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Previous Article | Next Article 
Antimicrobial Agents and Chemotherapy, December 2000, p. 3317-3321, Vol. 44, No. 12
0066-4804/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Interactions of Bacterial Cationic Peptide
Antibiotics with Outer and Cytoplasmic Membranes of
Pseudomonas aeruginosa
Lijuan
Zhang,
Pawandeep
Dhillon,
Hong
Yan,
Susan
Farmer, and
Robert E. W.
Hancock*
Department of Microbiology and Immunology, University
of British Columbia, Vancouver, British Columbia, Canada V6T 1Z3
Received 17 March 2000/Returned for modification 5 July
2000/Accepted 11 September 2000
 |
ABSTRACT |
Polymyxins B and E1 and gramicidin S are bacterium-derived cationic
antimicrobial peptides. The polymyxins were more potent than gramicidin
S against Pseudomonas aeruginosa, with MICs of 0.125 to
0.25 and 8 µg/ml, respectively. These peptides differed in their
affinities for binding to lipopolysaccharide, but all were able to
permeabilize the outer membrane of wild-type P. aeruginosa PAO1 strain H103, suggesting differences in their mechanisms of self-promoted uptake. Gramicidin S caused rapid depolarization of the
bacterial cytoplasmic membrane at concentrations at which no killing
was observed within 30 min, whereas, conversely, the concentrations of
the polymyxins that resulted in rapid killing resulted in minimal
depolarization. These data indicate that the depolarization of the
cytoplasmic membrane by these peptides did not correlate with bacterial
cell lethality.
| |
INTRODUCTION |
Gramicidin S and the polymyxins are
nonribosomally produced peptides obtained from bacteria. They have
achieved widespread usage as topical agents (4). Gramicidin
S is a dibasic cyclic decapeptide with a two-stranded antiparallel
-sheet structure with the strands interconnected by two type II
-turns (4, 6). The polymyxins, including polymyxin B and
colistin (a mixture of polymyxins E1 and E2), are a family of closely
related pentabasic peptide antibiotics containing a cycloheptapeptide
ring with a C-8 or C-9 fatty acid attached through an amide bond
(17). Both classes of antibiotics are active against many
gram-negative and a few gram-positive microorganisms (13,
15). Colymycin M, a derivative of colistin with the positive
charges neutralized by methane sulfonate, has been developed as an
anti-Pseudomonas aeruginosa prodrug that is being used in
aerosol formulations to treat patients with cystic fibrosis
(8). Although these antibiotics were discovered more than 50 years ago, their mode of action is still not precisely known. A number
of studies have suggested that polymyxins (4, 18), related
octapeptins (14), and gramicidin S (6) can act on
bacterial cytoplasmic membranes, although many of these studies,
especially those performed with the polymyxins and octapeptins, were
done at high multiples of the MIC. Polymyxin B was also shown to have
pleiotropic effects on respiration, uptake, and efflux of
-methylglucopyranoside and RNA and DNA synthesis (13,
20).
Gramicidin S and the polymyxins share two unique features with the
antimicrobial peptides from animals, plants, and insects in that they
are polycationic and amphipathic (4, 5). Such characteristics have been thought to contribute to the mechanism of
killing of gram-negative bacteria by cationic antimicrobial peptides by
promoting the initial interaction with the negatively charged surface
molecule lipopolysaccharide (LPS), leading to self-promoted uptake
across the outer membrane and subsequently promoting the interaction
with and insertion into the negatively charged cytoplasmic membrane of
bacteria (4, 20). This can lead to membrane perturbation and
probably the translocation of the peptide across the membrane
(21). The actual mechanism of action is not yet fully
understood but has been proposed variously to involve cell lysis,
breakdown of the cytoplasmic membrane barrier, or interaction with a
cytoplasmic target such as DNA (4). Evidence arguing against
cell lysis or membrane breakdown as the sole mechanism of action has
been presented elsewhere (20, 21).
In the killing of gram-negative bacteria, a cationic antimicrobial
peptide must interact with both bacterial cell envelope membranes. In
the study described in this report, we studied the abilities of
bacterium-derived antimicrobial peptides to bind to LPS, to depolarize
both the outer and cytoplasmic membranes, to interact with lipid
monolayers, and to kill a wild-type P. aeruginosa PAO1
strain, strain H103. To explore the hypothesis that permeabilization of
the cytoplasmic membrane is responsible for killing, we monitored cell
viability and cytoplasmic membrane permeabilization at the same time.
Our data strongly suggest that cytoplasmic membrane depolarization did
not correlate well with bacterial cell lethality.
| |
MATERIALS AND METHODS |
Strains and reagents.
P. aeruginosa PAO1 strain
H103 (3) was grown in Mueller-Hinton (MH) broth (Difco
Laboratories, Detroit, Mich.) at 37°C unless otherwise indicated.
Polymyxin E1 and colymycin M were provided by Pathogenesis Corp.
(Seattle, Wash.), whereas gramicidin S and polymyxin B were purchased
from Sigma Chemical Co., (St. Louis, Mo.). The LPS of P. aeruginosa H103 was isolated as described by Moore et al.
(11). 1-N-Phenylnaphthylamine (NPN), carbonyl cyanide-m-chlorophenylhydrazone, and Re LPS from
Salmonella enterica serovar Minnesota R595 (Re mutant) were
purchased from Sigma Chemical Co. Dansyl polymyxin B was synthesized as
described previously (11). The lipids
1-pamitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC),
1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol,
1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-ethanolamine (POPE), and phosphatidylglycerol from egg yolk (egg-PG) were purchased from Avanti Polar Lipids Inc. (Alabaster, Ala.). The fluorescent dye
3,3-dipropylthiacarbocyanine (diSC35) was purchased from
Molecular Probes (Eugene, Oreg.)
MIC assay.
The MICs of the peptides for a range of
microorganisms was determined by the modified broth microdilution
method in MH medium in polypropylene microtiter plates (19).
The MIC was determined as the lowest peptide concentration at which
growth was completely inhibited after overnight incubation of the
plates at 37°C. MICs were determined three times on different
occasions, and the median values are shown. The minimal bactericidal
concentration (MBC) was taken as the lowest concentration of peptide
that produced less than 10
3 survivors.
Dansyl polymyxin B displacement assay.
The relative binding
affinity of each peptide for LPS was determined by the dansyl polymyxin
B displacement assay of Moore et al. (11) with LPS isolated
from P. aeruginosa H103. Maximal displacement of LPS was
expressed as a percentage in which 100% displacement of dansyl
polymyxin B was taken as the displacement observed with polymyxin B. IC50 was defined as the concentration that led to
half-maximal displacement of dansyl polymyxin B.
Membrane permeabilization assay.
The outer membrane
permeabilization activity of the peptide variants was determined by the
NPN uptake assay of Loh et al. (9) with intact cells of
P. aeruginosa H103. The concentration of peptide that led to
a 50% maximal increase in NPN uptake (PC50) was recorded.
The cytoplasmic membrane depolarization activities of the peptides were
determined with the membrane potential-sensitive dye diSC35
(16) and P. aeruginosa H103. Bacterial cells in
the mid-logarithmic phase were centrifuged, washed in 5 mM HEPES (pH
7.8), and resuspended in the same buffer to an optical density at 600 nm of 0.05. The cells were first treated with 0.2 mM EDTA (pH 8.0) in
order to permeabilize the outer membrane to allow dye uptake, and then a stock solution of diSC35 was added to a final
concentration of 0.4 µM and quenching was allowed to occur at room
temperature for 20 to 30 min. KCl was then added to the cell suspension
to a final concentration of 100 mM to equilibrate the cytoplasmic and
external K+ concentrations. A 2-ml cell suspension was
placed in a 1-cm cuvette, and the desired concentration of the peptide
to be tested was added. Changes in fluorescence due to the disruption
of the membrane potential gradient (
) across the cytoplasmic
membrane were continuously recorded with a Perkin-Elmer model 650-10S
spectrofluorimeter at an excitation wavelength of 622 nm and an
emission wavelength of 670 nm. At regular intervals, the surviving
cells were plated on MH agar plates, and the plates were incubated at
37°C overnight to assess the residual numbers of CFU.
Langmuir monolayer assay.
Lipid monolayers were formed by
applying the appropriate lipids dissolved in hexane or chloroform onto
water contained in a circular Teflon trough (diameter, 4.5 cm; total
volume, 11.5 ml). Monolayers were allowed to equilibrate until a stable
surface pressure was obtained (drift in surface pressure
[
], <0.2 mN/m). A small port in the side of the trough enabled
injection of reagents into the subphase without disruption of the
monolayer. The subphase was gently mixed with a magnetic stir bar at 45 rpm. Surface pressure measurements were obtained by using the Whilhelmy
plate method (10). The plate was cleaned with methanol three
times and was thoroughly rinsed with double-distilled water prior to
each surface pressure measurement. The experiments were run at 23°C.
An LPS monolayer film on the air-water interface was obtained by
applying S. enterica serovar Minnesota Re LPS (0.5 mg/ml in chloroform-methanol-H2O [17/7/1; vol/vol])
(2) onto buffer alone (5 mM HEPES, 150 mM NaCl) or in the
presence of either 2 or 5 mM MgCl2. The monolayer was
spread to achieve an initial pressure of 18 ± 1 mN/m and was
allowed to stabilize for 5 min before addition of peptide. Peptides
were injected to a final concentration of 0.8 µg/ml for polymyxin B,
polymyxin E1, and colymycin M and 0.32 µg/ml for gramicidin S into
the subphase without disruption of the monolayer.
| |
RESULTS AND DISCUSSIONS |
Antimicrobial activity.
The characteristics of the peptides
included in this study are listed in Table
1. The two polymyxins were highly active
against strain H103, with polymyxin B and E1 MICs of 0.125 and 0.25 µg/ml, respectively (Table 2), whereas
gramicidin S and colymycin M were substantially less active, with MICs
of 8 and 4 µg/ml, respectively. Direct plating of samples from each
well in which bacterial cell growth was inhibited by peptides permitted
an assessment of the MBC. For polymycin B, gramicidin S, and colymycin
M, the MIC and MBC were the same concentration. For polymyxin E1, only
2 logarithms of killing occurred at the MIC and the MBC was twofold
higher than the MIC. Thus, all of these peptides were bactericidal. The MICs for Escherichia coli were found to be within
twofold of the MICs for P. aeruginosa reported in Table 2.
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TABLE 2.
MICs, relative binding of peptides to LPS isolated from
membrane of P. aeruginosa H103, and ability to permeabilize
the outer membrane of P. aeruginosa H103
|
|
Ability to bind to LPS and interaction with LPS monolayers.
A
number of studies have shown that cationic peptides are taken up by the
self-promoted uptake route, for which the initial step involves an
electrostatic interaction with Mg2+ binding sites on LPS
molecules (11). Thus, the ability of each peptide to bind to
P. aeruginosa LPS was determined by the dansyl polymyxin B
displacement assay (11). The polymyxins showed relatively high affinities for purified LPS, as judged by their low
IC50s, whereas gramicidin S bound to LPS somewhat more
weakly and colymycin M showed no significant displacement of dansyl
polymyxin B at concentrations up to 300 µg/ml (Table 2).
Interaction of peptides with LPS monolayers.
Both polymyxin B
and polymyxin E1 induced an increase in surface pressure () (Fig.
1), indicating that these peptides
penetrated the LPS monolayer. The surface pressure increases mediated
by the polymyxins decreased as the external Mg2+
concentration increased, consistent with the ability of divalent cations to competitively inhibit polymyxin binding to LPS
(11). Gramicidin S at concentrations above 0.5 µg/ml
tended to disrupt the LPS monolayers within seconds. Therefore, the
value was measured with a 0.32-µg/ml concentration. As shown
in Fig. 1, it penetrated the LPS monolayers rapidly, resulting in a
of 17 mN/m, which was comparable to that induced by the
polymyxins at a concentration of 1 µg/ml. This effect was not
significantly reduced by the addition of Mg2+. Colymycin M,
on the other hand, showed no interaction with the LPS monolayers (Fig.
1).
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FIG. 1.
Influence of peptide addition to the aqueous subphase
bathing the LPS monolayers on the surface pressure, as measured in a
Langmuir balance. Peptide (0.8 µg/ml [0.32 µg/ml in the case of
gramicidin S]) was added to the aqueous subphase bathing the S. enterica serovar Minnesota Re LPS monolayers. The Mg2+
concentration of the subphase was varied from 0 (filled bars) to 2 mM
(open bars) to 5 mM (hatched bars). The results shown are the averages
of two independent experiments.
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|
Interaction with the outer and cytoplasmic membranes of P. aeruginosa H103.
The ability of each peptide to permeabilize
the outer membrane of P. aeruginosa H103 was determined with
intact cells by the NPN uptake assay (9). NPN is a small
hydrophobic molecule that is excluded by the intact bacterial outer
membrane but that exhibits increased fluorescence after partitioning
into disrupted outer membranes. Thus, an increase in fluorescence in
the presence of a peptide indicates the ability of the peptide to
permeabilize the bacterial outer membrane. As shown in Table 1, both
polymyxins promoted NPN uptake across the outer membrane of P. aeruginosa H103 to similar extents, with PC50s of
about a 0.7 µg/ml. Gramicidin S had a higher PC50 of 5 µg/ml, indicating a weaker ability to permeabilize the outer membrane
than polymyxins. Colymycin M, however, mediated NPN uptake only at
extremely high concentrations (PC50, >300 µg/ml), and
the fluorescence intensity never reached that observed with the
polymyxins (data not shown).
Direct and spectrophotometric observation of cells indicated that
neither gramicidin S nor the polymyxins were bacteriolytic at modest
concentrations above the MBC. Thus, we assessed their abilities to
permeabilize the cytoplasmic membrane to ions as a function of time and
residual cell viability. A major component of the energy-generating
mechanisms of bacteria involves the establishment of a
trans-cytoplasmic membrane proton motive force, the largest component of which is an electrical potential gradient, 
, of 
140 mV. This gradient was assessed with a membrane
potential-sensitive fluorescent probe, diSC35
(16). Providing it can cross the outer membrane,
diSC35 is taken up by bacterial cells according to the magnitude of the electrical potential gradient of the cytoplasmic membrane and becomes concentrated in the cytoplasmic membrane, where it
self-quenches its own fluorescence. Any compound that permeabilizes the
cytoplasmic membrane and thus depolarizes the will lead to the
release of diSC35 and a consequent increase in
fluorescence. The diSC35 assay was used previously in
conjunction with an outer membrane hyperpermeable mutant of E. coli, mutant DC2, to assess the interactions of cationic peptides
with the cytoplasmic membrane (20). We were able to
establish this assay with wild-type P. aeruginosa H103 by
permeabilizing the outer membrane with 0.2 mM EDTA (pH 8.0). At this
concentration, EDTA did not interfere with the diSC35
fluorescence or influence the MICs of the peptides included in this
study (data not shown).
We also assessed cell viability by sampling bacteria at various time
intervals during the diSC35 assay and plating for CFU determination to monitor bacterial cell death. Gramicidin S, at its MIC
of 8 µg/ml, caused rapid depolarization of the cytoplasmic membrane,
resulting in more than 90% maximal release of diSC35 in 2 to 4 min (Fig. 2). However, killing was
slow, with only about a 1-logarithm decrease in cell viability in 30 min. In contrast, at their MICs, neither polymyxin B nor polymyxin E1
showed any detectable level of diSC35 release. Polymyxin B
at 2 µg/ml (15-fold the MIC) caused only a trace amount of
diSC35 release after a lag time of 12 min; however, more
than 4 log units of killing was observed in the first 8 min, during
which no indication of cytoplasmic membrane depolarization occurred
(Fig. 2). Similarly, polymyxin E1 at 2 µg/ml caused about 2 log units
of decrease in cell viability, but no release of diSC35 was
detected and only a trace amount of diSC35 release was
detected at 10 µg/ml, under which conditions more than 3 log units of
killing by polymyxin E1 were observed (Fig. 2). Colymycin M showed no
cytoplasmic membrane depolarization within 30 min, even at
concentrations greater than 64 µg/ml, and no cell killing was
observed after 60 min of treatment with colymycin M at 64 µg/ml.
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FIG. 2.
Relationship between cytoplasmic membrane
permeabilization, as assessed by the diSC35 assay, and cell
killing, as measured by determination of the numbers of CFU at the same
time as the permeabilization assay. Dotted curves represent data
obtained from the diSC35 assay, and solid curves represent data
obtained from killing assays.  , polymyxin E1 at 2 µg/ml;  ,
polymyxin E1 at 10 µg/ml;  , gramicidin S at 8 µg/ml;  ,
polymyxin B at 2 µg/ml;  , colymycin M at 64 µg/ml.
Abbreviations: PXB, polymyxin B; PXE1, polymyxin E1; GS, gramicidin S;
CM, colymycin M. The number following the abbreviation is the
concentration applied (in micrograms per milliliter).
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|
To ensure that these results were not due to the use of EDTA in the
modified assay with P. aeruginosa, control experiments were
performed with E. coli. Using the same diSC35
assay previously reported for E. coli DC2 (20),
we confirmed that gramicidin S at concentrations at or below the MIC
caused maximal cytoplasmic membrane depolarization, whereas polymyxins
B and E1 at 4-fold the MIC had no effect within 9 min and at
concentrations equal to 25-fold the MICs had a minimal effect on
cytoplasmic membrane permeabilization (<10% maximal increase in
diSC35 fluorescence in 4 min) (data not shown). Colymycin M
at 100-fold the MIC had no effect (data not shown). These results
suggested that the peptides exerted the same characteristics, in terms
of their ability to permeabilize the E. coli cytoplasmic
membrane, observed with P. aeruginosa H103 after EDTA
treatment, indicating that EDTA has little effect on this particular
characteristic. In addition, another cytoplasmic membrane
permeabilization assay was used to assess the abilities of the peptides
to promote the uptake of the chromogenic substrate
ortho-nitrophenyl--D-galactopyranoside (ONPG), in which it could be cleaved by the constitutive
-galactosidase of E. coli ML-35. In that assay,
gramicidin S at 1.5- to 2-fold the MIC showed maximal unmasking of
cytoplasmic -galactosidase. In contrast, polymyxins B and E1 at
4-fold the MICs had no effect, and even at 200-fold the MICs they
caused a minimal permeabilization of the cytoplasmic membrane to ONPG
(i.e., the rate of ONPG hydrolysis by cytoplasmic -galactosidase was
150-fold lower than that caused by 1.5-fold the MIC of gramicidin S).
Interaction with lipid monolayers.
The use of lipid monolayers
(10) created at an air-water interface with a Langmuir
balance apparatus is a simple means of mimicking biological membranes.
Such monolayers provide a powerful tool for the assessment of membrane
insertion. A molecule that interacts only with the head groups of a
given lipid monolayer will not increase the surface pressure of the
monolayer. Thus, when a protein or peptide is injected into the aqueous
subphase bathing the monolayer, the corresponding surface pressure
change () can be interpreted as a result of protein or peptide
insertion into the fatty acid chains of the monolayer. To further
assess the interactions of these peptides with membranes, we made
monolayers with phosphatidylethanolamine (PE)-egg phosphatidylglycerol
(PG)-cardiolipin (CL) at a ratio of 78:4.7:14.7 (Avanti Polar Lipids
Inc.) to mimic the phospholipid content of the bacterial cytoplasmic
membrane and tested the abilities of the peptides to interact with such monolayers (Fig. 3). Gramicidin S caused
a large and rapid increase in the monolayer surface pressure even with
a single addition of 0.1 µg of peptide per ml into the subphase. The
reached a plateau at about 17 mN/m after the second addition of
the same amount of peptide. In contrast, at the same concentration,
both polymyxins induced surface pressure increases as a sigmoidal
function of the peptide concentration, suggesting cooperativity, and
the maximum increase in was about 5 mN/m for both polymyxin B
and polymyxin E1. The use of colymycin M resulted in no detectable change in surface pressure of the same monolayer during the 2-h observation period.
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FIG. 3.
Influence of gramicidin S (GS), polymyxin B (PXB),
polymyxin E1 (PXE1), or colymycin M (CM) addition to the aqueous
subphase on the surface pressure of phospholipid monolayers, as
measured in a Langmuir balance. (A) Plot of the surface pressure
increase as a function of peptide concentration. Titration of the
surface pressure increase was accomplished by adding successive amounts
of peptide to the subphase while continuously monitoring the surface
pressure of the film. (B) Influence on surface pressure of the addition
of 1 µg of a peptide per ml to the aqueous phase of a monolayer made
from POPC, POPE, egg-PG, or CL. The results shown are the averages of
two independent experiments.
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|
Lipid specificity was monitored by determination of the ability of a
peptide to cause maximal surface pressure changes upon injection of 1 µg of peptide per ml into the subphase of pure lipid monolayers.
Gramicidin S showed a good ability to insert into both neutral and
negatively charged phospholipids, although it seemed to intercalate
more efficiently into negatively charged PG or CL monolayers than into
neutral phosphatidylcholine (PC) or PE monolayers. Gramicidin S
interacted most strongly with PG monolayers. Polymyxins, however, did
not cause a surface pressure increase with neutral PC or PE monolayers,
indicating their low affinity for neutral lipids, but penetrated to
similar extents into negatively charged PG or CL monolayers.
Polymyxins, gramicidin S, and colymycin M are distinctive antimicrobial
peptides on the basis of their abilities to interact with
phospholipids, LPS, and bacterial cytoplasmic and outer membranes. For
example, these peptides displayed somewhat different methods of
overcoming the barriers of the outer membranes of gram-negative bacteria. Gramicidin S had a relatively low affinity for binding to
isolated LPS but penetrated well into LPS monolayers in a manner that
was not strongly inhibitable by Mg2+ ions, and at higher
concentrations (0.5 µg/ml) gramicidin S completely disrupted these
monolayers. Consistent with its ability to interact with LPS
monolayers, gramicidin S at concentrations at about the MIC
permeabilized the outer membrane of P. aeruginosa H103. In contrast, polymyxins bound strongly to LPS. Although they penetrated LPS monolayers to a similar extent as gramicidin S, Mg2+
ions were clearly antagonistic, consistent with the antagonism of
polymyxin action by Mg2+ ions (9, 11, 12, 13,
15). Hancock and Chapple (4) recently suggested that
there are two separate mechanisms of self-promoted uptake: a classical
mechanism such as that used by the polymyxins and a variant mechanism
involving weak LPS binding and reasonable permeabilization, as is
apparently used by gramicidin S (and also, for example, by the
bactenecins [19]). Polymyxins, which have a strong positive charge
and a hydrophobic acyl chain, have a high binding affinity for LPS
molecules and permeabilize the outer membrane by disrupting the
negatively charged (surface) head groups through displacement of
divalent cations from their binding sites on LPS. In contrast,
gramicidin S, which is more weakly positively charged and amphipathic,
presumably binds diffusely to the negatively charged surface of the
outer membrane and inserts into and passes across the outer membrane by
using hydrophobic interactions. By examination of the higher MICs and
weaker permeabilizing activity of gramicidin S, the classical mechanism
of self-promoted uptake appears to be considerably more efficient. We
are unable to explain, on the basis of the current data, how the
neutral lipopeptide colymycin M is taken up across either membrane,
unless this colistin methane sulfonate acts as a slow-release prodrug
for polymyxin E1 and polymyxin E2.
We also examined uptake across the cytoplasmic membrane. As evident
from lipid monolayer assays, the polymyxins interacted only with
negatively charged lipids, which represent about 20% of the
cytoplasmic membrane phospholipids, while gramicidin S interacted well
with both neutral and anionic phospholipids. The differences in the
lipid affinities of these peptides seemed to correlate well with their
abilities to depolarize bacterial cytoplasmic membranes. It has been
proposed and is widely cited that the sole target for killing of
bacteria by antimicrobial cationic peptides (4, 7),
including gramicidin S and the polymyxins (4, 5, 18), is the
cytoplasmic membrane. We have previously presented studies that argue
against this hypothesis for cationic antimicrobial peptides (20,
21), and the data in this paper extend this to the
bacterium-derived antimicrobial peptides. Thus, there was little
correlation between membrane permeabilization and bacterial cell death.
For example, the concentration required for gramicidin S to cause a
50% maximal release of diSC35 (i.e., to decrease the
membrane potential by half) was about 2 µg/ml (one-quarter the MIC).
However, at such concentrations, there were no differences in the
viabilities of peptide-treated and nontreated cells during a 30-min
observation period. In contrast, the use of polymyxin B at 2 µg/ml
(16-fold the MIC) resulted in a greater than 4-logarithm decrease in
the numbers of CFU within 6 min, but only a 5% maximal release of
diSC35 from the cells was detected. This is consistent with
data obtained from diSC35 and cytoplasmic -galactosidase unmasking assays of E. coli cytoplasmic membrane
permeability in response to the same peptides. Thus, it appears that
polymyxin-mediated cell death takes place prior to cytoplasmic membrane
depolarization, whereas for gramicidin S the situation is reversed. We
believe that interaction with membranes must be part of the action of such antimicrobial peptides but is not per se the lethal event. This is
consistent with the observation that polymyxin B and polymyxin B
nonapeptide cause rather similar perturbations of the E. coli cytoplasmic membrane (at high concentrations), even though
the latter does not demonstrate antibiotic activity (1).
Rather, given the large number of functions of bacteria that are
simultaneously inhibited by polymyxins (18) and other
cationic antimicrobial peptides (7), we favor a multihit
hypothesis, in which there are multiple potential anionic targets, some
of which are cytoplasmic.
| |
ACKNOWLEDGMENTS |
This work was supported by funding from the Canadian Bacterial
Diseases Network and Canadian Cystic Fibrosis Foundation's SPARx
program to R.E.W.H., who is also the recipient of Medical Research
Council of Canada Distinguished Scientist Award.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Microbiology and Immunology, University of British Columbia, #300-6174 University Blvd., Vancouver, BC, Canada V6T 1Z3. Phone: (604) 822-2682. Fax: (604) 822-6041. E-mail: bob{at}cmdr.ubc.ca.
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Antimicrobial Agents and Chemotherapy, December 2000, p. 3317-3321, Vol. 44, No. 12
0066-4804/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
