All reagents used in electrophoresis and buffers components were purchased from Bio-Rad (Hercules, CA, USA) and Merck (Darmstaldt, Germany). Human serum albumin (HSA), 1,10-phenanthroline, resazurin, AgClO₄, Cu(ClO₄)₂·6H₂O, dimethyl sulfoxide (DMSO), 3-(N-morpholino) propanesulfonic acid (MOPS), itraconazole (ITC), amphotericin B (AMB) ketoconazole (KTC), ergosterol, lanosterol, silica gel 60 plates, Czapek-Dox and Sabouraud-dextrose agar (SDA) components were obtained from Sigma-Aldrich Chemical Co (St Louis, MO, USA). Roswell Park Memorial Institute (RPMI) 1640 medium was purchased from Invitrogen (Camarillo, CA, USA). 1,10-Phenanthroline-5,6-dione (phendione), [Cu(phendione)₃](ClO₄)₂·4H₂O ([Cu(phendione)₃]²⁺) and [Ag(phendione)₂]ClO₄ ([Ag(phendione)₂]⁺) were prepared in accordance with published procedures (McCann et al., 2004).

Phialophora verrucosa (FMC.2214 strain) isolated from a human patient with CBM was grown on SDA medium. Fungal cells were cultivated for 7 days under constant agitation (130 rpm) at 26°C in 100 ml of Czapek-Dox, a chemically defined medium containing: 3 g sucrose; 0.3 g NaNO₃; 0.05 g MgSO₄.7H₂O; 0.05 g KCl; 0.1 g KH₂PO₄; 0.001 g FeSO₄.7H₂O, pH 5.5. Conidia were obtained after gauze filtration followed by centrifugation (4.000 × g/10 min). Then, conidia were washed three times in 0.85% NaCl and cell density estimated by counting in a Neubauer chamber (Granato et al., 2015). All the experiments with P. verrucosa were conducted under Biosafety Level 2 (BSL-2) conditions.

Antifungal susceptibility testing was performed using the M38-A2 document for filamentous fungi as described by Clinical and Laboratory Standards Institute (CLSI, 2008) with some modifications (Granato et al., 2015). Briefly, the broth microdilution method was carried out using 96-well microtiter assay plates containing RPMI 1640 medium at pH 7.0 buffered with 0.16 M MOPS. All the test compounds were dissolved in DMSO and the maximum concentration of organic solvent was 2.0%. Serial dilution was made following CLSI guidelines in order to obtain final concentrations ranging from 0.01 to 20 mg/L of phendione and its derivatives, as well as the simple salts, AgClO₄ and Cu(ClO₄)₂·6H₂O. The minimum inhibitory concentration (MIC) for each test compound was determined after 5 days of incubation by visual inspection and resazurin staining assay (Liu et al., 2007). The lowest concentration capable in inhibiting 100% of fungal growth was recorded as the MIC. ITC (0.01 to 100 mg/L) was used as reference antifungal drug. In addition, the minimum fungicidal concentration (MFC) was established before microtiter plate (MIC assay) spectrophotometric reading, in which the contents of the plate wells were homogenized and an aliquot from each well was transferred onto SDA drug-free plates. The plates were incubated at 30°C for 10 days and MFC was determined as the lowest concentration without visual fungal growth. A fungicidal effect was defined as the MFC value equal or up to four times the MIC value, as proposed by Pfaller et al. (2004). The IC₅₀ value, defined as the drug concentration (μM) able to cause a 50% reduction in fungal viability, was also calculated by using logarithmic regression after MIC determination, as detailed by Granato et al. (2015).

The fungal ultrastructure was evaluated using scanning electron microscopy (SEM). Briefly, conidia (5 × 10⁷ cells) were incubated for 20 h at 26°C in the absence (control) or in the presence of phendione, [Ag(phendione)₂]⁺ or [Cu(phendione)₃]²⁺ at concentrations corresponding to MIC and 2 × MIC. Subsequently, the conidia were washed and fixed with 4% paraformaldehyde and 2.5% glutaraldehyde in 0.15 M sodium cacodylate buffer (pH 7.2) at 26°C for 2 h. Cells were washed and then post-fixed for 1 h at 26°C with 1% OsO₄ in the same buffer. Next, samples were dehydrated using a graded series of ethanol (50–100%), and dried by the critical point method. Finally, the samples were mounted on stubs, coated with gold and observed using a Jeol JSM 6490LV scanning electron microscope (Abi-chacra et al., 2013).

All the test compounds were dissolved in DMSO. P. verrucosa conidia (1 × 10³/ml) were exposed to individual clinically used antifungal drugs [AMB (3.12 mg/L), KTC (1.56 mg/L) and ITC (0.78 mg/L) at concentration values corresponding to ½ × MIC of each drug] and also to [Cu(phendione)₃]²⁺ (½ × MIC). In addition, combinations of separate samples of AMB, KTC and ITC with [Cu(phendione)₃]²⁺ at these concentrations were also screened for 1 h at 26°C. After exposure, 0.1 ml of each system was plated onto solid Czapek-Dox medium without drugs and incubated for 5 days at 26°C. Fungal growth was estimated by counting colony-forming units (CFU) (Palmeira et al., 2008) and the results were compared to the untreated control.

P. verrucosa conidial cells (5 × 10⁹) were resuspended in 0.2 ml of sterile phosphate-buffered saline (PBS, pH 7.2) supplemented with 2% glucose and incubated with constant agitation (130 rpm). After 2 h, conidia were removed by centrifugation (4000 × g/10 min) and the cell-free PBS-glucose supernatant was subjected to a peptidase activity assay as described by Granato et al. (2015). Briefly, 15 μl of cell-free PBS-glucose supernatant (10 μg of protein) and 1.5 μl HSA (1 mg/ml) were incubated for 20 h at 37°C in the absence (control) or in the presence of the classical metallopeptidase inhibitor, 1,10-phenanthroline (10 mM), and 8 × MIC of the test compounds, phendione, [Ag(phendione)₂]⁺ and [Cu(phendione)₃]²⁺. The reaction mixtures were then added to 15 μl sample buffer (125 mM Tris, pH 6.8, 4% SDS, 20% glycerol, 0.002% bromophenol blue and 10% β-mercaptoethanol), boiled at 100°C for 5 min, and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Electrophoresis was carried out at 4°C, 120 V for 1.5 h. The degradation protein profiles were detected by silver staining (Granato et al., 2015). Densitometric quantification was performed using the free ImageJ software.

Conidia (1 × 10⁷/ml) were incubated in Czapek-Dox medium in the absence (control) or in the presence of sub-inhibitory concentrations (½ × MIC and ¼ × MIC) of phendione, [Ag(phendione)₂]⁺ or [Cu(phendione)₃]²⁺. After 48 h, conidia were washed in PBS and total lipids were extracted with chloroform:methanol (2:1, 1:1 and 1:2). The combined extracts were mixed, dried and Folch partition was then performed (Folch et al., 1957). The lower phase containing the neutral lipids was recovered, evaporated and subjected to high performance thin layer chromatography (HPTLC). Chromatography was carried out on silica gel 60 plates, which were developed with a solvent system containing hexane:ether:acetic acid (60:30:1.5). The spots (violet-red color) were visualized after spraying the plate with a reagent (comprising 50 mg iron chloride, 5 ml sulfuric acid, 5 ml acetic acid and 90 ml distilled water) and subsequent heating (Larsen et al., 2004). The sterol standards, ergosterol (4 μg) and lanosterol (1 μg) were used. Sterol quantitative determination was performed using ImageJ software.

Conidia (1 × 10⁶/ml) were incubated at 26°C in the absence (control) or in the presence of sub-inhibitory concentrations (½ × MIC and ¼ × MIC) of phendione, [Ag(phendione)₂]⁺ or [Cu(phendione)₃]²⁺ in RPMI medium, pH 7.0 (without agitation), in order to induce the filamentation in P. verrucosa. The fungal cells were then observed using a Carl Zeiss MicroImaging GmbH optical microscope and images obtained every 24 h (Granato et al., 2015). Conidial viability after the treatment with the test compounds was performed using the resazurin assay (Liu et al., 2007).

All experiments were performed in triplicate in three independent experimental sets. The graphics and data were constructed and analyzed statistically by means of Student's t-test using GraphPad Prism 5.01 software. P values of 0.05 or less were assumed as significant.

The in vitro antifungal activities of phendione and its Ag⁺ and Cu²⁺ complexes were evaluated against P. verrucosa. All of the test compounds inhibited conidial cell growth with the following activity order based on the IC₅₀ values (μM): [Cu(phendione)₃]²⁺ > [Ag(phendione)₂]⁺ > phendione (Figure 1). Only [Cu(phendione)₃]²⁺ showed a fungicidal effect. These data corroborate previously published results, which revealed that phendione-based metal complexes had higher antimicrobial activity than metal-free phendione toward different classes of microorganisms (McCann et al., 2012a; Viganor et al., 2016).

In order to confirm that the cytotoxic effect observed was due to the complexes, rather than the free Ag⁺ and Cu²⁺ ions, the antifungal activities of the simple perchlorate salts, Cu(ClO₄)₂·6H₂O and AgClO₄, were also assessed (Figure 1). While AgClO₄ showed moderate anti-P. verrucosa activity (MIC 24.0 μM), Cu(ClO₄)₂·6H₂O did not affect fungal growth, even at the maximum test concentration (MIC > 54.0 μM). The results showed that Ag⁺ coordinated to phendione ([Ag(phendione)₂]⁺) was about 6 times more effective (MIC 4.0 μM) than simple AgClO₄ (MIC 24.0 μM). Although, Cu(ClO₄)₂·6H₂O was essentially inactive (MFC > 54.0 μM), Cu²⁺ coordinated to phendione ([Cu(phendione)₃]²⁺) was able to inhibit 100% P. verrucosa proliferation at 5.0 μM. It is believed that the phendione metal complexes have a higher lipophilicity than the simple metal salts (Viganor et al., 2016). This property is due to the total electron density reduction on the free ligand upon complexation to the metal ion, and also to the sharing of the positive charge of the metal cation with N-donor atoms of the phendione ligand, which promotes an electron delocalization all over the chelate ring (Raman et al., 2014; Viganor et al., 2016). Aqueous DMSO, which was used as the solvent for all of the test compounds, was inactive against P. verrucosa conidial proliferation (data not shown). Altogether, the present P. verrucosa growth inhibition results suggest that the activity of the metal complexes as a whole is superior to that of either the free metal ion or metal-free phendione ligand. Ag⁺ (d¹⁰ outer electron configuration) complexes have zero ligand field stabilization energy (LFSE) whilst octahedral Cu²⁺ (d⁹) has only a small amount of LFSE. As such, Ag⁺ and Cu²⁺ complexes are expected to be labile and the original chelating phendione ligands are expected to be rapidly exchanged for biological ligands present within the fungal cells and also in the growth medium (amino acids, proteins, ammonia, chloride etc.). It is thus likely that cell growth inhibition arises due to the destructive interference of cellular processes by the metal ion coupled with inactivation of other cell events by the phendione ligand. It also appears that it is the phendione ligand component of the administered metal complex that plays the dominant role in the demise of the conidia.

Our previous results described that P. verrucosa was also sensitive to 1,10-phenanthroline treatment, showing MIC equal to 4.4 μM (0.8 μg/ml) (Granato et al., 2015). Thus, the antifungal activity of 1,10-phenanthroline was superior to metal-free phendione, but similar to its Ag⁺ and Cu²⁺ complexes. However, neither 1,10-phenanthroline nor phendione presented fungicidal effect against P. verrucosa (Granato et al., 2015).

The effect of phendione, [Ag(phendione)₂]⁺ and [Cu(phendione)₃]²⁺ on P. verrucosa ultrastructure was probed using SEM. In contrast to control cells, which had typical spherical-to-oval morphology (Figures 2A,B), cells exposed to the test compounds exhibited several surface alterations, such as detachment of cell wall components, invaginations, cellular disruptions and shrinkages (Figures 2C–O), which are indicative of cell death. [Cu(phendione)₃]²⁺ was the most aggressive in promoting changes on both conidial architecture and morphology, for example, inducing cell size increases when treated at the MIC concentration (Figure 2H) and cellular debris following incubation with 2 × MIC (Figures 2I,J).

Our previous study showed that 1,10-phenanthroline (25 μM) also caused irreversible ultrastructure alterations on P. verrucosa conidia, including cell wall detachment, intense intracellular vacuolization and reduction in the cytoplasm electron density (Granato et al., 2015). Similarly, phendione and [Ag(phendione)₂]+, both at 10 μg/ml, promoted changes in the internal structure of C. albicans, as observed by transmission electron microscopy (McCann et al., 2004). In that work, phendione promoted an increase in size as well as a reduction in the budding process of C. albicans yeast cells. Furthermore, C. albicans exhibited a diffuse cell wall, rupture of internal organelles and nucleus enlargement following treatment with phendione. C. albicans yeast cells treated with [Ag(phendione)₂]⁺ had a distended cell wall, rupture of membranous organelles and, in some cases, a fragmented nucleus. Phendione and its Ag⁺ complex were both able to cause a withdrawal of the cytoplasmic membrane from within the cell wall in C. albicans (McCann et al., 2004).

In this set of experiments, the most potent test compound, [Cu(phendione)₃]²⁺, was chosen to be combined with a selection of classical antifungal agents in an attempt to check their ability to control P. verrucosa growth. In this context, the combination of [Cu(phendione)₃]²⁺ with AMB, both of which were deployed at (½ × MIC) concentrations, was able to significantly inhibit the fungal proliferation by around 40% (Figure 3). However, at the concentrations used, [Cu(phendione)₃]²⁺ did not positively compliment the activities of either KTC or ITC, since P. verrucosa growth was not affected by these combinations (Figure 3).

Eshwika and coworkers [22] showed that pre-growth of C. albicans with sub-inhibitory concentrations (¼ × MIC₉₀) of [Ag(phendione)₂]⁺ affected the subsequent susceptibility of this yeast to miconazole and AMB, reducing the concentrations of these clinical antifungal agents required to achieve MIC₉₀. Benefits of combination therapy are well-known and include broad spectrum efficacy, greater potency compared to monotherapy, improvements in both safety and tolerability as well as a reduction in the emergence of resistance (Cuenca-Estrella, 2004; Spitzer et al., 2016). Metal-based drugs can have modes of action distinct from classical antifungal agents, allowing their use in cases where there is resistance to conventional therapies. Furthermore, different mode(s) of action can be used by employing such metal-containing drugs in combination with existing antifungals in order to target two (or more) sites in the fungal cells. Thus, there is the potential of achieving the same therapeutic effect by reducing the concentration of clinical drugs used (Eshwika et al., 2004).

[Cu(phendione)₃]²⁺ and [Ag(phendione)₂]⁺ were capable of inhibiting P. verrucosa extracellular metallopeptidase activity by around 85 and 40%, respectively (Figure 4). However, the metal-free ligand, phendione, did not affect this enzymatic activity (Figure 4). The presence of metallopeptidase in P. verrucosa PBS-glucose supernatant was confirmed by its inhibition by 10 mM of 1,10-phenanthroline (Figure 4).

Metal-chelating-type compounds may affect typical functions of several eukaryotic proteins, such as various metallo-type enzymes, including metallopeptidases (McCann et al., 2012b; Santos et al., 2012; Granato et al., 2015). In fact, the inhibition of metallopeptidases by 1,10-phenanthroline occurs mainly due to its ability of promoting Zn²⁺ chelation, which is required for catalytic activity and which leaves an inactive apoenzyme (Santos et al., 2012). Our research group showed previously that Zn²⁺ ions were able to stimulate the enzymatic activity of P. verrucosa extracellular peptidase in a typical dose-dependent manner, suggesting the presence of Zn²⁺-metallopeptidase in this fungus (Granato et al., 2015). It is well-known that metallopeptidase inhibition can prevent fungal cells to obtain necessary peptides and amino acids for nutrition, leading to reduction or complete inhibition of cell growth (Santos et al., 2012).

The treatment of conidial cells with phendione (¼ × MIC and ½ × MIC) and also with [Ag(phendione)₂]⁺ (½ × MIC) promoted a reduction in ergosterol content by approximately 50% in relation to the control (untreated) cells (Figure 5). On the contrary, [Cu(phendione)₃]²⁺ did not affect the sterol content of P. verrucosa (Figure 5).

Previous studies showed that sterol synthesis in C. albicans was also disturbed by phendione and its metal complexes (Eshwika et al., 2004). Interestingly, and in contrast to our results, [Cu(phendione)₃]²⁺ diminished the ergosterol content in C. albicans yeast cells, while [Ag(phendione)₂]⁺ enhanced the amount of this lipid level (Eshwika et al., 2004). It is well-known that sterol deficiency disturbs crucial cell membrane properties, leading to an increased fluidity and permeability, which may cause severe structural aberrations that contributes to cell death (Kathiravan et al., 2012).

The present studies demonstrated that all the test compounds were able to effectively block to a large extent the morphological transition (conidia-into-mycelial transformation) when compared to untreated fungal cells (Figure 6). It is important to notice that hyphal formation was mostly inhibited by phendione, followed by [Ag(phendione)₂]⁺ and then by [Cu(phendione)₃]²⁺ (Figure 6). In fact, several conidia (instead of hyphae form) were observed after the treatment with phendione and [Ag(phendione)₂]⁺, both used at a sub-inhibitory concentration (¼ × MIC). It was also possible to observe some conidial cells in the [Cu(phendione)₃]²⁺ system (¼ × MIC); however, the highest effect of this compound was its considerable ability to reduce hyphal branch growth (Figure 6).

Morphological transition is one of the strategies used by several fungi to survive in environment and in vertebrate hosts (Wang and Lin, 2012). In this context, several studies have shown that cell differentiation is an essential step in the establishment and success of fungal infection (Jacobsen et al., 2012; Wang and Lin, 2012). We have previously demonstrated that chelating compounds, such as 1,10-phenanthroline, were able to inhibit the conidia-into-hyphae transformation in P. verrucosa (Granato et al., 2015). Other studies also reported the same capacity of 1,10-phenanthroline to affect cell differentiation in C. albicans, Pseudallescheria boydii, and Fonsecaea pedrosoi (Santos et al., 2012). The mechanisms involved in the morphological transition of P. verrucosa have not been established. It is well-known that several factors are associated with fungal morphogenesis, including nutritional elements, temperature and aerobic conditions (Wang and Lin, 2012). Mendoza et al. (1993) showed that CBM fungi, including P. verrucosa produced large numbers of sclerotic bodies after inoculation into a defined pH 2.5 medium containing the metal ion Ca²⁺ at 0.1 mM concentration. While higher concentrations of Ca²⁺ (1 mM) reversed this pattern and promoted maintenance of P. verrucosa hyphal growth. Addition of the Ca²⁺ chelator, ethylene glycol tetraacetic acid (EGTA, 8 mM) to the culture medium induced P. verrucosa sclerotic bodies, demonstrating the importance of metal ion Ca²⁺ in this fungal essential process (Mendoza et al., 1993). In this context, these results revealed that metal ion chelating agents can modulate morphological transitions in CBM fungi.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.