Cryptococcus neoformans Site-2 protease is required for virulence and survival in the presence of azole drugs

In the human fungal pathogen Cryptococcus neoformans, the SREBP orthologue Sre1 is important for adaptation and growth in nutrient-limiting host tissues. In this study, we characterize the C. neoformans serotype A Sre1 and its activating protease, Stp1. We demonstrate that Stp1 is a functionally conserved orthologue of the mammalian Site-2 protease and that Stp1 cleaves Sre1 within its predicted first transmembrane segment. Gene expression analysis revealed that Stp1 is required for both Sre1-dependent and Sre1-independent gene transcription, indicating that other substrates of Stp1 may exist. Using gas chromatography, we showed that Sre1 and Stp1 are required for both normoxic and hypoxic ergosterol biosynthesis, and therefore cells lacking SRE1 or STP1 are defective for growth in the presence of low levels of the ergosterol biosynthesis inhibitors, itraconazole and 25-thialanosterol. Importantly, our studies demonstrated fungicidal effects of itraconazole and 25-thialanosterol towards sre1Δ and stp1Δ cells, demonstrating that the Sre1 pathway is required for both growth and survival in the presence of sterol biosynthesis-inhibiting antifungal drugs. Given the need for fungicidal drugs, we propose that inhibitors of Stp1, Sre1, or other regulators of Sre1 function administered in combination with a sterol synthesis inhibitor could prove an effective anticryptococcal therapy.