Proteolipid of vacuolar H(+)-ATPase of Plasmodium falciparum: cDNA cloning, gene organization and complementation of a yeast null mutant.

Vacuolar H(+)-ATPase (V-ATPase), an electrogenic proton pump, is highly expressed in Plasmodium falciparum, the human malaria parasite. Although V-ATPase-driven proton transport is involved in various physiological processes in the parasite, the overall features of the V-ATPase of P. falciparum, including the gene organization and biogenesis, are far less known. Here, we report cDNA cloning of proteolipid subunit c of P. falciparum, the smallest and most highly hydrophobic subunit of V-ATPase. RT-PCR analysis as well as Northern blotting indicated expression of the proteolipid gene in the parasite cells. cDNA, which encodes a complete reading frame comprising 165 amino acids, was obtained, and its deduced amino acid sequence exhibits 52 and 57% similarity to the yeast and human counterparts, respectively. Southern blot analysis suggested the presence of a single copy of the proteolipid gene, with 5 exons and 4 introns. Upon transfection of the cDNA into a yeast null mutant, the cells became able to grow at neutral pH, accompanied by vesicular accumulation of quinacrine. In contrast, a mutated proteolipid with replacement of glutamate residue 138 with glutamine did not lead to recovery of the growth ability or vesicular accumulation of quinacrine. These results indicated that the cDNA actually encodes the proteolipid of P. falciparum and that the proteolipid is functional in yeast.

Plasmodium falciparum serine-repeat antigen (SERA) forms a homodimer through disulfide bond.

Plasmodium falciparum serine-repeat antigen (SERA) is one potential blood-stage vaccine candidate and is expressed as a protomer that is subsequently processed into four fragments (P47, P50, P6, and P17). Although recent evidence shows that P50 exhibits chymotrypsin-like protease activity, the function of SERA is still largely unknown. Here, we found that apart from cathepsin L-like cysteine protease, P50 showed significant homology to silicatein-alpha and testin which were shown to bind to cellular components, suggesting that SERA may have similar function. Immunoprecipitation of schizont lysate and molecular assignment of its precipitate by mass spectrometry provided evidence that SERA forms a homodimer through disulfide bond. Moreover, analysis of the fate of SERA using cell-free system revealed that the kinetics of conversion of SERA dimer into monomer is faster than that of processing of SERA monomer into various fragments. These findings may contribute to elucidate a possible function of SERA other than a protease.

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.

Evidence that Plasmodium falciparum diacylglycerol acyltransferase is essential for intraerythrocytic proliferation.

In triacylglycerol (TAG)-accumulating organisms, the physiological roles of diacylglycerol acyltransferase (DGAT), a principal enzyme in the major biosynthetic pathway for TAG, appear to be diverse. Apicomplexan parasite, Plasmodium falciparum, shows unique features in TAG metabolism and trafficking during intraerythrocytic development, and unlike most eukaryotes, only one open reading frame (ORF) encoding a candidate DGAT could be found in its genome. However, whether this candidate ORF encodes P. falciparum DGAT and its physiological relevance have not been assessed. Here, we demonstrate that the ORF is transcribed as a approximately 3.6 kb single mRNA throughout intraerythrocytic development, markedly elevated at trophozoite, schizont, and segmented schizont, and indeed encodes a protein exhibiting DGAT activity. Further, we provide evidence that the parasite in which the ORF was disrupted via double crossover recombination cannot be enriched, implying a fundamental role of PfDGAT in intraerythrocytic proliferation.

Developmental-stage-specific triacylglycerol biosynthesis, degradation and trafficking as lipid bodies in Plasmodium falciparum-infected erythrocytes.

Triacylglycerol (TAG) serves as a major energy storage molecule in eukaryotes. In Plasmodium, however, this established function of TAG appears unlikely, despite detecting previously considerable amount of TAG associated with intraerythrocytic parasites, because plasmodial cells have very little capacity to oxidize fatty acids. Thus, it is plausible that TAG and its biosynthesis in Plasmodium have other functions. As a first step in understanding the biological significance of TAG and its biosynthesis to the intraerythrocytic proliferation of Plasmodium falciparum, we performed detailed characterization of TAG metabolism and trafficking in parasitized erythrocyte. Metabolic labeling using radiolabeled-oleic and palmitic acids in association with serum albumin, which have been shown to be among the serum essential factors for intraerythrocytic proliferation of P. falciparum, revealed that accumulation of TAG was strikingly pronounced from trophozoite to schizont, whereas TAG degradation became active from schizont to segmented schizont; the consequent products, free fatty acids, were released into the medium during schizont rupture and/or merozoite release. These results were further supported by visualization of lipid bodies through immunofluorescence and electron microscopy. At the schizont stages, there is some evidence that the lipid bodies are partly localized in the parasitophorous vacuole. Interestingly, the discrete formation and/or trafficking of lipid bodies are inhibited by brefeldin A and trifluoperazine. Inhibition by trifluoperazine hints at least that a de novo TAG biosynthetic pathway via phosphatidic acid contributes to lipid body formation. Indeed, biochemical analysis reveals a higher activity of acyl-CoA:diacylglycerol acyltransferase, the principal enzyme in the sn-glycerol-3-phosphate pathway for TAG synthesis, at trophozoite and schizont stages. Together, these results establish that TAG metabolism and trafficking in P. falciparum-infected erythrocyte occurs in a stage-specific manner during the intraerythrocytic cycle and we propose that these unique and dynamic cellular events participate during schizont rupture and/or merozoite release.

Human malaria parasite orotate phosphoribosyltransferase: functional expression, characterization of kinetic reaction mechanism and inhibition profile.

Plasmodium falciparum, the causative agent of the most lethal form of human malaria, relies on de novo pyrimidine biosynthesis. A gene encoding orotate phosphoribosyltransferase (OPRT), the fifth enzyme of the de novo pathway catalyzing formation of orotidine 5'-monophosphate (OMP) and pyrophosphate (PP(i)) from 5-phosphoribosyl-1-pyrophosphate (PRPP) and orotate, was identified from P. falciparum (pfOPRT). The deduced amino acid sequence for pfOPRT was compared with OPRTs from other organisms and found to be most similar to that of Escherichia coli. The catalytic residues and consensus sequences for substrate binding in the enzyme were conserved among other organisms. The pfOPRT was exceptional in that it contained a unique insertion of 20 amino acids and an amino-terminal extension of 66 amino acids, making the longest amino acid sequence (281 amino acids with a predicted molecular mass of 33kDa). The cDNA of the pfOPRT gene was cloned, sequenced and functionally expressed in soluble form. The recombinant pfOPRT was purified from the E. coli lysate by two steps, nickel metal-affinity and gel-filtration chromatography. From 1l E. coli culture, 1.2-1.5mg of pure pfOPRT was obtained. SDS-PAGE revealed that the pfOPRT had a molecular mass of 33kDa and analytical gel-filtration chromatography showed that the enzyme activity eluted at approximately 67kDa. Using dimethyl suberimidate to cross-link neighboring subunits of the pfOPRT, it was confirmed that the native enzyme exists in a dimeric form. The steady state kinetics of initial velocity and product inhibition studies indicate that the enzyme pfOPRT follows a random sequential kinetic mechanism. Compounds aimed at the pfOPRT nexus may act against the parasite through at least two mechanisms: by directly inhibiting the enzyme activity, or be processed to an inhibitor of thymidylate synthase. This study provides a working system with which to investigate new antimalarial agents targeted against P. falciparum OPRT.

Relative frequencies of polymorphisms of variation in Block 2 repeats and 5' recombinant types of Plasmodium falciparum msp1 alleles.

The mechanisms producing the genetic polymorphism at Plasmodium falciparum merozoite surface antigen-1 locus (pfmsp1) include the insertion and deletion of the different type of dimorphic Block 2 9-nucleotide repeat units as well as the intragenic recombination. To study relative occurrence frequencies of these two distinct mechanisms, we have developed a sensitive PCR strategy to identify both 5' recombinant types and the number of Block 2 repeats from the same sample. This method can specifically detect the target 5' recombinant type (Blocks 2-6) at the sensitivity of 1-4 copies of the pfmsp1. Applying the new method to field isolates from the Solomon Islands enabled us to identify six different 5' recombinant types and variation in Block 2 repeat number in three of them, thus distinguishing 10 different alleles. Distribution of these alleles in local three villages in the study area suggests that frequencies of variation in the number of Block 2 9-bp repeats and recombination events within Blocks 2-6 are mutually independent and the frequency of repeat variation is relatively high as compared to that of recombination events at the pfmsp1 locus in P. falciparum populations from the Solomon Islands.

Lipid metabolism in Plasmodium falciparum-infected erythrocytes: possible new targets for malaria chemotherapy.

The emergence and spread of drug-resistant parasites coupled with the absence of an effective vaccine makes malaria treatment more complicated, and thus the development of new antimalarial drugs is one of the urgent tasks in malaria research. This review highlights lipid metabolism in Plasmodium parasite cells, the study of which would lead to providing new targets for therapeutic intervention.

Differential localization of processed fragments of Plasmodium falciparum serine repeat antigen and further processing of its N-terminal 47 kDa fragment.

The serine repeat antigen (SERA) of Plasmodium falciparum is a blood stage malaria vaccine candidate. It has been shown that 120 kDa SERA was proteolytically processed into N-terminal 47 kDa fragment (P47), central 56 kDa fragment (P56) that was further converted to 50 kDa (P50), and C-terminal 18 kDa fragment (P18). Here, we have examined the processing of SERA and the localization of its processed fragments by using mouse antibodies directed against recombinant proteins corresponding to different domains of SERA. Western blot analysis showed that all the processing events occurred inside parasitized erythrocytes at the stage just prior to the schizont rupture, that P47 was further processed into two 25 kDa fragments and that the two fragments, which were linked to P18 through disulfide bonds, were associated with the merozoite. In contrast, P50 was completely shed into culture medium and absent from the merozoite. This observation was further supported by the results of indirect immunofluorescence assay. These results could account for the findings that antibodies against P47 were inhibitory to the parasite growth in vitro but those against P50 were not. Finally, we demonstrated that the further processing of P47 is allelic type-dependent. The results of the present study would help in vaccine designing based on SERA.

Serine repeat antigen (SERA5) is predominantly expressed among the SERA multigene family of Plasmodium falciparum, and the acquired antibody titers correlate with serum inhibition of the parasite growth.

The Plasmodium falciparum serine repeat antigen (SERA) is one of the blood stage malaria vaccine candidates. The malaria genome project has revealed that SERA is a member of the SERA multigene family consisting of eight SERA homologues clustered on chromosome 2 and one SERA homologue on chromosome 9. Northern blotting and real time quantitative reverse transcription-PCR with five independent parasite strains, including three allelic representative forms of the SERA gene, have shown that all of the SERA homologues are transcribed most actively at trophozoite and schizont stages and that SERA5 (SERA/SERP) is transcribed predominantly among the family. Polyclonal antibodies were raised against recombinant proteins representing the N-terminal portions of four significantly transcribed SERA homologues (SERA3 to -6) in the center of the cluster on chromosome 2. Using these antibodies, indirect immunofluorescence microscopy detected the expression of SERA3 to -6, with similar localization, in all trophozoite- and schizont-infected erythrocytes. We have examined 40 sera from Ugandan adults for their antibody reactivity and found that enzyme-linked immunosorbent assay titer against SERA5 N-terminal domain, but not against other SERA proteins, is positively correlated with the inhibition of in vitro parasite growth by individual sera. Our data confirm the usefulness of the N-terminal domain of SERA5 as a promising malaria candidate vaccine.

Characterization of proteases involved in the processing of Plasmodium falciparum serine repeat antigen (SERA).

The Plasmodium falciparum serine repeat antigen (SERA), a malaria vaccine candidate, is processed into several fragments (P73, P47, P56, P50, and P18) at the late schizont stage prior to schizont rupture in the erythrocytic cycle of the parasite. We have established an in vitro cell-free system using a baculovirus-expressed recombinant SERA (bvSERA) that mimics the SERA processing that occurs in parasitized erythrocytes. SERA processing was mediated by parasite-derived trans-acting proteases, but not an autocatalytic event. The processing activities appeared at late schizont stage. The proteases are membrane associated, correlating with the secretion and accumulation of SERA within the parasitophorous vacuole membrane (PVM). The activity responsible for the primary processing step of SERA to P47 and P73 was inhibited by serine protease inhibitor DFP. In contrast, the activity responsible for the conversion of P56 into P50 was inhibited by each of the cysteine protease inhibitors E-64, leupeptin and iodoacetoamide. Moreover, addition of DFP, E-64 or leupeptin to the cultures of schizont-stage parasites blocked schizont rupture and release of merozoites from PVM. These results indicate that SERA processing correlates to schizont rupture and the processing is mediated by at least three distinct proteases.

Plasmodium falciparum phospholipase C hydrolyzing sphingomyelin and lysocholinephospholipids is a possible target for malaria chemotherapy.

Sphingomyelinase (SMase) is one of the principal enzymes in sphingomyelin (SM) metabolism. Here, we identified a Plasmodium falciparum gene (PfNSM) encoding a 46-kD protein, the amino acid sequence of which is approximately 25% identical to that of bacteria SMases. Biochemical analyses of the recombinant protein GST-PfNSM, a fusion protein of the PfNSM product with glutathione-S-transferase, reveal that this enzyme retained similar characteristics in various aspects to SMase detected in P. falciparum-infected erythrocytes and isolated parasites. In addition, the recombinant protein retains hydrolyzing activity not only of SM but also of lysocholinephospholipids (LCPL) including lysophosphatidylcholine and lysoplatelet-activating factor, indicating that PfNSM encodes SM/LCPL-phospholipase C (PLC). Scyphostatin inhibited SM/LCPL-PLC activities of the PfNSM product as well as the intraerythrocytic proliferation of P. falciparum in a dose-dependent manner with ID(50) values for SM/LCPL-PLC activities and the parasite growth at 3-5 microM and approximately 7 microM, respectively. Morphological analysis demonstrated most severe impairment in the intraerythrocytic development with the addition of scyphostatin at trophozoite stage than at ring or schizont stages, suggesting its effect specifically on the stage progression from trophozoite to schizont, coinciding with the active transcription of PfNSM gene.