Erg251 has complex and pleiotropic effects on azole susceptibility, filamentation, and stress response phenotypes

Abstract

Ergosterol is essential for fungal cell membrane integrity and growth, and numerous antifungal drugs target ergosterol. Inactivation or modification of ergosterol biosynthetic genes can lead to changes in antifungal drug susceptibility, filamentation and stress response. Here, we found that the ergosterol biosynthesis gene ERG251 is a hotspot for point mutations during adaptation to antifungal drug stress within two distinct genetic backgrounds of Candida albicans. Heterozygous point mutations led to single allele dysfunction of ERG251 and resulted in azole tolerance in both genetic backgrounds. This is the first known example of point mutations causing azole tolerance in C. albicans. Importantly, single allele dysfunction of ERG251 in combination with recurrent chromosome aneuploidies resulted in bona fide azole resistance. Homozygous deletions of ERG251 caused increased fitness in low concentrations of fluconazole and decreased fitness in rich medium, especially at low initial cell density. Dysfunction of ERG251 resulted in transcriptional upregulation of the alternate sterol biosynthesis pathway and ZRT2, a Zinc transporter. Notably, we determined that overexpression of ZRT2 is sufficient to increase azole tolerance in C. albicans. Our combined transcriptional and phenotypic analyses revealed the pleiotropic effects of ERG251 on stress responses including cell wall, osmotic and oxidative stress. Interestingly, while loss of either allele of ERG251 resulted in similar antifungal drug responses, we observed functional divergence in filamentation regulation between the two alleles of ERG251 (ERG251-A and ERG251-B) with ERG251-A exhibiting a dominant role in the SC5314 genetic background. Finally, in a murine model of systemic infection, homozygous deletion of ERG251 resulted in decreased virulence while the heterozygous deletion mutants maintain their pathogenicity. Overall, this study provides extensive genetic, transcriptional and phenotypic analysis for the effects of ERG251 on drug susceptibility, fitness, filamentation and stress responses.

Fig. 1. The point mutation of ERG251 leads to the partial dysfunction of ERG251 causing acquisition of azole tolerance.

Liquid microbroth drug susceptibility assay. Fluconazole (FLC) resistance quantified as the MIC₅₀ at 24hr in increasing centrations of FLC (left) and FLC tolerance quantified as the Supra-MIC growth at 48hr (SMG, right) which is the average growth above the MIC₅₀ for: A. the wildtype SC5314 (ERG251/ERG251), engineered heterozygous ERG251 point mutations strains in the SC5314 background, and both heterozygous deletion mutants of ERG251 in the SC5314 background; and B. the wildtype SC5314 (ERG251/ERG251), ERG251 overexpression strain, both heterozygous deletion mutants of ERG251 and their corresponding complementation strains, an ERG251 heterozygous deletion in the P75063 background and wildtype P75063 (P75063-ERG251/ERG251) as a control. C. Rhodamine 6G efflux kinetics of two heterozygous deletion mutants and the homozygous deletion of ERG251 in SC5314 with SC5314 (ERG251/ERG251) as the control in YPAD (left) and YPAD+1µg/ml FLC (right). Plots indicate average fluorescence intensity changes of Rhodamine 6G (R6G) from three biology replicates over 90 min. D. 24hr MIC (left, µg/ml) and 48hr SMG (right, tolerance) in FLC with or without radicicol (Hsp90 inhibitor) treatment for two het deletion mutants of ERG251 with SC5314 (ERG251/ERG251) and a positive control strain known to be resistant to FLC as the controls. A&B&D. Each bar represents the average of three technical replicates of a single strain.

Fig 2.. Single allele dysfunction of ERG251 in combination with concurrent aneuploidy causes azole resistance.

A. Representative whole genome sequencing (WGS) data of the FLC-evolved strains 1.1/1.2, 2.1/2.2, and 3.1/3.2 that acquired heterozygous point mutations at ERG251 and Chr3 and Chr6 concurrent aneuploidy. B. WGS data of FLC-evolved strain 4.1 that had wild-type alleles of ERG251/ERG251 and Chr3 and Chr6 concurrent aneuploidy, plus two ERG251 heterozygous deletion mutants engineered in the Evolved 4.1 aneuploid background. A&B WGS data are plotted as the log2 ratio and converted to chromosome copy number (y-axis, 1–4 copies) as a function of chromosome position (x-axis, Chr1-ChrR). The baseline ploidy was determined by propidium iodide staining (S1 Table). Haplotypes relative to the reference genome SC5314 are indicated. C. 24hr MIC (left, µg/ml) and 48hr SMG (right, tolerance) in FLC for SC5314 (ERG251/ERG251), ERG251 heterozygous deletion mutant in the SC5314 background, FLC-evolved strain 4.1, and two ERG251 heterozygous deletion mutants engineered in the Evolved 4.1 aneuploid background (two independent transformations). Each bar represents the average of three technical replicates per strain. D. Rhodamine 6G efflux kinetics of ERG251 heterozygous deletion mutant in evolved strain 4.1 background with evolved strain 4.1 and SC5314 (ERG251/ERG251) as the controls in YPAD (left) and YPAD+1µg/ml FLC (right). Plots indicate fluorescence intensity changes of Rhodamine 6G (R6G) over 90 min.

Fig 3.. Homozygous deletion of ERG251 results in decreased fitness at low initial cell density and increased fitness in the presence of low concentrations of FLC (≤1µg/ml).

A. 48hr growth curve analysis of erg251∆/∆ started at three different initial cell densities (OD₆₀₀=0.001, 0.005, or 0.01) with ERG251/ERG251 (SC5314, OD₆₀₀=0.001) as the control. B. Carrying capacity (K) and doubling time (Td, hrs) determined from growth curve analysis in Fig 2A. C&D. X-Y growth curve assay of (C) erg251∆/∆ and (D) ERG251/ERG251 in the presence of increasing concentrations of FLC ( X-axis, 0–256 µg/ml, 2-fold dilutions) and/or increasing concentrations of farnesol (FAR) (Y-axis, 0–1000 µM, 2-fold dilutions). Growth was estimated with the area under the curve (AUC heatmap) of the 48hr growth curve. E&F. Cell viability of (E) erg251∆/∆ and (F) ERG251/ERG251 after 48 hr exposure to FLC or/and FAR. Cells from Fig 3B were plated on YPAD agar and imaged after 24hr incubation. G. Relative fitness calculated from head-to-head competitive assay for erg251∆/ERG251, ERG251/erg251∆, erg251∆/∆, erg251∆/∆+ERG251-A, and erg251∆/∆+ERG251-B compared to the fluorescent control strain (ERG251/ERG251). B&G: Values are mean ± SEM calculated from three technical replicates. Data were assessed for normality by Shapiro-Wilk, and significant differences between the ERG251/ERG251 and mutants were calculated using two-way ANOVA with Dunnett’s multiple comparisons test. ****p<0.0001, **p<0.01. A-G: At least three biological replicates were performed.

Fig 4.. Homozygous deletion of ERG251 leads to increased sensitivity to cell wall and osmotic stress but decreased sensitivity to oxidative stress.

A. Volcano plot for differentially expressed genes (log2 fold change ≥ 1 or ≤-1 and adjusted p-value < 0.05) in the erg251∆/∆ mutant compared to ERG251/ERG251 in YPAD with both fold change and p-value indicated. B. Gene Ontology (GO) terms for differentially expressed genes (log2 fold change ≥ 1 or ≤ −1 and adjusted p-value < 0.05) in the erg251∆/∆ mutant compared to ERG251/ERG251 in YPAD. C. Spot plates growth of ERG251/ERG251, erg251∆/ERG251, ERG251/erg251∆, and erg251∆/∆ on YPAD (30°C), YPAD (37°C), 1.2M NaCl, 0.03% SDS and 7.5mM H₂O₂ agar plates. A-C: At least three biological replicates were performed.

Fig 5.. Deletion of ERG251-A but not ERG251-B leads to decreased filamentation.

A. Representative filamentation images of wildtype ERG251/ERG251, erg251∆/ERG251, erg251∆/ERG251+ERG251-A, ERG251/erg251∆, ERG251/erg251∆+ERG251-B, erg251∆/∆, erg251∆/∆+ERG251-A, and erg251∆/∆+ERG251-B. Cells were induced in RPMI supplemented with 10% FBS for 4 hrs. Scale bar, 20 μm. B. Quantification of the yeast (<6μm), pseudohyphae (15–36 μm), and hyphae (>36 μm) from genotypes in Fig 5A. 150 to 500 cells were counted for each strain, and at least two biological replicates were performed. Error bars indicate SEM. Statistical significance for filamentation was compared to ERG251/ERG251 and assessed using two-way ANOVA with uncorrected Fisher’s LSD, ***P <0.001, **P <0.01,* P ≤ 0.05, ns: P >0.05. C. Principal component analysis of transcriptional data in YPAD and YPAD+FLC (1µg/ml) for ERG251/ERG251, erg251∆/ERG251, ERG251/erg251∆, and erg251∆/∆. D. Venn diagrams comparing the genes that are differentially expressed in erg251∆/ERG251 and ERG251/erg251∆ (log2 fold change ≥ 0.5 or ≤-0.5 and adjusted p-value < 0.1) relative to ERG251/ERG251 in YPAD. E. The relative expression level (log2 fold change) of genes associated with filamentation in erg251∆/ERG251, ERG251/erg251∆, and erg251∆/∆compared to ERG251/ERG251 in YPAD.

Fig 6.. Homozygous deletion of ERG251 leads to the downregulation of ergosterol biosynthesis genes.

A. Overview of the ergosterol biosynthetic pathway in C. albicans, including the mevalonate, late ergosterol, and alternate pathways. Genes that were down-regulated (blue) and up-regulated (red) in the erg251∆/∆ under YPAD conditions relative to SC5314 [,,–71]. B. The relative gene expression levels (log2-fold change) for all ERG genes in the heterozygous and homozygous mutants erg251∆/ERG251, ERG251/erg251∆, and erg251∆/∆ grown in YPAD or YPAD+1µg/ml FLC conditions, compared to the wildtype ERG251/ERG251 in the same condition. C. The relative expression level (log2 fold change) of ERG genes in the wildtype ERG251/ERG251, and mutants erg251∆/ERG251, ERG251/erg251∆, and erg251∆/∆ grown in YPAD+1µg/ml FLC compared to YPAD condition.

Fig 7.. Dysfunction of ERG251 activates a Zinc transporter contributing to decreased azole susceptibility.

A. The relative expression level (log2 fold change) of CDR1, CDR2, MDR1 and ZRT2 in erg251∆/ERG251, ERG251/erg251∆, and erg251∆/∆ compared to ERG251/ERG251 under YPAD+1µg/ml FLC condition. B. Venn diagrams comparing the genes that differentially expressed in erg251∆/ERG251, ERG251/erg251∆ and erg251∆/∆ related to ERG251/ERG251 under YPAD+1µg/ml FLC condition. C. 24hr MIC (left, µg/ml) and 48hr SMG (right, tolerance) in FLC for two ZRT2 overexpression strains (tetO-ZRT2-1 and tetO-ZRT2-2) in SC5314 background together with SC5314 (ERG251/ERG251) and erg251∆/ERG251 as the controls. At least three biological replicates were performed.

Fig 8.. Heterozygous deletion of ERG251 maintains pathogenicity in a murine model.

A. ICR mice were injected via the tail vein with 5x10⁵ cells of ERG251/ERG251 (SC5314), erg251∆/ERG251, ERG251/erg251∆, and erg251∆/∆+ERG251-A and erg251∆/∆+ERG251-B and survival was presented over the time. The erg251∆/∆ mutant survival curves were significantly attenuated from that of the ERG251/ERG251 (Log-rank (Mantel-Cox) test; **, p = 0.0015). Eight mice per strain were used.