Strains H99 of C. neoformans (sorotype A) and R265 of C. gattii (sorotype B) were maintained in Sabouraud’s agar. For capsule size determination and fluorescence microscopy, fungal cells were cultivated in a minimal medium composed of glucose (15 mM), MgSO₄ (10 mM), KH₂PO₄ (29.4 mM), glycine (13 mM), and thiamine-HCl (3 μM), pH5.5 for 48 h at 37°C with shaking. The C. gattii mutant library was maintained in 96-well plates containing yeast peptone-dextrose (YPD) broth with 30% glycerol at -20°C. The cell line J774.16 (murine macrophages) was maintained in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% of Fetal Bovine Serum (FBS), and 1% of penicillin-streptomycin at 37°C in 5% CO₂ atmosphere_. After four passages in culture medium, the macrophages were plated into 96 well plates for tests of mebendazole intracellular activity.

The National Institutes of Health (NIH) clinical collection (NCC) was screened for antifungal activity against C. neoformans. The NCC consists in a small molecule repository of 727 compounds arrayed in 96-well plates at 10 mM solution in DMSO. These compounds are part of screening library for the NIH Roadmap Molecular Libraries Screening Centers Network (MLSCN) and correspond to a collection of chemically diverse compounds that have been in phase I-III clinical trials. Each compound was first diluted to 1 mM in DMSO and stored at -20°C until use. For initial screening, all compounds were used at 10 μM in 100 μl of RPMI 1640 (two times concentrated) medium buffered with morpholinepropanesulfonic acid (MOPS) at pH 7 containing 2% of glucose, in 96-well plates. Final concentration of DMSO in all samples corresponded to 1%. C. neoformans cells (10⁴) suspended in 100 μl of water were added to each well. The plates were incubated at 37°C with shaking for 48 h. The optical density at 540 nm (OD₅₄₀) was recorded using the FilterMax 5 microplate reader (Molecular Devices, Sunnyvale, CA, USA). Compounds producing values of OD₅₄₀ smaller than 0.05 after fungal growth were selected for further studies. As further detailed in the section “Results,” mebendazole was the NCC compound selected for most of the analysis performed in this study.

Values of minimum inhibitory concentrations (MICs) were determined using the methods proposed by the European Committee on Antimicrobial Susceptibility Testing (EUCAST) with minor modifications. NCC compounds showing antifungal activity were serially diluted (20 to 0.03 μM) in RPMI 1640 (two times concentrated, pH 7; 2% glucose) buffered with MOPS in 96-well plates. The inocula of C. neoformans (strain H99) and C. gattii (strain R265) were prepared following the EUCAST protocol (Subcommittee on Antifungal Susceptibility Testing of the EECfAST, 2008; Arendrup et al., 2012). The plates were incubated at 37°C with shaking for 48 h. MIC values corresponded to the lowest compound concentration producing inhibition of fungal growth. For determination of fungicidal activity, C. neoformans cells were grown overnight in YPD broth at 30°C, washed in PBS and suspended in RPMI 1640 buffered with MOPS, pH 7. The yeast suspension was adjusted to 2 × 10⁴ cells per 10 ml of RPMI 1640 (pH 7; 2% glucose) buffered with MOPS and supplemented with 1.25, 0.3125, and 0.078 μM of antifungal compounds. Final concentration of DMSO corresponded to 0.6% in all samples. The samples were then incubated at 37°C in a rotary shaker at 200 rpm. Aliquots were taken at different time points and plated onto YPD agar plates that were incubated at 37°C for 48 h. The numbers of CFU were then counted and recorded. The minimum fungicidal concentration (MFC) was defined as the lowest drug concentration inhibiting CFU formation in at least 90% in comparison to systems containing no antifungals.

To assess antifungal activity against intracellular C. neoformans, the fungus was first opsonized by incubation (20 min, 37°C, with shaking) in DMEM containing 10% FBS and 10 μg/ml of the 18B7 IgG1, an opsonic monoclonal antibody to GXM (Casadevall et al., 1998) kindly donated by Dr. Arturo Casadevall (Johns Hopkins University). The fungus was washed with fresh DMEM and incubated with J774.16 macrophages (1:1 ratio, 5 × 10⁵ cells/well of 96 well-plates) for 2 h in DMEM containing 10% FBS, 0.3 μg/ml LPS and 0.005 μg/ml IFNγ (37°C, 5% CO₂). After interaction of C. neoformans with macrophages, the systems were washed with DMEM to remove extracellular fungal cells and fresh DMEM containing 10% FBS and variable concentrations of mebendazole (0.25, 0.5, and 1 μM, 200 μl/well) was added to each well. The plates were incubated at 37°C with 5% CO₂. After 8 or 24 h, supernatants were collected for inoculation of YPD agar plates and subsequent CFU counting. Alternatively, infected macrophages were lysed with cold, distillated water and the resulting lysates were plated onto YPD agar plates for CFU counting. To evaluate the toxicity of mebendazole for J774.16 macrophages, 5 × 10⁵ macrophages suspended in DMEM containing 10% FBS were plated in each well of 96 wells plates and incubated overnight at 37°C with 5% CO₂. The medium was supplemented with fresh DMEM containing 10% FBS and mebendazole (0.25, 0.5, and 1 μM) or 0.5% DMSO. The plate was incubated at 37°C in a 5% CO₂ atmosphere. After 48 h, the systems were washed with DMEM and 50 μl of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) at 5 mg/ml was added to each well, for further incubation for 4 h (37°C, 5% CO₂) under light protection (Mosmann, 1983). Supernatants were removed and 200 μl DMSO was added for dissolution of formazan crystals. Absorbances were recorded using the FilterMax 5 microplate reader (Molecular Devices, Sunnyvale, CA, USA) at 570 nm.

Synergistic activity between mebendazole and standard antifungal drugs was determined on the basis of the calculation of the fractional inhibitory index (FIC) (Mor et al., 2015). Briefly, mebendazole (denominated drug A) was serially diluted (0.38–0.006 μg/ml, 8 dilutions) in 96-well plates. Standard antifungals (denominated drugs B) were serially diluted (11 dilutions) from 16 to 0.015 μg/ml (amphotericin B) or 64 to 0.06 μg/ml (fluconazole). The FIC was defined as:

Synergism was categorized as follows: strongly synergistic effect, FIC < 0.5; synergistic effect, FIC < 1; additive effect, FIC = 1; no effect, 1 < FIC < 2; antagonistic effect, FIC > 2 (Mor et al., 2015).

Cryptococcus neoformans cells (10⁵/well of 96-well plates, final volume of 200 μl, duplicates) were cultivated in RPMI 1640 buffered with MOPS, pH 7. The medium was supplemented with mebendazole (0.3125–0.001 μM). After 48 h of incubation at 37°C with shaking, the optical density of 540 nm (OD₅₄₀) was recorded using the FilterMax 5 microplate reader (Molecular Devices, Sunnyvale, CA, USA). The plate was centrifuged for 10 min and supernatants were used for GXM quantification by ELISA using the protocol described by Casadevall et al. (1992). C. neoformans viability was monitored by propidium iodide (PI) staining of fungal cells. For this analysis, fungal cells obtained after exposure to 0.3125, 0.15625, and 0.078 μM mebendazole as described above were stained with 1 mg/ml PI for 5 min on ice and analyzed by flow cytometry in a FACS Cabibur (BD Biosciences, CA, USA). The percentage of stained (non-viable) cells was obtained with the FlowJo 7 software.

Cryptococcus neoformans was grown overnight in YPD broth and washed twice with PBS. The fungus was then suspended in minimal medium containing sub-inhibitory concentrations of mebendazole and incubated for 48 h at 37°C with shaking. C. neoformans cells were collected by centrifugation, washed in PBS and analyzed by microscopic approaches. For capsule size determination, the suspension was counterstained with India ink and placed onto glass slides. The suspensions were covered with glass coverslips and analyzed with an Axioplan 2 (Zeiss, Germany) microscope. Capsule size, calculated with the ImageJ Software, was defined as the distance between the cell wall and the outer border of the capsule. Cell diameters were determined using the same software. For additional analysis of capsular morphology, cellular suspensions were processed for fluorescence microscopy. Staining reagents used in this analysis-included calcofluor white (cell wall chitin, blue fluorescence) and the monoclonal antibody 18B7 (Casadevall et al., 1998). C. neoformans cells were prepared for fluorescence microscopy following the protocols established by our laboratory for routinely analysis of surface architecture (Rodrigues et al., 2008).

Cryptococcus neoformans was grown in Sabouraud’s dextrose broth for 24 h at 30°C with shaking. The cells were centrifuged at 3,000 g for 5 min, washed twice with PBS, suspended in minimal medium and adjusted to a density of 10⁷ cells/ml. Cell suspensions (100 μl) were added into quadruplicate wells of polystyrene 96-well plates (Greiner Bio-One, Australia), following incubation at 37°C for 48 h. The wells containing biofilms were washed three times with PBS to remove non-adhered cryptococcal cells. Fungal cells that remained attached to the wells were considered mature biofilms. To evaluate the susceptibility of C. neoformans biofilms to mebendazole, 100 μl solutions (31.25, 15.63, 3.13, 1.56, 0.31, and 0.16 μM) were added to each well. Amphotericin B and fluconazole (2 and 8 μg/ml, respectively) were used as control systems of antifungal activity. Negative controls corresponded to wells containing only water and untreated biofilms. Mature biofilms and antifungal drugs were incubated at 37°C for 24 h, washed three times with PBS and the biofilm metabolic activity quantified by the 2,3-bis (2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino) carbonyl]-2H-tetrazolium hydroxide (XTT) reduction assay (Meshulam et al., 1995). Prior studies demonstrated that the XTT reduction assay measurements correlate with biofilm and fungal cell number (Martinez and Casadevall, 2007). In addition to testing the effects of mebendazole on established biofilms, we evaluated whether this compound would inhibit biofilm formation. Cryptococcal cells were suspended in minimal medium and adjusted to a density of 10⁷ cells/ml in the presence or absence of mebendazole (31.25, 15.63, 3.13, 1.56, 0.31, and 0.16 μM). These suspensions were added in quadruplicates to the wells of polystyrene 96-well plates, following incubation at 37°C for 48 h. Amphotericin B and fluconazole (2 and 8 μg/ml, respectively) were used as antifungal controls. In negative controls, wells contained only ultrapure water. The wells were washed three times with PBS and biofilm formation was quantified by the XTT assay.

A collection of randomly generated C. gattii mutants was screened for identification of possible cellular targets for antifungal activity. Mutants (n = 7,569) were generated by insertional mutagenesis after incubation of C. gattii with Agrobacterium tumefaciens as previously described (Idnurm et al., 2004). All colonies that grew on YPD hygromycin plates were selected and maintained at -20°C in 96 wells plates containing 200 μl/well of YPD broth. Before exposure to mebendazole, mutant cells were first grown for 72 h (30°C) in 200 μl of YPD distributed into the wells of 96 wells plates. The antifungal activity of mebendazole against C. neoformans was reproduced in the C. gattii R265 strain. The mutants were tested for their ability to grow in the presence of RPMI 1640 supplemented with MOPS, 2% of glucose, 1% DMSO and 10 μM mebendazole. Resistance phenotypes (A₅₄₀ > 0.3) were selected for dose-response tests of antifungal activity as described above and potential target identification was performed as detailed below.

Based on clear resistant phenotypes, two C. gattii mutant strains were selected for target identification by polymerase chain reaction (PCR). The fungus was cultivated overnight in 10 ml YPD at 30°C with constant rotation. Each culture (2 ml) was centrifuged for 2 min at 4,000 × g and cell pellets were washed twice with PBS and collected for DNA extraction (Bolano et al., 2001). The cells were suspended in 400 μl of lysis buffer (50 mM Tris-HCl 1 mM EDTA, 200 mM NaCl, 2% Triton X100, 0.5% SDS, pH 7.5), for further addition of 1 volume of a phenol-chloroform mixture (pH 8) and 100 μl of 2-μm, acid washed glass beads. Mechanical disruption was performed by alternate 1-min cycles of vortexing and ice incubation. Lysates were centrifuged 4,000 × g for 20 min at 4°C. Supernatants were collected and DNA was ethanol-precipitated overnight at -20°C for subsequent treatment with RNAse (Bolano et al., 2001). Identification of missing genes in the mutants was performed using inverse PCR (Pavlopoulos, 2011). DNA was quantified using the Qubit reagent (Invitrogen) and 1 μg of each sample was cleaved separately with BglII, SalI, or StuI (Promega) restriction enzymes. The cleavage product was submitted to T4 DNA ligase reaction (New England) followed by PCR using primers for inverse PCR. Amplicons were gel-purified with the QIAquick Gel Extraction Kit (Qiagen). For the DNA sequencing reaction, 50 ng of each sample and 5 pmol of each primer were used. Sequences were obtained in an ABI-Prism 3500 Genetic Analyzer (Applied Biosystems) and their qualities were determined by a electropherogram analysis based on phred¹. The identification of genes interrupted by the T-DNA was performed by comparison of each sequenced DNA fragment, which correspond to the T-DNA flanks, with the genomic sequence of C. neoformans H99 strain, available in the Cryptococcus genome databases (Broad Institute²) using BLASTn. Orthologs distribution was evaluated using the OrthoMCL database (Chen et al., 2006).

Mutants showing resistance to mebendazole were analyzed for their ability to produce melanin and extracellular GXM as previously described by our group (Rodrigues et al., 2015).

Statistics were obtained with the GraphPad Prism 6.0 software. Unpaired t student test was used for mebendazole toxicity analysis. The variance two-way ANOVA was carried out using Tukey’s and Bonferroni’s comparisons test for fungicidal activity and intracellular activity of mebendazole in macrophages. For biofilm analyses, one-way ANOVA was performed using Dunnett’s multiple comparisons.

Of the 727 drugs tested at 10 μM, 17 compounds were active against C. neoformans, including antibacterials (chloroxine, cycloserine, and linezolid), a neuroleptic drug (mezoridazine), calcium channel blockers (nisoldipine and enalaprilat), antiarrhythmic agents (flecainide acetate), drugs for gastrointestinal malignances (irsogladine maleate and cisapride), gynecologic regulators (medroxyprogesterone acetate and clomid), an anti-histaminic (olopatadine) and the anti-helminthic benzimidazoles (mebendazole, albendazole, flubendazole, and triclabendazole) (Figure 1). Noteworthy, none of the molecules showing antifungal activity were structurally related, except the benzimidazoles. Considering the currently observed efficacy in inhibiting the growth of C. neoformans, their structural similarity and previously described anti-cryptococcal activity (Cruz et al., 1994), we selected benzimidazoles for further investigation in our model.

We extended the results obtained with the compound collection to dose-response tests using the four benzimidazoles showing antifungal activity and other related molecules, including thiabendazole, oxibendazole, and fenbendazole. The most active compounds were mebendazole, fenbendazole, and flubendazole (Figure 2). Mebendazole and flubendazole produced the same MIC values (0.3125 μM). Fenbendazole was the most potent compound, with a MIC corresponding to 0.039 μM. Mebendazole, however, efficiently penetrates the brain in animal models (Bai et al., 2015) and is in clinical trial for the treatment of pediatric gliomas in humans (ClinicalTrials.gov, 2013). Considering that the worse clinical outcome of cryptococcosis includes colonization of the brain, we selected mebendazole as the molecular prototype for our subsequent analyses of fungicidal effects, ability to kill intracellular cryptococci, effects on fungal morphology, interference on fungal biofilms and identification of cellular targets.

To test the fungicidal activity of mebendazole, C. neoformans was exposed to different drug concentrations for different periods of time. Mebendazole concentrations lower than 0.3125 μM had no effects on C. neoformans (Figure 3). At 0.3125 μM or higher, however, significant antifungal activity was observed after 6 to 12 h of exposure of C. neoformans to the drug. After 48 h, mebendazole killed 100% of C. neoformans cells.

Cryptococcus neoformans is a facultative intracellular pathogen and this characteristic likely has a negative impact on the anti-cryptococcal treatment (Feldmesser et al., 2000). We then evaluated the ability of mebendazole to kill intracellular fungi. J774.16 macrophages were first infected with C. neoformans and then the cultures were treated with mebendazole for different periods (8 and 24 h) at variable drug concentrations (1, 0.5, and 0.25 μM). Macrophage viability was monitored by treating non-infected J774.16 cells with mebendazole alone (Figure 4A). After 8 h, no differences were observed between the viability of untreated macrophages and mebendazole-treated cells (P > 0.1). After 24 h, macrophage viability was affected by higher mebendazole concentrations (P = 0.0071 for 1 μM mebendazole), but no differences were observed between untreated phagocytes and cells that were exposed to 0.25 μM mebendazole (P = 0.3225). In infected macrophages, all mebendazole concentrations showed antifungal activity against both extracellular and intracellular fungi (Figure 4B). The concentration showing the lowest impact on macrophage viability (0.25 μM) was efficient in killing both intracellular and extracellular C. neoformans.

To evaluate whether the association of mebendazole with amphotericin B or fluconazole results in improved anti-cryptococcal activity, checkerboard assays were performed for calculation of the FIC index (Table 1). We found that mebendazole had additive activity against C. neoformans when combined with amphotericin B. Fluconazole did not show any improved effect in combination with mebendazole.

To evaluate whether mebendazole affected key cellular structures of C. neoformans during regular growth, we cultivated the fungus for 48 h in the presence of a sub-inhibitory concentration (IC50; 0.22 μM) of the drug for further analysis of morphology and capsule size by a combination of fluorescence microscopy and India ink counterstaining. Fungal cells cultivated in the presence of mebendazole presented marked morphological alterations, including loss of spherical shape, intracellular furrows (Figure 5A) and reduced capsular dimensions (Figure 5B, P < 0.05).

Since mebendazole interfered with capsule size (Figure 5B), we asked whether GXM release was affected by exposure of C. neoformans to the drug. Supernatants of fungal cells cultivated in the presence of mebendazole were used for GXM quantification by ELISA (Figure 6). Unexpectedly, supernatants of mebendazole-treated cells had increased GXM concentration, mainly in the dose range required for fungal killing. Since the polysaccharide is synthesized intracellularly (Yoneda and Doering, 2006), we hypothesized that the increased GXM detection would result from leakage induced by membrane damage. To address this possibility, mebendazole-treated cells were stained with PI. Stained cells varied from 70 to 90% in the dose range generating cell death, which was compatible with membrane damage and polysaccharide leakage. Noteworthy, these results and those described in Figure 5 are also compatible with the hypothesis of GXM release from the cell surface, so we cannot rule out the possibility that mebendazole promotes detachment of capsular structures in C. neoformans.

Biofilm formation causes well-known difficulties in the treatment of a number of infectious diseases, including cryptococcosis (Martinez and Casadevall, 2006; Borghi et al., 2016). Based on this observation, we evaluated whether co-incubation of mebendazole with yeast cells prevented C. neoformans biofilm formation or caused damage to mature biofilms. The metabolic activity was measured by XTT reduction assay (Figure 7). Fluconazole is known to have no effects on mature biofilms, in contrast to amphotericin B (Martinez and Casadevall, 2006). Therefore, these drugs were used as negative and positive controls, respectively. Mebendazole at MIC (0.3125 μM) affected biofilm formation (Figure 7A; P < 0.0001) and damaged mature biofilms (Figure 7B; P < 0.0001). Lower concentrations of mebendazole similarly affected C. neoformans mature biofilms (Figure 7B; P < 0.0001). As expected, higher concentrations of mebendazole had even clearer impacts on C. neoformans biofilms.

Antifungal activities of benzimidazoles were described before (Cruz et al., 1994), but the mechanisms by which these drugs affected cryptococcal growth remained unknown. In order to identify potential targets for mebendazole in Cryptococcus spp., we first evaluated whether the drug affected the growth of C. gattii and C. neoformans in a similar fashion. In fact, growth inhibition curves were identical for both pathogens (Figure 8A).

A mutant collection generated by co-incubation of C. gattii and A. tumefaciens (Idnurm et al., 2004) was produced by our group for general purposes involving development of antifungals and pathogenic studies. In the present study, these mutants were screened for resistance phenotypes in the presence of 10 μM mebendazole, based on the assumption that, in the absence of a cellular target required for antifungal activity, the drug would lack anti-cryptococcal properties. Most of the mutants were sensitive to mebendazole and a number of strains were partially resistant to the drug (Figure 8B). However, two of the mutant strains clearly stood out from the entire collection, showing highest levels of resistance even in higher mebendazole concentrations (Figure 8C). Interrupted regions in these two mutants were identified by inverse PCR (Figure 8D). In the most mebendazole-resistant mutant, the protein codified by the interrupted gene contained a scramblase domain (PF03803 – CNBG_3981). In the second most resistant mutant, the gene coding for ribosome biogenesis protein Nop16 (PF09420 – CNBG_3695) was interrupted. These domains were found in diverse phylogenetic groups, according to the PFAM database (Finn et al., 2016). However, orthologs for these genes were found in a narrow group of organisms and absent in human cells (Figure 8D), according to OrthoMCL database (Chen et al., 2006). These results indicate that at least two novel cellular targets are involved in the antifungal activity of mebendazole.

Neutralizing cryptococcal virulence factors is also likely to be beneficial for the control of cryptococcosis. In this context, we evaluated whether the mutants lacking potential targets for activity of mebendazole had normal production of the most-well characterized cryptococcal virulence factors. The two mutants had normal urease activity (not shown). Analysis of extracellular GXM and pigmentation, however, showed important differences between WT and mutant cells (Figure 9). Mutants disrupted for expression of the putative scramblase and of nucleolar protein 16 had decreased contents of extracellular GXM, in comparison with WT cells (P < 0.001). The kinetics of melanin production was also negatively affected in the mutants.