Thymol Induces Conidial Apoptosis inAspergillus flavus via

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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)

ACS Paragon Plus Environment


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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

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

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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

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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)

15

could significantly alleviate thymol-mediated conidial apoptosis, indicating that

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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

26

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

27

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

31

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

32

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

34

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

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

36

etc. Mechanisms underlying its antimicrobial activity has been extensively studied

37

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

38

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

40

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

45

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

50

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%

52

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

53

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

55

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

56

analytical grade.

57

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

59

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

60

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.

62

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

63

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

68

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).

70

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

71

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

74

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

75

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

77

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

78

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

79

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

81

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

82

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

85

(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

87

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

89

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

93

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

94

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

95

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

100

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

101

structures of these homologues were obtained through homology modeling using

102

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

103

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

104

interactions between these homologues and thymol were modelled by Autodock 4.2

105

27

106

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

107

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

108

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

109

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

114

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

115

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

116

KCNAB was mostly expressed in inclusion bodies, debris containing inclusion

117

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

118

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

119

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

123

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

124

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

125

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

126

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

127

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

128

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



132

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

137

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

139

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

143

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

145

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

147

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,

149

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

150

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

151

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

152

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

157

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

160

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

170

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

171

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

172

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

173

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

174

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

175

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



176

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

177

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

178

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

180

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

184

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

187

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

194

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

195

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

196

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

197

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

203

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

204

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

207

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

208

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

209

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

213

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

215

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

216

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

217

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

218

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

219

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



220

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

221

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

222

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

223

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

224

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

225

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

226

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

227

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

228

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

229

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

230

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

231

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

232

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

234

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

235

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

236

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

237

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

238

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

240

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

241

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

244

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

245

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

246

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

247

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

248

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

249

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

250

leading to apoptosis.

251

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

252

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

253

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

254

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

255

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

256

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

257

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

258

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

259

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

260

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

261

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

262

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

263

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



264

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

265

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

266

(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

268

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

269

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

270

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

271

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

272

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

273

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

274

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

275

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

276

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

277

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

278

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

279

(Figure S6).

280

DISCUSSION

281

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

282

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

283

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

284

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

285

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

288

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

289

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

290

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

291

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

292

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

293

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

295

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

296

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

299

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

300

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

301

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

302

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

303

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.



308

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

309

of the purified recombinant protein KCNAB in vitro, demonstrating that

310

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+

315

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

318

Kv system containing KCNAB was the endogenous receptor targeted by thymol

319

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

320

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

321

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

322

potential of thymol as a therapeutic agent.

323

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

324

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

325

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

326

through the cell membrane. Electrophysiological techniques based on

327

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

331

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

332

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

333

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

334

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

335

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

336

implicated in mitochondrial dysfunction, which leads to the release of

337

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

338

interesting that blocking ROS production with the inhibitors of NADPH oxidase

339

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

340

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

341

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

342

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

343

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

344

23

345

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

346

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

347

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

348

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

349

apoptotic pathway.

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

350 351

ACKNOWLEDGEMENTS



352

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

353

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

354

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

355

for Science & Technology Innovation Team in Universities of Henan Province

356

(16IRTSTHN007).

357

Supporting Information

358

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

359

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

360

(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

362

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

363

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

364

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

365

(Predication of acceptors targeted by thymol leading to apoptosis through

366

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

367

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

368

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

369

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

370

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

371

http://pubs.acs.org

372


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

513

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

514

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

515

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

516

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

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

518

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519

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

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

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

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Thymol Induces Conidial Apoptosis inAspergillus flavus via

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