Exploration of novel mechanisms of azole resistance in Candida auris

Abstract

Candida auris is a pathogenic yeast of particular concern because of its ability to cause nosocomial outbreaks of invasive candidiasis (IC) and to develop resistance to all current antifungal drug classes. Most C. auris clinical isolates are resistant to fluconazole, an azole drug that is used for the treatment of IC. Azole resistance may arise from diverse mechanisms, such as mutations of the target gene (ERG11) or upregulation of efflux pumps via gain of function mutations of the transcription factors TAC1 and/or MRR1. To explore novel mechanisms of azole resistance in C. auris, we applied an in vitro evolutionary protocol to induce azole resistance in a TAC1A/TAC1B/MRR1 triple-deletion strain. Azole-resistant isolates without ERG11 mutations were further analyzed. In addition to a whole chromosome aneuploidy of chromosome 5, amino acid substitutions were recovered in the transcription factor Upc2 (N592S, L499F), the ubiquitin ligase complex consisting of Ubr2 (P708T, H1275P) and Mub1 (Y765*), and the mitochondrial protein Mrs7 (D293H). Genetic introduction of these mutations in an azole-susceptible wild-type C. auris isolate of clade IV resulted in significantly decreased azole susceptibility. Real-time reverse transcription PCR analyses were performed to assess the impact of these mutations on the expression of genes involved in azole resistance, such as ERG11, the efflux pumps CDR1 and MDR1 or the transcription factor RPN4. In conclusion, this work provides further insights in the complex and multiple pathways of azole resistance of C. auris. Further analyses would be warranted to assess their respective role in azole resistance of clinical isolates.

Keywords: drug transporters; fluconazole; mitochondria; transcription factors.

Fig 1

Schematic representation of the in vitro evolution experiment. The tac1ab∆/mrr1∆ strain was grown in the absence of FLC (control) or with FLC at 4 µg/mL (group A), 8 µg/mL (group B), or 16 µg/mL (group C). The following days, 500 µL of the overnight culture was transferred to 5 mL of fresh RPMI medium without (control) or with FLC (groups A, B, and C). The FLC concentration was determined according to the overnight growth (see formula). The same experiment was repeated for 15 days until stable resistance was acquired. Single colonies of the FLC-resistant strains were isolated and selected for further analyses. Illustration created using BioRender.

Fig 2

Relative expression of targeted genes in the strains with UPC2 mutations. Results are expressed as fold-change compared with the wild-type IV.1 strain (A) or the tac1ab∆/mrr1∆ (JLY0045) strain (B). Bars represent means with standard deviations of three biological replicates. Statistically significant: *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, and ****P ≤ 0.0001; ns, not statistically significant.

Fig 3

Relative expression of targeted genes in the strains with UBR2/MUB1 mutations. Results are expressed as fold-change compared with the wild-type IV.1 strain (A) or the tac1ab∆/mrr1∆ (JLY0045) strain (B). Bars represent means with standard deviations of three biological replicates. Statistically significant: *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, and ****P ≤ 0.0001; ns, not statistically significant.

Fig 4

Relative expression of targeted genes in the strains with MRS7 mutation. Results are expressed as fold-change compared with the wild-type IV.1 strain (A) or the tac1ab∆/mrr1∆ (JLY0045) strain (B). Bars represent means with standard deviations of three biological replicates. Statistically significant: *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, and ****P ≤ 0.0001; ns, not statistically significant.