Resistance mutations reveal the atovaquone-binding domain of cytochrome b in malaria parasites

Atovaquone represents a class of antimicrobial agents with a broad-spectrum activity against various parasitic infections, including malaria, toxoplas mosis and Pneumocystis pneumonia. In malaria parasites, atovaquone inhibits mitochondrial electron transport at the level of the cytochrome bc(1) comp lex and collapses mitochondrial membrane potential. In addition, this drug is unique in being selectively toxic to parasite mitochondria without affec ting the host mitochondrial functions. A better understanding of the struct ural basis for the selective toxicity of atovaquone could help in designing drugs against infections caused by mitochondria-containing parasites. To t hat end, we derived nine independent atovaquone-resistant malaria parasite lines by suboptimal treatment of mice infected with Plasmodium yoelii; thes e mutants exhibited resistance to atovaquone-mediated collapse of mitochond rial membrane potential as well as inhibition of electron transport. The mu tants were also resistant to the synergistic effects of atovaquone/proguani l combination. Sequencing of the mitochondrially encoded cytochrome b gene placed these mutants into four categories, three with single amino acid cha nges and one with two adjacent amino acid changes. Of the 12 nucleotide cha nges seen in the nine independently derived mutants 11 replaced A:T basepai rs with G:C basepairs, possibly because of reactive oxygen species resultin g from atovaquone treatment. Visualization of the resistance-conferring ami no acid positions on the recently solved crystal structure of the vertebrat e cytochrome bc(1) complex revealed a discrete cavity in which subtle varia tions in hydrophobicity and volume of the amino acid side-chains may determ ine atovaquone-binding affinity, and thereby selective toxicity. These stru ctural insights may prove useful in designing agents that selectively affec t cytochrome bc(1) functions in a wide range of eukaryotic pathogens.