Naturally-occurring and recombinant forms of the aspartic proteinases plasmepsins I and II from the human malaria parasite Plasmodium falciparum.

Comparable kinetic parameters were derived for the hydrolysis of peptide substrates and the interaction of synthetic inhibitors with recombinant and naturally-occurring forms of plasmepsin II. In contrast, recombinant plasmepsin I was extended by 12 residues at its N-terminus relative to its naturally-occurring counterpart and a 3-10-fold diminution in the k(cat) values was measured for substrate hydrolysis by the recombinant protein. However, comparable Ki values were derived for the interaction of two distinct inhibitors with both forms of plasmepsin I, thereby validating the use of recombinant material for drug screening. The value of plasmepsin I inhibitors was determined by assessing their selectivity using human aspartic proteinases.     

Activity and inhibition of plasmepsin IV, a new aspartic proteinase from the malaria parasite, Plasmodium falciparum

A new aspartic proteinase from the human malaria parasite Plasmodium falciparum is able to hydrolyse human haemoglobin at a site known to be the essential primary cleavage site in the haemoglobin degradation pathway. Thus, plasmepsin IV may play a crucial role in this critical process which yields nutrients for parasite growth. Furthermore, synthetic inhibitors known to inhibit parasite growth in red cells in culture are able to inhibit the activity of this enzyme in vitro. As a result, plasmepsin IV appears to be a potential target for the development of new antiparasitic drugs.     

Circumsporozoite protein gene from Plasmodium reichenowi, a chimpanzee malaria parasite evolutionarily related to the human malaria parasite Plasmodium falciparum

We have cloned and sequenced the gene encoding the circumsporozoite (CS) protein of Plasmodium reichenowi a Plasmodium falciparum-like malaria parasite of chimpanzees. Comparison of the two CS proteins reveals both similarities and differences in these two evolutionarily related parasites that have adapted to different hosts. The P. reichenowi CS protein has a new repeat sequence, NVNP, in addition to the P. falciparum-like NANP and NVDP repeats. In the immunodominant TH2R and TH3R regions of the CS protein, the amino acid sequences are similar in both parasite proteins. The differences in the two proteins exist in domains around the conserved regions, Region I and Region II, which are otherwise conserved in the CS proteins of P. falciparum analyzed to date. Studies of parasite protein genes of evolutionarily related malaria parasites, together with other immunologic and biologic characteristics, will help better understand the evolution and host parasite relationship of malaria parasites and may provide a tool for identifying protein determinants for malaria vaccine development.     

The membrane potential of the intraerythrocytic malaria parasite Plasmodium falciparum

The membrane potential (Deltapsi) of the mature asexual form of the human malaria parasite, Plasmodium falciparum, isolated from its host erythrocyte using a saponin permeabilization technique, was investigated using both the radiolabeled Deltapsi indicator tetraphenylphosphonium ([(3)H]TPP(+)) and the fluorescent Deltapsi indicator DiBAC(4)(3) (bis-oxonol). For isolated parasites suspended in a high Na(+), low K(+) solution, Deltapsi was estimated from the measured distribution of [(3)H]TPP(+) to be -95 +/- 2 mV. Deltapsi was reduced by the specific V-type H(+) pump inhibitor bafilomycin A(1), by the H(+) ionophore CCCP, and by glucose deprivation. Acidification of the parasite cytosol (induced by the addition of lactate) resulted in a transient hyperpolarization, whereas a cytosolic alkalinization (induced by the addition of NH(4)(+)) resulted in a transient depolarization. A decrease in the extracellular pH resulted in a membrane depolarization, whereas an increase in the extracellular pH resulted in a membrane hyperpolarization. The parasite plasma membrane depolarized in response to an increase in the extracellular K(+) concentration and hyperpolarized in response to a decrease in the extracellular K(+) concentration and to the addition of the K(+) channel blockers Ba(2+) or Cs(+) to the suspending medium. The data are consistent with Deltapsi of the intraerythrocytic P. falciparum trophozoite being due to the electrogenic extrusion of H(+) via the V-type H(+) pump at the parasite surface. The current associated with the efflux of H(+) is countered, in part, by the influx of K(+) via Ba(2+)- and Cs(+)-sensitive K(+) channels in the parasite plasma membrane.     

Purification and characterization of dihydroorotate dehydrogenase from the rodent malaria parasite Plasmodium berghei.

Dihydroorotate dehydrogenase (DHODase) has been purified 400-fold from the rodent malaria parasite Plasmodium berghei to apparent homogeneity by Triton X-100 solubilization followed by anion-exchange, Cibacron Blue F3GA-agarose affinity, and gel filtration chromatography. The purified enzyme has a molecular mass of 52 +/- 2 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and of 55 +/- 6 kDa by gel filtration chromatography, and it has a pI of 8.2. It is active in monomeric form, contains 2.022 mol of iron and 1.602 acid-labile sulfurs per mole of enzyme, and does not contain a flavin cofactor. The purified DHODase exhibits optimal activity at pH 8.0 in the presence of the ubiquinone coenzyme CoQ6, CoQ7, CoQ9, or CoQ10. The Km values for L-DHO and CoQ6 are 7.9 +/- 2.5 microM and 21.6 +/- 5.5 microM, respectively. The kcat values for both substrates are 11.44 min-1 and 11.70 min-1, respectively. The reaction product orotate and an orotate analogue, 5-fluoroorotate, are competitive inhibitors of the enzyme-catalyzed reaction with Ki values of 30.5 microM and 34.9 microM, respectively. The requirement of the long-chain ubiquinones for activity supports the hypothesis of the linkage of pyrimidine biosynthesis to the electron transport system and oxygen utilization in malaria by DHODase via ubiquinones [Gutteridge, W. E., Dave, D., & Richards, W. H. G. (1979) Biochim. Biophys. Acta 582, 390-401].     

Mitosis in the human malaria parasite Plasmodium falciparum

Malaria is caused by intraerythrocytic protozoan parasites belonging to Plasmodium spp. (phylum Apicomplexa) that produce significant morbidity and mortality, mostly in developing countries. Plasmodium parasites have a complex life cycle that includes multiple stages in anopheline mosquito vectors and vertebrate hosts. During the life cycle, the parasites undergo several cycles of extreme population growth within a brief span, and this is critical for their continued transmission and a contributing factor for their pathogenesis in the host. As with other eukaryotes, successful mitosis is an essential requirement for Plasmodium reproduction; however, some aspects of Plasmodium mitosis are quite distinct and not fully understood. In this review, we will discuss the current understanding of the architecture and key events of mitosis in Plasmodium falciparum and related parasites and compare them with the traditional mitotic events described for other eukaryotes.     

Proteomics of the human malaria parasite Plasmodium falciparum

The lethal species of malaria parasite, Plasmodium falciparum, continues to exact a huge toll of mortality and morbidity, particularly in sub-Saharan Africa. Completion of the genome sequence of this organism and advances in proteomics and mass spectrometry have opened up unprecedented opportunities for understanding the complex biology of this parasite and how it responds to drug challenge and other interventions. This review describes recent progress that has been made in applying proteomics technology to this important pathogen and provides a look forward to likely future developments.     

Characterization of adenine phosphoribosyltransferase from the human malaria parasite, Plasmodium falciparum

Because of their inability to synthesize purines de novo, malaria parasites rely on purine phosphoribosyltransferases (PRTases) to convert purine bases salvaged from the host cell (the erythrocyte) into the corresponding purine nucleoside monophosphates. Our studies with late trophozoites of the human malaria parasite, Plasmodium falciparum, showed that virtually all of the purine PRTase activity is accounted for by two distinct enzymes. One enzyme utilizes hypoxanthine, guanine and xanthine (Queen, S.A., Vander Jagt, D. and Reyes, P. (1988) Mol. Biochem. Parasitol. 30, 123-134). The second enzyme utilizes only adenine and is the subject of this paper. This latter enzyme exhibits a biphasic pH-activity profile and is moderately to weakly inhibited by several divalent metal ions. Several of the properties of the P. falciparum enzyme were found to differ significantly from those of human erythrocyte adenine PRTase. (1) The molecular weight (18,000) of the parasite enzyme is smaller than that of the host cell enzyme. (2) The parasite enzyme, unlike the erythrocyte enzyme, is not significantly inhibited by sulfhydryl reagents. (3) 6-Mercaptopurine and 2,6-diaminopurine proved to be competitive inhibitors of the parasite enzyme (Ki 0.70 and 1.0 mM, respectively); on the other hand, the human enzyme is not inhibited by these agents. (4) The Km for adenine (0.80 microM) and 5-phosphoribosyl-1-pyrophosphate (0.70 microM) displayed by the parasite enzyme are significantly smaller than the corresponding Km values shown by the erythrocyte enzyme. These distinctions between the parasite and host enzymes point to the possibility that adenine PRTase of P. falciparum may represent a potential target for chemotherapeutic attack.     

Detection of a system of microsomal monooxygenases in the rodent malaria parasite Plasmodium berghei

A microsomal fraction from the cells of the malaria parasite of rodent Plasmodium berghei was obtained. The spectral properties of microsomal preparations suggest that P. berghei microsomes contain cytochromes b5 and P-420. Electrophoretic separation of microsomal proteins revealed the presence of proteins whose molecular mass corresponds to NADPH-cytochrome c reductase, cytochrome P-450 and epoxide hydratase. The activities of NADPH-cytochrome c reductase and benzpyrene hydroxylase were determined. The spectral parameters, electrophoretic data and enzymatic activities of microsomal proteins indicate that P. berghei cells contain a cytochrome P-450 monooxygenase system. The interrelationship between the activity of the microsomal monooxygenase system and the resistance of P. berghei cells to the antimalaria preparation chloroquine is discussed.     

The multifunctional autophagy pathway in the human malaria parasite,Plasmodium falciparum

Autophagy is a catabolic pathway typically induced by nutrient starvation to recycle amino acids, but can also function in removing damaged organelles. In addition, this pathway plays a key role in eukaryotic development. To date, not much is known about the role of autophagy in apicomplexan parasites and more specifically in the human malaria parasite Plasmodium falciparum. Comparative genomic analysis has uncovered some, but not all, orthologs of autophagy-related (ATG) genes in the malaria parasite genome. Here, using a genome-wide in silico analysis, we confirmed that ATG genes whose products are required for vesicle expansion and completion are present, while genes involved in induction of autophagy and cargo packaging are mostly absent. We subsequently focused on the molecular and cellular function of P. falciparum ATG8 (PfATG8), an autophagosome membrane marker and key component of the autophagy pathway, throughout the parasite asexual and sexual erythrocytic stages. In this context, we showed that PfATG8 has a distinct and atypical role in parasite development. PfATG8 localized in the apicoplast and in vesicles throughout the cytosol during parasite development. Immunofluorescence assays of PfATG8 in apicoplast-minus parasites suggest that PfATG8 is involved in apicoplast biogenesis. Furthermore, treatment of parasite cultures with bafilomycin A₁ and chloroquine, both lysosomotropic agents that inhibit autophagosome and lysosome fusion, resulted in dramatic morphological changes of the apicoplast, and parasite death. Furthermore, deep proteomic analysis of components associated with PfATG8 indicated that it may possibly be involved in ribophagy and piecemeal microautophagy of the nucleus. Collectively, our data revealed the importance and specificity of the autophagy pathway in the malaria parasite and offer potential novel therapeutic strategies.     

The multifunctional autophagy pathway in the human malaria parasite,Plasmodium falciparum

Autophagy is a catabolic pathway typically induced by nutrient starvation to recycle amino acids, but can also function in removing damaged organelles. In addition, this pathway plays a key role in eukaryotic development. To date, not much is known about the role of autophagy in apicomplexan parasites and more specifically in the human malaria parasite Plasmodium falciparum. Comparative genomic analysis has uncovered some, but not all, orthologs of autophagy-related (ATG) genes in the malaria parasite genome. Here, using a genome-wide in silico analysis, we confirmed that ATG genes whose products are required for vesicle expansion and completion are present, while genes involved in induction of autophagy and cargo packaging are mostly absent. We subsequently focused on the molecular and cellular function of P. falciparum ATG8 (PfATG8), an autophagosome membrane marker and key component of the autophagy pathway, throughout the parasite asexual and sexual erythrocytic stages. In this context, we showed that PfATG8 has a distinct and atypical role in parasite development. PfATG8 localized in the apicoplast and in vesicles throughout the cytosol during parasite development. Immunofluorescence assays of PfATG8 in apicoplast-minus parasites suggest that PfATG8 is involved in apicoplast biogenesis. Furthermore, treatment of parasite cultures with bafilomycin A₁ and chloroquine, both lysosomotropic agents that inhibit autophagosome and lysosome fusion, resulted in dramatic morphological changes of the apicoplast, and parasite death. Furthermore, deep proteomic analysis of components associated with PfATG8 indicated that it may possibly be involved in ribophagy and piecemeal microautophagy of the nucleus. Collectively, our data revealed the importance and specificity of the autophagy pathway in the malaria parasite and offer potential novel therapeutic strategies.     

Transfection of the human malaria parasite Plasmodium falciparum.

In the past few years, methods have been developed which allow the introduction of exogenous DNA into the human malaria parasite Plasmodium falciparum. This important technical advance known as parasite transfection, provides powerful new tools to study the function of Plasmodium proteins and their roles in biology and disease. Already it has allowed the analysis of promoter function and has been successfully applied to establish the role of particular molecules and/or mutations in the biology of this parasite. This review summarises the current state of the technology and how it has been applied to dissect the function of the P. falciparum genome.     

Genetic mapping in the human malaria parasite Plasmodium falciparum

The Plasmodium falciparum genome sequence has boosted hopes for a new era of malaria research and for the application of comprehensive molecular knowledge to disease control, but formidable obstacles remain: approximately 60% of the predicted P. falciparum proteins have no known functions or homologues, and most life cycle stages of this haploid eukaryotic parasite are relatively intractable to cultivation and biochemical manipulation. Genetic mapping based on high-resolution maps saturated with single-nucleotide polymorphisms or microsatellites is now providing effective strategies for discovering candidate genes determining important parasite phenotypes. Here we review classical linkage studies using laboratory crosses and population associations that are now amenable to genome-wide approaches and are revealing multiple candidate genes involved in complex drug responses. Moreover, mapping by linkage disequilibrium is practicable in cases where chromosomal segments flanking drug-selected genes have been preserved in populations during relatively recent P. falciparum evolution. We discuss the advantages and limitations of these various genetic mapping strategies, results from which offer complementary insights to those emerging from gene knockout experiments and/or high-throughput genomic technologies.     

FULL-malaria: a database for a full-length enriched cDNA library from human malaria parasite, Plasmodium falciparum

FULL-malaria is a database for a full-length-enriched cDNA library from the human malaria parasite Plasmodium falciparum (http://133.11. 149.55/). Because of its medical importance, this organism is the first target for genome sequencing of a eukaryotic pathogen; the sequences of two of its 14 chromosomes have already been determined. However, for the full exploitation of this rapidly accumulating information, correct identification of the genes and study of their expression are essential. Using the oligo-capping method, we have produced a full-length-enriched cDNA library from erythrocytic stage parasites and performed one-pass reading. The database consists of nucleotide sequences of 2490 random clones that include 390 (16%) known malaria genes according to BLASTN analysis of the nr-nt database in GenBank; these represent 98 genes, and the clones for 48 of these genes contain the complete protein-coding sequence (49%). On the other hand, comparisons with the complete chromosome 2 sequence revealed that 35 of 210 predicted genes are expressed, and in addition led to detection of three new gene candidates that were not previously known. In total, 19 of these 38 clones (50%) were full-length. From these observations, it is expected that the database contains approximately 1000 genes, including 500 full-length clones. It should be an invaluable resource for the development of vaccines and novel drugs.     

Crystal structure of S-adenosyl-L-homocysteine hydrolase from the human malaria parasite Plasmodium falciparum

The human malaria parasite Plasmodium falciparum is responsible for the death of more than a million people each year. The emergence of strains of malarial parasite resistant to conventional drug therapy has stimulated searches for antimalarials with novel modes of action. S-Adenosyl-L-homocysteine hydrolase (SAHH) is a regulator of biological methylations. Inhibitors of SAHH affect the methylation status of nucleic acids, proteins, and small molecules. P.falciparum SAHH (PfSAHH) inhibitors are expected to provide a new type of chemotherapeutic agent against malaria. Despite the pressing need to develop selective PfSAHH inhibitors as therapeutic drugs, only the mammalian SAHH structures are currently available. Here, we report the crystal structure of PfSAHH complexed with the reaction product adenosine (Ado). Knowledge of the structure of the Ado complex in combination with a structural comparison with Homo sapiens SAHH (HsSAHH) revealed that a single substitution between the PfSAHH (Cys59) and HsSAHH (Thr60) accounts for the differential interactions with nucleoside inhibitors. To examine roles of the Cys59 in the interactions with nucleoside inhibitors, a mutant PfSAHH was prepared. A replacement of Cys59 by Thr results in mutant PfSAHH, which shows HsSAHH-like nucleoside inhibitor sensitivity. The present structure should provide opportunities to design potent and selective PfSAHH inhibitors.     

The human malaria parasite Plasmodium falciparum has distinct organelle-specific lipoylation pathways

Lipoic acid is an essential cofactor of alpha-keto acid dehydrogenase complexes (KADHCs). This study shows that Plasmodium falciparum possesses two distinct lipoylation pathways that are found in separate subcellular localizations. Lipoic acid synthesis comprising lipoic acid synthase and lipoyl-ACP:protein N-lipoyl transferase is present in the parasite's apicoplast, whereas the second pathway consisting of lipoic acid protein ligase is located in the parasite's mitochondrion. The two localizations were established by overexpressing green fluorescent protein fusions of the N-terminal sequences of lipoic acid synthase and lipoic acid protein ligase in intraerythrocytic stages of P. falciparum. Northern and Western blot analyses revealed that the genes/proteins encoding lipoic acid synthase, lipoyl-ACP:protein N-lipoyl transferase and lipoic acid protein ligase are expressed maximally in the early and late stages of P. falciparum erythrocytic development. The functionality of the three proteins was proven by complementation of bacteria deficient in lipA and lipB. Our results show that P. falciparum possesses two independent pathways, with different locations, responsible for the post-translational modification of KADHCs. Both pathways fundamentally differ from those in the human host. As KADHCs provide metabolites that are required for essential biosynthetic processes such as fatty acid biosynthesis and haem biosynthesis, the two lipoylation pathways of P. falciparum might be attractive therapeutic targets against malaria.