Enzymes that Counteract Nitrosative Stress Promote Fungal Virulence

Enzymes that protect cells from reactive oxygen species (superoxide dismutase, catalase, peroxidase) have well-established roles in mammalian biology and microbial pathogenesis. Two recently identified enzymes detoxify nitric oxide (NO)-related molecules; flavohemoglobin denitrosylase consumes NO, and S-nitrosoglutathione (GSNO) reductase metabolizes GSNO. Although both enzymes protect microorganisms from nitrosative challenge in vitro, their relevance has not been established in physiological contexts. Here we studied their biological functions in Cryptococcus neoformans, an established human fungal pathogen that replicates in macrophages and whose growth in vitro and in infected animals is controlled by NO bioactivity. We show that both flavohemoglobin denitrosylase and GSNO reductase contribute to C. neoformans pathogenesis. FHB1 and GNO1 mutations abolished NO- and GSNO-consuming activity, respectively. Growth of fhb1 mutant cells was inhibited by nitrosative challenge, whereas that of gno1 mutants was not. fhb1 mutants showed attenuated virulence in a murine model, and virulence was restored in iNOS(-/-) animals. Survival of the fhb1 mutant was also reduced in activated macrophages and restored to wild-type by inhibition of NOS activity. Combining mutations in flavohemoglobin and GSNO reductase, or flavohemoglobin and superoxide dismutase, further attenuated virulence. These studies illustrate that fungal pathogens elaborate enzymatic defenses against nitrosative stress mounted by the host.