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Perillaldehyde, a Promising Antifungal Agent Used in Food Preservation, Triggers Apoptosis through a Metacaspase-dependent Pathway in Aspergillus flavus Jun Tian, Yanzhen Wang, Zhaoqun Lu, Chunhui Sun, Man Zhang, Aihua Zhu, and Xue Peng J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b03546 • Publication Date (Web): 13 Sep 2016 Downloaded from http://pubs.acs.org on September 15, 2016

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Journal of Agricultural and Food Chemistry

Graphical abstract

ACS Paragon Plus Environment


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Running Title:

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Perillaldehyde Induces Apoptosis in Aspergillus flavus

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Article Title:

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Perillaldehyde, a Promising Antifungal Agent Used in Food Preservation,

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Triggers Apoptosis through a Metacaspase-dependent Pathway in Aspergillus

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flavus

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Jun Tian1,2#,*, Yanzhen Wang1#, Zhaoqun Lu1, Chunhui Sun1, Man Zhang1, Aihua

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Zhu1, Xue Peng1**

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College of Life Science, Jiangsu Normal University, Xuzhou 221116, Jiangsu

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Province, PR China

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for Viro-Biotech, Research Institute of Tsinghua University in Shenzhen, Shenzhen

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518057, Guangdong, People’s Republic of China

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* Correspondence to: Jun Tian, College of Life Science, Jiangsu Normal University,

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Xuzhou 221116, Jiangsu Province, PR China. Tel.: +86-516-83403172; Fax: +86-

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516-83403173

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** Corresponding author: Tel: +86-516-83500033; Fax: +86-516-83403173

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E-mail addresses: [email protected] (J. Tian), [email protected] (X. Peng)

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#

Key Lab for New Drug Research of TCM and Shenzhen Branch, State R&D Centre

Jun Tian and Yanzhen Wang contributed equally to this work

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ABSTRACT

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In the present study, we provide detailed insights into perillaldehyde(PAE)’s

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mechanisms of action on Aspergillus flavus and offer evidence in favor of the

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induction of an apoptosis-like phenotype. Specifically, PAE’s antifungal mode of

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action was investigated through the detection of mitochondrial membrane potential

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(Mt∆Ψ) and phosphatidylserine (PS) exposure, as well as intracellular Ca2+ level,

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reactive oxygen species (ROS) accumulation, and metacaspase activation. This was

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done by way of fluorometry, measuring DNA fragmentation, and condensation by

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fluorescent microscopy. Furthermore, we searched for phenotypic changes

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characteristic of apoptosis by transmission electron microscopy (TEM) and flow

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cytometry, determining the amount of cytochrome c (cyt c) released using Western

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blotting. Results indicated that cultivation of A. flavus in the presence of PAE caused

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depolarization of Mt∆Ψ, rapid DNA condensation, large-scale DNA fragmentation,

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and an elevation of intracellular Ca2+ level. The percentage of early apoptotic cells

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with exposure of phosphatidylserine were 27.4% and 48.7%, respectively, after 9 h

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incubations with 0.25 and 0.5 µl/ml of PAE. The percentage of stained cells with

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activated intracellular metacaspases exposed to PAE at concentrations of 0.25 and 0.5

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µl/ml compared with control subjects were increased by 28.4±3.25% and

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37.9±4.24%, respectively. The above results has revealed that PAE induces fungal

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apoptosis through a caspase-dependent mitochondrial pathway. In all, our findings

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provide a novel mechanism for exploring a possible antifungal agent used in food

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preservation. 2



Aspergillus

flavus;

Apoptosis;

Antifungal;

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Perillaldehyde;

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KEYWORDS:

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Metacaspase

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ABBREVIATIONS:

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PAE, perillaldehyde; Mt∆Ψ, Mitochondrial membrane potential; ROS, reactive

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oxygen species; PS, phosphatidylserine; PI, propidium iodide; cyt c, cytochrome c;

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Rh123, rhodamine 123; DCFH-DA, 2',7'-dichlorofluorescein diacetate; DAPI, 4′,6-

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diamidino-2-phenylindole; TUNEL, terminal deoxynucleotidyl transferase dUTP nick

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end labeling;

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INTRODUCTION

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Aspergillus flavus, a filamentous fungus, is one of the three most common species

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that is frequently the primary cause of food contamination resulting in the production

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of aflatoxins, which are highly toxic compounds that can lead to both acute and

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chronic toxicity in humans and many other animals 1. Unfortunately, despite the fact

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that antibiotics are widely used in treating such fungal infections, increasingly-

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resistant strains continue to emerge, and various other drawbacks in terms of toxicity,

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drug interactions, and high costs still exist 2. Therefore, growing public concern has

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been directed towards natural antifungal agents due to the multiple potential

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resistances and side effects of the synthetic antifungal agents 3. Plant-derived natural

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antimycotics have been regarded as ideal alternatives to traditional chemical

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preservatives for improving food quality 4. In fact, the antifungal activities of natural

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products derived from plants have formed the basis of several applications, including 3


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many in the realms of pharmaceuticals and food preservation .

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Perillaldehyde (PAE) is a natural organic compound found most abundantly in the

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annual herb perilla (Perilla frutescens), which has been historically used as a

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medicinal agent and a popular leafy vegetable 5. It is used extensively as a food

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additive for flavoring, and as an ingredient in perfume 6. PAE could be developed into

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a natural preservative for controlling the infection of table grapes and cherry tomatoes

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by spoilage fungi, according to our previously published paper

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information exists about the exact mechanism of its action on A. flavus, as this is yet

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to explored.

5, 7

. However, no

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Recent evidence has shown that apoptosis occurs not only in multicellular

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organisms, but also in unicellular organisms, such as fungi 8. For example, fungi can

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undergo apoptotic-type programmed cell death upon exposure to antifungal agents,

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developmental signals, weak acids, oxidative stress, salt stress, and UV irradiation 9. A

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better understanding of cell death pathways might be the basis of developing novel

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antifungal therapeutics to control A. flavus.

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Thus, the mechanisms of the antifungal action of PAE were investigated in the

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present study. Furthermore, we investigated apoptotic effects caused by PAE,

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including increased reactive oxygen species (ROS), decreased mitochondrial

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membrane potential, phosphatidylserine externalization, cytochrome (cyt c) release,

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the activation of metacaspases, DNA condensation and fragmentation, and increased

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intracellular Ca2+ levels, in A. flavus cells, in order to elucidate its mode of action.

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MATERIALS AND METHODS 4



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Chemicals

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The PAE (CAS-No. 18031-40-8) used in this study was purchased from TCI Co.

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Ltd., and prepared as a stock solution in 0.1% (v/v) Tween-80 and added to

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experimental soloutions to achieve final concentrations (0, 0.125, 0.25, 0.5, and 0.75

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µl/ml). Other chemicals, such as rhodamine 123 (Rh123), 2',7'-dichlorofluorescein

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diacetate (DCFH-DA), 4',6-diamidino-2-phenylindole (DAPI), and Fluo-3/AM were

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obtained from Sigma Chemical Co. Ltd. The CaspACETM FITC-VAD-FMK In Situ

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marker was acquired from Promega Co. Ltd, and an FITC-Annexin V apoptosis

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detection kit and TUNEL Apoptosis Detection Kit were purchased from Nanjing

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Jiancheng Bioengineering Institute.

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Fungus Strain and CultivationA. flavus CGMCC 3.06434 was purchased from the

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China General Microbiological Culture Collection Center (CGMCC). The A. flavus

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was cultured at 28 ℃ in a potato dextrose agar (PDA) and was stored in a refrigerator

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at 4 ℃.

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Intracellular Reactive Oxygen Species Accumulation

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Intracellular production of reactive oxygen species (ROS) was measured using a

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fluorescent dye DCFH-DA 10. A. flavus cell suspensions adjusted to 5×106 spores/ml

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in PBS-2%G were treated with various concentrations of PAE (0, 0.125, 0.25, 0.5, and

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0.75 µl/ml) for 12 h at 28 ℃. After incubation, the cells were washed with PBS before

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being stained with 10 µM of DCFH-DA for 4 h at 28 ℃. The cells were then washed,

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re-suspended in PBS, and analyzed by an Accuri C6 flow cytometer (BD

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Biosciences). 5


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Determination of Mitochondrial Membrane Potential (Mt∆Ψ)

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The fluorescent dye Rh123 was used to investigate the changes in Mt∆Ψ in A.

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flavus cells after PAE treatment 11. These cells (5×106 spores/ml) were suspended in

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PBS-2%G and treated with different concentrations of PAE (0.125, 0.25, 0.5, and 0.75

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µl/ml). Samples without any PAE treatment were considered to be controls. The

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suspensions were mixed with 100 ng/ml of Rh123 before shake-culturing them at 28

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℃ for 12 h in the dark. Following this, the cells were washed in PBS and analyzed

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using an Accuri C6 flow cytometer (BD Biosciences).

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Double Staining of Annexin V and Propidium Iodide (PI)

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Phosphatidylserine (PS) exposure was detected by a Fourier infrared transform

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chromatography (FITC)-coupled annexin V reaction with the Annexin V-FITC kit, as

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previously outlined

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incubated as described; they were shaken at 200 rpm for 8 h and treated with 1%

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cellulase and snailase, as well as 100 U Lyticase, for 2 h at 30 ℃. The protoplasts of A.

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flavus were washed twice and re-suspended in PBS containing 1 M sorbitol, then

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shake-cultured with different consistencies of PAE (0, 0.25, and 0.5 µl/ml) for 3, 6,

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and 9 h at 28 ℃. Protoplasts (5×106 spores/ml) were obtained in an annexin-binding

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buffer and stained with 5 µl of PI and 5 µl of Annexin V-FITC for 30 min at room

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temperature in the dark. Finally, samples were then analyzed with an Accuri C6 flow

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cytometer (BD Biosciences).

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Determination of the Effect of PAE on the Cell Ultrastructure of A. flavus

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. To prepare viable protoplasts of A. flavus, the cells were

The effect of PAE on the cell ultrastructure of A. flavus was observed using 6



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transmission electron microscopy (TEM) according to the previously described

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method 13. In this process, A. flavus cell suspensions were seeded in PDB. After 24 h

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of incubation at 28 °C, hyphae were exposed to different concentrations of PAE (0.25

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and 0.5 µl/ml) for 3 h at room temperature. After treatment, the samples were washed

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twice in PBS and post-fixed with 2.5% glutaraldehyde before being dehydrated then

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embedded and polymerized in Spurr’s resin. Lastly, TEM (Tecnai G2 T12, FEI

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Company, Hillsboro, USA) was applied in order to observe the section of samples.

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Determination of the Effect of PAE on Morphological Changes of A. flavus

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The effect of PAE on morphological changes of A. flavus was measured by an

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Accuri C6 flow cytometer (BD Biosciences) 14. The A. flavus cells were treated with

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0.25 and 0.5 µl/ml of PAE at 28 ℃ and shaken constantly (200 rpm). After incubation

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for 12 h, the cells were washed and re-suspended in PBS. Cells were then illuminated

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by 488 nm of light, and forward light scatter (FSC) and side scatter (SSC) were

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measured.

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Determination of Cyt c Release from Mitochondria

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The mitochondria of A. flavus were isolated according to the method of Wu et al. 15.

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The A. flavus cells were cultured in PDA at 28 ℃ for 3 d, collected, and adjusted to

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5×106 spores/ml. The cells were then treated with PAE at 30 ℃ for 12 h, collected, and

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lysed with lysis buffer (150 mM sodium chloride, 1% triton X-100, 1 mM EDTA, 1

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mM EGTA, 50 mM Tris, pH 8). After treatment, a sonifier cell disrupter (XC-CD,

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XianChang Electronics Technology Co., Ltd., Ningbo, China) was used to shatter

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cells constantly. The treated homogenates were collected by centrifugation at 2000×g 7


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for 10 min to remove the cell debris and unbroken cells. Subsequently, the

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supernatants were collected and centrifuged at 12000×g for 40 min in order to test the

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cyt c release from the mitochondria to cytoplasm. The pellets were incubated for 5

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min with 50 mM Tris (pH 5.0) and 2 mM EDTA, and centrifuged at 5000×g for 30 s

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to measure the remaining cyt c in the mitochondria. Next, the UV-visible

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spectrophotometer (TU-1810C, Persee, Beijing, China) was used to measure protein

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content of these sample. Each sample containing 50 µg protein was resolved on 12%

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SDS-PAGE. The separated proteins were transferred to a polyvinylidene fluoride

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membrane and blocked with 5% nonfat dry milk. Rabbit anti-cyt c was used as a

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primary antibody and HRP-linked goat anti-rabbit IgG was applied as the secondary

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antibody. GAPDH and COX IV primary antibody, HRP-linked goat anti-rabbit IgG

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and HRP-linked rabbit anti-goat IgG secondary antibody were used in loading control

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analysis for normalization. The signal was detected on polyvinylidene fluoride

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membranes using an enhanced chemiluminescence substrate.

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Determination of Metacaspase Activation

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The change in metacaspase activation in A. flavus after treatment with PAE was

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examined using the CaspACETM FITC-VAD-FMK In Situ Marker

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flavus cell suspensions adjusted to 5×106 spores/ml in PBS-2%G were treated with

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different concentrations of PAE (0, 0.25, and 0.5 µl/ml) at 28 ℃. After incubation for

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12 h, the cells were washed with PBS, re-suspended in 500 µl of staining solution—

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including 5 µg/ml CaspACETM FITC-VAD-FMK In Situ Marker—and incubated for

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30 min at room temperature in the dark. All tests were performed in triplicate. The 8


16

. Briefly, the A.


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cells were quantitatively analyzed by an Accuri C6 flow cytometer (BD Biosciences).

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Determination of DNA damages

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In order to obtain further proof that apoptosis was induced in PAE-treated A. flavus

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cells, DNA and nuclear fragmentation were measured using TUNEL (terminal

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deoxynucleotidyl transferase dUTP nick-end labeling) and DAPI (4',6-diamidino-2-

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phenylindole) assays

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and 0.5 µl/ml) for 12 h were collected. For the TUNEL assay, the cells were washed

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with PBS and re-suspended in 4% formaldehyde (v/v in 10 mM PBS) at room

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temperature for fixation. After incubation for 30 min, the cells were again washed

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three times with PBS, then digested with 1% cellulase and nailase and 100 U Lyticase

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for 2 h at 30 ℃. The cells washed with PBS contained 1 M sorbitol and were stained

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with TUNEL. For DAPI staining, the cells were washed twice with PBS,

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permeabilized and fixed in 70% ethanol, and treated with 1 mg/ml of DAPI for 20

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min in the dark. Finally, the stained cells were placed on a slide and examined using

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an Olympus fluorescence microscope (Tokyo, Japan).

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Determination of intracellular Ca2+

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. The cells treated with various concentrations of PAE (0.25

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To investigate intracellular Ca2+ levels due to PAE treatment, the non-fluorescent

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dye fluo-3 AM was used 18. A. flavus cells were cultured in a PDA medium at 28 ℃

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and suspended in PBS. The cell suspensions were then mixed with PAE for 12 h at the

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following concentrations: 0, 0.125, 0.25, 0.5, and 0.75 µl/ml. At the end of the

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treatments, the cells were washed and loaded with 5 µM Fluo-3/AM supplemented

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with 0.1% pluronic F-127 for 40 min, at 37 ℃. After loading, the cells were washed 9


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twice and re-suspended in PBS, and the fluorescence intensity of the cells were

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analyzed with an Accuri C6 flow cytometer (BD Biosciences).

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Statistical analysis

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All experiments were carried out in triplicate and results were reported as means ±

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standard deviations (n=3). The significant differences between mean values were

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determined using Duncan’s Multiple Range test (p

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