Phaseolus vulgaris L. seeds were supplied by the Empresa de Pesquisa Agropecuária do Estado do Rio de Janeiro – Pesagro, Campos dos Goytacazes, RJ, Brazil.
The cDNA encoding the defensin PvD1 was obtained as described by .
Chemically competent cells of the Escherichia coli bacterial strains JM 109 [genotype: endA1, recA1, gyrA96, thi, hsdR17, (rk
+), relA1, supE44, λ-, Δ(lac-proAB), [F’, traD36, proAB, lacIqZΔM15]] (Promega Corporation, USA) and Rosetta-gami 2 (DE3) pLysS [genotype: Δ(ara–leu)7697 ΔlacX74 ΔphoA PvuII phoR araD139 ahpC galE galK rpsL (DE3) F’[lac
pro] gor522::Tn10 trxB pLysSRARE23 (CamR, StrR, TetR)4)] (Novagen) were used. Luria-Bertani (LB) medium was used as a routine bacterial growth and expression medium.
Candida albicans mutant strain (Δ)GCS1, deficient on glucosyl ceramide synthase (GCS) enzyme, was kindly provided by Dr. Dirk Warnecke from the Institut fur Allgemeine Botanik, University of Hamburg, Germany, and was obtained from the Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil. Yeast cultures were maintained on potato dextrose agar (potato infusion 0.4%, dextrose 2.0%, agar 1.5%).
Extraction and purification of the PvD1 from P. vulgaris seeds
The natural defensin from P. vulgaris seeds, PvD1, was purified as follows. Fine flour (100 g) was prepared from the seeds of P. vulgaris in a mill. A protein extract was prepared from this flour using 500 mL of extraction buffer (10 mM Na2HPO4, 15 mM NaH2PO4, 100 mM KCl, 1.5% EDTA, pH 5.4) for 2 h at 4°C with constant agitation. This protein extract was centrifuged at 15,000 g, and the supernatant was fractionated at a 70% relative ammonium sulfate saturation at 4°C for 18 h. After centrifugation under the same conditions, the precipitate was redissolved in distilled water and heated at 80°C for 15 min in a water bath. This heated protein extract was centrifuged at 10,000 g. The supernatant was recovered and extensively dialyzed against distilled water for three days and then recovered by freeze drying. For peptides, purification was initially performed on a DEAE-Sepharose column (with 100 mL of resin) equilibrated with 20 mM Tris–HCl (pH 8.0) at flow rate of 60 mL/h. The freeze dried protein extract (50 mg) was reconstituted in 5 mL of the equilibrium buffer and centrifuged (16,000 g, 3 min at 4°C), and the supernatant was loaded onto the column. A non-retained fraction (D1) was eluted in the equilibrium buffer, and a bound fraction (D2) was eluted in the same buffer with 1 M NaCl. The D1 fraction was freeze dried to concentrate, resuspended in 0.1% (v/v) trifluoroacetic acid (TFA) and injected into an HPLC C2C18 reversed-phase column (μRPC C2/C18 ST 4.6/100, GE Healthcare) attached to a C8 guard column (Pelliguard, Sigma). The bound peptides were eluted with an acetonitrile gradient starting with 100% solvent A (0.1% TFA) for 10 min followed by a mixture of solvent A and solvent B (80% acetonitrile containing 0.1% TFA) from 0 to 100% for 48 min and was then washed with 100% solvent B for 10 min. The concentration of solvent A rose to 100% following the 60 min total chromatography run, as shown by Games et al. .
Construction of the recombinant expression vector
The strategic design for the expression vector was based on the amplification of the cDNA encoding PvD1 using a specific set of primers that were designed to allow the amplicon to be cloned into a pET-32 EK/LIC expression vector (Novagen). This vector is part of a system for the expression of recombinant proteins that are fused to the thioredoxin (Trx tag), which facilitates protein solubility. Additionally, this vector provides six consecutive histidines (His) and glutathione (S) tags for the purification and identification of recombinant proteins. After purification, all tags can be completely removed via a cleavage reaction with the endoproteinase enterokinase (EK), and the proteolytic site is provided by the vector.
Fragment preparation for cloning into the expression vector
One set of primers was designed for the cloning of PvD1 into the pET-32 EK/LIC vector, according to the manufacturer’s instruction. The primers were as follows: 5′-GACGACGACAAGATGAAGACGTGCGAGAACCTG-3′ for the sense primer (Ds) and 5′-GAGGAGAAGCCCGGT
TTAACAGTTTTTGGT-3′ for the antisense primer (Das). The bold letters correspond to the sequence that anneals to the coding sequence of the PvD1 cDNA, and the underlined letters correspond to the sequence that anneals to the vector and were introduced to generate specific overhangs that would anneal with the pET-32 EK/LIC vector after treatment with T4 DNA polymerase. The double waves indicate a methionine codon, which was included according to the instructions furnished by the manufacturer (the sequence coding for the defensin does not include this codon naturally). This strategy permits directional cloning without the need for restriction digestion or ligation by a process called ligation-independent cloning (LIC). The primers were synthesized by Invitrogen, USA/Life Technologies, Brazil.
PCRs for fragment amplification were performed with a Mastercycler gradient 22331 (Eppendorf), and the PCR mixture contained 1× FideliTaq buffer (GE Healthcare), 0.2 mM dNTPs, 20 μM Ds, 20 μM Das, 1 μL of cDNA, and 0.5 units of FideliTaq DNA polymerase I (GE Healthcare) in a final volume of 20 μL per reaction. The reactions were initially warmed at 95°C for 1.5 min, followed by 45.3°C for 45 s, 68°C for 1.5 min, 35 cycles of the following program: 95°C for 45 s, 58.7°C for 45 s, and 68°C for 1.5 min.
The PCR products were directly purified with Wizard SV gel and a PCR clean-up system (Promega), according to the manufacturer’s instructions, and were treated with T4 DNA polymerase to generate overhang ends that were compatible with the vector. The T4 DNA polymerase reactions contained the following reagents: 300 ng of the purified fragment, 1× T4 DNA polymerase buffer (33 mM Tris-acetate (pH 7.9), 66 mM sodium acetate, 10 mM magnesium acetate, 1 mM DTT), 2.5 mM dATP, 5 mM DTT, and 5 units of T4 DNA polymerase in a 20 μL reaction. The reactions were incubated at 22°C for 30 min, and subsequently, the T4 DNA polymerase was inactivated by incubating the reactions at 75°C for 20 min.
pET-32 EK/LIC preparation and annealing
The pET-32 EK/LIC vector was furnished by the manufacturer and annealed to the fragment encoding the defensin, which was prepared as described above. The annealing reaction consisted of 50 ng of pET-32 EK/LIC and 2 μL of the T4 DNA polymerase-treated fragment. The reaction was incubated at 22°C for 5 min, and 1 μL of 25 mM EDTA was subsequently added, followed by a second incubation at 22°C for 5 min. From this reaction, 1 μL was used for bacterial (E. coli JM 109) transformation.
Transformation and colony screening
The transformation of JM 109 competent cells was performed as described by Inoue et al. , and the screening was performed by a plasmid extraction and digestion (Eco RI and Bgl II). The resulting DNA construct was named pET-PvD1. After successful cloning was confirmed, the construct pET-PvD1 was used to transform the super-expression strain of E. coli Rosetta-gami 2 (DE3) pLysS competent cells, according to the manufacturer’s instructions. Screening was performed by PCR directly from the bacterial colonies as follows: three colonies were randomly selected from the agar plate using sterile pipette tips, transferred to PCR tubes that contained 10 μL of sterile water, and homogenated with the pipette tips. Ten microliters of a mixture that contained 1× Taq buffer (GE Healthcare), 0.2 mM dNTPs, 20 μM Ds, 20 μM Das, and 0.5 units FideliTaq DNA polymerase I (GE Healthcare) were added to these tubes to yield a final volume of 20 μL. These reactions were submitted for PCR analysis. The PCR products were loaded onto a 1% agarose gel.
PvD1r expression and purification
The transformed cells were grown at 37°C in liquid LB medium (0.5% yeast extract, 1.0% tryptone, 1.0% NaCl) supplemented with ampicillin (50 μg.mL-1) and chloramphenicol (34 μg.mL-1) to an optical density (at 600 nm) of 0.5 before induction with 1 mM IPTG. After 3 h of induction, the cells were harvested by centrifugation (4,000 g for 15 min at 4°C); suspended in 50 mM phosphate buffer (pH 7.4) containing 500 mM NaCl, 40 mM imidazole, 1 mM PMSF, and a protease inhibitor cocktail for general use (Sigma, USA), according to the manufacturer’s manual; and ruptured by sonication (10 pulses of 30 sec at 10 watts) (R2D91109, Unique). Next, the cell lysate was clarified by centrifugation (5,000 g for 10 min at 4°C) (5430R, Eppendorf), and the supernatant was heated at 90°C for 30 min. The resulting suspension was centrifuged (7,200 g for 10 min at 4°C), and the supernatant was purified further by chromatographic methods.
Initially, Ni+-NTA agarose resin (Qiagen) was used to purify the soluble fusion protein by affinity chromatography. The Ni+-NTA agarose resin was equilibrated with a 50 mM phosphate buffer (pH 7.4) that contained 500 mM NaCl and 40 mM imidazole. The bound fraction was eluted with 500 mM imidazole in a 50 mM phosphate buffer (pH 7.4). To separate PvD1r from the tags (Trx, S and His) provided by the vector, cleavage of the fusion protein was performed by incubating the bound fraction with recombinant bovine enterokinase (Sigma, Co.) for 16 h at 25°C, according to the manufacturer’s instruction. After cleavage, this fraction was resubmitted for affinity chromatography on Ni+-NTA agarose resin under the conditions described above. At this point, the recombinant defensin had lost affinity to the Ni+ because it did not have the His tag and was therefore found in the unbounded fraction. This purified PvD1r was submitted to the C2C18 reversed-phase column exactly as described in the purification of the natural PvD1 and the retention time was recorded. In the second round of purification of the PvD1r, the cleavage sample was applied directly into the C2C18 reversed-phase column, and the peak with the same retention time of the PvD1r, as previously recorded, was collected.
The purification process was monitored by SDS-tricine gel electrophoresis performed according to the method of Schägger and von Jagow .
PvD1r amino acid sequencing
The purified PvD1r that was obtained after Ni+ affinity chromatography underwent Edman automated N-terminal amino acid sequencing  on a Shimadzu PPSQ-10 Automated Protein Sequencer. The sequence was determined from the purified peptide that was blotted onto PVDF membranes after SDS-tricine gel electrophoresis. PTH-amino acids were detected at 269 nm after separation on a reverse-phase C4 column (4.6 × 2.5 mm) under isocratic conditions, according to the manufacturer’s instructions. The sequences were compared to sequences reported in amino acid data banks and were submitted for automatic alignment using the NCBI-BLAST search system.
The three-dimensional structure of PvD1r was modeled with the Modeller Program . Initially, a search of a protein sequence databank was performed using the sequence of the PvD1r as a query in BLASTP . Based on this analysis, the Vigna radiata defensin 2 (VrD2; pdb CODE 2GL1) was selected as the template. The PvD1r model was generated, and minimization of energy (approximately 10) was performed to optimize the geometric parameters of the model. The minimization of the model was achieved using the Gromos96 Swiss-PDB Viewer program. The stereochemical quality of the models was checked using the Ramachandran plots (PROCHECK program) and Profile 3D, both of which were available on the PARMODEL web server [19, 20], and several parameters were analyzed, including the peptide bonds, planarity of the rings of the side chains, and twist angles ϕ and ψ of the main chain.
Candida albicans growth inhibition assay
For the preparation of the yeast cell cultures, an inoculum was transferred to Petri dishes that contained potato dextrose agar, and the inoculates were allowed to grow on the plates at 30°C for 2 days. Next, the cells were transferred to sterile culture medium (10 mL). The yeast cells were quantified in a Neubauer chamber for further calculations of appropriate dilutions. A quantitative assay for yeast growth inhibition was performed according to the protocol developed previously by Broekaert et al. , with some modifications. To monitor the effects of PvD1 and PvD1r on the growth of fungal cells, the cells were incubated in microplates at 30°C, at a final volume of 200 μL (10.000 cells in 1 mL of potato dextrose broth), and in the presence or absence (general control) of PvD1 and PvD1r (100 μg.mL-1). Optical readings at 620 nm were taken at timepoint zero and at every 6 h for the following 24 h. The readings were taken against a blank that contained only the culture medium. All of the experiments were run in triplicate, and the reading averages, standard errors and coefficients of variation were calculated.
Optical microscopy analysis and localization of PvD1r conjugated to FITC in Candida albicans cells
For optical microscopy analysis, after a 24 h yeast growth inhibition assay with PvD1 and PvD1r, the yeast cells were separated from the growth medium by centrifugation (4,000 g for 5 min at 4°C), washed in potato dextrose broth and visualized with an optical microscope (Axiovert 135, Zeiss). The yeast cells that were grown in the absence of defensins were also analyzed.
For localization analysis, PvD1r was conjugated to fluorescein isothiocyanate (FITC), according to the manufacturer’s instructions for FITC (Sigma). The nuclei were stained with 50 μg.mL-1 of 4’,6-diamidino-2-phenylindole dihydrochloride (DAPI) for 10 min, which was followed by fluorescence analysis with image analysis software (Axiovision®) and an Axioplan Zeiss microscope.