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Applied and Environmental Microbiology, September 1998, p. 3530-3532, Vol. 64, No. 9
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Influence of Lipid Composition on Pediocin PA-1
Binding to Phospholipid Vesicles
Yuhuan
Chen,
Richard D.
Ludescher, and
Thomas J.
Montville*
Department of Food Science, Cook College,
Rutgers, The State University of New Jersey, New Brunswick, New
Jersey 08901
Received 15 April 1998/Accepted 22 June 1998
 |
ABSTRACT |
Pediocin PA-1 bound to anionic lipid vesicles with saturated or
unsaturated fatty acid chains in a lipid concentration-dependent fashion. Little change in binding parameters was observed for zwitterionic lipid vesicles. Decreasing the anionic lipid content of
the vesicles gave a higher relative dissociation constant for the
peptide-lipid interactions and further supports the electrostatic interaction model of binding.
| |
TEXT |
Pediocin PA-1 is a class IIa
bacteriocin that contains a YGNGV consensus motif. It has 44 amino
acids with two disulfide bonds, which may account for its broad range
of antimicrobial activity (11, 12). Like other bacteriocins
from lactic acid bacteria, pediocin PA-1 acts at the cytoplasmic
membranes of target cells through a multistep process of binding,
insertion, and pore formation (1, 6, 10, 16). How various
factors, such as pH, membrane potential, and lipid composition,
influence poration is a major focus of research on bacteriocins,
especially nisin, a class I bacteriocin (3, 7, 8, 14, 15).
Studies which have focused on the charge of the peptide demonstrated
that pediocin PA-1 functions in the absence of a protein receptor and
that the initial binding step is mediated by electrostatic interactions between positively charged amino acid residues in the peptide and
negatively charged phospholipids in the target membrane (4, 5). In this study, we examined the influence of lipid composition on the initial binding step, and we present further evidence to support
the electrostatic interaction model.
Homogeneous lipid vesicles were prepared by the extrusion method, as
previously described (4). The phospholipids (Avanti Polar
Lipids, Alabaster, Ala.) used were dimyristoyl-phosphatidylglycerol (DMPG), dimyristoyl-phosphatidylcholine (DMPC),
dioleoyl-phosphatidylglycerol (DOPG), and dioleoyl-phosphatidylcholine
(DOPC). Binding of pediocin PA-1 (a kind gift of P. Vandenbergh and
J. Henderson, Quest International, Sarasota, Fla.) to lipid
vesicles with different compositions was determined as described
previously (4). Changes in the fluorescence of tryptophan
residues were measured by titrating a fixed concentration of peptide
(3.0 or 3.75 µM) with increasing amounts of lipid vesicles.
Tryptophan emission spectra were recorded at room temperature with a
spectrofluorometer (model F1T11; Spex Industries, Metuchen, N.J.). Each
emission spectrum was analyzed to determine the maximum emission
wavelength (
max). A change in max (blue
shift) upon addition of lipid vesicles was determined as follows:
max = maxo 
max, where the subscript "o" denotes the
parameter in the absence of lipid vesicles. In some cases, the maximum
intensity increase (I/Io) was determined, where
Io and I were the intensity of the
spectrum in buffer and in the presence of a saturating amount of lipid
vesicles, respectively.
Pediocin PA-1 binding to anionic lipid vesicles.
The binding
of pediocin PA-1 to DOPG vesicles with negatively charged head groups
is shown in Fig. 1A (curve a). An
increase in blue shift (or a decrease in max) reflects
the translocation of a tryptophan residue(s), and thus the binding of a
peptide, to a lipid membrane(s) (4, 13, 14, 17). Upon
addition of DOPG vesicles to a fixed concentration of pediocin PA-1,
there was a blue shift in max for the tryptophan
emission spectrum. As progressively higher concentrations of DOPG
vesicles were added, there was a greater blue shift in
max. This indicated that progressively more pediocin
PA-1 molecules became bound to the vesicles. The binding pattern of
pediocin PA-1 for DOPG vesicles was similar to that for DMPG vesicles
reported previously (Fig. 1B, curve a). The binding curves reached a
plateau as the lipid/pediocin ratio increased.
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FIG. 1.
Influence of lipid composition on binding of pediocin
PA-1 to lipid vesicles. (A) DOPG ( ) and DOPC ( ). (B and C) Molar
ratios of DMPG to DMPC were 1:0 (), 1:1 ( ), 1:3 ( ), and 0:1
(). In panels A and B, the x-axis was calculated as the
ratio of total lipid concentration to peptide concentration. In panel
C, the x-axis was calculated by using the anionic lipid
concentration instead of the total lipid concentration in curves b and
c, while curves a and d were the same as curves a and d in panel B.
|
|
Pediocin PA-1 binding to zwitterionic lipid vesicles.
There
was little blue shift upon addition of PC vesicles with zwitterionic
head groups to pediocin PA-1 (Fig. 1A and B, curves d). The spectra of
pediocin PA-1 were recorded at room temperature. Figure 1A (curve d)
shows the binding of pediocin PA-1 to DOPC vesicles in a liquid
crystalline phase (melting temperature [Tm] = 20°C), and Fig. 1B (curve d) shows binding to DMPC vesicles around
the transition temperature (Tm = 23°C)
(2). Regardless of the saturation state of lipid chains,
incubation of pediocin PA-1 and PC vesicles did not change the
fluorescence emission maximum for lipid/pediocin molar ratios as high
as 250. Although there are no reports for other class IIa bacteriocins,
data for the class I bacteriocin nisin show no significant binding to
zwitterionic PC vesicles at lipid/nisin ratios up to 50 (9).
Pediocin PA-1 binding to lipid vesicles with mixed head
groups.
To further examine the influence of lipid composition on
pediocin PA-1-membrane interactions, binding titration curves were determined for lipid vesicles prepared by using a mixture of DMPG and
DMPC at PG-to-PC molar ratios of 1:1 (Fig. 1B, curve b) and 1:3 (Fig.
1B, curve c). The final extent of the blue shift decreased as DMPG
content decreased. Furthermore, the magnitude of blue shift increased
markedly at low lipid concentrations for vesicles containing 100% DMPG
and less markedly for vesicles containing 50 and 25% DMPG.
Binding parameters.
Based on previously described procedures
(4, 14), Fig. 2 illustrates
the determination of Kd/n (where n is
the number of binding sites per lipid molecule), the relative
dissociation constant, for DOPG vesicles. The nonlinear plot was
analyzed with KaleidaGraph version 3.09 (Synergy Software, Reading,
Pa.). Other binding curves were analyzed similarly to obtain
Kd/n values.
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FIG. 2.
Determination of Kd/n. The datum
points derived from Fig. 1A (curve a) were fitted by using the equation
( 1) = (b 1)m/(Kd/n + m). was defined as maxo/max,
and m was the total lipid concentration.
|
|
The anionic lipid content of the vesicles influenced the relative
dissociation constant, Kd/n, and the maximum
blue shift, max (Table
1). A higher max
indicates a more hydrophobic environment for the tryptophan residues,
which reflects a deeper insertion of the peptide into the membrane;
meanwhile, a smaller Kd/n indicates a stronger
affinity of the peptide for the membrane (4, 13, 14).
Binding of pediocin PA-1 to the vesicles with different lipid
compositions correlates well with the amount of PG in the vesicles: the
maximum blue shift was highest for vesicles containing 100% DMPG, and
as DMPG content decreased to 50 and 25%, there was a progressively
smaller maximum blue shift. There was no change in
max upon addition of zwitterionic lipid vesicles, which strongly suggests that anionic lipids are essential to induce the
fluorescence parameter changes associated with binding in pediocin
PA-1. The influence of anionic lipid content on tryptophan fluorescence
intensity followed the same pattern, where the amount of intensity
increase was lower for the vesicles with a smaller molar ratio of DMPG
to DMPC, and little change was observed upon addition of PC vesicles
(data not shown).
The Kd/n for vesicles with 50% DMPG was more
than an order of magnitude higher than that for the 100% DMPG vesicles
(Table 1, column 3). On the other hand, the Kd/n
for vesicles with 25% DMPG was fivefold higher than that for 100%
DMPG vesicles. This suggests that pediocin PA-1's affinity for the
vesicles with 25% DMPG is weaker than that for pure DMPG vesicles but
stronger than that for vesicles with 50% DMPG. These results were
obtained based on the premise that m is conventionally
defined as total lipid concentration. However, the binding titration
curves in Fig. 1A and B clearly indicate that the zwitterionic lipids
could not induce a blue shift. Therefore, it may be more appropriate to use PG alone, instead of the combination of PG and PC, as the lipid
concentration.
The titration curves in Fig. 1C, where lipid concentrations included
only anionic lipid, show binding patterns similar to those in Fig. 1B,
except that saturation of binding occurred at a lower ratio of lipid
concentration to peptide concentration. Based on the curves in Fig. 1C,
the Kd/n for the vesicles containing 50% DMPG
was about sixfold higher than that for 100% DMPG vesicles, while the
constant for vesicles with 25% DMPG was only slightly increased (Table
1, column 4). These results suggest that the presence of a small amount
of DMPG contributed significantly to the binding of pediocin PA-1 to
mixed DMPG-DMPC vesicles. While a larger amount of DMPG in these
vesicles might cause better insertion of pediocin PA-1 into the lipid
bilayer (a bigger max), the affinity of pediocin PA-1
for the 50% DMPG vesicles was weaker than that for pure DMPG vesicles.
The increase in the Kd/n value when DMPG content
increased from 25 to 50% might reflect a change in the parameter
n rather than the dissociation constant,
Kd, per se. In addition, vesicles with different
lipid compositions might have heterogeneous surfaces, due to phase
segregation, that could also account for the trend in
Kd/n. Although these data clearly indicate that
anionic lipid is required for the binding of pediocin PA-1 to lipid
vesicles as measured by changes in tryptophan fluorescence parameters,
there is a complex aspect of the peptide-membrane interaction which
remains elusive.
The Kd/n values for DMPG and DOPG vesicles were
the same. This suggests that pediocin PA-1 has a similarly strong
affinity for both types of anionic lipid vesicles. Compared to the
changes caused by lipid head groups, the saturation state of the fatty acid chains has minor, if any, influence on pediocin PA-1 binding. The
blue shift in the presence of saturating amount of DMPG was higher than
that with DOPG (Table 1). In addition, the binding of pediocin PA-1 to
saturating amount of DMPG vesicles caused a 30.3% increase in
fluorescence intensity, while for DOPG vesicles the increase was
17.2%. These results indicated a less hydrophobic environment for the
tryptophan residue(s) of DOPG-bound pediocin molecules than for those
of DMPG-bound molecules (4, 13, 17). The matrix of DOPG
fatty acid chains may provide a less hydrophobic environment than that
of DMPG due to a higher fluidity (Tm of DOPG is
lower than that of DMPG), which allowed water molecules to diffuse
further into the matrix. In addition, double bonds in oleoyl chains
(18:1c9; DOPG) are less hydrophobic than their single bond counterparts
in myristoyl chains (14:0; DMPG).
We reported previously that the net positive charge of pediocin PA-1's
N-terminal fragments parallels their binding to DMPG vesicles
(4). Altering the membrane vesicle composition modifies the
charge properties of the target, instead of the properties of the
peptides. Both types of manipulation give similar information about how
electrostatic interactions modulate bacteriocin binding to membranes.
Results from the present study demonstrated that binding of pediocin
PA-1 to the membrane strongly depended on the negative charge on the
membrane surface. Similar findings have been reported for other
bacteriocins. The binding of the cationic class I bacteriocin epilancin
K7 to DOPG vesicles involves electrostatic interactions (8).
Nisin binds strongly to dipalmitoyl-phosphatidylglycerol and DOPG
vesicles but very weakly to PC vesicles with the same fatty acid
(9). Furthermore, the proportion of anionic phospholipids in
the membrane is a major determinant for nisin insertion and pore
formation. Nisin penetrates more strongly into monolayers of anionic
phospholipids than those of zwitterionic phospholipids (7).
In lipid vesicles with varying ratios of anionic and zwitterionic phospholipids, nisin-induced release of vesicular contents increases with an increasing proportion of anionic phospholipid (3). Results from this study provide the first evidence that the initial binding step for pediocin PA-1, a class IIa bacteriocin, displays a
similar anionic lipid dependency.
In conclusion, the lipid composition of the target membrane plays an
important role in modulating pediocin PA-1 action. A higher content of
negatively charged phospholipids increases the affinity of pediocin
PA-1 for the membrane. Together with previous evidence obtained by
altering the charge properties of the pediocin molecules, this makes a
compelling case that electrostatic interactions are responsible for the
initial binding of pediocin PA-1 to target membranes.
| |
ACKNOWLEDGMENTS |
Research in the authors' laboratory and preparation of the
manuscript were supported by state appropriations, U.S. Hatch Act Funds, and the U.S.-Israel Binational Agricultural Research and Development Fund (no. US-2113-92).
We thank K. Schaich for the use of instruments in her laboratory and Z. Rajfur for assistance with spectrofluorometer operations.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Food Science, Cook College, Rutgers, The State University of New
Jersey, 65 Dudley Rd., New Brunswick, NJ 08901. Phone: (732) 932-9611, ext. 201. Fax: (732) 932-6776. E-mail:
montville{at}aesop.rutgers.edu.
Report D-10131-1-98 of the New Jersey Agricultural Experiment
Station.
| |
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Applied and Environmental Microbiology, September 1998, p. 3530-3532, Vol. 64, No. 9
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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