The Hsp90 Co-chaperones Sti1, Aha1, and P23 Regulate Adaptive Responses to Antifungal Azoles

Abstract

Heat Shock Protein 90 (Hsp90) is essential for tumor progression in humans and drug resistance in fungi. However, the roles of its many co-chaperones in antifungal resistance are unknown. In this study, by susceptibility test of Neurospora crassa mutants lacking each of 18 Hsp90/Calcineurin system member genes (including 8 Hsp90 co-chaperone genes) to antifungal drugs and other stresses, we demonstrate that the Hsp90 co-chaperones Sti1 (Hop1 in yeast), Aha1, and P23 (Sba1 in yeast) were required for the basal resistance to antifungal azoles and heat stress. Deletion of any of them resulted in hypersensitivity to azoles and heat. Liquid chromatography-mass spectrometry (LC-MS) analysis showed that the toxic sterols eburicol and 14α-methyl-3,6-diol were significantly accumulated in the sti1 and p23 deletion mutants after ketoconazole treatment, which has been shown before to led to cell membrane stress. At the transcriptional level, Aha1, Sti1, and P23 positively regulate responses to ketoconazole stress by erg11 and erg6, key genes in the ergosterol biosynthetic pathway. Aha1, Sti1, and P23 are highly conserved in fungi, and sti1 and p23 deletion also increased the susceptibility to azoles in Fusarium verticillioides. These results indicate that Hsp90-cochaperones Aha1, Sti1, and P23 are critical for the basal azole resistance and could be potential targets for developing new antifungal agents.

Keywords: Hsp90; aha1; azole; co-chaperone; drug resistance; p23; sti1.

Figure 1

Susceptibility tests of wild-type N. crassa and the knockout mutants of Hsp90 orchestrates member genes [hsp80, hsp70-1, hsp70-2, hsp70-3, p23, aha1, sti1, cdc37, nup-17 (PPIase), nup-5 (PPIase B), nup-13 (PPIase H), and fkr-5 (PPIase FKBP-type)], calcineurin encoding genes (cna1 and cnb1), Hsp90/calcineurin-dependent stress responses genes (crz1 and hsf1) and other heat shock protein family genes (hsp88 and hsp98) to antifungal drug ketoconazole (KTC). Two microliters of conidial suspension (2 × 10⁶ conidia/ml) were inoculated in the center of plates (Φ 90 mm) with or without antifungal drug, then incubated at 28°C for the indicated times. Each test was duplicated and the experiment was independently repeated at least three times.

Figure 2

Susceptibility tests of N. crassa to antifungal drugs, oxidants and heat stress. (A) Susceptibility tests of wild-type N. crassa, the knockout mutants of p23, aha1, sti1 (Δp23, Δaha1, and Δsti1) and double deletion mutants (Δp23Δaha1 and Δp23Δsti1) to antifungal drugs, oxidants and heat stress; (B) Susceptibility tests of the N. crassa knockout mutants Δp23, Δaha1, and Δsti1 and their complemented strains Δp23;p23, Δaha1;aha1, and Δsti1;sti1 to Ketoconazole. Two microliters of conidial suspension (2 × 10⁶ conidia/ml) were inoculated in the center of plates (Φ 90 mm) with or without antifungal drugs or oxidants, then incubated at 28 or 42°C (heat tests) for the indicated times. Each test was duplicated and the experiment was independently repeated at least three times. KTC, ketoconazole; FLU, fluconazole; ITC, itraconazole; CSP, caspofungin; AMB, amphotericin B; Me, menadione.

Figure 3

Susceptibility tests of N. crassa to ketoconazole, the Hsp90 inhibitor geldanamycin and the two drugs combined. (A) Susceptibility tests with geldanamycin (GA, 2 mg/L), ketoconazole (KTC, 1 mg/L) or the two drugs combined. Two microliters of conidial suspension (2 × 10⁶ conidia/ml) were inoculated in the center of plates (Φ 90 mm) with or without antifungal drugs or oxidants, and then incubated at 28 or 42°C (heat tests) for the indicated time. (B) Relative growth inhibition rates were calculated based on colony diameters at 24 h after drug treatment. Values from three replicates were used for a statistical analysis. Means of the inhibition rates are shown, and standard deviations are marked with error bars. Differences between the mutants and the WT were statistically analyzed by the analysis of variance. Values with P < 0.0001, 0.0001 < P < 0.001, 0.001 < P < 0.01, and 0.01 < P < 0.05 are marked with ^****, ^***, ^** and ^*, respectively.

Figure 4

Differential expressions of genes in the Δsti1, Δaha1, and Δp23 strains relative to the wild type (WT) strain was determined by quantitative RT-PCR. Strains were grown in liquid Vogel's medium at 28°C with shaking at 180 rpm for 12 h. Then ketoconazole (KTC) was added to the medium to reach 2.5 mg/L. After 24 h incubation, the following genes were analyzed by quantitative RT-PCR: hsp80, erg11, erg6, and cdr4. Values shown are the means of three independent replicates. Standard deviations are indicated by error bars, and differences between the mutants and WT were statistically analyzed by analysis of variance. Values with P < 0.0001, 0.0001 < P < 0.001, 0.001 < P < 0.01, and 0.01 < P < 0.05 are marked with ^****, ^***, ^** and ^*, respectively.

Figure 5

Schematic representations of the ergosterol biosynthetic pathway (A) and quantification of sterol accumulations (B) in wild-type N. crassa and the knockout mutants. Strains were grown in liquid Vogel's medium at 28°C with shaking at 180 rpm for 12 h. Then ketoconazole (KTC) was added to the medium to reach 2.5 mg/L. After 24 h incubation, ergosterol, eburicol, and 14α-methyl-3,6-diol were analyzed by LC-MS with fluconazole as a standard reference. Values shown are the means of three independent replicates. Standard deviations are indicated by error bars. Differences between the mutants and the WT were statistically analyzed by analysis of variance. Values with P < 0.0001, 0.0001 < P < 0.001, 0.001 < P < 0.01, and 0.01 < P < 0.05 are marked with ^****, ^***, ^**, and ^*, respectively.

Figure 6

The Fusarium verticillioides sti1 homolog FVEG_00423 and p23 homolog FVEG_11505 knockout mutants were hypersensitive to ketoconazole (KTC). Two microliters of conidial suspensions of different concentrations (1 × 10⁷, 1 × 10⁶, 1 × 10⁵, 1 × 10⁴, and 1 × 10³ conidia/ml) were inoculated onto the plates (Φ 150 mm) of potato dextrose agar medium with or without 2 mg/L KTC, and incubated at 28°C for 72 h. Each test had three replicates and the experiment was independently repeated twice.