Antisense oligonucleotides targeting malarial aldolase inhibit the asexual erythrocytic stages of Plasmodium falciparum

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Abstract

A major obstacle in the global effort to control malaria is the paucity of anti-malarial drugs. This is compounded by the continuing emergence and spread of resistance to old and new anti-malarial drugs in the malarial parasites. Here we describe the anti-malarial effect of phosphorothioate antisense (AS) oligodeoxynucleotides (ODNs) targeting the aldolase enzyme of Plasmodium falciparum, using the asexual blood stages of the parasite grown in vitro. The blood stages of P. falciparum depend almost entirely on the energy produced by their own glycolysis. Aldolase, the fourth enzyme of the glycolytic pathway, is highly upregulated during the malarial 48-h life cycle. We found that the mRNA of this enzyme can be inhibited, in a sequence specific manner, using AS-ODN to the splice sites on the pre-mRNA of malarial aldolase. At the enzyme level, both specific AS-ODNs for the splice sites, as well as for the translation inititation site on mature mRNA, can inhibit aldolase enzyme activity within the trophozoites of P. falciparum. Furthermore, this downregulation of the malarial aldolase results in a reduction in the production of ATP within the parasite. Finally, the treatment reduces parasitemia. In summary, AS-ODNs targeting the aldolase gene of P. falciparum can interfere with the blood-stage life cycle of this parasite in vitro by inhibiting the expression of the enzyme aldolase which results in decreased malarial glycolysis and energy production. Thus, we conclude that blockade of the expression of malarial glycolytic enzymes using specific AS-ODNs has the potential of a new anti-malarial strategy.

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)

  • K.E. Hicks et al.

    Glycolytic pathway of the human malaria parasite Plasmodium falciparum: primary sequence analysis of the gene encoding 3-phosphoglycerate kinase and chromosomal mapping studies

    Gene

    (1991)
  • M. Grall et al.

    Plasmodium falciparum: identification and purification of the phosphoglycerate kinase of the malaria parasite

    Exp Parasitol

    (1992)
  • D.J. Bzik et al.

    Expression of Plasmodium falciparum lactate dehydrogenase in Escherichia coli

    Mol Biochem Parasitol

    (1993)
  • K.A. Creedon et al.

    Plasmodium falciparum S-adenosylhomocysteine hydrolase. cDNA identification, predicted protein sequence, and expression in Escherichia coli

    J Biol Chem

    (1994)
  • J.J. Carroll et al.

    A colorimetric serum glucose determination using hexokinase and glucose-6-phosphate dehydrogenase

    Biochem Med

    (1970)
  • P. Chomczynski et al.

    Single-step method of RNA isolation by acid guanidine thiocyanate–phenol–chloroform extraction

    Anal Biochem

    (1987)
  • U. Certa

    Regular initiation of translation of Plasmodium berghei aldolase-2 after pre-mRNA splicing

    Mol Biochem Parasitol

    (1994)
  • P. Olliaaro et al.

    Malaria, the submerged disease

    J Am Med Assoc

    (1996)
  • N.J. White

    The treatment of malaria

    New Engl J Med

    (1996)
  • D.J. Wyler

    Malaria chemophrophylaxis for the traveler

    New Engl J Med

    (1993)
  • D.L. Longworth

    Drug-resistant malaria in children and in travelers

    Pediatr Clin North Am

    (1995)
  • C.A. Stein et al.

    Antisense oligonucleotides as therapeutic agents—is the bullet really magical?

    Science

    (1993)
  • B. Calbretta et al.

    Antisense strategies in the treatment of leukemias

    Semin Oncol

    (1996)
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