The pathogenic yeast Candida glabrata is intrinsically resilient to azoles and rapidly acquires resistance to these antifungals, in vitro and in vivo. In most cases azole-resistant C. glabrata clinical strains encode hyperactive CgPdr1 variants, however, resistant strains encoding wild-type CgPDR1 alleles have also been isolated, although remaining to be disclosed the underlying resistance mechanism. In this work we scrutinized the mechanisms underlying resistance to azoles of eight resistant clinical C. glabrata strains, identified along the course of epidemiological surveys undertaken in Portugal. Seven of the strains were found to encode CgPdr1 GOF variants (I392M, E555K, G558C and I803T) with the substitutions I392M and I803T being herein identified as hyper-activating mutations for the first time. While cells expressing the wild-type CgPDR1 allele required the mediator subunit Gal11A to enhance tolerance to fluconazole, this was dispensable for cells expressing the I803T variant clearly indicating that the CgPdr1 interactome is shaped by different GOF substitutions. Genomic and transcriptomic profiling of the sole azole-resistant C. glabrata isolate encoding a wild-type CgPDR1 allele (ISTB218) revealed that under fluconazole stress this strain over-expresses various genes described to provide protection against this antifungal, while also showing reduced expression of genes described to increase sensitivity to these drugs. The overall role in driving the azole-resistance phenotype of the ISTB218 C. glabrata isolate played by these changes in the transcriptome and genome of the ISTB218 isolate are discussed shedding light into mechanisms of resistance that go beyond the CgPdr1-signalling pathway and that may alone, or in combination, pave the way for the acquisition of resistance to azoles in vivo.

The relevance of C. glabrata as a human pathogen is linked with its poor susceptibility to azoles as well as its extreme genomic plasticity that allows the rapid acquisition of resistance. Extensive characterization of azole-resistant C. glabrata strains unveiled the central role of the transcriptional regulator CgPdr1 in the resistance phenotype, with many strains encoding hyperactive (or gain-of-function; GOF) CgPdr1 alleles. Large scale profiling of a collection of clinical C. glabrata isolates recovered in hospitals of the Lisbon area, in Portugal, led to the identification of 11 strains exhibiting resistance to fluconazole and voriconazole, while 2 were only resistant to fluconazole. Among these strains, 10 were found to encode alleles of the CgPDR1 gene harbouring multiple non-synonymous SNPs that were not found in the alleles encoded by susceptible strains, including K274Q, I392M and I803T not previously described as GOF mutations. The isolates encoding these alleles were found to over-express several CgPdr1 target genes including the azole efflux pump CgCDR1 sustaining the idea that these represent new gain-of-function CgPdr1 alleles. Only one of the identified azole-resistant strains was found to encode a CgPDR1 allele fully identical to the one encoded by susceptible strains. To better understand the resistance phenotype of this strain, its transcriptome was compared with the one of a susceptible strain and of strains encoding CgPdr1 GOF alleles. The results of this comparative transcriptomic analysis will be discussed shedding light into the different azole-resistance mechanisms evolved by C. glabrata, including those independent of CgPdr1 GOF strains.