Molecular cloning and characterization of the Plasmodium falciparum cytidine triphosphate synthetase gene.

Using degenerate oligonucleotides derived from conserved amino acid regions of cytidine triphosphate synthetase, a fragment of the gene from the malarial parasite, Plasmodium falciparum, was isolated by polymerase chain reaction (PCR). This fragment was used as a probe in the isolation of genomic clones containing the entire pfCTP synthetase coding region (2580 bp). The gene encodes the largest CTP synthetase found in any organism to date due to the presence of two additional sequences which are part of the continuous open reading frame and are not introns as their presence in the mRNA was confirmed by reverse transcriptase-PCR. These features distinguish the parasite enzyme from that of the host making it an attractive target for structure based drug design.

Inhibition of Plasmodium falciparum proliferation in vitro by ribozymes.

Catalytic RNA (ribozymes) suppressed the growth of the human malarial parasite Plasmodium falciparum in vitro. The phosphorothioated hammerhead ribozymes targeted unique regions of the P. falciparum carbamoyl-phosphate synthetase II gene. The P. falciparum carbamoyl-phosphate synthetase II gene encodes the first and limiting enzyme in the pathway, and its mRNA transcript contains two large insert regions absent in other carbamoyl-phosphate synthetases, including that from humans. These inserts are ideal targets for nucleic acid therapy. Exogenous delivery of ribozymes to cultures reduced malarial viability up to 55% at 0.5 microM ribozyme concentrations, which is significantly greater than control levels (5-15% reduction), suggesting a sequence-specific inhibition. This inhibition was shown to be stage-specific, with optimal inhibitions being detected after 24 h, coincident with maximal production of the carbamoyl-phosphate synthetase enzyme in the course of the life cycle of the parasite. A decrease in total carbamoyl-phosphate synthetase activity was observed only in cultures treated with the ribozymes. The task of developing alternative therapeutic agents against malaria is urgent due to the evolution of drug-resistant strains of P. falciparum, the most virulent of all human malarial parasites. Another critical issue to be addressed is the possibility of eliminating or reducing any systemic toxicity to the host, which can potentially be provided by nucleic acid therapy. This work is the first reported assessment of the ability of ribozymes as antimalarials. Ribozyme inhibition assays can also aid in identifying important antimalarial loci for chemotherapy. The malarial parasite can, in turn, be a useful in vivo host to study the catalysis and function of new ribozyme designs.

Laboratory and field comparisons of adenosine influx in Plasmodium falciparum and Plasmodium vivax infected erythrocytes with genetic abnormalities from patients in Myanmar.

Influx of the purine nucleoside, adenosine, was assessed in erythrocytes from both normal subjects and from subjects with a range of genetically determined erythrocyte disorders from Myanmar. The latter included alpha-thalassemia major (Myanmar variant), beta-thalassemia major (Myanmar variant), beta-thalassemia trait, HbEE and HbAE erythrocytes and two variants of glucose-6-phosphate dehydrogenase (G6PDH) deficiency. Significant reductions (p < 0.01) of adenosine influx were observed in erythrocytes from individuals with alpha- and beta-thalassemia major and severe G6PDH deficiency. Abnormal erythrocytes infected with the malarial parasites, Plasmodium falciparum or Plasmodium vivax, demonstrated a reduction in adenosine transport which correlated with the proportion of abnormal erythrocytes present in the samples obtained. The effect of nitrobenzylthioinosine (NBMPR) on adenosine influx was explored in normal and abnormal erythrocytes. In all these cases, NBMPR completely inhibited the transport of adenosine. However, transport of adenosine into P. falciparum and P. vivax-infected normal erythrocytes and abnormal cells was only inhibited 50-60% by NBMPR. The combination of tubercidin and NBMPR completely blocked adenosine transport into both normal and abnormal erythrocytes infected with either P. falciparum or P. vivax.

Isolation, characterization and expression of the gene encoding cytidine triphosphate synthetase from Giardia intestinalis.

The cytidine triphosphate synthetase gene from Giardia intestinalis was cloned using a PCR-based strategy. A 519 bp PCR product was obtained from the amplification of genomic DNA using two oligonucleotides derived from the CTP synthetase amino acid consensus sequences DPYINVDPG and KTKPTQ. This product was used to probe restriction endonuclease digested genomic DNA and the respective plasmid mini-libraries. Two genomic clones were obtained one with a 3.6 kb HindIII DNA fragment, containing approximately three-quarters of the 5'-end of the synthetase gene and subsequently, a 5.8 kb PstI DNA fragment which contained the whole gene. The intronless gene has a 1863 bp open reading frame encoding 620 amino acids (M(r) of 68.3 kDa). A well conserved catalytic glutamine aminotransferase (GAT) domain was identified. In addition, three insert sequences were found which are not present in CTP synthetase from other species. Alignment and comparison of the deduced amino acid sequence relative to CTP synthetases from other species revealed a high degree of identity (34%) with a greater resemblance to prokaryotes than eukaryotes. The gene is located on chromosome 6 and the messenger RNA encoding it is estimated to be 1.9 kb. The coding region of G. intestinalis CTP synthetase was generated by PCR and subsequently cloned into the pQE30 vector for expression in E. coli. This construct yielded a soluble and enzymatically active recombinant protein which was purified by a Ni-NTA affinity column. The purified recombinant protein had a subunit molecular weight of 69.5 kDa and a native molecular weight of approximately 274 kDa. Kinetic studies of the partially purified recombinant G. intestinalis CTP synthetase gave apparent K(m) values of 0.1 mM and approximately 0.5 mM for the substrates UTP and L-glutamine respectively in accord with previously reported values for the native enzyme.

Properties of uracil phosphoribosyltransferase from Giardia intestinalis.

Incorporation of pyrimidine ribonucleotides in Giardia intestinalis occurs via uracil phosphoribosyltransferase (UPRTase). The enzyme was purified over 1000-fold to apparent homogeneity from parasite extracts, using Fast Protein Liquid Chromatography, namely Mono Q anion exchange, Mono P chromatofocusing and Superose 12 chromatography. The specific activity of the purified enzyme was 3100 nmol min-1 mg protein-1. The enzyme was found to be a dimer of mol. wt. 76,000. Kinetic analysis, including initial velocity and product inhibition studies, indicated that it obeyed a rapid-random equilibrium mechanism. GTP and dGTP caused a dramatic increase in the activity of the enzyme, though there was no effect on the Michaelis constants. All other nucleotides tested were without effect or were inhibitory. The effect of GTP is similar to that observed for UPRTase from E. coli but not from other eukaryotes.

Genetic red cell disorders and severity of falciparum malaria in Myanmar.

A hospital-based survey was undertaken to investigate the relationship between the incidence and severity of malaria infection and various red cell disorders in Myanmar. The mean parasitaemia levels of patients with alpha- or beta-thalassaemia trait or with severe glucose-6-phosphate dehydrogenase (G6PD) deficiency were lower than those of individuals with normal haemoglobin AA or with heterozygous haemoglobin E. The double genetic defect of thalassaemia trait and severe G6PD deficiency appeared to confer some degree of protection against malaria.

Salvage synthesis of purine nucleotides by Helicobacter pylori.

The incorporation of purine nucleotide precursors into Helicobacter pylori and the activities of enzymes involved in nucleotide salvage biosynthetic pathways were investigated by radioactive tracer analysis and nuclear magnetic resonance spectroscopy. The organism took up the nucleobases adenine, guanine and hypoxanthine, and the nucleosides adenosine, guanosine and deoxyadenosine. Any incorporation of deoxyguanosine by the cells was below the detection limits of the methods employed. The activities of adenine-, guanine- and hypoxanthine-phosphoribosyl transferases were established. The bacterium showed high levels of adenosine and guanosine nucleosidase activities and lesser activity for deoxyadenosine; no hydrolysis of deoxyguanosine was detected. Phosphorylase activities were not observed with any of the nucleosides. Phosphotransferase activities with similar rates were demonstrated for adenosine, guanosine and deoxyadenosine; and a weaker activity was detected for deoxyguanosine. No nucleoside kinase activities were observed with any of the nucleosides. The presence of adenylate kinase was established, but no guanylate kinase activity was observed. The study provided evidence for the presence in H. pylori of salvage pathways for the biosynthesis of purine nucleotides.

CTP synthetase and enzymes of pyrimidine ribonucleotide metabolism in Giardia intestinalis.

The presence of the enzyme, CTP synthetase was detected in G. intestinalis and appears to be the major route by which the parasite obtains its cytidine nucleotides, though a low phosphoribosyltransferase activity which directly converted cytosine to CMP, was also detected. The giardial CTP synthetase was substantially purified and appeared to be a dimer of molecular weight of approximately 260,000. The enzyme was activated by the purine ribonucleotide, GTP, as was uracil phosphoribosyltransferase (UPRTase), an earlier enzyme in the pyrimidine ribonucleotide pathway. Detection of other enzyme activities in the present study together with results from previous studies has allowed the delineation of the pathway by which G. intestinalis synthesizes its major pyrimidine ribonucleotides.

De novo synthesis of pyrimidine nucleotides by Helicobacter pylori.

The incorporation of pyrimidine nucleotide precursors into Helicobacter pylori and the activities of enzymes involved in their synthetic pathways were investigated by radioactive tracer analysis and 31P nuclear magnetic resonance spectroscopy. The bacterium was found to take up aspartate and bicarbonate and to incorporate carbon atoms from these precursors into its genomic DNA. Orotate, an intermediate of de novo pyrimidine biosynthesis, and uracil and uridine, precursors for pyrimidine pathways, were also incorporated by the micro-organism. Radiolabelled substrates were used to assess the activities of aspartate transcarbamoylase, orotate phosphoribosyltransferase, orotidylate decarboxylase, CTP synthetase, uracil phosphoribosyltransferase, thymidine kinase and deoxycytidine kinase in bacterial lysates. The study provided evidence for the presence in H. pylori of an operational de novo pathway, and a less active salvage pathway for the biosynthesis of pyrimidine nucleotides.

Adenosine analogues as antimetabolites against Plasmodium falciparum malaria.

Analogues of purine nucleosides and deoxynucleosides were tested for toxicity against the intraerythrocytic parasite Plasmodium falciparum in vitro culture. Sangivamycin (7-deaza-7-amido-adenosine) (IC37 of 0.3 microM), tubercidin (7-deaza-adenosine) (IC37 of 0.7 microM), 6-methylamino-deoxyadenosine (IC37 of 10 microM), 8-aza-2-amino-deoxy-adenosine (IC37 of 11 microM) and 2-chloro-adenosine (IC37 of 11 microM) were found to be the most toxic towards the parasite. Structure-activity analysis suggested that alteration of the purine ring at the 7 or 8 position significantly increased the toxicity of the compound against P. falciparum. Analysis by HPLC of parasite lysates which had been subjected to the cytotoxic compounds confirmed that alterations in the flux of the purine salvage pathways of the parasite had occurred. Comparison of the toxicity of these compounds against P. falciparum with the toxicity against a similar intraerythrocytic parasite, Babesia bovis, or human melanoma cell lines indicated a differential toxicity, in that many of the compounds toxic towards P. falciparum were relatively non-toxic towards human melanoma cell lines or B. bovis and vice versa. The mechanism of toxicity of the deoxyadenosine and adenosine analogues, whose normal metabolism involves transport, metabolism and incorporation into nucleic acids appears to vary significantly between P. falciparum, B. bovis and mammalian cells.

A cytidine triphosphate synthetase gene in Plasmodium falciparum.

The malarial parasite Plasmodium falciparum is dependent on de novo synthesis for its pyrimidine nucleotide requirements. However, the activity of the key enzyme in cytidine nucleotide synthesis, CTP synthetase (EC 6.3.4.2), has not been reported. We present evidence for a CTP synthetase gene in P. falciparum, having isolated a PCR product obtained using 2 primers derived from the CTP synthetase amino acid consensus sequences DPYINVDPG and GICLGMQ. The amplified DNA segment encodes an amino acid sequence with considerable homology to CTP synthetases from several other species including human and yeast.

Deoxynucleoside kinases of Giardia intestinalis.

Giardia intestinalis lacks the ability to synthesise deoxyribonucleotides de novo and must rely on salvage synthesis. Two separate kinases, specific for purines (deoxyadenosine and deoxyguanosine) and pyrimidines (thymidine and deoxycytidine), respectively, are responsible for the incorporation of deoxyribonucleosides. A substantial degree of purification was achieved for the purine deoxynucleoside kinase by the combination of Mono Q anion exchange chromatography, preparative gel electrophoresis and Superose 12 gel filtration. An overall recovery of 4%, with 186- and 174-fold purification, for deoxyguanosine kinase and deoxyadenosine kinase activities, respectively, was observed. The molecular weight was found to be approximately 80,000 by gel filtration. Only a partial purification of thymidine/deoxycytidine kinase was achieved. However, both pyrimidine activities remained associated throughout various purification procedures and appeared to be associated with a protein of 44 kDa.

Reduced transport of adenosine in erythrocytes from patients with beta-thalassaemia.

The transport of adenosine into blood from beta-thalassaemia subjects was measured to provide a background to the relationship between resistance of malaria infection and beta-thalassaemia. Adenosine transport was significantly reduced in the abnormal cells in the blood samples. As adenosine is one of the major purines salvaged by P. falciparum malaria, we suggest that the resistance to malaria in beta-thalassaemia subjects may be due to a nutrient deficiency in the abnormal red cells.

Arginine metabolism during culture of Giardia intestinalis.

The effect of arginine on the growth and metabolism of Giardia intestinalis trophozoites was determined. Supplementation of the normal growth medium (Diamond's TYI-S-33) with 5 or 10 mM arginine accelerated trophozoite growth over the first 2 days. There was a corresponding rapid utilisation of arginine, with none being detectable after this time. The decrease was associated with the appearance in the growth medium of 1 mol of ornithine and 2 mol of ammonia per mol of arginine utilised, the stoichiometry being consistent with the operation of the arginine dihydrolase pathway. Subsequently, there was a decrease in the ammonia concentration in the medium. Removal of arginine from the medium by pretreatment with arginase substantially decreased cell growth. In TYI-S-33 medium containing no added glucose, instead of the normal 50 mM glucose concentration, arginine supplementation also increased cell growth over the first 2 days, with concurrent stoichiometric production of ornithine and ammonia. However, in these conditions, the ammonia concentration remained elevated. This suggests that under normal conditions there is re-uptake of ammonia, which is glucose dependent. The observations confirm the operation of a functional arginine dihydrolase pathway in G. intestinalis. The concordance of cessation of rapid growth with the depletion of arginine, and the beneficial effect on growth of arginine supplementation suggests that arginine availability is a limiting factor during the initial stages of rapid growth. It would appear that arginine is a major potential energy source during the initial stages of giardial growth, and that supplementation of Diamond's TYI-S-33 medium with additional arginine may provide an improved in vitro culture medium.

Mitochondrial function in Babesia bovis.

A variety of anti-mitochondrial drugs that had previously been found to inhibit the growth of the malarial parasite Plasmodium falciparum were tested on Babesia bovis in vitro. Several of these drugs were found to be non-toxic towards B. bovis. However, those drugs that were found to inhibit babesial growth included compounds (shown in parentheses) that have the following putative mitochondrial targets in the parasite: ATP synthetase complex (rhodamine 123, oligomycin, Janus Green); ATP-ADP translocase (bongkrekic acid); electron transport (rotenone, n-heptyl-4-hydroxyquinoline-N-oxide (HQNO), antimycin A); ubiquinone (CoQ) function (BW58C, menoctone); protein synthesis (tetracycline); and the proton pump (CCCP). We have also investigated the effects of some of these drugs on pyrimidine biosynthesis de novo by following the incorporation of [14C]bicarbonate into pyrimidine nucleotides and into the pyrimidine moieties of nucleic acids. The ubiquinone analogues BW58C and menoctone inhibited this pathway in the nM-microM range of concentrations. Inhibitors of electron transport (antimycin A and oligomycin) and an uncoupler (CCCP) were also effective inhibitors of pyrimidine biosynthesis de novo. We conclude that B. bovis has a functional mitochondrion that contributes significantly to pyrimidine biosynthesis de novo and to the overall energy metabolism of the parasite.

Studies on phosphoribosylpyrophosphate synthetase from Giardia intestinalis.

A substantial degree of purification, up to 3200-fold, with recoveries of 8-11% of phosphoribosylpyrophosphate (P-Rib-PP) synthetase from Giardia intestinalis extracts was achieved by the high resolution techniques of anion exchange chromatography and chromatofocusing columns on a fast protein liquid chromatography system. A Mono P chromatofocusing column gave rise to an enzyme peak eluting from the column at pH 4.5, indicating that the enzyme has an isoelectric point (pI) at approximately this value. The molecular weight of the enzyme was found to be 150,000 from a Sephacryl S-200 column. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis of the purified enzyme gave a single protein band with a subunit molecular weight of 38,000, indicating that the enzyme existed as a tetramer. The properties of G. intestinalis P-Rib-PP synthetase in terms of pH optimum, isoelectric point, subunit structure, phosphate requirement, metal and nucleotide specificity, appear to be very similar to those of the enzyme from other sources.

A simple method for the purification of mitochondrial DNA from Plasmodium falciparum.

Mitochondrial DNA (mtDNA) from Plasmodium falciparum was isolated by conventional differential centrifugation in an SS34 rotor, a simpler method than CsC1 centrifugation of total DNA as employed by other workers. The nature of the sample was verified by sequencing a polymerase chain reaction (PCR) product obtained using oligonucleotide primers derived from known malarial mtDNA sequence.