Characterisation of cDNA clones for hypoxanthine-guanine phosphoribosyltransferase from the human malarial parasite, Plasmodium falciparum: comparisons to the mammalian gene and protein.

The isolation of cDNA clones for hypoxanthine-guanine phosphoribosyltransferase (HPRT) from the human malarial parasite, Plasmodium falciparum, is described. Northern analysis indicates that P. falciparum HPRT mRNA is the same size as that coding for mammalian HPRT. The predicted amino acid sequence of the P. falciparum HPRT protein shows extensive homology to the mammalian enzyme. Homology between the two proteins occurs in distinct blocks and a putative catalytic binding domain in the centre of the protein is also conserved. Five out of the seven characterised mammalian HPRT missense mutations map to regions which are conserved in the P. falciparum protein.     

Drug-resistant genotypes and multi-clonality in Plasmodium falciparum analysed by direct genome sequencing from peripheral blood of malaria patients

Naturally acquired blood-stage infections of the malaria parasite Plasmodium falciparum typically harbour multiple haploid clones. The apparent number of clones observed in any single infection depends on the diversity of the polymorphic markers used for the analysis, and the relative abundance of rare clones, which frequently fail to be detected among PCR products derived from numerically dominant clones. However, minority clones are of clinical interest as they may harbour genes conferring drug resistance, leading to enhanced survival after treatment and the possibility of subsequent therapeutic failure. We deployed new generation sequencing to derive genome data for five non-propagated parasite isolates taken directly from 4 different patients treated for clinical malaria in a UK hospital. Analysis of depth of coverage and length of sequence intervals between paired reads identified both previously described and novel gene deletions and amplifications. Full-length sequence data was extracted for 6 loci considered to be under selection by antimalarial drugs, and both known and previously unknown amino acid substitutions were identified. Full mitochondrial genomes were extracted from the sequencing data for each isolate, and these are compared against a panel of polymorphic sites derived from published or unpublished but publicly available data. Finally, genome-wide analysis of clone multiplicity was performed, and the number of infecting parasite clones estimated for each isolate. Each patient harboured at least 3 clones of P. falciparum by this analysis, consistent with results obtained with conventional PCR analysis of polymorphic merozoite antigen loci. We conclude that genome sequencing of peripheral blood P. falciparum taken directly from malaria patients provides high quality data useful for drug resistance studies, genomic structural analyses and population genetics, and also robustly represents clonal multiplicity.     

Recombinant Plasmodium falciparum NADP-dependent isocitrate dehydrogenase is active and harbours a unique 26 amino acid tail

During infection, Plasmodium spp. require reducing equivalents such as NADPH to support the function of specific enzymes in overcoming oxidative stress. The catalysis of isocitrate by the NADP-dependent isocitrate dehydrogenase of Plasmodium falciparum (pfICDH) generates NADPH and is thus crucial for the parasite's survival and pathogenecity. In this study, pfICDH was cloned from a clinical isolate of P. falciparum. This was facilitated by designing primers based on the P. falciparum genome sequence resource PlasmoDB. DNA sequence of the cloned gene revealed an ORF that encodes a protein of 468 aa. Furthermore, after expression in Esherichia coli BL21, enzyme assays of cell-free extracts confirmed the overexpression and function of pfICDH. Further, pfICDH purified by affinity chromatography retained its enzyme activity. Substitution of NADP for NAD, or the use of EDTA, in enzyme assays abolished pfICDH activity. ATP and chloroquine, as well as cupric and argentic ions, inhibited pfICDH activity. Phylogenetic analysis revealed high primary structure homology (45-97%) among genes coding for eukaryal NADP-dependent ICDH, and the occurrence of three subfamilies of ICDH genes. Interestingly, there were significant sequence dissimilarities between pfICDH and its mammalian or bacterial homologs, particularly at the N- and C-termini. Confirming the functionality of the cloned pfICDH, and asserting its distance from the human homolog by molecular definitions, are important prerequisites for promoting this gene as a drug target screen.     

Discovery and biochemical characterization of Plasmodium thioredoxin reductase inhibitors from an antimalarial set

Plasmodium falciparum is the most prevalent and deadly species of the human malaria parasites, and thioredoxin reductase (TrxR) is an enzyme involved in the redox response to oxidative stress. Essential for P. falciparum survival, the enzyme has been highlighted as a promising target for novel antimalarial drugs. Here we report the discovery and characterization of seven molecules from an antimalarial set of 13533 compounds through single-target TrxR biochemical screens. We have produced high-purity, full-length, recombinant native enzyme from four Plasmodium species, and thioredoxin substrates from P. falciparum and Rattus norvegicus. The enzymes were screened using a unique, high-throughput, in vitro native substrate assay, and we have observed selectivity between the Plasmodium species and the mammalian form of the enzyme. This has indicated differences in their biomolecular profiles and has provided valuable insights into the biochemical mechanisms of action of compounds with proven antimalarial activity.     

Amplification of a Cryptosporidium parvum gene fragment encoding thymidylate synthase.

Currently, there is no effective therapy for cryptosporidiosis and it is unclear why antifolate drugs which are effective treatments for infections caused by closely related parasites are not also effective against Cryptosporidium parvum. In protozoa, the target of these drugs, dihydrofolate reductase (DHFR), exists as a bifunctional enzyme also manifesting thymidylate synthase (TS) activity and is encoded by a fused DHFR-TS gene. In order to prepare a probe to isolate the C. parvum DHFR-TS gene we have used degenerate oligonucleotides whose sequences are based on strongly conserved regions of TS protein sequence to prime the polymerase chain reaction (PCR) with C. parvum DNA. The PCR amplified a 375-bp DNA fragment which was cloned and sequenced; the deduced amino acid sequence had significant identity with known TS sequences, including strict conservation of all phylogenetically invariant TS amino acid residues. The cloned PCR fragment was used as a probe to isolate a number of overlapping clones from a C. parvum genomic library which were definitively shown to be of cryptosporidial origin by genomic Southern and molecular karyotype analyses. The deduced protein sequence of C. parvum TS was most similar to the bifunctional TS enzymes of Plasmodium chabaudi and Plasmodium falciparum.     

Characterisation of carbonic anhydrase in Plasmodium falciparum

Here we report the existence, purification and characterisation of carbonic anhydrase in Plasmodium falciparum. The infected red cells contained carbonic anhydrase approximately 2 times higher than those of normal red cells. The three developmental forms of the asexual stages, ring, trophozoite and schizont were isolated from their host red cells and found to have stage-dependent activity of the carbonic anhydrase. The enzyme was purified to homogeneity from the crude extract of P. falciparum using multiple steps of fast liquid chromatographic techniques. It had a Mr of 32 kDa and was active in a monomeric form. The human red cell enzyme was also purified for comparison with the parasite enzyme. The parasite enzyme activity was sensitive to well-known sulfonamide-based inhibitors of both bacterial and mammalian enzymes, sulfanilamide and acetazolamide. The kinetic properties and the amino terminal sequences of the purified enzymes from the parasite and host red cell were found to be different, indicating that the purified protein most likely exhibited the P. falciparum carbonic anhydrase activity. In addition, the enzyme inhibitors had antimalarial effect against in vitro growth of P. falciparum. Moreover, the vital contribution of the carbonic anhydrase to the parasite survival makes the enzyme an attractive target for therapeutic evaluation.     

Fatty acid acylation regulates trafficking of the unusual Plasmodium falciparum calpain to the nucleolus

The Plasmodium falciparum genome encodes a single calpain. By generating P. falciparum clones expressing C-terminally tagged calpain, we localized this protein to the nucleolus. Pf_calpain possesses an unusual and long N-terminal domain in which we identified three subregions that are highly conserved among Plasmodium species. Two have putative targeting signals: a myristoylation motif and a nuclear localization sequence. We assessed their functionality. Our data show that the nuclear localization sequence is an active nuclear import motif that contains an embedded signal conferring nucleolar localization on various chimeras. The N-terminus is myristoylated at Gly2 and palmitoylated at Cys3 and Cys22. Palmitoylation status has an important role in dictating P. falciparum calpain localization. The targeting signals function in mammalian cells as well as in the parasite. P. falciparum calpain is a unique nucleolar protein with an interesting mechanism of targeting.     

Plasmodium falciparum: expression of the adenine phosphoribosyltransferase gene in mouse L cells

Genomic libraries of Plasmodium falciparum were constructed in the pBR322 plasmid. Using the DNA-mediated gene transfer technique, the genomic libraries were introduced into tissue-cultured mouse cells lacking the enzyme adenine phosphoribosyltransferase. Following selection for the adenine phosphoribosyltransferse phenotype, several colonies were isolated. All clones were shown to possess adenine phosphoribosyltransferase activity and pBR322 sequences. In addition, the Km value of adenine phosphoribosyltransferase (for adenine) from a transformant was found to be identical to that from P. falciparum. These results indicate that the adenine phosphoribosyltransferase gene of P. falciparum was successfully cloned and expressed in a mammalian system.     

Glucose-6-phosphate metabolism in Plasmodium falciparum

Malaria is still one of the most threatening diseases worldwide. The high drug resistance rates of malarial parasites make its eradication difficult and furthermore necessitate the development of new antimalarial drugs. Plasmodium falciparum is responsible for severe malaria and therefore of special interest with regard to drug development. Plasmodium parasites are highly dependent on glucose and very sensitive to oxidative stress; two observations that drew interest to the pentose phosphate pathway (PPP) with its key enzyme glucose-6-phosphate dehydrogenase (G6PD). A central position of the PPP for malaria parasites is supported by the fact that human G6PD deficiency protects to a certain degree from malaria infections. Plasmodium parasites and the human host possess a complete PPP, both of which seem to be important for the parasites. Interestingly, there are major differences between parasite and human G6PD, making the enzyme of Plasmodium a promising target for antimalarial drug design. This review gives an overview of the current state of research on glucose-6-phosphate metabolism in P. falciparum and its impact on malaria infections. Moreover, the unique characteristics of the enzyme G6PD in P. falciparum are discussed, upon which its current status as promising target for drug development is based.     

Origins of sequence diversity in the malaria vaccine candidate merozoite surface protein-2 (MSP-2) in Amazonian isolates of Plasmodium falciparum

The recent evolution of Plasmodium falciparum is at odds with the extensive polymorphism found in most genes coding for antigens. Here, we examined the patterns and putative mechanisms of sequence diversification in the merozoite surface protein-2 (MSP-2), a major malarial repetitive surface antigen. We compared the msp-2 gene sequences from closely related clones derived from sympatric parasite isolates from Brazilian Amazonia and used microsatellite typing to examine, in these same clones, the haplotype background of chromosome 2, where msp-2 is located. We found examples of msp-2 sequence rearrangements putatively created by nonreciprocal recombinational events, such as replication slippage and gene conversion, while maintaining the chromosome haplotype. We conclude that these nonreciprocal recombination events may represent a major source of antigenic diversity in MSP-2 in P. falciparum populations with low rates of classical meiotic recombination.     

Triclosan offers protection against blood stages of malaria by inhibiting enoyl-ACP reductase of Plasmodium falciparum

The antimicrobial biocide triclosan [5-chloro-2-(2,4-dichlorophenoxy)phenol] potently inhibits the growth of Plasmodium falciparum in vitro and, in a mouse model, Plasmodium berghei in vivo. Inhibition of [14C]acetate and [14C]malonyl-CoA incorporation into fatty acids in vivo and in vitro, respectively, by triclosan implicate FabI as its target. Here we demonstrate that the enoyl-ACP reductase purified from P. falciparum is triclosan sensitive. Also, we present the evidence for the existence of FabI gene in P. falciparum. We establish the existence of the de novo fatty acid biosynthetic pathway in this parasite, and identify a key enzyme of this pathway for the development of new antimalarials.     

Gene encoding a deubiquitinating enzyme is mutated in artesunate- and chloroquine-resistant rodent malaria parasites

Artemisinin- and artesunate-resistant Plasmodium chabaudi mutants, AS-ART and AS-ATN, were previously selected from chloroquine-resistant clones AS-30CQ and AS-15CQ respectively. Now, a genetic cross between AS-ART and the artemisinin-sensitive clone AJ has been analysed by Linkage Group Selection. A genetic linkage group on chromosome 2 was selected under artemisinin treatment. Within this locus, we identified two different mutations in a gene encoding a deubiquitinating enzyme. A distinct mutation occurred in each of the clones AS-30CQ and AS-ATN, relative to their respective progenitors in the AS lineage. The mutations occurred independently in different clones under drug selection with chloroquine (high concentration) or artesunate. Each mutation maps to a critical residue in a homologous human deubiquitinating protein structure. Although one mutation could theoretically account for the resistance of AS-ATN to artemisinin derivates, the other cannot account solely for the resistance of AS-ART, relative to the responses of its sensitive progenitor AS-30CQ. Two lines of Plasmodium falciparum with decreased susceptibility to artemisinin were also selected. Their drug-response phenotype was not genetically stable. No mutations in the UBP-1 gene encoding the P. falciparum orthologue of the deubiquitinating enzyme were observed. The possible significance of these mutations in parasite responses to chloroquine or artemisinin is discussed.     

Overproduction of Arabidopsis thaliana FeSOD confers oxidative stress tolerance to transgenic maize.

Transgenic maize (Zea mays L.) plants have been generated by particle gun bombardment that overproduce an Arabidopsis thaliana iron superoxide dismutase (FeSOD). To target this enzyme into chloroplasts, the mature Fesod coding sequence was fused to a chloroplast transit peptide from a pea ribulose-1,5-bisphosphate carboxylase gene. Expression of the chimeric gene was driven by the CaMV 35S promoter. Growth characteristics and in vitro oxidative stress tolerance of transgenic lines grown in control and chilling temperatures were evaluated. The transgenic line with the highest transgenic FeSOD activities had enhanced tolerance toward methyl viologen and had increased growth rates.     

Crystals of peptide deformylase from Plasmodium falciparum reveal critical characteristics of the active site for drug design

Peptide deformylase catalyzes the deformylation reaction of the amino terminal fMet residue of newly synthesized proteins in bacteria, and most likely in Plasmodium falciparum, and has therefore been identified as a potential antibacterial and antimalarial drug target. The structure of P. falciparum peptide deformylase, determined at 2.8 A resolution with ten subunits per asymmetric unit, is similar to the bacterial enzyme with the residues involved in catalysis, the position of the bound metal ion, and a catalytically important water structurally conserved between the two enzymes. However, critical differences in the substrate binding region explain the poor affinity of E. coli deformylase inhibitors and substrates toward the Plasmodium enzyme. The Plasmodium structure serves as a guide for designing novel antimalarials.     

Human T cells recognize polymorphic and non-polymorphic regions of the Plasmodium falciparum circumsporozoite protein.

In order to characterize T cell epitopes in the Plasmodium falciparum circumsporozoite (CS) protein sequence, we isolated T cell clones, from non-immune donors, which reacted with synthetic peptides corresponding to two predicted CS protein T cell epitopes. Peptide CS.T3 (corresponding to a non-polymorphic region of the CS protein, residues 378-398) was recognized in association with either DR2 or DRw9 restriction elements. T cell clones recognizing CS.T3 also reacted with the sporozoite-derived CS protein. Peptide CS.T2 corresponds to a polymorphic region (residues 325-341) of the CS protein. Unlike the CS.T3-specific clones, the CS.T2-specific clones did not recognize the CS protein. Since the CS.T2 peptide includes residues which are polymorphic in different P. falciparum isolates, we investigated whether these residues were critical for recognition of the peptide. We show here that a single amino acid substitution at a position of the CS protein which shows genetic polymorphism affects recognition of the sequence by human T cells. The implications of these data for malaria vaccine development are discussed.     

The expression of Plasmodium falciparum bloodstage antigens in Escherichia coli

A library of cDNA clones expressing proteins of the asexual blood stages of a Papua New Guinean isolate of Plasmodium falciparum (isolate FCQ27/PNG (FC27] was constructed in the bacteriophage vector lambda gt11-Amp3. In an in situ colony immunoassay, human serum was used to identify colonies producing natural immunogens. Sera from donors of defined clinical status, or reactive to a defined subset of natural immunogens were used to identify clones of particular interest (for example, clones reacting with convalescent but not with acute serum or clones expressing the isolate specific S-antigen of FC27). Antisera raised by immunizing mice and rabbits with cloned antigens were used to characterize the P. falciparum proteins corresponding to the antigen-positive clones. Nucleotide sequence analysis of an antigen found on the surface of cells infected with ring stage parasites revealed an unusual sequence coding for eight, four and three amino acid repeats rich in acidic amino acids. The discussion centres on the use of cloned antigens as tools for the analysis of the host-protective immune response and selection of candidate vaccine molecules.     

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.     

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.     

Genetic analysis of polymorphic proteins of the human malaria parasite Plasmodium falciparum

Five polymorphic proteins, detected by two-dimensional electrophoresis, were analysed in the parents and progeny of a cross between two clones of the malaria parasite Plasmodium falciparum. The information obtained showed that different forms of each protein were determined by allelic variants of each respective gene. One protein was identified as the parasite enzyme adenosine deaminase. Recombinant parasites were produced at a higher than expected frequency.