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Membrane Destruction and DNA Binding of Staphylococcus aureus Cells Induced by Carvacrol, and its Combined Effect with Pulsed Electric Field Langhong Wang, man sheng wang, Xin-An Zeng, Zhihong Zhang, Deming Gong, and Yanbo Huang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b02507 • Publication Date (Web): 15 Jul 2016 Downloaded from http://pubs.acs.org on July 18, 2016

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Journal of Agricultural and Food Chemistry

Carvacrol exerted its antibacterial effect by increasing the permeability of the bacterial cell membrane and binding directly to genomic DNA. 47x26mm (300 x 300 DPI)

ACS Paragon Plus Environment


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Membrane Destruction and DNA Binding of Staphylococcus

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aureus Cells Induced by Carvacrol, and its Combined Effect

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with Pulsed Electric Field

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Lang-Hong Wangab, Man-Sheng Wangab, Xin-An Zengab*, Zhi-Hong Zhangab,

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De-Ming Gongc, and Yan-Bo Huanga

a

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School of Food Science and Engineering, b Food Green Processing and Nutrition

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Regulation Research Center of Guangdong Province, South China University of

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Technology, Guangzhou 510641, China c

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School of Biological Sciences, The University of Auckland, Auckland 1142, New Zealand

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Running title: Carvacrol-induced Membrane Destruction and DNA Binding in

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Staphylococcus aureus

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________________________

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*

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[email protected] (Professor Xin-An Zeng, PhD).

Corresponding author. Tel: +86 2087113668, Fax: +86-2039381191. E-mail address:

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ABSTRACT

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Carvacrol (5-isopropyl-2-methylphenol, CAR) is an antibacterial ingredient that

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occurs naturally in the leaves of the plant Origanum vulgare. The antimicrobial

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mechanism of CAR against Staphylococcus aureus ATCC 43300 was investigated in

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the study. Analysis of the membrane fatty acids by gas chromatography-mass

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spectrometry (GC-MS) showed that exposure to CAR at low concentrations induced a

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marked increase in the level of unbranched fatty acids (from 34.90 ± 1.77% to 62.37 ±

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4.26%). Moreover, CAR at higher levels severely damaged the integrity and

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morphologies of S. aureus cell membrane. The DNA binding properties of CAR were

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also investigated using fluorescence, circular dichroism, molecular modeling and

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atomic force microscopy. The results showed that CAR bound to DNA via minor

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groove mode, mildly perturbed the DNA secondary structure, and induced DNA

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molecules to be aggregated. Furthermore, a combination of CAR with pulsed electric

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field was found to exhibit strong synergistic effects on S. aureus.

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Keywords: Staphylococcus aureus; Gas chromatography-mass spectrometry; DNA

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binding; Molecular modeling; Synergistic effect

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INTRODUCTION

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Food spoilage is one of the major problems in food storage and transportation, posing

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serious risks to people’s health.

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pathogenic and spoilage microorganisms in food, including thermal treatment,

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dehydration and use of chemical preservatives. However, safety concern about the use

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of chemical preservatives has attracted widespread attention as they are potentially

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toxic to human health. (2) For these reasons, ‘green’ or natural alternative means for

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the treatment of microbial contamination in foods has been increasingly explored in

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recent years. Carvacrol (5-isopropyl-2-methylphenol, CAR) is a natural antimicrobial

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component in the leaves of the plant Origanum vulgare. (3) It was reported that CAR

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enhanced flavor and shelf-life, (4) and prevented the growth of foodborne pathogens

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and spoilage microorganisms.(5-8) Importantly, CAR was generally safe and can be

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directly added to human food as a food additive. (3) Previous studies found that the

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antimicrobial action of CAR may be through disruption of structure and function of

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the cytoplasmic membrane.

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antimicrobial substance might have additional intracellular targets. (11) However, the

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underlying mechanisms of the antibacterial action of CAR are still not fully

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understood, and thus additional research is necessary before CAR can be well used to

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control foodborne pathogens and spoilage microorganisms in human food.

(9, 10)

(1)

There are many strategies for preventing

Recent findings have indicated that this natural

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As a rapidly developing non-thermal technique, the pulsed electric field (PEF) can

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effectively inactivate bacterial and quality-degrading enzymes in liquid foods within a

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few seconds. (12) In addition, PEF used a much lower temperature than conventional

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thermal pasteurisation, avoiding additional food quality losses and retaining the colour

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and flavour of products. (13) Generally, two strategies can be explored to enhance the

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inactivation efficiency obtained by PEF technology: 1) Increasing PEF treatment

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duration or electric field intensity, which would increase energy costs;

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Combination of PEF with non-thermal technologies, mild temperature and

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antibacterial substances, in accordance with the “hurdle technology” concept. (15, 16)

(14)

2)

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The aim of the study was to investigate the antimicrobial mechanisms of CAR,

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including the alteration of cell membrane fatty acid composition, the permeability and

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damage of CAR on cells membrane and morphology of Staphylococcus aureus ATCC

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43300 (S. aureus). Moreover, the binding properties of CAR to genomic DNA of S.

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aureus cells were also investigated to explore the possible intracellular targeting

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behaviors. Considering the growing interest in the application of PEF in the food

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industry, the synergetic sterilization effect of CAR with PEF was also determined.

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This study is expected to provide a comprehensive view of the antimicrobial

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mechanism of CAR on S. aureus.

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MATERIALS AND METHODS

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Chemicals. CAR (purity, 99.0%) was purchased from Sigma–Aldrich Co. (St.

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Louis, MO, USA). A stock solution of CAR (0.12 M) was prepared in ethanol under

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sterile conditions, and stored at 4 °C. O-nitrophenyl-β-d-galactoside (ONPG, purity ≥

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99%) was obtained from Sigma–Aldrich Co. (St. Louis, MO, USA). A 23.0 mM stock

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solution of ONPG was made in potassium phosphate buffer (pH 7.2, 0.01 M).

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Strains and Growth Conditions. Gram-positive Staphylococcus aureus ATCC 4



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43300 (S. aureus) was obtained from the Microbiology Laboratory, South China

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University of Technology (Guangzhou, China). The strain was stored at −80 °C in

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tryptic soy broth containing 30% (v/v) of glycerol (TSB, Huankai Microbial Sci. &

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Tech. Co., Guangzhou, China) and activated by inoculating with a loopful of

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inoculum onto a plate containing tryptone soy agar supplemented with 0.6% yeast

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extract (TSA-YE) and incubated at 37 °C for 18 to 24 h. A loopful of a single colony

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was then transferred to a 1000 mL conical flask containing 200 mL of sterile tryptic

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soy broth with 0.6% of yeast extract (TSB-YE), and incubated on an orbital shaker

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(120 rpm; HY-5, JinBo Equipment Industry Co., Jiangsu, China) at 37 °C for 12 h.

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Then, the pre-cultured S. aureus cells were transferred to a fresh TSA-YE liquid

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medium (100 mL) in which the initial cell density was determined to be

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approximately 0.15 (OD600) by a Shimadzu UV-1800 spectrophotometer (Shimadzu,

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Japan). The minimum inhibitory concentration (MIC) of CAR toward S. aureus was

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2.06 mM (0.31 g/L). The cells were cultivated at 37 °C on a rotary shaker at 120 rpm

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with CAR at different concentrations (0, 0.26, 0.52 and 0.77 mM) until the

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mid-stationary phase was achieved, and the optical density of culture was about 1.60.

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These cultures were then centrifuged at 4000 × g for 5 min at 4 °C in a refrigerated

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centrifuge (JW-3021HR, Anhui Jiaven Equipment Industry Co., Anqing, China). The

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resulting pellets were washed twice with sterile water and prepared for the analysis of

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their membrane fatty acid composition.

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Analysis of Membrane Fatty Acid Composition. The extraction and methylation

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of cell membrane fatty acids were simply described in the following way: (17, 18) (i)

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approximately 40 mg of fresh pellets were treated with 1.0 mL of saponification

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solution (4.5 g NaOH, 15.0 mL methanol and 15.0 mL distilled water), and mixed

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thoroughly for 30 min while immersed in boiling water bath; (ii) after cooling down to

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room temperature, 2.0 mL of methylation solution (32.5 mL of 6.0 M HCl and 27.5

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mL of methanol) were added, followed by incubation for 10 min at 80 °C; (iii) The

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resulting suspension was mixed with 1.25 mL of extraction solution (hexane and

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methyl-tert-butyl ether, v/v = 1:1), and the aqueous phase (lower) was discarded; (iv)

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3.0 mL of 0.3 M NaOH were added, and two-thirds of the organic phase was pipetted

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into a vial for GC-MS analysis.

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Analysis of fatty acid methyl esters was performed on a gas chromatograph-mass

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spectrometer (Agilent 7820A gas chromatograph-Agilent MS-5975C, Agilent

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Technologies, Palo Alto, CA, USA) equipped with an capillary column HP-5MS (5%

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phenyl methylsiloxane) (30 m × 0.25 mm i.d × 0.25 µm film thickness; Agilent

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Technologies, Palo Alto, CA, USA) as a previous report.

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identified by comparing the mass spectra with the National Institute of Standards and

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Technology mass spectral library 2011, involving a similitude index (SI ≥ 90), and

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comparing their retention time with a standard mixture of bacterial acid methyl esters

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(Sigma-Supelco, Bellefonte, PA, USA).

(18)

Fatty acids were

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Cell Membrane Permeability Assay. The permeability of S. aureus cell

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membranes was determined by measuring the amount of cytoplasmic β-galactosidase

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released. (19) Briefly, S. aureus cells were cultured in M9 lactose medium at 37 °C for

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12 h. After centrifugation at 4,000 × g for 5 min, bacterial cells were collected,

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washed twice with sterile saline and resuspended to an OD600 of 0.30. Then, 1.0 mL of

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23.0 mM ONPG was added to the bacterial suspension. Subsequently, these samples

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were treated with CAR at different concentrations (0, 1.03, 2.06, 4.12 and 8.24 mM)

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and incubated at 37 °C. The production of o-nitrophenol over time was monitored

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using a Shimadzu UV-1800 spectrophotometer at 420 nm. The ethanol contents of

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samples were kept at equal concentrations to eliminate the possible influence of

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ethanol.

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Scanning Electron Microscopy. Morphology of the S. aureus cells was observed

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using a scanning electron microscope (SEM, Zeiss EVO18, Germany) operating at

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10.0 kV, based on a previously reported method.

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glutaraldehyde (2.5% in 0.01 M phosphate buffer, pH 7.2) overnight at 4 °C, the

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untreated and CAR-treated cell samples were dehydrated using a gradient 30–100% of

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ethanol solution (30 min each time) and incubated in tertiary-butanol twice for 30 min.

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The resulting samples were placed on a silicon wafer and subjected to vacuum

149

freeze-drying. Finally, the dried samples were mounted on aluminum stubs with

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sticky double-side conductive metal tape, and gold-coated by ion sputtering (Jeol

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JFC1100, Japan) for approximately 2 min.

(18)

After being fixed in

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Interaction between CAR and DNA. The interaction between CAR and genomic

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DNA of S. aureus was investigated by fluorescence and circular dichroism (CD)

154

measurements. S. aureus cells at mid-stationary phase were treated with lysozyme

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(purity ≥ 98%, Sigma-Aldrich, USA) to remove the cell wall peptidoglycan, and then

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used to extract genomic DNA by a GenElute™ bacterial genomic DNA kit

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(Sigma-Aldrich, USA). The extracted genomic DNA was free of proteins as the ratio

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of A260/A280 > 1.80. The extracted DNA was dissolved in a TE solution (10 mM

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Tris-HCl, 0.1 mM EDTA, pH 8.0) at a final concentration of 2.55 mM, as measured

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by UV absorption at 260 nm using a molar absorption coefficient ε260 = 6600 M−1

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cm−1.

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spectrofluorimeter equipped with a 150 W xenon lamp and a thermostat bath. A CAR

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solution (3.0 mL, 32.0 µM) was added to a 1.0 cm quartz cell. The DNA solution was

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then gradually titrated to the quartz cell using a micropipette and allowed to stand for

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5 min to equilibrate. The CD spectra of DNA in the absence and presence of CAR at

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different concentrations were measured on a Bio-Logic MOS 450 CD spectrometer

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(Bio-Logic, Claix, France) in pH 8.0 TE buffer under a nitrogen atmosphere. All

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observed CD spectra were the average of three scans recorded at a speed of 120 nm

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min−1 and corrected for buffer signal.

(20)

All fluorescence spectra were obtained on a Hitachi Model F–7000

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Molecular Modeling. The molecular docking studies were carried out by

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AutoGrid4 and AutoDock4 with the aid of the MGL tools 1.5.6rc3 according to a

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previous report.

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Protein Data Bank (ID: 4LLN), and polar hydrogen atoms and Gasteiger charges were

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added to the macromolecule file in the preparation for the docking. The structure of

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CAR was generated by Sybyl-x 2.0 and subsequently optimized to minimal energy

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with the aid of the MMFF94 force field using MMFF94 charges. (22) To determine the

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preferred binding sites in each DNA sequence, the CAR molecule was allowed to

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move over the whole region of DNA with 100 runs to obtain all the possible binding

(21)

The crystal structure of the B-DNA was downloaded from the

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positions, and the binding mode of the CAR–DNA complex with the lowest energy

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was displayed.

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Atomic Force Microscopy (AFM) Measurements. AFM measurements were

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carried out in tapping mode using a Multimode 8 SPM AFM (Bruker, Karlsruhe,

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Germany) with SCANASYST-AIR probes having a nominal spring constant and tip

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radius of 0.4 N m−1 and 2 nm, respectively. In order to assure comparability between

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height determinations, the peak force set point was maintained at a constant value of

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2.5 nN for all samples. A 10 µL mixed solution containing 0.5 mM DNA in the

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absence and presence of CAR was incubated for 3.0 h at room temperature, and then 5

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µL samples were dropped onto a freshly cleaved mica surface and dried overnight.

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Images were processed with the Nanoscope Analysis v150r1sr3 software package and

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are presented unfiltered.

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Effects of a Combination of CAR with PEF. The following treatments were

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performed to establish whether synergy between CAR and PEF treatment occurred

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during and/or after the PEF treatment: (a) CAR without PEF treatment; (b) PEF

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treatment without CAR; (c) CAR during PEF treatment; (d) CAR after PEF treatment

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in recovery agar; (f) CAR during and after PEF treatment. S. aureus cells were

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exposed to PEF using a bench-scale continuous PEF system (SCUT PEF Team, South

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China University of Technology, China) using a bipolar square wave with different

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PEF strengths (0, 10.0, 20.0 and 30.0 kV/cm) as reported earlier. (23, 24) The conditions

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of PEF inactivation process were as the following: electrode distance, 0.30 cm;

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chamber volume, 0.02 mL; sample flow rate, 13.1 mL/min; the pulse frequency, 1.0

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kHz; pulse width, 40 µs. Before each treatment, the PEF system was disinfected with

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75% (v/v) ethanol and then rinsed with sterile distilled water. The temperature of the

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samples was maintained at below 30 °C in order to avoid thermal effects. After PEF

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or mock treatment, the samples were immediately plated on two recovery agars

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containing either no CAR or CAR at a specific concentration (i.e., 0.26 mM, 0.52 mM,

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or 0.77 mM). Each treatment was performed in triplicate, and all samples were

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examined to determine the total number of viable cells. The effects of a combination

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of CAR with PEF were determined from the relationship:

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−log ( N1 / N0 )PEF-CAR = −log ( N1 / N0 )PEF×CAR − [−log ( N1 / N0 )CAR − log ( N1 / N0 )PEF ]

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where N1 is the number of viable cells after treatment (CFU/mL), N0 is the number of

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cells before treatment. The treatment was considered synergistic if the net log

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reduction [−log(N1/N0)PEF-CAR] was > 0; additive, if = 0; and antagonistic, if < 0.

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Statistical Analysis. Results were expressed as means ± SD. Data were analysed

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using OriginPro 8.0 (OriginLab, Northampton, MA, USA). Differences were

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considered statistically significant at p < 0.05. Principal component analysis (PCA)

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was conducted to resolve the GC-MS data, using the program Unscrambler® 10.1

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(CAMO AS, Oslo, Norway) via mean centering and scaling-to-unit-variance methods

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(25)

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cells and the differences between them.

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RESULTS AND DISCUSSION

, and obtain an overview of fatty acids from untreated and CAR-treated S. aureus

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PCA of Cell Fatty Acid Data. A total of 14 fatty acids were identified in the S.

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aureus cell membrane by GC-MS. To investigate which fatty acids were mainly 10



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responsible for discrimination between the control and CAR-treated groups, PCA

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analysis, an unsupervised clustering method, was used to process the GC-MS data.

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The scores plot was generated to show the clustering of cell membrane samples

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according to their fatty acid composition, while the corresponding loading plot was

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produced to identify fatty acids that had a significant influence on the separation or

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clustering of data. As shown in Figure 1, the first two principal components accounted

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for 98.0% of the total variance in S. aureus cell membrane, distributed between PC1

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(94.0%) and PC2 (4.0%) positions. Distinct clustering was found in different groups,

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except the control and ethanol group (Figure 1A), suggesting that the exposure of S.

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aureus cells to CAR may lead to systematic changes in the membrane fatty acid

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composition of this bacterium, while ethanol (0.64%) had no significant effect on the

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membrane fatty acid composition compared to the control cells.

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In the PCA model, the contribution of each fatty acid to a specific component is

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reflected in the loading value in the loading plot. The fatty acids with the highest

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loading values account for the biggest differences among cells grown at different

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concentration of CAR. As shown in Figure 1B, three variables, namely

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13-methyltetradecanoic acid (iso C15:0), 12-methyltetradecanoic acid (anteiso C15:0)

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and 14-methylhexadecanoic acid (anteiso C17:0), had positive loading values on PC1,

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implying that their contents in cell membrane were considerably high after S. aureus

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cells were cultured in the absence or presence of CAR at low concentration (0.26

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mM). In contrast, hexadecanoic acid (C16:0) was on the negative side of both PC1

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and PC2, which may reflect a high content in S. aureus cells exposed to CAR at the

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highest concentration (0.77 mM). Stearic acid (C18:0) was distributed on positive side

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of PC2. This observation may be because there was a high proportion of this kind of

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fatty acid in the cell membrane of S. aureus cultivated in 0.52 mM CAR. These five

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fatty acids were correlated in PC1 and/or PC2 space, suggesting that they were the

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best markers for differentiating CAR-treated cells from the control.

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Effect of CAR on Fatty Acid Profiles of S. aureus. The effects of CAR on

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membrane fatty acids of S. aureus cells were investigated by using GC-MS analysis.

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A total of 14 fatty acids, including lauric acid (C12:0), 12-methyltridecanoic acid (iso

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C14:0), 11-methyltridecanoic acid (anteiso C14:0), tetradecanoic acid (C14:0),

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13-methyltetradecanoic acid (iso C15:0), 12-methyltetradecanoic acid (anteiso C15:0),

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hexadecanoic

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14-methylhexadecanoic acid (anteiso C17:0), oleic acid (C18:1ω9), stearic acid

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(C18:0), cis-9,10-Methyleneoctadecanoic acid (C19:0△9,10), nonadecanoic acid (C19:0)

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and eicosanoic acid (C20:0) were identified (Figure 2A and B). Branched 15 and 17

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carbon in these fatty acids accounted for a high proportion. These findings were

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consistent with an earlier study showing that the major fatty acids in S. aureus species

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were 15 and 17 carbon molecules with methyl branches located on either the third or

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second carbon from the terminal methyl group (anteiso or iso) at the methyl end of the

263

alkyl chain. (26)

acid

(C16:0),

15-methylhexadecanoic

acid

(iso

C17:0),

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The fatty acids of S. aureus can be divided into two groups; i.e., branched-chain

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fatty acids (BCFAs) and unbranched fatty acids (UBFAs). The BCFAs constituted

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59.04% of the total fatty acids in the cells grown in the absence of CAR (Figure 2C).

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This figure decreased to 32.89% as S. aureus was grown in increasing concentrations

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of CAR (from 0 to 0.77 mM). Most of this decrease was a result of the decrease in the

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level of branched iso C15:0 and anteiso C15:0. For example, the contents of branched

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iso C15:0 and anteiso C15:0 decreased from 8.39% and 36.61% to 6.62% and 33.10%,

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respectively, when S. aureus cells were exposed to CAR at 0.26 mM. Their

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proportions continued to decrease (to 2.65% and 23.34%, respectively) when CAR

273

concentration rose to 0.77 mM. In contrast, the content of UBFAs, consisting mainly

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of lauric acid (C12:0), tetradecanoic acid (C14:0), hexadecanoic acid (C16:0), stearic

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acid (C18:0), nonadecanoic acid (C19:0) and eicosanoic acid (C20:0) increased

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significantly from 34.90% in the absence of CAR to 62.37% in the presence of 0.77

277

mM CAR.

278

The proportion of UBFAs to BCFAs increased with an increase in CAR

279

concentration (Figure 2D). Maintenance of a proper proportion of saturated UBFAs to

280

BCFAs and/or unsaturated fatty acids within the microbial membrane is often

281

identified as an adaptive response to environmental disturbances.

282

studies have shown that exposure to higher temperatures, acidic extremes or certain

283

chemicals can fluidize or solidify the membrane by alterring the fatty acid

284

composition. For instance, membranes of Listeria monocytogenes cells were found to

285

be less fluid as a result of an increased level of UBFAs that reduced the ability of

286

weak acid preservatives to pass through the membrane. (29) An increase in the level of

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saturated UBFAs in the membranes of Escherichia coli cells was observed after

288

addition of phenol to the growth medium.

(30)

(27, 28)

Previous

It was also reported that some organic

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compounds, including chain alcohols and phenols can pass through the cell wall and

290

accumulate in the cytoplasmic membrane. (31) An increase in the level of saturated

291

UBFAs can help not only compensate for the fluidizing effects of phenols but also

292

block access of these molecules to the inner membrane due to the greater packing

293

efficiency of the saturated straight chains. (27) Our results were in good agreement with

294

these earlier studies, suggesting that alterations in membrane fatty acid composition

295

might contribute to the self-protection of S. aureus cells by maintaining the normal

296

membrane structure and function in effectively response to CAR-induced toxic stress.

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S. aureus Cell Membrane Integrity. The endoenzyme, β-galactosidase, is often

298

used as an indicator of cell membrane damage because its activity can be determined

299

by measuring the level of o-nitrophenol after leaking through damaged cell membrane.

300

(18)

301

concentrations are shown in Figure 3. The absorbance at 420 nm increased with an

302

increase in the concentration of CAR or incubation time, indicating that the CAR

303

evoked a release of the intracellular components, including β-galactosidase into the

304

culture medium. This observation was consistent with earlier studies. (32, 33)

The permeabilities of inner membranes of S. aureus cells exposed to various CAR

305

Morphological Changes. As shown in Figure 4A, the untreated S. aureus cells had

306

a normal smooth surface. However, after the treatment with CAR at concentrations of

307

1.03, 2.06 and 4.12 mM for 4 h, the cells became rough and wrinkled, with depression

308

appearing on their surfaces. Specifically, Some S. aureus treated with 1.03 mM CAR

309

became rough and crumpled in shape (Figure 4B). The cells treated with 2.06 and

310

4.12 mM CAR showed further distorted, with some of them even collapsed (Figure

14



311

4C and 4D). These observations showed that CAR treatment affected the integrity of S.

312

aureus cell membrane, which likely resulted in a decrease in cell viability. (34) Together

313

with β-galactosidase release study, these findings also suggest that CAR may

314

damage the cell membranes of S. aureus in a concentration-dependent manner.

315

Interaction between CAR and Genomic DNA of S. aureus Cells. Besides

316

affecting the cell membrane, CAR may affact the biological functions of intracellular

317

biomacromolecules, such as DNA.

318

intracellular DNA, the interaction between CAR and genomic DNA of S. aureus cells

319

was studied by fluorescence and circular dichroism (CD) techniques. As shown in

320

Figure 5A, the fluorescence emission peak of CAR at 304 nm decreased gradually

321

without any apparent shift in wavelength with the increasing amounts of DNA,

322

indicating that CAR can bind to DNA. (20) Modified Stern−Volmer equation was then

323

used to determine the binding constant (K): (20)

324

(19, 35)

To determine if CAR directly affects

F0 1 1 1 = + F0 − F f a K [Q ] f a

325

where F0 and F are the fluorescence intensities of CAR in the absence and presence of

326

DNA; fa and [Q] are the fraction of accessible fluorescence and the concentration of

327

DNA, respectively. The ratio of intercept to the slope of 1/fa versus 1/faK (Figure 5A

328

inset) gave a binding constant, K, which was estimated to be (1.42 ± 0.09) × 104 M−1

329

(R2 = 0.9980). The K value obtained here (in the order of 104 M−1) was lower than that

330

reported for classical intercalators, such as ethidium bromide (2.6 × 106 M−1) and

331

acridine orange (4.0 × 105 M−1)

332

implying that CAR may bind DNA via groove binding.

(36)

, but similar to the groove binders of DNA, (37)

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CD spectroscopy is an effective way to study the properties of binding between

334

small molecules and DNA and corresponding DNA conformational change. (38) As

335

shown in Figure 5B, the CD spectra of DNA exhibited a negative band at 246 nm due

336

to the right-handed helicity and a positive band at 275 nm due to base stacking,

337

indicating that the genomic DNA of S. aureus has a typical B-DNA structure. (39) It

338

was reported that simple groove binding of small molecules to DNA shows less or no

339

perturbation in the base stacking and helicity bands while intercalators alter the

340

intensities of both bands.

341

negative band at 246 nm shifted slightly towards shorter wavelengths, with a mild

342

decrease in intensity, and inconspicuous change was observed at the positive band.

343

This type of change in the CD spectra has been regarded as an indicator of a groove

344

binding mode in the case of small ligand binding to DNA. (40) Interaction of ligands

345

with DNA may affect cell function by interfering gene expression and protein

346

synthesis, thereby playing an important role in antibacterial action of some ligands

347

when they alter DNA function. In short, at least some of the antibacterial actions of

348

CAR may be related to its ability of binding to DNA here.

(39, 40)

In the presence of CAR (1.0 and 2.0 mM), the

349

Computational Modeling of the CAR–DNA Interaction. After the 100 docking

350

runs were successfully performed, a total of 22 multimember conformational clusters

351

were formed. As shown in Figure 6A, the highest populated cluster (marked in blue)

352

contained 14 of the 100 conformations, and possessed the lowest binding energy

353

(−4.62 kcal M−1). Thus, this predicted binding model was selected for binding

354

position analysis. The result showed that CAR entered into the DNA minor groove

16



355

and formed two hydrogen bonds (green dashed lines). One hydrogen bond was

356

generated between the hydrogen atom H21 associated with N2 of G6 and the oxygen

357

atom O12 of CAR, with a bond length of 1.748 Å, and the other hydrogen bond was

358

formed with a bond length of 2.05 Å between the oxygen atom O4’ associated with

359

deoxyribose of T7 on chain B and hydrogen atom H13 of CAR (Figure 6B). The

360

docking results clearly showed that CAR binding in the minor groove of DNA, and

361

the hydrogen bonds may play an important role in the interaction between CAR and

362

DNA.

363

Surface Morphological Changes in DNA. As shown in Figure 7A, the free DNA

364

molecules were adsorbed evenly on the mica surface, and its AFM morphological

365

images showed smooth lines with good distribution and had no marked cross-linking.

366

The mean height of the individual DNA molecule was determined to be

367

approximately 1.5 nm (Figure 7B). DNA molecules appeared to be kinked and

368

cross-linked after complexation with CAR (2.0 mM) (Figure 7C), where the mean

369

height of the individual DNA molecule increased to 2.5 nm (Figure 7D). These

370

findings along with the CD results suggested that CAR interacted with DNA in

371

solution, slightly perturbed the DNA secondary structure, and even induced DNA

372

molecules to be aggregated here.

373

Effect of a Combination of CAR with PEF. The effects of a combination of CAR

374

with PEF on S. aureus was assessed using different electric field intensities (0, 10, 20

375

and 30 kV/cm) and CAR concentrations (0, 0.26, 0.52 and 0.77 mM). As shown in

376

Tables 1 and 2, the combination of CAR with PEF produced a synergistic effect

17


Page 18 of 37

Page 19 of 37


377

against S. aureus, especially when the concentrations of CAR or field intensities were

378

relatively high. For example, when CAR was added at a concentration of 0.52 mM

379

and the field intensity was 20.0 kV/cm, the log reduction values of S. aureus were

380

0.96 and 1.23, respectively (recovery condition), and so −log(N1/N0)CAR plus

381

−log(N1/N0)PEF was 2.19. However, when CAR and PEF were combined, the observed

382

log

383

−log(N1/N0)CAR-PEF = 0.37 > 0, suggesting that CAR and PEF had a synergistic effect

384

on the inactivation of S. aureus. The cell membrane was reported to be a critical target

385

in PEF inactivation, and that this technique can reduce microbial cell membrane

386

thickness and even result in the formation of hydrophilic pores. (41) Moreover, PEF

387

may make the plasma membranes permeable to small molecules due to the

388

destabilization of the lipid bilayer and proteins in cell membranes.

389

proposed that PEF may facilitate the access of CAR to the cytoplasm membrane and,

390

allow entry of CAR into the cells to interact directly with DNA.

reduction

value

[−log(N1/N0)CAR×PEF]

of

S.

aureus

was

(42)

2.56,

and

Thus, it is

391

In summary, this work has shown that CAR exerted its antibacterial effect by two

392

key mechanisms: increasing the permeability of the bacterial cell membrane and

393

binding directly to genomic DNA. S. aureus cell membrane integrity and morphology

394

were damaged when the cells were exposed to higher levels of CAR. Fatty acid

395

composition analysis showed that there was a change in the fatty acid composition of

396

S. aureus cell membranes an an increase in the UBFAs content, which may modify

397

the cell’s fluidity and block access of CAR to the inner membrane when grown in low

398

concentrations of CAR. In intro studies showed that CAR can bind to the monor

18



399

groove of the DNA helix, forming a CAR−DNA complex which resulted in changes

400

in the secondary structure and surface morphology of DNA and its molecular

401

aggregation. The combination of CAR exposure and PEF treatment showed strong

402

synergistic effects on S. aureus. In conclusion, CAR was found to have excellent

403

antimicrobial activity against S. aureus, and a combination of CAR with PEF may be

404

a valid strategy for increasing the safety of foods. Furthermore, CAR may have

405

valuable applications in various fields, such as in agricultural and food industries.

406

ACKNOWLEDGEMENTS

407

This research was supported by the National Natural Science Foundation of

408

China (21576099, 21376094) as well as S&T projects of Guangdong Province

409

(2015A030312001 and 2013B020203001).

410 411 412 413 414 415 416 417 418 419

19


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420

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41. Silvius, J. R., Role of cholesterol in lipid raft formation: lessons from lipid model systems. BBA Biomembranes 2003, 1610, 174–183.

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555

constituents exert a charge effect on electroporation threshold. Biochimica et

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557 558 559 560

Figure captions

561

Figure 1. Principal component analysis of membrane fatty acid composition of S.

562

aureus cells. (A) Scores plot of fatty acid composition using the first two principal

563

component analysis in relation to ethanol (0.64%) and different concentrations of

564

CAR. (B) Loadings plot of fatty acid composition in different concentrations of CAR

565

defined by the first two principal components.

566

Figure 2. (A) and (B) The relative proportions of different fatty acid species. (C) Total

567

BCFAs and UBFAs proportions, and (D) ratios of UBFAs to BCFAs in the

568

cytoplasmic membrane of S. aureus grown in the medium with different

569

concentrations of CAR, c(CAR) = 0, 0.26 mM, 0.52 mM, 0.77 mM, respectively.

570

Figure 3. Activity of cytoplasmic β-galactosidase (measured by absorbance at 420 nm)

571

on S. aureus cells treated with various CAR concentrations, c(CAR) = 0, 1.03, 2.06,

572

4.12 and 8.24 mM, respectively.

573

Figure 4. SEM images of S. aureus treated with different concentrations of CAR (A)

26



574

0, showing a smooth cell membrane of a normal cell; (B) 1.03 mM, cells changed and

575

distorted to oval; (C) 2.06 mM, distortion and even collapse in cell morphology, and

576

(D) 4.12 mM, cell debris due to the bursting of cells.

577

Figure 5. (A) Effect of DNA on fluorescence spectra of CAR (λex = 275 nm, λem =

578

304 nm); c(CAR) = 21.40 µM; and c(DNA) = 0, 8.50, 17.0, 25.5, 34.0, 42.5, 51.0,

579

59.5 and 68.0 µM, for curves 1→9, respectively; (B) CD spectra of DNA in the

580

presence of increasing amounts of CAR. c(DNA) = 0.50 mM. The molar ratios of

581

CAR to DNA were 0:1, 2:1, and 4:1 for curves 1→3, respectively. c(CAR) = 0.50

582

mM (dashed line).

583

Figure 6. (A) Cluster analyses of the AutoDock docking runs of CAR with DNA; (B)

584

Molecular modeling results of the energy-minimized structure of the CAR–DNA

585

system. The green dashed lines stand for hydrogen bonds.

586

Figure 7. AFM images of two-dimensional and three-dimensional graphs of DNA (A)

587

and (C), the CAR−DNA complex (B) and (D) with the scan size of the image is 2.0

588

µm × 2.0 µm, respectively. c(DNA) = 0.5 mM, c(CAR) = 2.0 mM.

27


Page 28 of 37

Page 29 of 37


Table 1. Effects of CAR during and/or after PEF with different electric fields for 1.6 ms on log reduction of S. aureus. −log(N1/N0) CAR(mM)

0 kV/cm

10.0 kV/cm

20.0 kV/cm

30.0 kV/cm

A

B

C

A

B

C

A

B

C

A

B

C

0

-

-

-

0.86±0.06

0.78±0.02

0.91±0.03

1.20±0.12

1.26±0.11

1.31±0.30

1.43±0.32

1.52±0.20

1.54±0.18

0.26

0.15±0.01

0.45±0.02

0.42±0.01

1.06±0.11

1.45±0.31

1.51±0.27

1.39±0.18

1.81±0.21

1.92±0.22

1.69±0.22

2.33±0.18

2.22±0.15

0.52 0.77

0.19±0.03 0.17±0.04

0.96±0.05 1.34±0.21

0.89±0.02 1.43±0.15

1.09±0.20 1.20±0.16

1.95±0.26 2.56±0.54

2.23±0.45 2.61±0.35

1.44±0.31 1.54±0.22

2.59±0.15 3.18±0.32

2.43±0.16 3.51±0.24

1.81±0.14 1.79±0.16

2.76±0.21 3.44±0.45

2.91±0.42 3.81±0.33

A. presence of CAR only during PEF treatment; B. recovery, presence of CAR only after PEF treatment in the recovery agar medium; C. presence of CAR during PEF treatment and in the recovery agar medium.

28



Table 2. Net effect of CAR during and/or after PEF for 1.6 ms on log reduction of S. aureus.

CAR(mM) 0.26 0.52 0.77

10.0 kV/cm A B C 0.05 0.22 0.18 0.04 0.21 0.43 0.17 0.44 0.27

−log(N1/N0)CAR-PEF 20.0 kV/cm A B C 0.04 0.10 0.19 0.05 0.37 0.23 0.17 0.58 0.77

30.0 kV/cm A B C 0.11 0.36 0.26 0.21 0.28 0.48 0.19 0.58 0.84

A. presence of CAR only during PEF treatment; B. recovery, presence of CAR only after PEF treatment in the recovery agar medium; C. presence of CAR during PEF treatment and in the recovery agar medium.

29


Page 30 of 37

Page 31 of 37


Figure 1 (A)

(B)

30



Figure 2 (A)

(B)

(C)

(D)

31


Page 32 of 37

Page 33 of 37


Figure 3

32



Figure 4 (A)

(B)

(C)

(D)

33


Page 34 of 37

Page 35 of 37


Figure 5 (A)

(B)

34



Figure 6 (A)

(B)

35


Page 36 of 37

Page 37 of 37


Figure 7 (A)

(B)

(C)

(D)

36


Membrane Destruction and DNA Binding of ... - ACS Publications

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