Abstract

Azole antifungal drugs directly inhibit lanosterol 14-α-demethylase and indirectly affect the expression of metabolic, transmembrane transporter, and cell wall organization genes in fungal pathogens. It is not known how these indirect azole effects depend on dose, timing, and specific azole used, or how they influence host interactions. Candida glabrata (recently renamed Nakaseomyces glabratus) is the second leading cause of candidiasis, and clinical strains have high rates of intrinsic resistance to azoles. We investigated the early responses of reference strains BG2 and CBS138 to sub-inhibitory doses of fluconazole and voriconazole, and particularly, how these responses affect host-pathogen interactions. Cell wall profiling and transcriptomic data revealed highly similar responses for each strain to both azoles, including the upregulation of several virulence factors, such as yapsins. We also observed significant increases in CBS138 survival in macrophages and increased virulence in Galleria mellonella after voriconazole exposure. Using a combination of pharmacological inhibition of calcium ion channels and deletion strains, we determined that voriconazole-enhanced virulence requires a yapsin protease, YPS1, and is regulated via the calcineurin pathway and the cell wall integrity pathway, both of which regulate YPS1 expression. We also observed that voriconazole treatment significantly reduced the virulence of the bck1Δ strain in G. mellonella, suggesting that inhibitors of the cell wall integrity pathway might potentiate azole activity by improving susceptibility to host killing. Our study provides new insight into short-term azole adaptation in C. glabrata, and importantly demonstrates that sub-inhibitory azole exposure can induce virulence factors and alter fungal pathogenesis.