Antibacterial Effects of a Cell-Penetrating Peptide Isolated from Kefir

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Antibacterial effects of a cell-penetrating peptide isolated from kefir Jianyin Miao, Haoxian Guo, Feilong Chen, Lichao Zhao, Liping He, Yangwen Ou, Manman Huang, Yi Zhang, Baoyan Guo, Yong Cao, and Qingrong Huang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b00730 • Publication Date (Web): 22 Mar 2016 Downloaded from http://pubs.acs.org on March 25, 2016

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Journal of Agricultural and Food Chemistry

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Antibacterial effects of a cell-penetrating peptide isolated from kefir

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Jianyin Miao†,‡,§,#, Haoxian Guo†,§, Feilong Chen†,‡, Lichao Zhao†,‡, Liping He†,‡, Yangwen

3

Ou#, Manman Huang†, Yi Zhang†, Baoyan Guo†, Yong Cao†,‡, *, Qingrong Huang¶,*

4 5

†

6

Republic of China

7

‡

8

Guangzhou 510642, People’s Republic of China

9

§

10

¶

11

08901, USA

12

#

13

410000, People’s Republic of China

College of Food Science, South China Agricultural University, Guangzhou 510642, People’s

Guangdong Provence Engineering Research Center for Bioactive Natural Products,

Qinzhou University, Qinzhou 535000, People’s Republic of China Department of Food Science, Rutgers University, 65 Dudley Road, New Brunswick, NJ

School of Pharmacy, Hunan University of Chinese Medicine, Changsha

14

15

Corresponding Authors

16

* Telephone: +86-20-85286234. Fax: +86-20-85286234. E-mail: [email protected].

17

* Telephone: 848-932-5514. Fax: 732-932-6776. E-mail: [email protected].

18 19

20

ACS Paragon Plus Environment


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ABSTRACT: Kefir is a traditional fermented milk beverage used throughout the

22

world for centuries. A cell-penetrating peptide F3 was isolated from kefir by

23

Sephadex G-50 gel filtration, DEAE-52 ion exchange and reverse-phase high

24

performance liquid chromatography. F3 was determined to be a low molecular weight

25

peptide containing one leucine and one tyrosine with two phosphate radicals. This

26

peptide displayed antimicrobial activity across a broad spectrum of organisms

27

including several Gram-positive and Gram-negative bacteria as well as fungi, with

28

minimal inhibitory concentration (MIC) values ranging from 125 to 500 µg/mL.

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Cellular penetration and accumulation of F3 were determined by confocal laser

30

scanning microscopy. The peptide was able to penetrate the cellular membrane of

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Escherichia coli and Staphylococcus aureus. Changes in cell morphology were

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observed by scanning electron microscopy (SEM). The results indicate that peptide F3

33

may be a good candidate for use as an effective biological preservative in agriculture

34

and food industry.

35 36

KEYWORDS: Kefir, antimicrobial peptide, purification, identification, antibacterial

37

effects

38 39 40 41 42

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INTRODUCTION

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Foodborne illness caused by microbial contamination results in large economic

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losses in food industry each year.1-3 Although traditional heat sterilization can

46

effectively inhibit microbial growth in foods, the obtained sterile food products are

47

always partly decreased in the quality of sensory, nutrition and biological function.

48

Compared with traditional heat sterilization technology, the synthetic preservatives

49

can fully retain food nutrients and the original flavor, however consumers are

50

increasingly concerned about the safety of the synthetic chemicals used as

51

preservatives in food. Therefore, development of effective, natural antimicrobial

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substances for food preservation and food safety is in great demand in food industry.

53

Extensive research has investigated the potential application of natural antimicrobial

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agents in food preservation, such as plant essential oils and their constituents,4,5

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animal origin antimicrobial agents,6,7 and microbial origin antimicrobial agents.8,9

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Among these natural antimicrobial agents, antimicrobial peptides have been under

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consideration as promising candidates because they provide nutrition, no special

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flavor, and have special mechanism of action compared with traditional antibiotics.10

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Nisin, a bacteriocin produced by Lactococcus lactis subsp. lactis was the first natural

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antimicrobial peptide approved for food use and is the only antimicrobial peptide

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widely employed as a food preservative in over 40 countries.11,12

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Kefir is a traditional, natural fermented milk beverage fermented with a starter kefir

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grains, which mainly composed of three types of microorganisms: lactic acid bacteria,

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acetic acid bacteria, and yeasts.13,14 Kefir has been used for centuries in many 2



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countries. As a traditional safe fermented milk, numerous studies have focused on the

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biological functions of kefir in recent years. Kefir is believed to have a number of

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beneficial properties,15 including the clinical treatment of metabolic diseases,

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hypertension, ischemic heart disease (IHD), and allergies.16 Among these properties,

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the antimicrobial activity displayed by kefir has attracted more attention.17-20 The

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substances in kefir that may be responsible for its antimicrobial activity include lactic

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acid, volatile acids, and inorganic compounds and antimicrobial peptides produced by

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lactic acid bacteria.17 Some studies reported the antimicrobial activity of kefir,

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however the exact substance compositions were not clear, because the antibacterial

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effect were contributed by fermentation broth,18,19,21 a mixture made up of many

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different substances ( such as polysaccharides, proteins, peptides, lactic acid or acetic

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acid). Therefore, purification and obtaining pure antimicrobial substance are key

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processes in researching the antimicrobial activity of kefir, which are preconditions to

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identify the chemical structure and to further study the antibacterial effects and

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antibacterial mechanisms.

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In the present study, antimicrobial peptide F3 was purified from kefir by a

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three-step purification procedure. The chemical structure of F3 was identified by

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MALDI–TOF MS, NMR experiments and X-ray fluorescence analysis. The effect of

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F3 on the integrity of the bacterial membrane was investigated by the outer membrane

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/inner membrane permeability assay and confocal laser scanning microscopy assay.

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The effect of F3 on the bacterial morphology was analyzed using scanning electron

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microscope (SEM). Our results provided fundamental information on the purification, 3


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identification and antimicrobial activity of the natural antimicrobial peptide F3 from

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kefir.

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MATERIALS AND METHODS

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Preparation of the kefir fermentation broth. Tibetan kefir grains were obtained

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from the laboratory of food microorganisms in the College of Food Science at South

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China Agricultural University (Guangzhou, China). Kefir grains (2%, m/v) were

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added to sterilized milk and kept hermetically at 37 °C for 24 h. After fermentation,

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the kefir grains were filtered. The fermentation broth was obtained to analyze its

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antibacterial activity. The broth was centrifuged at 3500 g for 20 min and the

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supernatant was filtered through a 0.22-µm millipore filter. The cell-free supernatant

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was used for subsequent purification process.

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Screening for antimicrobial activity components. To confirm the antimicrobial

99

activity of each purified fraction from the cell-free supernatant, the agar spot-test 22

100

method

was applied where the diameter of the inhibitory zone was used as an

101

indicator of antimicrobial activity. Escherichia coli (E. coli) and Staphylococcus

102

aureus (S. aureus) were used as test microorganisms.

103

Purification of fraction with the highest antimicrobial activity. All the eluted

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fractions were recorded at 214 nm during the purification. The cell-free supernatant

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was initially purified via a Sephadex G-50 gel filtration column (70 cm × 1.6 cm) at

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20 °C. The eluent used was doubly distilled water with the flow rate of 1.5 mL/min. 4



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The eluted fractions (SP-1, SP-2 and SP-3) were then collected, concentrated, and

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lyophilized before the antimicrobial activity assay.

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After purification by gel filtration chromatography, the dried fraction displaying the

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highest antimicrobial activity was redissolved in doubly distilled water and loaded

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onto a DEAE-52 ion exchange column (40 cm × 2.6 cm) at 20 °C. The eluent gradient

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used was doubly distilled water (0-3 min), 5.0 mM NH4OAc (4-10 min), 10 mM

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NH4OAc (10-15 min), 20 mM NH4OAc (16-25 min), and 100 mM NH4OAc (26-60

114

min). The flow rate was 1.5 mL/min. The eluted fractions (W-1 and Y-2) were

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collected, concentrated, and lyophilized for the antimicrobial assay.

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The fraction obtained from ion exchange chromatography displaying the highest

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antimicrobial activity was redissolved in doubly distilled water and loaded onto a

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reversed-phase (RP) Shim-pack PRC-ODS (K) column (250 mm × 30 mm, 15µm,

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Shimadzu, Kyoto, Japan). Solvent A was 0.1% trifluoroacetic acid in double-distilled

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water, and solvent B was 100% methanol. A linear gradient of 5%–20% solvent B was

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applied at a flow rate of 1 mL/min for 80 min. Eluted peaks (F0, F1, F2 and F3) were

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collected, concentrated, and lyophilized for use in the antimicrobial activity assay. The

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highest antimicrobial activity fraction was then analyzed on a C18 column (300 mm ×

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3.9 mm, 4 µm) (Waters, Boston, MA) using a 5% methanol isocratic elution at a flow

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rate of 1 mL/min to determine the purity.

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Identification of the fraction with the highest antimicrobial activity. Ninhydrin

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test was applied to determine whether the fraction with the highest antimicrobial

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activity contains peptide or protein.23 One drop of the fraction was added onto a silica 5


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gel plate and dried using a hair drier. A ninhydrin solution (0.25% ninhydrin dissolved

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in ethanol) was then sprayed on the silica gel plate. The silica gel plate was then kept

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at 100 °C for 5 min. A color change to purple indicates the test material may contain a

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peptide or protein. Molecular weight of the fraction displaying the largest

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antimicrobial activity isolated from RP-HPLC was determined using an ABI 4800

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MALDI-TOF-MS (Shimadzu, Kyoto, Japan). 1H and

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AV-600 spectrometer (Bruker Spectrospin AG, Rheinstetten, Germany) at room

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temperature (1H NMR, 400 MHz;

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were measured in ppm. The fraction with the highest antimicrobial activity (6 mg)

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was dissolved in 0.6 ml D2O in a 5 mm NMR tube. The element compositions were

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then characterized with an S4 PIONEER X-ray fluorescence spectrometer (Bruker,

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Karlsruhe, Germany).24

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Determination of the antimicrobial spectrum and minimum inhibitory

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concentration (MIC). The spectrum of antimicrobial activity and minimum

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inhibitory concentration (MIC) of the fraction displaying the largest antimicrobial

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activity was determined using a microdilution technique.20,25 The bacterial strains

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tested were Escherichia coli (ATCC 25922), Staphylococcus aureus (ATCC 63589),

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Salmonella enterica (CMCC 9812), Shigella dysenteriae (CMCC（B）50071) and

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Bacillus thuringiensis (CMCC 9812), and the fungi strains were Aspergillus niger

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(ACCC 30005), Aspergillus flavus (CGMCC 3. 2890), Rhizopus nigricans (AS3.4997)

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and Penicillium glaucum (STL 3501). Samples were dissolved in sterilized water and

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diluted to an initial concentration of 2 mg/mL.

13

13

C NMR were recorded on an

C NMR, 100 MHz). Chemical shifts (δ scale)

6



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Membrane permeability assay. E. coli and S. aureus were chosen to assess the cell

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membrane damaging ability of fraction peptide F3. The outer membrane permeability

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assay for E. coli was performed using a synergistic growth inhibition assay in the

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presence of erythromycin and F3. E. coli was cultured to logarithmic phase, washed,

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and re-suspended in LB broth at 106 CFU/mL. 0.5 MIC (62.5 µg/mL) of F3 with

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different concentrations of erythromycin (1 µg/mL, 2 µg/mL, 4 µg/mL, 7 µg/mL, 13

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µg/mL, or 25 µg/mL) were incubated with the cells at 37°C for 10 h.26 Synergistic

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growth inhibition was monitored by detecting a decrease in absorbance at 630 nm.

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Permeability of the E. coli inner cell membrane and the S. aureus cell membrane

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was

determined

by

detection

of

β-Galactosidase

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O-nitrophenyl-β-D-galactopyranoside

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compound, as a substrate.27 E. coli and S. aureus were cultured to logarithmic phase,

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washed, and re-suspended in 10 mM sodium phosphate buffer (pH 7.4). ONPG (1.5

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mM) was dissolved in the same buffer and F3 (final concentration of F3 was 1 MIC)

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was incubated with E. coli or S. aureus. The degree of permeability was determined

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every 30 min by monitoring the hydrolysis of ONPG to o-nitrophenol at 405 nm. A

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cell suspension without F3 was used as control.

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Confocal laser Scanning Microscopy. Fluorescein isothiocyanate (FITC)-labeled

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peptide F3 was prepared as described previously.28 An E. coli cell suspension grown

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until the exponential phase (108 CFU/mL) was mixed with FITC- labeled F3 to a final

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concentration equal to 1MIC (125 µg/mL). Samples were kept in the dark at 37 °C. At

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time points of 10, 30, and 180 min, cells were washed with PBS buffer three times

(ONPG),

a

activity

non-membrane

7


using

permeative

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and observed using a confocal laser-scanning microscope (Zeiss, Berlin, Germany).

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Examination of bacterial membrane damage by scanning electron microscopy

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(SEM). The test strain was grown to logarithmic phase in LB broth. Cell suspension

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(106 CFU/mL) was incubated with F3 (final concentration of 1 MIC) at 37°C for

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various time periods (0.5, 1, and 4 h). After pelleted by centrifugation at 3,000 g for 5

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min, cell morphology was observed under an XL30 ESEM scanning electron

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microscope (Philips, Eindhoven, Netherlands) according to a previously published

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method.29

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RESULTS AND DISCUSSION

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Purification and molecular weight determination of the fraction with the highest

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antimicrobial activity. The results from the purification process are shown in Figure

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1 and Table 1. The highest antimicrobial activity fraction was obtained by a three-step

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purification procedure (Figure 1A, B and C). The most active peak, F3, which eluted

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at 48.02 min during the third step of the three-step purification procedure (Figure 1C)

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exhibited the highest antimicrobial activity against E.coli and S. aureus with

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diameters measured to be 7.4±0.18 mm and 8.1±0.41 mm, respectively. This active

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peak (F3) was then loaded on a reversed-phase (RP) C18 column to evaluate its purity.

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A single peak was found at 19.24 min (Figure 1D), highlighting the high purity of F3.

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Fraction F3 was then collected and subjected to MALDI–TOF MS to identify its

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molecular mass (Figure 1E). The m/z ratio of the major peak was found to be 453.1.

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The more accurate mass of peptide F3 determined by high resolution mass 8



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spectrometry was 453.16861 Da, which is similar to the result of 453.18 Da by

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MALDI–TOF MS. Compared to previously reported antimicrobial peptides isolated

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from kefir and other materials, such as bacST8KF (3.5 kDa),17 bacteriocin F1

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(2113.842 kDa),20 sakacin C2 (2113.842 kDa),30 bacteriocin A5-11A (5206 Da) and

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A5-11B (5218 Da),31 fraction F3 did not match with any of these known antimicrobial

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peptides.

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Identification of the highest antimicrobial activity fraction. The ninhydrin test is a

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classic method used to confirm and characterize amino acids, peptides, and

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proteins.32-35 This test was used to determine whether F3, the fraction with the highest

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antimicrobial activity, was composed of amino acids, peptides or proteins. The single

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purple spot (positive color change) on the silica gel plate corresponding to fraction F3

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confirmed that it was composed of amino acids, peptides, or proteins. With the

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ninhydrin test and molecular weight determination, we proposed that F3 is a low

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molecular weight peptide. 1

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H and

13

C NMR spectra were then used to identify the chemical structure of F3

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(Figure 2). The proton peaks at 6.9 and 7.1 ppm (each 2H, d, J = 6.6 Hz) and at 6.5–

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7.5 ppm in the 1H NMR spectra (Figure 2A) were attributed to the proton signals of

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the benzene ring of tyrosine. Peaks at 157.7, 133.7, 118.7 and 120.2 ppm in the

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NMR spectra (Figure 2B) were attributed to the carbon signals of the benzene ring of

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tyrosine. This confirmed the presence of a tyrosine residue in F3. Peaks at 3-4 ppm in

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the 1H NMR spectra (Figure 2A) and peaks at 23.8, 24.9, 27.0, 42.6 and 56.3 in the

215

13

13

C

C NMR spectra (Figure 2B) corresponded to methyl and methylene groups of 9


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leucine, therefore F3 contains a leucine. Considering the determined molecular weight

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of 452.1 Da, F3 should contain other post-translational modification groups except for

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tyrosine and leucine.

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The elemental composition of F3 was measured by X-ray fluorescence. This

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experiment showed that, except for O, C, and N, F3 contains other elements including

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P (2.107%), Sr (0.001%), Cl (0.031%), Si (0.066% SiO2), Al (0.01%), Na (0.173%),

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Ca (0.009%), Ni (0.002%), Zn (0.001%), and Ga (0.001%). After eliminating system

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errors caused by equipment, we confirmed that F3 contained the phosphorus element.

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In the present study, no signals for other amino acids were found in the 1H and

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NMR spectra aside from tyrosine and leucine. With the molecular weight, NMR

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experiments and X-ray fluorescence analysis in hand, we concluded that there are two

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phosphate radicals on F3. The proposed chemical structure of F3 contains two amino

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acids (leucyl-tyrosine or tyrosyl-leucine) with two phosphate radicals connected to the

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benzene ring of tyrosine. Based on the analysis by Edman degradation, the tentative

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molecular formula of F3 is considered to be tyrosyl-leucine with two phosphate

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radicals connected to the benzene ring of tyrosine (Figure 3). Several reported short

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cationic antibacterial peptides, such as some dipeptides or tripeptides, were

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surprisingly active compared to many far larger size antibacterial peptides, especially

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against multi-resistant strains.36, 37 Post-translational modifications have been found in

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several antimicrobial peptides,38 and some modifications are more complex and

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extensive.39,40 The biological role of post-translational modiﬁcations probably make

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the peptides resistance to endogenous proteolytic enzymes, and increase their activity 10


13

C


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stability. 38 The proposed chemical structure showed that F3 is a dipeptide with two

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phosphate radicals connected to the C-terminal, which indicated F3 is a novel

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antimicrobial peptide and worthy of further study.

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Antimicrobial spectrum and minimum inhibitory concentration (MIC). The

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microdilution technique was used to further assess the antimicrobial activity of F3.

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Table 2 shows that F3 displayed antimicrobial activity against several bacteria and

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fungi with MIC values ranging from 125 to 500 µg/mL. E. coli and Bacillus

245

thuringiensis exhibited the highest sensitivity to F3 with a MIC value of 125 µg/mL.

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Although F3 was initially screened with E. coli, a Gram-negative bacterial strain and

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S. aureus, a Gram-positive bacterial after purification, antimicrobial activity against

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several fungi was also observed. These results were similar to an antimicrobial

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peptide reported in a previous study from our group.20 Antimicrobial peptides showing

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antimicrobial activity against Gram-positive and Gram-negative bacteria, and fungi

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were not common in previous reports, since the majority of antimicrobial peptides

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only preserved antibacterial activity against Gram-positive bacteria.30,41 Our results

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suggested that F3 was an antimicrobial peptide with a broad antimicrobial spectrum.

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Effect of the peptide F3 on the permeability of bacterial membranes. Unlike

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Gram-positive bateria, Gram-negative bacteria possess an outer membrane attached to

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the peptidoglycan layer. This membrane acts as a selective permeability barrier,

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reinforcing the shape of the cell and providing a protective barrier against harmful

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agents in the external environment.42 Erythromycin is less effective against 11


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Gram-negative bacteria because of its poor permeation of the outer membrane,

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especially at the lower concentrations, while it can quickly cross through a damaged

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outer membrane and exert detrimental effects on cells.43 In our previous experiment,

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we found that 0.5 MIC of F3 did not cause any significant inhibition to E. coli growth,

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therefore we chose erythromycin as a probe in conjunction with 0.5 MIC F3. This

264

would allow us to determine whether F3 has the penetration enhancing ability. The

265

results are shown in Figure 4. In the absence of F3, erythromycin showed

266

dose-dependent inhibition on E. coli growth. When a low concentration of

267

erythromycin and 0.5 MIC F3 were added to the cell culture, an enhanced inhibitory

268

effect was observed on the growth of E. coli. This result indicated that F3 increased

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the permeability of the outer membrane of E. coli to allow erythromycin to enter cells

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and kill the bacteria at a lower concentration. Several reported antimicrobial

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substances also exhibit similar outer membrane permeability activity in E. coli.26,44

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O-nitrophenyl-β-D-galactopyranoside (ONPG) is an analog of lactose which can

273

be hydrolyzed into a yellow product, ο-nitrophenol, by β-Galactosidase, a hydrolase

274

inside the cytoplasm.45 β-Galactosidase can be released from cells when the

275

cytoplasmic membrane is permeable. Therefore, ONPG was chosen as a probe to

276

determine the permeability of the cytoplasmic membrane. As shown in Figure 4,

277

β-Galactosidase leakage was observed from E. coli (Figure 4B) and S. aureus (Figure

278

4C) in the presence of F3 in a time-dependent manner. No significant change of

279

β-Galactosidase activity was detected in the culture medium composed of control

280

cells. These results suggested that F3 had the ability to cause permeability of the 12



281

cytoplasmic membrane of E. coli and S. aureus.

282

Internalization of FITC-labeled F3 in Escherichia coli cells. The results of the cell

283

membrane permeability test described above indicated that F3 was able to increase

284

bacterial membrane permeability. To determine the localization of F3, FITC-labeled

285

F3 was incubated with E. coli cells and was visualized by confocal laser scanning

286

microscopy. From the observed results showed in Figure 5, F3 was found to localize

287

in the cytoplasm of bacterial cells in a time dependent manner between 10 and 180

288

minutes. This further confirms the membrane permeability activity of F3.

289

Examination of morphologic changes in cells by scanning electron microscopy.

290

The surface morphology of E. coli and S. aureus in the presence of F3 were evaluated

291

using SEM. As shown in Figure 6, untreated E. coli (Figure 6A) and S. aureus (Figure

292

6E) cells showed a normal, smooth, intact surface. After treatment with F3 for 2 h, E.

293

coli (Figure 6D) and isolated S. aureus (Figure 6H) cells showed a significant change

294

in morphology indicated by the presence of wrinkles and deformation on cellular

295

surfaces. These results indicated that F3 damages the cellular integrity and caused

296

significant deformation in cell morphology. The ultrastructure changes observed on

297

cell surfaces caused by F3 were similar to other reported results.46-47

298

The present study demonstrates that peptide F3 is a novel dipeptide, especially

299

with the post-translational modification of phosphate radicals, providing a new

300

reference in the field of short peptide antibiotics. The special chemical structure of F3

301

may contribute to its broad antimicrobial spectrum against Gram-positive bacteria, 13


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Gram-negative bacteria and fungi, which is definitely worth further exploring. In

303

addition, the membrane-penetrating activity of F3 provides further insight into the

304

interaction between antimicrobial peptides and bacterial membranes. F3 has the

305

potential to serve as a valuable natural food preservative in food industry.

306

AUTHOR INFORMATION

307

Corresponding Author

308

* Telephone: +86-20-85286234. Fax: +86-20-85286234. E-mail:

309

[email protected].

310

* Telephone: 848-932-5514. Fax: 732-932-6776. E-mail: [email protected].

311

Funding

312

The authors would like to express their gratitude to the National Natural Science

313

Foundation of China (No. 31171768) and the Scientific Research Project of

314

Guangdong Province Office of Education (No. 2013gjhz0003) for the financial

315

support.

316

Notes

317

The authors declare no competing financial interest.

318

319

320

321

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against

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465 466 467 468 469 470 471 472 473 474 475 476 21


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Page 23 of 32


477

FIGURE CAPTIONS

478

Figure 1. Purification of peptide F3. A. Analysis of active fractions in the supernatant;

479

B. Analysis of active fraction SP-3 obtained from A; C. Analysis of active fraction

480

W-1 obtained from B; D. Analysis of active fraction F3 obtained from C; E.

481

MALDI-TOF MS analysis of F3.

482

Figure 2. A. 1H, and B. 13C NMR spectra of F3.

483

Figure 3. The tentative chemical structure of F3.

484

Figure 4. Effects of F3 on the membrane permeability of E. coli and S. aureus cells.

485

A. Effects on the outer membrane permeability of Escherichia coli cells. ( )

486

Erythromycin (1 µg/mL, 2 µg/mL, 4 µg/mL, 7 µg/mL, 13 µg/mL, or 25 µg/mL); (

487

0.5 MIC F3 and erythromycin (1 µg/mL, 2 µg/mL, 4 µg/mL, 7 µg/mL, 13 µg/mL, or

488

25 µg/mL); and ( ) sterile water. B. Effects on the inner membrane permeability of E.

489

coli cells. (

490

of S. aureus cells. ( ) 1 MIC F3; (×) control. The mean and standard deviation of

491

triplicate values are shown.

492

Figure 5. E. coli cells treated with FITC-conjugated F3. A. 10 min; B. 30 min; and C.

493

180 min.

494

Figure 6. Scanning electron microscopy observation in E.coli (A, B, C, and D) and S.

495

aureus (E, F, G, and H) treated with 1 MIC of F3 for 0 h (A and E), 0.5 h (B and F), 1

496

h (C and G) and 2 h (D and H).

)

) 1 MIC F3; (×) control. C. Effects on the cell membrane permeability

497

22



Page 24 of 32

Tables Table 1 Antimicrobial activity of each purified fraction.

Purified fraction

Concentration(mg/mL)

Inhibition zone diameter (mm) Staphylococcus Escherichia coli aureus ATCC 25922 ATCC 63589 – – 3.5±0.21 3.3±0.23

SP-1 SP-2

300 300

SP-3

300

6.9±0.23*

6.5±0.24*

W-1 Y-1 F0 F1 F2

300 300 50 50 50

8.5±0.32** 6.6±0.22 – 4.6±0.17 3.2±0.37

7.2±0.27** 5.8±0.23 – 4.1±0.28 2.9±0.25

F3

50

7.4±0.18***

8.1±0.41***

–: No inhibition zone recorded. * indicates statistical significance between SP-1, SP-2and SP-3 (p < 0.05, n = 3). ** indicates statistical significance between W-1and Y-1 (p < 0.05, n = 3). *** indicates statistical significance between F0, F1, F2 and F3 (p < 0.05, n = 3). The mean and standard deviation of triplicate values are shown.

23


Page 25 of 32


Table 2 Minimum inhibitory concentrations (MIC) of the peptide F3.

Microorganism

MIC values (µg/mL) F3

a

Escherichia coli ATCC 25922

125±1.29 a

Staphylococcus aureus ATCC 63589

500±3.23 b

Salmonella enterica CMCC 9812

500±4.14 b

Shigella dysenteriae CMCC（B）50071

500±1.93 b

Bacillus thuringiensis CMCC 9812

125±2.21 a

Aspergillus niger ACCC 30005

500±3.43 b

Aspergillus flavus CGMCC 3. 2890

500±4.73 b

Rhizopus nigricans AS3.4997

500±4.11 b

Penicillium glaucum STL 3501

500±0.23 b

Differences were considered significant at probability levels of p< 0.05, indicated

by different letters. The mean and standard deviation of triplicate values are shown.

24



Page 26 of 32

Figure 1.

A

1.6 1.4

W-1

1.0

SP-2

Abs at 214nm

1.2

Abs at 214nm

B

1.2

SP-3

1.0 0.8 0.6

0.8

0.6

0.4

SP-1 0.4

Y-1

0.2 0.2

0.0

0.0 0

20

40

60

80

100

120

140

160

0

180

20

40

60

80

Fraction number

Fraction number

F1

C

D F3

F0 F3 F2

E

25


Page 27 of 32


Figure 2.

A

B

26



Figure 3.

27


Page 28 of 32

Page 29 of 32


Figure 4.

B

OD（405 nm）

A

0.2 0.15 0.1 0.05 0 0

1

2 Time（h）

C

28


3

4


Page 30 of 32

Figure 5.

A

B

29


C

Page 31 of 32


Figure 6. B

C

E

F

G

H

30



Table of Contents Graphic

31


Page 32 of 32

Antibacterial Effects of a Cell-Penetrating Peptide Isolated from Kefir

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