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Membrane disruption and DNA binding of Fusarium graminearum cell induced by C16-Fengycin A produced by Bacillus amyloliquefaciens
Food Control 102 (2019) 206–213
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Membrane disruption and DNA binding of Fusarium graminearum cell induced by C16-Fengycin A produced by Bacillus amyloliquefaciens
T
Yanan Liu, Jing Lu, Jing Sun, Fengxia Lu, Xiaomei Bie, Zhaoxin Lu∗ College of Food Science and Technology, Nanjing Agricultural University, Weigang 1, Nanjing, 210095, PR China
A R T I C LE I N FO
A B S T R A C T
Keywords: C16-Fengycin A Fusarium graminearum Antifungal activity Membrane damage DNA binding
Fusarium graminearum causes Fusarium head blight in wheat, barley, oats and stem rot of corn, and produces mycotoxins. C16-Fengycin A is a natural product produced by Bacillus amyloliquefaciens. In this work, the antifungal activity and mechanism of C16-Fengycin A against F. graminearum were investigated. The minimum inhibitory concentration (MIC) of C16-Fengycin A against F. graminearum was 64 μg/mL, and the growth inhibitory assay also demonstrated that C16-Fengycin A exhibited a potent antifungal activity against F. graminearum. Membrane permeability, flow cytometric analysis and transmission electron microscope demonstrated that C16-Fengycin A disrupted the membrane integrity of F. graminearum. Meanwhile, C16-Fengycin A could lower the content of ergosterol, affecting the membrane structure and stability. Moreover, a gel retardation assay, spectroscopic approaches and simulative docking were used to assess the interaction of C16-Fengycin A and genomic DNA. Overall, the results revealed potential for development of C16-Fengycin A as the basis for novel agricultural food preservatives with fungicidal activity owing to its ability to damage cellular membranes and bind to DNA.
1. Introduction Diseases caused by Fusarium graminearum, such as seedling blight, root and crown rot, stalk rot as well as head blight, are the most important and devastating diseases of small grains (Goswami & Kistler, 2004). When F. graminearum attacks sensitive wheat, the yield loss is up to 17%. (Pereyra & Dill-Macky, 2010). F. graminearum is a devastating fungal pathogen that not only causes yield losses, but also produces mycotoxins, such as Deoxynivalenol (DON), Nivalenol (NIV) and other mycotoxins (McMullen et al., 2012; Trail, 2009). It is not suitable for food after the grain is infected, which will cause serious food safety problems and can adversely affect human health. Plant cultivars that are completely resistant to this pathogenic fungus have not been reported thus far. In this regard, the disease caused by F. graminearum remains an important challenge for the agricultural products and food. Many approaches have been taking to limit pathogenic microbial growth in agricultural products, with the majority of these strategies relying upon synthetic chemicals. These compounds, however, can have residual toxicity and potential carcinogenic activity which has resulted in significant debate regarding the safety of their use for food production (Ye et al., 2013). It is therefore critical to find novel approaches to reduce or eliminate pathogenic fungus during the storage period of agricultural products. Lipopeptides are a class of antibiotics that are ∗
effective in combatting many pathogenic microorganisms (Ongena & Jacques, 2008). Most of lipopeptides are produced by bacteria and can inhibit the activity of both bacteria and fungi. Lipopeptides are known to be important for combatting pathogens, due to its benefits to the environment. The research on lipopeptide-based drugs have gradually increased since the approval of daptomycin (J. Sun et al., 2018; Yang et al., 2016). Fengycin, along with surfactin and iturin, is a member of a family of cyclic lipopeptides, and it is produced by Bacillus subtilis. As a fungicide, fengycin are capable of resisting a variety of pathogenic fungi, such as Plasmodiophora monoliforme, Fusarium moniliforme, Fusarium graminearum and Podosphaera fusca (Chan et al., 2009; Hu, Shi, Zhang, & Yang, 2007; Li et al., 2013; Romero, De, Olmos, Dávila, & Pérez-García, 2007). Compared with traditional antimicrobial peptides, fengycin features low hemolytic activity, besides, the combination of D-amino acid and cyclic structure dramatically reduces the vulnerability of the compound to degradation by peptidases (Vanittanakom, Loeffler, Koch, & Jung, 1986). Fengycin, as a biological control agent, does not have the same adverse properties as do chemical pesticides, and is not easy to develop drug resistance. Fengycin and its related compounds show good prospects to the development of antifungal drugs. Fungicidal mechanism of fengycin may be linked with the disruption of cell membrane structure and function. Specifically, fengycin
Corresponding author. Weigang 1, Nanjing, 210095, Jiangsu Province, PR China. E-mail address: [email protected] (Z. Lu).
https://doi.org/10.1016/j.foodcont.2019.03.031 Received 20 November 2018; Received in revised form 23 March 2019; Accepted 25 March 2019 Available online 26 March 2019 0956-7135/ © 2019 Elsevier Ltd. All rights reserved.
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added (64 or 128 μg/mL C16-Fengycin A) and incubated at 30 °C for 0, 0.5, 1, 2, 3, 4 or 5 h. Aliquots were then filtered using filter paper, and the remaining supernatant was 0.22 μm filtered, after which plasma atomic emission spectrometry was used to assess K+ levels (Optima 8000, PerkinElmer, Inc., USA). A Microplate BCATM Protein AssayReducing Agent Compatible Kit (Shermo Scientific, USA) was used to determine the protein concentrations. As controls cultures not containing any antifungal agents were used.
disrupts the lipid bilayer, resulting in changes in permeability, fluidity and integrity of the cell membrane, and causing the cell leakage and death (Deleu, Paquot, & Nylander, 2008; Heerklotz & Seelig, 2007; Patel, Tscheka, Edwards, Karlsson, & Heerklotz, 2011; Tang et al., 2014). However, at present few studies investigating how fengycin mediates antifungal activity have been conducted. As such, it is important that more work be performed to elucidate these mechanisms in order to improve the application of fengycin as a means of controlling fungal growth and thereby protecting and preserving agricultural food products. C16-Fengycin A (purity, 95.0%) was isolated and purified in our laboratory, and it is a single substance in the Fengycin family. In this study, the antifungal activity and mode of action of C16-Fengycin A against F. graminearum were investigated. After evaluating the antifungal effects of C16-Fengycin A, we further elucidated how C16Fengycin A interacts with the cell membrane to act against F. graminearum. Moreover, the direct binding interaction of C16-Fengycin A to the important intracellular biomacromolecules, genomic DNA, was also evaluated. Therefore, this work offered novel insights into the antifungal behaviors of C16-Fengycin A on F. graminearum.
2.5. Flow cytometric analysis
2. Materials and methods
C16-Fengycin A was added to the spore suspension of 1.0 × 106 CFU/mL of F. graminearum, and spore suspensions with C16Fengycin A concentration of 32 and 64 μg/mL were prepared. The spore suspension treated without C16-Fengycin A was used as control. The spore culture medium was allowed to stand for 1–3 h at 30 °C and centrifuged (3000×g for 5 min). Spore cells were resuspended two times with 0.02 M PBS after discarding supernatants. Spore cells were stained protected from light with 15 μL propidium iodide (PI) for 15 min. The spore cells were analysed using Becton Dickinson FACS Calibur (USA), and the obtained data were processed using FCS Express 4 software (USA).
2.1. Reagents, strains and growth conditions
2.6. Transmission electron microscope
The microbial strain F. graminearum (2021) was obtained from the Enzyme Engineering Laboratory, Nanjing Agriculture University (Nanjing, China) and stored at −80 °C in potato dextrose broth (PDB) containing 25% (v/v) glycerol. Following activation, thawed microbial stock suspensions were inoculated onto a potato dextrose agar (PDA) plate and incubated at 30 °C for 24 h–36 h. One colony was then chosen and inoculated on a PDA medium plate that was then placed for 18 h in an orbital shaker at 30 °C.
F. graminearum was inoculated into PDB liquid medium, cultured at 30 °C for 36 h, and filtered to obtain mycelium. After washing with sterile PBS buffer for 3 times, 500 mg mycelium were redissolved in 50 mL sterile PBS buffer and treated with C16-Fengycin A at the final concentration of 1 × MIC at 30 °C for three different periods (0, 2, and 4 h). The treated mycelium were filtered and washed with sterile PBS buffer. The pellets were fixe with 2.5% buffered glutaraldehyde overnight at 4 °C. The morphological changes of mycelium were observed by transmission electron microscopy (JEM-1011, Japan).
2.2. Determination of MIC 2.7. Determination of ergosterol F. graminearum was incubated for 3 days at 30 °C on a PDA medium plate. Sterile phosphate-buffered saline (PBS) (5 mL; 10 mM; Na2HPO4 and NaH2PO4; pH 7.4) was then used as a means of washing the plate, and a suspension containing 1.5 × 108 CFU/mL spores was prepared. Next, the MIC of C16-Fengycin A against F. graminearum was determined by conducting 2-fold dilutions of the peptides, as previously described (Diao, Hu, Zhang, & Xu, 2014).
The content of plasma membrane ergosterol in F. graminearum was determined as reported by Kocsis et al. (Kocsis et al., 2009). 100 μl of a 107 spore/mL F. graminearum suspension was added to PDB media containing 0, 32, 64, and 128 μg/mL of C16-Fengycin A for 4 days at 30 °C. After incubation, centrifugation was used to collect cells, which were then suspended in alcoholic KOH (25% w/v, 3 mL) for 1 h in an 85 °C water bath. Samples were cooled to ambient temperature, and sterols were isolated via adding 1 mL distilled water and 3 mL n-heptane. After vortexing for 3 min, the heptane layer was isolated and ethanol was used to dilute it prior to a spectrophotometric scan between 240 nm and 300 nm.
2.3. Effect of C16-Fengycin A on F. graminearum growth The effect of C16-Fengycin A on F. graminearum growth was evaluated using the dry weight method of mycelium. Briefly, the bevelpreserved F. graminearum was transferred to a PDA plate and incubated at 30 °C for 48 h. The sterile punch was used to punch a 5 mm diameter cake and transferred to PDB medium. The filtered C16-Fengycin A (with 0.22 μm membrane) was used to supplement cultures, ensuring a final 1 × MIC or 2 × MIC concentration. The F. graminearum culture without C16-Fengycin A served as control. Then, the culture was further incubated at 180 rpm and at 30 °C. The fermentation broth was spun at 10000×g for different time intervals. The supernatant was removed and washed thrice with sterile water. The mixture was dried at 65 °C until constant weight and weighed. Considering time as the abscissa and mycelium mass as the ordinate, the growth curve of F. graminearum was plotted.
2.8. Gel retardation assay F. graminearum genomic DNA was extracted with the Shanghai Shenggong Fungal Genomic DNA Rapid Extraction kit (SK8229, Sangon Biotech, Shanghai, China). This DNA was dissolved in Tris-ethylenediaminetetraacetic acid (TE) buffer, and concentration was assessed at 260 nm with a molar absorption coefficient ε260 = 6600 M−1 cm−1 (Tao, Zhang, Pan, & Xiong, 2016). The concentration was 2.7 mM, and A260/A280 > 1.80 confirmed the purity of this DNA. DNA was mixed with C16-Fengycin A at 128, 256, 512 and 1024 μg/mL and incubated at room temperature for 1 h. Gel blocking experiments were performed on 1% agarose gels. The C16-Fengycin A used in the experiments was dissolved in the binding buffer.
2.4. Determination of K+ and proteins
2.9. Ultraviolet spectroscopy
F. graminearum was maintained in PDB media, with hyphae being collected via Buchner funnel using PBS. 500 mg of the collected hyphae were then resuspended using 50 mL PBS, and antimicrobials were then
Interaction of C16-Fengycin A with F. graminearum genomic DNA 207
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shown in Fig. 1 C1 and C2, there was a time-dependent increase in supernatant K + and protein levels after F. graminearum hyphae were treated with C16-Fengycin A at varying concentrations (1.0 and 2.0 × MIC). The result indicated that the intracellular material leakage rate was significantly increased when F. graminearum cells were exposed to C16-Fengycin A. This result demonstrated that after treatment with antimicrobial agents, membrane permeability increased. Such permeability could lead to leakage of cytoplasm, including ions and proteins.
was assessed using a variation on previous methods (Ebrahimipour et al., 2015). Absorption spectral titrations were performed by keeping the concentration of C16-Fengycin A (5 μg/mL) constant in 0.01 M PBS (pH 7.2) while increasing the concentration of DNA gradually. Samples were allowed to stand at 25 °C for 10 min for equilibration purposes, after which the C16-Fengycin A spectrum was measured over the 200 nm–320 nm range (Shimadzu UV-2450 spectrometer, Tokyo, Japan). 2.10. Fluorescence spectroscopy
3.2.2. Flow cytometric analysis Flow cytometry was used to investigate how C16-Fengycin affects F. graminearum cell integrity. Propidium iodide (PI), DNA binding dye, was used, as it can only enter cells that have damaged plasma membranes and are dying or already dead (Bauer, 1993). The results of staining F. graminearum cells with PI were presented in Fig. 2. Different concentrations of C16-Fengycin A treatment of F. graminearum resulted in different degrees of damage and the cell damage increased with the increase in C16-Fengycin A concentration. Relative to controls, the cell damage rate increased from 36.9% to 53.1% when F. graminearum cells were given 32 and 64 μg/mL C16-Fengycin A for 2 h. This dose-dependent C16-Fengycin A activity against F. graminearum cells may be a consequence of direct damage induced to the plasma membrane, and not a consequence of metabolic disruption leading to secondary damage of the membrane. C16-Fengycin A may alter plasma membrane structure, thereby compromising fungal cell integrity. Given these results, C16-Fengycin A acted via creating primary lesions in membranes of target cells.
All binding studies were performed as previously described (Ebrahimipour et al., 2015). Briefly, C16-Fengycin A, keeping the concentration of C16-Fengycin A (6.4 μg/mL) constant in 0.01 M PBS, while increasing the concentration of DNA gradually. After being incubated at 25 °C for 30 min, the emission spectra was assessed for these samples over the 300–450 nm range. 2.11. Molecular modelling of C16-Fengycin A with DNA The AutoDock 4.2 package was used to assess molecular docking, using the Lamarckian genetic algorithm as stochastic search algorithm (Karami et al., 2016; Zhang, Zhang, Zhou, & Li, 2013). The 3D structure of C16-Fengycin A was constructed by TRIPOS Sybyl-x 2.0 (Tripos Inc., St. Louis, US), and B-DNA (CGCGAATTCGCG)2 was downloaded from the Protein Data Bank (PDB ID: 5E9H). To prepare for docking, according the method reported by Ricci and Netz (Ricci & Netz, 2009), water molecules were omitted, while both Gasteiger charges and polar hydrogens were added using MGL tools 1.5.6 rc3.
3.3. Effect of C16-Fengycin A on morphology and structure of F. graminearum
2.12. Statistical analysis
The results of transmission electron microscopy (TEM) revealed that untreated F. graminearum cell walls were clear and distinguishable, the cytoplasm was full, and the morphology of organelle was complete (Fig. 3 A). After being treated with C16-FengcyinA for 2 h, the cell wall electron density was not uniform, the defect of the cell wall could be observed, the cytoplasmic structure was disordered, the organelle and the vacuole disappeared. (Fig. 3 B). After 4 h treatment, severe cell necrosis was observed, the structure of the cytoplasm disappeared, and the residual cell contents flocculate or gather sideways. (Fig. 3 C). This result was consistent with the results of intracellular material leakage and flow cytometric experiments, indicating that there were different degrees of burst on the surface of F. graminearum with the increasing concentrations of C16-Fengycin A (Figs. 1 and 2). These observations demonstrated that C16-Fengycin A could cause concentration-dependent damage to F. graminearum cell membrane, and may decrease viability (Chatterjee, Chatterjee, Majumdar, & Chakrabarti, 2015; Kangwansupamonkon, Lauruengtana, Surassmo, & Ruktanonchai, 2009).
OriginPro 8.0 (Origin Lab, Northampton, MA, USA) was used for all statistical testing. Data are means ± standard deviation (SD), and differences of p < 0.05 were considered to be significant. 3. Results and discussion 3.1. Effect of C16-Fengycin A on F. graminearum growth The MIC of C16-Fengycin A against F. graminearum was determined as a means of investigating the effect of C16-Fengycin A on F. graminearum growth. When the concentration of C16-Fengycin was < 64 μg/ mL, the growth of F.graminearum was almost unaffected. Furthermore, when the concentration of C16-Fengycin A was ≥ 64 μg/mL, F. graminearum growth was inhibited completely (Fig. 1 A). The MIC of C16Fengycin A against F. graminearum was thus 64 μg/mL. The antibacterial activity of C16-Fengycin A against F. graminearum was also illustrated by developing growth curves in the presence of a range of concentrations. Relative to controls, C16-Fengycin A significantly affected F. graminearum growth on PDB (Fig. 1 B). When treated with 1 × MIC C16-Fengycin A, the lag phase growth of F. graminearum was prolonged from 2d (control) to 3d, and this was increased even more if F. graminearum was treated with a higher concentration of C16-Fengycin A (2 × MIC). These findings demonstrated that C16Fengycin A could block F. graminearum growth at relatively low concentrations.
3.4. Ergosterol content determination Ergosterol is a unique sterol in fungi that is a significant component of their cell membranes, allowing them to preserve cellular integrity and membrane liquidity (Rodriguez, Low, Bottema, & Parks, 1985). To confirm the C16-Fengycin A target in the plasma membrane, the effect of C16-Fengycin A on the amount of ergosterol was assessed. The method described by Kocsis et al. (Kocsis et al., 2009) uses the characteristic absorption spectrum of the fungal sterols between 240 and 300 nm. Specifically, these sterols, which are composed of both ergosterol and 24 (28)-dehydroergosterol, exhibit maximum absorption at 281.5 nm with 24 (28)-dehydroergosterol also having a separate 230 nm maximum. Using these two values, it is therefore possible to determine ergosterol levels. The efficacies of C16-Fengycin A on the ergosterol content in the
3.2. Effect of C16-Fengycin A on membrane integrity 3.2.1. K + and protein leakage It is commonly known that the plasma membrane is a complex mixture of lipids and proteins (Bo et al., 2016). Given that it maintains separation between the intracellular and extracellular environments, many antifunal agents often target this membrane. Therefore, we assessed membrane permeability after C16-Fengycin A treatment. As 208
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Fig. 1. Effect of C16-Fengycin A on cell growth (A, B) and K
+
and protein release (C1, C2) of F. graminearum. Data are mean ± SD of three replicates.
Fig. 2. Flow cytometry graphs of cell damage of F. graminearum. Cells of F. graminearum were treated with 0, 32 and 64 μg/mL of C16-Fengycin A. M, events of dead cells; 10000 cells were calculated.
Fig. 3. The images of transmission electron microscopy (TEM) of F. graminearum cells exposed to C16-Fengycin A. Note: A, the hypha treated without C16-Fengycin A; B, the hypha treated with 64 μg/mL of bacillomycin for 2 h; C, the hypha treated with 64 μg/mL of bacillomycin for 4 h. 209
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Fig. 4. Spectrometric analysis of ergosterol profiles of F. graminearum.
in natamycin. These polar amino acids bind to DNA bases. Therefore, it was concluded that the antibacterial effect of C16-Fengycin A not only acts on the cell membrane but also possibly on other intracellular substances. This finding provided a new perspective to further exploitation of the antifungal mechanism of C16-Fengycin A.
plasma membrane of F. graminearum were highlighted in Fig. 4. The cells were treated with 0, 32, 64, and 128 μg/mL of C16-Fengycin A, and the results showed that the ergosterol levels (at 282 nm) in F. graminearum membranes decreased significantly in a dose-dependent manner with maximal inhibition at 128 μg/mL. Our observations suggested that C16-Fengycin A substantially disrupts ergosterol biosynthesis by F. graminearum. Previous work has also found that certain other compounds can disrupt and decrease ergosterol levels (Pinto et al., 2006; Pinto, Vale-Silva, Cavaleiro, & Salgueiro, 2009). Indeed, Kelly et al. found that azole fungicidal drugs act by means of disrupting sterol biosynthesis, thereby compromising ergosterol production and fungal growth (Kelly, Lamb, Corran, Baldwin, & Kelly, 1995). The plasma membrane was therefore a major target of C16-Fengycin A.
3.5.2. UV spectroscopy UV absorption spectroscopy is an optimal means of assessing interactions between DNA and specific ligands (Ahmadi, Alizadeh, Bakhshandeh-Saraskanrood, Jafari, & Khodadadian, 2010; Shahabadi, Fili, & Kheirdoosh, 2013). There are two mechanisms by which small molecular weight compounds interact with DNA: intercalation, wherein dipole-dipole interactions and π-π stacking stabilize planar molecules that are inserted between DNA base pairs, and groove binding, wherein different types of interactions between molecules such as electrostatic or hydrogen bonding stabilize compound binding (Liu et al., 2015; Palchaudhuri & Hergenrother, 2007). Fig. 5 B showed the C16-Fengycin A absorption spectra after combination with a range of DNA concentrations. As DNA was slowly added, C16-Fengycin A absorption decreased slightly at 205 nm, without any remarkable shift, and there was additionally a single isobestic point near 240 nm. This limited absorption decline couple with a lack of major change to λ-max are consistent with groove binding (Ebrahimipour et al., 2015). The isobestic point further indicated that there was a complex of drug-DNA formed (Fei, Lu, Fan, & Wu, 2009). This therefore confirmed that C16Fengycin A could bind to DNA, with groove binding being the likely mode of interaction.
3.5. Effect of C16-fengycin A on DNA 3.5.1. Gel retardation assay In addition to targeting the F. graminearum cell membrane, C16Fengycin A can interact with nucleic acids, potentially allowing for antimicrobial action by interfering with DNA. Gel retardation experiments are usually used to study the binding of polypeptides to DNA. (Miao et al., 2016). In this study, the binding conditions of C16-Fengycin A and the polyene antifungal compound natamycin were compared, as shown in Fig. 5 A1-A3. As C16-Fengycin A concentration increased, F. graminearum DNA migration rate reduced, and the band dimmed, beause part of the DNA stayed in the sample well. When the concentration of C16-Fengycin A was 128 or 256 μg/mL, as genomic DNA combined with C16-Fengycin A, the migration rate changed, a portion of the DNA was retained in the sample well, and band brightness was less than that of the control. When the concentration of C16Fengycin A reached 512 μg/mL, both genomic DNA and C19-Fengycin A remained in the sample well owing to given their strong combination (lane 2 in Fig. 5 A1). The results showed that C16-Fengycin A could bind non-specifically to DNA in vitro, but natamycin cannot (lane 5 in Fig. 5 A1), presumably due to the different compositions of these compounds. C16-Fengycin A contains polar amino acids, such as Tyr, Glu and Thr, which are absent
3.5.3. Fluorescence spectroscopy The groove binding mode of C16-Fengycin A with DNA was further confirmed by the fluorescence absorption spectroscopy. As shown in Fig. 5 C1, C16-Fengycin A showed a strong fluorescence emission peak at 340 nm following 260 nm excitation, and the addition of DNA to C16-Fengycin A substantially reduced the fluorescence intensity. The Stern-Volmer equation was used in order to establish the binding constant (Kq): F0/F = 1 + Kq τ0 [DNA] = 1 + Ksv [DNA] (Zhang & Tang, 2004), where F0 and F indicate the fluorescence 210
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Fig. 5. C16-Fengycin A interaction with F. graminearum genomic DNA. A1, Gel retardation assay by C16-Fengycin A and the DNA of F. graminearum, 1–4, concentrations of C16-Fengycin A were 1024, 512, 256, and 128 μg/mL; 5, DNA+30 μg/mL natamycin; 6, DNA + TE buffer, respectively. A2, Brightness of different bands. A3, Migration distance of different bands. B, Ultraviolet spectra of C16-Fengycin A with increasing concentrations of DNA. C1, Effect of DNA on fluorescence spectra of C16-Fengycin A (λex = 260 nm, λem = 340 nm). C2, the Stern–Volmer plots of C16-Fengycin A by DNA (Room temperature).
corresponding binding energy of the distributed conformation using a RMSD (Root-Mean-Square Deviation) tolerance of 2.0 Å. The lowest energy cluster contained 13 conformations, with a binding energy of −3.35 kcal M−1. The cluster with an energy of −2.9 kcal M− 1 had the most analysed conformations (20/100). The cluster was thus used for binding orientation analysis, with the highest energy ranking being selected to predict interactions between C16-Fengycin A and DNA (Fig. 6B, C, 6D). As shown in Fig. 6B, C and 6D, C16-Fengycin A entered the DNA major groove in AT-rich regions, surrounded by A5, A6, T19 and T20 base pairs. These results suggested that C16-Fengycin A could bind to the DNA major groove of DNA, with hydrogen binding likely facilitating this interaction.
intensities of free C16-Fengycin A in the absence and presence of DNA, [DNA] is the concentration of genomic DNA, τ0 is the average fluorescence lifetime of the molecule and its value is 10−8 s, and Kq is the rate constant for the bimolecular quenching process. Kq (6.96 ± 0.22 × 1011 L mol−1s−1, R2 = 0.9928) was calculated based on the ratio of intercept to slope (Fig. 5 C2). In general, the maximum diffusion collision quenching rate constant (Kq) of quenching agents on biomolecules is not more than 2 × 1010 L mol−1s−1 (Sun, Zhou, Hou, Liu, & Xiang, 2006). The calculation results showed that the quenching rate constant (Kq) of C16Fengycin A was greater than the diffusion control constant (2 × 1010 L mol−1s−1), which proved that the above quenching was not caused by dynamic collision, but should be static quenching. The binding constant (Ka) values of ethidium bromide (2.6 × 106 M−1), and acridine orange (4.0 × 105 M−1), which are both classic intercalating agents, are 105–106 M−1 (Cao & He, 1998). Our identified Ka value of C16-Fengycin A with DNA was much lower than that expected for an intercalating compound, but was consistent with one that undergoes groove binding (Ahmadi et al., 2010; Shahabadi et al., 2013), further supporting that C16-Fengycin A undergoes groove binding to DNA.
4. Conclusions This study had demonstrated that C16-Fengycin A possesses a potent of antifungal activity against F. graminearum via cell membrane disruption and direct genomic DNA binding. C16-Fengycin A showed a antifungal effect against F.graminearum at relative low concentrations, and it could increase cell membrane permeability, potentially causing a leakage of intracellular materials and a disruption in the cellular morphology. C16-Fengycin A could bind to the major groove of DNA, potentially slightly altering DNA secondary structure and morphology. These results revealed that C16-Fengycin A may serve as a good DNA binder, and its antifungal activity could be partly due to its DNAbinding ability. Our findings have suggested that C16-Fengycin A may be a promising natural preservative ideal for use in the agriculture and
3.5.4. Molecular modelling of C16-Fengycin A–DNA interaction Molecular docking was often used as a means of visualizing the binding mode of small ligands with DNA (Wang, Zhang, Zeng, Gong, & Wang, 2017). After 100 successful dockings, eight energy clusters were present and could be ranked on the basis of a scoring function relating to docking free energy. Fig. 6A showed the cluster analysis and 211
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Fig. 6. A, Cluster analyses of the AutoDock docking runs of C16-Fengycin A with DNA; B–D, molecular modelling results of the energy-minimised structure of the C16-Fengycin A–DNA system.
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Membrane disruption and DNA binding of Fusarium graminearum cell induced by C16-Fengycin A produced by Bacillus amyloliquefaciens
T
Yanan Liu, Jing Lu, Jing Sun, Fengxia Lu, Xiaomei Bie, Zhaoxin Lu∗ College of Food Science and Technology, Nanjing Agricultural University, Weigang 1, Nanjing, 210095, PR China
A R T I C LE I N FO
A B S T R A C T
Keywords: C16-Fengycin A Fusarium graminearum Antifungal activity Membrane damage DNA binding
Fusarium graminearum causes Fusarium head blight in wheat, barley, oats and stem rot of corn, and produces mycotoxins. C16-Fengycin A is a natural product produced by Bacillus amyloliquefaciens. In this work, the antifungal activity and mechanism of C16-Fengycin A against F. graminearum were investigated. The minimum inhibitory concentration (MIC) of C16-Fengycin A against F. graminearum was 64 μg/mL, and the growth inhibitory assay also demonstrated that C16-Fengycin A exhibited a potent antifungal activity against F. graminearum. Membrane permeability, flow cytometric analysis and transmission electron microscope demonstrated that C16-Fengycin A disrupted the membrane integrity of F. graminearum. Meanwhile, C16-Fengycin A could lower the content of ergosterol, affecting the membrane structure and stability. Moreover, a gel retardation assay, spectroscopic approaches and simulative docking were used to assess the interaction of C16-Fengycin A and genomic DNA. Overall, the results revealed potential for development of C16-Fengycin A as the basis for novel agricultural food preservatives with fungicidal activity owing to its ability to damage cellular membranes and bind to DNA.
1. Introduction Diseases caused by Fusarium graminearum, such as seedling blight, root and crown rot, stalk rot as well as head blight, are the most important and devastating diseases of small grains (Goswami & Kistler, 2004). When F. graminearum attacks sensitive wheat, the yield loss is up to 17%. (Pereyra & Dill-Macky, 2010). F. graminearum is a devastating fungal pathogen that not only causes yield losses, but also produces mycotoxins, such as Deoxynivalenol (DON), Nivalenol (NIV) and other mycotoxins (McMullen et al., 2012; Trail, 2009). It is not suitable for food after the grain is infected, which will cause serious food safety problems and can adversely affect human health. Plant cultivars that are completely resistant to this pathogenic fungus have not been reported thus far. In this regard, the disease caused by F. graminearum remains an important challenge for the agricultural products and food. Many approaches have been taking to limit pathogenic microbial growth in agricultural products, with the majority of these strategies relying upon synthetic chemicals. These compounds, however, can have residual toxicity and potential carcinogenic activity which has resulted in significant debate regarding the safety of their use for food production (Ye et al., 2013). It is therefore critical to find novel approaches to reduce or eliminate pathogenic fungus during the storage period of agricultural products. Lipopeptides are a class of antibiotics that are ∗
effective in combatting many pathogenic microorganisms (Ongena & Jacques, 2008). Most of lipopeptides are produced by bacteria and can inhibit the activity of both bacteria and fungi. Lipopeptides are known to be important for combatting pathogens, due to its benefits to the environment. The research on lipopeptide-based drugs have gradually increased since the approval of daptomycin (J. Sun et al., 2018; Yang et al., 2016). Fengycin, along with surfactin and iturin, is a member of a family of cyclic lipopeptides, and it is produced by Bacillus subtilis. As a fungicide, fengycin are capable of resisting a variety of pathogenic fungi, such as Plasmodiophora monoliforme, Fusarium moniliforme, Fusarium graminearum and Podosphaera fusca (Chan et al., 2009; Hu, Shi, Zhang, & Yang, 2007; Li et al., 2013; Romero, De, Olmos, Dávila, & Pérez-García, 2007). Compared with traditional antimicrobial peptides, fengycin features low hemolytic activity, besides, the combination of D-amino acid and cyclic structure dramatically reduces the vulnerability of the compound to degradation by peptidases (Vanittanakom, Loeffler, Koch, & Jung, 1986). Fengycin, as a biological control agent, does not have the same adverse properties as do chemical pesticides, and is not easy to develop drug resistance. Fengycin and its related compounds show good prospects to the development of antifungal drugs. Fungicidal mechanism of fengycin may be linked with the disruption of cell membrane structure and function. Specifically, fengycin
Corresponding author. Weigang 1, Nanjing, 210095, Jiangsu Province, PR China. E-mail address: [email protected] (Z. Lu).
https://doi.org/10.1016/j.foodcont.2019.03.031 Received 20 November 2018; Received in revised form 23 March 2019; Accepted 25 March 2019 Available online 26 March 2019 0956-7135/ © 2019 Elsevier Ltd. All rights reserved.
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added (64 or 128 μg/mL C16-Fengycin A) and incubated at 30 °C for 0, 0.5, 1, 2, 3, 4 or 5 h. Aliquots were then filtered using filter paper, and the remaining supernatant was 0.22 μm filtered, after which plasma atomic emission spectrometry was used to assess K+ levels (Optima 8000, PerkinElmer, Inc., USA). A Microplate BCATM Protein AssayReducing Agent Compatible Kit (Shermo Scientific, USA) was used to determine the protein concentrations. As controls cultures not containing any antifungal agents were used.
disrupts the lipid bilayer, resulting in changes in permeability, fluidity and integrity of the cell membrane, and causing the cell leakage and death (Deleu, Paquot, & Nylander, 2008; Heerklotz & Seelig, 2007; Patel, Tscheka, Edwards, Karlsson, & Heerklotz, 2011; Tang et al., 2014). However, at present few studies investigating how fengycin mediates antifungal activity have been conducted. As such, it is important that more work be performed to elucidate these mechanisms in order to improve the application of fengycin as a means of controlling fungal growth and thereby protecting and preserving agricultural food products. C16-Fengycin A (purity, 95.0%) was isolated and purified in our laboratory, and it is a single substance in the Fengycin family. In this study, the antifungal activity and mode of action of C16-Fengycin A against F. graminearum were investigated. After evaluating the antifungal effects of C16-Fengycin A, we further elucidated how C16Fengycin A interacts with the cell membrane to act against F. graminearum. Moreover, the direct binding interaction of C16-Fengycin A to the important intracellular biomacromolecules, genomic DNA, was also evaluated. Therefore, this work offered novel insights into the antifungal behaviors of C16-Fengycin A on F. graminearum.
2.5. Flow cytometric analysis
2. Materials and methods
C16-Fengycin A was added to the spore suspension of 1.0 × 106 CFU/mL of F. graminearum, and spore suspensions with C16Fengycin A concentration of 32 and 64 μg/mL were prepared. The spore suspension treated without C16-Fengycin A was used as control. The spore culture medium was allowed to stand for 1–3 h at 30 °C and centrifuged (3000×g for 5 min). Spore cells were resuspended two times with 0.02 M PBS after discarding supernatants. Spore cells were stained protected from light with 15 μL propidium iodide (PI) for 15 min. The spore cells were analysed using Becton Dickinson FACS Calibur (USA), and the obtained data were processed using FCS Express 4 software (USA).
2.1. Reagents, strains and growth conditions
2.6. Transmission electron microscope
The microbial strain F. graminearum (2021) was obtained from the Enzyme Engineering Laboratory, Nanjing Agriculture University (Nanjing, China) and stored at −80 °C in potato dextrose broth (PDB) containing 25% (v/v) glycerol. Following activation, thawed microbial stock suspensions were inoculated onto a potato dextrose agar (PDA) plate and incubated at 30 °C for 24 h–36 h. One colony was then chosen and inoculated on a PDA medium plate that was then placed for 18 h in an orbital shaker at 30 °C.
F. graminearum was inoculated into PDB liquid medium, cultured at 30 °C for 36 h, and filtered to obtain mycelium. After washing with sterile PBS buffer for 3 times, 500 mg mycelium were redissolved in 50 mL sterile PBS buffer and treated with C16-Fengycin A at the final concentration of 1 × MIC at 30 °C for three different periods (0, 2, and 4 h). The treated mycelium were filtered and washed with sterile PBS buffer. The pellets were fixe with 2.5% buffered glutaraldehyde overnight at 4 °C. The morphological changes of mycelium were observed by transmission electron microscopy (JEM-1011, Japan).
2.2. Determination of MIC 2.7. Determination of ergosterol F. graminearum was incubated for 3 days at 30 °C on a PDA medium plate. Sterile phosphate-buffered saline (PBS) (5 mL; 10 mM; Na2HPO4 and NaH2PO4; pH 7.4) was then used as a means of washing the plate, and a suspension containing 1.5 × 108 CFU/mL spores was prepared. Next, the MIC of C16-Fengycin A against F. graminearum was determined by conducting 2-fold dilutions of the peptides, as previously described (Diao, Hu, Zhang, & Xu, 2014).
The content of plasma membrane ergosterol in F. graminearum was determined as reported by Kocsis et al. (Kocsis et al., 2009). 100 μl of a 107 spore/mL F. graminearum suspension was added to PDB media containing 0, 32, 64, and 128 μg/mL of C16-Fengycin A for 4 days at 30 °C. After incubation, centrifugation was used to collect cells, which were then suspended in alcoholic KOH (25% w/v, 3 mL) for 1 h in an 85 °C water bath. Samples were cooled to ambient temperature, and sterols were isolated via adding 1 mL distilled water and 3 mL n-heptane. After vortexing for 3 min, the heptane layer was isolated and ethanol was used to dilute it prior to a spectrophotometric scan between 240 nm and 300 nm.
2.3. Effect of C16-Fengycin A on F. graminearum growth The effect of C16-Fengycin A on F. graminearum growth was evaluated using the dry weight method of mycelium. Briefly, the bevelpreserved F. graminearum was transferred to a PDA plate and incubated at 30 °C for 48 h. The sterile punch was used to punch a 5 mm diameter cake and transferred to PDB medium. The filtered C16-Fengycin A (with 0.22 μm membrane) was used to supplement cultures, ensuring a final 1 × MIC or 2 × MIC concentration. The F. graminearum culture without C16-Fengycin A served as control. Then, the culture was further incubated at 180 rpm and at 30 °C. The fermentation broth was spun at 10000×g for different time intervals. The supernatant was removed and washed thrice with sterile water. The mixture was dried at 65 °C until constant weight and weighed. Considering time as the abscissa and mycelium mass as the ordinate, the growth curve of F. graminearum was plotted.
2.8. Gel retardation assay F. graminearum genomic DNA was extracted with the Shanghai Shenggong Fungal Genomic DNA Rapid Extraction kit (SK8229, Sangon Biotech, Shanghai, China). This DNA was dissolved in Tris-ethylenediaminetetraacetic acid (TE) buffer, and concentration was assessed at 260 nm with a molar absorption coefficient ε260 = 6600 M−1 cm−1 (Tao, Zhang, Pan, & Xiong, 2016). The concentration was 2.7 mM, and A260/A280 > 1.80 confirmed the purity of this DNA. DNA was mixed with C16-Fengycin A at 128, 256, 512 and 1024 μg/mL and incubated at room temperature for 1 h. Gel blocking experiments were performed on 1% agarose gels. The C16-Fengycin A used in the experiments was dissolved in the binding buffer.
2.4. Determination of K+ and proteins
2.9. Ultraviolet spectroscopy
F. graminearum was maintained in PDB media, with hyphae being collected via Buchner funnel using PBS. 500 mg of the collected hyphae were then resuspended using 50 mL PBS, and antimicrobials were then
Interaction of C16-Fengycin A with F. graminearum genomic DNA 207
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shown in Fig. 1 C1 and C2, there was a time-dependent increase in supernatant K + and protein levels after F. graminearum hyphae were treated with C16-Fengycin A at varying concentrations (1.0 and 2.0 × MIC). The result indicated that the intracellular material leakage rate was significantly increased when F. graminearum cells were exposed to C16-Fengycin A. This result demonstrated that after treatment with antimicrobial agents, membrane permeability increased. Such permeability could lead to leakage of cytoplasm, including ions and proteins.
was assessed using a variation on previous methods (Ebrahimipour et al., 2015). Absorption spectral titrations were performed by keeping the concentration of C16-Fengycin A (5 μg/mL) constant in 0.01 M PBS (pH 7.2) while increasing the concentration of DNA gradually. Samples were allowed to stand at 25 °C for 10 min for equilibration purposes, after which the C16-Fengycin A spectrum was measured over the 200 nm–320 nm range (Shimadzu UV-2450 spectrometer, Tokyo, Japan). 2.10. Fluorescence spectroscopy
3.2.2. Flow cytometric analysis Flow cytometry was used to investigate how C16-Fengycin affects F. graminearum cell integrity. Propidium iodide (PI), DNA binding dye, was used, as it can only enter cells that have damaged plasma membranes and are dying or already dead (Bauer, 1993). The results of staining F. graminearum cells with PI were presented in Fig. 2. Different concentrations of C16-Fengycin A treatment of F. graminearum resulted in different degrees of damage and the cell damage increased with the increase in C16-Fengycin A concentration. Relative to controls, the cell damage rate increased from 36.9% to 53.1% when F. graminearum cells were given 32 and 64 μg/mL C16-Fengycin A for 2 h. This dose-dependent C16-Fengycin A activity against F. graminearum cells may be a consequence of direct damage induced to the plasma membrane, and not a consequence of metabolic disruption leading to secondary damage of the membrane. C16-Fengycin A may alter plasma membrane structure, thereby compromising fungal cell integrity. Given these results, C16-Fengycin A acted via creating primary lesions in membranes of target cells.
All binding studies were performed as previously described (Ebrahimipour et al., 2015). Briefly, C16-Fengycin A, keeping the concentration of C16-Fengycin A (6.4 μg/mL) constant in 0.01 M PBS, while increasing the concentration of DNA gradually. After being incubated at 25 °C for 30 min, the emission spectra was assessed for these samples over the 300–450 nm range. 2.11. Molecular modelling of C16-Fengycin A with DNA The AutoDock 4.2 package was used to assess molecular docking, using the Lamarckian genetic algorithm as stochastic search algorithm (Karami et al., 2016; Zhang, Zhang, Zhou, & Li, 2013). The 3D structure of C16-Fengycin A was constructed by TRIPOS Sybyl-x 2.0 (Tripos Inc., St. Louis, US), and B-DNA (CGCGAATTCGCG)2 was downloaded from the Protein Data Bank (PDB ID: 5E9H). To prepare for docking, according the method reported by Ricci and Netz (Ricci & Netz, 2009), water molecules were omitted, while both Gasteiger charges and polar hydrogens were added using MGL tools 1.5.6 rc3.
3.3. Effect of C16-Fengycin A on morphology and structure of F. graminearum
2.12. Statistical analysis
The results of transmission electron microscopy (TEM) revealed that untreated F. graminearum cell walls were clear and distinguishable, the cytoplasm was full, and the morphology of organelle was complete (Fig. 3 A). After being treated with C16-FengcyinA for 2 h, the cell wall electron density was not uniform, the defect of the cell wall could be observed, the cytoplasmic structure was disordered, the organelle and the vacuole disappeared. (Fig. 3 B). After 4 h treatment, severe cell necrosis was observed, the structure of the cytoplasm disappeared, and the residual cell contents flocculate or gather sideways. (Fig. 3 C). This result was consistent with the results of intracellular material leakage and flow cytometric experiments, indicating that there were different degrees of burst on the surface of F. graminearum with the increasing concentrations of C16-Fengycin A (Figs. 1 and 2). These observations demonstrated that C16-Fengycin A could cause concentration-dependent damage to F. graminearum cell membrane, and may decrease viability (Chatterjee, Chatterjee, Majumdar, & Chakrabarti, 2015; Kangwansupamonkon, Lauruengtana, Surassmo, & Ruktanonchai, 2009).
OriginPro 8.0 (Origin Lab, Northampton, MA, USA) was used for all statistical testing. Data are means ± standard deviation (SD), and differences of p < 0.05 were considered to be significant. 3. Results and discussion 3.1. Effect of C16-Fengycin A on F. graminearum growth The MIC of C16-Fengycin A against F. graminearum was determined as a means of investigating the effect of C16-Fengycin A on F. graminearum growth. When the concentration of C16-Fengycin was < 64 μg/ mL, the growth of F.graminearum was almost unaffected. Furthermore, when the concentration of C16-Fengycin A was ≥ 64 μg/mL, F. graminearum growth was inhibited completely (Fig. 1 A). The MIC of C16Fengycin A against F. graminearum was thus 64 μg/mL. The antibacterial activity of C16-Fengycin A against F. graminearum was also illustrated by developing growth curves in the presence of a range of concentrations. Relative to controls, C16-Fengycin A significantly affected F. graminearum growth on PDB (Fig. 1 B). When treated with 1 × MIC C16-Fengycin A, the lag phase growth of F. graminearum was prolonged from 2d (control) to 3d, and this was increased even more if F. graminearum was treated with a higher concentration of C16-Fengycin A (2 × MIC). These findings demonstrated that C16Fengycin A could block F. graminearum growth at relatively low concentrations.
3.4. Ergosterol content determination Ergosterol is a unique sterol in fungi that is a significant component of their cell membranes, allowing them to preserve cellular integrity and membrane liquidity (Rodriguez, Low, Bottema, & Parks, 1985). To confirm the C16-Fengycin A target in the plasma membrane, the effect of C16-Fengycin A on the amount of ergosterol was assessed. The method described by Kocsis et al. (Kocsis et al., 2009) uses the characteristic absorption spectrum of the fungal sterols between 240 and 300 nm. Specifically, these sterols, which are composed of both ergosterol and 24 (28)-dehydroergosterol, exhibit maximum absorption at 281.5 nm with 24 (28)-dehydroergosterol also having a separate 230 nm maximum. Using these two values, it is therefore possible to determine ergosterol levels. The efficacies of C16-Fengycin A on the ergosterol content in the
3.2. Effect of C16-Fengycin A on membrane integrity 3.2.1. K + and protein leakage It is commonly known that the plasma membrane is a complex mixture of lipids and proteins (Bo et al., 2016). Given that it maintains separation between the intracellular and extracellular environments, many antifunal agents often target this membrane. Therefore, we assessed membrane permeability after C16-Fengycin A treatment. As 208
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Fig. 1. Effect of C16-Fengycin A on cell growth (A, B) and K
+
and protein release (C1, C2) of F. graminearum. Data are mean ± SD of three replicates.
Fig. 2. Flow cytometry graphs of cell damage of F. graminearum. Cells of F. graminearum were treated with 0, 32 and 64 μg/mL of C16-Fengycin A. M, events of dead cells; 10000 cells were calculated.
Fig. 3. The images of transmission electron microscopy (TEM) of F. graminearum cells exposed to C16-Fengycin A. Note: A, the hypha treated without C16-Fengycin A; B, the hypha treated with 64 μg/mL of bacillomycin for 2 h; C, the hypha treated with 64 μg/mL of bacillomycin for 4 h. 209
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Fig. 4. Spectrometric analysis of ergosterol profiles of F. graminearum.
in natamycin. These polar amino acids bind to DNA bases. Therefore, it was concluded that the antibacterial effect of C16-Fengycin A not only acts on the cell membrane but also possibly on other intracellular substances. This finding provided a new perspective to further exploitation of the antifungal mechanism of C16-Fengycin A.
plasma membrane of F. graminearum were highlighted in Fig. 4. The cells were treated with 0, 32, 64, and 128 μg/mL of C16-Fengycin A, and the results showed that the ergosterol levels (at 282 nm) in F. graminearum membranes decreased significantly in a dose-dependent manner with maximal inhibition at 128 μg/mL. Our observations suggested that C16-Fengycin A substantially disrupts ergosterol biosynthesis by F. graminearum. Previous work has also found that certain other compounds can disrupt and decrease ergosterol levels (Pinto et al., 2006; Pinto, Vale-Silva, Cavaleiro, & Salgueiro, 2009). Indeed, Kelly et al. found that azole fungicidal drugs act by means of disrupting sterol biosynthesis, thereby compromising ergosterol production and fungal growth (Kelly, Lamb, Corran, Baldwin, & Kelly, 1995). The plasma membrane was therefore a major target of C16-Fengycin A.
3.5.2. UV spectroscopy UV absorption spectroscopy is an optimal means of assessing interactions between DNA and specific ligands (Ahmadi, Alizadeh, Bakhshandeh-Saraskanrood, Jafari, & Khodadadian, 2010; Shahabadi, Fili, & Kheirdoosh, 2013). There are two mechanisms by which small molecular weight compounds interact with DNA: intercalation, wherein dipole-dipole interactions and π-π stacking stabilize planar molecules that are inserted between DNA base pairs, and groove binding, wherein different types of interactions between molecules such as electrostatic or hydrogen bonding stabilize compound binding (Liu et al., 2015; Palchaudhuri & Hergenrother, 2007). Fig. 5 B showed the C16-Fengycin A absorption spectra after combination with a range of DNA concentrations. As DNA was slowly added, C16-Fengycin A absorption decreased slightly at 205 nm, without any remarkable shift, and there was additionally a single isobestic point near 240 nm. This limited absorption decline couple with a lack of major change to λ-max are consistent with groove binding (Ebrahimipour et al., 2015). The isobestic point further indicated that there was a complex of drug-DNA formed (Fei, Lu, Fan, & Wu, 2009). This therefore confirmed that C16Fengycin A could bind to DNA, with groove binding being the likely mode of interaction.
3.5. Effect of C16-fengycin A on DNA 3.5.1. Gel retardation assay In addition to targeting the F. graminearum cell membrane, C16Fengycin A can interact with nucleic acids, potentially allowing for antimicrobial action by interfering with DNA. Gel retardation experiments are usually used to study the binding of polypeptides to DNA. (Miao et al., 2016). In this study, the binding conditions of C16-Fengycin A and the polyene antifungal compound natamycin were compared, as shown in Fig. 5 A1-A3. As C16-Fengycin A concentration increased, F. graminearum DNA migration rate reduced, and the band dimmed, beause part of the DNA stayed in the sample well. When the concentration of C16-Fengycin A was 128 or 256 μg/mL, as genomic DNA combined with C16-Fengycin A, the migration rate changed, a portion of the DNA was retained in the sample well, and band brightness was less than that of the control. When the concentration of C16Fengycin A reached 512 μg/mL, both genomic DNA and C19-Fengycin A remained in the sample well owing to given their strong combination (lane 2 in Fig. 5 A1). The results showed that C16-Fengycin A could bind non-specifically to DNA in vitro, but natamycin cannot (lane 5 in Fig. 5 A1), presumably due to the different compositions of these compounds. C16-Fengycin A contains polar amino acids, such as Tyr, Glu and Thr, which are absent
3.5.3. Fluorescence spectroscopy The groove binding mode of C16-Fengycin A with DNA was further confirmed by the fluorescence absorption spectroscopy. As shown in Fig. 5 C1, C16-Fengycin A showed a strong fluorescence emission peak at 340 nm following 260 nm excitation, and the addition of DNA to C16-Fengycin A substantially reduced the fluorescence intensity. The Stern-Volmer equation was used in order to establish the binding constant (Kq): F0/F = 1 + Kq τ0 [DNA] = 1 + Ksv [DNA] (Zhang & Tang, 2004), where F0 and F indicate the fluorescence 210
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Fig. 5. C16-Fengycin A interaction with F. graminearum genomic DNA. A1, Gel retardation assay by C16-Fengycin A and the DNA of F. graminearum, 1–4, concentrations of C16-Fengycin A were 1024, 512, 256, and 128 μg/mL; 5, DNA+30 μg/mL natamycin; 6, DNA + TE buffer, respectively. A2, Brightness of different bands. A3, Migration distance of different bands. B, Ultraviolet spectra of C16-Fengycin A with increasing concentrations of DNA. C1, Effect of DNA on fluorescence spectra of C16-Fengycin A (λex = 260 nm, λem = 340 nm). C2, the Stern–Volmer plots of C16-Fengycin A by DNA (Room temperature).
corresponding binding energy of the distributed conformation using a RMSD (Root-Mean-Square Deviation) tolerance of 2.0 Å. The lowest energy cluster contained 13 conformations, with a binding energy of −3.35 kcal M−1. The cluster with an energy of −2.9 kcal M− 1 had the most analysed conformations (20/100). The cluster was thus used for binding orientation analysis, with the highest energy ranking being selected to predict interactions between C16-Fengycin A and DNA (Fig. 6B, C, 6D). As shown in Fig. 6B, C and 6D, C16-Fengycin A entered the DNA major groove in AT-rich regions, surrounded by A5, A6, T19 and T20 base pairs. These results suggested that C16-Fengycin A could bind to the DNA major groove of DNA, with hydrogen binding likely facilitating this interaction.
intensities of free C16-Fengycin A in the absence and presence of DNA, [DNA] is the concentration of genomic DNA, τ0 is the average fluorescence lifetime of the molecule and its value is 10−8 s, and Kq is the rate constant for the bimolecular quenching process. Kq (6.96 ± 0.22 × 1011 L mol−1s−1, R2 = 0.9928) was calculated based on the ratio of intercept to slope (Fig. 5 C2). In general, the maximum diffusion collision quenching rate constant (Kq) of quenching agents on biomolecules is not more than 2 × 1010 L mol−1s−1 (Sun, Zhou, Hou, Liu, & Xiang, 2006). The calculation results showed that the quenching rate constant (Kq) of C16Fengycin A was greater than the diffusion control constant (2 × 1010 L mol−1s−1), which proved that the above quenching was not caused by dynamic collision, but should be static quenching. The binding constant (Ka) values of ethidium bromide (2.6 × 106 M−1), and acridine orange (4.0 × 105 M−1), which are both classic intercalating agents, are 105–106 M−1 (Cao & He, 1998). Our identified Ka value of C16-Fengycin A with DNA was much lower than that expected for an intercalating compound, but was consistent with one that undergoes groove binding (Ahmadi et al., 2010; Shahabadi et al., 2013), further supporting that C16-Fengycin A undergoes groove binding to DNA.
4. Conclusions This study had demonstrated that C16-Fengycin A possesses a potent of antifungal activity against F. graminearum via cell membrane disruption and direct genomic DNA binding. C16-Fengycin A showed a antifungal effect against F.graminearum at relative low concentrations, and it could increase cell membrane permeability, potentially causing a leakage of intracellular materials and a disruption in the cellular morphology. C16-Fengycin A could bind to the major groove of DNA, potentially slightly altering DNA secondary structure and morphology. These results revealed that C16-Fengycin A may serve as a good DNA binder, and its antifungal activity could be partly due to its DNAbinding ability. Our findings have suggested that C16-Fengycin A may be a promising natural preservative ideal for use in the agriculture and
3.5.4. Molecular modelling of C16-Fengycin A–DNA interaction Molecular docking was often used as a means of visualizing the binding mode of small ligands with DNA (Wang, Zhang, Zeng, Gong, & Wang, 2017). After 100 successful dockings, eight energy clusters were present and could be ranked on the basis of a scoring function relating to docking free energy. Fig. 6A showed the cluster analysis and 211
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Fig. 6. A, Cluster analyses of the AutoDock docking runs of C16-Fengycin A with DNA; B–D, molecular modelling results of the energy-minimised structure of the C16-Fengycin A–DNA system.
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