Tài liệu Báo cáo khoa học: Transient silencing of Plasmodium falciparum bifunctional

Transient silencing of Plasmodium falciparum

bifunctional glucose-6-phosphate dehydrogenase)

6-phosphogluconolactonase

Almudena Crooke1,*, Amalia Diez1

, Philip J. Mason2,† and Jose´ M. Bautista1

1 Department of Biochemistry and Molecular Biology IV, Universidad Complutense de Madrid, Facultad de Veterinaria, Madrid, Spain

2 Haematology Department, Imperial College School of Medicine, Hammersmith Hospital, Du Cane Road, London, UK

Malaria is a major health hazard in tropical and sub￾tropical areas around the world. In Africa alone, every

year over a million children under the age of 5 years

die of malaria and around 300–500 million people are

infected by the parasite [1,2]. Added to this, the

appearance of parasites resistant to antimalarial drugs

is on the increase and it is not proving easy to develop

an efficient vaccine against Plasmodium falciparum.

There is thus a need for new therapeutic targets.

The sequencing of the P. falciparum genome [3–5]

has revealed a large amount of molecular information.

This information, coupled to microarray mRNA ana￾lysis [6,7] and specific expression proteomic analysis of

the parasite’s developmental stages [8], is allowing the

molecular exploration of new strategies to fight against

malaria.

Based on their equivalent functions in other organ￾isms, the search for genes thought to be essential for

Keywords

antisense RNA; dsRNA; gene silencing;

glucose-6-phosphate dehydrogenase;

malaria; Plasmodium falciparum

Correspondence

J.M. Bautista, Departamento de Bioquı´mica

y Biologı´a Molecular IV, Universidad

Complutense de Madrid, Facultad de

Veterinaria, Ciudad Universitaria, 28040

Madrid, Spain

Fax: +34 91 3943824

Tel: +34 91 3943823

E-mail: [email protected]

*Present address

Department of Biochemistry and Molecular

Biology IV, Universidad Complutense de

Madrid, Escuela de O´ ptica, Madrid, Spain

†Present address

Division of Hematology, Department of

Internal Medicine, Washington University

School of Medicine, St Louis, USA

(Received 5 August 2005, revised 2 February

2006, accepted 10 February 2006)

doi:10.1111/j.1742-4658.2006.05174.x

The bifunctional enzyme glucose-6-phosphate dehydrogenase-6-phospho￾gluconolactonase (G6PD-6PGL) found in Plasmodium falciparum has

unique structural and functional characteristics restricted to this genus.

This study was designed to examine the effects of RNA-mediated PfG6PD￾6PGL gene silencing in cultures of P. falciparum on the expression of para￾site antioxidant defense genes at the transcription level. The highest degree

of G6PD-6PGL silencing achieved was 86% at the mRNA level, with a

recovery to almost normal levels within 24 h, indicating only transient

diminished expression of the PfG6PD-6PGL gene. PfG6PD-6PGL silencing

caused arrest of the trophozoite stage and enhanced gametocyte formation.

In addition, an immediate transcriptional response was shown by thiore￾doxin reductase suggesting that P. falciparum G6PD-6PGL plays a physio￾logical role in the specific response of the parasite to intracellullar oxidative

stress. P. falciparum transfection with an empty DNA vector also promoted

intracellular stress, as determined by mRNA up-regulation of antioxidant

genes. Collectively, our findings point to an important role for this enzyme

in the parasite’s infection cycle. The different characteristics of G6PD￾6PGL with respect to its homologue in the host make it an ideal target for

therapeutic strategies.

Abbreviations

CT, cycle threshold; FeSOD, iron superoxide dismutase; G6PD-6PGL, glucose-6-phosphate dehydrogenase-6-phosphogluconolactonase; GPx,

glutathione peroxidase; GR, glutathione reductase; PPP, pentose phosphate pathway; TrxR, thioredoxin reductase.

FEBS Journal 273 (2006) 1537–1546 ª 2006 The Authors Journal compilation ª 2006 FEBS 1537