Multifactorial Role of Mitochondria in Echinocandin Tolerance Revealed by Transcriptome Analysis of Drug-Tolerant Cells

Fungal infections cause significant mortality and morbidity worldwide, and the limited existing antifungal reservoir is further weakened by the emergence of strains resistant to echinocandins, a first line of antifungal therapy. Candida glabrata is an opportunistic fungal pathogen that rapidly develops mutations in the echinocandin drug target β-1,3-glucan synthase (GS), which are associated with drug resistance and clinical failure. Although echinocandins are considered fungicidal in Candida sp., a subset of C. glabrata cells survive echinocandin exposure, forming a drug-tolerant cell reservoir, from which resistant mutations are thought to emerge. Despite their importance, the physiology of rare drug-tolerant cells is poorly understood. We used fluorescence-activated cell sorting to enrich for echinocandin-tolerant cells, followed by modified single-cell RNA sequencing to examine their transcriptional landscape. This analysis identified a transcriptional signature distinct from the stereotypical yeast environmental stress response and characterized by upregulation of pathways involved in chromosome structure and DNA topology and downregulation of oxidative stress responses, of which the latter was observed despite increased levels of reactive oxygen species. Further analyses implicated mitochondria in echinocandin tolerance, wherein inhibitors of mitochondrial complexes I and IV reduced echinocandin-mediated cell killing, but mutants lacking various mitochondrial components all showed an echinocandin hypotolerant phenotype. Finally, GS enzyme complexes purified from mitochondrial mutants exhibited normal in vitro inhibition kinetics, indicating that mitochondrial defects influence cell survival downstream of the drug-target interaction. Together, these results provide new insights into the C. glabrata response to echinocandins and reveal a multifactorial role of mitochondria in echinocandin tolerance.