Lysine deacetylases Hda1 and Rpd3 regulate Hsp90 function thereby governing fungal drug resistance

Abstract

The molecular chaperone Hsp90 is a hub of protein homeostasis and regulatory circuitry. Hsp90 function is regulated by posttranslational modifications including acetylation in mammals; however, whether this regulation is conserved remains unknown. In fungi, Hsp90 governs the evolution of drug resistance by stabilizing signal transducers. Here, we establish that pharmacological inhibition of lysine deacetylases (KDACs) blocks the emergence and maintenance of Hsp90-dependent resistance to the most widely deployed antifungals, the azoles, in the human fungal pathogen Candida albicans and the model yeast Saccharomyces cerevisiae. S. cerevisiae Hsp90 is acetylated on lysine 27 and 270, and key KDACs for drug resistance are Hda1 and Rpd3. Compromising KDACs alters stability and function of Hsp90 client proteins, including the drug-resistance regulator calcineurin. Thus, we establish acetylation as a mechanism of posttranslational control of Hsp90 function in fungi, functional redundancy between KDACs Hda1 and Rpd3, as well as a mechanism governing fungal drug resistance with broad therapeutic potential.

Graphical abstract

Figure 1

Trichostatin A Blocks the Emergence and Maintenance of Drug Resistance in C. albicans and S. cerevisiae (A) A total of 1 × 10⁵ cells of a wild-type C. albicans strain was plated onto YPD with no drug (−), 800 nM of the azole miconazole (ML), 1 μM of the Hsp90 inhibitor radicicol (RAD), 6 μg/ml of the KDAC inhibitor trichostatin A (TSA), or a combination of azole and inhibitor, as indicated. (B) Fluconazole (FL) resistance of C. albicans clinical isolates is abrogated when KDACs are inhibited. MIC assays were conducted in YPD with no inhibitor (−), with the Hsp90 inhibitor geldanamycin (GdA, 5 μM), or with TSA (6 μg/ml). C. albicans clinical isolates (CaCis) are ordered sequentially with those recovered early in treatment at the top. Data are quantitatively displayed with color using TreeView (see bar). (C) TSA specifically reduces FL resistance of C. albicans and S. cerevisiae Hsp90-dependent azole-resistant mutants. MIC assays were conducted in YPD with no inhibitor (−), with GdA (1 μM for C. albicans, 5 μM for S. cerevisiae), or with TSA (6 μg/ml). Growth and analysis are as in (B). PDR5⇑ represents a S. cerevisiae strain that acquired FL resistance by upregulation of the drug efflux pump Pdr5. erg3 represents a S. cerevisiae strain that acquired FL resistance due to a loss-of-function mutation in ERG3. See also Table S1.

Figure 2

Trichostatin A Does Not Decrease the Expression of Hsp90 or Key Drug-Resistance Determinants (A) Treatment of C. albicans or S. cerevisiae with trichostatin A (TSA) does not reduce Hsp90 gene expression. Strains were grown in rich medium to log phase without (−) or with TSA. Transcript levels of gene(s) encoding Hsp90 were measured by quantitative RT-PCR and normalized to GPD1 (C. albicans) or ACT1 (S. cerevisiae). Levels are expressed relative to the untreated sample, which is set to 1. Data are mean ± SD for triplicates. (B) Treatment of S. cerevisiae or C. albicans with TSA does not alter Hsp90 protein levels. Cultures were grown as in (A). Total protein was resolved by SDS-PAGE, and blots were hybridized with α-Hsp90 or α-tubulin. (C) TSA does not block the expression of azole-resistance determinants. Transcript levels of C. albicans or S. cerevisiae erg3 mutants were measured by quantitative RT-PCR after growth to log phase with no treatment, TSA, fluconazole (FL), or the combination of TSA and FL. Transcript levels are normalized as in (A). See also Table S2.

Figure 3

Deletion of RPD3 and HDA1, Encoding Catalytic Subunits of Distinct KDAC Complexes, Decreases Azole Resistance of Hsp90-Dependent Resistant Mutants in S. cerevisiae (A) To identify the key targets of trichostatin A (TSA) that influence fluconazole (FL) resistance, we tested the impact of deletion of KDACs individually and in combination in an erg3 mutant in an FL MIC assay. The multiple erg3Δhda1Δrpd3Δ strains shown are independent meiotic progeny with the confirmed genotype. Assays are performed as in Figure 1. See also Figure S1. (B) Deletion of both HDA1 and RPD3 has no effect on azole resistance caused by drug pump overexpression. Growth is as in (A) and analysis as in Figure 1.

Figure 4

Hsp90 Is Acetylated in S. cerevisiae, and Compromising KDAC Function Results in Hypersensitivity to Further Hsp90 Inhibition (A) Deletion of HDA1 and RPD3 in S. cerevisiae causes hypersusceptibility to geldanamycin (GdA) without altering Hsp90 protein levels. A titration of GdA was generated, and growth was measured as in Figure 1 (left panel). Total protein was resolved by SDS-PAGE, and blots were hybridized with α-Hsp90 or α-tubulin (right panels). (B) Hsp90 is acetylated in S. cerevisiae. Immunoprecipitation of 6×His-Hsp82 with NiNTA agarose pulled down acetylated Hsp90 when immunoprecipitated samples were hybridized with α-Hsp90 or α-acetylated lysine (AcK). Reciprocally, Hsp90 was immunoprecipitated when protein extracts were incubated with protein A agarose coupled to an AcK antibody. C, a control lane where protein extracts were incubated with protein A agarose in the absence of AcK antibody; IB, immunoblot; IP, immunoprecipitate. (C) Schematic of Hsp90 domain structure indicating candidate acetylated lysine residues. Mass spectrometry analysis identified a putative acetylation site at K27 of S. cerevisiae Hsp82 in an hda1Δrpd3Δ mutant, indicated by an asterisk. K270 has been identified in mammalian cells as acetylated. Sequences containing the putative acetylated lysines (bolded and underlined) are shown below. Hsp90 was immunoprecipitated with an AcK antibody as described in (B). (D) K27 and K270 are functionally important Hsp90 residues that are modified by acetylation. Growth curves were conducted in YPD with or without GdA over 48 hr, and the area under the curve (AUC) was calculated. Data are mean ± SD of quadruplicates. See also Figure S2 and Table S3.

Figure 5

Inhibition of KDACs Impairs Calcineurin Activation and Blocks the Interaction between Hsp90 and Calcineurin (A) Geldanamycin (GdA) and trichostatin A (TSA) block calcium-induced calcineurin activation. Log phase cultures of a S. cerevisiae strain containing a 4×CDRE-lacZ reporter or a C. albicans strain harboring a UTR2p-lacZ construct were untreated (−), treated with 200 mM CaCl2 (Ca), treated with Ca and GdA, or treated with Ca and TSA. Data are mean ± SD from technical triplicates and are representative of biological duplicates. ^∗p < 0.001. (B) Deletion of HDA1 and RPD3 blocks the physical interaction between Hsp90 and calcineurin. Protein from S. cerevisiae strains harboring a HA-tagged catalytic subunit of calcineurin (Cna1) was immunoprecipitated with anti-HA agarose beads, resolved by SDS-PAGE, and blots were hybridized with α-HA or α-Hsp90 antibodies. Experiment was quantified using ImageJ, and data are mean ± SD of biological duplicates.

Figure 6

Inhibition of KDACs Impairs Hsp90 Client Protein Activation and Stability (A) Geldanamycin (GdA) and trichostatin A (TSA) induce the heat shock response in S. cerevisiae and C. albicans. Log phase cultures of strains with an HSE-lacZ reporter (S. cerevisiae) or HSP70p-lacZ reporter (C. albicans) were untreated (−) or treated with GdA or TSA. β-Galactosidase data are mean ± SD for technical triplicates and are representative of biological duplicates. (B) Deletion of HDA1 and RPD3 enhances the heat shock response. Log phase S. cerevisiae harboring an HSE-lacZ reporter was grown at 42°C for 3 hr. (C) TSA blocks glucocorticoid receptor (GR) maturation in S. cerevisiae. Wild-type S. cerevisiae containing a GR expression plasmid and a GR reporter plasmid with a GR response element (GRE) driving lacZ expression was used. Maturation of GR upon addition of deoxycorticosterone (DOC, 10 μM) was assessed in the absence and presence of GdA or TSA. (D) TSA leads to destabilization of Ste11. Wild-type S. cerevisiae was grown in SD with glucose (glu) as a negative control or galactose (gal) to induce 6×His-Ste11 expression. Cultures grown in galactose were grown without inhibitor (−) or with GdA or TSA. Total protein was resolved by SDS-PAGE, and blots were hybridized with α-His or with α-Hsp90 as a loading control. (E) TSA does not rescue v-Src-induced lethality in S. cerevisiae. S. cerevisiae containing a vector with v-Src under the control of a galactose-inducible promoter was spotted in 5-fold dilutions onto SD with 2% glucose or 2% galactose with no drug (−), GdA, or TSA. Images are representative of biological duplicates. ^∗p < 0.001.

Figure S1

Genetic Deletion of HOS1, HOS2, or HOS3 Has No Effect on erg3-Mediated Azole Resistance, Related to Figure 3 To determine if other KDACs contribute to fluconazole (FL) resistance, we tested the impact of the deletion of HOS1, HOS2 or HOS3 individually and in combination with HDA1 in an erg3 mutant background in a FL MIC assay. Cultures were grown in YPD at 30° for 48 hr and growth was measured as in Figure 1.

Figure S2

K27 and K270 Point Mutations in Hsp90-Mimicking Acetylation or Deacetylation Do Not Alter Fluconazole Susceptibility, Related to Figure 4 To test the functional importance of candidate acetylated lysines, site-directed mutagenesis was performed to mutate K27 and K270 of Hsp90 to mimic permanent acetylation (lysine (K) to glutamine (Q)) or deacetylation (lysine (K) to arginine (R)). These mutant alleles were expressed as the only source of Hsp90 in the cell. We tested the impact of these mutations on fluconazole (FL) susceptibility in an MIC assay. Cultures were grown in YPD at 30°C for 48 hr and growth was measured as in Figure 1.