Fungal strain and Carum carvi
Carum carvi seeds were prepared from the herbarium of the Research Institute of Forests and Rangelands (RIFR), Tehran, Iran. The plant material was identified by V. Mozaffarian and a voucher specimen was deposited at RIFR under the Voucher Number of TARI-51257. Aspergillus species including A. fumigatus Af293, A. niger ATCC 9029, and A. flavus PFCC 100C were provided by the Pathogenic Fungi Culture Collection of the Pasteur Institute of Iran (http://fa.pasteur.ac.ir/VisitDetails.aspx?Id=1311). The strains were kept in Sabouraud dextrose broth (SDB, Merck, Germany) including 20% glycerol at 70ºC. The strains were subcultured in Sabouraud dextrose agar (SDA, Merck, Germany). Carum carvi seeds were purchased from the market and stored at room temperature.
Peptide extraction and purification
In brief, Carum carvi seeds were ground to a fine flour, and extracted with an extraction buffer (100 mM KCl, 15 mM NaH2PO4, 10 mM Na2HPO4, and 1.5% EDTA) pH 5.4, in the shaker at 4°C for 4 h 33. Subsequently, the supernatant was filtered (Whatman filter paper, USA) and saturated with 85% ammonium sulphate ((NH4)2SO4). The precipitate, formed overnight, was dialyzed against distilled by using benzoylated membrane performance (MWCO 2,000 Da) (Sigma Aldrich-USA). For isolating low molecular weight peptides, the protein extract was filtered through AmiconUltra-15ml 10,000 MWCO centrifugal filters (Millipore- USA) followed by lyophilization. The reverse phase HPLC column (C18 column, 7.8 × 300 mm; Tosoh, Tokyo, Japan) with the gradient of 5%–65% (v/v) solution B (0.098% TFA in acetonitrile) and A (0.1% TFA in water) at a flow rate of 1 mL/min for 85 min was employed for purifying the antifungal peptides from lyophilized extracts 34. According to the absorbance at 220 nm, the peaks were collected and then lyophilized for determining the antifungal effect and those that had the highest activity were collected. For checking the peak with antifungal activity purity, this peak was re-chromatographed in the same column using the similar solvent system in the same conditions.
For evaluating the molecular weight as well as the purity of the peak with antifungal activity, we performed the tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Tricine-SDS-PAGE) under reducing conditions on a Bio-Rad electrophoresis apparatus based on the Schagger approach 35. Products were run on a 12% tris-tricine gel with tricine-SDS running buffer for overnight in 25 V tension and then the gel was stained with silver staining. Protein ladder (2–250 KDa) was used as a standard for determining proximate molecular mass.
Radial diffusion assay
The antifungal activity of the collected peaks was assessed using a radial diffusion assay (RDA) according to the Wang approach 36. The Aspergillus species spore suspension was obtained a scraping the culture surface slightly by a sterile glass rod following the addition of enough 0.1% aqueous solution of Tween 80. Briefly, fungal cells were added to Sabouraud dextrose agar fixed at 42°C followed by a quick dispensing into a Petri dish. The wells were punched and the peptide was loaded into the wells. Following incubation (35°C/ 48 h), the clear zones around the wells were assessed. Amphotericin B was considered as a positive control. All the experiments were
Minimum inhibitory concentrations (MICs) and minimum fungicidal concentrations (MFCs)
The minimum inhibition concentration assay (MIC) of the purified and active peptide was measured as described by Li et al 37 after some corrections. In brief, the assay was determined by an inoculum of 105 conidia/ mL in Sabouraud Dextrose Broth and evaluated in 96-well micro plates. Serial 2-fold dilutions were provided for obtaining final levels (1 to 250 μg/mL). Amphotericin B (0.016 to 4μg/mL) was used as control. Incubation of the plates was done (35°C/ 48 h). The minimum inhibitory concentration (MIC) was defined as 99% inhibition of fungal growth in 96-well micro plates by visual assay. 20 μL of the specimens of each well was then isolated and plated on Sabouraud dextrose agar plates. The plates were incubated at 35°C for 48 h. The minimum fungicidal concentration (MFC) was defined as the lowest peptide level to kill fungal cells.
Mass spectrometry and N‑terminal sequence analysis
Molecular masse of the isolated antifungal peptide was determined by electro-spray ionization mass spectrometry (MS) at a mass to charge (m/z). Amino acid sequence of the purified antifungal peptide was determined using Edman degradation. Accordingly, an ABI Procise Edman Micro Sequencer (Model 492) was connected online to the 140C ABI PTH Amino Acid Analyzer.
Sequence alignment and phylogenetic tree
The search for similar sequences was performed by database search (for find similar peptides with highest similarity to new peptide) and CLC main workbench software. In this section, 13 peptides with highest similarity to new peptide were obtained after database search. Program blast was done for comparison of the sequences of these peptides with new peptide. Alignment was manually adjusted and phylogenetic tree was acquired by CLC main workbench software. For evaluation of reproducibility of the tree topology, phylogenetic tree was measured using bootstrap analysis with 100 replications.
For general AMP prediction, APD3 prediction server (https://wangapd3.com/main.php) was used. AMP probability of the peptide was anticipated through machine learning algorithms support vector machine (SVM), artificial neural network, random forest, and discriminant analysis using the CAMPR3 server (http://http://www.camp3.bicnirrh.res). Each algorithm threshold was from 0.5 to 1. Peptides with the threshold number of >0.5 were AMP. The physicochemical characteristics of antifungal peptide were predicted using ExPASy Proteomics server (http://www.expasy.org/tools/protparam. html) for the net charge, hydrophobic ratio, Boman index, isoelectric point (pI), values of the instability index, aliphatic index, and molecular weight. To predict the antifungal activity of the expected peptide the online server iAMPpred (http://cabgrid.res.in:8080/amppred/server.php) was used. Helical wheel projection was done for predicting the amino acid position in peptides using Heliquest server (https://heliquest.ipmc.cnrs.fr/cgi-bin/ComputPara ms.py). The three-dimensional structure of the peptide was estimated online by the I-TASSER server (http://zhanglab.ccmb.med.umich.edu/ITASSER/). The model quality was measured through Accelrys, DS visualizer ver. 1.7. BLAST indicated the similarity between the peptide and other AMPs in APD3 database. To predict the hemolytic property of the peptide the HemoPI server (https: //crdd.osdd.net/ragha va/hemop i/design n.php) was used (SVM score ranges between 0 and 1, i.e., 1 very likely to be hemolytic, 0 very unlikely to be hemolytic) 38.
The peptide was chemically synthesized by Biomatik Co. (Ontario, Canada) using 9-fluorenylmethoxycarbonyl (F-moc) solid-phase chemistry 39. Peptide purity and mass identity were confirmed by reversed-phase high-performance liquid chromatography (RP-HPLC) and electrospray ionization-mass spectrometry (ESI-MS, Waters ZQ2000, Milford, MA, USA), respectively.
The peptide hemolytic activity was calculated according to Wu et al. 40. Fresh heparinized human whole blood prepared from Department of Cell Bank of the Pasteur Institute of Iran was centrifuged for 5 min at 4ºC. The resultant erythrocytes were washed three times followed by suspending in PBS. Then, the peptide serial dilution (1- 128 µg/mL) was mixed with erythrocytes and subjected to incubation (1 h/ 37ºC). Erythrocytes after treatment with PBS were applied as a negative control and 0.1% Triton X-100 as positive control. Hemoglobin release was measured through the measurement of the absorption at 567 nm using the ELISA reader. The amount of hemolysis was calculated by the following formula:
Hemolysis (%) = [test OD − negative control OD)/( positive control OD – negative control OD)] × 100.
The cytotoxic effects of the antifungal peptide on Human Embryonic Kidney cell line 293 (HEK293) prepared from Department of Cell Bank of the Pasteur Institute of Iran was calculated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays 41. In brief, Dulbecco's modified Eagle's medium (DMEM) treated with 10% (v/v) fetal bovine serum (FBS) was used for growing of the cell lines and was subjected to incubation with 5% CO2 at 37°C. Seeding the cells was done by plates of 96 wells with about 5,000 cells per well. Following overnight culture at 37ºC in 5% CO2, different peptide levels (1-128 µg/mL) were used for the treatment of the cells for 24 h. Next, incubation was done c using 50 ml of MTT (0.5 mg/mL) for 4 h. Following incubation, the plates were centrifuged (5 min), and the supernatants were removed, and for dissolving the formazan crystals, DMSO (dimethyl sulfoxide; 150 mL) was added. At the end, the ELISA reader was used to measure the absorbance at 570 nm. The findings were obtained from three separate experiments, each conducted in triplicate.
Temperature and pH stability assay
The peptide stability at different temperatures and pH values was calculated as defined by 42. Heat sensitivity was evaluated after incubation at various temperatures (10-100°C) for 1 h. The peptide without heating at various temperatures was considered as a control. For pH stability testing, the samples were adjusted to 2-10 with 1 mol/l HCl or 1 mol/l NaOH and placed at 25°C for 1 h. Then, the pH value was fixed at 7.0 before antifungal assays. The peptide dissolving in solution (pH: 7.2) was considered as a control. The tests were performed three times. A. fumigatus was applied as an indicator for detecting the antifungal activity that was assessed using radial diffusion assay.
Determination of fungal cell integrity using PI uptake
To determine the membrane permeability of the antifungal peptide on A. fumigatus hyphae, the propidium iodide (PI) uptake assay was used according to the Fluorescence microscopy assay.
In brief, a conidia suspension of A. fumigatus (106 conidia/ mL) in SDB was poured on a 6-well micro plate and incubated (35°C/ 24 h). Next, the hyphae were incubated with the antifungal peptide at the concentration of MIC and 2 × MIC for 4 h at 35°C via constant shaking (120 rpm). Subsequently, the PI solution with a final concentration of 50 μg/mL was added to each well for 15 min at room temperature in the dark. Next, the stained hyphae specimens were visualized by fluorescence microscopy (Eclipse 80i- Nikon-Japan) with appropriate filters (excitation/emission at 530/590 nm). Untreated fungal hypha was used as negative controls.
Transmission Electron Microscopy (TEM)
TEM was performed as described previously 43 with some corrections. For evaluating the peptide effect on hyphae morphology, we cultured the conidia suspension of A. fumigatus (106conidia/mL) in SDB (1 mL) in microplates of 24 wells (24 h/ 35°C) followed by exposing to antifungal peptide at the concentration of 2× MIC and incubation at 35°C for 4 h. Then the specimens were fixed using 2.5% (v/v) glutaraldehyde (3 h/ 4ºC). Following three times washing in 0.1% PBS, the samples were then fixed using 1% osmium tetroxide in PBS within 70 min and were subjected to washing for two times with PBS dehydrated in gently increasing acetone solutions, and embedding in Epon 812. The ultra-microtome was used to cut ultrathin sections and staining with uranyl acetate and lead citrate was done, and observed by a Zeiss EM-900 TEM device at 80 kV. The non-treated hypha was considered as controls.
Values were expressed as mean ± SD. Statistical analysis was done with graph pad prism5 Statistical software (GraphPad Software, Inc., La Jolla, CA, USA).