Identification of a Key Lysine Residue in Heat Shock Protein 90 Required for Azole and Echinocandin Resistance in Aspergillus fumigatus

Abstract

f Heat shock protein 90 (Hsp90) is an essential chaperone involved in the fungal stress response that can be harnessed as a novel antifungal target for the treatment of invasive aspergillosis. We previously showed that genetic repression of Hsp90 reduced Aspergillus fumigatus virulence and potentiated the effect of the echinocandin caspofungin. In this study, we sought to identify sites of posttranslational modifications (phosphorylation or acetylation) that are important for Hsp90 function in A. fumigatus. Phosphopeptide enrichment and tandem mass spectrometry revealed phosphorylation of three residues in Hsp90 (S49, S288, and T681), but their mutation did not compromise Hsp90 function. Acetylation of lysine residues of Hsp90 was recovered after treatment with deacetylase inhibitors, and acetylation-mimetic mutations (K27A and K271A) resulted in reduced virulence in a murine model of invasive aspergillosis, supporting their role in Hsp90 function. A single deletion of lysine K27 or an acetylationmimetic mutation (K27A) resulted in increased susceptibility to voriconazole and caspofungin. This effect was attenuated following a deacetylation-mimetic mutation (K27R), suggesting that this site is crucial and should be deacetylated for proper Hsp90 function in antifungal resistance pathways. In contrast to previous reports in Candida albicans, the lysine deacetylase inhibitor trichostatin A (TSA) was active alone against A. fumigatus and potentiated the effect of caspofungin against both the wild type and an echinocandin-resistant strain. Our results indicate that the Hsp90 K27 residue is required for azole and echinocandin resistance in A. fumigatus and that deacetylase inhibition may represent an adjunctive anti-Aspergillus strategy. Invasive aspergillosis (IA) is a life-threatening infection in the increasing population of patients with depressed immune systems, such as cancer patients or transplant recipients (1). Three antifungal drug classes are approved for the treatment of IA: the triazoles (e.g., voriconazole), the polyenes (e.g., amphotericin B), and the echinocandins (e.g., caspofungin), all of them targeting the fungal cell membrane or cell wall (2). The emergence in Aspergillus fumigatus of resistance to triazoles, the preferred primary therapy against IA, is a growing concern (3). Moreover, the toxicity of polyenes often limits their use, and the echinocandins possess only fungistatic activity against Aspergillus spp. Combination antifungal therapy with existing classes of agents has shown limited promise (4), suggesting the need for new therapeutic options.One novel therapeutic approach consists of the development of drugs targeting intracellular signaling proteins involved in compensatory mechanisms of the cell wall. The heat shock protein 90 (Hsp90)-calcineurin axis is a crucial element governing stress adaptation processes in fungi (5). Calcineurin inhibitors (FK506 and cyclosporine) and Hsp90 inhibitors (geldanamycin,, and 17-demethylaminoethylamino-17-demethoxygeldanamycin [17-DMAG]) are active a...

The Paradoxical Effect of Echinocandins in Aspergillus fumigatus Relies on Recovery of the β-1,3-Glucan Synthase Fks1

“…One possibility could be the activation of a chaperone that protects or stabilizes the ␤-1,3-glucan synthase. Interestingly, it was previously shown that the chaperone Hsp90 is required for the paradoxical effect and that it localizes to the cell wall upon echinocandin exposure (17,(21)(22)(23). Although highly speculative, this could provide a possible mechanism for the restoration of ␤-1,3-glucan synthase activity.…”

Heat Shock Protein 90 (Hsp90) in Fungal Growth and Pathogenesis

“…Trichostatin A has no intrinsic antifungal activity against C. albicans, but potentiates the effect of azoles [40]. However, it is active against A. fumigatus, reaching more than 90 % inhibition at a concentration of 4 μg/ml, and interestingly, has a positive interaction with caspofungin, but not voriconazole [29]. MGCD290 (MethylGene Inc.), another HDAC inhibitor with a different anti-HDAC spectrum, had modest antifungal activity, but was synergistic against a series of azole-resistant yeasts and molds [41].…”

“…An increase of Hsp90 expression is also essential for the paradoxical response, and is mediated by a 100-bp proximal region of the hsp90 promoter [17•]. Furthermore, a conserved lysine residue and putative acetylation site of Hsp90 (K27) was also found to be important for capsofungin resistance [29]. Another study suggested a role of Hsp90 in basal resistance to polyenes in Aspergillus terreus, showing a synergistic effect between Hsp90 inhibitors and amphotericin B [30].…”

Mechanisms of Antifungal Drug Resistance

“…Hsp90 function was also found to be regulated by acetylation such that pharmacological inhibition of lysine deacylases phenocopies inhibition of Hsp90, blocking the evolution of azole resistance and reducing resistance of C. albicans that evolved in a human host (Robbins et al 2012). Regulation of Hsp90 function by acetylation is conserved in A. fumgiatus, in which the key residue for azole and echinocandin resistance is K27 (Lamoth et al 2014b). In S. cerevisiae, the key lysine deacetylases that modulate Hsp90 function are Hda1 and Rpd3 (Robbins et al 2012), although the relevant players remain enigmatic in other fungi.…”