Redefiningpleiotropic drug resistance in a pathogenic yeast: Pdr1 functions as a sensor of cellular stresses in Candida glabrata

Candida glabrata is a prominent opportunistic fungal pathogen of humans. The increasing incidence of C. glabrata infections is attributed to both innate and acquired resistance to antifungals. Previous studies suggest the transcription factor Pdr1 and several target genes encoding ABC transporters are critical elements of pleiotropic defense against azoles and other antifungals. This study utilizes Hermes transposon insertion profilingto investigate Pdr1-independent and Pdr1-dependent mechanisms that alter susceptibility to the frontline antifungal fluconazole.Several new genes were found to alter fluconazolesusceptibility independent of Pdr1 (CYB5, SSK1, SSK2, HOG1, TRP1). A bZIP transcription repressor of mitochondrial function (CIN5) positively regulated Pdr1 while hundreds of genes encoding mitochondrial proteins were confirmedas negative regulators of Pdr1. The antibiotic oligomycin activated Pdr1 and antagonized fluconazoleefficacylikely by interfering with mitochondrial processes in C. glabrata. Unexpectedly, disruption of many 60S ribosomal proteins also activated Pdr1, thus mimicking the effectsof the mRNA translation inhibitors. Cycloheximide failed to fully activate Pdr1 in a cycloheximide-resistant Rpl28-Q38E mutant. Similarly, fluconazolefailed to fully activate Pdr1 in a strain expressing a low-affinityvariant of Erg11. Fluconazole activated Pdr1 with very slow kinetics that correlated with the delayed onset of cellular stress. These findingsare inconsistent with the idea that Pdr1 directly senses xenobiotics and support an alternative hypothesis where Pdr1 senses cellular stresses that arise only after engagement of xenobiotics with their targets.