Báo cáo khoa học: The antibiotic activity of cationic linear amphipathic peptides: lessons from the action of leucine/lysine copolymers on bacteria of the class Mollicutes doc

The antibiotic activity of cationic linear amphipathic peptides:
lessons from the action of leucine/lysine copolymers on bacteria
of the class
Mollicutes
Laure Be
´
ven
1
, Sabine Castano
2
, Jean Dufourcq
2
,A
˚
ke Wieslander
3
and Henri Wro
´
blewski
1
1
UMR CNRS 6026, Universite
´
de Rennes 1, France;
2
Centre de Recherche Paul Pascal, CNRS, Pessac, France;
3
Department of Biochemistry and Biophysics, Stockholm University, Sweden
Peptides composed of leucyl and lysyl residues (ÔLK pep-
tidesÕ) with different compositions and sequences were
compared for their antibacterial activities using cell wall-less

bacteria of the class Mollicutes (acholeplasmas, mycoplas-
mas and spiroplasmas) as targets. The antibacterial activity
of the amphipathic a-helical peptides varied with their size,
15 residues being the optimal length, independent of the
membrane hydrophobic core thickness and the amount of
cholesterol. The 15-residue ideally amphipathic a helix with
a +5 positive net charge (KLLKLLLKLLLKLLK) had the
strongest antibacterial activity, similar to that of melittin. In
contrast, scrambled peptides devoid of amphipathy and the
less hydrophobic b-sheeted peptides [(LK)
n
K], even those
15-residue long, were far less potent than the helical ones.
Furthermore, the growth inhibitory activity of the peptides
was correlated with their ability to abolish membrane
potential. These data are fully consistent with a
predominantly flat orientation of LK peptides at the lipid/
water interface and strongly supports that these peptides and
probably the linear polycationic amphipathic defence pep-
tides act on bacterial membranes in four main steps
according to the ÔcarpetÕ model: (a) interfacial partitioning
with accumulation of monomers on the target membrane
(limiting step); (b) peptide structural changes (conformation,
aggregation, and orientation) induced by interactions with
the lipid bilayer (as already shown with liposomes and
erythrocytes); (c) plasma membrane permeabilization/
depolarization via a detergent-like effect; and (d) rapid
bacterial cell death if the extent of depolarization is main-
tained above a critical threshold.
Keywords: amphipathic peptides; antibacterial activity;

bacterial cell death; membrane depolarization; mollicutes.
The amphipathic a helix concept helps in understanding the
behaviour of very different classes of proteins and peptides,
especially those acting on membranes [1–4]. This concept,
which has been useful in the field of peptidic cytotoxins to
develop new analogues, and to better understand their
mode of action [5–7], is still the basis for the rational design
of new antimicrobial compounds, i.e. analogues or chimeras
of natural products [8], or radically new molecules [9,10].
The need to understand their mode of action and improve
their efficacy and/or selectivity towards microorganisms led
to the synthesis of new active peptides, made possible largely
by the progress in solid state synthesis. As a result, a large
wealth of information was obtained on many natural
peptides endowed with cytotoxic (including antimicrobial)
activity. However, answers are still missing regarding: (a) the
requirements for optimized activity and selectivity; and (b)
the mechanisms of action, especially in bacterial cells. In
an effort of rationalization, a minimalistic approach was
initiated by the pioneering work of De Grado and Lear [11]
using residue substitutions or designing simplified sequences
[2,12–14]. Using the very minimal requirement of an
amphipathic structure able to associate properly with either
hydrophilic or hydrophobic side chains, several families of
peptides were designed including those composed of only
leucines and lysines (LK peptides) [11,14–17]. Ideally secon-
dary amphipathic structures (helices or b sheets) can thus be
obtained by playing only with the composition (i.e. the L/K
molar ratio) and charge periodicity [11,18–21]. The lytic
activities of these peptides vary according to the L/K ratio

[22,23] and a specific sequence is not required to get an
adequate polar/apolar topology and a strong membranolytic
activity, matching those of the stronger natural toxins [20,24].
In homologous series of such LK peptides, the observed
lytic activities on zwitterionic liposomes and erythrocytes
are similar having an optimum at a length of 15 residues, i.e.
there is a delicate balance between hydrophobicity and
charge repulsion (two antagonistic forces) [14,22,25]. Due to
the lipid affinity of such peptides, there is an increase of lytic
activity with chain length. Moreover, in contrast with earlier
studies [26], neither a threshold in length nor a matching
between peptide length and membrane thickness was
observed [21,24,25]. This strongly supports a mechanism
Correspondence to H. Wro
´
blewski, UMR CNRS 6026,
Universite
´
de Rennes 1, Campus de Beaulieu,
35042 Rennes Cedex, France. Fax: + 33 2 23 23 50 52,
E-mail:
Abbreviations:CFU,colonyformingunit;Dns,dansyl;MIC,minimal
inhibitory concentration; MDC, minimal deforming concentration;
DpH, transmembrane pH gradient (DpH ¼ pH
in
) pH
out
);
DY, membrane electrical potential (DY ¼ Y
in

– Y
out
).
(Received 12 December 2002, revised 19 March 2003,
accepted 24 March 2003)
Eur. J. Biochem. 270, 2207–2217 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03587.x
of action based upon the invasion of the outer membrane
leaflet by the peptides and their insertion in a flat orientation
to form ÔraftsÕ or ÔcarpetsÕ [21,24,25,27,28].
In this work, we studied the antibacterial activities of a
homologous series of 8–22-residue LK peptides having
different sequences, hydrophobicities and secondary struc-
tures. Our goal was to assess whether or not the rules drawn
from the studies with model membranes and erythrocytes
[21,25] are also valid for bacteria. Mollicutes were chosen as
targets because these bacteria are devoid of both a cell wall
and an outer membrane [29] making them sensitive to many
natural membrane-active peptides. Peptide activities were
monitored at different physiological levels: growth inhibi-
tion, plasma membrane depolarization, and cell shape
modification [30]. Furthermore, to get a better insight into
the mechanism of action, we compared the activities of a
series of peptides differing in length on Acholeplasma
laidlawii cells whose membrane thickness can be tuned via
the lipid diet [31,32].
Materials and methods
Chemicals
Ultrapure mellitin was from Sigma. Dansylated (Dns)
peptides were synthesized by Fournier Pharma (Heidelberg,
Germany) and all the other peptides were from Neosystem

(Strasbourg, France) [24,25]. The purity of the peptides was
% 97% as estimated by HPLC and their mass was consistent
with that expected from the sequence. They were stored as
dry powders at )20 °C and dissolved just before use in
methanol to give 1 or 10 m
M
stock solutions. The peptides
were named according to their length and K-residue
periodicity (2.0, 3.0 or 3.6 indicating whether they were
designed as amphipathic b strands, 3.
10
helices or a helices,
respectively; Table 1). A scrambled 15-residue peptide [scr-
LK15(W14)] was used as a nonamphipathic control peptide,
in which the K residues are distributed to achieve a
hydrophobic moment close to zero [25]. This peptide has
free N and C termini, and leucine 14 is substituted by a
tryptophan residue.
HPLC
Retention times of the peptides were measured by reversed-
phase HPLC on a C18 Purospher RP-18 end-capped
semipreparative column (125 · 4 mm, 5-lm particle size) in
conjunction with a Waters Millenium HPLC system, as
described previously [25].
Antimicrobial assays
The mollicutes Acholeplasma laidlawii A-EF22, Myco-
plasma gallisepticum S6, M. mycoides ssp. mycoides SC
KH3J, Spiroplasma citri R8A2, S. floricola BNR1, and
S. melliferum BC3 were cultured as described previously
[30]. The minimal inhibitory concentrations (MICs) were

determined in 96-well microtitre plates by growing the
Table 1. Listing of the peptides used in this work. Dansylation of peptides is indicated by Dns and D. L, length of the peptide (residuesÆmol
)1
);
S, membrane-bound peptide conformation (note that although peptides LK8(3.6), LK9(3.6) and DnsLK9(3.6) were designed to form ideally
amphipathic a helices, they adopt an extended conformation when interacting with lipid bilayers; similarly, peptide LK16(W15)(3.0) which was
designed to form an ideally amphipathic 3
10
helix is in fact a-helical when interacting with spiroplasma lipids; C, Charge. Lysine periodicity (2.0, 3.0
or 3.6) is indicated in parentheses to help peptide identification in the text.
Name L Composition S C Sequence
Alpha-amphi series
LK8(3.6) 8 L5K3 b 3 KLLLKLLK
LK9(3.6) 9 L6K3 b 3 LKLLLKLLK
DnsLK9(3.6) 9 L6K3 b 2 DLKLLLKLLK
LK12(3.6) 12 L8K4 a 4 KLLLKLLLKLLK
DnsLK12(3.6) 12 L8K4 a 3 DKLLLKLLLKLLK
LK15(3.6) 15 L10K5 a 5 KLLKLLLKLLLKLLK
LK15(W14)(3.6) 15 L9K5W a 5 KLLKLLLKLLLKLWK
DnsLK15(3.6) 15 L10K5 a 4 DKLLKLLLKLLLKLLK
DnsLK18(3.6) 18 L13K5 a 4 DLLLKLLKLLLKLLLKLLK
DnsLK19(3.6) 19 L13K6 a 5 DKLLLKLLKLLLKLLLKLLK
DnsLK21(3.6) 21 L15K6 a 5 DLLKLLLKLLKLLLKLLLKLLK
DnsLK22(3.6) 22 L15K7 a 6 DKLLKLLLKLLKLLLKLLLKLLK
3.
10
-amphi
LK16(W15)(3.0) 16 L9K6W a 6 KLLKLLKLLKLLKLWK
Beta-amphi series
DnsLK9(2.0) 9 L4K5 b 4 DKLKLKLKLK–CONH

2
DnsLK11(2.0) 11 L5K6 b 5 DKLKLKLKLKLK–CONH
2
DnsLK15(2.0) 15 L7K8 b 7 DKLKLKLKLKLKLKLK–CONH
2
Scrambled
Scr-LK15(W14)(3.6) 15 L9K5W a 5 LKLLLLKLLKLKLWK
Natural
Mellitin 26 a 6 GIGAVLKVLTTGLPALISWIKRKRQQ–CONH
2
2208 L. Be
´
ven et al. (Eur. J. Biochem. 270) Ó FEBS 2003
bacteria in the presence of twofold serial dilutions of
peptide. The starting cell concentration in each well was 10
6
colony-forming units (CFU)ÆmL
)1
. All assays were per-
formed in triplicate. Bactericidal activities were assessed by
spreading on agar plates cells treated for 2 h with different
peptide concentrations. The minimal lethal concentration
was defined as the lowest peptide concentration capable of
killing 99% of the cells in a suspension containing
10
6
CFUÆmL
)1
. In addition, to determine the influence of
the hydrophobic core thickness of the cell membrane on

peptide antibiotic activity, A. laidlawii A-EF22 was adapted
to grow in a lipid-free medium supplemented with appro-
priate fatty acids, as described previously [32,33].
Light microscopy
All of the experiments were performed on spiroplasma cell
suspensions containing 10
10
CFUÆml
)1
(A
600
¼ 1.0) in
50 m
M
sodium phosphate buffer pH 7.0, 50 m
MD
-glucose
and 549 m
MD
-sorbitol. Dark-field optics were used to
analyse the effects of the peptides on spiroplasma motility
and cell morphology, as described previously [30,33–35].
Microphotographs were taken using Kodak T-Max 35-mm
film (ISO 400 or 3200).
Membrane potential measurement
Alterations of membrane potential by peptides in A. laid-
lawii and S. melliferum were probed spectrofluorometrically
using the fluorescent dye 3,3¢-dipropyl-2,2¢-thiadicarbocya-
nine iodide [36]. A detailed description of the experimental
conditions and of the calibration method of fluorescence

signal vs. membrane potential is given in previous reports
[33,34].
Determination of intracellular pH
The intracellular pH of A. laidlawii and S. melliferum cells
was determined by spectrofluorometry using the internally
conjugated fluorescent probe 5(6-)-carboxyfluorescein suc-
cinimidyl ester [37] as described previously [33].
Protein and cholesterol determination
Protein was determined with the bicinchoninic acid method
[38] using BSA as standard. Peptide concentrations
were estimated from absorbance measurements using
e
340
¼ 4640
M
)1
Æcm
)1
for dansylated peptides and
e
280
¼ 5600
M
)1
Æcm
)1
for tryptophan-containing peptides.
Total cholesterol was determined in the chloroform/meth-
anol membrane lipid fraction with the Sigma Diagnostics
Cholesterol reagent kit.

Results
Antibacterial activity of the peptides as a function
of length and structure
The first step of our work was to screen the growth
inhibition activities of 15 LK peptides towards six different
species of mollicutes. Melittin was used as a reference
because it is a well known cytotoxic peptide which is lethal
for mollicutes [30,33]. The diversity of the LK peptides used
in this study (Table 1) allowed us to assess the importance
of both peptide length and structure for antibacterial
activity.
Table 2 shows that the susceptibility of mollicutes to
melittin and the LK peptides was independent of the
amount of cholesterol in the membranes of these bacteria.
Table 2. Antibacterial activities of LK peptides and melittin against several species of mollicutes.
Cholesterol (%)
a
MICs (l
M
)
Al
b
(2.1)
Mg
(7.2)
Mm
(3.5)
Sc
(25.2)
Sf

(16.8)
Sm
(22.2)
Peptides
LK8(3.6) 100 R R R R R
LK9(3.6) 100 R R 100 R R
DnsLK9(3.6) 50 100 100 50 R 100
LK12(3.6) 25 50 25 25 50 50
DnsLK12(3.6) 3.12 12.5 6.25 6.25 12.5 12.5
LK15(3.6) 1.56 3.12 6.25 6.25 6.25 6.25
LK15(W14)(3.6) 1.56 3.12 6.25 6.25 6.25 6.25
DnsLK15(3.6) 3.12 6.25 12.5 12.5 12.5 12.5
DnsLK19(3.6) 50 50 R R R R
DnsLK22(3.6) 25 50 100 25 25 25
LK16(W15)(3.0) 0.78 12.5 12.5 6.25 6.25 6.25
Scr-LK15(W14) 3.12 12.5 R 100 100 100
DnsLK9(2.0) 6.25 6.25 R 50 100 50
DnsLK11(2.0) 12.5 25 R R R R
DnsLK15(W14)(2.0) 12.5 25 R 50 100 100
Melittin 0.78 6.25 12.5 0.39 3.12 1.56
a
The percentage (by mass) of cholesterol in the whole membrane lipid fraction of the different bacteria (% chol.) is indicated below the
abbreviation of their name.
b
Bacterial targets: A. laidlawii (Al), M. gallisepticum (Mg), M. mycoides ssp. mycoides SC (Mm), S. citri (Sc),
S. floricola (Sf), and S. melliferum (Sm). R, No activity at concentrations £ 100 l
M
.
Ó FEBS 2003 Antibacterial activity of LK peptides (Eur. J. Biochem. 270) 2209
The most potent of the LK peptides was, overall,

LK15(3.6), i.e. the peptide designed to adopt an ideal
amphipathic a-helical conformation (KLLKLLLKLLLK
LLK). With MICs ranging from 1.56 to 6.25 l
M
,this
molecule exhibited a growth inhibition activity similar to
that of melittin (MIC, 0.39–12.5 l
M
). In this series, the
12-residue peptide was much less potent but, interestingly,
dansylation increased its activity whilst the same modifi-
cation decreased the activity of the 15-residue peptide.
Shorter peptides (8 or 9 residues) displaying a b confor-
mation in the membrane-bound state [24] were poorly
effective or harmless, even at concentrations up to 100 l
M
.
Longer peptides [DnsLK19(3.6) and DnsLK22(3.6)] were
also less active, but the relationship between length and
activity was less clear than in the case of the shorter
peptides. In addition, the L14W substitution in LK15(3.6)
had no effect on activity. Collectively, these observations
indicate that for ideally amphipathic a helices composed of
only leucine and lysine, a sequence of 15 residues
constitutes the optimal length to inhibit the growth of
mollicutes.
Table 2 also shows that with MICs ranging from 0.78 to
12.5 l
M
, LK16(W15)(3.0), the peptide designed to form

ideally amphipathic 3.
10
helices but which, in fact, is
a-helical in membranes (data not shown), was as effective
as LK15(3.6) (L/K ¼ 2). In comparison, peptides of the
beta-amphi series designed to form ideally amphipathic b
strands, were significantly less effective. The MICs of Dns-
LK15(2.0) (L/K ¼ 1) were similar to those of DnsLK9(3.6).
So, here again a decrease of the length in the same series
below 15 residues resulted in a loss of activity. Finally,
scrambling the sequence of LK15(W14)(3.6) [peptide
scr-LK15(W14)] strongly decreased its activity, notably
against M. mycoides ssp. mycoides SC and the three
spiroplasmas. This second set of data pinpoints the import-
ance of peptide charge topology, showing this time that
provided they have the optimum length (i.e. 15 residues),
ideally amphipathic helices are significantly more potent in
inhibiting the growth of mollicutes than irregular sequences
with the same composition and than ideally amphipathic
b sheets.
It should also be noted that plating peptide-treated
mollicutes on agar broth revealed that the most active
peptides (MICs < 50 l
M
) were bactericidal as no surviving
cells could be detected by this method after a 2 h peptide
treatment.
Spiroplasma cell deformation by the peptides
In contrast with other mollicutes (e.g. acholeplasmas and
mycoplasmas), spiroplasmas exhibit a helical shape and

motility which are altered by ÔmembranotropicÕ peptides, a
phenomenon easy to observe by dark-field light microscopy
[30,33–35]. In the second step of this work, we have thus
assessed, using S. melliferum as a target, the cell deforming
activity of LK15(3.6) compared with melittin. Upon
treatment with the latter, the cells lost their motility and
helical shape. Deformation of 100% of the cells could be
achieved within seconds with 1 l
M
melittin, which ham-
pered a reliable microphotographic recording. However, as
the effects were both concentration- and time-dependent, it
was possible to find conditions compatible with the
technique. Hence, 100% of the cells (10
10
CFUÆmL
)1
)were
deformed with 0.1 l
M
melittin within 5 min whilst only
50% were deformed within the same time with 0.01 l
M
melittin. At this latter concentration, a limit of 55–60% was
reached after 10 min (Fig. 1). In the same conditions,
LK15(W14)(3.6) was less efficient than melittin at the lowest
concentration (20% cells deformed after 10 min) but
equally active at the highest one.
As the spiroplasma cell deformation test proved to be
relevant and reliable to investigate the ÔmembranotropicÕ

activity of antibacterial peptides, this technique was used to
assess the importance of the structure of the LK peptides.
Table 3 [columns MDC (minimal deforming concentra-
tion)
50
and MDC
100
] reveals that, consistent with the results
of growth inhibition tests, the activity of melittin was
matched by LK15(3.6), LK15(W14)(3.6), and to a lesser
extent by LK16(W15)(3.0). In contrast, scr-LK15(W14)
exhibited a deforming activity about two orders of magni-
tude weaker and the Dns(LK)
n
-series peptides were harm-
less independently of their length.
Hence, the secondary structure and amphiphilicity of
model peptides composed of L and K residues were also
critical parameters in the spiroplasma cell deformation test.
Fig. 1. Time-course of S. melliferum cell deformation by melittin and
LK15(3.6). Spiroplasma cells (10
10
CFUÆmL
)1
)energizedwith50m
M
D
-glucose in 50 m
M
sodium phosphate buffer (pH 7.0, 32 °C) con-

taining 549 m
MD
-sorbitol as osmoprotectant were treated with
different peptide concentrations and pictures were recorded with a light
microscope equipped with dark-field optics. The cells were considered
deformed as soon as they lost their helicity. Points on the curves are the
average of three determinations (SD £ 4.5%).
2210 L. Be
´
ven et al. (Eur. J. Biochem. 270) Ó FEBS 2003
The 15-residue ideally amphipathic helix LK15(3.6) proved
again to be the optimal structure in contrast with b-sheeted
structures or a scrambled sequence.
Membrane depolarization by the peptides
We have previously shown that there is a correlation
between the ability of peptides to inhibit the growth of
mollicutes, to abolish the membrane potential, and to
deform spiroplasma cells [30,33–35]. The next step of this
work was to investigate the depolarizing activity of the LK
peptides. Fig. 2 shows that the relative efficacy of LK15(3.6)
compared with melittin was the same in A. laidlawii and
S. melliferum with respect to the extent of membrane
depolarization and the delay necessary to reach a steady-
state level of depolarization. Actually, 0.1 l
M
melittin
totally depolarized the A. laidlawii membrane within
5minandthatofS. melliferum by 41%. Under the same
conditions (10
9

CFUÆmL
)1
), 0.1 l
M
LK15(3.6) depolarized
the A. laidlawii membrane by 64% and that of S. melliferum
by 16%.
S. melliferum being more robust than A. laidlawii was
then used to compare the depolarizing activities of 15- and
16-residue LK peptides having different structures. Table 3
(DDY columns) shows that the depolarization of the
S. melliferum membrane mirrored the spiroplasma cell
deformation phenomenon described above. Indeed, here
again melittin, LK15(3.6), LK15(W14)(3.6), and LK16
(W15)(3.0) proved to be more effective than scr-LK15(W14)
whilst the three Dns(LK)
n
peptides produced a negligible
effect at best.
Abolition of the transmembrane pH gradient
by the peptides
After having analysed the effects of melittin and LK15(3.6)
on the membrane potential of A. laidlawii and S. melli-
ferum, we have investigated the ability of these peptides to
alter (RT/F) · DpH, i.e. the second component of the
protonmotive force. The transmembrane pH gradient
(DpH ¼ pH
in
) pH
out

) was thus measured at 37 °Cfor
A. laidlawii and 32 °CforS. melliferum vs. extracellular pH
(pH
out
) (Fig. 3). When energized with 50 m
MD
-glucose,
A. laidlawii and S. melliferum cells (10
9
CFUÆmL
)1
) gener-
ated in slightly buffered solutions a D
1
pH ‡ 0 which proved
to be stable for at least 20 min. This transmembrane
gradient increased linearly from 0.23 to 1.31 in A. laidlawii
(Fig. 3A) and from 0 to 1.34 in S. melliferum (Fig. 3B)
when pH
out
decreased from 7.5 to 5.0. Immediately after the
addition of 0.1 l
M
melittin or LK15(3.6), the DpH increased
transiently for about 2 min and then dropped within 2 min
to reach a steady value, always lower than that observed in
the absence of peptide. As expected from DY measurements
(see above), 0.1 l
M
LK15(3.6) diminished the DpH in both

mollicutes almost as efficiently as melittin. The linear
relationship of DpH vs. pH
out
indicates that the activity of
both peptides was independent of the extracellular pH
within the explored range (5.0–7.5) which largely covers the
conditions found by bacteria either in their animal hosts or
in culture media.
Table 3. Effect of melittin and LK peptides on Spiroplasma mel liferum
cell shape and membrane potential. MDC
50
and MDC
100
are the min-
imal concentrations (l
M
) required for the deformation of 50% and
100% of the cells, respectively, upon 5 min of action. No stands for no
observed effect. DDY is the percentage of membrane depolarization
induced by 0.1 and 1 l
M
peptide concentrations (unperturbed poten-
tial: 68 ± 5 mV, inside negative; SD, 4%) upon 10 min of action.
Peptides MDC
50
MDC
100
DDY
0.1 l
M

1 l
M
DnsLK9 (2.0) No No 0 0
DnsLK11(2.0) No No 0 7
DnsLK15(2.0) No No 0 7
LK15(3.6) 0.05 0.1 16 65
LK15(W14)(3.6) 0.05 0.1 17 65
Scr-LK15(W14) 10 50 3 16
LK16(W15)(3.6) 0.1 0.2 16 65
Melittin 0.01 0.1 41 75
Fig. 2. Time-course of A. laidlawii and S. melliferum plasma mem-
branes depolarization by melittin and LK15(3.6). The cells (10
9
CFUÆmL
)1
) were energized with 50 m
MD
-glucose in 5 m
M
Hepes
buffer (pH 7.0) containing either 150 m
M
NaCl (A. laidlawii)or
128 m
M
NaCl (S. melliferum). Measurements were performed at 37 °C
for A. laidlawii and 32 °CforS. melliferum. The arrows indicate the
time at which the peptides were injected into the cell suspensions. The
curves are the means of three determinations (SD £ 4%). A. laidlawii
(- -) and S. melliferum (—).

Ó FEBS 2003 Antibacterial activity of LK peptides (Eur. J. Biochem. 270) 2211
Influence of membrane thickness on the antibacterial
activity of the peptides
The possibility of modifying the A. laidlawii membrane
lipid bilayer via the fatty acids incorporated into the
growth medium [29,31,32] was exploited to study the
influence of membrane thickness on the antibacterial
activity of L
i
K
j
a-helical amphipathic peptides (i ¼ 2j).
A. laidlawii was thus grown under conditions allowing
23, 25, 26.3 or 28-A
˚
membrane hydrophobic core thick-
ness to be obtained (Table 4). As previously shown [33],
the MIC of honey bee melittin increased from 0.78 to
3.12 l
M
when increasing the hydrophobic thickness from
23 to 28 A
˚
. However, even with the thickest cell
membrane, the activity of melittin was still very high.
With activities similar to those of mellitin, LK15(3.6) and
Dns-LK15(3.6) were the most efficient of the LK
peptides for the four types of membranes, i.e. independ-
ent of the membrane thickness. Their inhibitory activities
decayed as peptide length was either decreased or

increased but, similar to the results obtained in the
growth inhibition experiments (Table 2), the loss of
activity due to peptide lengthening was less sharp than
that due to peptide shortening. It should also be stressed
that the 25-A
˚
hydrophobic core membrane which
contained exclusively 16 : 1c fatty acyl chains and no
cholesterol was overall more sensitive to the peptides
than the three other membranes.
Fig. 3. Effect of melittin and LK15(3.6) on the transmembrane pH gradient of A. laidlawii (A) and S. melliferum (B). DpH ¼ pH
in
) pH
out
.
Measurements were performed at 37 °CforA. laidlawii and 32 °CforS. melliferum. Each point on the curves is the mean of three independent
determinations (SD £ 3%).
Table 4. Influence of Acholeplasma laidlawii membrane thickness on the
antibacterial activity of melittin and LK peptides of the alpha-amphi
series. Data are expressed as l
M
MIC. R, No growth inhibition for
concentrations £ 100 l
M
. A. laidlawii (strain A-EF22) was grown in
lipid-free medium supplemented with: (a) 75 l
M
tetradecanoic acid
(14 : 0) + 75 l
M

Dcis-9-tetradecanoic acid (14 : 1c); (b) 150 l
M
Dcis-
9-hexadecanoic acid (16 : 1c); (c) 150 l
M
Dcis-9-octadecanoic acid
(18 : 1c); and (d) 150 l
M
Dcis-9-octadecanoic acid (18 : 1c) + 20 l
M
cholesterol. These conditions give the following compositions in
membrane lipids: (a) 14 : 0 + 14 : 1, molar ratio, 50/50; (b) 100%
16 : 1c; (c) 100% 18 : 1c; and (d) 18 : 1c + cholesterol, molar ratio,
75/25. The average thickness of the A. laidlawii A-EF22 membrane
hydrophobic core, in these conditions, was previously determined to be
23 A
˚
,25A
˚
, 26.3 A
˚
and 28 A
˚
[32].
Peptide
Membrane thickness
23 A
˚
25 A
˚

26.3 A
˚
28 A
˚
LK8(3.6) 50 100 100 100
LK9(3.6) R 50 100 100
DnsLK9(3.6) 3.12 0.78 12.5 50
LK12(3.6) 12.5 25 100 R
LK15(3.6) 0.39 0.78 1.56 0.78
DnsLK15(3.6) 0.78 0.78 3.12 1.56
DnsLK18(3.6) R 100 R R
DnsLK19(3.6) 50 25 25 25
DnsLK21(3.6) R 25 50 100
DnsLK22(3.6) 6.25 6.25 12.5 12.5
Melittin 0.78 0.78 1.56 3.12
2212 L. Be
´
ven et al. (Eur. J. Biochem. 270) Ó FEBS 2003
Discussion
Previous studies based on the action of minimalist LK
peptides on model membranes and erythrocytes showed
that the critical parameters governing activity are peptide
length, total hydrophobicity, amphipathy, and secondary
structure, which collectively determine peptide membrane
affinity [21,24,25]. However, due to the complexity of
bacterial cells compared to liposomes and erythrocytes, it
was necessary to check whether these rules hold also for the
antimicrobial activity of these peptides. In this work, we
have taken advantage of the fact that bacteria of the class
Mollicutes are devoid of an outer membrane and of a cell

wall, to avoid possible interferences of these structures in the
interactions between peptides and the bacterial plasma
membrane.
Most of the LK peptides studied here exhibited an
antibacterial activity in agreement with previous studies on
closely related compounds [15,23]. The activity varied with
peptide length, the optimum occurring for the 15-residue
ideally amphipathic a-helical structure [LK15(3.6)] the
activity of which was similar to that of melittin, a bee
venom peptide known as one of the most efficient natural
peptides in killing bacteria [8,39–41].
Lengthening peptides of the alpha-amphi series (generic
composition L
i
K
j
with i ¼ 2j) over 15 residues did not result
in a parallel increase in activity. This was previously found
for their efficacy to induce leakage in lipid vesicles and
erythrocytes, and was shown to be due to the self-
association of such a helices in solution [25]. However,
unlike haemolysis the bactericidal activity dropped more
severely from 18- to 21-residue length before a slight
increase for the 22-residue peptide (Fig. 4). Thus, two
competing processes probably occur in bacteria and eryth-
rocytes as in the case of artificial membranes [25,42,43]: (a) a
progressive increase of the membrane affinity with peptide
length; and (b) a drop of activity due to decreased free
energy in solution upon oligomer formation. The opposite
effects produced by dansylating short or long peptides are

consistent with this interpretation. Indeed, for short pep-
tides the dansyl group promotes activity by increasing
hydrophobicity, and thus membrane affinity, whilst for
peptides longer than 15 residues, already too hydrophobic
to remain in the monomeric state in buffer, dansylation
favours oligomerization at the expense of lipid affinity. The
total lack of activity of LK18(3.6) and LK21(3.6) compared
to LK22(3.6) does not contradict this interpretation because
these peptides have no K at the N terminus but, instead,
several L residues increasing hydrophobicity and thus
oligomerization tendency.
The beta-amphi series peptides [i.e. (LK)
n
Kpeptides]are
much less hydrophobic and have a larger charge repulsion.
They are thus monomeric in water and have a lower affinity
for lipids than the alpha-amphi series peptides (i.e. L
i
K
j
peptides with i ¼ 2j) of same length [21]. This explains why
these peptides are less active against bacteria (as observed in
this work) and also less haemolytic [21,25].
In addition to total charge and hydrophobicity, the
topology of the distribution of L and K residues proved to
be important: the more the peptides were amphipathic, the
more they were active against mollicutes. This is clearly
shown for L10K5 peptides by comparing the MICs of the
ideally amphipathic peptide L10K5(3.6) and of the
scrambled one [scr-LK15(W14)], although LK16W15(3.0)

which folds into a nonamphipathic a helix, keeps also quite
a high activity. These secondary amphipathic peptides
proved to be more active than previously studied primary
amphipathic ones in which the positive charges were
clustered at the C termini of the molecules (see e.g. [33]).
Hence, contrary to haemolysis, the antibacterial activity
seems to be more sensitive to peptide aggregation and less
sensitive to amphipathy.
LK peptides have different secondary structures when
bound to lipids: the L
i
K
j
peptides (with i ¼ 2j and n > 12)
are a-helical [24] whilst the shorter and/or alternated ones
[(KL)
n
K] fold into antiparallel b sheets [25]. It is also
noteworthy that the scrambled peptide scr-LK15(W14) is
b-sheeted when bound to dimyristoyl phosphatidyl choline
[24] but a-helical in the presence of spiroplasma lipids [44].
This might be due to the fact that dimyristoyl phospha-
tidylcholine is zwitterionic whilst the spiroplasma mem-
brane contains anionic lipids. The beta-amphi series
peptides were less active against mollicutes than their more
hydrophobic ideally amphipathic a-helical homologues.
However, both a-helical and b-sheeted peptides acted on
Fig. 4. Comparison of the effects of alpha-amphi series peptide length on
antibacterial and haemolytic activities. Antibacterial activities (black
curve) are expressed as MIC

)1
. The data were normalized in such a
way that the highest activity, corresponding to a MIC of 0.78 l
M
,was
given a value of 100. Haemolytic activities (grey curve) were taken
from [25] and expressed as the inverse of the concentration inducing
50% of lysis (LC
À1
50
).
Ó FEBS 2003 Antibacterial activity of LK peptides (Eur. J. Biochem. 270) 2213
different species of mollicutes with the same ranking within
their respective series: A. laidlawii > M. gallisepti-
cum > S. citri % S. floricola % S. melliferum % M. myco-
mycoides ssp. mycoides SC, while for melittin S. citri and
S. floricola were much more sensitive. Such a ranking seems
therefore more relevant to peculiarities of these bacteria
whose lipid composition varies according to the species,
than to properties of the peptides. Despite the fact they are
less efficient than their a-helical homologues, peptides of the
beta-amphi series are also intrinsically capable of killing
mollicutes. This suggests that within this series, longer
peptides should prove more efficient in growth inhibition
tests than those used in this work.
Measurements of DY and DpH in A. laidlawii and
S. melliferum revealed that the antibacterial activities of
the peptides were correlated with their ability to depolarize
the plasma membrane (Tables 2 and 3, and Figs 2 and 3). In
the case of S. melliferum, the loss of cell motility and helicity

induced by the action of LK peptides was also correlated
with membrane depolarization as previously observed with
several natural antibacterial peptides [30]. However, MICs
were about one order of magnitude higher than depolarizing
concentrations. This difference is probably due to lipopro-
teins present in the culture medium used for growth
inhibition assays. As serum components compete with
membranes for the binding of membrane-active peptides
[45–47], they should indeed increase their apparent MICs.
Our data indicate that the bactericidal activity of LK
peptides towards mollicutes is due to their ability to
permeabilize the plasma membrane; this raises the question
of the molecular mechanism governing their action. In the
case of hydrophobic peptides such as alamethicin, experi-
mental data indicate that permeabilization occurs through
the formation of ion-conducting transmembrane channels
in accordance with the Ôbarrel-staveÕ model [35,48]. How-
ever, such a mechanism hardly fits LK peptides, even the
a-helical ones, because of their polycationic nature and
charge periodicity (+1 per a-helix turn). Indeed, the
transfer of five or more positive charges per molecule from
water into the hydrophobic core of the lipid bilayer is
energetically extremely unfavourable unless they are pro-
perly counterbalanced by a set of negative charges in register
with them. Hence, if transient transmembrane bundles of
LK peptide helices were to exist, such bundles would be very
unstable because of K
+
/K
+

electrostatic repulsions [49,50].
It should also be stressed that the a helix LK15(3.6) is
anyway too short to span the membrane bilayer hydropho-
bic core, even in the case of the thinnest A. laidlawii
membrane (see Table 4). A 15-residue helix would be
22.5 A
˚
long, i.e. very close to the thickness of the membrane
hydrophobic core (23 A
˚
), but polarity of the N and C
termini should hamper their localization within an apolar
environment. In contrast, with a length of % 50 A
˚
,the
b strand DnsLK15(2.0) is too long for a correct transmem-
brane fit. In fact, PM-IRRAS spectra show unambiguously
that both a and b ideally amphipathic LK peptides are
laying flat on the interface between water and lipids
including those of S. melliferum [44]. In the same conditions,
scr-LK15(W14) and LK16W15(3.0) exhibited a mainly
a-helical folding, without amphipathy, and a slightly tilted
orientation with respect to the lipid/water interface plane
[44]. Such a flat orientation should thus be considered the
most stable one for LK peptides, even if other orientations
are possible (see below). As suggested by the ÔcarpetÕ model,
aggregation on the membrane surface should enhance
peptide dynamic reorientations and the subsequent forma-
tion of transient transmembrane pores [51].
Among the different mechanisms proposed to explain

peptide action on membranes, the interfacial models such as
ÔraftsÕ or ÔcarpetsÕ [28,52,53] thus seem to be more relevant to
polycationic amphipathic molecules than the Ôbarrel-staveÕ
model. This view is strongly supported by the data of
Table 4 and Fig. 5 showing that the activity of the LK
peptides is essentially independent of membrane thickness.
Indeed, the helical 15-residue peptides are the most active
ones independent of the mollicute species and, for the same
peptide, independent of the membrane thickness, whilst the
formation of transmembrane bundles of helices would
require longer molecules for a better match between
membrane thickness and peptide length. Hence, the anti-
bacterial action of these peptides comprises four main steps:
step 1, interfacial partitioning and exofacial accumulation of
Fig. 5. Graphical illustration of the effects on antibacterial activity of the
length of alpha-amphi series peptides vs. A. laidlawii membrane thick-
ness. Antibacterial activities are expressed as MIC
)1
.Thedatawere
normalized in such a way that the highest activity, corresponding to a
MIC of 0.78 l
M
, was given a value of 100.
2214 L. Be
´
ven et al. (Eur. J. Biochem. 270) Ó FEBS 2003
monomers on the target membrane (limiting step); step 2,
peptide structural changes (conformation, aggregation, and
orientation) induced by interactions with the lipid bilayer, as
indicated by previous studies with liposomes and erythro-

cytes [17,25]; step 3, plasma membrane permeabilization/
depolarization via detergent-like effects; step 4, rapid
bacterial cell death if the extent of depolarization is
maintained above a critical threshold. At step 3, bound
peptides can modifiy the membrane curvature stress above a
certain peptide/lipid ratio which should also contribute to
their toxicity (see for example [54]). Steps 3 and 4
(permeabilization and physiological consequences, respect-
ively) are identical to those induced by ion-channel forming
peptides such as alamethicin although the molecular
mechanisms of permeabilization (step 3) differ. Whilst for
alamethicin and analogues there is strong evidence that
membrane permeabilization is due to the formation of
dynamic barrel-staves [35,48], carpet-forming peptides
behave rather like detergents disrupting the lipid bilayer
when a threshold concentration of peptide monomer is
reached; at this stage, transient transmembrane pores might
be formed [53]. Some cationic peptides such as magainin are
also capable of forming transient toroidal pores composed
of dynamic, peptid/lipid supramolecular complexes [55].
Whatever the permeabilization mechanism, a sudden
membrane depolarization should prevent bacteria from
setting up appropriate countermeasures and lead to a rapid
cell death if the peptide concentration is maintained above a
critical threshold. In the case of bacteria like mollicutes
which are devoid of a cell wall (murein), cell death can occur
still faster upon the action of the most efficient peptides
since in this case massive entry of water into the cytoplasm
can lead to cell burst.
Mechanisms other than membrane permeabilization have

also been proposed to explain the antimicrobial activity of
some cationic peptides. In some cases, the inhibition of
intracellular proteins might indeed be responsible for
bacterial cell death (see for example [56,57] for brief
discussions). We believe that these different views are not
contradictory but rather reflect, even for a same peptide,
differences in the context of its action, the variables (notably
the properties of the target cell) being probably too many to
allow for a single and general mechanism of action.
Acknowledgements
We are pleased to thank Dr. K. Bu
¨
tner, now at Neosystem, for kindly
providing peptides and W. Ne
´
ri for peptide purification.
This work was supported by the GDR CNRS 790 (ÔPeptides et
Prote
´
ines MembranotropesÕ)andtheÔMiniste
`
re de lÕEnseignement
Supe
´
rieur et de la Recherche’ (ACC ÔPhysico-Chimie des Membranes
BiologiquesÕ and the ÔProgramme de Recherche Fondamentale en
Microbiologie et Maladies Infectieuses et ParasitairesÕ).
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