat shock protein DnaJ homologue Pfj4, a type II Hsp40 protein. We analysed the ability of Pfj4 to functionally replace Escherichia coli Hsp40 proteins in a dnaJ cbpA mutant strain. Western analysis on cellular fractions of P. falciparum-infected erythrocytes revealed that Pfj4 expression increased upon heat shock. Localisation studies using immunofluorescence and immuno-electron microscopy suggested that Pfj4 and P. falciparum Hsp70, PfHsp70-1, were both localised to the parasites nucleus and cytoplasm. In some cases, Pfj4 was also detected in the erythrocyte cytoplasm of infected erythrocytes. Immunoprecipitation studies and size exclusion chromatography indicated that Pfj4 and PfHsp70-1 may directly or indirectly interact. Our results suggest a possible involvement of Pfj4 together with PfHsp70-1 in cytoprotection, and therefore, parasite survival inside the erythrocyte.     

Purification and properties of recombinant Plasmodium falciparum S-adenosyl-L-homocysteine hydrolase

Recombinant S-adenosyl-L-homocysteine (SAH) hydrolase of the malaria parasite Plasmodium falciparum was expressed in Escherichia coli, purified to homogeneity and characterized. Comparison of the malaria parasite SAH hydrolase with that derived from the human gene indicated marked differences in kcat values. The values of both forward and reverse reactions of P. falciparum SAH hydrolase are more than 21-fold smaller than those of the human enzyme. Km values of the parasite and human SAH enzymes are 1.2 and 7.8 microM, respectively. On the other hand, IC50 values of neplanocin A, a strong inhibitor of SAH hydrolase and a growth inhibitor of P. falciparum, are 101 nM for the parasite enzyme and 47 nM for human enzyme. P. falciparum SAH hydrolase has been thought to be a target for a chemotherapeutic agent against malaria. This study may make it possible to develop a specific inhibitor for the parasite SAH hydrolase.     

A novel enzyme complex of orotate phosphoribosyltransferase and orotidine 5'-monophosphate decarboxylase in human malaria parasite Plasmodium falciparum: physical association, kinetics, and inhibition characterization

Human malaria parasite, Plasmodium falciparum, can only synthesize pyrimidine nucleotides using the de novo pathway, whereas mammalian cells obtain pyrimidine nucleotides from both the de novo and salvage pathways. The parasite's orotate phosphoribosyltransferase (PfOPRT) and orotidine 5'-monophosphate decarboxylase (PfOMPDC) of the de novo pyrimidine pathway are attractive targets for antimalarial drug development. Previously, we have reported that the two enzymes in P. falciparum exist as a multienzyme complex containing two subunits each of 33-kDa PfOPRT and 38-kDa PfOMPDC. In this report, the gene encoding PfOPRT has been cloned and expressed in Escherichia coli. An open reading frame of PfOMPDC gene was identified in the malaria genome database, and PfOMPDC was cloned from P. falciparum cDNA, functionally expressed in E. coli, purified, and characterized. The protein sequence has <20% identity with human OMPDC and four microbial OMPDC for which crystal structures are known. Recombinant PfOMPDC was catalytically active in a dimeric form. Both recombinant PfOPRT and PfOMPDC monofunctional enzymes were kinetically different from the native multienzyme complex purified from P. falciparum. Oligomerization of PfOPRT and PfOMPDC cross-linked by dimethyl suberimidate indicated that they were tightly associated as the heterotetrameric 140-kDa complex, (PfOPRT)2(PfOMPDC)2. Kinetic analysis of the PfOPRT-PfOMPDC associated complex was similar to that of the native P. falciparum enzymes and was different from that of the bifunctional human enzymes. Interestingly, a nanomolar inhibitor of the yeast OMPDC, 6-thiocarboxamido-uridine 5'-monophosphate, was about 5 orders of magnitude less effective on the PfOMPDC than on the yeast enzyme. Our results support that the malaria parasite has unique structural and functional properties, sharing characteristics of the monofunctional pyrimidine-metabolizing enzymes in prokaryotes and bifunctional complexes in eukaryotes.     

Molecular characterization and expression of an alternate proliferating cell nuclear antigen homologue,PfPCNA2, in Plasmodium falciparum

The malaria parasite Plasmodium falciparum genome sequencing has revealed the existence of a second gene for proliferating cell nuclear antigen (PCNA), a key factor in a variety of DNA metabolic events. The alternate copy of PCNA (PfPCNA2) shows only 23% identity to an earlier reported P. falciparum PCNA homologue (PfPCNA1). Our analysis indicated structural conservation of PfPCNA2 compared to eukaryotic PCNAs. PfPCNA1 and 2 polypeptides showed differential expression in the intraerythrocytic cell cycle of the malaria parasite. PfPCNA1 expression slowly increases about threefold from the ring to the late schizont stage. In contrast PfPCNA2 showed robust expression in trophozoites and early schizonts with a sudden drop in expression in the late schizont stage, suggesting that the two PfPCNAs may function under different physiological conditions. Chemical cross-linking indicated the presence of a trimeric PfPCNA2 protein, indicating the possible existence of a functional ring-like PfPCNA2 structure.     

Unraveling the ubiquitome of the human malaria parasite

Malaria is one of the deadliest infectious diseases worldwide. The most severe form is caused by the eukaryotic protozoan parasite Plasmodium falciparum. Recent studies have highlighted the importance of post-translational regulations for the parasite's progression throughout its life cycle, protein ubiquitylation being certainly one of the most abundant. The specificity of its components and the wide range of biological processes in which it is involved make the ubiquitylation pathway a promising source of suitable targets for anti-malarial drug development. Here, we combined immunofluorescent microscopy, biochemical assays, in silico prediction, and mass spectrometry analysis using the multidimensional protein identification technology, or MudPIT, to describe the P. falciparum ubiquitome. We found that ubiquitin conjugates are detected at every morphological stage of the parasite erythrocytic cycle. Furthermore, we detected that more than half of the parasite's proteome represents possible targets for ubiquitylation, especially proteins found to be present at the most replicative stage of the asexual cycle, the trophozoite stage. A large proportion of ubiquitin conjugates were also detected at the schizont stage, consistent with a cell activity slowdown to prepare for merozoite differentiation and invasion. Finally, for the first time in the human malaria parasite, our results strongly indicate the presence of heterologous mixed conjugations, SUMO/UB. This discovery suggests that sumoylated proteins may be regulated by ubiquitylation in P. falciparum. Altogether, our results present the first stepping stone toward a better understanding of ubiquitylation and its role(s) in the biology of the human malaria parasite.     

Plasmodium falciparum carbonic anhydrase is a possible target for malaria chemotherapy

Plasmodiumfalciparum is responsible for the majority of life-threatening cases of human malaria. The global emergence of drug-resistant malarial parasites necessitates identification and characterization of novel drug targets. Carbonic anhydrase (CA) is present at high levels in human red cells and in P. falciparum. Existence of at least three isozymes of the alpha < class was demonstrated in P. falciparum and a rodent malarial parasite Plasmodium berghei. The major isozyme CA1 was purified and partially characterized from P. falciparum (PfCA1). A search of the malarial genome database yielded an open reading frame similar to the alpha-CAs from various organisms, including human. The primary amino acid sequence of the PfCA1 has 60% identity with a rodent parasite Plasmodium yoelii enzyme (PyCA). The single open reading frames encoded 235 and 252 amino acid proteins for PfCA1 and PyCA, respectively. The highly conserved active site residues were also found among organisms having alpha-CAs. The PfCA1 gene was cloned, sequenced and expressed in Escherichia coli. The purified recombinant PfCA1 enzyme was catalytically active. It was sensitive to acetazolamide and sulfanilamide inhibition. Kinetic properties of the recombinant PfCA1 revealed the authenticity to the wild type enzyme purified from P. falciparum in vitro culture. Furthermore, the PfCA1 inhibitors acetazolamide and sulfanilamide showed good antimalarial effect on the in vitro growth of P. falciparum. Our molecular tools developed for the recombinant enzyme expression will be useful for developing potential antimalarials directed at P. falciparum carbonic anhydrase.     

Co-ordinated programme of gene expression during asexual intraerythrocytic development of the human malaria parasite Plasmodium falciparum revealed by microarray analysis

Plasmodium falciparum is a protozoan parasite responsible for the most severe forms of human malaria. All the clinical symptoms and pathological changes seen during human infection are caused by the asexual blood stages of Plasmodium. Within host red blood cells, the parasite undergoes enormous developmental changes during its maturation. In order to analyse the expression of genes during intraerythrocytic development, DNA microarrays were constructed and probed with stage-specific cDNA. Developmental upregulation of specific mRNAs was found to cluster into functional groups and revealed a co-ordinated programme of gene expression. Those involved in protein synthesis (ribosomal proteins, translation factors) peaked early in development, followed by those involved in metabolism, most dramatically glycolysis genes. Adhesion/invasion genes were turned on later in the maturation process. At the end of intraerythrocytic development (late schizogony), there was a general shut-off of gene expression, although a small set of genes, including a number of protein kinases, were turned on at this stage. Nearly all genes showed some regulation over the course of development. A handful of genes remained constant and should be useful for normalizing mRNA levels between stages. These data will facilitate functional analysis of the P. falciparum genome and will help to identify genes with a critical role in parasite progression and multiplication in the human host.     

Cloning, overexpression, purification and characterization of Plasmodium knowlesi lactate dehydrogenase

Plasmodial lactate dehydrogenase, key enzyme of anaerobic glycolysis, has been shown to be a potential immunodiagnostic marker as well as a novel target for chemotherapy. We have cloned, overexpressed and immunochemically characterized the recombinant lactate dehydrogenase of Plasmodium knowlesi, the fifth human malaria parasite. The P. knowlesi lactate dehydrogenase (PkLDH) gene was PCR amplified and 0.9 kb PCR product was cloned into pGEM-T Easy vector. Sequencing and BLAST analysis revealed open reading frame of 316 amino acids of PkLDH showing 96.8% homology with Plasmodium vivax LDH and around 90% with Plasmodium falciparum, Plasmodium malariae and Plasmodium ovale LDHs. The PkLDH gene was subcloned into pGEX-6P1 expression vector and the SDS-PAGE analysis revealed that about 70% of fusion protein was present in the soluble fraction. The fusion protein was cleaved with PreScission protease and recombinant PkLDH (34 kDa) was affinity purified to homogeneity. The purified PkLDH exhibited high reactivity with polyclonal and monoclonal antibodies against plasmodial LDH. The polyclonal antibody produced against purified recombinant PkLDH in rabbits showed high ELISA reactivity with both native and recombinant PkLDH and could detect parasite LDH in malaria infected blood samples by sandwich ELISA. The purified recombinant PkLDH can be used to produce P. knowlesi specific monoclonal antibodies for specific diagnosis of P. knowlesi infection in humans.     

Genetic evidence strongly support an essential role for PfPV1 in intra-erythrocytic growth of P. falciparum

Upon invading the host erythrocyte, the human malaria parasite P. falciparum lives and replicates within a membrane bound compartment referred to as the parasitophorous vacuole. Recently, interest in this compartment and its protein content has grown, due to the important roles these play in parasite egress and protein traffic to the host cell. Surprisingly, the function of many proteins within this compartment has not been experimentally addressed. Here, we study the importance of one of these proteins, termed PfPV1, for intra-erythrocytic parasite survival. Despite numerous attempts to inactivate the gene encoding PfPV1, we were unable to recover deletion mutants. Control experiments verified that the pv1 gene locus was per se open for gene targeting experiments, allowing us to exclude technical limitations in our experimental strategy. Our data provide strong genetic evidence that PfPV1 is essential for survival of blood stage P. falciparum, and further highlight the importance of parasitophorous vacuole proteins in this part of the parasite's life cycle.     

Short odds for malaria vaccines

The immunology of falciparum malaria, the lethal type of human malaria, has been transformed by two developments. First, a culture system for the asexual blood stages of Plasmodium falciparum.¹ Secondly, the cloning and expression of genes coding for a large number of the protein antigens of this malaria parasite over the past two years. Data on proteins, protein antigens and epitopes of P. falciparum supplied by gene cloning techniques have been supplemented by monoclonal antibody approaches, peptide synthesis, and high-resolution immunochemistry.     

Bilirubin inhibits Plasmodium falciparum growth through the generation of reactive oxygen species

Free heme is very toxic because it generates highly reactive hydroxyl radicals ((.)OH) to cause oxidative damage. Detoxification of free heme by the heme oxygenase (HO) system is a very common phenomenon by which free heme is catabolized to form bilirubin as an end product. Interestingly, the malaria parasite, Plasmodium falciparum, lacks an HO system, but it forms hemozoin, mainly to detoxify free heme. Here, we report that bilirubin significantly induces oxidative stress in the parasite as evident from the increased formation of lipid peroxide, decrease in glutathione content, and increased formation of H(2)O(2) and (.)OH. Bilirubin can effectively inhibit hemozoin formation also. Furthermore, results indicate that bilirubin inhibits parasite growth and induces caspase-like protease activity, up-regulates the expression of apoptosis-related protein (Gene ID PFI0450c), and reduces the mitochondrial membrane potential. (.)OH scavengers such as mannitol, as well as the spin trap alpha-phenyl-n-tert-butylnitrone, effectively protect the parasite from bilirubin-induced oxidative stress and growth inhibition. These findings suggest that bilirubin, through the development of oxidative stress, induces P. falciparum cell death and that the malaria parasite lacks an HO system probably to protect itself from bilirubin-induced cell death as a second line of defense.     

Bipolar, Dual Plasmodium falciparum helicase 45 expressed in the intraerythrocytic developmental cycle is required for parasite growth

Helicases are ubiquitous molecular motor proteins that have an important role in the metabolism of nucleic acids. The gene encoding a helicase was cloned from the human malaria parasite Plasmodium falciparum. The polypeptide of 398 amino acid residues has a molecular mass of 45 kDa, contains striking homology to eukaryotic translation initiation factor 4A (eIF4A) and all the conserved domains of the DEAD-box family. The recombinantly expressed and homogeneous P. falciparum protein PfH45 is an ATP-dependent DNA and RNA helicase, with ATPase and ATP-binding activities. PfH45 is a unique bipolar helicase that contains both the 3' to 5' and 5' to 3' directional helicase activities and anti-PfH45 antibodies curtail all its activities. PfH45 is expressed in all the intraerythrocytic developmental stages of the parasite and has a role in translation. Parasite cultures treated with PfH45 double-stranded RNA or purified immunoglobulins against PfH45 exhibited approximately 60% and approximately 55% growth inhibition, respectively. This inhibitory effect was due to interference with expression of the cognate messenger and down-regulation of synthesis of PfH45 protein in the parasite culture and was associated with morphologic deformation of the parasite. These studies indicate that PfH45 is an indispensable enzyme that is essential for growth, and probably survival, of P. falciparum.     

Histone lysine methyltransferases and demethylases in Plasmodium falciparum

Dynamic histone lysine methylation, regulated by methyltransferases and demethylases, plays fundamental roles in chromatin structure and gene expression in a wide range of eukaryotic organisms. A large number of SET-domain-containing proteins make up the histone lysine methyltransferase (HKMT) family, which catalyses the methylation of different lysine residues with relatively high substrate specificities. Another large family of Jumonji C (JmjC)-domain-containing histone lysine demethylases (JHDMs) reverses histone lysine methylation with both lysine site and methyl-state specificities. Through bioinformatic analysis, at least nine SET-domain-containing genes were found in the malaria parasite Plasmodium falciparum and its sibling species. Phylogenetic analysis separated these putative HKMTs into five subfamilies with different putative substrate specificities. Consistent with the phylogenetic subdivision, methyl marks were found on K4, K9 and K36 of histone H3 and K20 of histone H4 by site-specific methyl-lysine antibodies. In addition, most SET-domain genes and histone methyl-lysine marks displayed dynamic changes during the parasite asexual erythrocytic cycle, suggesting that they constitute an important epigenetic mechanism of gene regulation in malaria parasites. Furthermore, the malaria parasite and other apicomplexan genomes also encode JmjC-domain-containing proteins that may serve as histone lysine demethylases. Whereas prokaryotic expression of putative active domains of four P. falciparum SET proteins did not yield detectable HKMT activity towards recombinant P. falciparum histones, two protein domains expressed in vitro in a eukaryotic system showed HKMT activities towards H3 and H4, respectively. With the discovery of these Plasmodium SET- and JmjC-domain genes in the malaria parasite genomes, future efforts will be directed towards elucidation of their substrate specificities and functions in various cellular processes of the parasites.     

Structure and non-essential function of glycerol kinase in Plasmodium falciparum blood stages

Malaria pathology is caused by multiplication of asexual parasites within erythrocytes, whereas mosquito transmission of malaria is mediated by sexual precursor cells (gametocytes). Microarray analysis identified glycerol kinase (GK) as the second most highly upregulated gene in Plasmodium falciparum gametocytes with no expression detectable in asexual blood stage parasites. Phosphorylation of glycerol by GK is the rate-limiting step in glycerol utilization. Deletion of this gene from P. falciparum had no effect on asexual parasite growth, but surprisingly also had no effect on gametocyte development or exflagellation, suggesting that these life cycle stages do not utilize host-derived glycerol as a carbon source. Kinetic studies of purified PfGK showed that the enzyme is not regulated by fructose 1,6 bisphosphate. The high-resolution crystal structure of P. falciparum GK, the first of a eukaryotic GK, reveals two domains embracing a capacious ligand-binding groove. In the complexes of PfGK with glycerol and ADP, we observed closed and open forms of the active site respectively. The 27° domain opening is larger than in orthologous systems and exposes an extensive surface with potential for exploitation in selective inhibitor design should the enzyme prove to be essential in vivo either in the human or in the mosquito.     

A principal target of human immunity to malaria identified by molecular population genetic and immunological analyses

New strategies are required to identify the most important targets of protective immunity in complex eukaryotic pathogens. Natural selection maintains allelic variation in some antigens of the malaria parasite Plasmodium falciparum. Analysis of allele frequency distributions could identify the loci under most intense selection. The merozoite surface protein 1 (Msp1) is the most-abundant surface component on the erythrocyte-invading stage of P. falciparum. Immunization with whole Msp1 has protected monkeys completely against homologous and partially against non-homologous parasite strains. The single-copy msp1 gene, of about 5 kilobases, has highly divergent alleles with stable frequencies in endemic populations. To identify the region of msp1 under strongest selection to maintain alleles within populations, we studied multiple intragenic sequence loci in populations in different regions of Africa and Southeast Asia. On both continents, the locus with the lowest inter-population variance in allele frequencies was block 2, indicating selection in this part of the gene. To test the hypothesis of immune selection, we undertook a large prospective longitudinal cohort study. This demonstrated that serum IgG antibodies against each of the two most frequent allelic types of block 2 of the protein were strongly associated with protection from P. falciparum malaria.     

Molecular mechanism of host specificity in Plasmodium falciparum infection: role of circumsporozoite protein

Plasmodium falciparum sporozoites invade liver cells in humans and set the stage for malaria infection. Circumsporozoite protein (CSP), a predominant surface antigen on sporozoite surface, has been associated with the binding and invasion of liver cells by the sporozoites. Although CSP across the Plasmodium genus has homology and conserved structural organization, infection of a non-natural host by a species is rare. We investigated the role of CSP in providing the host specificity in P. falciparum infection. CSP from P. falciparum, P. gallinaceum, P. knowlesi, and P. yoelii species representing human, avian, simian, and rodent malaria species were recombinantly expressed, and the proteins were purified to homogeneity. The recombinant proteins were evaluated for their capacity to bind to human liver cell line HepG2 and to prevent P. falciparum sporozoites from invading these cells. The proteins showed significant differences in the binding and sporozoite invasion inhibition activity. Differences among proteins directly correlate with changes in the binding affinity to the sporozoite receptor on liver cells. P. knowlesi CSP (PkCSP) and P. yoelii CSP (PyCSP) had 4,790- and 17,800-fold lower affinity for heparin in comparison to P. falciparum CSP (PfCSP). We suggest that a difference in the binding affinity for the liver cell receptor is a mechanism involved in maintaining the host specificity by the malaria parasite.