All assays involving mammals were conducted in two laboratories with GLP compliance status: Covance Laboratories (England), and Eurofins PSL – Product Safety Labs (United States). At Covance, the following studies were performed in accordance with the requirements of the Animals (Scientific Procedures) Act 1986 and all protocols were previously subjected to the site Ethical Review Process (EPR). For the 21-day study, animal testing was performed under the approval of the Research Ethics Committee (UK Health Departments’ Research Ethics Service, National Health Service, England), with the project license number PPL 70/7602-2. At Eurofins PSL, all acute toxicological studies were performed in accordance with the requirements of 40 CFR Part 160: U.S. EPA (FIFRA), 1989, OECD (as revised in 1997) published in ENV/MC/CHEM (98)17, OECD, Paris, 1998; and EC Directive 2004/10/EC, Official Journal of the European Union, L50/44, Feb. 20, 2004. EUROFINS Product Safety Labs (PSL, United States), is AAALAC accredited, GLP compliant and USDA registered. All protocols of the acute toxicological studies were reviewed and unanimously approved by the Internal Animal Care and Use Committee (IACUC) on the fourteen of May 2010: acute dermal toxicity (PSL protocol #P322), primary eye irritation (PSL protocol #324), dermal sensitization (PSL protocol #P328) and primary skin irritation (PSL protocol #P326).

Saccharomyces cerevisiae BY4743 and Candida albicans CBS 562 were grown in Glucose-Yeast extract-Peptone (GYP) medium (0.5% w/v peptone, 0.5% w/v yeast extract, 2% w/v glucose, and 1.5% w/v agar), for 24 h, at 30 and 34°C, respectively. For the assays described here, the media used were: Synthetic Complete (SC) broth with 2% (w/v) glucose (FORMEDIUM) for S. cerevisiae, and Potato-Dextrose-Broth (PDB, DIFCO) buffered at pH 7.5 and Yeast extract-peptone-Dextrose medium (YPD: 2% w/v peptone, 1% w/v yeast extract, and 2% w/v glucose) for C. albicans.

Lupinus albus L. seeds were purchased from Inveja SAS (France) and were germinated and grown in growth chambers with a photoperiod of 16 h light/8 h dark at 18°C, for periods up to 10 days. The seed coats were removed and the intact cotyledons were dissected from the axes and stored frozen at -80°C until needed.

Blad-containing oligomer (BCO) was extracted and purified from 8-days-old cotyledons as previously described (Monteiro et al., 2010) and stored lyophilized at room temperature. Stock solutions of AMB, FLC, and caspofungin (all from SIGMA) were prepared and stored frozen at -20°C until used.

The transcriptome profiling of C. albicans was analyzed by RNA-sequencing (RNA-seq). Two cultures of C. albicans were grown as described below in the ‘Determination of endogenous ROS production’ section, one in the presence of BCO, and the other in its absence (control). After 4 h of incubation, cells were harvested and subjected to a treatment with 0.4 mg/mL lyticase in 50 mM Tris-HCl pH 7.5, 1 M sorbitol, 0.8 M KCl, 10 mM MgSO₄, and 15 mM β-mercaptoethanol for 1 h at 30°C with gentle stirring to digest the cell wall. Spheroplasts were pelleted by centrifugation and their RNA extracted according to the instructions of the RNA isolation kit (RNeasy Mini Kit – QIAGEN) and stored at -80°C.

Poly(A) mRNA was purified from ca. 25 μg total RNA with two rounds of Dynabeads mRNA DIRECT Micro Purification Kit (Invitrogen). cDNA libraries were constructed with the Ion Total RNA-Seq Kit v2 (Life Technologies) and quantified with Agilent DNA 1000 Kit in the Agilent 2100 Bioanalyzer (Agilent Technologies). The fragments of six barcoded libraries were pooled and clonally amplified by emulsion PCR using the Ion PI Template OT2 200 kit v2 and the Ion OneTouch 2 System (Life Technologies), and the positive Ion Sphere Particles enriched with Ion OneTouch ES (Life Technologies). This procedure was performed twice for a total of two groups of clonally amplified library spheres. Finally, the positive ion spheres were loaded into two Ion PI chip v2 and sequenced in the Ion Proton System (Life Technologies) and Ion PI Sequencing 200 Kit v2 at Genoinseq (Biocant, Cantanhede, Portugal). Ion Proton adapter sequences and low-quality bases were trimmed using the Torrent Suite software (Life Technologies). Duplicate reads were removed using PRINSEQ (Schmieder and Edwards, 2011) and rRNA reads were removed using RiboPicker (Schmieder et al., 2012). The remaining reads were mapped using TMAP version 4.0.6 (Life Technologies) against the reference transcriptome of C. albicans (genome C. albicans SC5314 A22-s06-m01-r05) (Skrzypek et al., 2016). Read count was performed by eXpress¹ for each condition (Roberts and Pachter, 2012). Gene name and description for each reference transcript were extracted from the Candida Genome Database annotation files (Skrzypek et al., 2016).

Read counts were uploaded to the Degust² and analyzed using Limma/Voom method, which was incorporated into Degust. Coding sequences with a false discovery rate (FDR) < 0.1 and a fold change greater than 1, were considered significant.

In this study we used S. cerevisiae as model organism, rather thanC. albicans, since this is the only yeast species for which a complete deletion mutant strain collection exists (Giaever and Nislow, 2014). BCO was tested on S. cerevisiae gene deletion strains, purchased from EUROSCARF (Supplementary Table S1). The 27 selected strains have been previously determined to be characteristic for common modes of action in which a specific target protein is lacking (Hoepfner et al., 2014) and constitute a mini HIP HOP assay (Prescott and Panaretou, 2017). Heterozygous diploids: nmd3Δ/NMD3, rei1Δ/REI1, lsg1Δ/LSG1, ssl2Δ/SSL2, neo1Δ/NEO1, tim54Δ/TIM54, pik1Δ/PIK1, cmd1Δ/CMD1, tda10Δ/TDA10, mia40Δ/MIA40, tom40Δ/TOM40, and mrpl19Δ/MRPL19. Homozygous diploids: lem3Δ/lem3Δ, fen1Δ/fen1Δ, lro1Δ/lro1Δ, aft1Δ/aft1Δ, ftr1Δ/ftr1Δ, ctr1Δ/ctr1Δ, fet3Δ/fet3Δ, ire1Δ/ire1Δ, hac1Δ/hac1Δ, sur4Δ/sur4Δ, slt2Δ/slt2Δ, aro1Δ/aro1Δ, trp4Δ/trp4Δ, bck1Δ/bck1Δ, and rim101Δ/rim10 Δ. The diploids have the following isogenic background: MATa/α; his3Δ1/his3Δ1; leu2Δ0/leu Δ0; met15Δ0/MET15; LYS2/lys2Δ0; ura3Δ0/ura3Δ0 with genes deleted with KANMX4. Cultures of each strain were grown to late log phase and their optical density determined with a 1 cm path length spectrophotometer. The cultures were then adjusted to OD₆₀₀ 0.00015 in SC medium and transferred to a transparent polystyrene 384 well microplate. Each microplate well contained 50 μl yeast culture in liquid SC medium with or without BCO. The microplate with lid was then incubated at 30°C in a Tecan Infinite M200 plate reader recording OD₆₀₀ readings in 9 separate locations per well every 20 min between 5 and 27 h. Growth inhibition over 24 h was determined for each strain relative to the corresponding untreated control. The same procedure was carried out for the isogenic control strain BY4743. Each curve is an average of 12 replicate wells.

To test the effect of metallic ions on BCO antifungal activity, susceptibility tests were performed on C. albicans in the presence of increasing concentrations of these elements. The susceptibility tests were made according to the CLSI – Clinical and Laboratory Standards Institute (former NCCLS – National Committee for Clinical Laboratory Standards) guideline M27-A2 (NCCLS, 2002), with some adjustments, using broth microdilution method, as previously described (Pinheiro et al., 2016). Six different cations were supplied, separately, in the culture medium (PDB): copper (CuCl₂ and CuSO₄.5H₂O), calcium [CaCl₂.2H₂O, CaSO₄ and Ca(NO₃)₂], magnesium (MgCl₂.10H₂O and MgSO₄.7H₂O), iron (FeCl₂ and FeSO₄.7H₂O), zinc (ZnCl₂ and ZnSO₄.7H₂O), and manganese (ZnCl₂ and ZnSO₄.7H₂O). Each cation was tested under different salt forms (described above in round brackets) separately, in order to determine any possible influence of the anion in the results. First, the minimum toxic concentration for each of these cations was determined with concentrations ranging from 4.88 μM to 10 mM. After determining the toxicity lower limit, progressively lower concentrations of each cation were tested with BCO. A culture medium without supplemented cations and with BCO was used as control. All assays were performed in triplicate. The content of each cation in the unsupplemented (original) PDB medium was quantified prior to the addition of the salts. A mixture of three different batches of PDB was homogenized and a single analysis was performed. The mixture was previously acid digested with micro-waves, and the content of the cations was determined by inductively coupled plasma optical emission spectrometry (ICP-OES) (ISO 11885, 2007).

A fresh culture of C. albicans was inoculated in 50 mL YPD medium for 24 h at 34°C, without shaking containing a sub-inhibitory concentration of BCO (2.4 μM). Sub-inhibitory concentrations of FLC (208 μM), AMB (0.27 μM), and caspofungin (26 μM) were also tested as references of ergosterol targeted modes of action (positive control – FLC; negative control – caspofungin; multitarget mode of action – AMB). Cells were harvested by centrifugation and washed with distilled water. Total intracellular sterols were extracted and quantified as previously described (Arthington-Skaggs et al., 1999). Statistical analysis was performed using GraphPad Prism software version 5.02, La Jolla, CA, United States. P-values were calculated using one-way ANOVA and Bonferroni multiple comparison post-test.

A fresh culture of C. albicans was inoculated (1 × 10⁵ CFU/mL) in PDB pH 7.5 with 2.4 μM BCO and incubated at 34°C without shaking. After 4 h incubation, intracellular reactive oxygen species (ROS) levels were measured by a fluorometric assay using the fluorescent dye 2,7-dichloro-dihydro-fluorescein diacetate (DCFH-DA; Sigma) as a ROS indicator, as previously described (Favre et al., 2008). PDB pH 7.5 was used as negative control and PDB pH 7.5 with 20 mM H₂O₂ (added 30 min before ROS analysis) as positive control.

Annexin V/propidium iodide (PI) binding assays were performed according to the FITC Annexin V/Dead Cell Apoptosis Kit (Molecular Probes), with minor modifications, as previously described (Du et al., 2008). Two cultures of C. albicans were grown as described in the ‘Determination of endogenous ROS production’ section, one in the presence of BCO, and the other in its absence (control).

All samples were observed under a fluorescence microscope (Axioscope A1 with phase contrast and epi-fluorescence, Zeiss) equipped with a camera (AxioCam ICm1, Zeiss), using three different filters: Filter Set 49 DAPI (Excitation G 365, Emission BP 420/470); Filter Set 10 FITC/GFP (Excitation BP 450–490, Emission BP 515–565); and Filter Set 15 Rhodamine (Excitation BP 540–552, Emission LP 590).

To analyze BCO genome-wide effects and to identify the mechanisms underlying its fungal growth inhibition (Pinheiro et al., 2016), RNA-seq was employed to analyze C. albicans transcriptome upon exposure to BCO for 4 h. A total of 124 genes were found to be differentially expressed. Of these, 80 were up-regulated and 44 were down-regulated comparatively to untreated yeast cultures. The functional distribution of BCO responsive genes is shown in Figure 1.

To confirm some of these results, a yeast chemical genetics approach was carried out using a short list of S. cerevisiae mutant strains based on their known hypersensitive profiles. The selected 27 profiles corresponded to 10 common modes of action described for antifungal drugs (Supplementary Table S1). Each yeast deletion strain was screened for hypersensitivity to BCO, as well as the parental strain, using a previously determined (Supplementary Figure S1) sublethal dose of BCO (0.024 μM). The results presented in Figure 2 clearly show that, under these conditions, the only deletion strain displaying a marked hypersensitivity to the BCO sublethal concentration corresponded to the mutant lacking the transcription factor Aft1, a gene involved on iron utilization and homeostasis (Yamaguchi-lwai et al., 1995).

Considering the results obtained in the experiments above, namely the hypersensitivity of the yeast mutant strain aft1Δ/aft1Δ and the effect on ion transport/homeostasis responsive genes, susceptibility tests to BCO were conducted with C. albicans cells in PDB medium supplemented with six divalent cations. The following concentrations of each cation was quantified in the original (unsupplemented) PDB medium: 8 μM Cu²⁺, 18 μM Ca²⁺, 59 μM Mg²⁺, 2 μM Fe²⁺, 3 μM Zn²⁺, and 0.09 μM Mn²⁺. These concentrations are negligible compared to the concentrations added and therefore, only the supplemented concentration was considered for the analysis of the results (Table 1). Considering the inherent toxicity of each cation to the cells (Table 1), sub-toxic concentrations were tested to assess their individual effect on the activity of BCO against C. albicans. As shown in Table 1, all cations induced a marked increase in both MIC and MFC values, thus expressing a clear decrease in the antifungal activity of BCO. Ultimately, albeit at different concentrations, all cations were able to neutralize the activity of BCO. The cation potency to neutralize BCO was lower for magnesium, followed by copper, calcium, and iron. Manganese and specially zinc were the cations with the strongest influence on BCO. No differences were found for distinct salts of the same cation nor between replicates.

The set of genes in the category of lipid, fatty acid, and sterol metabolism was among the most responsive in the RNA-seq experiment (Figure 1) and, therefore, the effect of BCO on the ergosterol content of C. albicans plasma membrane was assessed. As a positive control, total ergosterol content was also determined after exposing the cells to FLC. AMB was used as a reference control for a cell membrane disruption mode of action without direct interference in the biosynthesis of ergosterol. Caspofungin was used as negative control. The results (Figure 3) show that C. albicans suffered a 91% reduction in its ergosterol contact when exposed to FLC, as compared to the control. This reduction was significantly lower when the culture was exposed to AMB (71%) or BCO (69%) whereas caspofungin did not interfere with the ergosterol content at all. No statistical differences were observed in the reduction of the ergosterol content between AMB and BCO.

The third interesting set of responsive genes obtained with the RNA-seq study was the oxidative stress associated genes (Figure 1). This gene family has long been associated to cell apoptosis and previous studies have already shown the induction of cell death by BCO (Pinheiro et al., 2016). To investigate whether BCO lethal effect occurs via induction of apoptosis, and/or necrosis, annexin V and PI staining were performed (Figure 4). An early marker of apoptosis is the exposure of phosphatidylserine (PS) on the cell surface, since it is normally located in the luminal layer of the cytoplasmic membrane (Martin et al., 1995; Madeo et al., 1997). Annexin V displays a high affinity for PS and its previous labeling with fluorescein isothiocyanate (FITC) allows the visual identification of apoptotic cells under a fluorescence microscope. Staining with PI allows detection of damaged membranes. Therefore, differential staining patterns discriminate among live cells (annexin V-/PI-), early apoptosis (annexin V+/PI-), necrosis (annexin V-/PI+), and late apoptosis/necrosis (annexin V+/PI+) (Hao et al., 2013). The majority of C. albicans cells exposed for 4 h to 2.4 μM BCO were only green in color, which is indicative of early apoptosis (Figure 4A). Some cells were stained with both dyes, meaning that, at this point, a minority of cells was undergoing late apoptosis (Figures 4A,B). Cells grown in the absence of BCO were not stained by annexin V or PI (data not shown).

Production of reactive oxidative species (ROS) by C. albicans exposed for 4 h to 2.4 μM BCO was analyzed with DCFH-DA, a non-fluorescent ROS indicator which diffuses across the cell membrane, undergoes oxidation and emits green fluorescence (Figure 5). Appropriate controls were cells grown in the absence of BCO (negative control) and cells exposed to 20 mM H₂O₂ for 30 min (positive control). After 4 h of incubation with BCO, ROS accumulation was already detectable in some cells (Figure 5A). The negative control did not present DCFH-DA staining, whereas in the positive control 100% cells were stained (data not shown).

In the 22-day dermal test, no clinical observations of toxicological importance, no deaths and no clinical signs of toxicity at necropsy were noted in all groups of rats tested [Crl:WI (Han) rats, 5 sex^-1 dose^-1].