Characterization of cytochrome b from Toxoplasma gondii and Q(o) domain mutations as a mechanism of atovaquone-resistance

Atovaquone is active in vitro against the tachyzoites of Toxoplasma gondii at nanomolar concentrations and is used clinically to treat acute cases of human toxoplasmosis. In pursuit of the mechanism of action of atovaquone ag ainst T. gondii and to understand how resistance might arise, drug-resistan t mutants Evert generated and examined. The previously uncloned cytochrome b gene of T. gondii was cloned and sequenced from wild type and resistant s trains as this was a likely candidate for the target of the drug and thus a source of resistance. Mutations are present within the cytochrome b gene o f atovaquone-resistant parasites (M 129L and 1254L) and represent alteratio ns in two different regions of the ubiquinol-binding pocket (Q(o) domain) o f cytochrome b: suggesting that atovaquone interferes with electron transpo rt at the cytochrome be, complex in T. gondii. A structural model for how t his hydroxynaphthoquinone is binding within the Q(o) domain is presented. F urther analysis of the cytochrome b gene suggested that the protein may dif fer from other homologues by terminating within the mitochondrial membrane. Cytochrome b becomes the first complete mitochondrial gene and cognate pro tein to be described for T. gondii. (C) 2000 Elsevier Science B.V. All righ ts reserved.