Loss of Upc2p-Inducible ERG3 Transcription Is Sufficient To Confer Niche-Specific Azole Resistance without Compromising Candida albicans Pathogenicity

Azole-inducible ERG3 transcription is lost in the shprERG3 strain. Wild-type (WT), erg3Δ/Δ, shprERG3, and lnprERG3 strains were grown for 24 h in YPD at 37°C in the presence or absence of 5 µg/ml fluconazole. RNA was then extracted, ERG3 transcript abundance was quantified by qRT-PCR using the ΔC_T method, and ERG3 transcript levels were normalized to that of ACT1. The mean plus standard error of the mean (error bar) for three biological replicates are shown for each strain. Each treatment group was compared using a two-way analysis of variance (ANOVA) and Tukey’s multiple-comparison test, and the P values are indicated.

Loss of azole-inducible ERG3 transcription does not affect C. albicans stress tolerance or hyphal growth. (A) C. albicans cell suspensions were prepared at 1 × 10⁷ cells/ml, and serial 1:5 dilutions prepared in sterile water. Cell suspensions were then applied to YPD agar plates or YPD agar supplemented with the indicated concentrations of fluconazole (Fluc), SDS, NaCl, or CaCl₂ using a sterile multipronged applicator. The plates were incubated at 30°C for 48 h and then imaged. (B) C. albicans strains were suspended at 1 × 10⁷ cells/ml in sterile water, and 2.5 µl spotted onto either an M199 or 10% FBS agar plate. The resulting colonies were imaged after 96-h incubation at 37°C. (C) The capacity to form biofilms was also evaluated in flat-bottomed 96-well plates. Each C. albicans strain was grown in RPMI 1640 medium at 37°C for 24 h and stained with crystal violet before biofilm mass was quantified as OD₅₇₀. Values are means plus standard errors of the means (SEM) from four independent experiments. The biofilm mass of each group was compared using one-way ANOVA and Tukey’s multiple-comparison test. The value for the erg3Δ/Δ mutant group was significantly different (P < 0.0001) from the values for the WT, shprERG3, and lnprERG3 groups as indicated by the two asterisks.

Azole-inducible ERG3 transcription is dependent upon the Upc2p transcription factor. The GFPγ coding sequence was cloned downstream of 1 kb of the ERG3 promoter sequence, and the resulting construct introduced into C. albicans wild-type strain CAI4 (WT) or a upc2Δ/Δ mutant. The resulting strains were grown in YNB broth in the presence or absence of 5 µg/ml fluconazole for 24 h at 35°C with shaking, before GFPγ fluorescence was quantified (excitation wavelength of 488 nm [9 nm bandpass]; emission wavelength of 507 nm [9 nm bandpass]). Fluorescence intensity was then normalized to the cell density (OD₆₀₀) and expressed relative to the DMSO control. The means ± the standard deviations for four biological replicates are shown. The relative fluorescence of each treatment group was then compared using a two-way ANOVA and Tukey’s multiple-comparison test, and the P values are indicated.

The shprERG3 strain is virulent but susceptible to fluconazole treatment in a mouse model of disseminated candidiasis. (A) BALB/c mice (six mice in each group) were inoculated with ~5 × 10⁵ CFU of either the WT (GP1), erg3Δ/Δ, shprERG3, or lnprERG3 strain via lateral tail vein injection. The mice were then monitored three times daily for 14 days, and those showing distress were humanely euthanized. The survival of each group was compared using the log rank test (P = 0.0051). (B) Groups of BALB/c mice were inoculated with ~5 × 10⁵ CFU of either WT (GP1), erg3Δ/Δ, shprERG3, or lnprERG3 strain as described above and then split into two treatment groups (four mice in each group). One group was treated with 25 mg/kg fluconazole (Fluc), and another group was treated with vehicle alone (PBS) by daily intraperitoneal injection starting 24 h after infection. Animals were monitored for 8 days, and the survival of each group was compared using the log rank test (P < 0.0001).