Food Control 33 (2013) 433e439

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Food Control
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A study of the antibacterial mechanism of catechins: Isolation

and identification of Escherichia coli cell surface proteins that interact
with epigallocatechin gallate
Motokazu Nakayama a, *, Kanami Shimatani a, Tadahiro Ozawa a, Naofumi Shigemune a,
Takashi Tsugukuni a, Daisuke Tomiyama a, Masahiro Kurahachi b, Ai Nonaka b,
Takahisa Miyamoto b
a
Kao Corporation, Global R＆D-Safty Science, Akabane 2606, Haga-Gun, Ichikai-Machi, Tochigi, Japan
b
Division of Food Science and Biotechnology, Department of Bioscience and Biotechnology, Faculty of Agriculture, Graduate School, Kyushu University,
6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan

a r t i c l e i n f o a b s t r a c t

Article history: Catechins have high anti-bacterial activity against various microorganisms. In this study, the mechanism
Received 23 December 2012 of anti-bacterial activity of catechins was investigated using Escherichia coli. Transmission electron mi-
Received in revised form croscope analysis revealed that deposits containing EGCg were found only on the outer membrane,
3 March 2013
which is the outermost layer of the cell surface, in E .coli cells treated with EGCg. Based on this obser-
Accepted 9 March 2013
vation, we focused on outer membrane proteins as targets of EGCg in E. coli. Two-dimensional electro-
phoresis identified 16 spots that had disappeared or showed markedly reduced intensity after treatment
Keywords:
with EGCg compared to the control. Of these, an outer membrane porin protein, OmpG, acids suggested
Catechins
Epigallocatechin gallate
that the basic amino acids Lys, Arg, and His strongly interacted with EGCg. The docking simulation with
Escherichia coli EGCg and OmpG revealed that EGCg enters into the porin pore and binds to Arg residues present on the
Porin inner surface of the pore channel through hydrogen bonding, resulting in inhibition of the porin function.
Furthermore, glucose uptake by E. coli was inhibited in cells treated with EGCg. Taken together, these
results suggest that EGCg inhibits the major function of porin proteins, namely the passive transport of
small hydrophilic molecules such as glucose, leading to growth inhibition of E. coli.
Ó 2013 Elsevier Ltd. All rights reserved.

1. Introduction The known antimicrobial activities of catechins include inhibi-

Catechins are a class of polyphenols found in green tea, which is
a popular beverage since ancient times (Stagg, 1980). Catechins
have several useful properties, including antibacterial activity
(Hamilton-Miller, 1995; Juneja, Bari, Inatsu, Kawamoto, & Friedman,
2007), antioxidant activity (Cao, Sofic, & Prior, 1996; Rice-Evans,
Miller, & Paganga, 1996), anticarcinogenic effects (Ahmad &
Mukhtar, 1999; Buschman, 1998), and fat reduction activity. (Maki
et al., 2009; Murase, Nagasawa, Suzuki, Hase, & Tokimitsu, 2002)
Catechins are expected to have wide applications as additives in
foodstuffs as they are extremely safe and tolerable, making them
readily acceptable to consumers (Mendel, 2007).
Abbreviations: ECg, epicatechin gallate; EGCg, epigallocatechin gallate; MIC,
minimum inhibitory concentration; PMF, Peptide mass fingerprinting; SCD, soy-
bean-casein digest; TA, tannic acid (TA).
* Corresponding author. Tel.: þ81 285687845.
E-mail address: nakayama.motokazu@kao.co.jp (M. Nakayama).
tory effects against food poisoning bacteria, antiviral activity
(Mendel, 2007; Nance, Siwak, & Shearer, 2009), and inhibition of
bacterial toxins (Mendel, 2007). Among catechins, epicatechin
gallate (ECg) and epigallocatechin gallate (EGCg) have particularly
high antibacterial activity (Mabe, Yamada, Oguni, & Takahashi,
1999) and are more effective against Gram-positive than Gram-
negative bacteria (Mabe et al., 1999). It has been reported that
catechins interact with the cell wall and membrane of bacterial
cells (Bernal, Zloh, & Taylor, 2009; Caturla, Vera-Samper, Villalaín,
Mateo, & Micol, 2003; Tsuchiya, 2001) and cause lethal damage to
bacterial cells through the production of hydrogen peroxide
(Akagawa, Shigemitsu, & Suyama, 2003; Arakawa, Maeda, Ookubo,
& Shimamura, 2004; Nakayama, Ichiba, Kuwabara, Kajiya, &
Kumazawa, 2002). However, the detailed mechanisms underlying
the antibacterial properties of catechins are not fully understood.
One approach to clarifying the antibacterial mechanisms of
catechins is to identify their target site in bacteria by analyzing the
localization of catechin molecules on bacterial cell surfaces. We
previously found that the hydrogen peroxide produced by

0956-7135/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.foodcont.2013.03.016
434 M. Nakayama et al. / Food Control 33 (2013) 433e439
catechins in an alkaline environment reacts with cerium to form a
precipitate (3H2O2 þ2Ce3þ þ 6OH/2Ce(OH)3OOH þ 2H2O) that
further reacts with catechin to form a larger deposit (Nakayama,
Shigemune, Tsugukuni, Tokuda, & Miyamoto, 2010). We devel-
oped an electron microscopy technique for visualizing this cerium
deposit and were able to determine the localization of catechins on
a bacterial surface (Nakayama, Shigemune, Tsugukuni, Tokuda, &
Miyamoto, 2011). SDS-PAGE analysis of cell proteins showed that
catechins attached to proteins on the bacterial surface to form high-
molecular weight complexes, suggesting the possibility that cate-
chins inhibit the uptake and secretion of substrates, thereby
inhibiting enzyme activity (Nakayama, Shigemune, Tsugukuni,
et al., 2011).
In the present study, we analyzed the localization of EGCg on the
surface of Escherichia coli cells using the visualization technique
previously developed in our laboratory (Nakayama, Shigemune,
Tsugukuni, Tokuda, et al., 2011). In addition, total proteins ob-
tained from bacterial cells pretreated with EGCg were analyzed by
SDS-PAGE and two-dimensional electrophoresis to isolate and
identify the proteins that directly interact with EGCg.
2. Materials and methods
2.1. Bacterial strain and culture conditions
The bacterial strain used in this study was E. coli NBRC 3972. The
bacterium was cultured in tryptic soy broth (TSB; Becton, Dickinson
& Co., Franklin Lakes, NJ, USA) and soybean-casein digest (SCD) agar
(Nihon Pharmaceutical Co., Ltd., Tokyo, Japan) for preparing sam-
ples for two-dimensional (2-D) electrophoresis and for trans-
mission electron microscopy (TEM) and DOX analyses, respectively.
2.2. TEM examination
E. coli NBRC 3972 cells were collected after cultivation on SCD
agar at 35  C for 24 h and were suspended in 10 mM HEPES buffer
(pH 6.5) to a final cell concentration of 108 cfu/ml. EGCg dissolved at
2 g/L in 10 mM HEPES buffer (pH 6.5) and 0.45-ml aliquots of this
solution were added to 1.5-ml microtubes. To this solution, 0.05 ml
of the E. coli NBRC 3972 suspension was added, and the resulting
mixture was then incubated for 1 h at room temperature with
shaking. The suspension was then centrifuged at 14,000  g for
10 min and the pellets were washed three times with 0.5 ml of
10 mM HEPES buffer (pH 6.5). The EGCg-treated cells were then
suspended in 0.5 ml of 10 mM HEPES buffer (pH 8) containing 0.1%
CeCl3 and mixed thoroughly to allow the hydrogen peroxide pro-
duced by EGCg to react with cerium. After 15 min, the bacteria were
collected by centrifugation at 14,000  g for 10 min and washed
three times with 0.5 ml of 10 mM HEPES buffer (pH 7.0). The pre-
cipitate was suspended in 0.5 ml of 10 mM HEPES buffer (pH 7.0)
containing 2.5% glutaraldehyde and incubated at 4  C for 24 h for
fixation. The cells were harvested by centrifugation at 14,000  g
for 10 min and were washed twice with 0.5 ml of 10 mM HEPES
buffer (pH 7.0). The bacterial cells were dehydrated by serial
ethanol washing (0.5 ml each; 50%, 70%, 80%, 90%, 95%, 100%, and
100%) for 15 min at room temperature. Following dehydration,
0.02 ml of the bacterial suspension was aliquoted into BEEM
embedding capsules (Nissin EM Corp., Tokyo, Japan) and then
washed with 0.02 ml propylene oxide (Nissin EM Corp.). The cap-
sules were dried in a desiccator for 15 min, washed again with
0.02 ml propylene oxide, and dried again for 15 min; this procedure
was repeated three times to substitute the ethanol with propylene
oxide. Following propylene oxide substitution, 0.05 ml resin (Q-
615; Nissin EM Corp.) was added to the capsules. Samples were
degassed in a sonicator (Branson 5200; Yamato Scientific Co., Ltd.,
Tokyo, Japan) for 30 s and then dried in a desiccator under negative
pressure at room temperature for 48 h to embed each sample in the
resin. The resin was subsequently hardened by heating the capsules
in a block heater (Cool Thermo Unit CTU-N Neo; Taitec Co., Ltd.,
Tokyo, Japan) at 40  C for 24 h followed by 70  C for 48 h. The resin
was removed, and ultrathin sections (70e90 nm) were then cut
with a diamond knife (Sumi Knife; Okenshoji Co., Ltd., Tokyo,
Japan) using a microtome (Ultracut-S; Leica Microsystems, Tokyo,
Japan) and collected on copper mesh for TEM examination (Sheet
Mesh 150-A; Nissin EM Corp.). After drying, the mesh containing
the collected samples was examined under a TEM (H-7650; Hitachi
High-Technologies Corp., Tokyo, Japan).
2.3. 2-D electrophoresis
E. coli was cultured overnight in 100 ml TSB at 37  C with
shaking at 130 rpm. The bacterial culture was suspended in sterile
water at an OD660 of 1.0. Bacterial cells were harvested from 30 ml
of the resulting cell suspension by centrifugation at 6000  g for
10 min at room temperature. The cells were then resuspended in
300 ml sterile water to give a final concentration of approximately
1  1010 cfu/ml.
Three ml of EGCg dissolved in 0.1 M phosphate buffer was added
to 27 ml of 0.1 M phosphate buffer solution to give a 30 ml EGCg
solution at a final concentration of 1 g/L, which is the MIC against
E. coli. A 30 ml phosphate buffer solution was used as a control. The
bacterial suspension (300 ml) was added to the test and control
solutions to give a final concentration of 108 cfu/ml. The samples
were mixed thoroughly and then incubated at 37  C for 1 h. The
mixture was transferred to a FastPROTEINÔ BLUE tube containing
glass beads (MP Biomedicals, LLC), which were cooled in advance
to 20  C, and the bacterial cells were disrupted using a FastPrep-
24 Instrument (MP Biomedicals, LLC) at 6.0 m/s for 20 s. After
cooling the sample for 1 min on ice, the disruption was repeated a
second time and the supernatants were collected by centrifugation
at 10,000  g for 1 min at 4  C. The concentration of proteins in the
supernatant was measured using a Coomassie brilliant blue (CBB)
protein assay, and the supernatant was then diluted with 50 mM
phosphate buffer solution to give a concentration of approximately
3 mg protein/ml. The supernatant was filtered through a Millex-HV
Filter (Millipore Corporation, Billerica, MA, USA), and the filtrate
was used as a source of total cellular proteins. Protein samples used
for isoelectric focusing (IEF) were prepared as follows by reference
to protein purification method of Suzuki, et al (2011). Trifluoro-
acetic acid was added to 250 ml of the extracted proteins to give a
final concentration of 15%, and the precipitate was collected by
centrifugation (20,000  g, 15 min, 4  C). Cold ethanol/ether solvent
(1 ml) was added to the precipitate, and the solution was strongly
agitated. The precipitate collected from the suspension by centri-
fugation at 20,000  g for 20 min at 4  C was suspended in 1 ml
ethanol/ether solvent. After repeating the washing process three
times, the collected precipitate was dried at room temperature, and
then dissolved in 0.25 ml IEF lysis buffer (6 M urea, 2 M thiourea, 3%
CHAPS, 1% Triton X-100, and DeStreak reagent [GE Healthcare]) and
sonicated at 20 W for 45 min at room temperature. The IEF sample
thus prepared was centrifuged at 20,000  g for 20 min at room
temperature and the resulting supernatant was loaded onto an
Immobiline DryStrip gel (pH 3e10, non-linear, 13 cm; GE Health-
care) by swelling the gel for 5 h. IEF was performed at 5000 V for
15 h. The Immobiline DryStrip gel was then equilibrated in 2-D
electrophoresis sample buffer (6 M urea, 20% glycerol, 2% DTT, 2%
SDS, and 100 mM TriseHCl, pH 8.8) for 45 min. After equilibration,
electrophoresis of the Immobiline DryStrip gel was carried out
using a 10e18%, 16  16 cm polyacrylamide gradient gel (Bio Craft
Co., Ltd., Tokyo, Japan) at a constant current of 25 mA/strip. After
 M. Nakayama et al. / Food Control 33 (2013) 433e439
electrophoresis, the gel was stained for 16 h in 0.1% CBB G-250
solution and then decolorized in 5% acetic acid. Imaging was per-
formed with a GS-800 Calibrated Imaging Densitometer (Bio-Rad
Laboratories, Inc.). Spots that decreased to 1/3 or less compared to
control and those that disappeared as a result of EGCg treatment
were judged to be the spots of proteins that interacted with EGCg.
The proteins in these spots were identified by Peptide Mass
Fingerprinting (PMF) analysis or MS/MS ion searches. For the
analysis, the samples were enzymatically digested with trypsin and
then desalted and purified with a ZipTip C18 column (Millipore
Corp.) according to the protocol of the manufacturer.
The extracted samples were applied to a Matrix Assisted Laser
Desorption/Ionization (MALDI) plate and allowed to dry. A matrix
solution (5 mg/ml a-cyano-4-hydroxycinnamic acid [CHCA] in 0.1%
trifluoroacetic acid/50% acetonitrile) was applied to the sample and
after drying, mass spectrometry analysis was carried out using a
MALDI-TOF-MS fitted with a PMF 337-nm nitrogen laser (AXIMA
Performance; Shimadzu Corp.) in positive-ion reflectron mode. The
proteins were identified using the MASCOT search engine (Matrix
Science, Ltd.) against the National Center for Biotechnology Infor-
mation (NCBI) database.
In the MS/MS ion search, peptides were obtained in the same
way as for the PMF analysis. The peptide fragments were isolated
by nano-liquid chromatography (nano LC) and individual frag-
ments were then analyzed by mass spectrometry. Nano LC-MS/MS
analysis was performed using a DiNa-2A Nano LC system (KYA
Technologies, Tokyo, Japan) coupled online to a LCMS-IT-TOF mass
spectrometer (Shimadzu, Kyoto, Japan). Separation of peptides
was performed using a Pico Frit Beta Basic C18 column (New
Objective, USA) at a constant flow rate of 300 nl/min. Peptides
were eluted from the column with gradients of 2e40% solvent B
(0.1% formic acid in 80% ACN)/0e30 min, 40e100% solvent B/30e
40 min, and 100% solvent B/40e60 min. The LCMS-IT-TOF mass
spectrometer was operated in the data-dependent MS/MS mode
at scan ranges (Hyakkoku, Hamanaka, Tsuruma, Shimazawa, &
Hara, 2010).
2.4. Interaction between amino acids and EGCg
Twenty amino acids (Gly, Ala, Val, Leu, Ile, Phe, Thr, Cys, Met, Trp,
Tyr, Gln, Asp, Glu, Arg, His [Wako Pure Chemical Industries, Ltd.],
Pro, Ser, Asn [Kanto Chemical Co., Inc.], and Lys [Tokyo Chemical
Industry Co., Ltd.]) were added to separate solutions of 1% EGCg in
50 mM sodium phosphate buffer solution (pH 6.5) to give a final
amino acid concentration of 2%. After incubation for a fixed time at
25  C to allow reaction with EGCg, precipitate formation was
confirmed by the naked eye.
Fig. 1. TEM micrographs of Escherichia coli NBRC 3972 cells treated with EGCg. Bacteria were treated with EGCg in HEPES buffer at pH 6.5 for 1 h at room temperature. Following
centrifugation and washing, cells were treated with 0.1% cerium chloride at pH 8.0, sectioned, and examined by TEM. A, Ultra-thin section of bacteria treated with 0.50 mg/mL EGCg.
B, Magnification of the boxed area in A. Bar ¼ 100 nm.
435
2.5. Analysis of interaction between porin proteins and EGCg
The interaction between EGCg and porin proteins was analyzed
using the CDOCKER module of Discovery Studio software (Accelrys,
Inc.) on a PC running Windows XP. The crystal structures of the
porin proteins OmpC (Baslé, Rummel, Storici, Rosenbusch, &
Schirmer, 2006) and OmpG (Korkmaz-Özkan, Köster, Kühlbrandt,
Mäntele, & Yildiz, 2010) registered in the Protein Data Bank were
used for the calculations. Docking simulations were conducted
with EGCg positioned in the center of the inner part of the porin
pore. In this section of the pore, there are three residues of the basic
amino acid Arg, which reacts with EGCg, and the pore size is at its
narrowest.
2.6. Measurement of oxygen consumption rate accompanying
uptake of glucose
E. coli colonies that formed on SCD agar were collected by
scraping, suspended in a sterile saline solution, and aliquoted into
1.5-ml tubes (Sarstedt K.K., Tokyo, Japan). The bacterial cells were
washed by centrifugation and suspension in 1.0 ml of a sterile saline
solution. The resulting suspension was centrifuged to collect bac-
terial cells, which were then resuspended to approximately
3  109 cfu/ml in 1.0 ml of 10 mM HEPES buffer (pH 6.0) (Irvine
Scientific, Santa Ana, CA, USA) containing either 0.5 g/L EGCg or no
EGCg. The samples were incubated for 1 h at 35  C. After incubation,
the EGCg-treated and non-treated cells were collected by centri-
fugation and suspended in 1.0 ml of 10 mM HEPES buffer (pH 6.0).
The sample solutions (1 ml) were added to a DOX s-electrode cell
(Bio-Theta, Ltd., Osaka, Japan), to which 1 ml of a 0.2% glucose so-
lution in 10 mM HEPES buffer (pH 6.0) was added. The DOX cells
were promptly set in a DOX-60F cell cartridge with electrodes (Bio-
Theta, Ltd., Osaka, Japan) and the electric current due to dissolved
oxygen in the fluid of the DOX cell was measured using an oxygen
electrode.
3. Results
3.1. Identification of EGCg attachment sites on E. coli by TEM
E. coli cells treated with EGCg were reacted with cerium chlo-
ride, and ultrathin sections of bacterial cells embedded in resin
were then examined by TEM (Fig. 1). Deposits containing EGCg
were found only on the outer membrane, which is the outermost
layer of the cell surface. The deposits were unevenly distributed
across the outer membrane, and were attached to the cell surface in
sparse patches. No deposits were found inside of cells.
436 M. Nakayama et al. / Food Control 33 (2013) 433e439

3.2. 2-D electrophoresis Table 1

Identification of proteins on the membrane surface of Escherichia coli NBRC 3972
which were markedly down-regulated in response to EGCg treatment.
2-D electrophoresis patterns of bacterial cell proteins prepared
by disrupting EGCg-treated and non-treated bacterial cells are Gene ID Annotation tMWa tpIb
shown in Fig. 2. The migration patterns of the total cellular proteins no. of ortholog

were analyzed by imaging, which showed that numerous spots gij157160451 Outer membrane protein F (Porin) 39.9 4.8
gij6650193 Outer membrane protein OmpC (Porin) 40.4 4.5
present in the non-EGCg-treated cells had either disappeared or
gij170019471 Outer membrane porin protein C 41.4 4.6
showed a marked reduction in intensity in the EGCg-treated cells. gij12517874 Penicillin- binding protein 3 39.1 5.3
The proteins corresponding to these spots had likely formed de- gij16130858 Periplasmic L-asparaginase II 36.9 5.9
posits as a result of EGCg treatment and thus did not migrate in the Includes proteins whose spots were decreased in intensity by two-thirds or greater
gel. The 16 spots identified in the gel (Fig. 2) that had either reduced in the two-dimensional electrophoresis.
a
to one third or less of their original intensity or had completely Theoretical mass.
b
disappeared were selected. Of the 16 spots, proteins with a mo- Theoretical pI.
lecular mass of 20 kDa or less were identified by MS/MS ion
searches, while those with a molecular mass greater than 20 kDa
Table 2
were identified by PMF. A total of 64 proteins with significant scores EGCg precipitation reactions with individual amino acids.
were found, of which five are present on the cell surface (Table 1).
Amino Precipitation Amino Precipitation Amino Precipitation
Of these, only porin proteins were identified as outer membrane
acid acid acid
proteins. The cell membrane and periplasm proteins MreB, peri-
Gly e Ser e Gln e
plasmic asparaginase, and periplasmic oligopeptide-binding pro- Ala e Thr e Asp e
tein were also identified. Val e Cys e Glu e
Leu e Met e Lys þ
Ile e Trp e Arg þþþ
3.3. Interaction between amino acids and EGCg
Phe e Tyr e His þþ
Pro e Asn e
The reactivity of amino acids towards EGCg is shown in Table 2.
20 amino acids were added to a final concentration of 2% to a solution of 1% EGCg in
The amino acids that reacted strongly with EGCg to form a pre- 50 mM sodium phosphate buffer (pH 6.5) and allowed to react. After incubation for a
cipitate were the basic amino acids Lys, Arg, and His. fixed time at 25  C, each solution was checked for precipitate.
e, no reaction producing precipitate; þ, reaction producing trace amounts of
precipitate; þþ, reaction producing a precipitate; þþþ, reaction producing a strong
3.4. Interaction between EGCg and outer membrane porin proteins
precipitate.

Fig. 3 shows the three-dimensional structure of the E. coli porin

protein OmpG (Outer membrane protein G) revealed by X-ray crystal
and control groups with respect to the dissolved oxygen con-
analysis. The basic amino acids in the porin protein molecule are
sumption rate was proportionate to differences in the glucose
located in the inner surface of the pore and have side chains that
consumption rate. The effect of EGCg treatment on the oxygen
extend into the pore channel. The section of the pore channel where
consumption rate due to glucose uptake is shown in Fig. 5. The
the three Arg residues are present is particularly narrow. The results
oxygen consumption rate was lower in the EGCg-treated group
of the docking simulation show that EGCg entered the porin protein
than in the control cells, indicating that glucose uptake was lower
and was stably bonded to the Arg residues at the center of the pore
in the former group.
channel through a hydrogen bond, thus blocking the pore (Fig. 4).

3.5. Measurement of glucose uptake 4. Discussion

As the only nutrient source in the fluid in the DOX 60-F cell By TEM observation and 2-D electrophoresis analysis, we found
cartridge was glucose, the difference between the EGCg-treated that EGCg interacts with the channel of the E. coli outer membrane

Fig. 2. Two-dimensional electrophoresis gel images of proteins extracted from EGCg-treated (A) and untreated Escherichia coli NBRC 3972 cells (B). A, Cells treated with 1.0 mg/ml
EGCg; B, Untreated cells. Arrows indicate protein spots of lower intensity in the sample treated with 1.0 mg/ml EGCg compared to the untreated sample.
 M. Nakayama et al. / Food Control 33 (2013) 433e439
Fig. 3. Crystal structure of the porin protein OmpG of Escherichia coli K-12. The three-
dimensional structure of the porin protein was based on the crystal analysis
performed.
porin protein OmpG, thereby inhibiting the major function of the
porin, namely the transport of small hydrophilic molecules.
Numerous studies have examined the interaction between cate-
chins and bacterial proteins (Hernández et al., 1993) and have
found that EGCg interacts with lysine residues in casein and with a
nitrogen atom in caffeine (Aurélie et al., 2010). The results of the
present study have revealed that EGCg interacts most strongly with
basic amino acids and poorly with other types of amino acids. Based
on these findings, it seems reasonable to conclude that the hydroxyl
groups of EGCg interact with the nitrogen atom of basic amino acid
side chains through hydrogen bonding. Docking simulations also
showed that EGCg stably interacted with Arg residues localized on
the inner wall of the porin barrel through hydrogen bonding.
As EGCg appeared to block the pore channel of OmpG, it is
possible that the passive transport of small hydrophilic molecules
through the outer membrane was inhibited. Thus, we conducted a
glucose uptake analysis using the DOX system to detect changes in
the dissolved oxygen concentration of cell cultures. In the study on
the interaction of EGCg with cellular proteins or amino acids, the
ECGg treatment of bacterial cells was performed at pH 6.5, because
EGCg shows high antibacterial activity at this pH and attaches to
cells in large amounts, although H2O2 is only generated at low
levels. However, the glucose uptake analysis was performed at pH
6.0 to minimize the stress response by cells in response to H2O2
generated from EGCg molecules. The results of the DOX analysis
clearly indicate that cells treated with EGCg have a decreased rate
of oxygen consumption caused by the lower uptake of glucose
Fig. 4. Structure of OmpC (2J1N.pdb) of E. coli K-12 without (left) and with (right) a
docked EGCg molecule.
437
Fig. 5. Measurement of glucose uptake using a DOX-based assay. Glucose uptake by
Escherichia coli NBRC 3972 cells in 10 mM HEPES buffer (pH 6.0) treated with or
without 0.5 mg/ml EGCg was measured by monitoring changes in electric current
corresponding to dissolved oxygen consumption. :: Bacteria cultured in 10 mM
HEPES buffer containing 0.1% glucose and treated with 0.5 mg/ml EGCg; C: Bacteria
cultured in 10 mM HEPES buffer containing 0.1% glucose; O: Bacteria cultured in
10 mM HEPES buffer not containing glucose and treated with 0.5 mg/ml EGCg; B:
Bacteria cultured in 10 mM HEPES buffer without glucose.
compared to untreated cells. Notably, the reduction in oxygen
consumption was detected within 30 min after the EGCg treatment
was started, indicating X and Y.
According to the TEM analysis of EGCg-treated cells, no EGCg is
found intracellularly. However, we also identified an intracellular
protein that appeared to interact with EGCg based on the 2-D
electrophoresis analysis, presumably due to free EGCg that was
only absorbed weakly on the cell surface and was capable of
interacting and inhibiting the function of this intracellular protein
following disruption of the cells and preparation of total cellular
proteins. Outer and inner cell membrane damage of Pseudomonas
aeruginosa by tea polyphenol treatment has been reported by Yi,
Zhu, Fu., and Li (2010), who showed that proteins involved in the
TCA cycle (dihydrolipoamide dehydrogenase, succinyl Co-A syn-
thetase beta subunit) and protein synthesis (50S ribosomal protein,
Elongation factor-Ts, glycine cleavage system protein T2, chaperon
protein DnaJ, and polyamine transport protein) are down-regulated
in cells by such treatment, which may have induced a metabolic
disorder resulting in death. Cho, Oh, and Oh (2011) examined the
synergistic anti-bacterial effects of imipenem in combination with
EGCg for imipenem-resistant Klebsiella pneumoniae (IRKP) by 2-D
electrophoresis analysis and identified 12 down and up-regulated
proteins. It has been reported that intracellular and outer mem-
brane proteins involved in energy and DNA metabolism are down-
regulated in response to EGCg (Cho et al., 2011).
In contrast to the reports described above, our SDS-PAGE analysis
of proteins extracted from E. coli after treatment with EGCg iden-
tified proteins that did not migrate in the stacking or separation gels,
and which had decreased band intensity, even for proteins that
entered the gel. Based on the observed localization of EGCg on the
outer membrane surface (Fig. 1), combined with our previous re-
sults on the SDS-PAGE profiles of proteins from EGCg-treated cells, it
was presumed that incorporation and secretion of substances
through the outer membrane is inhibited following exposure to
EGCg. The results obtained by the substrate-uptake experiment
indicate the possibility that the incorporation of hydrophilic sub-
strates into E. coli cells is inhibited after treatment with EGCg (Fig. 5).
Several bacteria species, including E. coli, have two glucose up-
take pathways. One is a phosphotransferase system by which
glucose is phosphorylated and concomitantly transferred into the
cytoplasm. The second is a glucose dehydrogenase-linked
438 M. Nakayama et al. / Food Control 33 (2013) 433e439
Fig. 6. The presumed mechanism of antibacterial activity of EGCg in E. coli. (a) Porin functions as a pore for passive transport of small hydrophilic molecules across the outer
membrane. (b) EGCg interacts with basic amino acid residues localized on the inner wall of the pore formed by the porin protein, and results in the inhibition of the function of
porin. Consequently, EGCg seems to inhibit the uptake of hydrophilic nutrients such as glucose.
gluconate transport system, in which glucose is directly oxidized in
the periplasm and the produced electrons are transferred to the
respiratory chain to form electrochemical gradients across the
cytoplasmic membrane. As the protein spots related to glucose
uptake and metabolism were not changed in the 2-DE gels of EGCg-
treated cells, we conclude that the inhibition of glucose uptake was
due to the EGCg-mediated inhibition of transport of hydrophilic
low-molecular-weight substrates through the outer membrane,
and not by down-regulated transcription of the genes encoding
outer membrane proteins. The scheme of presumed mechanism of
antibacterial activity of EGCg in E. coli is showed in Fig. 6.
The effect of tannic acid (TA) on Lactobacillus plantarum was
previously evaluated using a 2-DE/MS approach (Cecconi et al.,
2009). In the study, a total of 15 protein spots from TA-treated
cells were found to be down-regulated and 21 were up-regulated,
indicating that TA affects proteins involved in several cellular and
metabolic pathways, including glycolysis, amino acid metabolism,
and protein translation and folding. Based on these findings, the
authors concluded that modulation of these specific pathways
correlates with the positive effect of TA on the survival of tannase-
positive L. plantarum. Additionally, Bossi et al. (2006) has reported
that several intracellular enzymes involved in metabolism are
down-regulated by TA in Lactobacillus hilgardii. Although these
results in Lactobacillus suggest that several intracellular proteins
are down-regulated by EGCg treatment, our present findings sug-
gest that EGCg only interacts with proteins in the outer membrane,
and does not interact with cytoplasmic proteins in the E. coli strain
analyzed in this study. We plan to investigate whether this obser-
vation was due to structural differences of the cell envelope be-
tween Gram-positive and Gram-negative bacteria. The results of
the 2-DE analysis suggest that EGCg reacted with the MreB protein
located in the peptidoglycan layers, although the exact quantity of
EGCg entering the peptidoglycan layers was unclear. A previous
study that treated the Gram-negative bacterium Salmonella typhi-
murium and Gram-positive bacterium Bacillus cereus with ECg and
EGCg showed that these two catechins altered the cell morphology
from long rods to short rods or cocci, a change that may have
resulted from disturbed cell division (Si et al., 2006). Thus, it is
highly possible that the MreB protein is one of the targets of EGCg.
In future studies, we plan to quantify the amount of EGCg that
enters into the periplasmic layer through outer membrane porin
proteins and clarify whether EGCg inhibits cell division. We will
also investigate the antibacterial mechanism of EGCg in Gram-
positive bacteria in the near future.
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