Antisense oligonucleotides targeting malarial aldolase inhibit the asexual erythrocytic stages of Plasmodium falciparum
Introduction
Malaria is one of the most prevalent human infectious diseases and a leading cause of morbidity and mortality world-wide [1]. Despite the tremendous impact of malaria on the human population since antiquity, only a few effective anti-malarial drugs have been developed, several of which have become ineffective because of the continuing emergence and spread of drug resistance in the malarial parasites [2], [3], [4].
It is now necessary to develop new strategies in anti-malarial drug design. In this regard, antisense (AS) technology offers a great potential. It is currently possible to synthesize derivatives of oligonucleotides that are chemically stable in vitro and in vivo, that can sufficiently enter target cells and act on target genes, producing the desired pharmacological effects within favorable therapeutic indices [5], [6], [7], [8], [9], [10], [11], [12], [13], [14], [15], [16]. Synthetic AS oligonucleotides in the forms of phosphorothioate oligodeoxynucleotides (ODNs) or ribozymes have been shown to have efficacy against various cancers and infectious organisms including Plasmodium falciparum [5], [6], [7], [10], [17], [18], [19]. Importantly, unlike conventional drug design which requires determination of the structure of target proteins, antisense efficacy is governed by Watson–Crick base-pairing, making the design of antisense compounds much simpler and more feasible [5], [7], [9], [13].
Malarial glycolysis is a potential target of antisense drug strategy. It has been established that the energy needs of the blood-stage malaria parasite are met entirely by anaerobic glycolysis because the parasite lacks a functional tricarboxylic acid cycle [20], [21], [22], [23]. The fact that glycolysis is critical to blood-stage malaria is illustrated by the high levels of glucose consumption in P. falciparum-infected erythrocytes (IE) which may reach 100 times that of normal erythrocytes (E) [20]. Consistent with the glucose consumption by P. falciparum during blood-stage development, levels of ten of the 11 glycolytic enzymes are increased as much as 11–18-fold in the blood-stage parasites [20], [22], [23]. Moreover, biochemical and molecular studies have established that these malarial enzymes are different from their human equivalents in their molecular structures, electrophoretic mobility and kinetics [20], [22], [23], [24], [25], [26], [27], [28], [29], [30], [31], [32].
The genes for malarial fructose biphosphate aldolase (aldolase) have been cloned in P. falciparum and Plasmodium berghei. In P. falciparum, the gene is single-copy and has two exons, the first encoding only the initial methionine residue and the second exon encoding the rest of the protein [25]. The protein has 60% sequence homology with known vertebrate aldolases, including human [25]. Northern blot analyses indicate high abundance of P. falciparum aldolase mRNA in blood-stage trophozoites and schizonts [26], [27]. Extremely high aldolase enzyme activity has been found in P. falciparum IE in culture [22]. Peak activity is at 32–36 h of the 48-h blood-stage life cycle, corresponding to the mature trophozoite stage [22]. The peak enzymatic activity coincides with the peak levels of aldolase mRNA and protein expression suggesting that this enzyme is transcriptionally regulated [22], [26], [27]. Sequence analysis shows that, although the gene for aldolase has been highly conserved throughout evolution, the sequence at and near the translation initiation site of the mRNA of P. falciparum aldolase differs from the host sequence [25]. Thus, malarial aldolase is physiologically important to the parasite and its gene is substantially different from all isozymes of human aldolase. We determined the antiparasitic effect of phosphorothioate AS-ODNs directed at several target sites on the gene of P. falciparum aldolase.
Section snippets
Culture of P. falciparum in human E
P. falciparum clonal derivative 3D7 of the strain NF54 was obtained from Dr L. Pologe (New York University Medical Center). P. falciparum IE, maintained in 5–10% hematocrit of human E in RPMI 1640 supplemented with AlbuMAX I (5 mg/ml; Gibco, Shady Grove, MD), 1% O2 and 5% CO2 humidified environment [33] were used in all experiments. Fresh human E (group O, Rh positive) blood cells, obtained from anonymous donors, were used as host cells. Parasites were synchronized as previously described [33].
Oligonucleotide primers for PCR
AS-ODNs inhibit the growth of blood-stage P. falciparum
To demonstrate the anti-malarial effect of AS-ODNs targeting the P. falciparum aldolase, we measured parasite growth in P. falciparum grown in human E in vitro in the presence or absence of specific ODNs. Synchronous ring-stage P. falciparum IE at 2% parasitemia were aliquoted into wells and incubated in the presence of various concentrations of ODNs. After 72 h of incubation, which corresponds to the late trophozoite/schizont stages of the subsequent schizogonic cycle, parasitemia was
Discussion
Our data show that the growth of asexual blood-stage P. falciparum can be inhibited when malarial glycolysis is inhibited using antisense ODNs. These ODNs targeted the gene for aldolase, the fourth enzyme of glycolytic pathway. The reduction in parasitemia is sequence specific and correlates with reduction in the abundance of enzyme specific mRNA and enzyme activity of aldolase within the parasites. Inhibition of glycolysis is confirmed by the concomitant reduction in total energy production by
Acknowledgements
This work was supported by NIH grants AI34064 and HC38655.
References (47)
Antisense oligonucleotides: toward clinical trials
Trends Biotechnol
(1996)- et al.
Molecular therapy for renal diseases
Am J Kidney Dis
(1996) - et al.
Inhibition of Plasmodium falciparum proliferation in vitro by ribozyme
J Biol Chem
(1997) - et al.
The enzymes of the glycolytic pathway in erythrocytes infected with Plasmodium falciparum malaria parasites
Blood
(1988) Malarial parasite hexokinase and hexokinase-dependent glutathione reduction in the Plasmodium falciparum-infected human erythrocyte
J Biol Chem
(1987)- et al.
Plasmodium falciparum aldolase: gene structure and localization
Mol Biochem Parasitol
(1990) - et al.
Molecular analysis of Plasmodium falciparum hexokinase
Mol Biochem Parasitol
(1992) - et al.
Expression and cellular localization of hexokinase during the bloodstage development of Plasmodium falciparum
Mol Biochem Parasitol
(1994) - et al.
Identification and purification of glucose phosphate isomerase of Plasmodium falciparum
Mol Biochem Parasitol
(1992) - et al.
Cloning of the triosephosphate isomerase gene of Plasmodium falciparum and expression in Escherichia coli
Mol Biochem Parasitol
(1993)
Glycolytic pathway of the human malaria parasite Plasmodium falciparum: primary sequence analysis of the gene encoding 3-phosphoglycerate kinase and chromosomal mapping studies
Gene
Plasmodium falciparum: identification and purification of the phosphoglycerate kinase of the malaria parasite
Exp Parasitol
Expression of Plasmodium falciparum lactate dehydrogenase in Escherichia coli
Mol Biochem Parasitol
Plasmodium falciparum S-adenosylhomocysteine hydrolase. cDNA identification, predicted protein sequence, and expression in Escherichia coli
J Biol Chem
A colorimetric serum glucose determination using hexokinase and glucose-6-phosphate dehydrogenase
Biochem Med
Single-step method of RNA isolation by acid guanidine thiocyanate–phenol–chloroform extraction
Anal Biochem
Regular initiation of translation of Plasmodium berghei aldolase-2 after pre-mRNA splicing
Mol Biochem Parasitol
Malaria, the submerged disease
J Am Med Assoc
The treatment of malaria
New Engl J Med
Malaria chemophrophylaxis for the traveler
New Engl J Med
Drug-resistant malaria in children and in travelers
Pediatr Clin North Am
Antisense oligonucleotides as therapeutic agents—is the bullet really magical?
Science
Antisense strategies in the treatment of leukemias
Semin Oncol
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