Plasmodium falciparum: Functional mitochondrial ADP/ATP transporter in Escherichia coli plasmic membrane as a tool for selective drug screening Vale ´rie Razakantoanina, Isabelle Florent, Ginette Jaureguiberry * Biologie Fonctionnelle des Protozoaires, USM504-EA3335, De ´partement Re ´gulations, De ´veloppement, Diversite ´ Mole ´ culaire, Muse ´um National d’Histoire Naturelle, 61 rue Buffon, 75005 Paris, France Received 3 May 2007; received in revised form 27 July 2007; accepted 31 July 2007 Available online 28 August 2007 Abstract Plasmodium falciparum mitochondrial ADP/ATP transporter or adenylate translocase (PfAdT) was previously characterised at the molecular level and intracellularly located by immuno-electromicroscopy. Inhibition of this transporter blocks parasite development in erythrocytes. In this study, PfAdT was expressed in C43 (DE3) Escherichia coli strain under isopropyl beta-D-thiogalacto-pyranoside (IPTG) induction to screen inhibitory molecules. PfAdT was integrated directly into the bacterial cytoplasmic membrane. Whereas IPTG-induced bacterial cells imported radioactively labelled ATP, non-induced cells did not. The transporter bound specifically ADP and ATP, but not AMP. IPTG-induced cells preloaded with labelled ATP exported ATP after exogenous addition of unlabelled ADP or ATP, indicating a counter exchange transport mechanism. Bongrekic acid and atractyloside, two well-known specific inhibitors of mitochondrial ADP/ATP transporter, were tested. This experimental model was evaluated using three Malagasy crude plants extracts which have shown antiplasmodial activity on in vitro parasite cultures. Ó 2007 Elsevier Inc. All rights reserved. Index Descriptors and Abbreviations: Malaria, Plasmodium falciparum; Rickettsia prowazekii; Arabidopsis thaliana; Brachylaena ramiflora; Phyllarthron bernierianum; Strychnos sp.; C43 (DE3) E. coli; Natural plant extracts; ADP/ATP carrier; Bongkrekic acid; Plants extracts; ADP, adenosine diphosphate; AMP, adenosine monophosphate; ATP, adenosine triphosphate; ATR, atractyloside; BA, bongkrekic acid; EDTA, ethylene diamine tetraacetate; IPTG, isopropyl beta-D-thiogalacto-pyranoside; PfAdT, P. falciparum adenylate translocase or ADP/ATP transporter; PB, phosphate buffer; SDS, sodium dodecyl sulphate; YT, yeast tryptone 1. Introduction Malaria is still the most serious and prevalent cause of morbidity and mortality in tropical and sub-tropical coun- tries, constituting a major health problem for individuals and communities and impairing economic development. At present, at least 300 million people are affected by malaria and about 1.5 million people die each year from this disease. Pregnant women and children are especially vulnerable to the disease and one child in 20 is killed by it before the age of five. Of the four species specific for humans Plasmodium falciparum is the most virulent one, developing drug resistance in many parts of the world and continuing to spread. To overcome this drug resis- tance, new preventive and curative antimalarial molecules need to be synthesised and new molecular therapeutic tar- gets characterised. Although P. falciparum is considered a homolactate fer- mentor and possesses an acristate mitochondrion, parasite mitochondrion plays an important role in ATP production (Ginsburg et al., 1986; Van Dooren et al., 2006). Initially, it was suggested that Plasmodium, like typhus Rickettsia, another obligate intracellular parasite (Winkler, 1976) may utilise an ADP/ATP exchange system to facilitate 0014-4894/$ - see front matter Ó 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.exppara.2007.07.015 * Corresponding author. Fax: +33 1 40 79 34 99. E-mail address: jauregui@mnhn.fr (G. Jaureguiberry). www.elsevier.com/locate/yexpr Available online at www.sciencedirect.com Experimental Parasitology 118 (2008) 181–187