Ergosterol-depleted clinical isolates of Nakaseomyces glabratus can develop multi-drug resistance without apparent fitness and virulence defects

Objectives: Nakaseomyces glabratus (formerly Candida glabrata) is a leading cause of invasive candidiasis and rapidly develops antifungal drug resistance during treatment. An increasing number of clinical isolates shows reduced susceptibility to echinocandins and azoles, leaving amphotericin B (AMB) as a last therapeutic option. Resistance of N. glabratus to this drug is rare and its underlying mechanisms are still not fully understood. Here, we describe two independent multidrug resistant (MDR) bloodstream isolates displaying resistance to AMB and anidulafungin (ANF) as well as a reduced susceptibility to azoles. Methods: Whole-genome sequencing and sterol profiling were performed on nine clinical N. glabratus isolates which were resistant to ANF and displayed resistance or low susceptibility to fluconazole (FLU) and AMB. The transcriptional response of reference strain CBS138 and an AMBR+ANFR isolate was analyzed by RNA-seq. Furthermore, PDR1 was deleted in the latter isolate to examine its influence on efflux pump gene expression. Additionally, fitness and virulence of the AMBR+ANFR isolate were examined in growth assays and a Galleria mellonella infection model. Results: Loss of function mutations in the genes ERG3 and ERG4 is linked to ergosterol depletion and AMB resistance. Ergosterol depletion also contributed to a Pdr1-mediated up-regulation of ERG and ABC transporter genes which was associated with low FLU susceptibility. The AMBR isolates displayed no fitness defects and one of them was fully virulent in a G. mellonella infection model. Conclusions: These findings demonstrate that ergosterol depletion in N. glabratus leads to AMB resistance without affecting fitness or virulence.