Increasing the fungicidal action of amphotericin B by inhibiting the nitric oxide-dependent tolerance pathway

Amphotericin B (AmB) induces oxidative and nitrosative stress, characterized by production of reactive oxygen and nitrogen species, in fungi. Yet, how these toxic species contribute to AmB-induced fungal cell death is unclear. We investigated the role of superoxide and nitric oxide radicals in AmB fungicidal activity in Saccharomyces cerevisiae, using a digital microfluidic platform, which enabled monitoring individual cells at a spatiotemporal resolution, and plating assays. The nitric oxide synthases inhibitor L-NAME was used to interfere with nitric oxide radical production. L-NAME increases and accelerates AmB-induced accumulation of superoxide radicals, membrane permeabilization and loss of proliferative capacity in S. cerevisiae. In contrast, the nitric oxide donor S-nitrosoglutathione inhibits AmB’s action. Hence, superoxide radicals are important for AmB’s fungicidal action, whereas nitric oxide radicals mediate tolerance towards AmB. Finally, also the human pathogens Candida albicans and Candida glabrata are more susceptible to AmB in the presence of L-NAME, pointing to the potential of AmB – L-NAME combination therapy to treat fungal infections.status: publishe