Thymol Induces Conidial Apoptosis in Aspergillus flavus via

risk of hepatocellular carcinoma or aspergillosis in human. Thymol, as a food. 2 preservative, could efficiently kill conidia of A. flavus. However, t...
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Bioactive Constituents, Metabolites, and Functions

Thymol Induces Conidial Apoptosis in Aspergillus flavus via Stimulating K+ Eruption Liang-bin Hu, Fang-Fang Ban, hongbo li, Pan-Pan Qian, QingShan Shen, Yan-Yan Zhao, Hai-Zhen Mo, and Xiaohui Zhou J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b02117 • Publication Date (Web): 25 Jul 2018 Downloaded from http://pubs.acs.org on July 30, 2018

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Journal of Agricultural and Food Chemistry

Thymol Induces Conidial Apoptosis in Aspergillus flavus via Stimulating K+ Eruption Liang-Bin Hua , Fang-Fang Bana, Hong-Bo Lia, Pan-Pan Qiana, Qing-Shan Shena, Yan-Yan Zhaoa, Hai-Zhen Mo a*, Xiaohui Zhoub* a

Department of Food Science, Henan Institute of Science and Technology, Xinxiang

453003, China b

Department of Pathobiology & Veterinary Science, The University of Connecticut;

61 N. Eagleville Rd, Storrs, CT *Corresponding authors E-mail: [email protected] (HM) or [email protected] (XZ)

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ABSTRACT: Aspergillus flavus is a notorious foodborne fungus, posing significant

2

risk of hepatocellular carcinoma or aspergillosis in human. Thymol, as a food

3

preservative, could efficiently kill conidia of A. flavus. However, the underlying

4

mechanisms by which thymol kills A. flavus are not completely understood. With

5

specific fluorescent dyes, we detected several apoptotic hallmarks including

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chromatin condensation, phosphatidylserine externalization, DNA damage,

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mitochondrial depolarization and caspase 9 activation in conidia exposed to

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200µg/ml of thymol, indicating that thymol induced a caspase-dependent conidial

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apoptosis in A. flavus. Chemical-protein interactome (CPI) and autodock analyses

10

showed that KCNAB, homologue to the β-subunit of voltage gated potassium

11

channel (Kv) and aldo-keto reductase, was the potential target of thymol. Following

12

studies demonsrated that thymol could activate the aldo-keto reductase activity of

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KCNAB in vitro and stimulate a transient K+ efflux in conidia as determined using a

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Port-a-Patch. Blocking K+ eruption by 4-aminopyridine (a universal inhibitor of Kv)

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could significantly alleviate thymol-mediated conidial apoptosis, indicating that

16

activation of Kv was responsible for the apoptosis. Taken together, our results

17

revealed a K+ efflux-mediated apoptotic pathway in A. flavus, which greatly

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contributed to the development of alternative strategy to control this pathogen.

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KEYWORDS: Aspergillus flavus, thymol, conidia, apoptosis, K+ eruption,

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Voltage-gated potassium channel

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INTRODUCTION

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Aspergillus flavus is notorious as a biological pollutant in our food material, where it

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produces the carcinogenic aflatoxin. More than 28% of the total worldwide cases of

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hepatocellular carcinoma is associated with the intake of aflatoxins 1, 2. Aflatoxins

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are also responsible for some acute poisoning, immune-system dysfunction and

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stunted growth in children 3. In addition, A. flavus causes aspergillosis in

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immunocompromised individuals 4, and is responsible for fungal rhino-sinusitis and

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fungal eye infections (endopthalmitis and keratitis) in some tropical countries (e.g.

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India, Sudan, Kuwait, and Iran) 5, 6. As one of major constituents of essential oil in

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Monarda punctate, thymol (2-isopropyl-5-methylphenol) has been identified with

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antioxidant, antiviral, antitumor, and anti-inflamatory properties 7-10. Nevertheless,

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thymol has been mainly applied as antimicrobial agent to control a broad spectrum

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of microorganisms including Escherichia 11, Salmonella 12, Listeria 13,

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Staphylococcus 14, Actinobacillus 15, Saccharomyces 16, Candida 17, Fusarium 18 and

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etc. Mechanisms underlying its antimicrobial activity has been extensively studied

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involving the inhibition of H(+)-ATPase 19, the interruption of surface electrostatics

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of cell membrane and membrane integrity 20, the reversal of drug efflux pumps 21,

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the inhibition of telomerase activity 16 and the induction of destabilization in the

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DNA secondary structure 22. It should be noted that thymol leads to different death

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mode in different organisms via different ways 16. Thymol has been shown to

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efficiently kill conidia of A. flavus via triggering reactive oxygen species (ROS) and

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reactive nitrogen species (RNS) 23. However, the target of thymol and the death

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mode in conidia of A. flavus are unknown. In this study, we showed that thymol

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targets KCNAB to trigger K+ efflux and consequently induces A. flavus apoptosis.

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Understanding the target of thymol and the mode of cell death would greatly

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contribute to the development of alternative strategy to control A. flavus.

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MATERIALS AND METHODS

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Strains and Chemicals. Aspergillus flavus CGMCC3.2890 was purchased

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from China General Microbiological Culture Collection Center. All spores in this

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study were cultured in Sabourand Dextrose (SD) Medium containing 4% glucose, 1%

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peptone, 1.5% agar power at 30°C. For the drug treatment experiments, spores were

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harvested post thymol (Sigma) treatment for 3~9h at the concentration of 200 µg/ml.

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Thymol was dissolved in dimethylsulfoxide (DMSO) with the concentration of

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DMSO kept below 0.1% in the treatment groups. All other chemical agents were

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analytical grade.

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Detection of Conidial Apoptosis. Conidial apoptosis in A. flavus was evaluated

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by using an Apoptosis and Necrosis Assay Kit (C1056, Beyotime Institute of

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Biotechnology, China). Briefly, the conidia with different treatments were washed

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once with phosphate-buffered saline (PBS, pH 7.4), and then stained with Hoechst

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33342 (10 µg/ml) and propidium iodide (PI) (1 µg/ml) for 30min at 4°C in the dark.

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The apoptotic cells were observed with an inverted fluorescent microscope (Zeiss

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Axio Vert A1) and a flow cytometer (Beckman CytoFLEX FCM).

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Phosphatidylserine Externalization Assay and TUNEL Assay. Spores

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suspension at a density of 2×106 was exposed to 200 µg/ml thymol for 6h, and then

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collected for following assay. Conidial phosphatidylserine externalization (PS) was

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analyzed using an annexin V-FITC apoptosis detection kit (C1063, Beyotime

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Institute of Biotechnology, China), and DNA damage was analyzed using One Step

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TUNEL Apoptosis Assay Kit (C1086, Beyotime Institute of Biotechnology, China).

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All the procedures were performed following the manufacturer’s instructions. Flow

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cytometry and fluorescence microscopy were utilized to monitor the spores with

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positive fluorescence staining.

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Assay for Mitochondrial Membrane Potential. A fluorescent dye JC-1

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(C2005, Institute of Biotechnology, China) was used to analyze mitochondrial

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membrane potential (∆ψm) in conidia of A. flavus. JC-1 dye is capable of

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accumulating potential-dependently in mitochondria. At high ∆ψm, JC-1 aggregates

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and gives a red fluorescence. At low ∆ψm, it depolymerizes to monomers and emits

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green fluorescence. Mitochondrial depolarization is indicated by an increase in the

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green (529 nm)/red (590 nm) fluorescence intensity ratio. JC-1 was uploaded in the

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conidia according to its manual instruction. Corresponding fluorescence assays were

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performed by using a microreader (Thermol, Varioskan Flash) and a fluorescent

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microscope.

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Caspase 9 Activity Assay. Conidial caspase 9 activity was detected by using a

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Kit (C1157, Beyotime Institute of Biotechnology, China) containing the substrate

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(Ac-LEHD- acetyl-Leu-Glu-His-Asp p-nitroanilide) for caspase 9. Spore suspension

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of A. flavus was inoculated into the SD liquid medium with final concentration of

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2×106/mL, and then exposed to 200 µg/mL of thymol for 6h. In parallel, another

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group of spores was exposed to 20 µM of Z-VAD-FMK (a caspase inhibitor) prior to

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the addition of thymol. Subsequently, the spores were washed using PBS, and

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suspended with lysis buffer, followed by ultra-sonication on the ice for 20min. After

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centrifugation at 16,000g, 4°C for 15min, the supernatant was transferred to the

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precooling tube to determine the increase in absorbance at 400 nm indicating

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caspase-9 activity. The release of p-nitroanilide (pNA) was calculated based on a

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standard curve between its amount and its absorbance at 400 nm, and 1U of

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caspase-9 activity was defined as the facilitation of 1 µM of pNA release. Bioinformatics Analysis for the Potential Targets of Thymol. To identify the

96 97

targets of thymol leading to apoptosis in A. flavus, we firstly predicted the proteins in

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human that have potential to interact with thymol through the server of DRAR-CPI

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(http://202.120.31.160/drar/) 24, 25. Some proteins associated with apoptosis were

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picked and then their homologues in A. flavus were identified. Three-dimensional

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structures of these homologues were obtained through homology modeling using

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I-TASSER (http://zhanglab.ccmb.med.umich.edu/I-TASSER/) 26. The structure of

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thymol was obtained from Pubchem database (CID: 6989). The predicted

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interactions between these homologues and thymol were modelled by Autodock 4.2

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27

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. Protein Expression and Purification. The coding sequence of KCNAB was

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cloned from the cDNA library of A. flavus which was prepared by using the Kit

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according to its instructions. Then, it was constructed into pET28a (+) vector with

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Ncol and BamHl, and transformed to E.coli Arctic Express strain by chemical

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transformation. To induce KCNAB expression in the recombination strain, 0.5 mM

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of isopropyl-β-d-thiogalactoside (IPTG) was added into bacterial culture of

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OD600=0.6~0.8, continuing incubation at 220 r/min, 37°C for 4h. The bacterial cells

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were harvested and broken by ultrasonication (400w for 20min) in lysate solution

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(20 mM Tris-HCl containing 1 mM phenylmethanesulfonyl fluoride (PMSF)

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supplemented with one tablet of bacteria protease inhibitor cocktail, pH 8.0). As

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KCNAB was mostly expressed in inclusion bodies, debris containing inclusion

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bodies was collected and washed 3 times with scrubbing solution (20 mM Tris, 1

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mM EDTA, 2 M urea, 1 M NaCl, 1% Triton X-100, pH 8.0). Inclusion bodies were

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resolved with Solution buffer (20 mM Tris, 5 mM dithiothreitol (DTT), 8 M Urea,

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pH 8.0) and dialyzed in a buffer (20 mM Tris-HCl, 0.15 M NaCl, pH 8.0) overnight.

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The purification for KCNAB was carried with the nickel ion affinity

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chromatography column (Ni-IDA -Sepharose CL-6B). KCNAB was eluted by

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elution-Buffer (20 mM Tris-HCl, 250 mM imidazole, 0.15 M NaCl, pH 8.0) and

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dialyzed in 1 M phosphate buffer (PB) (pH 7.4) overnight. The purity of KCNAB

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was evaluated by 12% SDS-PAGE, and its identity was confirmed through detecting

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the His-tag of recombinant KNCAB by western blotting with His antibody (AH158,

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Beyotime Institute of Biotechnology, China) according to its instructions.

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Aldo-keto Reductase Activity of KCNAB. The aldo-keto reductase activity of

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KCNAB was determined according to Weng et al 28 with slight modification. The

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reaction mixture of 0.4 mM NADPH, 10 mM of 4-formylbenzonitrile and 300 µg/ml

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KCNAB protein in PB (pH 7.4) was incubated at 37°C for 2h. The absorption at 340

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nm was measured by a microreader with a µDropTM plate (No. N12391). The

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reductase activity of KCNAB was indicated as a decrease of OD340nm (1U=0.01).

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Blank controls with no KCNAB were incorporated routinely and the background

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NADPH consumption was subtracted.

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Intracellular K+ Assay. Intracellular K+ level in conidia was evaluated by

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applying a K+ fluorescent indicator of PBFI-AM (C270, GeneCopoeia, USA). The

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relative intensity of fluorescence at 500nm excited by 340 nm and 380 nm were

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time-course recorded by a microreader, respectively. The ration between them at

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each time point was calculated, and its curves indicated the dynamical changes of K+

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level in conidia. Flow cytometry was also used to evaluate the changes of K+ level in

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conidia. To decipher the K+ distribution in conidia, double staining including PBFI

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and JC-1 was performed and K+ distribution was evaluated by using a confocal laser

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scanning microscope (Zeiss LSM 180). All these fluorescent probes were loaded into

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conidia 1h prior to the addition of thymol and incubated in the dark at 37°C.

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Automated Patch-clamp Recording. The protoplasts of A. flavus were

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prepared according to Woloshuk 29. Whole cell patch clamp recordings were

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conducted according to Nanion’s standard procedure for the Port-a-Patch (Nanion,

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Germany). Chips with single-hole medium resistance (~10 MΩ) were used for the

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recording of protoplasts. The external perfusion system was also used. After

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initiating the experiment, cell catching, sealing, whole-cell formation, liquid

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application, recording, and data acquisition were performed sequentially. Currents

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were recorded every 0.7s by 500ms voltage ramps from -80 mV to + 80 mV.

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Currents were elicited by application of thymol via the external perfusion system.

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Thymol was made as a 200 mg/ml stock in DMSO and diluted in external recording

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solution to the concentrations indicated. A representative trace of three repeats of

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each experiment was shown.

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Statistics Analysis. All assay were repeated independently at least 3 times and

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experimental date were represented as mean ± SD. The data between two different

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treatments were compared statistically by Student’s t-test. The differences were

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considered significant at P < 0.05.

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RESULTS

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Thymol Induced Conidial Apoptosis in A. flavus.

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In order to explore the conidial death mode in response to thymol, we employed

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a conidial staining with Hoechst 33342/PI. Usually, PI can only enter the dying cells

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leading to a positive staining, and Hoechst 33342 staining is denser in dying or

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apoptotic cells due to condensed conidial chromatin. Thus, Hoechst 33342-enhanced,

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but not PI-positive conidia, were judged as apoptosis. As shown by flow cytometry,

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very limited shifting in red fluorescence (PI-DNA excitation ) distribution of conidia

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occurred when exposed to thymol for even 9h (Figure 1A). However significant

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shifting in blue fluorescence (Hoechst 33342-DNA excitation) distribution could be

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observed since 3h post the exposure to thymol (Figure 1B). By counting, blue

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fluorescent cells were induced in approximately 40% of conidia with 3h exposure to

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thymol, and in over 80% of conidia within 6 h exposure (Figure 1C). Fluorescent

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microscopy further confirmed that thymol led to weak changes in red fluorescence

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but a significantly enhanced blue fluorescence of conidia (Figure S1). Besides

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chromatin condensation, PS as one of characteristics of apoptosis was analyzed with

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an Annexin V-FITC assay. Through flow cytometry, we showed that conidia with

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Annexin staining (FITC fluorescence) was significantly increased when exposed to

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thymol for 6h (Figure 1D). Enhanced conidial green fluorescence was also observed

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by fluorescent microscopy (Figure S2) after exposure to thymol. These results

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indicated positive PS in conidia exposed to thymol for 6h. During apoptosis

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chromosomal DNA could be cleaved into oligo-nucleosomal size fragments, which

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is also a biochemical hallmark of apoptosis 30. By the TUNEL assay in situ,

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positively-shifted conidial distribution in fluorescence (FITC) (Figure 1E) and much

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stronger green fluorescence (Figure S3) could be observed on conidia exposed to

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thymol through flow cytometry and fluorescent microscopy, respectively. This

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indicated the occurrence of DNA cleavage in conidia exposed to thymol for 6h. The

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mitochondrion plays an important role in the execution phase of apoptosis 31. A

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distinctive feature of the early stages of apoptosis is the disruption of active

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mitochondria leading to alteration in membrane potential 32, 33. JC-1 dye is utilized to

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manifest mitochondrial depolarization occurring in apoptosis indicated as an increase

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in the green (529 nm)/red (590 nm) fluorescence intensity ratio. Our results showed

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that thymol treatment led to a significant increase in conidial green/red fluorescence

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intensity ratio (Figure 1F). Fluorescence microscopy showed that conidia was almost

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green after treatment with thymol (Figure S4), indicating that exposure to thymol led

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to the occurrence of mitochondrial depolarization in conidia. Taken together, these

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results showed that conidia of A. flavus undergo apoptosis after treatment with

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thymol.

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Thymol-mediated Apoptosis in A. flavus is Caspase-dependent.

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In fungi, apoptosis can be either caspase (metacaspase) dependent or

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independent 33. Caspase-9, is specific to the mitochondrial (intrinsic) pathway, and

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can act directly as an apoptotic effector molecule 33, 34. Considering mitochondrial

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disruption in conidia, we determined the activity of caspase 9 in conidia exposed to

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thymol. Our results showed that caspase 9 activity in conidia was significantly

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activated when exposed to thymol for 6h (Figure 2). The addition of its inhibitor

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(Z-VAD-FMK) prior to the exposure to thymol significantly blocked the activation

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of caspase 9 activity (Figure 2A) and alleviated the occurrence of conidial apoptosis

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in response to thymol treatment (Figure 2B). These results indicated that conidial

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apoptosis induced by thymol was caspase-dependent.

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KCNAB is a Target of Thymol Mediating Conidial Apoptosis.

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Identification of the endogenous molecular switches that trigger fungal

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apoptosis is of paramount importance 35. Receptors targeted by thymol leading to

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apoptosis were predicted through several steps as shown in Figure S5A. Firstly, we

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used chemical-protein interactome (CPI) profiles through the server DPDR-CPI to

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predict potential targets of thymol in humans. This analysis provided a list of 393

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proteins with the potential to interact with thymol. We selected voltage-gated

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potassium channel (Kv) subunit beta-2 (encoded by KCNAB2 gene; PBD ID: 1ZSX)

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for further investigation because (1) KCNAB2 has been reported involved in

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apoptosis in cancer cells, and (2) KCNAB2 has a higher predictive score to interact

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with thymol. In addition, KCNAB2 harbors dual activities including Kv regulation

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and aldo-keto reductase, and thus KCNAB2 would be more like a molecular switch

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to regulate metabolic state in response to external stimulation. More importantly, a

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KCNAB2 homologue (KCNAB; Gene ID: 7908969) exists in A. flavus. Using

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I-TASSER, the three three-dimensional structures of KCNAB were constructed

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(Figure S5B). The quality of model was assessed by SAVES, and the results showed

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that 97.7% residues of KCNAB model was in the favored and allowed region, and

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96.57% of the residues had an averaged 3D-1D score≥0.2. The overall quality factor

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of ERRAT was 94.135. The potential interactions between KCNAB and thymol were

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predicated by Autodock 4.2. The results showed that thymol potentially interacted

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with 4 amino acids of KCNAB, which were listed in the annotation tables in Figure

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S5B. To further determine the interaction between KCNAB and thymol, KCNAB was

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cloned into a pET-28a (+) vector for expression in E. coli DE3. Expressed KCNAB

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was purified on a Ni+-IDA-Sepharose CL-6B affinity column. Through SDS-PAGE,

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we showed that KCNAB was obtained at electrophoretic purity, and further western

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blotting confirmed the identity of recombinant KCNAB (Figure 3A). With NADPH

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and 4-formylbenzonitrile as substrates, the aldo-keto activity of KCNAB could be

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determined by detecting the decreasing absorbance at 340nm due to the consumption

239

of NADPH. The results showed that the addition of thymol significantly increased

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the activity of KCNAB (Figure 3B), indicating a direct interaction between KCNAB

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and thymol. Considering potential regulation of Kv by KCNAB, we monitored the

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changes of intracellular K+ level in conidia exposed to thymol with PBFI-AM, a

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cell-permeable fluorescent K+ binding probe. It was interesting that a transient

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eruption of intracellular K+ was observed after treatment with thymol using both

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microreader (Figure 4A) and flow cytometry (Figure 4B). Treatment with

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4-aminopyridine (4-Ap), a specific inhibitor of Kv, significantly mitigated the K+

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eruption in conidia in response to thymol (Figure 4A,B). What’s more important,

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4-Ap also significantly alleviated thymol-induced conidial apoptosis (Figure 4C,D).

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This strongly supported that thymol targeted a Kv system in conidia of A. flavus

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leading to apoptosis.

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Thymol Promotes K+ Efflux in Conidia of A. flavus.

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The opening of Kv channels would result in an efflux of positive charge due to

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the concentration gradient of K+ that exists across the cell surface membrane 36.

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However, we observed an increasing intracellular K+ level using K+ probe, PBFI.

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Using confocal microscope, we were surprised to see that K+-fluorescence probe

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PBFI was circularly distributed around the cellular surface (Figure 5A). If high

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concentration of K+ gathers around the cellular surface, there should form a layer of

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high potential similar to mitochondrial intermembrane space full of H+. Therefore,

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we employed both PBFI (probe for K+) and JC-1 (membrane potential) to detect the

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K+ distribution in conidia in response to thymol. The results showed both green

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fluorescence from PBFI and red fluorescence from accumulated JC-1 were

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distributed around the cell surface after addition of thymol (Figure 5A), strongly

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suggesting that K+-PBFI probe gathered circularly inside of conidial envelope.

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Interestingly, in the absence of PBFI staining, thymol treatment led to few changes in

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JC-1 distribution in conidia in which red fluorescence was scattered in the cell space

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(Figure 5B), indicating that high potential layer was not formed upon thymol

267

treatment. Therefore, we reasoned that gathering of K+ in conidia exposed to thymol

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was due to the existence of its fluorescent probe PBFI. In order to explore the true

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movement of K+, we prepared the protoplast of A. flavus and monitored the K+ flow

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in protoplast exposed to thymol by using an automated patch-clamp recorder. A.

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flavus protoplast showed an outward K+ current under certain positive voltages,

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which showed the voltage-gated properties of Kv (Figure 6A). The I-V curves

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indicated a significant activation of Kv leading to more dramatic outward K+ current

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in response to thymol (Figure 6B). Taken together it could be concluded that thymol

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induced conidial apoptosis in A. flavus by stimulating K+ efflux via activating Kv. As

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for K+ gathering in the presence of K+ fluorescent probe, we interpreted that it is

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attributed to binding of K+ with probe. K+-probe complex could not pass the Kv

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channel, resulting in the trapped K+ gathering at the inside of the plasma membrane

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(Figure S6).

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DISCUSSION

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A. flavus vigorously produces conidia, leading to food contamination and

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human infection 4,37. Usually these conidia are relatively-resistant to stress

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including desiccation, heat, and UV stress 38, 39, which poses a great challenge to

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eliminate them. Our results revealed that thymol could induce conidial death via

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apoptosis, which provided not only new insights into the development of strategy

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to control A. flavus contamination, but also an opportunity to further study the

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conidial death mechanism in A. flavus. To our knowledge, there are few reports

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on fungal apoptosis induced by thymol. We have detected necrosis instead of

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apoptosis in S. cerevisiae exposed to thymol 40. Thus, the mechanism underlying

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conidial apoptosis in A. flavus is different from those in other fungi by thymol.

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Sharon et al 33 proposed an apoptotic pathways in fungi wherein caspases

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cascades mediate apoptosis. However, no caspase has been identified in A. flavus

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except for metacaspase CasA and CasB. It has been reported that perillaldehyde

294

activated metacapase and mediated apoptosis in A. flavus 41. However, there was

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no evidence in that study to support the roles of metacaspase in apoptosis

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induced by perillaldehyde. Although there has been no caspase 9 present in the

297

genome of A. flavus, we still detected the activity of caspase 9 with its specific

298

substrate in vitro. This suggested there should be some homologues of caspase 9

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in A. flavus with low sequence similarities to the known caspase 9 sequences.

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Caspase 9 is considered as an important executor in mitochondrial pathway of

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apoptosis 33, and JC-1 dying indicated mitochondrial disruption in conidia

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exposed to thymol. Thus, we hypothesize that thymol induces conidial apoptosis

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via a classical mitochondrial pathway. This hypothesis was supported by our

304

results that Z-VAD-FMK that specifically inhibited the activation of caspase 9

305

could alleviate thymol-mediate conidial apoptosis.

306 307

How does thymol trigger conidial apoptosis? We identified KCNAB, a potential β-subunit of Kv in A. flavus, with great potential to interact with thymol.

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Further analysis showed that thymol could activate aldo-keto reductase activity

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of the purified recombinant protein KCNAB in vitro, demonstrating that

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KCNAB directly targets KCNAB. In mammals, Kv with such a subunit is a

311

tetrameric integral membrane protein that, upon membrane depolarization, allow

312

potassium ions to flow out of a cell42. Activation of Kv and a loss of intracellular

313

K+ is necessary for caspase-mediated neuronal apoptosis induced by

314

oxidant-liberated Zn2+ 43. Our results showed that thymol could stimulate K+

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efflux and blocking K+ efflux via 4-Ap could significantly alleviate

316

thymol-mediated conidial apoptosis, indicating that activation of Kv was

317

responsible for this apoptosis. Based on these results, we concluded that some

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Kv system containing KCNAB was the endogenous receptor targeted by thymol

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triggering apoptosis. Thymol-based compounds have been proved as agonists of

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transient receptor potential (TRP) family of ion channel which involved in a

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wide array of physiological functions 44. Kv activation attributed to novel

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potential of thymol as a therapeutic agent.

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It is worth to note that K+ fluorescent probes could indicate the intracellular

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K+ concentration. However, such probe may lead to a misleading interpretation

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on K+ influx or efflux as the K+-probe complex is too large to be transported

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through the cell membrane. Electrophysiological techniques based on

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auto-clamp-patch could provide more reliable data on ion influx or efflux.

328 329

Our results also confirmed that KCNAB was an aldo-keto reductase which could be activated by thymol. Such NADPH-dependent redox enzymes are

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suggested to be involved in cellular metabolism by being coupled to electrical

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signaling through ion channels 28. Although we had no evidence for the roles of

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aldo-keto reductase activity of KCNAB in conidial apoptosis, such a redox

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enzyme could sense the intracellular redox state and lead to long-lasting effects

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on Kv-α 45. Our previous studies showed that thymol induced ROS eruption in

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conidia, which was proved to involve in the conidial death 23. ROS have been

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implicated in mitochondrial dysfunction, which leads to the release of

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pro-apoptotic proteins triggering the caspase activation and apoptosis 46. It was

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interesting that blocking ROS production with the inhibitors of NADPH oxidase

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attenuated the conidial apoptosis by thymol (Figure S7), while alleviation of K+

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eruption by the inhibitor of Kv also blocked the ROS generation by thymol

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(Figure S8), suggesting that K+ efflux leads to ROS production and subsequent

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apoptosis. This was further supported by the fact that K+ eruption was stimulated

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instantly by thymol, while ROS was produced 30min after the addition of thymol

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23

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is a molecular switch that controls K+ efflux and mediates apoptosis in A. flavus

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induced by thymol, which greatly contributed to the development of alternative

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strategy against this fungal pathogen. However, little information on α-subunit of

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this Kv is available, and further studies are required to elucidate this valuable

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apoptotic pathway.

. Taken together, our research revealed an apoptotic pathway in which KCNAB

350 351

ACKNOWLEDGEMENTS

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This work is financially supported by General Project of National Natural Science

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Foundation of China (Grant No.31671952), Program for Science & Technology

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Innovation Talents in Universities of Henan Province (18HASTIT037), and Program

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for Science & Technology Innovation Team in Universities of Henan Province

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(16IRTSTHN007).

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Supporting Information

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Supplemental data including Figure S1 (Fluorescent microscopy of conidia with 6h

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exposure to thymol through double staining with Hoechst 33342 and PI), Figure S2

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(PS externalization in conidia exposed to thymol for 6h detected through fluorescent

361

microscopy with an Annexin V-FITC assay), Figure S3 (DNA damage in conidia

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exposed to thymol for 6h detected by TUNEL staining through fluorescent

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microscopy.), Figure S4 (Variation of mitochondrial potential in conidia exposed to

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thymol for 6h indicated by JC-1 through fluorescent microscopy), Figure S5

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(Predication of acceptors targeted by thymol leading to apoptosis through

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DRAR-CPI and autodock analysis), Figure S6 (Model on K+-probe complex trapped

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inside of the cells), Figure S7 (The effects of ROS blockers (PY and IMZ) on the

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conidial apoptosis induced by thymol) and Figure S8 (The effects of a Kv inhibitor

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(4-Ap) on the ROS generation in the conidia induced by thymol) (Supplemental

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Material). This material is available free of charge via the Internet at

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http://pubs.acs.org

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REFERENCES

374

1. Liu, Y.; Wu, F. Global burden of aflatoxin-induced hepatocellular carcinoma: a

375

risk assessment. Environ. Health Persp. 2010, 118, 818-824.

376

2. Liu, Y.; Chang, C. C.; Marsh, G. M.; Wu, F. Population attributable risk of

377

aflatoxin-related liver cancer: systematic review and meta-analysis. Eur. J. Cancer.

378

2012, 48, 2125-2136.

379

3. Wu, F. Perspective: time to face the fungal threat. Nature 2014, 516, S7.

380

4. Amaike, S.; Keller, N. P. Aspergillus flavus. Annu. Rev.Phytopathol. 2011, 49,

381

107-133.

382

5. Chakrabarti, A.; Singh, R. The emerging epidemiology of mould infections in

383

developing countries. Curr. Opin. Infect. Dis. 2011, 24, 521-526.

384

6. Chakrabarti, A.; Rudramurthy, S. M.; Panda, N.; Das, A.; Singh, A. Epidemiology

385

of chronic fungal rhinosinusitis in rural India. Mycoses 2015, 58, 294-302.

386

7. Luna, A.; Lema-Alba, R. C.; Dambolena, J. S.; Zygadlo, J. A.; Labaque, M. C.;

387

Marin, R. H. Thymol as natural antioxidant additive for poultry feed: oxidative

388

stability improvement. Poultry. Sci. 2017, 96, 3214-3220.

389

8. Lai, W. L.; Chuang, H. S.; Lee, M. H.; Wei, C. L.; Lin, C. F.; Tsai, Y. C. Inhibition

390

of herpes simplex virus type 1 by thymol-related monoterpenoids. Planta. Med. 2012,

391

78, 1636-1638.

392

9. Gunes-Bayir, A.; Kocyigit, A.; Guler, E. M.; Kiziltan, H. S. Effects of thymol, a

393

natural phenolic compound, on human gastric adenocarcinoma cells In vitro. Altern.

394

Ther. Health.M. 2018. PMID: 29477139

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

395

10. Yao, L.; Hou, G.; Wang, L.; Zuo, X. S.; Liu, Z. Protective effects of thymol on

396

LPS-induced acute lung injury in mice. Microb. Pathogenesis 2018, 116, 8-12.

397

11. Burt, S. A.; Vlielander, R.; Haagsman, H. P.; Veldhuizen, E. J. Increase in

398

activity of essential oil components carvacrol and thymol against Escherichia coli

399

O157:H7 by addition of food stabilizers. J Food Protect. 2005, 68, 919-926.

400

12. Chauhan, A. K.; Kang, S. C. Thymol disrupts the membrane integrity of

401

Salmonella ser. typhimurium in vitro and recovers infected macrophages from

402

oxidative stress in an ex vivo model. Res. Microbiol. 2014, 165, 559-565.

403

13. Guevara, L.; Antolinos, V.; Palop, A.; Periago, P. M. Impact of moderate heat,

404

carvacrol, and thymol treatments on the viability, injury, and stress response of

405

Listeria monocytogenes. Biomed. Res. Int. 2015, 2015, 548930.

406

14. Nostro, A.; Blanco, A. R.; Cannatelli, M. A.; Enea, V.; Flamini, G.; Morelli, I.;

407

Sudano Roccaro, A.; Alonzo, V. Susceptibility of methicillin-resistant staphylococci

408

to oregano essential oil, carvacrol and thymol. FEMS Microbiol Lett. 2004, 230,

409

191-195.

410

15. Wang, L.; Zhao, X.; Zhu, C.; Xia, X.; Qin, W.; Li, M.; Wang, T.; Chen, S.; Xu,

411

Y.; Hang, B.; Sun, Y.; Jiang, J.; Richard, L. P.; Lei, L.; Zhang, G.; Hu, J. Thymol

412

kills bacteria, reduces biofilm formation, and protects mice against a fatal infection

413

of Actinobacillus pleuropneumoniae strain L20. Vet Microbiol. 2017, 203, 202-210.

414

16. Darvishi, E.; Omidi, M.; Bushehri, A. A.; Golshani, A.; Smith, M. L. Thymol

415

antifungal mode of action involves telomerase inhibition. Med. Mycol. 2013, 51,

416

826-834.

ACS Paragon Plus Environment

Page 20 of 32

Page 21 of 32

Journal of Agricultural and Food Chemistry

417

17. Shu, C.; Sun, L.; Zhang, W. Thymol has antifungal activity against Candida

418

albicans during infection and maintains the innate immune response required for

419

function of the p38 MAPK signaling pathway in Caenorhabditis elegans. Immunol.

420

Res. 2016, 64, 1013-1024.

421

18. Gao, T.; Zhou, H.; Zhou, W.; Hu, L.; Chen, J.; Shi, Z. The fungicidal activity of

422

thymol against Fusarium graminearum via inducing lipid peroxidation and

423

disrupting ergosterol biosynthesis. Molecules 2016, 21.PMID: 27322238

424

19. Ahmad, A.; Khan, A.; Yousuf, S.; Khan, L. A.; Manzoor, N. Proton translocating

425

ATPase mediated fungicidal activity of eugenol and thymol. Fitoterapia 2010, 81,

426

1157-1162.

427

20. Ahmad, A.; Khan, A.; Akhtar, F.; Yousuf, S.; Xess, I.; Khan, L.; Manzoor, N.

428

Fungicidal activity of thymol and carvacrol by disrupting ergosterol biosynthesis and

429

membrane integrity against Candida. Eur. J. Clin. Microbiol Infect. Dis. 2011, 30,

430

41-50.

431

21. Ahmad, A.; Khan, A.; Manzoor, N. Reversal of efflux mediated antifungal

432

resistance underlies synergistic activity of two monoterpenes with fluconazole. Eur.

433

J. Pharm. Sci. 2013, 48, 80-86.

434

22. Wang, L. H.; Zhang, Z. H.; Zeng, X. A.; Gong, D. M.; Wang, M. S. Combination

435

of microbiological, spectroscopic and molecular docking techniques to study the

436

antibacterial mechanism of thymol against Staphylococcus aureus: membrane

437

damage and genomic DNA binding. Anal. Bioanal. Chem. 2017, 409, 1615-1625.

438

23. Shen, Q.; Zhou, W.; Li, H.; Hu, L.; Mo, H. ROS Involves the fungicidal actions

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

439

of thymol against spores of Aspergillus flavus via the induction of nitric oxide. PLoS

440

One 2016, 11, e0155647.

441

24. Yang, L.; Chen, J.; He, L. Harvesting candidate genes responsible for serious

442

adverse drug reactions from a chemical-protein interactome. PLoS Comput. Biol.

443

2009, 5, e1000441.

444

25. Luo, H.; Chen, J.; Shi, L.; Mikailov, M.; Zhu, H.; Wang, K.; He, L.; Yang, L.

445

DRAR-CPI: a server for identifying drug repositioning potential and adverse drug

446

reactions via the chemical-protein interactome. Nucleic. Acids. Res. 2011, 39,

447

W492-8.

448

26. Yang, J.; Yan, R.; Roy, A.; Xu, D.; Poisson, J.; Zhang, Y. The I-TASSER Suite:

449

protein structure and function prediction. Nat. Methods. 2015, 12, 7-8.

450

27. Morris, G. M.; Huey, R.; Lindstrom, W.; Sanner, M. F.; Belew, R. K.; Goodsell,

451

D. S.; Olson, A. J. AutoDock4 and AutoDockTools4: Automated docking with

452

selective receptor flexibility. J. Comput. Chem. 2009, 30, 2785-2791.

453

28. Weng, J.; Cao, Y.; Moss, N.; Zhou, M. Modulation of voltage-dependent Shaker

454

family potassium channels by an aldo-keto reductase. J. Biol. Chem. 2006, 281,

455

15194-15200.

456

29. Woloshuk, C. P.; Seip, E. R.; Payne, G. A.; Adkins, C. R. Genetic transformation

457

system for the aflatoxin-producing fungus Aspergillus flavus. Appl. Environ.

458

Microbiol. 1989, 55, 86-90.

459

30. Wyllie, A. H. Glucocorticoid-induced thymocyte apoptosis is associated with

460

endogenous endonuclease activation. Nature 1980, 284, 555-556.

ACS Paragon Plus Environment

Page 22 of 32

Page 23 of 32

Journal of Agricultural and Food Chemistry

461

31. Carmody, R. J.; Cotter, T. G. Signalling apoptosis: a radical approach. Redox.

462

Rep. 2001, 6, 77-90.

463

32. Ito, S.; Ihara, T.; Tamura, H.; Tanaka, S.; Ikeda, T.; Kajihara, H.; Dissanayake,

464

C.; Abdel-Motaal, F. F.; El-Sayed, M. A. alpha-Tomatine, the major saponin in

465

tomato, induces programmed cell death mediated by reactive oxygen species in the

466

fungal pathogen Fusarium oxysporum. FEBS Lett. 2007, 581, 3217-3722.

467

33. Sharon, A.; Finkelstein, A.; Shlezinger, N.; Hatam, I. Fungal apoptosis: function,

468

genes and gene function. FEMS. Microbiol. Rev. 2009, 33, 833-854.

469

34. D'Sa-Eipper, C.; Leonard, J. R.; Putcha, G.; Zheng, T. S.; Flavell, R. A.; Rakic,

470

P.; Kuida, K.; Roth, K. A. DNA damage-induced neural precursor cell apoptosis

471

requires p53 and caspase 9 but neither Bax nor caspase 3. Development (Cambridge,

472

England) 2001, 128, 137-146.

473

35. Ramsdale, M. Programmed cell death in pathogenic fungi. Biochim.Biophys.

474

Acta. 2008, 1783, 1369-1380.

475

36. Wulff, H.; Castle, N. A.; Pardo, L. A. Voltage-gated potassium channels as

476

therapeutic targets. Nat. Rev. Drug Discov. 2009, 8, 982-1001.

477

37. Adhikari, A.; Sen, M. M.; Gupta-Bhattacharya, S.; Chanda, S. Volumetric

478

assessment of airborne fungi in two sections of a rural indoor dairy cattle shed.

479

Environ. Int. 2004, 29, 1071-1078.

480

38. Hagiwara, D.; Sakamoto, K.; Abe, K.; Gomi, K. Signaling pathways for stress

481

responses and adaptation in Aspergillus species: stress biology in the post-genomic

482

era. Biosci. Biotech.Bioch. 2016, 80, 1667-1680.

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

483

39. Hagiwara, D.; Sakai, K.; Suzuki, S.; Umemura, M.; Nogawa, T.; Kato, N.;

484

Osada, H.; Watanabe, A.; Kawamoto, S.; Gonoi, T.; Kamei, K. Temperature during

485

conidiation affects stress tolerance, pigmentation, and trypacidin accumulation in the

486

conidia of the airborne pathogen Aspergillus fumigatus. PLoS One 2017, 12,

487

e0177050.

488

40. Qian, P.; Li, H.; Zhao, Y.; Zhou, W.; Mo, M.; Hu, L. Double function yeast cell

489

wall extract and its capability of adsorbing aflatoxin B1. Sci. Tech. Food. Ind. 2017,

490

25-29. (In Chinese)

491

41. Tian, J.; Wang, Y.; Lu, Z.; Sun, C.; Zhang, M.; Zhu, A.; Peng, X. Perillaldehyde,

492

a promising antifungal agent used in food preservation, triggers apoptosis through a

493

metacaspase-dependent pathway in Aspergillus flavus. J Agr. Food. Chem. 2016, 64,

494

7404-7413.

495

42. Pan, Y.; Weng, J.; Kabaleeswaran, V.; Li, H.; Cao, Y.; Bhosle, R. C.; Zhou, M.

496

Cortisone dissociates the Shaker family K+ channels from their beta subunits. Nat.

497

Chem. Biol. 2008, 4, 708-714.

498

43. Redman, P. T.; Hartnett, K. A.; Aras, M. A.; Levitan, E. S.; Aizenman, E.

499

Regulation of apoptotic potassium currents by coordinated zinc-dependent signalling.

500

J. Physiol. 2009, 587, 4393-4404.

501

44. Ortar, G.; Morera, L.; Moriello, A. S.; Morera, E.; Nalli, M.; Di, M. V.; De, P. L.,

502

Modulation of thermo-transient receptor potential (thermo-TRP) channels by

503

thymol-based compounds. Bioorg.Med. Chem. Lett. 2012, 22, 3535-3539.

504

45. Heinemann, S. H.; Hoshi, T. Multifunctional potassium channels: electrical

ACS Paragon Plus Environment

Page 24 of 32

Page 25 of 32

Journal of Agricultural and Food Chemistry

505

switches and redox enzymes, all in one. Science's STKE 2006, 2006, pe33.

506

46. Orrenius, S., Reactive oxygen species in mitochondria-mediated cell death.

507

Drug. Metab. Rev. 2007, 39, 443-455.

508 509

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

510

511 512

Figure 1. Conidial apoptosis in A. flavus induced by thymol. (A) and (B) Histograms

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of flow cytometry of thymol-exposed conidia stained by PI and Hoechst 33342,

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respectively; (C) Percentage of apoptotic conidia by counting cells with

515

Hoechst-positive and PI negative staining; (D) PS externalization in conidia exposed

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to thymol for 6h detected through flow cytometry with an Annexin V-FITC assay; (E)

517

DNA damage in conidia exposed to thymol for 6h detected by TUNEL staining

518

through flow cytometry; (F) Mitochondrial potential in conidia with or without the

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exposure to thymol for 6h was evaluated by the calculating ratio of green

520

fluorescence intensity (529 nm) to red fluorescence (590 nm) intensity as determined

521

by a microreader. Each data bar was indicated as the means of three replicates ±

522

standard deviation. Different letter indicated a significant difference between them (P

523