Advanced
Identification and Characterization of an Antifungal Protein, AfAFP<sub>R9</sub>, Produced by Marine-Derived Aspergillus fumigatus R9
Identification and Characterization of an Antifungal Protein, AfAFPR9, Produced by Marine-Derived Aspergillus fumigatus R9
Journal of Microbiology and Biotechnology. 2015. May, 25(5): 620-628
Copyright © 2015, The Korean Society For Microbiology And Biotechnology
  • Received : September 23, 2014
  • Accepted : November 13, 2014
  • Published : May 28, 2015
Download
PDF
e-PUB
PubReader
PPT
Export by style
Article
Author
Metrics
Cited by
TagCloud
About the Authors
Qi Rao
Wenbin Guo
Xinhua Chen
chenxinhua@tio.org.cn

Abstract
A fungal strain, R9, was isolated from the South Atlantic sediment sample and identified as Aspergillus fumigatus . An antifungal protein, AfAFP R9 , was purified from the culture supernatant of Aspergillus fumigatus R9. AfAFP R9 was identified to be restrictocin, which is a member of the ribosome-inactivating proteins (RIPs), by MALDI-TOF-TOF-MS. AfAFP R9 displayed antifungal activity against plant pathogenic Fusarium oxysporum, Alternaria longipes, Colletotrichum gloeosporioides, Paecilomyces variotii , and Trichoderma viride at minimum inhibitory concentrations of 0.6, 0.6, 1.2, 1.2, and 2.4 μg/disc, respectively. Moreover, AfAFP R9 exhibited a certain extent of thermostability, and metal ion and denaturant tolerance. The iodoacetamide assay showed that the disulfide bridge in AfAFP R9 was indispensable for its antifungal action. The cDNA encoding for AfAFP R9 was cloned from A. fumigatus R9 by RT-PCR and heterologously expressed in E. coli . The recombinant AfAFP R9 protein exhibited obvious antifungal activity against C. gloeosporioides, T. viride , and A. longipes . These results reveal the antifungal properties of a RIP member (AfAFP R9 ) from marine-derived Aspergillus fumigatus and indicated its potential application in controlling plant pathogenic fungi.
Keywords
Introduction
There are a vast number and diversity of microorganisms living in the oceans. The interaction between the marine microorganisms and their unique environments causes the development of special metabolic pathways in these microorganisms. The antifungal substances from marine microorganisms are becoming an important part of discovering new antifungal antibiotics and developing marine drugs in recent years. Many antimicrobial fungi have been isolated by culture-dependent methods from various marine organisms such as sponges and algae [35] . Rateb and Ebel [24] gave an overview of new natural products from marine fungi and their biological activities during 2006 to mid-2010, and 690 structures were presented.
Now, a great variety of antifungal proteins with different antifungal characteristics have been identified from various species of fungus. Aspergillus giganteus was the first filamentous fungus to potently produce an antifungal peptide (AFP) [18 , 31] . Subsequently, a number of different antifungal proteins have been derived from ascomycetes, such as PAF from Penicillium chrysogenum [6 , 15 , 19 , 25] , NAF from Penicillium nalgiovense [7] , AcAFP from Aspergillus clavatus [27 , 28] , AnAFP from Aspergillus niger [9] , NFAP from Neosartorya fisheri [5 , 8] , and Pc-Actin from Penicillium chrysogenum A096 [3] . According to their structure, molecular mass, and antifungal mechanism, the antifungal proteins are classified into ribosome-inactivating proteins (RIPs), pathogenesis-related proteins (PR), defensins, glycine/histidine-rich proteins, lipid transfer proteins (LTPs), protease inhibitors, and other proteins [26] .
RIPs have been found in bacteria, fungi, mushrooms, and plants, and have a broad spectrum of biological activities, including antitumor, antivirus, antifungus, and anti-insect activities. The RIPs have a glycosidase or phosphatase activity, resulting in the arrest of protein synthesis due to the ribosome damage caused by these two enzymes [23 , 30] . RIPs have been classified into three types. Type 1 RIPs are single-chain N -glycosidases with a molecular mass of 11 to 30 kDa. Type 2 RIPs contain two chains, a cell-binding lectin (B chain) and an N-glycosidase (A chain), with a molecular mass of 60 kDa [34] . Type 3 RIPs contain only one chain, which covers both the cell-binding lectin and N -glycosidase [22] . Type 1 RIPs are much less toxic, as they lack the B-chain, and thus they do not bind and enter cells [29] .
In this study, an antifungal strain, Aspergillus fumigatus R9, was isolated from a South Atlantic sediment sample. Its antifungal protein AfAFP R9 was purified and identified as a member of RIPs. The antifungal properties of the natural and recombinant AfAFP R9 proteins were characterized. The results obtained suggest that AfAFP R9 may represent a potential candidate of fungicide controlling plant pathogenic fungi.
Materials and Methods
- Tested Strains
The tested fungi, including Colletotrichum gloeosporioides (ACCC 31200, Agricultural Cultural Collection of China), Fusarium oxysporum (ACCC 31352), Trichoderma viride (ACCC 30902), Rhizoctonia solani (ACCC 36316), Alternaria longipes (ACCC 30002), and Sclerotinia sclerotiorum (ACCC 36081), were provided by Agricultural Cultural Collection of China. Paecilomyces variotii (CGMCC 3.776, China General Microbiological Culture Collection Center) was obtained from CGMCC, Institute of Microbiological Chinese Academy of Sciences. These seven tested fungi are important plant pathogenic fungi in agriculture.
- Isolation and Identification of an Antifungal Strain
The sediment samples used for strain isolation were collected from the South Atlantic (W 14.87°, S 12.12°; depth of water: 2,647 m). Isolation and identification of the strains as well as analysis of their antifungal activity were carried out as previously described [3] . Briefly, the sediment samples were diluted with sterilized seawater and approximately 200 µl of the diluted sample was spread on plates containing different types of medium, such as GPY (glucose 1%, peptone 0.2%, and yeast extract 0.05%) and YTM (0.5% yeast extract, 0.3% tryptone, and 2.5% mannitol). Plates were incubated at 28℃ for growth. The strains were selected based on their morphological features and inoculated into the corresponding liquid media for further growth to evaluate their antifungal potential. For the identification of the antifungal strain, the ribosomal internal transcribed spacer (ITS) DNA sequence was amplified using the primers ITS5 (5’-GGAAGTAAAAGTCGTAACAAGG-3’) and ITS4 (5’-TCCTCCGCTTATTGATATGC-3’), sequenced at Sangon Biotech (Shanghai, China) and analyzed for similarity using BLAST ( http://blast.ncbi. nlm.nih.gov/Blast.cgi ).
- Purification and Identification of Antifungal Protein fromA. fumigatusR9
A. fumigatus R9 was cultured in GPY medium at 28℃ for 7 days. The purification of the antifungal protein was performed as previously presented [3] . Briefly, the culture supernatant of A. fumigatus strain R9 was obtained by vacuum filtration through qualitative filter paper. The culture supernatant was fully saturated with ammonium sulfate and then centrifugated at 12,000 × g for 30 min at 4℃. The precipitate containing crude proteins was dissolved in distilled water and dialyzed at 4℃ for 24 h, and finally lyophilized.
The crude proteins and the fractions separated by ion exchange chromatography were tested for antifungal activity against the tested fungi. Ion-exchange chromatography was performed with an AKTA FPLC system (GE Healthcare, USA). The crude protein solution was loaded onto a DEAE Sepharose Fast Flow column (GE Healthcare), which was pre-equilibrated with starting buffer A (pH 8.1, 20 mM Tris–HCl) for the primary purification step. The elution program was as follows: 0% elution buffer B (pH 8.1, 20 mM Tris–HCl, 1 M NaCl), 3 CV (coloum volume); 0~40% elution buffer B, 10 CV; 100% elution buffer B, 2 CV; 0% elution buffer B, 2 CV. The bioactive fraction was concentrated by ultrafiltration in a Vivaspin 15R (molecular weight cutoff 5,000; Sartorius, Germany) and further purified on a CM Sepharose Fast Flow column (GE Healthcare, USA) under the same program. The resulting active component was concentrated by ultrafiltration in a Vivaspin 15R and its purity was assessed by 15% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and silver nitrate staining. The purified antifungal protein, named AfAFP R9 ; was identified by MALDI-TOF-TOF-MS at Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. The database search was performed on the Mascot server ( http://www.matrixscience.com/search_from_select.html ) with MALDI-TOF-TOF-MS data.
- Assay of Antifungal Activity
The assay for antifungal activity toward the seven phytopathogenic fungal species was carried out in PDA plates. One 0.6 cm diameter piece of tested phytopathogenic fungal strains’ cylinder agar with mycelial growth was placed on the center of a PDA plate. After the mycelial colony had developed, sterile blank paper discs of 0.65 cm diameter were placed at a distance of 0.8 cm away from the rim of the growing mycelial colony. Fifty microliter aliquots of the supernatant of A. fumigatus R9 and the fractions of ionexchange chromatography were added to each paper disc. Fifty microliters of GPY medium or starting buffer A, which dissolved the antifungal protein, was used as blank controls. The plates were incubated at 28℃ until mycelial growth enveloped discs containing the control disc, or formed crescents of inhibition around discs containing samples with antifungal activity.
- MIC Determination of Antifungal Protein AfAFPR9
The minimum inhibitory concentration (MIC) of AfAFP R9 against different pathogenic fungi was determined by the paper disc dilution method [32] . Two-fold serial dilutions of AfAFP R9 solution ranging from 0.4 to 0.00625 µg/µl were prepared, and 50 µl of each diluted solution was added onto paper discs placed 0.8 cm from the edge of growing mold on a PDA plate. The plates were placed at 28℃ for several days, depending on the tested pathogen. The MIC was determined as the lowest concentration of active protein that could inhibit visible mold growth and calculated as the total protein added on each paper disc (microgram per disc). Starting buffer A without active protein was used as the blank control.
- Physiochemical Properties of Antifungal Protein
To examine the effects of metal ions on AfAFP R9 activity, several metal ions, such as Na + , K + , Mg 2+ , Cu 2+ , and Ag + , were dissolved in starting buffer A to a final concentration of 10 mM. AfAFP R9 (0.4 µg/µl) was treated with the different ion solutions at room temperature for 1 h before being tested for antifungal activity. Fifty microliters of the treated AfAFP R9 was used for the antifungal tests. The AfAFP R9 protein without metal ion treatment was used as the positive control, and starting buffer A and ion solutions were used as blank and negative controls.
To evaluate the thermostability of AfAFP R9 , AfAFP R9 solution (0.4 µg/µl) was treated at 100℃ for 20, 40, 60, and 80 min, respectively. After cooling to room temperature, the residual antifungal activity of AfAFP R9 was tested against A. longipes . Fifty microliters of the heat-treated AfAFP R9 was used for the antifungal test. AfAFP R9 solution without heat treatment and starting buffer A were used as the positive and blank controls, respectively.
For denaturant-resistant test, AfAFP R9 (0.4 µg/µl) was treated with 0.1% SDS, 0.1% carbamide, and 0.1% guanidine hydrochloride at 28℃ for 24 h. The treatment effect was analyzed by observing the residual antifungal activity of AfAFP R9 . Fifty microliters of the treated antifungal protein was used for the antifungal tests. The AfAFP R9 without denaturant treatment was used as the positive control. Only denaturants and starting buffer A were used as blank controls. All experiments, including metal-ion-resistant, thermostability, and denaturant-resistant tests, were done in triplicate and the results are shown as mean ± standard deviation (SD) of three experiments.
- Cloning of AfAFPR9cDNA
Total RNA was extracted from strain R9 by using TRIzol reagent (Invitrogen, USA), according to the manufacturer’s instructions. First-strand cDNA was synthesized from 1 µg of total RNA using oligo-dT-adaptor primer (TaKaRa, Dalian, China) and used as the template for PCR amplification of AfAFP R9 cDNA. Based on the restrictocin gene sequence (GenBank Accession No. AAA32707.1), two primers were designed: forward primer 5’-GCGACCTGGACATGCATCAACCAA-3’ and reverse primer 5’-CTAATGAGAACACAGTCTCAAGTC-3’. PCR was performed with an initial denaturation step of 3 min at 94℃ and then 35 cycles were run as follows: 30 sec denaturation at 94℃, 30 sec annealing at 55℃, and 30 sec extension at 72℃, followed by a cycle of 72℃ for 10 min. The amplified product was sequenced at Sangon Biotech. Sequence homology search was performed using the BLAST program ( http://blast.ncbi.nlm.nih.gov/Blast.cgi ). Multiple sequence alignment was carried out using the DNAMAN tool (Lynnon Biosoft).
- Heterologous Expression of AfAFPR9inEscherichia coliand Antifungal Activity Analysis of Its Recombinant Protein
AfAFP R9 was expressed as a fusion protein with 6× His-tag and thioredoxin (TRX) using a pET-32a vector in the E. coli Rosetta strain (Amersham Pharmacia Biotech). The AfAFP R9 cDNA was amplified using the following primers: forward primer 5’-CCG GAATTC GCGACCTGGACATGC-3’ with an Eco RI digestion site (underlined), and reverse primer 5’-CCG CTCGAG CTAATGAGAACACAGTC-3’ with an Xho I digestion site (underlined). The PCR product was cloned into the Eco RI/ Xho I-digested pET-32a. The resulting plasmid pET-32a-AfAFP R9 was transformed into the competent cells of E. coli Rosetta using pET-32a as a vector control. The positive colonies were identified by PCR and DNA sequencing. AfAFP R9 fusion protein was expressed by 1 mM isopropy l-β-D-thiogalactopyranoside (IPTG) induction at 16℃ for 48 h. The recombinant proteins were then purified using Ni 2+ IDA affinity chromatography (Novagen, USA) as described in the supplier’s instructions. SDS-PAGE was performed for analysis of recombinant protein expression and purification. The antifungal activity of the purified recombinant protein was determined using a microtiter plate assay as previously described [10] .
- Effect of the Disulfide Bridge on the Antifungal Activity of AfAFPR9
AfAFP R9 (0.4 µg/µl) was reduced by β-mercaptoethanol, and the partially reduced AfAFP R9 was alkylated by iodoacetamide [2 , 13] . Freshly prepared β-mercaptoethanol was added to the AfAFP R9 solution to a final concentration of 1 mM. The mixture was incubated at 37℃ for 10 min followed by dialyzing against distilled water at 4℃ for 24 h with several changes of water. After that, 100 µl of the partially reduced AfAFP R9 was placed on a paper disc and tested for its antifungal activity against A. longipes . Another 400 µl of the AfAFP R9 was diluted into the same volume of iodoacetamide solution and then kept in the dark for 3 min. Five micrograms of iodoacetamide was dissolved in 400 µl of Tris-HCl buffer (50 mM, pH 8.5). After alkylation, the mixture was dialyzed against distilled water at 4℃ for 24 h with several changes of water. Finally, 200 µl of the alkylated AfAFP R9 was placed on a paper disc for antifungal test. The antifungal experiments were done in triplicate and the results are shown as the mean± SD.
Results
- Strain Identification and Antifungal Activity Detection
A total of 24 strains were purified based on their morphological features and inoculated into corresponding liquid media for further growth to evaluate their antifungal potential. The supernatant of the strain R9 culture showed obvious inhibitory activity against several plant pathogenic fungi, including C. gloeosporioides, F. oxysporum, T. viride, R. solani, A. longipes, S. sclerotiorum , and P. variotii . The R9 strain produced a large number of green spores. Its ITS gene sequence showed the highest identity of 99% with that of A. fumigatus KARVS04 (GenBank Accession No. KC119200.1) and has been submitted to the NCBI GenBank with an accession number of KF985037. Based on the morphological characteristics and molecular information, strain R9 was identified as A. fumigatus . The A. fumigatus R9 strain has now been deposited at China Center for Type Culture Collection (CCTCC, Wuhan, China) with a preservation number of M2013206.
- Purification of Antifungal Protein AfAFPR9
Further analysis showed that the precipitate from A. fumigatus R9 culture supernatant by saturation with ammonium sulfate and some fractions of ion-exchange chromatography also displayed antifungal activity against several tested fungi, including F. oxysporum, A. longipes, C. gloeosporioides, P. variotii , and T. viride . The precipitate was dissolved in water and first separated into 12 different fractions on a DEAE Sepharose Fast Flow column ( Fig. 1 A). Evaluation the of antifungal activity of the fractions revealed that only the second fraction AP2 had obvious antifungal activity. The AP2 fraction was then collected and further separated on a CM Sepharose Fast Flow column, and two main fractions (BP1, BP2) were obtained ( Fig. 1 B). Fraction BP2 showed antifungal activity against the tested fungi above ( Fig. 2 ). SDS-PAGE analyses showed that only one protein band corresponding to about 18 kDa was observed in fraction BP2 ( Fig. 3 ). This purified antifungal protein produced by A. fumigatus R9 was named AfAFP R9 .
PPT Slide
Lager Image
Isolation of the antifungal protein by ion-exchange chromatography. (A) The crude protein was loaded on a DEAE Sepharose Fast Flow column. (B) Antifungal fraction AP2 was loaded on a CM Sepharose Fast Flow column.
PPT Slide
Lager Image
Antifungal activity of fraction BP2 (disc 2) against the sensitive tested fungi with starting buffer A (disc 1) as blank control. (A) A. longipes. (B) T. viride. (C) C. gloeosporioides. (D) P. variotii. (E) F. oxysporum.
PPT Slide
Lager Image
15% SDS-PAGE of the purified antifungal protein. Lane 1. Standard protein marker; Lane 2. Antifungal fraction BP2 collected from CM Sepharose Fast Flow column.
- Identification and cDNA Cloning of AfAFPR9
After being treated with trypsin, four peptide sequences of AfAFP R9 were identified with MALDI-TOF-TOF. All of them were contained in a protein named restrictocin (GenBank Accession No. AAA32707.1) ( Table 1 ), which was a member of the RIPs. The AfAFP R9 cDNA was then cloned using RT-PCR. It is 450 bp long, encoding a protein of 149 amino acids. It has been submitted to NCBI GenBank with an accession number of KJ081439. The deduced AfAFP R9 protein contained all the four peptides identified by MALDI-TOF-TOF ( Fig. 4 ), indicating that the cDNA cloned here encodes the antifungal protein AfAFP R9 . Multiple sequence alignment showed that AfAFP R9 shared 84.66% amino acid sequence identity with restrictocin from Aspergillus restricts (AAA32707.1), 84.09% with mitogillin from Aspergillus restrictus (P67876.1), 72.32% with α-sarcin from Aspergillus giganteus (CAA43180.1), 71.75% with clavin from Aspergillus clavatus (ACC49407.1), 5.66% with RIP from Momordica charantia L (AAS17014.1), and 8.15% with antiviral protein 1 from Bougainvillea xbuttiana (AAY34283.2). These results indicate that the antifungal protein AfAFP R9 is a member of the RIP family from marine fungi, belonging to type 1 RIP.
Mass spectrum identification of antifungal protein AfAFPR9.
PPT Slide
Lager Image
Mass spectrum identification of antifungal protein AfAFPR9.
PPT Slide
Lager Image
Nucleotide sequence of AfAFPR9 cDNA and deduced amino acid sequence. The regions underlined show the four matched peptides identified by MALDI-TOF-TOF. ▲: indicates the first and last amino acid residues of the characteristic microbial RNases superfamily sequence (21-148 aa); △: indicates the five active sites, Tyr47, His49, Glu95, Arg120, and His136; ☆: indicates the two cysteine residues in AfAFPR9.
- Physiochemical Properties of AfAFPR9
In order to investigate the MICs of AfAFP R9 against the five tested strains, the paper disc dilution method was performed. The MICs of AfAFP R9 were 0.6, 0.6, 1.2, 1.2, and 2.4 µg/disc against F. oxysporum, A. longipes, C. gloeosporioides, P. variotii , and T. viride , respectively. In addition, metal ions, such as Na + , K + , Mg 2+ , Cu 2+ , and Ag + were tested for their effects on the antifungal activity of AfAFP R9 . The antifungal activity of AfAFP R9 against all five tested strains was not reduced when it was treated with Na + ( Table 2 ). When AfAFP R9 was treated with K + , its antifungal activity against F. oxysporum was 87.16 ± 13.31% retained but was largely impaired against the other four strains ( Table 2 ). AfAFP R9 was sensitive to metal ions Mg 2+ , Cu 2+ , and Ag + , since its antifungal activity was largely or even completely lost after being treated with 10 mM Mg 2+ , Cu 2+ , or Ag + ( Table 2 ). However, the antifungal activity of AfAFP R9 against P. variotii was almost not reduced when it was treated with Mg 2+ ( Table 2 ). Different metal ions thus have different effects on the antifungal activity of AfAFP R9 . Finally, the denaturant-resistant test was also carried out. Relatively, the antifungal activity of AfAFP R9 against all five tested strains was slightly or not affected by 0.1% SDS, 0.1% carbamide, and 0.1% guanidine hydrochloride ( Table 3 ).
Antifungal activity of AfAFPR9against the sensitive tested fungi after treatment with metal ions (10 mM).
PPT Slide
Lager Image
Antifungal activity of AfAFPR9 against the sensitive tested fungi after treatment with metal ions (10 mM).
Antifungal activity of AfAFPR9against the sensitive tested fungi after treatment with several denaturants.
PPT Slide
Lager Image
Antifungal activity of AfAFPR9 against the sensitive tested fungi after treatment with several denaturants.
AfAFP R9 maintained undamaged activity against A. longipes when it was heated at 100℃ for 20 min ( Fig. 5 ). However, with the extension of heating time, the activity of AfAFP R9 was gradually lost. When heated at 100℃ for 80 min, its antifungal activity was completely lost ( Fig. 5 ). These results indicated that AfAFP R9 exhibited a certain extent of thermostability.
PPT Slide
Lager Image
Antifungal activity of AfAFPR9 against A. longipes after heat treatment at 100℃ for 20, 40, 60, and 80 min. The antifungal activity was indicated as the average radius, with standard deviation, of the inhibition zone forming on the plates by AfAFPR9 against A. longipes.
- The Disulfide Bridge in AfAFPR9Contributing to Its Antifungal Activity
In order to investigate whether the two cysteine residues in AfAFP R9 form a disulfide bridge that contributes to its antifungal activity, the iodoacetamide assay was carried out. When AfAFP R9 was partially reduced by β-mercaptoethanol, its antifungal activity against A. longipes was reduced by 25.83 ± 10.10% ( Fig. 6 , middle colomn). When the mercapto groups of the two cysteine residues were fully alkylated by iodoacetamide, the antifungal activity of AfAFP R9 against A. longipes was almost lost completely ( Fig. 6 , right colomn). These results indicated that the disulfide bridge in AfAFP R9 contributed to its antifungal activity.
PPT Slide
Lager Image
Effect of the disulfide bridge on the antifungal activity of AfAFPR9 against A. longipes. The antifungal activity was indicated as the average radius, with standard deviation, of the inhibition zone forming in the plates by AfAFPR9 against A. longipes.
- Expression of AfAFPR9inEscherichia coli
The AfAFP R9 cDNA was cloned into the pET-32a vector and expressed in E. coli Rosetta after IPTG induction. Both induced and non-induced E. coli Rosetta/pET-32a-AfAFP R9 and E. coli Rosetta/pET-32a (the vector control) were analyzed by SDS-PAGE ( Fig. 7 ). A 35 kDa band ( Fig. 7 , lane 5) corresponding to the size of recombinant AfAFP R9 protein with 6× His-tag and thioredoxin (TRX) was only observed in the induced E. coli Rosetta/pET-32a-AfAFP R9 , indicating that the recombinant AfAFP R9 was specifically expressed in E. coli Rosetta/pET-32a-AfAFP R9 . The recombinant AfAFP R9 protein was then purified using Ni 2+ IDA affinity chromatography ( Fig. 7 , lane 6).
PPT Slide
Lager Image
SDS-PAGE analysis of recombinant AfAFPR9. Lane 1: protein marker; Lane 2: non-induced E. coli Rosetta/pET-32a; Lane 3: induced E. coli Rosetta/pET-32a; Lane 4: non-induced E. coli Rosetta/pET-32a-AfAFPR9; Lane 5: induced E. coli Rosetta/pET-32a-AfAFPR9; Lane 6: purified recombinant AfAFPR9 protein.
- Antifungal Activity of the Recombinant AfAFPR9
The antifungal activity of the recombinant AfAFP R9 was measured against the tested fungi in comparison with the natural AfAFP R9 , using culture medium and recombinant TRX protein as control. In all of the experiments, five replicates were prepared for each treatment and the concentrations of proteins were all at 400 ng/µl. The antifungal activity was observed by measuring the optical density at 600 nm (OD 600 ) of the tested fungi growing on microplates at 24 h after treatment with AfAFP R9 . The recombinant AfAFP R9 had obvious antifungal activity against C. gloeosporioides, T. viride , and A. longipes , and almost no effect on the growth of F. oxysporum ( Fig. 8 ). This method is not appropriate for P. variotii , whose hyphae entwined together, thereby resulting in an unchanged OD 600 ( Fig. 8 ). These results indicate that the recombinant AfAFP R9 produced in E. coli exhibits good antifungal activity.
PPT Slide
Lager Image
Analysis of antifungal activity of the recombinant AfAFPR9 protein. The antifungal activity was expressed as the growth inhibition of tested fungi by measuring the OD600. of the tested fungi at 24 h after treatment with AfAFPR9.
Discussion
The marine-derived RIP AfAFP R9 had a broad antifungal spectrum in that it could inhibit some plant pathogenic fungi, including F. oxysporum, A. longipes, C. gloeosporioides, P. variotii , and T. viride . AfAFP R9 was different from other RIPs in origin, function, and antimicrobial spectrum. Lyophyllin was isolated from mushroom Lyophyllum shimeji and exhibited good antifungal activity against Physalospora piricola and Coprinus comatus [11] . Luffacylin from sponge gourd seeds exerted antifungal activity against Mycosphaerella arachidicola and Fusarium oxysporum [21] . Hispin from seeds of the hairy melon was also found to have antifungal activity against C. comatus [17] . Bougainvillea xbuttiana antiviral protein 1 from E. coli exhibited antiviral activity against sunnhemp rosette virus [4] . As with some other RIPs expressed in E. coli , recombinant AfAFP R9 also exhibited antifugal activity. Recombinant Momordica charantia L RIP can inhibit the growth of the Sphaerotheca fuliginea in vitro [33] . Clavin from the filamentous fungus A. clavatus IFO 8605 was expressed in E. coli and its recombinant protein showed antitumor activity [20] .
The deduced AfAFP R9 protein of the cDNA has a characteristic microbial RNases superfamily sequence at 21-148 aa and five active sites, Tyr47, His49, Glu95, Arg120, and His136, as found in restrictocin from A. restrictus ( Fig. 4 ) [12] . This provided further evidence that AfAFP R9 is a member of the RIP family.
The MIC test results showed the difference in antifungal activities of AfAFP R9 against five tested strains, which could be attributed to the different sensitivity of these five strains to AfAFP R9 . In the metal ion tests, the antifungal activity of AfAFP R9 was maintained by treatment of monovalent ions, and partially or completely lost by treatment by divalent ions such as Mg 2+ and Cu 2+ . It was presumed that the divalent ions Mg 2+ and Cu 2+ could change the structure of AfAFP R9 , thus leading to the loss of activity. Although the divalent ions such as Mg 2+ and Cu 2+ are rich in soil, they have no restriction in practical field application since AfAFP R9 will be sprayed on the plants.
It was shown that AfAFP R9 exhibited a certain extent of thermostability. Two cysteine residues in AfAFP R9 may form a disulfide bridge, which would contribute to its thermostability ( Fig. 4 ). Previous reports showed that antifungal proteins AFP and PAF had eight and six cysteine residues, respectively, which might form disulfide bridges contributing to their heat stability [14 , 16] . Further study also revealed that the disulfide bridge in AfAFP R9 also contributed to its antifungal activity. This was consistent with the previous results that the disulfide bridges in antifungal PAF was indispensable for its antifungal action [1] .
The antifungal protein AfAFP R9 was purified from marinederived A. fumigatus and identified as a member of the RIP family. The broad antifungal spectrum, good antifungal activity, thermostability, and resistance to several metal ions and denaturants of AfAFP R9 suggest that it may represent a potential candidate of fungicide controlling plant pathogenic fungi.
Acknowledgements
This study was financially supported by the Scientific Research Foundation of Third Institute of Oceanography, SOA (No. 2013022), grants from China Ocean Mineral Resources Research and Development Association (DY125-15-T-07) Scientific Research Project of the Marine Public Welfare Industry of China (201205020), Natural Science Foundation of China (41306166), and Xiamen South Ocean Research Center (13GZP002NF08).
References
Batta G , Barna T , Gaspari Z , Sandor S , Kövér KE , Binder U 2009 Functional aspects of the solution structure and dynamics of PAF - a highly stable antifungal protein fromPenicillium chrysogenum. FEBS J. 276 2875 - 2890    DOI : 10.1111/j.1742-4658.2009.07011.x
Cao A , Hu D , Lai L 2004 Formation of amyloid fibrils from fully reduced hen egg white lysozyme. Protein Sci. 13 319 - 324    DOI : 10.1110/ps.03183404
Chen Z , Ao J , Yang W , Jiao L , Zheng T , Chen X 2013 Purification and characterization of a novel antifungal protein secreted byPenicillium chrysogenumfrom an Arctic sediment. Appl. Microbiol. Biotechnol. 97 10381 - 10390    DOI : 10.1007/s00253-013-4800-6
Choudhary N , Yadav O , Lodha M 2008 Ribonuclease, deoxyribonuclease, and antiviral activity ofEscherichia coli-expressedBougainvillea xbuttianaantiviral protein 1. Biochemistry (Moscow) 73 273 - 277    DOI : 10.1134/S000629790803005X
Galgóczy L , Kovács L , Karácsony Z , Virágh M , Hamari Z , Vágvölgyi C 2013 Investigation of the antimicrobial effect ofNeosartorya fischeriantifungal protein (NFAP) after heterologous expression inAspergillus nidulans. Microbiology 159 411 - 419    DOI : 10.1099/mic.0.061119-0
Galgóczy L , Virágh M , Kovács L , Tóth B , Papp T , Vágvölgyi C 2013 Antifungal peptides homologous to thePenicillium chrysogenumantifungal protein (PAF) are widespread amongFusaria. Peptides 39 131 - 137    DOI : 10.1016/j.peptides.2012.10.016
Geisen R 2000 P. nalgiovensecarries a gene which is homologous to thepafgene ofP. chrysogenumwhich codes for an antifungal peptide. Int. J. Food Microbiol. 62 95 - 101    DOI : 10.1016/S0168-1605(00)00367-6
Kovács L , Virágh M , Takó M , Papp T , Vágvölgyi C , Galgóczy L 2011 Isolation and characterization ofNeosartorya fischeriantifungal protein (NFAP). Peptides 32 1724 - 1731    DOI : 10.1016/j.peptides.2011.06.022
Lee DG , Shin SY , Maeng C-Y , Jin ZZ , Kim KL , Hahm K-S 1999 Isolation and characterization of a novel antifungal peptide fromAspergillus niger. Biochem. Biophys. Res. Commun. 263 646 - 651    DOI : 10.1006/bbrc.1999.1428
López-García B , Moreno AB , San Segundo B , De los Ríos V , Manning JM , Gavilanes JG , Martínez-del-Pozo Á 2010 Production of the biotechnologically relevant AFP fromAspergillus giganteusin the yeastPichia pastoris. Protein Expr. Purif. 70 206 - 210    DOI : 10.1016/j.pep.2009.11.002
Lam SK , Ng TB 2001 First simultaneous isolation of a ribosome inactivating protein and an antifungal protein from a mushroom (Lyophyllum shimeji) together with evidence for synergism of their antifungal effects. Arch. Biochem. Biophys. 393 271 - 280    DOI : 10.1006/abbi.2001.2506
Lamy B , Moutaouakil M , Latge JP , Davies J 1991 Secretion of a potential virulence factor, a fungal ribonucleotoxin, during human aspergillosis infections. Mol. Microbiol. 5 1811 - 1815    DOI : 10.1111/j.1365-2958.1991.tb01930.x
Li Y , Gong H , Sun Y , Yan J , Cheng B , Zhang X 2012 Dissecting the role of disulfide bonds on the amyloid formation of insulin. Biochem. Biophys. Res. Commun. 423 373 - 378    DOI : 10.1016/j.bbrc.2012.05.133
Marx F , Binder U , Leiter E , Pocsi I 2008 ThePenicillium chrysogenumantifungal protein PAF, a promising tool for the development of new antifungal therapies and fungal cell biology studies. Cell. Mol. Life Sci. 65 445 - 454    DOI : 10.1007/s00018-007-7364-8
Marx F , Haas H , Reindl M , Stöffler G , Lottspeich F , Redl B 1995 Cloning, structural organization and regulation of expression of thePenicillium chrysogenum pafgene encoding an abundantly secreted protein with antifungal activity. Gene 167 167 - 171    DOI : 10.1016/0378-1119(95)00701-6
Meyer V 2008 A small protein that fights fungi: AFP as a new promising antifungal agent of biotechnological value. Appl. Microbiol. Biotechnol. 78 17 - 28    DOI : 10.1007/s00253-007-1291-3
Ng TB , Parkash A 2002 Hispin, a novel ribosome inactivating protein with antifungal activity from hairy melon seeds. Protein Expr. Purif. 26 211 - 217    DOI : 10.1016/S1046-5928(02)00511-9
Olson B , Goerner GL 1965 Alpha sarcin, a new antitumor agent I. Isolation, purification, chemical composition, and the identity of a new amino acid. Appl. Microbiol. 13 314 - 321
Palicz Z , Jenes Á , Gáll T , Miszti-Blasius K , Kollár S , Kovács I 2013 In vivoapplication of a small molecular weight antifungal protein ofPenicillium chrysogenum(PAF). Toxicol. Appl. Pharmacol. 269 8 - 16    DOI : 10.1016/j.taap.2013.02.014
Parente D , Raucci G , Celano B , Pacilli A , Zanoni L , Canevari S 1996 Clavin, a type-1 ribosome-inactivating protein fromAspergillus clavatusIFO 8605. cDNA isolation, heterologous expression, biochemical and biological characterization of the recombinant protein. Eur. J. Biochem. 239 272 - 280    DOI : 10.1111/j.1432-1033.1996.0272u.x
Parkash A , Ng TB , Tso WW 2002 Isolation and characterization of luffacylin, a ribosome inactivating peptide with antifungal activity from sponge gourd (Luffa cylindrica) seeds. Peptides 23 1019 - 1024    DOI : 10.1016/S0196-9781(02)00045-1
Peumans WJ , Hao Q , van Damme EJ 2001 Ribosome-inactivating proteins from plants: more than RNA N-glycosidases? FASEB J. 15 1493 - 1506    DOI : 10.1096/fj.00-0751rev
Pu Z , Lu B-Y , Liu W-Y , Jin S-W 1996 Characterization of the enzymatic mechanismof γ-momorcharin, a novel ribosome-inactivating protein with lower molecular weight of 11,500 purified from the seeds of bitter gourd (Momordica charantia). Biochem. Biophys. Res. Commun. 229 287 - 294    DOI : 10.1006/bbrc.1996.1794
Rateb ME , Ebel R 2011 Secondary metabolites of fungi from marine habitats. Nat. Prod. Rep. 28 290 - 344    DOI : 10.1039/c0np00061b
Rodríguez-Martín A , Acosta R , Liddell S , Núñez F , Benito MJ , Asensio MA 2010 Characterization of the novel antifungal protein PgAFP and the encoding gene ofPenicillium chrysogenum. Peptides 31 541 - 547    DOI : 10.1016/j.peptides.2009.11.002
Selitrennikoff CP 2001 Antifungal proteins. Appl. Environ. Microbiol. 67 2883 - 2894    DOI : 10.1128/AEM.67.7.2883-2894.2001
Skouri-Gargouri H , Ali MB , Gargouri A 2009 Molecular cloning, structural analysis and modelling of the AcAFP antifungal peptide fromAspergillus clavatus. Peptides 30 1798 - 1804    DOI : 10.1016/j.peptides.2009.06.034
Skouri-Gargouri H , Gargouri A 2008 First isolation of a novel thermostable antifungal peptide secreted byAspergillus clavatus. Peptides 29 1871 - 1877    DOI : 10.1016/j.peptides.2008.07.005
Stirpe F 2013 Ribosome-inactivating proteins: from toxins to useful proteins. Toxicon 67 12 - 16    DOI : 10.1016/j.toxicon.2013.02.005
Taylor BE , Irvin JD 1990 Depurination of plant ribosomes by pokeweed antiviral protein. FEBS Lett. 273 144 - 146    DOI : 10.1016/0014-5793(90)81070-5
Wnendt S , Ulbrich N , Stahl U 1994 Molecular cloning, sequence analysis and expression of the gene encoding an antifungal-protein fromAspergillus giganteus. Curr. Genet. 25 519 - 523    DOI : 10.1007/BF00351672
Woo J-H , Kitamura E , Myouga H , Kamei Y 2002 An antifungal protein from the marine bacteriumStreptomycessp. strain AP77 is specific forPythium porphyrae, a causative agent of red rot disease inPorphyraspp. Appl. Environ. Microbiol. 68 2666 - 2675    DOI : 10.1128/AEM.68.6.2666-2675.2002
Xu J , Wang H , Fan J 2007 Expression of a ribosome-inactivating protein gene in bitter melon is induced bySphaerotheca fuligineaand abiotic stimuli. Biotechnol. Lett. 29 1605 - 1610    DOI : 10.1007/s10529-007-9433-3
Zhang G-P , Shi Y-L , Wang W-P , Liu W-Y 1999 Cation channel formed at lipid bilayer by Cinnamomin, a new type II ribosome-inactivating protein. Toxicon 37 1313 - 1322    DOI : 10.1016/S0041-0101(99)00078-1
Zhang Y , Mu J , Feng Y , Kang Y , Zhang J , Gu P-J 2009 Broad-spectrum antimicrobial epiphytic and endophytic fungi from marine organisms: isolation, bioassay and taxonomy. Marine Drugs 7 97 - 112    DOI : 10.3390/md7020097