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Applied and Environmental Microbiology, April 2001, p. 1689-1692, Vol. 67, No. 4
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.4.1689-1692.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Enterocin P Selectively Dissipates the
Membrane Potential of Enterococcus faecium
T136
C.
Herranz,1
Y.
Chen,2
H.-J.
Chung,2
L. M.
Cintas,1
P. E.
Hernández,1
T. J.
Montville,2 and
M. L.
Chikindas2,*
Departmento de Nutrición y
Bromatología III, Facultad de Veterinaria, Universidad
Complutense, 28040 Madrid, Spain,1 and
Department of Food Science, Rutgers, The State University
of New Jersey, New Brunswick, New Jersey 089012
Received 25 September 2000/Accepted 5 February 2001
 |
ABSTRACT |
Enterocin P is a pediocin-like, broad-spectrum
bacteriocin which displays a strong inhibitory activity
against Listeria monocytogenes. The bacteriocin was
purified from the culture supernatant of Enterococcus faecium P13, and its molecular mechanism of action against the sensitive strain E. faecium T136 was evaluated. Although
enterocin P caused significant reduction of the membrane potential
(
) and the intracellular ATP pool of the indicator organism, the pH gradient (pH) component of the proton motive force (p) was not
dissipated. By contrast, enterocin P caused carboxyfluorescein efflux
from E. faecium T136-derived liposomes.
| |
INTRODUCTION |
Bacteriocins produced by
bacteria are a heterogeneous group of ribosomally synthesized
antimicrobial proteins, which display antimicrobial activity against
other bacteria (16, 18, 29). The genus
Enterococcus is among the lactic acid bacteria (LAB) associated with foods that produce bacteriocins (2, 4, 6, 11,
12). Enterococcus faecium P13 and other E. faecium strains isolated from dry-fermented sausages produce
enterocin P, a bacteriocin with strong inhibitory activity
against Listeria monocytogenes (11,
14). Enterocin P is a pediocin-like bacteriocin
(27) with an N-terminal signal peptide which may allow it
to be secreted via the sec pathway (for a review, see reference
30). The enterocin P operon has been previously
characterized (11), and it consists of the bacteriocin
structural gene (entP) encoding a 71-amino-acid precursor
with a 27-amino-acid signal peptide and has a second open reading frame
(orf2) located immediately downstream of entP and
potentially encoding the immunity protein.
The increasing interest of consumers in minimally processed,
naturally preserved foods has prompted the proposal to use
bacteriocinogenic LAB or their bacteriocins as biopreservatives
to increase microbiological safety (19, 25, 31). However,
rational application of the bacteriocins requires an understanding of
the mechanisms underlying their specificity and activity
(24, 26), as well as knowledge of the structure-function
relationships, to develop new compounds with an improved
efficacy. In this context, the objective of this study was to
examine the mechanistic action of enterocin P on the sensitive strain
E. faecium T136.
| |
MATERIALS AND METHODS |
Bacterial strains and growth conditions.
E.
faecium P13 (11) and E. faecium T136
(6) were propagated in MRS broth (Difco Laboratories,
Detroit, Mich.) at 30°C and used as the enterocin P producer and
indicator microorganisms for bacteriocin activity, respectively.
Purification of enterocin P.
The bacteriocin was purified
from the supernatant of a 16-h E. faecium P13 culture by
hydrophobic interaction and cation-exchange chromatographies, basically
as described by Casaus et al. (6). Next, the sample was
dialyzed against distilled water in dialysis tubing (Spectrum, Houston,
Tex.; molecular weight cutoff, 1,000) and concentrated with
polyethylene glycol to half of its original volume. This sample was
further purified by preparative isoelectric focusing using a Rotofor
cell (Bio-Rad Laboratories, Melville, N.Y.), as previously described
(8, 10). Fractions displaying the highest antimicrobial
activity were pooled together, subjected to ultrafiltration
(Centricon-3 concentrators; Amicon; molecular weight cutoff, 3,000),
and stored at 4°C until used.
Activity assays.
The antimicrobial activity of the fractions
obtained throughout the purification process was calculated by a
microtiter plate assay performed basically as described by Holo et al.
(15). Briefly, 96-well plates containing 50 µl of
twofold diluted fractions and 150 µl of the freshly diluted indicator
organism (2.5 × 106 CFU ml
1) per well
were incubated at 30°C for 16 h. Growth inhibition was
measured spectrophotometrically at 620 nm with a microtiter plate
reader (Dynatech MR5000; Dynatech Laboratories), and bacteriocin activity was calculated in bacteriocin units (BU). One BU was defined
as the reciprocal of the highest dilution of bacteriocin causing 50%
growth inhibition (50% of the turbidity of the control culture without bacteriocin).
Measurements of proton motive force.
The membrane
potential () of E. faecium T136 cells was
qualitatively measured with the fluorescent probe
3,3'-dipropylthiadicarbocyanine iodide
[(DiSC3(5)] (Molecular Probes Inc., Eugene,
Oreg.). Cells were harvested in the log phase (optical density at 660 nm [OD660], 0.6), washed twice with ice-cold 50 mM
potassium HEPES (K-HEPES) buffer, pH 7.0, resuspended in the same
buffer to 1/100 of their initial volume, and stored on ice.
Glucose-energized E. faecium T136 cells (final
OD660, 0.3) were added to a stirred cuvette containing 2 ml
of the K-HEPES buffer and DiSC3(5) (5 µM).
Next, nigericin (1.5 nM), which dissipates the pH gradient (pH), and
enterocin P (200 BU/ml) or valinomicyn (1.5 nM) were added.
Fluorescence measurements were performed with a F1T11
spectrofluorometer (Spex Industries, Metuchen, N.J.) with a band-pass
width of 10 nm and wavelengths of 643 and 666 nm for excitation and
emission, respectively.
The transmembrane pH was measured by loading E. faecium
T136 cells (OD660, 0.6) with the fluorescent probe
2',7'-bis-(2-carboxyethyl)-5[and 6]-carboxyfluorescein acetoxymethyl
ester (BCECF AM) (Molecular Probes Inc.) by using an acid shock, as
described by Molenaar et al. (21). Glucose-energized,
BCECF-loaded cells (final OD660, 0.15) were added to a
stirred cuvette containing 2 ml of 50 mM KPi buffer, pH
6.0. Next, valinomycin (1.5 nM), which dissipates the , and
enterocin P (200 BU/ml) or nigericin (1.5 nM) were added. Fluorescence
was measured with band-pass widths of 5.0 and 15.0 nm and wavelengths
of 643 and 666 nm for excitation and emission, respectively.
Measurement of ATP.
E. faecium T136 cells grown
to an OD660 of 0.6 were collected by centrifugation, washed
once with 50 mM 2-(N-morpholino)ethanesulfonate (MES)
buffer, pH 6.5, and kept on ice until use. In order to energize the
cells prior to ATP measurements, they were resuspended to half of the
original volume in 50 mM MES buffer with 0.2% glucose and incubated
for 20 min. After treating the cells with 5, 20, 50, and 100 BU of
enterocin P/ml, total and external cellular ATP levels were determined
using the bioluminescence method described by Chen and Montville
(7) and an ATP bioluminescence assay kit (Sigma Chemical
Co., St. Louis, Mo.). Intracellular ATP was calculated by subtracting
external from total ATP. The assays were calibrated by using a standard
curve obtained by measuring the bioluminescence of ATP solutions of
known concentrations, and ATP levels were expressed as nmol
mg1 of cells (dry weight). Values are the mean of
two independent bioluminiscence measurements.
Lipid extraction, preparation of CF-loaded liposomes, and CF
leakage assay.
E. faecium T136 cells were grown to
mid-log phase (OD660, 0.7 to 0.8), harvested, and washed
with 0.1% peptone water. Total E. faecium T136 lipids were
extracted following the procedure of Bligh and Dyer (as described
by New in reference 28) with the modifications introduced
by Winkowski et al. (35). Extracted lipids were kept at
20°C in glass vials and were used within 2 weeks. Next, large
unilamellar vesicles composed of lipids from E. faecium T136
and loaded with 6-carboxyfluorescein (CF) (Sigma Chemical Co.) were
prepared as described by Chen et al. (8). CF-loaded
liposomes were stored on ice and used within 3 h.
The effect of enterocin P (90 and 450 BU/ml) on CF-loaded
E. faecium T136-derived liposomes was determined by monitoring the
fluorescence of the liposomal suspension upon bacteriocin addition.
Fluorescence measurements were performed with a band-pass width
of 0.8 nm and wavelengths of 516 and 490 nm for emission and excitation,
respectively. Results were expressed as the percentage of CF release,
calculated from the following equation: % efflux = (F
tF
0)/(F
F
0),
where F
t was the fluorescence at time
t,
F
0 was the control fluorescence
at time
t, and
F
was the fluorescence after the addition
of Triton
X-100 (10% [vol/vol] in distilled H
2O). The fluorescence
values were corrected by subtracting the fluorescence of the control
samples treated with 50 mM MES buffer instead of
bacteriocins.
| |
RESULTS |
Effect of enterocin P on and pH.
The effect of
enterocin P on the was qualitatively determined as quenching of
the fluorescent probe DiSC3(5) from the cells
treated with the bacteriocin (Fig. 1).
After glucose addition, an increase in the fluorescence occurred as a
result of the net proton extrusion by the membrane-bound
F0F1-ATPase. Upon addition of enterocin P to
energized, nigericin-treated cells, an increase in fluorescence
intensity was observed, which was an indication of a decrease in
internal membrane potential. After a short initial period, the
fluorescence increase induced by enterocin P was greater than that
provoked by valinomycin.
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FIG. 1.
Effect of enterocin P (200 BU/ml) (a) and valinomycin
(1.5 nM) (b) on the of E. faecium T136 cells.
Fluorescence levels before the addition of enterocin P or valinomycin
were arbitrarily designated zero, and the increase in fluorescence upon
the addition of bacteriocin or ionophore was expressed in arbitrary
units (a.u.).
|
|
Changes in intracellular pH caused by the addition of enterocin P to
the cellular suspension were qualitatively recorded by
measuring the
fluorescence of the probe BCECF AM (Fig.
2). After
the cells were energized, the
addition of valinomycin dissipated
the
and resulted in a
fluorescence increase that was maintained
until the addition of
nigericin, which rapidly reversed the pH
to a level equal to that of
the medium pH. The addition of 200
BU of enterocin P ml
1
did not dissipate
pH. Furthermore, an increase in the pH gradient
was observed upon bacteriocin treatment, which was more pronounced
in
cells that had not been previously treated with valinomycin.
Higher
enterocin P concentrations (i.e., 350 BU ml
1 [results
not shown]) produced similar behavior, i.e., a transient
increase
preceding the fluorescence decrease.
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FIG. 2.
Effect of enterocin P (200 BU/ml) on the pH of
valinomycin-treated (a) or untreated (b) E. faecium T136
cells and of nigericin (1.5 nM) (c). Fluorescence levels before the
addition of enterocin P or nigericin were arbitrarily designated zero,
and the variation in fluorescence upon the addition of bacteriocin or
ionophore was expressed in arbitrary units (a.u.).
|
|
Enterocin P depleted intracellular ATP levels of E. faecium T136 cells.
Upon energization, ATP levels of
E. faecium T136 cells increased to a mean value of 7.2 nmol
mg1 of cells (dry weight). This value was similar to that
reported by Chen and Montville (7) for energized L. monocytogenes cells. Enterocin P depleted intracellular ATP in a
time- and concentration-dependent manner (Fig.
3). After 20 min of treatment, ATP levels
were 39, 14, and 0.2% of the original ATP concentrations for cells
that had been treated with 5, 20, and 50 BU ml1,
respectively. Bacteriocin concentrations higher than 50 BU
ml1 (i.e., 100 BU ml1; data not shown) did
not induce any further intracellular ATP depletion. No significant
appearance of ATP was found in the external medium at any of the
enterocin P concentrations tested.
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FIG. 3.
Intracellular ATP levels of E. faecium T136
cells treated with 5 ( ), 10 ( ), and 50 ( ) BU of enterocin P/ml
and untreated ( ). No extracellular ATP was detected (data not
shown).
|
|
Enterocin P-induced CF efflux from E. faecium
T136-derived liposomes.
Exposure of E. faecium
T136-derived liposomes to enterocin P caused a gradual release of CF in
a concentration- and time-dependent fashion (Fig.
4). CF efflux plateaued earlier at the
lower enterocin P concentration (90 BU ml1). After a
600-s treatment, the percentages of CF efflux were 19 and 33% for 90 and 450 BU ml1 of enterocin P, respectively.
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FIG. 4.
Effect of 90 (a) and 450 (b) BU of enterocin P/ml on
E. faecium T136-derived liposomes. The CF efflux is
expressed as the percentage of the release induced by Triton X-100
(0.5%, wt/vol).
|
|
| |
DISCUSSION |
It is generally accepted that LAB bacteriocins act by
altering the permeability barrier of the cell membrane (13, 22, 33, 34, 35) and that one of the common mechanisms of inhibition of their target cells is the dissipation of the proton motive force
(PMF) (1, 5, 25). Since enterocin P is an amphipathic, cationic peptide (11), it may interact with the negatively
charged bacterial membranes of the sensitive cells and alter
their properties. In order to check this hypothesis, the components of
the PMF were measured in E. faecium T136 cells treated with
enterocin P. Enterocin P efficiently dissipated the of
nigericin-treated cells; the fact that the bacteriocin-induced
dissipation was greater than the valinomycin-induced one may
indicate that mechanisms different from the simple outward diffusion of
the dye in response to the decrease in could be
involved, for example, several degrees of membrane disruption.
When the same concentration of enterocin P was used, pH dissipation
of valinomycin-treated or untreated E. faecium T136 cells was not observed. Moreover, a slight increase in pH was recorded, which was greater in cells not treated with valinomycin. This contrasts
with the observation that, generally, the pH component of the PMF is
dissipated earlier than the one (5, 17, 32).
However, like enterocin P, lactococcin G, a monovalent cation-conducting bacteriocin (22, 23), and lacticin 3147 (20) exert a selective dissipation of and may cause
an increase in pH. Moll et al. (23) explained this
effect by the enhanced H+ extrusion by the
F0F1-ATPase as a consequence of
dissipation. In the case of lacticin 3147, a slow decrease in pH was
observed after its increase. This may be a secondary effect of the
diminished intracellular ATP pool, which was depleted in an attempt to
maintain the PMF (20).
The energetic state of the cells treated with enterocin P was evaluated
by measuring their ATP levels. The addition of enterocin P to energized
E. faecium T136 cells induced intracellular ATP depletion
without ATP efflux. This effect has been reported for several class II
bacteriocins, such as lactacin F (1), pediocin PA-1
(7), lactococcin G (22), and mundticin
(4). The ATP negative charge and size
(Mw, 507) may play a role in determining its
inability to pass through the pore. The fact that internal ATP may be
depleted in the absence of ATP efflux suggests that it is hydrolyzed
inside the sensitive cells. Two mechanisms have been proposed to
explain the hydrolysis: (i) a shift in the equilibrium of the ATP
hydrolysis reaction as a consequence of Pi loss through the
membrane with impaired permeability (1) or (ii)
accelerated hydrolysis due to the cell's attempt to regenerate the
electrochemical gradient by H+ extrusion driven by the
F0F1-ATPase energy-consuming pump (7, 23).
Finally, the effect of enterocin P in E. faecium
T136-derived liposomes was determined. The bacteriocin induced CF
efflux in liposomes in a time- and concentration-dependent fashion and in the absence of a PMF. CF release showed saturation kinetics and
plateaued far from 100% efflux. This suggests that the
permeabilization effect of enterocin P is transient in contrast with
the "all or none" disruptive action of detergent peptides. The
bacteriocin acts on liposomes in an energy-independent fashion.
The fact that enterocin P was able to act in a liposomal, protein-free
system indicated that a receptor of a proteinaceous nature is not
absolutely essential for bacteriocin action. The requirement of a
receptor-like factor to exert antimicrobial activity has been suggested
for the lactococcins A (33), B (34), and G
(22) and some other class II bacteriocins. Although the
broad antimicrobial spectrum of enterocin P (11) supports
the evidence that a protein receptor is not a requirement for the
activity, the activity enhancement in vivo by some proteinaceous
components of the membrane or cell surface, or even the whole membrane
constitution, should not be discarded (3, 9, 22).
Further studies will establish the ion specificity and the molecular
mechanism of pore formation for enterocin P, including energy
requirement and lipid dependence.
| |
ACKNOWLEDGMENTS |
Research in our laboratory and the preparation of the manuscript
were supported by the U.S. Department of Agriculture CSREES NRI Food
Safety Program (99-35201-8611), and other state and federal support was provided by the New Jersey Agricultural Experiment Station and by AGL2000-0707, Spain.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Food Science, Rutgers, The State University of New Jersey, 65 Dudley Rd., New Brunswick, NJ 08901-8520. Phone: (732) 932-9661, ext. 218. Fax: (732) 932-6776. E-mail: tchikindas{at}aesop.rutgers.edu.
| |
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Applied and Environmental Microbiology, April 2001, p. 1689-1692, Vol. 67, No. 4
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.4.1689-1692.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
