Heat shock protein 90 and calcineurin pathway inhibitors enhance the efficacy of triazoles against Scedosporium prolificans via induction of apoptosis

Scedosporium prolificans is a pathogenic mold resistant to current antifungals, and infection results in high mortality. Simultaneous targeting of both ergosterol biosynthesis and heat shock protein 90 (Hsp90) or the calcineurin pathway in S. prolificans may be an important strategy for enhancing the potency of antifungal agents. We hypothesized that the inactive triazoles posaconazole (PCZ) and itraconazole (ICZ) acquire fungicidal activity when combined with the calcineurin inhibitor tacrolimus (TCR) or Hsp90 inhibitor 17-demethoxy-17-(2-propenylamino) geldanamycin (17AAG). PCZ, ICZ, TCR and 17AAG alone were inactive in vitro against S. prolificans spores (MICs > 128 μg/ml). In contrast, MICs for PCZ or ICZ in combination with TCR or 17AAG (0.125-0.50 μg/ml) were much lower compared with drug alone. In addition PCZ and ICZ in combination with TCR or 17AAG became fungicidal. Because apoptosis is regulated by the calcineurin pathway in fungi and is under the control of Hsp90, we hypothesized that this synergistic fungicidal effect is mediated via apoptosis. This observed fungicidal activity was mediated by increased apoptosis of S. prolificans germlings, as evidenced by reactive oxygen species accumulation, decreased mitochondrial membrane potential, phosphatidylserine externalization, and DNA fragmentation. Furthermore, induction of caspase-like activity was correlated with TCR or 17AAG + PCZ/ICZ-induced cell death. In conclusion, we report for the first time that PCZ or ICZ in combination with TCR or 17AAG renders S. prolificans exquisitely sensitive to PCZ or ICZ via apoptosis. This finding may stimulate the development of new therapeutic strategies for patients infected with this recalcitrant fungus.


INTRODUCTION
Scedosporium prolificans is an emerging filamentous fungus that causes severe, frequently fatal pulmonary or disseminated opportunistic infections in immunocompromised patients [1]. S. prolificans is inherently resistant to treatment with a wide range of antifungals, including the new generation of broad-spectrum triazoles [1][2][3][4]. Hence, new therapeutic strategies for Scedosporium infections are urgently needed.
In pathogenic fungi, the calcineurin pathway and heat shock protein 90 (Hsp90) play major roles in maintaining fungal homeostatic cell responses, including resistance to antifungal agents [5][6][7][8][9][10]. The calcineurin inhibitor tacrolimus (TCR) is an immunosuppressive agent widely used in solid organ and hematopoietic stem cell transplant recipients to prevent graft rejection [11]. TCR binds to the intracellular protein immunophilin FKB12 and forms a complex, thereby inhibiting activation of the calcineurin pathway. In vitro studies have suggested synergy between triazoles and calcineurin inhibitors against Aspergillus spp. and the Mucorales [12][13][14]. Our group recently reported that treatment with the combination of TCR and posaconazole (PCZ) OPEN  improves control of invasive, necrotizing cutaneous mucormycosis in immunosuppressed mice compared with PCZ alone [15]. Hsp90 is a molecular chaperone involved in stress responses of Candida albicans and Aspergillus spp. and plays a major role in echinocandin resistance via regulation of the calcineurin pathway [6,16]. Specifically, pharmacological inhibition of Hsp90 by 17-demethoxy-17-(2propenylamino) geldanamycin (17AAG) prevents azole resistance and abrogates this resistance in C. albicans and A. fumigatus in a human host [6,16]. In addition, researchers recently suggested a role for the calcineurin pathway in regulation of apoptosis in fungi [17,18]. However, the role of Hsp90 in apoptosis remains unclear. Therefore, simultaneous targeting of both ergosterol biosynthesis and Hsp90 or calcineurin pathways in S. prolificans may be an important strategy for restoring the potency of antifungal agents. Specifically, we hypothesized that TCR or 17AAG in combination with the triazoles PCZ or itraconazole (ICZ) induces apoptosis in S. prolificans. Thus, we examined the effects of TCR and 17AAG co-administration on PCZ and ICZ activity using several in vitro methods to evaluate induction of apoptosis in S. prolificans.

RESULTS
PCZ and ICZ are inactive when used alone against S. prolificans, but exhibit significant fungicidal activity when combined with TCR or 17AAG Individually, PCZ, ICZ, TCR, and 17AAG were inactive against S. prolificans (isolates 1 to 3), with minimum inhibitory concentrations (MICs) ranging from 32 to128 µg/ml. In contrast, the combination of PCZ or ICZ with either TCR or 17AAG rendered S. prolificans exquisitely more sensitive to the triazoles than did use of the triazoles alone (Table 1). Specifically, in combination with TCR or 17AAG, PCZ and ICZ were synergistic, with a fractional inhibitory concentra-tion index (ΣFIC) of 0.5. In addition, bis-[1,3dibutylbarbituric acid] trimethine oxonol (DiBAC) vital staining revealed enhanced uptake of stain and plasma membrane damage in S. prolificans germlings (isolates 1 and 2) exposed to PCZ or ICZ in combination with TCR or 17AAG (Figure 1 A-C; Table S1). Use of PCZ or ICZ (0.125-0.25 µg/ml) in combination with TCR or 17AAG resulted in 2.0-to 2.5-fold greater plasma membrane damage than did the use of triazoles alone.

Detection of intracellular Reactive Oxygen Species (ROS)
accumulation and loss of mitochondrial membrane potential (∆Ψ m ) in S. prolificans (isolates 1 and 2) germlings in response to treatment with PCZ or ICZ combined with TCR or 17AAG Staining of S. prolificans germlings with dihydrorhodamine (DHR)-123 (red fluorescence) and rhodamine (Rh)-123 (green fluorescence) was most prominent in germlings treated with PCZ or ICZ in combination with TCR or 17AAG (Figures 2 and 3). A small percentage of control germlings and germlings treated with PCZ or ICZ alone exhibited positive staining for DHR-123 and Rh-123 (Figures 2 and 3). Staining with DHR123 and Rh-123 increased markedly when triazoles were combined with TCR or 17AAG, respectively (1.2-2.1 fold increase in fluorescence intensity), compared with triazoles alone (Figures 2 and 3 A-C). Isolate 2 in particular, had 1.0-2.1 fold and 1.3-2.1 fold increase in fluorescence for ROS accumulation and loss of mitochondrial potential, respectively, over germlings treated with triazoles alone (Table S1). Accumulation of intracellular ROS and disruption of ∆Ψ m are important steps in mitochondria-mediated apoptosis. These data indicate that treatment with PCZ or ICZ combined with TCR or 17AAG can trigger apoptosis in S. prolificans due to accumulation of ROS.  *MFC is given in parenthesis.

Evidence of apoptosis in S. prolificans (isolates 1 and 2) induced by treatment with PCZ or ICZ in combination with TCR or 17AAG
Because various drugs can induce both apoptosis and necrosis in mammalian cells [19], we sought to differentiate between apoptotic and necrotic S. prolificans protoplast using annexin V-fluorescein isothiocyanate (FITC)propidium iodide (PI) double staining, in which apoptotic cells are stained with annexin V-FITC (green), whereas the nuclei of necrotic cells are stained with PI (red) [20][21][22]. Incubation of S. prolificans (isolate 1) protoplasts in the presence of PCZ (0.25 µg/ml) or ICZ (0.125 µg/ml) in combination with TCR (0.060-0.125 µg/ml) at 37°C for 3 h led to annexin V-FITC staining of 35-50% of the protoplasts. We found that 30-40% of protoplasts exhibited annexin V-FITC staining, when incubated with PCZ or ICZ in combination with 17AAG (0.060-0.125 µg/ml) ( Table 2). In S. prolificans isolate 2, however, 40-65% of protoplasts were apoptotic after incubation with PCZ or ICZ in combination with TCR, and 35-70% were apoptotic after incubation with PCZ or ICZ with 17AAG (Table S1). We observed no annexin V-FITC staining in untreated protoplasts ( Table 2). These results suggested that a fungicidal property of PCZ and ICZ was due to induction of apoptosis in S. prolificans cells, especially in combination with TCR or 17AAG.

Induction of caspase-like activity in S. prolificans (isolate 1) germlings treated with PCZ or ICZ in combination with TCR or 17AAG
Caspases are activated in the early stages of apoptosis and play a central role in the apoptotic cascade [23,24]. Although caspases are not present in fungi, researchers have identified orthologs of mammalian caspases, called metacaspases in fungi (25). We stained S. prolificans germlings (isolate 1) pretreated with PCZ or ICZ in combination with TCR or 17AAG with the cell-permeable, broad-spectrum caspase inhibitor CaspACE-Z-VAD-FMK. In this staining, a green fluorescent signal is a direct measure of the amount of active caspase in a cell. S. prolificans germlings with activated metacaspases, treated with azoles in combination with TCR or 17 AAG were stained green, whereas germlings exposed to azoles alone remained unstained. This result indicated that treatment with PCZ or ICZ plus TCR or 17AAG triggered an apoptotic pathway in S. prolificans germlings via activation of metacaspases ( Figure 4).

DISCUSSION
We hypothesized that TCR and 17AAG enhance the negligible activity of the ergosterol biosynthesis inhibitors PCZ and ICZ, to the point that they become fungicidal, and that this fungicidal activity is mediated through apoptosis in S. prolificans. The calcineurin pathway and Hsp90 are important for the survival of pathogenic fungi because they  OPEN ACCESS | www.microbialcell.com have central roles in various cellular processes, including morphogenetic transition and development of antifungal tolerance and resistance [7,16]. Inhibition of the calcineurin pathway and Hsp90 in combination with administration of conventional antifungal agents may have broad therapeutic potential in patients with fungal infections [16,26].
Owing to the immunosuppressive properties of calcineurin inhibitors and the role of Hsp90 in controlling the calcineurin pathway, clinical use of a combination of TCR or 17AAG with triazole for treatment of S. prolificans infection would ultimately require a novel antifungal agent that selectively targets fungal stress pathways without having collateral effects on human immune cells.
We found evidence of synergy of PCZ and ICZ with TCR and 17AAG in S. prolificans in vitro, which is consistent with data on other fungal species [12,16,18,21]. In addition, we used multiple markers of cell death to show that apoptosis is a mechanism of PCZ/ICZ-and TCR/17AAGinduced cell death. We corroborated the rate of apoptosis in S. prolificans germlings using assays for detection of phosphatidylserine (PS) by annexin V-FITC, ROS accumulation by DHR-123 staining and decreased mitochondrial membrane potential by Rh123, DNA damage by TUNEL staining, and activation of caspase-like activity by CaspACE FITC-VAD-FMK. In each of the assays, apoptosis was evident at PCZ, ICZ, TCR, and 17AAG concentrations (0.125-0.250 µg/ml) that were below the MIC of triazoles. Taken together, these data indicate that PCZ or ICZ combined with TCR or 17AAG at concentrations below the MIC causes apoptosis in S. prolificans germlings.
We found that induction of apoptosis and the fungicidal activity of PCZ and ICZ in combination with TCR or 17AAG correlated with increased plasma and mitochondrial membrane disruption, PS externalization, DNA fragmentation, and ROS accumulation in S. prolificans germlings (isolates 1 and 2) (Tables 1, 2 and S1, Figures 1-4). Calcineurin activity is known to contribute to the fungicidal effects of Hsp90 inhibitors. 17-AAG in particular induces OPEN ACCESS | www.microbialcell.com apoptosis in colon carcinoma-derived cell lines (27), so determination of whether inhibition of Hsp90 can induce apoptotic cell death in fungi would be of interest. Dai et al. [28] demonstrated the role of Hsp90 in apoptosis in C. albicans and showed that inhibition of Hsp90 attenuated apoptosis by regulating the calcineurin pathway. Several fungi undergo apoptosis in response to antifungal treatment and various other stimuli [19]. Additional studies providing better understanding of fungal apoptotic pathways would promote the discovery of much-needed antifungal therapies.
Our results indicated that disruption of mitochondrial integrity by a combination of PCZ/ICZ with TCR or 17AAG induced apoptosis in S. prolificans. Our study in S. prolificans and studies in other fungi showed that 17AAG inhibits Hsp90, causing mitochondria-mediated apoptosis in rat histiocytomas [29]. Also, Shirazi and Kontoyiannis [18] showed that increased apoptosis after exposure to TCR was correlated with increased intracellular ROS accumulation in Mucorales. Furthermore, translocation of mitochondrial cyt c to the cytosol has led to binding of cyt c with apoptotic protease-activating factor to form a complex with caspase-9, resulting in caspase activation [23,24,30]. Release of cyt c requires an increase in mitochondrial membrane permeability during apoptosis [23]. As in Mucorales, our results in S. prolificans also demonstrated that, ROS formation, changes in ∆Ψ m , and cyt c release were associated with apoptosis [20][21][22]. Authors have also reported ROS-induced apoptosis in A. nidulans, Fusarium oxysporum, and C. albicans [31][32][33]. Sharon et al. [25] reported that apoptotic pathways in fungi seem to be mitochondrion-dependent, and can be powerful sources of superoxide radicals in cells undergoing miconazole and farnesol-induced apoptosis [34].
Authors have reported accumulating evidence that different stimuli induce different apoptotic pathways in yeasts and other fungi [35,36]. In mammals, apoptosis is regulated by activation of caspases, which cleave specific substrates and trigger apoptotic death [37]. Now it is evident that caspase-like proteolytic activity may exist not only in multicellular organisms but also unicellular organisms, such as fungi. In the current study, we observed caspase-like activity in S. prolificans germlings upon exposure to PCZ or ICZ with TCR or 17AAG. Further studies are needed to demonstrate how proteases contribute to apoptotic fungal death.
In conclusion, we have shown for the first time that coadministration of inhibitors of the ergosterol biosynthesis pathways with an inhibitor of calcineurin or Hsp90 induces apoptosis in the recalcitrant fungus S. prolificans. This fungicidal synergistic interaction requires further study, as it may be a useful therapeutic strategy for infections caused by pathogenic fungi for which treatment options are extremely limited.

Annexin V-FITC-PI double staining of S. prolificans (isolates 1 and 2)
The apoptosis marker PS is located on the inner leaflet of the lipid bilayer of the cytoplasmic membrane and is translocated to the outer leaflet at the onset of apoptosis [39][40][41]. PS can be detected using staining with annexin V-FITC, which binds to it. Germlings treated with PCZ (0.06-0.50 µg/ml) or ICZ (0.06-0.25 µg/ml) in combination with TCR or 17AAG (0.060 and 0.125 µg/ml) were digested with a lysing enzyme mixture (0.25 mg/ml chitinase, 15 U of lyticase, and 20 mg/ml lysing enzyme; Sigma) for 3 h at 30°C. After digestion, S. prolificans protoplasts were stained with annexin V-FITC (BD Pharmingen) and PI at room temperature for 15 min and ob-OPEN ACCESS | www.microbialcell.com served under a fluorescence microscope to assess the externalization of PS as described previously [39].

Measurement of DNA damage in S. prolificans (isolates 1 and 2)
DNA fragmentation, a characteristic of apoptosis, was detected in S. prolificans using a TUNEL assay. Germlings pretreated with PCZ (0.06-0.50 µg/ml) or ICZ (0.06-0.25 µg/ml) in combination with TCR or 17AAG (0.060 and 0.125 µg/ml) for 3 h at 37°C were fixed with 3.7% formaldehyde for 30 min on ice and digested using a lysing enzyme mixture. Enzyme-digested germlings were used to detect DNA fragmentation using a TUNEL assay as described by Madeo et al. [41]. The protoplasts were observed for fluorescence with excitation and emission wavelengths of 488 nm and 520 nm, respectively.

Statistical Analysis
For all assays, three independent experiments were performed in triplicate. Comparisons of multiple treatment groups were performed by using two-way analysis of variance with post-hoc paired comparisons using Dunnett's test. Calculations were made using the InStat software program (GraphPad Software). Two-tailed P values of less than 0.05 were considered statistically significant.