Genome-wide analysis of experimentally evolved Candida auris reveals multiple novel mechanisms of multidrug-resistance

Abstract

Candida auris is globally recognized as an opportunistic fungal pathogen of high concern, due to its extensive multidrug-resistance (MDR). Still, molecular mechanisms of MDR are largely unexplored. This is the first account of genome wide evolution of MDR in C. auris obtained through serial in vitro exposure to azoles, polyenes and echinocandins. We show the stepwise accumulation of multiple novel mutations in genes known and unknown in antifungal drug resistance, albeit almost all new for C. auris. Echinocandin resistance evolved through a codon deletion in FKS1 accompanied by a substitution in FKS1 hot spot 3. Mutations in ERG3 and CIS2 further increased the echinocandin MIC. Decreased azole susceptibility was acquired through a gain of function mutation in transcription factor TAC1b yielding overexpression of the drug efflux pump Cdr1; a segmental duplication of chromosome 1 containing ERG11; and a whole chromosome 5 duplication, which contains TAC1b. The latter was associated with increased expression of ERG11, TAC1b and CDR2, but not CDR1. The simultaneous emergence of nonsense mutations in ERG3 and ERG11, presumably leading to the abrogation of ergosterol synthesis, was shown to decrease amphotericin B susceptibility, accompanied with fluconazole cross resistance. A mutation in MEC3, a gene mainly known for its role in DNA damage homeostasis, further increased the polyene MIC. Overall, this study shows the alarming potential and diversity for MDR development in C. auris, even in a clade until now not associated with MDR (clade II), hereby stressing its clinical importance and the urge for future research. ImportanceC. auris is a recently discovered human fungal pathogens and has shown an alarming potential for multi- and pan-resistance towards all classes of antifungals most commonly used in the clinic. Currently, C. auris has been globally recognized as a nosocomial pathogen of high concern due to this evolutionary potential. So far, this is the first study in which the stepwise progression of MDR in C. auris is monitored in vitro. Multiple novel mutations in known resistance genes and genes previously not or vaguely associated with drug resistance reveal rapid MDR evolution in a C. auris clade II isolate. Additionally, this study shows that in vitro experimental evolution can be a powerful tool to discover new drug resistance mechanisms, although it has its limitations.