A member of a conserved Plasmodium protein family with membrane-attack complex/perforin (MACPF)-like domains localizes to the micronemes of sporozoites

Pore-forming proteins are employed by many pathogens to achieve successful host colonization. Intracellular pathogens use pore-forming proteins to invade host cells, survive within and productively interact with host cells, and finally egress from host cells to infect new ones. The malaria-causing parasites of the genus Plasmodium evolved a number of life cycle stages that enter and replicate in distinct cell types within the mosquito vector and vertebrate host. Despite the fact that interaction with host-cell membranes is a central theme in the Plasmodium life cycle, little is known about parasite proteins that mediate such interactions. We identified a family of five related genes in the genome of the rodent malaria parasite Plasmodium yoelii encoding secreted proteins all bearing a single membrane-attack complex/perforin (MACPF)-like domain. Each protein is highly conserved among Plasmodium species. Gene expression analysis in P. yoelii and the human malaria parasite Plasmodium falciparum indicated that the family is not expressed in the parasites blood stages. However, one of the genes was significantly expressed in P. yoelii sporozoites, the stage transmitted by mosquito bite. The protein localized to the micronemes of sporozoites, organelles of the secretory invasion apparatus intimately involved in host-cell infection. MACPF-like proteins may play important roles in parasite interactions with the mosquito vector and transmission to the vertebrate host.     

BDA-410: a novel synthetic calpain inhibitor active against blood stage malaria

Falcipains, the papain-family cysteine proteases of the Plasmodium falciparum, are potential drug targets for malaria parasite. Pharmacological inhibition of falcipains can block the hydrolysis of hemoglobin, parasite development, and egress, suggesting that falcipains play a key role at the blood stage of parasite life cycle. In the present study, we evaluated the anti-malarial effects of BDA-410, a novel cysteine protease inhibitor as a potential anti-malarial drug. Recombinant falcipain (MBP-FP-2B) and P. falciparum trophozoite extract containing native falcipains were used for enzyme inhibition studies in vitro. The effect of BDA-410 on the malaria parasite development in vitro as well as its anti-malarial activity in vivo was evaluated using the Plasmodium chabaudi infection rodent model. The 50% inhibitory concentrations of BDA-410 were determined to be 628 and 534nM for recombinant falcipain-2B and parasite extract, respectively. BDA-410 inhibited the malaria parasite growth in vitro with an IC(50) value of 173nM causing irreversible damage to the intracellular parasite. In vivo, the BDA-410 delayed the progression of malaria infection significantly using a mouse model of malaria pathogenesis. The characterization of BDA-410 as a potent inhibitor of P. falciparum cysteine proteases, and the demonstration of its efficacy in blocking parasite growth both in vitro and in vivo assays identifies BDA-410 is an important lead compound for the development of novel anti-malarial drugs.     

De novo search for non-coding RNA genes in the AT-rich genome of Dictyostelium discoideum: performance of Markov-dependent genome feature scoring

Genome data are increasingly important in the computational identification of novel regulatory non-coding RNAs (ncRNAs). However, most ncRNA gene-finders are either specialized to well-characterized ncRNA gene families or require comparisons of closely related genomes. We developed a method for de novo screening for ncRNA genes with a nucleotide composition that stands out against the background genome based on a partial sum process. We compared the performance when assuming independent and first-order Markov-dependent nucleotides, respectively, and used Karlin-Altschul and Karlin-Dembo statistics to evaluate the significance of hits. We hypothesized that a first-order Markov-dependent process might have better power to detect ncRNA genes since nearest-neighbor models have been shown to be successful in predicting RNA structures. A model based on a first-order partial sum process (analyzing overlapping dinucleotides) had better sensitivity and specificity than a zeroth-order model when applied to the AT-rich genome of the amoeba Dictyostelium discoideum. In this genome, we detected 94% of previously known ncRNA genes (at this sensitivity, the false positive rate was estimated to be 25% in a simulated background). The predictions were further refined by clustering candidate genes according to sequence similarity and/or searching for an ncRNA-associated upstream element. We experimentally verified six out of 10 tested ncRNA gene predictions. We conclude that higher-order models, in combination with other information, are useful for identification of novel ncRNA gene families in single-genome analysis of D. discoideum. Our generalizable approach extends the range of genomic data that can be searched for novel ncRNA genes using well-grounded statistical methods.     

Artificial linear episome-based protein expression system for protozoon Leishmania tarentolae

Plasmodium falciparum malaria is the most important parasitic disease worldwide, responsible for an estimated 1 million deaths annually. Two P. falciparum genes code for putative phosphoglycerate mutases (PGMases), a widespread protein group characterized by the involvement of histidine residues in their catalytic mechanism. PGMases are responsible for the interconversion between 2 and 3-phosphoglycerate, an intermediate step in the glycolysis pathway. We have determined the crystal structures of one of the P. falciparum's PGMases (PfPGM2) and a functionally distinct phosphoglycerate mutase from Cryptosporidium parvum, a related apicomplexan parasite. We performed sequence and structural comparisons between the two structures, another P. falciparum enzyme (PfPGM1) and several other PGM family members from other organisms. The comparisons revealed a distinct conformation of the catalytically active residues not seen in previously determined phosphoglycerate mutase structures. Furthermore, characterization of their enzymatic activities revealed contrasting behaviors between the PfPGM2 and the classical cofactor-dependent PGMase from C. parvum, clearly establishing PfPGM2 as a phosphatase with a residual level of mutase activity. Further support for this function attribution was provided by our structural comparison with previously characterized PGM family members. Genetic characterization of PGM2 in the rodent parasite Plasmodium berghei indicated that the protein might be essential to blood stage asexual growth, and a GFP tagged allele is expressed in both blood and zygote ookinete development and located in the cytoplasm. The P. falciparum PGM2 is either an enzyme implicated in the phosphate metabolism of the parasite or a regulator of its life cycle.     

Filling the gap of intracellular dephosphorylation in the Plasmodium falciparum vitamin B1 biosynthesis

Thiamine pyrophosphate (TPP), the active form of vitamin B1, is an essential cofactor for several enzymes. Humans depend exclusively on the uptake of vitamin B1, whereas bacteria, plants, fungi and the malaria parasite Plasmodium falciparum are able to synthesise thiamine monophosphate (TMP) de novo. TMP has to be dephosphorylated prior to pyrophosphorylation in order to obtain TPP. In P. falciparum the phosphatase capable to catalyse this reaction has been identified by analysis of the substrate specificity. The recombinant enzyme accepts beside vitamin B1 also nucleotides, phosphorylated sugars and the B6 vitamer pyridoxal 5'-phosphate. Vitamin B1 biosynthesis is known to occur in the cytosol. The cytosolic localisation of this phosphatase was verified by transfection of a GFP chimera construct. Stage specific Northern blot analysis of the phosphatase clearly identified an expression profile throughout the entire erythrocytic life cycle of P. falciparum and thereby emphasises the importance of dephosphorylation reactions within the malaria parasite.     

Novel antigen identification method for discovery of protective malaria antigens by rapid testing of DNA vaccines encoding exons from the parasite genome

We describe a novel approach for identifying target antigens for preerythrocytic malaria vaccines. Our strategy is to rapidly test hundreds of DNA vaccines encoding exons from the Plasmodium yoelii yoelii genomic sequence. In this antigen identification method, we measure reduction in parasite burden in the liver after sporozoite challenge in mice. Orthologs of protective P. y. yoelii genes can then be identified in the genomic databases of Plasmodium falciparum and Plasmodium vivax and investigated as candidate antigens for a human vaccine. A pilot study to develop the antigen identification method approach used 192 P. y. yoelii exons from genes expressed during the sporozoite stage of the life cycle. A total of 182 (94%) exons were successfully cloned into a DNA immunization vector with the Gateway cloning technology. To assess immunization strategies, mice were vaccinated with 19 of the new DNA plasmids in addition to the well-characterized protective plasmid encoding P. y. yoelii circumsporozoite protein. Single plasmid immunization by gene gun identified a novel vaccine target antigen which decreased liver parasite burden by 95% and which has orthologs in P. vivax and P. knowlesi but not P. falciparum. Intramuscular injection of DNA plasmids produced a different pattern of protective responses from those seen with gene gun immunization. Intramuscular immunization with plasmid pools could reduce liver parasite burden in mice despite the fact that none of the plasmids was protective when given individually. We conclude that high-throughput cloning of exons into DNA vaccines and their screening is feasible and can rapidly identify new malaria vaccine candidate antigens.     

Heterogeneity and expression of the Plasmodium falciparum 5.8S ribosomal RNA genes

The number and expression of some of the large ribosomal RNA (rRNA) gene classes present in the human malaria parasite Plasmodium falciparum was determined. Southern blot analyses, using the 5.8S rRNA coding region as a marker, indicate that the P. falciparum genome contains at least 5 distinct subclasses of large rRNA genes. Dideoxy sequencing of the 5.8S rRNA domain and Northern blot analyses demonstrate that only one subclass is transcribed during the parasite's asexual erythrocytic life cycle.     

PTRAMP; a conserved Plasmodium thrombospondin-related apical merozoite protein

A gene encoding a 352 amino acid protein with a putative signal sequence, transmembrane domain and thrombospondin structural homology repeat was identified in the genome of the human malaria parasite, Plasmodium falciparum and the rodent malaria parasite, Plasmodium berghei. The protein localises in the apical organelles of P. falciparum and P. berghei merozoites within intraerythrocytic schizonts and has, therefore, been termed the Plasmodium thrombospondin-related apical merozoite protein (PTRAMP). PTRAMP co-localises with the Apical Merozoite Antigen-1 (AMA-1) in developing micronemes and subsequently relocates onto the merozoite surface. Although the gene appears to be specific to the Plasmodium genus, orthologues are present in the genomes of all malaria parasite species examined suggesting a conserved function in host-cell invasion. PTRAMP, therefore, has all the features to merit further evaluation as a malaria vaccine candidate.     

Typing Plasmodium yoelii microsatellites using a simple and affordable fluorescent labeling method

The rodent malaria parasite Plasmodium yoelii has been an important animal model for studying malaria pathology and host-parasite interactions. Compared with other rodent malaria parasites such as Plasmodium chabaudi, however, genetic mapping studies on P. yoelii have been limited, partly due to the absence of genetic markers and the lack of well characterized phenotypes. Taking advantage of the available genome sequence, we initiated a project to develop a high-resolution microsatellite (MS) map for P. yoelii to study malaria disease phenotypes. Here we report screening the P. yoelii genome for simple sequence repeats and development of an inexpensive method (modified from a previously reported procedure) for typing malaria parasite MS: instead of labeling individual polymerase chain reaction primers, a single fluorescently labeled primer was used to type the MS markers. We evaluated various polymerase chain reaction cycling conditions and M13-tailed/labeled M13 primer ratios to establish a simple and robust procedure for typing P. yoelii MS markers. We also compared typing efficiencies between individually labeled primers and the M13-tailed single labeled primer method and found that the two approaches were comparable. Preliminary analyses of seven P. yoelii isolates deposited at MR4 with 77 MS showed that the markers were highly polymorphic and that the isolates belonged to two groups, suggesting potential common ancestry or laboratory contaminations among the isolates. The MS markers and the typing method provide important tools for genetic studies of P. yoelii. There is a good possibility that this method can be applied to type MS from other malaria parasites including important human pathogens Plasmodium falciparum and Plasmodium vivax.     

Molecular cloning and characterization of Plasmodium falciparum transportin

A cDNA encoding transportin, a protein involved in the nuclear import of M9 nuclear localization signal-bearing proteins, has been cloned from the malaria parasite Plasmodium falciparum. The complete cDNA consists of 3,667 bp encoding 1,136 amino acid residues. Amino acid sequence analysis revealed that Ran-GTP and M9 binding domains are highly conserved in P. falciparum, suggesting that the transportin-mediated nuclear transport pathway exists in this protozoan parasite. Southern blot analysis revealed that the transportin gene exists as a single copy in the malarial genome.     

Host vascular endothelial growth factor is trophic for Plasmodium falciparum-infected red blood cells

Plasmodium falciparum, the protozoan parasite responsible for severe malaria infection, undergoes a complex life cycle. Infected red blood cells (iRBC) sequester in host cerebral microvessels, which underlies the pathology of cerebral malaria. Using immunohistochemistry on post mortem brain samples, we demonstrated positive staining for vascular endothelial growth factor (VEGF) on iRBC. Confocal microscopy of cultured iRBC revealed accumulation of VEGF within the parasitophorous vacuole, expression of host VEGF-receptor 1 and activated VEGF-receptor 2 on the surface of iRBC, but no accumulation of VEGF receptors within the iRBC. Addition of VEGF to parasite cultures had a trophic effect on parasite growth and also partially rescued growth of drug treated parasites. Both these effects were abrogated when parasites were grown in serum-free medium, suggesting a requirement for soluble VEGF receptor. We conclude that P. falciparum iRBC can bind host VEGF-R on the erythrocyte membrane and accumulate host VEGF within the parasitophorous vacuole, which may have a trophic effect on parasite growth.     

A status report on the sequencing and annotation of the P. falciparum genome.

Almost 5 years ago, an international consortium of sequencing centers and funding agencies was formed to sequence the genome of the human malaria parasite Plasmodium falciparum. A novel chromosome by chromosome shotgun strategy was devised to sequence this very AT-rich genome. Two of the 14 chromosomes have been completed and the remaining chromosomes are in the final stages of gap closure. The consortium recently developed plans for the annotation and analysis of the complete genome sequence and its publication in 2002.