Strains and media
A minimal medium (MM: 2 % sucrose, 0.3 % NaNO3, 0.05 % KCl, 0.05 % MgSO4 × 7 H2O, 0.005 % FeSO4 × 7 H2O (w/v), 0.25 % 1 M potassium phosphate buffer pH 5.8, 0.01 % trace element solution (v/v); trace element solution: 0.1 % FeSO4 × 7 H2O, 0.9 % ZnSO4 × 7 H2O, 0.4 % CuSO4 × 5 H2O, 0.01 % MnSO4 × H2O, 0.01 % H3BO3, 0.01 % Na2MoO4 × 2 H2O (w/v)) was used to produce NFAP2 by N. fischeri NRRL 181 strain (Agricultural Research Service Culture Collection, National Center for Agricultural Utilization Research, Peoria, Illinois USA).
The antifungal activity of NFAP2, NFAP, and conventional antifungal agents was investigated against nine yeasts (Candida albicans American Type Culture Collection, Manassas, VA, USA, ATCC 10231; Candida glabrata Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands, CBS 138; Candida guilliermondii CBS 566; Candida krusei CBS 573; Candida lusitaniae CBS 6936; Candida parapsilosis CBS 604; Candida tropicalis CBS 94; Saccharomyces cerevisiae Szeged Microbiological Collection, Szeged, Hungary, SZMC 0644; and Schizosaccharomyces pombe SZMC 0142), and three NFAP-sensitive filamentous fungal isolates (Aspergillus nidulans Fungal Genetics Stock Center, Kansas, MO, USA, FGSC A4; Aspergillus niger SZMC 601; Rhizomucor miehei CBS 360.92) (Kovács et al. 2011; Virágh et al. 2015). Susceptibility tests were performed in low cationic broth medium (LCM: 0.5 % glucose, 0.025 % yeast extract, 0.0125 % peptone (w/v)).
Filamentous fungi were maintained on malt extract agar slants (MEA: 0.5 % malt extract, 0.25 % yeast extract, 1 % glucose, 2 % agar (w/v)), yeasts were maintained on yeast extract glucose medium (YEGK: 1 % glucose; 1 % KH2PO4; 0.5 % yeast extract, 2 % agar (w/v)) at 4 °C.
Isolation and purification of NFAP2
NFAP2 was isolated from the supernatant of N. fischeri NRRL 181 culture, which was grown in MM. Five 1 l-Erlenmeyer flasks each containing 200 ml MM was inoculated with 2 × 107 conidia and incubated for 7 days at 25 °C under continuous shaking at 210 rpm. Mycelia were removed with filtering the culture through paper filter (Rotilabo-round filters, type 111A; Carl Roth KG, Karlsruhe, Germany), then the mycelia-free supernatant was centrifuged (10,000×g, 17 °C) and filtered through paper filter (Fisherbrand QL115 folded filter paper, Fisher Scientific, Pittsburgh, PA, USA) again. NFAP2 was purified from this mycelia-free supernatant based on the slightly modified method described at NFAP previously (Virágh et al. 2014). The <30 kDa molecular fraction of the supernatant was separated by ultrafiltration (Ultracell 30 kDa Ultrafiltartion Discs, regenerated cellulose; Millipore, Billerica, MA, USA) then its protein content was purified by cation-exchange chromatography on a Bio-Scale™ Mini Macro-Prep® High S column (Bio-Rad Laboratories, Hercules, CA, USA) using the BioLogic Duo Flow™ system (Bio-Rad Laboratories, Hercules, CA, USA). The column was equilibrated with 10 mM sodium phosphate buffer (pH 6.6) containing 25 mM NaCl and 0.15 mM EDTA. Bound proteins were eluted with NaCl gradient (0.0–1.5 M) prepared in 10 mM sodium phosphate buffer (pH 6.6) at a flow rate of 1.2 ml min−1. The quality of the NFAP2 fractions was checked by SDS-PAGE (Novex™ 18 % Tris–Glycine Mini Protein Gels, 1.0 mm, 10-well; Thermo Fisher Scientific, Waltham, MA, USA). Protein bands were visualized applying Coomassie Brilliant Blue R-250 and silver staining. The pool of the pure NFAP2 fractions was dialyzed (Snake Skin™ dialysis tubing, 3.5 K MWCO, Thermo Scientific, Logan, UT, USA) against double distilled water, then lyophilized and dissolved in double distilled water. This protein solution was sterilized by syringe filtration (Millex-GV, PVDF, pore size: 0.22 µm; Millipore, Billerica, MA, USA).
Identification of NFAP2
Molar mass measurement of NFAP2 was performed on a Micromass Q-TOF Premier mass spectrometer (Waters MS Technologies, Manchester, UK) equipped with a nanoelectrospray ion source. Partial sequence of NFAP2 was determined from enzymatic digested protein sample. Ten microliter of protein solution containing 1 μg μl−1 protein was mixed with a buffer containing 25 mM NH4HCO3, pH 8.0, reduced with 10 mM DTT and alkylated with 55 mM iodoacetamide. The reduced and alkylated protein was purified with C4 containing ZipTip pipette tip (Millipore, Billerica, MA, USA) and it was subjected to enzymatic cleavage with 0.1 μg trypsin (Promega, Madison, WI, USA) solution (in 25 mM NH4HCO3) overnight at 37 °C. Then a mass spectrometric (MS) method was used, which was based on the database searching (Mascot Search Engine, NCBInr Database) of the protein fragment from the enzymatic digestion. The digested sample was analyzed on a Waters NanoAcquity UPLC (Waters MS Technologies, Manchester, UK) system coupled with a Micromass Q-TOF premier mass spectrometer. LC conditions were the followings: flow rate: 350 nl min−1; eluent A: water with 0.1 % (v/v) formic acid, eluent B: acetonitrile with 0.1 % (v/v) formic acid; gradient: 40 min, 3–40 % (v/v) B eluent; column: Waters BEH130 C18 75 lm 250 mm−1 column with 1.7 μm particle size C18 packing (Waters, Milford, MA, USA). The mass spectrometer was operated in MSE and DDA mode with lockmass correction (standard: Glu-1-Fibrinopeptide M + 2H + m/z = 785.842). Acquired data derived from the enzymatic cleavage were processed by the ProteinLynx Global Server (Waters, Milford, MA, USA).
In silico investigations
The SignalP1 4.1 server was used to predict the cleavage site of the signal sequence (Petersen et al. 2011). The molecular weight, pI, grand average of hydropathy (GRAVY) value, total net charge, and disulfide bridge pattern of the mature NFAP2 were predicted by ExPAsy ProtParam tool (Gasteiger et al. 2005), Protein Calculator v3.4 server (The Scripps Research Institute; http://www.scripps.edu/~cdputnam/protcalc.html), and DISULFIND Cysteines Disulfide Bonding State and Connectivity Predictor server (Ceroni et al. 2006), respectively.
Phylogenetic analysis
The BioEdit program (Hall 1999) was used to examine the antifungal protein sequences. Similarity searches to NFAP2 in the NCBI, EXPASY and JGI databases were performed using the Basic Local Alingment Search Tool (BLAST; Pevsner 2009). All previously described, isolated and characterized cysteine-rich antifungal proteins from filamentous ascomycetes, and the identified putative NFAP2 homologs were involved in the phylogenetic studies. Sequences were aligned by using the PRANK (Löytynoja and Goldman 2008). Ambiguously aligned positions were removed by GBlocks (Talavera and Castresana 2007). A maximum likelihood analysis (ML) was carried out under the WAG model of protein evolution with gamma distributed rate-heterogeneity and 1000 bootstrap replicates. Bootstrap percentages were summarized on the ML tree using the SumTrees script of the Dendropy package (Sukumaran and Holder 2010). Bootstrap proportions >70 % were considered as strong support.
Antifungal susceptibility tests
The in vitro antifungal effect of NFAP2, NFAP, and conventional antifungal agents (Sigma-Aldrich, St Louis, MO, USA) representing polyenes (amphotericin B, AMB), azoles (fluconazole, FLC and itraconazole, ITC), allylamines (terbinafine, TRB), and echinocandins (caspofungin, CSP) against mid-log phase yeast cells (grown up in LCM at 30 °C under continuous shaking at 210 rpm) and conidia or sporangiospores of 4-days-old filamentous fungi was examined in 96-well microtiter plate bioassays by measuring the optical density of the cultures. All conventional antifungal agents were dissolved in 96 % ethanol to prepare stock solutions (10.24 mg ml−1). One hundred microliter of purified NFAP2 (0.195–50 µg ml−1 in twofold dilution), or NFAP (25–400 µg ml−1 in twofold dilution), or antifungal drug (128–0.25 µg ml−1 in twofold dilution) diluted in LCM was mixed with 100 µl of 105 cells or conidia or sporangiospores ml−1 suspension prepared also in LCM. The flat-bottom plates were incubated for 0, 24, 48 and 72 h at 30 °C (yeasts), 25 °C (Aspergillus spp.), or 37 °C (R. miehei) without shaking, and then the absorbance (OD620) were measured in well scanning mode after shaking the plates for 5 s with a microtiter plate reader (SPECTROstar Nano, BMG Labtech, Ortenberg, Germany). Fresh medium (200 µl LCM) was used for background calibration. For calculation of the growth ability in the presence of antifungal proteins or drugs, the absorbance of the untreated control cultures (100 µl LCM mixed with 100 µl of 105 cells or conidia or sporangiospores ml−1 suspension prepared in LCM) were set to be 100 % growth. The minimal inhibitory concentration (MIC) was defined as the lowest antifungal protein or drug concentration at which growth was not detected after 24 (yeasts and R. miehei) or 48 h (Aspergillus spp.) of incubation on the basis of the OD620 values as compared to the untreated control. All susceptibility tests were repeated three times with three replicates.
Investigation of the manifestation of antifungal mechanism
All investigations were performed on mid-log phase S. cerevisiae cells grown up in LCM at 30 °C under continuous shaking at 210 rpm. To reveal the short- and long-term antifungal effect of NFAP2, 105 cells ml−1 were incubated in fresh LCM broth supplemented with the lethal (0.195 µg ml−1) or sublethal (0.098 µg ml−1) concentration of NFAP2 for 10, 30, 60 min and 4, 6 and 16 h at 30 °C. LCM without NFAP2 was used as control.
To compare the metabolic activity of the NFAP2-treated and untreated cells, FUN1 viability staining (Thermo Fisher Scientific, Waltham, MA, USA) was used based on the manufacturer’s instructions.
To determine the proportion of apoptotic and necrotic cells in NFAP2-treated and untreated samples the Annexin V-FITC (fluorescein isothiocyanate) Apoptosis detection kit (Sigma-Aldrich, St Louis, MO, USA) was used following the manufacturer’s instructions.
Plasma membrane disrupting activity of NFAP2 was investigated by applying the membrane impermeant, red-fluorescent nuclear and chromosome stain propidium iodide (PI). Cells were washed with LCM, and then stained with 5 µg ml−1 PI for 10 min at room temperature in the dark, and then washed again with LCM. Cells treated with 70 % (v/v) ethanol for 30 min at 4 °C were used as positive staining control.
Total and Annexin- or PI-positive cell numbers were determined in a Bürker chamber. All experiments were performed in three independent replicates.
Microscopy
Cells were visualized by light and fluorescence microscopy (Carl Zeiss Axiolab LR 66238C; Zeiss, Oberkochen, Germany) and photographed with a microscope camera (Zeiss AxioCam ERc 5 s; Zeiss, Oberkochen, Germany).
Heat stability investigation
Heat stability of NFAP2 was investigated on S. cerevisiae in microtiter plate bioassay. NFAP2 diluted in LCM (0.78–0.049 µg ml−1 in twofold dilution) was continuously heated from 25 to 95 °C, and then was incubated at the final temperature for 5 min. After cooling down to room temperature for 30 min, 100 µl treated protein solution was mixed with 100 µl 105 mid-log phase cells ml−1 grown up at 30 °C under continuous shaking at 210 rpm and diluted in LCM, then filled in the well of a flat-bottom microtiter plate. The microtiter plate was incubated at 30 °C for 24 h without shaking. The growth ability of S. cerevisiae was determined as described previously in the antifungal susceptibility tests. Untreated NFAP2 and S. cerevisiae culture (100 µl LCM mixed with 100 µl 105 ml−1 mid-log phase cells) were used as activity and growth controls, respectively. The test was repeated three times with two replicates.
Electronic circular dichroism spectroscopy and structural investigation
Secondary structure and thermal stability of NFAP2 was examined by electronic circular dichroism (ECD) spectroscopy. Measurements were performed in the 195–260 nm wavelength range using a Jasco-J815 spectropolarimeter (JASCO, Tokyo, Japan). The protein sample was presented in pure water in approximately 0.1 mg ml−1 concentration in a 0.1 cm path length quartz cuvette. First, the ECD spectrum of the sample was recorded at 25 °C with a scan speed of 100 nm s−1. The temperature was then gradually increased up to 95 °C at a rate of 1 °C min−1 using a Peltier thermo electronic controller (TE Technology, Traverse City, MI, USA), while ellipticity data was recorded as a function of temperature at three wavelengths, appointed by the extrema of the spectrum measured at 25 °C. The system was allowed to equilibrate for 1 min, before measurements were taken at each temperature point.
The resultant melting curves were fitted with a symmetrical sigmoidal function of which inflexion point corresponds to the melting temperature (Tm) of the protein structure.
At the final temperature, 95 °C, ECD spectrum in the 195-260 nm range was recorded again and then the sample was left to cool to 25 °C. Further spectrum acquisitions were done at 25 °C 1 min after cooling, then after 72 h and then 4 weeks later. The reported spectra are accumulations of ten scans, from which the similarly recorded, corresponding solvent spectrum was subtracted. Ellipticity data are given in mdeg units.
For determination of possible disulfide bond pattern of NFAP2, reversed-phase high performance liquid chromatography (RP-HPLC) runs were carried out on a Phenomenex Jupiter C18 column (250 × 4.6 mm; 10 μm particle size; 300 Å pore size; Phenomenex, Torrance, CA, USA) using an Agilent 1100 Series liquid chromatograph (Agilent Technologies, Little Falls, DE, USA). Linear gradient elution was carried out with 0.1 % (v/v) TFA in water (eluent A) and 80 % (v/v) acetonitrile and 0.1 % (v/v) TFA is water (eluent B) from 5 to 40 % (v/v) (B) over 35 min at a flow rate of 1.0 ml min−1.
Statistical analysis
Statistical analysis was performed using Microsoft Excel 2010 software (Microsoft, Edmond, WA, USA). Two sample t test was used to reveal significance between treated and untreated samples.