Oxy2 as a transcriptional activator gene for copper uptake in Cryptococcus neoformans

Cryptococcus neoformans is subject to oxidative attack by host immune cells; consequently, oxidant-resistant mechanisms may be important in pathogenesis. Mutations at the OXY2 locus confer decreased laccase and increased sensitivity to hyperbaric oxygen in the background of the oxyl mutation, but, alone, do not confer sensitivity to oxidants. Because metal deficiency can potentiate or ameliorate sensitivity to oxidants, and because the melanin-synthesizing laccase contains copper, we investigated copper acquisition in an oxy2 mutant. We found that its external Cu/Fe reductase activity was lower than that of wild type, and although copper deprivation induced the reductase in the wild type, it did not do so in oxy2. Oxy2 is sensitive to copper chelation but resistant to high copper, suggesting that copper transport is decreased. The strain expresses large amounts of alternate oxidase in response to Cu-chelation, perhaps in response to defective, Cu-deprived cytochrome oxidase, and is resistant to the oxidant, plumbagin, under this condition, perhaps due to the high alternate oxidase. These phenotypes are similar to those of the mac1- mutant of Saccharomyces cerevisiae and the melanin-deficient grisea mutant of Podospora anserina, in which homologous transcriptional activators for the reductase and copper transporter genes are mutated. They constitute physiologic evidence that oxy2 is mutated in a homologous copper-related transcriptional activator of C. neoformans.