Molecular basis of Toxoplasma gondii atovaquone resistance modeled in Saccharomyces cerevisiae

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0 d ondii is a widespread disease affecting primarily immunocomromised and pregnant individuals [1]. Atovaquone is a recently ntroduced anti-malarial compound with broad spectrum activty against various apicomplexan parasites [2–5] including T. ondii [6]. Approved by the FDA in 1995, this drug is a potent nd specific inhibitor of the cytochrome bc1 complex [7], an ssential respiratory enzyme present in the inner mitochondrial embrane. Two recent studies on treatment of malaria during regnancy show that atovaquone is a remarkably well tolerated olecule, safe for both the mother and the fetus [8,9]. Unforunately, there is strong evidence that the targeted parasites are ble to spontaneously develop drug resistance by mutation of mino acid residues located in or near the atovaquone-binding ite on cytochrome b. Because the yeast bc1 complex is also trongly inhibited by atovaquone, we have previously develped Saccharomyces cerevisiae as a model to study cytochrome mutations conferring atovaquone resistance in Pneumocystis 10,11] and Plasmodium species [12]. In the present study, we ave successfully transferred two mutations associated with atoaquone resistance in T. gondii [13] into the yeast cytochrome to the one recently found in the crystal structure of the yeast bc1 complex with a hydroxy-benzoxythiazol bound at center P [14]. The hydroxyl group of the hydroxy-naphthoquinone binds via a hydrogen bond to the nitrogen of His-181 of the Rieske iron–sulfur protein. On the opposite side of the ring system the carbonyl group at position 4 of the quinone ring of atovaquone forms a water-mediated hydrogen bond with Glu-272 of cytochrome b. The bulk of the molecular interactions between atovaquone and cytochrome b are essentially hydrophobic with a network of aromatic and aliphatic side chains surrounding the inhibitor. There is a high score of cytochrome b sequence identity (about 70%) within the atovaquone binding pocket between S. cerevisiae and T. gondii (Fig. 1A). We have thus chosen the yeast to study how mutations can affect the drug efficacy. Both atovaquone-resistant mutations in the cytochrome b sequence previously identified in T. gondii were localized within this binding site [13]. These two mutations (M129L and I254L in T. gondii numbering, equivalent to M139L and I269L in the yeast numbering system) associated with resistance in T. gondii gene. This has allowed us to biochemically confirm the linkge of atovaquone resistance to the cytochrome b mutations and o predict at the molecular level the mechanism by which T. ondiimay counter the efficacy of this potential anti-toxoplasma rug. were transferred into the S. cerevisiae cytochrome b gene by the biolistic method [11]. Growth of the two mutated strains was monitored in a nonfermentable medium in order to study the impact of the mutations on the yeast cells’ respiration. The growth curves revealed t s n o v m The molecular target of atovaquone is now known to be he ubiquinol oxidation pocket at center P of the cytochrome