Candida glabrata Transcription Factor Rpn4 Mediates Fluconazole Resistance through Regulation of Ergosterol Biosynthesis and Plasma Membrane Permeability

The ability to acquire azole resistance is an emblematic trait of the fungal pathogen Candida glabrata. Understanding the molecular basis of azole resistance in this pathogen is crucial for designing more suitable therapeutic strategies. This study shows that the C. glabrata transcription factor (TF) CgRpn4 is a determinant of azole drug resistance. RNA sequencing during fluconazole exposure revealed that CgRpn4 regulates the expression of 212 genes, activating 80 genes and repressing, likely in an indirect fashion, 132 genes. Targets comprise several proteasome and ergosterol biosynthesis genes, including ERG1, ERG2, ERG3, and ERG11. The localization of CgRpn4 to the nucleus increases upon fluconazole stress. Consistent with a role in ergosterol and plasma membrane homeostasis, CgRpn4 is required for the maintenance of ergosterol levels upon fluconazole stress, which is associated with a role in the upkeep of cell permeability and decreased intracellular fluconazole accumulation. We provide evidence that CgRpn4 directly regulates ERG11 expression through the TTGCAAA binding motif, reinforcing the relevance of this regulatory network in azole resistance. In summary, CgRpn4 is a new regulator of the ergosterol biosynthesis pathway in C. glabrata, contributing to plasma membrane homeostasis and, thus, decreasing azole drug accumulation.