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Antagonistic activities of novel peptides from Bacillus amyloliquefaciens PT14 against Fusarium solani and F. oxysporum YOUNG GWON KIM, Hee-Kyoung Kang, KEE-DEOK KWON, CHANG HO SEO, HYANG BURM LEE, and Yoonkyung Park J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b04068 • Publication Date (Web): 23 Oct 2015 Downloaded from http://pubs.acs.org on October 27, 2015

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Journal of Agricultural and Food Chemistry

1

Antagonistic

activities

of

novel

peptides

from

Bacillus

2

amyloliquefaciens PT14 against Fusarium solani and F. oxysporum

3 4

YOUNG GWON KIM†,#, HEE KYOUNG KANG†,#, KEE-DEOK KWON§, CHANG HO

5

SEO§, HYANG BURM LEE¶ AND YOONKYUNG PARK†, ‡, *

6 7

†

Department of Biomedical Science, Chosun University, Gwangju 61452, Korea

8

§

Department of Bioinformatics, Kongju National University, Kongju 32588, Korea

9

¶

College of Agriculture and Life Sciences, Chonnam National University, Gwangju

10

61186, Korea

11

‡

Research Center for Proteineous Materials, Chosun University, Gwangju 61452, Korea

12 13

TITLE RUNNING HEAD: Antagonistic activities of PT14 peptides

14 15

*Corresponding author (e-mail [email protected]; fax 82-62-225-6758).

16 17 18 19 20 21 22

†

Research Center for Proteineous Materials (RCPM), Chosun University. Department of Biomedical Science and BK21 Research Team for Protein Activity Control, Chosun University. §

#

These authors contributed equally to this work

1

ACS Paragon Plus Environment


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ABSTRACT: Bacillus species have recently drawn attention due to their potential use

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in the biological control of fungal diseases. Here we report on the antifungal activity of

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novel peptides isolated from Bacillus amyloliquefaciens PT14. Reverse-phase high

26

performance liquid chromatography revealed that B. amyloliquefaciens PT14 produces

27

five peptides (PT14-1, 2, 3, 4a and 4b) that exhibit antifungal activity but are inactive

28

against bacterial strains. In particular, PT14-3 and PT14-4a showed broad spectrum

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antifungal activity against Fusarium solani and F. oxysporum. The PT14-4a N-terminal

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amino acid sequence was identified through Edman degradation, and a BLAST

31

homology analysis showed it not to be identical to any other protein or peptide. PT14-4a

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displayed strong fungicidal activity with minimal inhibitory concentrations of 3.12

33

mg/L (F. solani) and 6.25 mg/L (F. oxysporum), inducing severe morphological

34

deformation in the conidia and hyphae. On the other hand, PT14-4a had no detectable

35

hemolytic activity. This suggests PT14-4a has the potential to serve as an antifungal

36

agent in clinical therapeutic and crop-protection applications.

37 38

KEYWORDS: Bacillus amyloliquefaciens, antifungal peptide, biological control,

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Fusarium oxysporum, Fusarium solani

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2


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■ INTRODUCTION

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Plant diseases caused by viruses, bacteria and fungi are responsible for significant crop

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losses and reductions in the quality and safety of agricultural products. They cause, for

44

example, wilt and rot diseases that diminish the commercial production of numerous

45

important agricultural and ornamental plant species, including tomatoes, bananas,

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cotton and tulip bulbs.1 One deadly pathogen infecting agricultural products is

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Fusarium oxysporum. Unfortunately, once F. oxysporum is established in the soil, it can

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be extremely difficult to eradicate, as its chlamydospores can remain dormant and infect

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the soil for many years. F. oxysporum f. sp. spinaciae is an important pathogen in

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spinach and is typically found in fields where the F. oxysporum population is high.2,3 It

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is also a major component of a seedling disease complex.2 Up to now, farmers have

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mainly relied on chemical pesticides to control this fungus; however, use of chemical

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pesticides inevitably leads to environmental pollution and microbial tolerance to the

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chemicals.4 For those reasons as well as for their potent bactericidal and/or fungicidal

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activities over a broad spectrum of microorganisms and their low propensity to induce

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tolerance, antimicrobial peptides have been attracting attention.

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In recent years, several groups have explored the potential use of rhizosphere-

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associated bacteria (Bacillus, Pseudomonas and Burkholderia spp.) as biological control

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agents.5-7 Among these, Bacillus spp., which occur ubiquitously in the soil, are

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considered to be natural “factories” for production of a wide array of biologically active

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compounds against soil-borne and post-harvest pathogens, including Fusarium spp.,

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Rhizoctonia spp., Pythium spp. and Aspergillus spp.8,10-12 In addition, they form

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endospores that are highly resistant to unfavorable environmental conditions.8,9

3



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Consequently, strains such as B. subtilis, B. megaterium, and B. cereus have already

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been developed as commercial biocontrol agents.13,14

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Bacillus amyloliquefaciens is closely related to B. subtilis and is able to produce a

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variety of structurally diverse antimicrobial compounds, more than half of which are

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cyclic or linear lipopeptides.9 The cyclic lipopeptides, which include the iturin group

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(iturins, mycosubtilins, and bacillomycins), the surfactin group and the fengycin group

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(fengycin A, fengycin B, and fengycin C),15 have well-recognized potential for use in

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biotechnology and biomedical applications related to their excellent antibacterial,

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antiviral, antitumoral, antifungal and antimycoplasmic activities.16 The objectives of the

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present study were to (a) investigate antagonistic effects of the peptides isolated from B.

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amyloliquefaciens PT14 against F. solani and F. oxysporum under in vitro conditions;

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(b) purify PT14 peptides by RP-HPLC, perform chemical profiling by MALDI-

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TOF/MS, and analyze their N-terminal sequences; (c) conduct antifungal assays with

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PT14 peptides, using disc diffusion, micro-well dilution, and scanning electron

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microscopy.

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4


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■ MATERIALS AND METHODS

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Bacterial strains and chemicals. A total of 14 Bacillus amyloliquefaciens

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strains isolated from soil were provided by the Environment Microorganism Laboratory,

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Chonnam National University (Gwangju, Korea).

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For antifungal and antimicrobial assays, the following strains were obtained from

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the Korea Collection for Type Cultures (KCTC) at the Korea Research Institute of

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Bioscience and Biotechnology (KRIBB, Daejon, Korea): F. solani (KCTC 6326), F.

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oxysporum (KCTC 16909), Trichoderma harzianum (KCTC 6043), Staphylococcus

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aureus (KCTC 1621) and Escherichia coli (KCTC 1682).

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A SPE 900 mg Lrg Pore C18 column was purchased from Maxi-CleanTM (USA). A

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reverse-phase C18 HPLC column for purification of peptides was purchased from

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Shimadzu Corporation (Kyoto, Japan). Acetonitrile and water (HPLC grade) were

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obtained from Burdick & Jackson Inc. (Muskegon, MI, USA), and trifluoroacetic acid

93

(TFA) was obtained from Merck. The Luria-Bertani (LB) medium used for B.

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amyloliquefaciens culture and other media were purchased from Difco Laboratories Inc.

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(Detroit, MI, USA). All other chemicals and reagents were of analytical grade.

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Screening B. amyloliquefaciens for antifungal activity. To isolate effective

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antagonists against F. oxysporum, F. solani, and Trichoderma harzianum, 14 B.

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amyloliquefaciens strains were screened using paper/agar disc assays for their antifungal

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activity. Of these, five strains (PT1, PT2, PT5, PT13, and PT14) exhibited strong

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antagonistic activity against F. oxysporum, F. solani, and T. harzianum in the assays

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(Supplementary Figure 1A). The strongest antagonistic activity was exhibited by PT14,

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which formed an 18-mm inhibition zone against F. solani in the agar diffusion assay

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(Supplementary Figure 1B, Supplementary Table 1). Thus, B. amyloliquefaciens 5



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PT14 was selected for subsequent physiological and biological analyses.

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Isolation and purification of antifungal proteins. B. amyloliquefaciens was

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grown at 37°C in 700 mL LB medium. After incubation for 24 h (to stationary growth

107

phase) in a shaking incubator (120 rpm), the culture medium was centrifuged at 14,000

108

× g for 30 min to remove the cells. The supernatants were then filtered through a 185-

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mm paper after SPE C18 column purification to remove remaining cells and LB medium.

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Peptides exhibiting antifungal activity were separated by RP-HPLC on a C18

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reverse phase column (VP-ODS, 4.6 × 250 mm) using a HPLC system (Shimadzu

112

Corporation, Japan) previously equilibrated with 0.1% (v/v) trifluoroacetic acid in

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HPLC-grade water containing 5% acetonitrile. Elution was performed using a linear

114

gradient of 0.1% (v/v) TFA in water (solvent A) and 0.1% (v/v) TFA in acetonitrile

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(solvent B) at a flow rate of 1 mL/min as follows: PT14 fractions were eluted in the

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presence of 40% solvent B for 15 min, 40-65% solvent B for 45 min and 65-95% for 65

117

min. Fractions eluted between 30 min and 45 min were pooled, dried using a vacuum

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concentrator, and then successively subjected for further purification under the same

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conditions. The eluate was monitored by measurement of the absorbance at 215 nm, and

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each peak fraction was collected and assayed for antifungal activity.

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Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis

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(SDS-PAGE). Electrophoresis of low-molecular-mass peptides was performed

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according to the method of Schagger and Jagow17 on 16.5% polyacrylamide gel. After

124

electrophoresis, the gel was stained with Coomassie Brilliant Blue G-250. The

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molecular weight markers in Tricine SDS-PAGE included triosephosphate isomerase

126

from rabbit muscle (26.6 kDa), myoglobin from horse heart (17 kDa), α-lactalbumin

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from bovine milk (14.2 kDa), aprotinin from bovine lung (6.5 kDa), oxidized insulin 6


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chain B from bovine pancreas (3.5 kDa) and bradykinin (1 kDa). Protein

determination.

Matrix-assisted

laser

desorption/ionization

130

(MALDI-MS) was performed in linear mode using an Axima-CFR MALDI-TOF mass

131

spectrometer (Kratos Analytical, Manchester, UK). The protein concentration was

132

determined according to the method of Bradford (1972)18 using bovine serum albumin

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(BSA) as the calibration standard.

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N-terminal amino acid sequence analysis. The N-terminal amino acid

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sequences of the purified peptides were analyzed using automated Edman degradation

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in a pulse liquid automatic sequencer (Applied Biosystems Inc., model 491) at the

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Korean Basic Science Institute (Seoul, Korea). The amino acid sequence was then

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compared to sequences from the NCBI database using the BLAST program.

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Antifungal assay. Assays for antifungal activity toward phytopathogenic fungi

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were carried out in petri dishes containing potato dextrose agar (PDA; Difco

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Laboratories Inc.). After development of a mycelial colony, sterile black paper disks (6

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mm in diameter) were placed 0.5 cm away from the rim of the colony. Aliquots of

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purified peptide in 10 mM PBS buffer (pH 7.4) were layered onto the disks, and the

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plates were incubated at 28°C for 18 h to allow mycelial growth.

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The minimal inhibitory concentrations (MICs) of the purified peptides against the

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fungal strains were determined as described by Moor et al.19 The MIC was defined in

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this study as the lowest concentration of peptide that inhibited fungal growth. After

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fungal strains were grown at 28°C in PDA agar plates, the fungal conidia were collected

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using 0.08% Triton X-100 and seeded at a density of 2 × 104 spore/mL (counted using a

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hemocytometer) in the wells of a 96-well plate (Nunc F96 microtiter plates, Denmark)

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containing YM medium. Serially diluted peptide solutions (final concentrations: 50, 25, 7



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12.5, 6.25 and 3.125 mg/L) were then added to the wells, and wells not receiving

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peptide were used as negative controls. The plate was then incubated overnight at 28°C,

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after which morphological changes to the conidia were examined using an ECLIPSE

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TE300 phase contrast microscope (Nikon, Japan). Identical assays examining the effects

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of the peptides on fungal hyphae were also performed.

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Antibacterial assay. Antibacterial activity was tested by measuring the

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inhibition of bacterial growth in sterile 96-well plates. Serial dilutions of sample

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solutions were added to the wells to final concentrations ranging from 0.78 to 100 µg.

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Then after adding bacteria to the wells (5 × 105 cfu/mL), the plate was incubated

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overnight at 37°C. Control assays contained all components, except the test sample.

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MICs were determined based on the increase in optical density at 600 nm measured

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using a Microtiter ELISA Reader after incubation for 18 h at 37°C.

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Circular dichroism (CD) spectroscopy. CD spectra were recorded at 25°C on

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a Jasco J-810 spectropolarimeter (JASCO Inc., Easton, MD, USA) equipped with a

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temperature control unit. A quartz cell with a 0.1-cm path length was used with a 50 µM

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peptide solution under the indicated conditions [various concentrations of peptide and

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1.5 mM liposomes (PC:CH, 10:1 w/w and PE:PG, 7:3 w/w) or 10 mM sodium

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phosphate buffer (pH 7.4). To improve the signal-to-noise ratio, five scans from 190-

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250 nm were acquired for each condition and averaged. The acquired CD signal spectra

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were then converted to the mean residue ellipticity using the following equation:

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[θ] = θobs /10ᆞlᆞc

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where θobs is the measured signal (ellipticity) in millidegrees, l is the optical path length

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of the cell in cm, and c is the concentration of peptide in mol/L [mean residue molar

8


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concentration: c = number of residues in the constructed of peptide × the molar

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concentration of the peptide].

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Hemolysis. Hemolytic activity was assessed using human red blood cells

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(hRBCs) collected from healthy donors. Fresh hRBCs were collected by centrifugation

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at 800 × g for 10 min and washed tree times with PBS. Peptides dissolved in PBS were

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then added to 100 µL of stock hRBCs suspended in PBS (final hRBC concentration, 8%

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v/v), after which the samples were incubated for 60 min at 37°C with agitation,

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followed by centrifugation for 10 min at 800 × g. The absorbance (A) of the

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supernatants was then assessed at 414 nm; hRBCs in PBS (Ablank) or 0.1% Triton X-100

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(Atriton) serving as negative and positive controls, respectively. Percent hemolysis was

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calculated according to the equation:

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% Hemolysis = [(Asample − Ablank)/(Atriton − Ablank)] × 100

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Each measurement was conducted in triplicate.

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9



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■ RESULTS AND DISCUSSION

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Recently reported effects of antifungal peptides from bacteria. In recent

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years, there has been increasing interest in the safety of agricultural products.20 For

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organic crops, in particular, biological disease controls are important.21-22 Reports from

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several studies provide strong evidence that strains of B. amyloliquefaciens12,

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significantly reduce disease severity in a variety of hosts,28 and they are currently being

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used commercially in biological control products, owing to their excellent antagonist

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effects and high stability.29

23-27

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Bacteria express a wide array of antifungal peptides. For instance, peptides with

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antifungal activity towards Alternaria brassicicola have been isolated from the

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fermentation broth of B. subtilis CL2T.30 In addition, iturin A and surfactin, two

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antifungal lipopeptides, have also been isolated from B. subtilis,31 while tensin is an

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antifungal cyclic lipopeptide from P. fluorescens.32 Tensin acts against F. solani by

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causing a reduction in radial mycelial extension and an increase in branching and rosette

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formation. Lipopeptides exhibiting fungicidal, bactericidal and insecticidal activities

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have most commonly been isolated from B. thuringiensis CMB26,33 though a novel

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antifungal peptide produced by another Bacillus strain, B-TL2, has been purified from

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tobacco stems.34 However, only few short linear antifungal/antimicrobial peptides

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produced by Bacillus strain are reported in the literature. Kening et al. (1999) pointed

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out that B. subtilis produces bacilysin, a small peptide containing an N-terminal alanine

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residue, and L-anticapsin, which is involved in synthesis of nucleotides, amino acids

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and coenzymes and causes the lysis of Candida albicans.35 Some strains of B. subtilis

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also produce chlorotetaine, a chlorinated derivative of bacilysin with similar antifungal

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activity.36 10


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For that reason, we tested B. amyloliquefaciens for its potential use against three

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plant fungal pathogens, F. solani, F. oxysporum and T. harzianum. Of those, F. solani is

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a phytopathogenic fungus and an important causal agent of several crop diseases,

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including root and stem rot in peas, sudden death syndrome in soybeans, foot rot in

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beans and dry rot in potatos,37-40 while F. oxysporum is a phytopathogenic fungus that

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affects tomato crops, causing huge losses to farmers.41 From B. amyloliquefaciens, we

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were able to isolate several small antifungal peptides that suppressed those fungi.

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Purification of antifungal peptides from B. amyloliquefaciens PT14. Using

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RP-HPLC we detected four main peaks that had retention times of 30 to 42 min and

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were eluted using a 40-65% gradient (Figure 1A). The peak fractions exhibiting

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antifungal activity were collected, pooled (Figure 1C), and confirmed by Tricine-SDS-

224

PAGE, which showed single bands of molecular weights ranging from 1 to 3.5 kDa

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(Figure 1B). However, exact molecular masses of the PT14 peptides could not be

226

determined by SDS-PAGE. In addition, peak 4 was separated into two peaks, 4a and 4b

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in the 2nd RP-HPLC (Figure 2A). Consistent with that finding, when subjected to

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MALDI-TOF/MS analysis, compounds 1, 2, 3, 4a and 4b exhibited m/z peaks at 1454.4,

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1478.5, 1463.2, 1514.0 and 1516.9, respectively (Supplementary Figure 2, Figure 2B).

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When these substances were tested in antifungal assays against F. oxysporum, F.

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solani and T. harzianum, compounds 1, 2, 3 and 4, corresponding to the peaks shown in

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Figure 1C, exhibited potent growth inhibiting activity against all three fungal strains.

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Note that peptides 4a and 4b exhibited antifungal activities at different concentrations

234

(Supplementary Figure 3). MALDI-TOF/MS analysis and antifungal activity assay

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confirmed PT-14a and PT-4b as different peptides.

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Antifungal activity assay. We next assessed the antifungal activities of PT14-1, 11



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2, 3, 4a and 4b by using spectrophotometrics and microscopy to evaluate their inhibitory

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capacity against F. solani and F. oxysporum conidial germination and hyphae growth.

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The MICs for each of the peptides are listed in Table 1. After incubation for 24 h, PT14-

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3 and PT14-4a at 3.12 mg/L (log10 = 0.49) for F. solani and 6.25 mg/L (log10 = 0.80) for

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F. oxysporum completely inhibited conidial germination (100% growth inhibition)

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(Figure 3). PT14-4b completely inhibited the germination of F. solani conidia at 25

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mg/L (log10 = 1.40), but F. oxysporum conidia were able to germinate and grow even in

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the presence of 25 µg/mL peptide, though 100% growth inhibition was achieved at 50

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mg/L (log10 = 1.70) (Figure 3). PT14-1 and PT14-2 also completely inhibited the

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growth of F. oxysporum at 50 mg/L; at 6.25 µg/mL, PT14-1 and PT14-2 inhibited

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growth of F. oxysporum by 59% and 74%, respectively (Figure 3). Notably, none of the

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peptides tested exhibited antibacterial activity against gram-positive S. aureus or gram-

249

negative E. coli (MIC >100 mg/L).

250

Photomicrographs of F. solani and F. oxysporum mycelia were taken after a 12 h

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growth period, and the effect of PT14-4a on hyphal morphology was monitored and

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compared with the morphology of untreated hyphae. As shown in Figure 4, in control

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cultures, without an antagonistic peptide, conidial spores had their typical canoe shape

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and hyphae grew actively, displaying equal widths, even surfaces and active branching.

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By contrast, treatment with PT14-4a resulted in conidia being greatly deformed and

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exhibiting a damaged morphology characterized by lateral expansion, budding and

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uneven surfaces. The antifungal activities of PT14-1, 2, 3, 4a, and 4b against F.

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oxysporum and F. solani were detected as hyphal swelling, indicating inhibition of the

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hyphal growth (Supplementary Figures 4 and 5). Treating the hyphae with PT14-4a

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caused their structures to become severely distorted and condensed with enlarged 12


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vacuoles and conglobated apical tips (Figure 4). The altered conidial morphology

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induced in F. oxysporum and F. solani by PT14-3 and 4a, including the hyphal swelling,

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suggests the peptides may irreversibly inactivate cellular machinery within the fungi.

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However, whether the peptides directly or indirectly inhibit the catalytic activities of

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cellular enzymes remains unknown.

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Toxicity of PT14 peptides. The toxicity of the peptides against eukaryotic cells

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was assessed based on their ability to lyse hRBCs at concentrations ranging from 3.52

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to 450 µg/mL (Figure 5). The percent hemolysis was determined by measuring the

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absorbance of the supernatant from a hRBC suspension incubated with PT14-1, 2, 3, 4a,

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4b or melittin. The PT14 peptides did not induce hemolysis, even at the highest

271

concentration tested. By contrast, melittin, which is known to be highly cytotoxic and

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hemolytic, induced lysis even at the lowest concentration tested.

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PT14-4a identification. In an effort to identify the peptides, the sequences of the

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N-terminal 14 residues were determined. The N-terminal amino acid sequence of the

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purified PT14-4a peptide was YLEALEAESITTGV. A BLAST homology analysis

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revealed the sequence of 4a to be most similar to the corresponding N-terminal amino

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acid sequences of Temporin-1Ca from a green frog Rana clamitans, North America

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(GenBank ID in NCBI: P82880, similarity: 46.66 %) and Caerin 3.5 from skin dorsal

279

glands of Australian dainty green tree frog, Litoria gracilenta (P0C2A9, 40.9%).

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However, the sequence of PT14-4a was not identical to that of any other protein,

281

suggesting it is a novel antifungal peptide. Figure 2B shows single charged potassium

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adducts [M+K]+ of PT14-4a with m/z value of 1514.0, corresponding to 1495 Da, the

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MALDI-TOF/MS result of PT14-4a. MALDI-TOF/MS result of PT14-4a and its

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theoretical molecular weight fit well with its amino acid sequence determined by Edman 13



285

degradation.

286

PT14-4a secondary structure. PT14-4a has an amphipathic helical structure, as

287

indicated by the helical wheel projection in Figure 6A, which also shows it carries a

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negative charge (-3) and low hydrophobicity (H, 0.469). CD spectral analysis of the

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secondary structures of PT14-3 and PT14-4a in a membrane environment showed that

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both PT14-3 and PT14-4a have little helicity in aqueous solution or in a bacterial

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membrane mimetic environment (PE:PG vesicles, 7:3 w/w), but exhibit a high degree of

292

α-helical structure in a fungal membrane mimetic environment (PC:CH vesicles, 10:1

293

w/w) (Figure 6B-D). PT14-4a displayed a positive band at 197 nm and a negative band

294

at 210 nm in PC:CH vesicles (Figure 6C), indicating that its side chains partitioned

295

preferentially into the more rigid and hydrophobic environment of the lipid bilayer.

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Using the secondary structure predictor server K2D3,42 the percentage of α-helix in both

297

PT14-3 and PT14-4a was calculated to be 80% in the presence of PC:CH vesicles (10:1

298

w/w), but much lower (

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