J. Microbiol. Biotechnol. (2015), 25(5), 620–628 http://dx.doi.org/10.4014/jmb.1409.09071 Research Article Review jmb

Identification and Characterization of an Antifungal Protein, AfAFPR9, Produced by Marine-Derived fumigatus R9 Qi Rao†, Wenbin Guo†, and Xinhua Chen*

Key Laboratory of Marine Biogenetic Resources, Third Institute of Oceanography, State Oceanic Administration, Xiamen 361005, P.R. China

Received: September 23, 2014 Revised: October 29, 2014 A fungal strain, R9, was isolated from the South Atlantic sediment sample and identified as Accepted: November 13, 2014 . An antifungal protein, AfAFPR9, was purified from the culture

supernatant of Aspergillus fumigatus R9. AfAFPR9 was identified to be restrictocin, which is a

member of the ribosome-inactivating proteins (RIPs), by MALDI-TOF-TOF-MS. AfAFPR9 First published online displayed antifungal activity against plant pathogenic oxysporum, Alternaria longipes, November 14, 2014 Colletotrichum gloeosporioides, Paecilomyces variotii, and Trichoderma viride at minimum

*Corresponding author inhibitory concentrations of 0.6, 0.6, 1.2, 1.2, and 2.4 µg/disc, respectively. Moreover, AfAFPR9 Phone: +86-592-2195297; exhibited a certain extent of thermostability, and metal ion and denaturant tolerance. The Fax: +86-592-2085376; iodoacetamide assay showed that the disulfide bridge in AfAFP was indispensable for its E-mail: [email protected] R9

antifungal action. The cDNA encoding for AfAFPR9 was cloned from A. fumigatus R9 by RT- † These authors contributed PCR and heterologously expressed in E. coli. The recombinant AfAFP protein exhibited equally to this work. R9 obvious antifungal activity against C. gloeosporioides, T. viride, and A. longipes. These results

reveal the antifungal properties of a RIP member (AfAFPR9) from marine-derived Aspergillus fumigatus and indicated its potential application in controlling plant pathogenic fungi. pISSN 1017-7825, eISSN 1738-8872 Keywords: South Atlantic, antifungal activity, plant pathogenic fungi, ribosome-inactivating Copyright© 2015 by The Korean Society for Microbiology protein and Biotechnology

Introduction to potently produce an antifungal peptide (AFP) [18, 31]. Subsequently, a number of different antifungal proteins There are a vast number and diversity of microorganisms have been derived from ascomycetes, such as PAF from living in the oceans. The interaction between the marine Penicillium chrysogenum [6, 15, 19, 25], NAF from Penicillium microorganisms and their unique environments causes nalgiovense [7], AcAFP from Aspergillus clavatus [27, 28], the development of special metabolic pathways in these AnAFP from Aspergillus niger [9], NFAP from Neosartorya microorganisms. The antifungal substances from marine fisheri [5, 8], and Pc-Actin from Penicillium chrysogenum microorganisms are becoming an important part of discovering A096 [3]. According to their structure, molecular mass, and new antifungal and developing marine drugs in antifungal mechanism, the antifungal proteins are classified recent years. Many antimicrobial fungi have been isolated into ribosome-inactivating proteins (RIPs), pathogenesis- by culture-dependent methods from various marine organisms related proteins (PR), defensins, glycine/histidine-rich such as sponges and algae [35]. Rateb and Ebel [24] gave an proteins, lipid transfer proteins (LTPs), protease inhibitors, overview of new natural products from marine fungi and and other proteins [26]. their biological activities during 2006 to mid-2010, and 690 RIPs have been found in , fungi, mushrooms, and structures were presented. plants, and have a broad spectrum of biological activities, Now, a great variety of antifungal proteins with different including antitumor, antivirus, antifungus, and anti-insect antifungal characteristics have been identified from various activities. The RIPs have a glycosidase or phosphatase of fungus. Aspergillus giganteus was the first filamentous activity, resulting in the arrest of protein synthesis due to

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the ribosome damage caused by these two enzymes [23, Purification and Identification of Antifungal Protein from 30]. RIPs have been classified into three types. Type 1 RIPs A. fumigatus R9 are single-chain N-glycosidases with a molecular mass of A. fumigatus R9 was cultured in GPY medium at 28°C for 11 to 30 kDa. Type 2 RIPs contain two chains, a cell-binding 7 days. The purification of the antifungal protein was performed lectin (B chain) and an N-glycosidase (A chain), with a as previously presented [3]. Briefly, the culture supernatant of A. fumigatus strain R9 was obtained by vacuum filtration through molecular mass of 60 kDa [34]. Type 3 RIPs contain only qualitative filter paper. The culture supernatant was fully saturated one chain, which covers both the cell-binding lectin and N- with ammonium sulfate and then centrifugated at 12,000 ×g for glycosidase [22]. Type 1 RIPs are much less toxic, as they lack 30 min at 4°C. The precipitate containing crude proteins was the B-chain, and thus they do not bind and enter cells [29]. dissolved in distilled water and dialyzed at 4°C for 24 h, and In this study, an antifungal strain, Aspergillus fumigatus finally lyophilized. R9, was isolated from a South Atlantic sediment sample. Its The crude proteins and the fractions separated by ion exchange antifungal protein AfAFPR9 was purified and identified as a chromatography were tested for antifungal activity against the member of RIPs. The antifungal properties of the natural tested fungi. Ion-exchange chromatography was performed with an AKTA FPLC system (GE Healthcare, USA). The crude protein and recombinant AfAFPR9 proteins were characterized. The solution was loaded onto a DEAE Sepharose Fast Flow column results obtained suggest that AfAFPR9 may represent a potential candidate of fungicide controlling plant pathogenic (GE Healthcare), which was pre-equilibrated with starting buffer fungi. 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% Materials and Methods elution buffer B, 10 CV; 100% elution buffer B, 2 CV; 0% elution buffer B, 2 CV. The bioactive fraction was concentrated by Tested Strains ultrafiltration in a Vivaspin 15R (molecular weight cutoff 5,000; The tested fungi, including Colletotrichum gloeosporioides (ACCC Sartorius, Germany) and further purified on a CM Sepharose Fast 31200, Agricultural Cultural Collection of China), Flow column (GE Healthcare, USA) under the same program. The (ACCC 31352), Trichoderma viride (ACCC 30902), Rhizoctonia solani resulting active component was concentrated by ultrafiltration in (ACCC 36316), Alternaria longipes (ACCC 30002), and Sclerotinia a Vivaspin 15R and its purity was assessed by 15% sodium sclerotiorum (ACCC 36081), were provided by Agricultural Cultural dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) Collection of China. Paecilomyces variotii (CGMCC 3.776, China and silver nitrate staining. The purified antifungal protein, named General Microbiological Culture Collection Center) was obtained AfAFP ; was identified by MALDI-TOF-TOF-MS at Shanghai from CGMCC, Institute of Microbiological Chinese Academy of R9 Institutes for Biological Sciences, Chinese Academy of Sciences. Sciences. These seven tested fungi are important plant pathogenic The database search was performed on the Mascot server (http:// fungi in agriculture. www.matrixscience.com/search_from_select.html) with MALDI- TOF-TOF-MS data. Isolation and Identification of an Antifungal Strain The sediment samples used for strain isolation were collected Assay of Antifungal Activity from the South Atlantic (W 14.87°, S 12.12°; depth of water: 2,647 m). The assay for antifungal activity toward the seven phytopathogenic Isolation and identification of the strains as well as analysis of fungal species was carried out in PDA plates. One 0.6 cm diameter their antifungal activity were carried out as previously described piece of tested phytopathogenic fungal strains’ cylinder agar with [3]. Briefly, the sediment samples were diluted with sterilized mycelial growth was placed on the center of a PDA plate. After seawater and approximately 200 µl of the diluted sample was the mycelial colony had developed, sterile blank paper discs of spread on plates containing different types of medium, such as 0.65 cm diameter were placed at a distance of 0.8 cm away from GPY (glucose 1%, peptone 0.2%, and extract 0.05%) and the rim of the growing mycelial colony. Fifty microliter aliquots of YTM (0.5% yeast extract, 0.3% tryptone, and 2.5% mannitol). the supernatant of A. fumigatus R9 and the fractions of ion- Plates were incubated at 28°C for growth. The strains were exchange chromatography were added to each paper disc. Fifty selected based on their morphological features and inoculated microliters of GPY medium or starting buffer A, which dissolved into the corresponding liquid media for further growth to the antifungal protein, was used as blank controls. The plates evaluate their antifungal potential. For the identification of the were incubated at 28°C until mycelial growth enveloped discs antifungal strain, the ribosomal internal transcribed spacer (ITS) containing the control disc, or formed crescents of inhibition DNA sequence was amplified using the primers ITS5 (5’- around discs containing samples with antifungal activity. GGAAGTAAAAGTCGTAACAAGG-3’) and ITS4 (5’-TCCTCC GCTTATTGATATGC-3’), sequenced at Sangon Biotech (Shanghai, MIC Determination of Antifungal Protein AfAFP China) and analyzed for similarity using BLAST (http://blast.ncbi. R9 The minimum inhibitory concentration (MIC) of AfAFP against nlm.nih.gov/Blast.cgi). R9

J. Microbiol. Biotechnol. Characterization of Antifungal Protein AfAFPR9 622

different pathogenic fungi was determined by the paper disc 30 sec extension at 72°C, followed by a cycle of 72°C for 10 min. dilution method [32]. Two-fold serial dilutions of AfAFPR9 solution The amplified product was sequenced at Sangon Biotech. Sequence ranging from 0.4 to 0.00625 µg/µl were prepared, and 50 µl of homology search was performed using the BLAST program (http:// each diluted solution was added onto paper discs placed 0.8 cm blast.ncbi.nlm.nih.gov/Blast.cgi). Multiple sequence alignment was from the edge of growing on a PDA plate. The plates were carried out using the DNAMAN tool (Lynnon Biosoft). placed at 28°C for several days, depending on the tested pathogen.

The MIC was determined as the lowest concentration of active Heterologous Expression of AfAFPR9 in Escherichia coli and protein that could inhibit visible mold growth and calculated as Antifungal Activity Analysis of Its Recombinant Protein the total protein added on each paper disc (microgram per disc). AfAFPR9 was expressed as a fusion protein with 6× His-tag and Starting buffer A without active protein was used as the blank thioredoxin (TRX) using a pET-32a vector in the E. coli Rosetta strain control. (Amersham Pharmacia Biotech). The AfAFPR9 cDNA was amplified using the following primers: forward primer 5’-CCGGAATTC Physiochemical Properties of Antifungal Protein GCGACCTGGACATGC-3’ with an EcoRI digestion site (underlined),

To examine the effects of metal ions on AfAFPR9 activity, several and reverse primer 5’-CCGCTCGAGCTAATGAGAACACAGTC- metal ions, such as Na+, K+, Mg2+, Cu2+, and Ag+, were dissolved in 3’ with an XhoI digestion site (underlined). The PCR product was starting buffer A to a final concentration of 10 mM. AfAFPR9 cloned into the EcoRI/XhoI-digested pET-32a. The resulting

(0.4 µg/µl) was treated with the different ion solutions at room plasmid pET-32a-AfAFPR9 was transformed into the competent temperature for 1 h before being tested for antifungal activity. Fifty cells of E. coli Rosetta using pET-32a as a vector control. The microliters of the treated AfAFPR9 was used for the antifungal positive colonies were identified by PCR and DNA sequencing. tests. The AfAFPR9 protein without metal ion treatment was used AfAFPR9 fusion protein was expressed by 1 mM isopropy l-β-D- as the positive control, and starting buffer A and ion solutions thiogalactopyranoside (IPTG) induction at 16°C for 48 h. The were used as blank and negative controls. recombinant proteins were then purified using Ni2+ IDA affinity

To evaluate the thermostability of AfAFPR9, AfAFPR9 solution chromatography (Novagen, USA) as described in the supplier’s (0.4 µg/µl) was treated at 100°C for 20, 40, 60, and 80 min, respectively. instructions. SDS-PAGE was performed for analysis of recombinant After cooling to room temperature, the residual antifungal activity protein expression and purification. The antifungal activity of the of AfAFPR9 was tested against A. longipes. Fifty microliters of the purified recombinant protein was determined using a microtiter heat-treated AfAFPR9 was used for the antifungal test. AfAFPR9 plate assay as previously described [10]. solution without heat treatment and starting buffer A were used as the positive and blank controls, respectively. Effect of the Disulfide Bridge on the Antifungal Activity of

For denaturant-resistant test, AfAFPR9 (0.4µg/µl) was treated AfAFPR9 with 0.1% SDS, 0.1% carbamide, and 0.1% guanidine hydrochloride AfAFPR9 (0.4µg/µl) was reduced by β-mercaptoethanol, and at 28°C for 24 h. The treatment effect was analyzed by observing the partially reduced AfAFPR9 was alkylated by iodoacetamide [2, the residual antifungal activity of AfAFPR9. Fifty microliters of the 13]. Freshly prepared β-mercaptoethanol was added to the AfAFPR9 treated antifungal protein was used for the antifungal tests. The solution to a final concentration of 1 mM. The mixture was incubated

AfAFPR9 without denaturant treatment was used as the positive at 37°C for 10 min followed by dialyzing against distilled water at control. Only denaturants and starting buffer A were used as blank 4°C for 24 h with several changes of water. After that, 100 µl of the controls. All experiments, including metal-ion-resistant, thermostability, partially reduced AfAFPR9 was placed on a paper disc and tested and denaturant-resistant tests, were done in triplicate and the for its antifungal activity against A. longipes. Another 400 µl of the results are shown as mean ± standard deviation (SD) of three AfAFPR9 was diluted into the same volume of iodoacetamide experiments. 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,

Cloning of AfAFPR9 cDNA pH 8.5). After alkylation, the mixture was dialyzed against distilled Total RNA was extracted from strain R9 by using TRIzol reagent water at 4°C for 24 h with several changes of water. Finally, 200 µl

(Invitrogen, USA), according to the manufacturer’s instructions. of the alkylated AfAFPR9 was placed on a paper disc for antifungal First-strand cDNA was synthesized from 1 µg of total RNA using test. The antifungal experiments were done in triplicate and the oligo-dT-adaptor primer (TaKaRa, Dalian, China) and used as the results are shown as the mean± SD. template for PCR amplification of AfAFPR9 cDNA. Based on the restrictocin gene sequence (GenBank Accession No. AAA32707.1), Results two primers were designed: forward primer 5’-GCGACCTGG ACATGCATCAACCAA-3’ and reverse primer 5’-CTAATGAGA Strain Identification and Antifungal Activity Detection ACACAGTCTCAAGTC-3’. PCR was performed with an initial A total of 24 strains were purified based on their denaturation step of 3 min at 94°C and then 35 cycles were run as morphological features and inoculated into corresponding follows: 30 sec denaturation at 94°C, 30 sec annealing at 55°C, and liquid media for further growth to evaluate their antifungal

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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. 1A). 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. Isolation of the antifungal protein by ion-exchange (Fig. 1B). Fraction BP2 showed antifungal activity against chromatography. the tested fungi above (Fig. 2). SDS-PAGE analyses showed (A) The crude protein was loaded on a DEAE Sepharose Fast Flow that only one protein band corresponding to about 18 kDa column. (B) Antifungal fraction AP2 was loaded on a CM Sepharose Fast Flow column. was observed in fraction BP2 (Fig. 3). This purified antifungal protein produced by A. fumigatus R9 was named AfAFPR9. them were contained in a protein named restrictocin

Identification and cDNA Cloning of AfAFPR9 (GenBank Accession No. AAA32707.1) (Table 1), which

After being treated with trypsin, four peptide sequences was a member of the RIPs. The AfAFPR9 cDNA was then of AfAFPR9 were identified with MALDI-TOF-TOF. All of cloned using RT-PCR. It is 450 bp long, encoding a protein

Fig. 2. 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.

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Fig. 4. 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 15% SDS-PAGE of the purified antifungal protein. Fig. 3. of the characteristic microbial RNases superfamily sequence (21-148 Lane 1. Standard protein marker; Lane 2. Antifungal fraction BP2 aa); △: indicates the five active sites, Tyr47, His49, Glu95, Arg120, collected from CM Sepharose Fast Flow column. and His136; ☆: indicates the two cysteine residues in AfAFPR9. of 149 amino acids. It has been submitted to NCBI GenBank with an accession number of KJ081439. The deduced performed. The MICs of AfAFPR9 were 0.6, 0.6, 1.2, 1.2, and

AfAFPR9 protein contained all the four peptides identified 2.4 µg/disc against F. oxysporum, A. longipes, C. gloeosporioides, by MALDI-TOF-TOF (Fig. 4), indicating that the cDNA P. variotii, and T. viride, respectively. In addition, metal + + 2+ 2+ + cloned here encodes the antifungal protein AfAFPR9. ions, such as Na , K , Mg , Cu , and Ag were tested for

Multiple sequence alignment showed that AfAFPR9 shared their effects on the antifungal activity of AfAFPR9. The

84.66% amino acid sequence identity with restrictocin from antifungal activity of AfAFPR9 against all five tested strains Aspergillus restricts (AAA32707.1), 84.09% with mitogillin was not reduced when it was treated with Na+ (Table 2). + from Aspergillus restrictus (P67876.1), 72.32% with α-sarcin When AfAFPR9 was treated with K , its antifungal activity from Aspergillus giganteus (CAA43180.1), 71.75% with clavin against F. oxysporum was 87.16 ± 13.31% retained but was from Aspergillus clavatus (ACC49407.1), 5.66% with RIP largely impaired against the other four strains (Table 2). 2+ 2+ + from Momordica charantia L (AAS17014.1), and 8.15% with AfAFPR9 was sensitive to metal ions Mg , Cu , and Ag , antiviral protein 1 from Bougainvillea xbuttiana (AAY34283.2). since its antifungal activity was largely or even completely 2+ 2+ + These results indicate that the antifungal protein AfAFPR9 lost after being treated with 10 mM Mg , Cu , or Ag is a member of the RIP family from marine fungi, belonging (Table 2). However, the antifungal activity of AfAFPR9 to type 1 RIP. against P. variotii was almost not reduced when it was treated with Mg2+ (Table 2). Different metal ions thus have

Physiochemical Properties of AfAFPR9 different effects on the antifungal activity of AfAFPR9.

In order to investigate the MICs of AfAFPR9 against the Finally, the denaturant-resistant test was also carried out. five tested strains, the paper disc dilution method was Relatively, the antifungal activity of AfAFPR9 against all

Table 1. Mass spectrum identification of antifungal protein AfAFPR9. GenBank Accession No. Species Protein name Protein score Protein mass Peptide sequences AAA32707.1 Aspergillus restrictus Restrictocin 288 849.4069 LLYNQAK 1186.5281 VFCGIVAHQR 1573.6360 ATWTCINQQLNPK 2164.9915 VIYTYPNKVFCGIVAHQR

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Table 2. Antifungal activity of AfAFPR9 against the sensitive tested fungi after treatment with metal ions (10 mM). Radius of inhibition zone (mm) Tested fungi Untreated Na+ K+ Mg2+ Cu2+ Ag+ A. longipes 7.13 ± 0.94 6.67 ± 0.56 1.53 ± 0.26 0 0 0 C. gloeosporioides 6.17 ± 0.48 5.63 ± 0.60 1.33 ± 0.26 0 0 0 F. oxysporum 6.77 ± 0.82 6.27 ± 0.46 6.17 ± 0.45 0 0 0 P. variotii 6.43 ± 0.97 4.77 ± 0.45 1.67 ± 0.39 4.73 ± 0.41 1.43 ± 0.47 0 T. viride 5.37 ± 0.54 4.13 ± 0.56 1.47 ± 0.45 0 0 0

Table 3. Antifungal activity of AfAFPR9 against the sensitive tested fungi after treatment with several denaturants. Radius of inhibition zone (mm) Tested fungi Untreated SDS (0.1%) Carbamide (0.1%) Guanidine hydrochloride (0.1%) A. longipes 7.13 ± 0.94 6.97 ± 0.17 6.67 ± 0.45 6.03 ± 0.31 C. gloeosporioides 6.17 ± 0.48 6.13 ± 0.52 5.03 ± 0.52 5.13 ± 0.25 F. oxysporum 6.77 ± 0.82 6.03 ± 0.56 5.27 ± 0.33 4.13 ± 0.25 P. variotii 6.43 ± 0.97 4.77 ± 1.03 4.93 ± 0.50 6.57 ± 0.45 T. viride 5.37 ± 0.54 3.93 ± 0.97 4.53 ± 0.40 3.53 ± 0.40

five tested strains was slightly or not affected by 0.1% SDS, The Disulfide Bridge in AfAFPR9 Contributing to Its 0.1% carbamide, and 0.1% guanidine hydrochloride (Table 3). Antifungal Activity

AfAFPR9 maintained undamaged activity against A. longipes In order to investigate whether the two cysteine residues when it was heated at 100°C for 20 min (Fig. 5). However, in AfAFPR9 form a disulfide bridge that contributes to its with the extension of heating time, the activity of AfAFPR9 antifungal activity, the iodoacetamide assay was carried was gradually lost. When heated at 100°C for 80 min, its out. When AfAFPR9 was partially reduced by β-mercaptoethanol, antifungal activity was completely lost (Fig. 5). These its antifungal activity against A. longipes was reduced by results indicated that AfAFPR9 exhibited a certain extent of 25.83 ± 10.10% (Fig. 6, middle colomn). When the mercapto thermostability. groups of the two cysteine residues were fully alkylated by

iodoacetamide, the antifungal activity of AfAFPR9 against

Effect of the disulfide bridge on the antifungal activity Fig. 5. Antifungal activity of AfAFPR9 against A. longipes after Fig. 6. heat treatment at 100°C for 20, 40, 60, and 80 min. of AfAFPR9 against A. longipes. The antifungal activity was indicated as the average radius, with The antifungal activity was indicated as the average radius, with standard deviation, of the inhibition zone forming on the plates by standard deviation, of the inhibition zone forming in the plates by AfAFP against A. longipes. AfAFPR9 against A. longipes. R9

J. Microbiol. Biotechnol. Characterization of Antifungal Protein AfAFPR9 626

Fig. 7. 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. Fig. 8. Analysis of antifungal activity of the recombinant

AfAFPR9 protein. A. longipes was almost lost completely (Fig. 6, right colomn). The antifungal activity was expressed as the growth inhibition of These results indicated that the disulfide bridge in AfAFP R9 tested fungi by measuring the OD600. of the tested fungi at 24 h after contributed to its antifungal activity. treatment with AfAFPR9.

Expression of AfAFPR9 in Escherichia coli

The AfAFPR9 cDNA was cloned into the pET-32a vector recombinant AfAFPR9 had obvious antifungal activity against and expressed in E. coli Rosetta after IPTG induction. Both C. gloeosporioides, T. viride, and A. longipes, and almost no induced and non-induced E. coli Rosetta/pET-32a-AfAFPR9 effect on the growth of F. oxysporum (Fig. 8). This method is and E. coli Rosetta/pET-32a (the vector control) were not appropriate for P. variotii, whose hyphae entwined analyzed by SDS-PAGE (Fig. 7). A 35 kDa band (Fig. 7, together, thereby resulting in an unchanged OD600 (Fig. 8). lane 5) corresponding to the size of recombinant AfAFPR9 These results indicate that the recombinant AfAFPR9 protein with 6× His-tag and thioredoxin (TRX) was only produced in E. coli exhibits good antifungal activity. observed in the induced E. coli Rosetta/pET-32a-AfAFPR9, indicating that the recombinant AfAFPR9 was specifically Discussion expressed in E. coli Rosetta/pET-32a-AfAFPR9. The recombinant 2+ AfAFPR9 protein was then purified using Ni IDA affinity The marine-derived RIP AfAFPR9 had a broad antifungal chromatography (Fig. 7, lane 6). spectrum in that it could inhibit some plant pathogenic fungi, including F. oxysporum, A. longipes, C. gloeosporioides,

Antifungal Activity of the Recombinant AfAFPR9 P. variotii, and T. viride. AfAFPR9 was different from other RIPs

The antifungal activity of the recombinant AfAFPR9 was in origin, function, and antimicrobial spectrum. Lyophyllin measured against the tested fungi in comparison with the was isolated from mushroom Lyophyllum shimeji and natural AfAFPR9, using culture medium and recombinant exhibited good antifungal activity against Physalospora TRX protein as control. In all of the experiments, five piricola and Coprinus comatus [11]. Luffacylin from sponge replicates were prepared for each treatment and the gourd seeds exerted antifungal activity against Mycosphaerella concentrations of proteins were all at 400 ng/µl. The arachidicola and Fusarium oxysporum [21]. Hispin from seeds antifungal activity was observed by measuring the optical of the hairy melon was also found to have antifungal density at 600 nm (OD600) of the tested fungi growing on activity against C. comatus [17]. Bougainvillea xbuttiana antiviral microplates at 24 h after treatment with AfAFPR9. The protein 1 from E. coli exhibited antiviral activity against

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sunnhemp rosette virus [4]. As with some other RIPs SOA (No. 2013022), grants from China Ocean Mineral expressed in E. coli, recombinant AfAFPR9 also exhibited Resources Research and Development Association (DY125- antifugal activity. Recombinant Momordica charantia L RIP 15-T-07) Scientific Research Project of the Marine Public can inhibit the growth of the Sphaerotheca fuliginea in vitro Welfare Industry of China (201205020), Natural Science [33]. Clavin from the filamentous fungus A. clavatus IFO Foundation of China (41306166), and Xiamen South Ocean 8605 was expressed in E. coli and its recombinant protein Research Center (13GZP002NF08). showed antitumor activity [20].

The deduced AfAFPR9 protein of the cDNA has a References characteristic microbial RNases superfamily sequence at 21- 148 aa and five active sites, Tyr47, His49, Glu95, Arg120, 1. Batta G, Barna T, Gaspari Z, Sandor S, Kövér KE, Binder U , and His136, as found in restrictocin from A. restrictus (Fig. 4) et al. 2009. Functional aspects of the solution structure and dynamics of PAF - a highly stable antifungal protein from [12]. This provided further evidence that AfAFPR9 is a member of the RIP family. Penicillium chrysogenum. FEBS J. 276: 2875-2890. The MIC test results showed the difference in antifungal 2. Cao A, Hu D, Lai L. 2004. Formation of amyloid fibrils from fully reduced hen egg white lysozyme. Protein Sci. 13: 319-324. activities of AfAFP against five tested strains, which could R9 3. Chen Z, Ao J, Yang W, Jiao L, Zheng T, Chen X. 2013. be attributed to the different sensitivity of these five strains Purification and characterization of a novel antifungal protein to AfAFP . In the metal ion tests, the antifungal activity of R9 secreted by Penicillium chrysogenum from an Arctic sediment. AfAFPR9 was maintained by treatment of monovalent ions, Appl. Microbiol. Biotechnol. 97: 10381-10390. and partially or completely lost by treatment by divalent 4. Choudhary N, Yadav O, Lodha M. 2008. Ribonuclease, ions such as Mg2+ and Cu2+. It was presumed that the deoxyribonuclease, and antiviral activity of Escherichia coli- divalent ions Mg2+ and Cu2+ could change the structure of expressed Bougainvillea xbuttiana antiviral protein 1. Biochemistry

AfAFPR9, thus leading to the loss of activity. Although the (Moscow) 73: 273-277. divalent ions such as Mg2+ and Cu2+ are rich in soil, they 5. Galgóczy L, Kovács L, Karácsony Z, Virágh M, Hamari Z, have no restriction in practical field application since Vágvölgyi C. 2013. Investigation of the antimicrobial effect of Neosartorya fischeri antifungal protein (NFAP) after AfAFPR9 will be sprayed on the plants. It was shown that AfAFP exhibited a certain extent of heterologous expression in Aspergillus nidulans. Microbiology R9 159: 411-419. thermostability. Two cysteine residues in AfAFP may R9 6. Galgóczy L, Virágh M, Kovács L, Tóth B, Papp T, Vágvölgyi form a disulfide bridge, which would contribute to its C. 2013. Antifungal peptides homologous to the Penicillium thermostability (Fig. 4). Previous reports showed that chrysogenum antifungal protein (PAF) are widespread among antifungal proteins AFP and PAF had eight and six cysteine Fusaria. Peptides 39: 131-137. residues, respectively, which might form disulfide bridges 7. Geisen R. 2000. P. nalgiovense carries a gene which is contributing to their heat stability [14, 16]. Further study homologous to the paf gene of P. chrysogenum which codes also revealed that the disulfide bridge in AfAFPR9 also for an antifungal peptide. Int. J. Food Microbiol. 62: 95-101. contributed to its antifungal activity. This was consistent 8. Kovács L, Virágh M, Takó M, Papp T, Vágvölgyi C, Galgóczy with the previous results that the disulfide bridges in L. 2011. Isolation and characterization of Neosartorya fischeri antifungal PAF was indispensable for its antifungal action antifungal protein (NFAP). Peptides 32: 1724-1731. [1]. 9. Lee DG, Shin SY, Maeng C-Y, Jin ZZ, Kim KL, Hahm K-S. The antifungal protein AfAFP was purified from marine- 1999. Isolation and characterization of a novel antifungal R9 peptide from Aspergillus niger. Biochem. Biophys. Res. Commun. derived A. fumigatus and identified as a member of the RIP 263: 646-651. family. The broad antifungal spectrum, good antifungal 10. López-García B, Moreno AB, San Segundo B, De los Ríos V, activity, thermostability, and resistance to several metal Manning JM, Gavilanes JG, Martínez-del-Pozo Á. 2010. ions and denaturants of AfAFPR9 suggest that it may Production of the biotechnologically relevant AFP from represent a potential candidate of fungicide controlling Aspergillus giganteus in the yeast Pichia pastoris. Protein Expr. plant pathogenic fungi. Purif. 70: 206-210. 11. Lam SK, Ng TB. 2001. First simultaneous isolation of a Acknowledgments ribosome inactivating protein and an antifungal protein from a mushroom (Lyophyllum shimeji) together with evidence This study was financially supported by the Scientific for synergism of their antifungal effects. Arch. Biochem. Research Foundation of Third Institute of Oceanography, Biophys. 393: 271-280.

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