Experimental Evolution Identifies Adaptive Aneuploidy as a Mechanism of Fluconazole Resistance in Candida auris

Abstract

Candida auris is a newly emerging fungal pathogen of humans and has attracted considerable attention from both the clinical and basic research communities. Clinical isolates of C. auris are often resistant to one or more antifungal agents. To explore how antifungal resistance develops, we performed experimental evolution assays using a fluconazole-susceptible isolate of C. auris (BJCA001). After a series of passages through medium containing increasing concentrations of fluconazole, fungal cells acquired resistance. By sequencing and comparing the genomes of the parental fluconazole-susceptible strain and 26 experimentally evolved strains of C. auris, we found that a portion of fluconazole-resistant strains carried one extra copy of chromosome V. In the absence of fluconazole, C. auris cells rapidly became susceptible and lost the extra copy of chromosome V. Genomic and transcriptome sequencing (RNA-Seq) analyses indicate that this chromosome carries a number of drug resistance-related genes, which were transcriptionally upregulated in the resistant, aneuploid strains. Moreover, missense mutations were identified in the genes TAC1B, RRP6, and SFT2 in all experimentally evolved strains. Our findings suggest that the gain of an extra copy of chromosome V is associated with the rapid acquisition of fluconazole resistance and may represent an important evolutionary mechanism of antifungal resistance in C. auris.

Keywords: Candida auris; aneuploidy; antifungal resistance; experimental evolution; fluconazole.

FIG 1

Schematics of the experimental evolution strategies of fluconazole resistance in C. auris. (A) Eight independent samples (containing 4 × 10⁵ cells) of C. auris BJCA001 were initially cultured in 0.2 ml RPMI 1640 medium (OD₆₀₀ ≈ 0.1) containing 32 mg/liter fluconazole (FLC) and grown at 35°C for 48 h. After two more passages under the same conditions, cells were inoculated and grown in YPD medium containing 64 mg/liter and then 128 mg/liter fluconazole for three passages each. Two and three samples failed to grow after treated with 32 mg/liter and 64 mg/liter fluconazole, respectively. Three isolates that grew in the medium containing 128 mg/liter fluconazole were then plated on YPD medium for 3 days of incubation at 37°C. Eighteen independent colonies (A1 to A18) with MICs of ≥32 mg/liter were picked and subjected to whole-genome sequencing (WGS). To strengthen the fluconazole-resistant feature, samples A1 to A8 were inoculated into RPMI 1640 medium containing 128 mg/liter fluconazole for four additional passages and then subjected to WGS (samples B1 to B8). (B) Induction of the loss of fluconazole resistance strain B1. Cells of the fluconazole-resistant strain B1 were inoculated and grown on YPD plates without fluconazole at 25°C for 1 month. Colonies were then subjected to MIC testing. (C) MICs of fluconazole for BJCA001 and strains A1 to A18, B1 to B8, and R1 to R3.

FIG 2

Copy-number variations (CNV) of C. auris after a series of passages in the fluconazole-containing medium. Fluconazole-susceptible strain BJCA001 served as a reference. Experimentally evolved strains A1 to A8 and B1 to B8 (Fig. 1) were tested. A comparative genomic analysis shows a copy number variation at scaffold 6 (chromosome V) using the B8441 genome assembly as a reference. The data for strains A9 to A18 are presented in Fig. S1. The x axis indicates scaffolds 1 to 10. The y axis shows the copy number of each scaffold. Each spot represents the sequence depth for a genomic segment of 500 bp across the chromosomes.

FIG 3

Genome-wide profile of differential gene expression between fluconazole-resistant strain B1 and fluconazole-susceptible strain BJCA001 and scaffold copy number variation for B1. Different scaffolds are shown outside the circle. The differential gene expression and copy number changes are shown in the middle and inner rings. Several genes that may be involved in drug resistance are shown between the outside and middle circles; genes in red and green are upregulated and downregulated, respectively, in B1 compared with BJCA001.

FIG 4

Growth curves of strain BJCA001, experimentally evolved fluconazole-resistant strains B1 to B8, and recovery strains R1 to R3 in YPD medium. Cells were cultured with an initial inoculum with an OD₆₀₀ of 0.2 at 30°C with shaking for 48 h. Three biological repeats were performed.

FIG 5

Differentially expressed global gene profiles between the fluconazole-resistant strain B1 and original strain BJCA001. (A) Box plot of distribution of gene expression values for each scaffold. (B) Differentially expressed genes (DEG) in strains B1 and BJCA001. (C) Upregulated and downregulated genes in strains B1 and BJCA001 for each scaffold. (D) Functional classification of DEG at scaffold 6. Genes were classified based on C. albicans gene annotation and ontology (http://www.candidagenome.org).