Andrea, Universita` La Sapienza, Rome, Italy 2 Dipartimento di Scienze e Tecnologie Biomediche, Universita` di Cagliari, Monserrato, Italy 3 Dipartimento di Scienze e Tecnologie Chimiche
Trang 1peptide that synergizes with antibiotics and modifies the expression level of a limited number of proteins in
Escherichia coli
Ludovica Marcellini1, Marina Borro1, Giovanna Gentile1, Andrea C Rinaldi2, Lorenzo Stella3,
Pierpaolo Aimola4, Donatella Barra1 and Maria Luisa Mangoni1
1 Istituto Pasteur-Fondazione Cenci Bolognetti, Dipartimento di Scienze Biochimiche, Azienda Ospedaliera S Andrea, Universita` La Sapienza, Rome, Italy
2 Dipartimento di Scienze e Tecnologie Biomediche, Universita` di Cagliari, Monserrato, Italy
3 Dipartimento di Scienze e Tecnologie Chimiche, Universita` di Roma Tor Vergata, Rome, Italy
4 Dipartimento di Biologia di Base ed Applicata, Universita` de L’Aquila, Italy
Keywords
frog skin antimicrobial peptides;
Gram-negative bacteria; mode of action;
peptide–membrane interaction; proteomics
Correspondence
M L Mangoni, Unita` di Diagnostica
Molecolare Avanzata, II Facolta` di Medicina
e Chirurgia, Azienda Ospedaliera S Andrea,
via di Grottarossa, 1035-00189 Roma, Italy
Fax: +39 06 33776664
Tel: +39 06 33775457
E-mail: marialuisa.mangoni@uniroma1.it
(Received 18 May 2009, revised 27 July
2009, accepted 4 August 2009)
doi:10.1111/j.1742-4658.2009.07257.x
Antimicrobial peptides constitute one of the main classes of molecular weapons deployed by the innate immune system of all multicellular organisms to resist microbial invasion A good proportion of all antimi-crobial peptides currently known, numbering hundreds of molecules, have been isolated from frog skin Nevertheless, very little is known about the effect(s) and the mode(s) of action of amphibian antimicrobial peptides
on intact bacteria, especially when they are used at subinhibitory concen-trations and under conditions closer to those encountered in vivo Here
we show that esculentin-1b(1–18) [Esc(1–18)] (GIFSKLAGKKLKNL-LISG-NH2), a linear peptide encompassing the first 18 residues of the full-length esculentin-1b, rapidly kills Escherichia coli at the minimal inhibitory concentration The lethal event is concomitant with the perme-ation of the outer and inner bacterial membranes This is in contrast to what is found for many host defense peptides, which do not destabilize membranes at their minimal inhibitory concentrations Importantly, prote-omic analysis revealed that Esc(1–18) has a limited ability to modify the bacterium’s protein expression profile, at either bactericidal or sublethal concentrations To the best of our knowledge, this is the first report on the effects of an antimicrobial peptide from frog skin on the proteome of its bacterial target, and underscores the fact that the bacterial membrane
is the major target for the killing mechanism of Esc(1–18), rather than intracellular processes
Abbreviations
CFU, colony-forming unit; Esc(1–18), esculentin-1b(1–18); DTE, dithioerythritol; FIC, fractional inhibitory concentration; FITC-D 4, fluorescein isothiocyanate–dextran of 4 kDa average molecular mass; FITC-D 10, fluorescein isothiocyanate–dextran of 10 kDa average molecular mass; FITC-D 40, fluorescein isothiocyanate–dextran of 40 kDa average molecular mass; FITC-D 70, fluorescein isothiocyanate–dextran of 70 kDa average molecular mass; Gal-ONp, 2-nitrophenyl b- D -galactoside; IM, inner membrane; LPS, lipopolysaccharide; LUV, large unilamellar vesicle; MIC, minimal inhibitory concentration; OM, outer membrane; OMP, outer membrane protein; PE, phosphatidylethanolamine; PG, phosphatidylglycerol; PMF, peptide mass fingerprinting; SEM, scanning electron microscopy; TEM, transmission electron microscopy; TF, trigger factor; TFA, trifluoroacetic acid.
Trang 2Numerous families of ribosomally synthesized
antimi-crobial peptides, from virtually all life forms, have been
described [1,2] They are conserved components of the
innate immune system in plants and animals, and
repre-sent the most ancient and efficient weapon against
microbial pathogens [3] In recent years, for several
anti-microbial peptides, additional chemokine-like and
immunomodulatory activities have been reported; these
are involved in infection processes leading to the
appro-priate activation of adaptive immune responses in higher
vertebrates [4] For this reason, these molecules are more
properly referred to as host defense peptides [5]
An increasing number of microorganisms have
become resistant to a multiplicity of clinically used
drugs, causing a severe crisis in the treatment and
man-agement of infectious diseases, with serious
conse-quences for human health [6] Therefore, substantial
efforts have been devoted to identifying new classes of
antibiotics displaying diverse mode(s) of action:
antimi-crobial peptides are currently considered to be some of
the most promising candidates for the development of
novel anti-infective preparations [7,8] Although
antimi-crobial peptides show marked variation in size, sequence,
and conformation, most of them are polycationic, and
fold into an amphipathic helical or b-sheet structure [9]
Numerous articles have provided compelling evidence
that many antimicrobial peptides penetrate microbes and
interfere with general intracellular functions (e.g DNA,
protein and cell wall synthesis or chaperone-assisted
pro-tein folding) without destabilizing their plasma
mem-brane Some examples are as follows: (a) buforin 2, from
histone H2A of Bufo bufo, and PR-39, from pig intestine
[10]; (b) derivatives of pleurocidin, a fish-derived
antimi-crobial peptide, and dermaseptin, from frog skin [11]; (c)
drosocin and pyrrhocoricin, from insects [12]; and (d)
Bac-7(1–35), corresponding to the 35-residue N-terminal
region of Bac-7 from bovine neutrophils [13]
However, very little is known about the effect(s) of
antimicrobial peptides at subinhibitory concentrations
Also, as reported in the literature, the antibacterial
activities of a vast repertoire of host defense peptides
have been assayed only in buffered or dilute media, and
these peptides have been found to be ineffective in the
presence of physiological ionic strength or biological
flu-ids such as serum [7] Hence, intense research focusing
on antimicrobial peptides is currently directed at
com-pleting our knowledge of their mode(s) of action at both
lethal and sublethal doses and at shedding light on their
antimicrobial properties under physiological conditions
Among the natural sources for antimicrobial peptides,
the granular glands of amphibian skin constitute one of
the richest [14–16] Studies on the mode of action of amphibian antimicrobial peptides have mainly addressed their interaction with phospholipid bilayers, but some have also dealt with intact microbes, and revealed that these antimicrobial peptides can perturb both model and biological membranes [17–19] We have recently com-pared the killing activities of antimicrobial peptides belonging to families that include esculentins, temporins, and bombinins H, extracted from three different species
of anurans, against multidrug-resistant clinical isolates [20] These studies showed that esculentin-1b(1–18) [Esc(1–18), GIFSKLAGKKLKNLLISG-NH2], the amidated form of a linear peptide encompassing the first
18 residues of the full-length esculentin-1b (46 amino acids) from the skin of Pelophylax lessonae⁄ ridibundus (previously classified as Rana esculenta [21]), was the most potent peptide, particularly towards Gram-negative species, with a minimal bactericidal concentration ranging from 0.5 to 1 lm, in sodium phosphate buffer [20] Here, to expand our knowledge of the activity of Esc(1–18) against Gram-negative bacteria, along with the underlying molecular mechanism, we analyzed the effect(s) of this peptide on Escherichia coli ATCC 25922
by investigating the following: (a) its microbicidal action and kinetics in different media; (b) its ability to permeate both artificial and bacterial membranes; (c) its affinity of binding to lipopolysaccharide (LPS); (d) its ability to synergize with conventional antibiotics; and (e) its effects
on bacterial morphology and the bacterial proteome Our data have shown that this unique amphibian-derived peptide: (a) kills E coli via membrane pertur-bation; (b) strongly synergizes with erythromycin, presumably by increasing the intracellular influx of this drug, as a result of increased membrane permeability; (c) elicits identical changes in the bacterium’s protein expression pattern at lethal and sublethal concentra-tions; and (d) preserves antibacterial activity under conditions closer to those encountered in vivo This is in contrast to many other host defense peptides, which kill microorganisms by altering intracellular processes, and become inactive in physiological solutions Importantly,
to the best of our knowledge, this is the first demonstra-tion of how an amphibian antimicrobial peptide can affect the protein expression profile of its bacterial target
Results
Structural analysis The secondary structure of Esc(1–18) was determined by using CD spectroscopy in 10 mm sodium phosphate
Trang 3buffer (pH 7.4) and when bound to
phosphatidyletha-nolamine (PE)⁄ phosphatidylglycerol (PG) vesicles of
composition 7 : 3 (w⁄ w), which is typical of the E coli
inner membrane (IM) [22] As indicated in Fig 1A, the
peptide conformation in buffer was predominantly
dis-ordered, whereas association of the peptide with lipid
vesicles induced a transition to a predominantly
a-heli-cal conformation Complete binding of the peptide
to the lipid vesicles was manifested by the absence of
significant changes in the CD spectrum when the
lipid⁄ peptide molar ratio was increased from 100 to 400 The helical wheel diagram of Esc(1–18) in a perfect a-helical conformation (Fig 1B) shows amphipathicity
of the peptide, with hydrophobic and hydrophilic residues segregating on opposite sides of the molecule
Antibacterial activity The activity of Esc(1–18) against E coli ATCC 25922 was first evaluated by the microdilution broth assay to determine the minimal inhibitory concentration (MIC), using both a standard inoculum of 1· 106 colony-forming units (CFUs)ÆmL)1and 4· 107CFUÆmL)1, as most of the experiments described below needed this higher number of bacterial cells As shown in Table 1, where the frog skin membrane-active peptide tempo-rin-1Tl [23] is included as a reference, the MIC of Esc(1–18) in culture medium (Mueller–Hinton broth) was found to be directly correlated with the number of microbes present in the inoculum Afterwards, to examine the killing activity of Esc(1–18) against E coli and to determine whether this process was affected by the ionic strength of the incubation medium, we assayed the peptide’s bactericidal effect, as defined in Experimental procedures, after 1.5 h of incubation with bacteria, either in Mueller–Hinton broth, sodium phosphate buffer (pH 7.4), or NaCl⁄ Pi (Table 1) Interestingly, in all cases, a reduction in the number of viable cells of ‡ 3 log10CFUÆmL)1 (99.9% mortality) was achieved at twice the MIC (16 lm) when a stan-dard inoculum was used In contrast, with the higher number of bacteria (4· 107CFUÆmL)1), Esc(1–18) displayed a bactericidal effect at 32 lm, a concentra-tion equal to the MIC, under these condiconcentra-tions (Table 1) Furthermore, to estimate the peptide’s abil-ity to retain such activabil-ity under experimental condi-tions closer to those encountered in vivo, antimicrobial assays were performed in the presence of human serum It is noteworthy that, unlike temporin-1Tl (Table 1) and other natural antimicrobial peptides, such as human b-defensin 2 and dermaseptin S, which lost their bacteriostatic effect in the presence of 20–30% serum (MIC‡ 200 lm) [24,25], Esc(1–18) was able to partially preserve its antibacterial activity at a higher serum percentage (70%), with MIC and bacteri-cidal concentration values of 32 and 64 lm, respec-tively (Table 1), using a standard inoculum As the peptide’s degradation by serum enzymes was prevented
by heating serum at 56C (see Experimental proce-dures), our findings suggest that serum components do not strongly bind to Esc(1–18) and therefore do not significantly affect the availability of active peptide molecules
λ (nm)
2 ·dmol
A
B
Fig 1 Secondary structure of Esc(1–18) (A) CD spectra of the
peptide in sodium phosphate buffer (pH 7.4) (solid line) and after
association with PE⁄ PG vesicles (dotted line, peptide 10 l M , lipid
1 m M ; broken line, peptide 5 l M , lipid 2 m M ) (B) Helical wheel plot
of Esc(1–18): hydrophilic, hydrophobic and potentially positively
charged residues are represented as circles, diamonds and
penta-gons, respectively The peptide is amidated at its C-terminus.
Trang 4The killing kinetics occurred on a quite fast time
scale, causing more than 90% microbial deaths within
10 min, at the MIC (Fig 2) The latter result indicates
a substantial difference from those antimicrobial
pep-tides that preferentially act on intracellular targets and
over a longer time scale, and do not manifest any
lethal activity at their MICs [11,26]
Mode of action studies
It is well known that the mode of action of
antimicro-bial peptides depends on the mode(s) of their
inter-action with the cell membrane However, before
reaching it, the peptide needs to bind and traverse the cell wall, which, in Gram-negative bacteria, is sur-rounded by an outer membrane (OM), composed mainly of the anionic LPS (or endotoxin), which forms
a barrier to protect bacteria from many hydrophilic and hydrophobic molecules, including some antimicro-bial peptides [27] Therefore, we first investigated the ability of Esc(1–18) to bind LPS and penetrate the
E coliOM
LPS binding properties LPS films have been used as suitable model systems to mimic the outer layer of the Gram-negative OM [28,29] To investigate the binding of Esc(1–18) to LPS,
we monitored the changes in surface pressure of mono-layers of commercially available LPS from E coli O111:B4 upon a peptide’s insertion, using the method described in Experimental procedures Esc(1–18) effi-ciently penetrated E coli LPS monolayers, as mani-fested by the increase in film surface pressure (Fig 3) Under experimental conditions, monolayer penetration reached a substantial stability around 1.0 lm Esc(1–18) (Fig 3A), which was then selected as the peptide con-centration for subsequent experiments When data from similar measurements were analyzed in terms of change in surface pressure (Dp) versus initial surface pressure (p0), the critical surface pressure correspond-ing to the LPS lateral packcorrespond-ing density preventcorrespond-ing the intercalation of Esc(1–18) into E coli LPS films could
be derived by extrapolating the Dp)p0 slope to
Dp = 0, yielding a value of 47 mNÆm)1 (Fig 3B) The kinetics of the insertion of the peptide into the LPS monolayer were characterized by a rapid and marked enhancement of surface pressure that followed soon after injection of the peptide into the subphase, the lag phase for this process being too short to be measurable with our instrumentation (Fig 3C) In a typical experiment, within the first 60 s after peptide injection, p attained a value that was slightly over 85%
1 x 10 7
1 x 10 6
1 x 10 5
Time (min)
1 x 10 4
Fig 2 Time-kill curves for E coli ATCC 25922 and Esc(1–18)
Bac-teria (4 · 10 7
CFUsÆmL)1) were grown in Mueller–Hinton broth at
37 C and diluted in sodium phosphate buffer (pH 7.4) About
4 · 10 6 CFUs in 100 lL were incubated with Esc(1–18) at the MIC
(32 l M ; ) and at a sublethal dose (0.25 l M ; ) The control (r)
consisted of bacteria incubated in the absence of peptide Aliquots
were withdrawn, diluted in Mueller-Hinton broth and plated on agar
plates for CFUs counting Data are the means ± standard
devia-tions of three independent experiments Similar results were
obtained when bacteria were suspended in Mueller–Hinton broth or
NaCl ⁄ P i , and therefore are not shown.
Table 1 Antibacterial activity of Esc(1–18) and temporin-1Tl on E coli ATCC 25922 The bactericidal activity is defined as the concentration
of peptide that is sufficient to reduce the number of viable bacteria by ‡ 3 log 10 CFUsÆmL)1after 1.5 h of incubation The values found for temporin-1Tl are in parentheses.
CFUÆmL)1
Mueller–Hinton
Mueller–Hinton broth
Sodium phosphate
Trang 5of the value recorded at the end of measurement (Fig 3C) This initial surge was then followed by a slower increase in p for approximately the next 19 min, when a plateau was reached, and no more significant variation in p was observed for at least the next
16 min This general kinetics pattern was apparently independent of the initial surface pressure and from peptide concentration, and was similar to that recorded for temporin-1Tl interacting with a monolayer made of the same type of LPS [30]
OM permeability The permeabilization of the OM was determined by investigating the periplasmic b-lactamase activity against its specific substrate CENTA [31] A plot of enzyme release, as a function of peptide concentration,
is shown in Fig 4A Interestingly, there was a dose-dependent perturbation of the OM, and the greatest perturbation was obtained at the MIC of the peptide (32 lm with 4· 107 CFUÆmL)1) The rate of CENTA hydrolysis, upon addition of 1· MIC of Esc(1–18) to the cells, was also monitored for 20 min, and the amount hydrolyzed was found to be 70% of the total within the first 5 min (Fig 4B)
IM permeability Next, the effect of the peptide on the E coli IM was analyzed by measuring the intracellular influx of SYTOX Green [32] This cationic dye, which is excluded by intact membranes, but not from those with lesions large enough to allow its entrance, dramatically increases its fluorescence when bound to intracellular nucleic acids (Fig 5) The data revealed that Esc(1–18) augmented the permeability of the IM, with kinetics superimposable on those of the OM permeation (although with a slightly longer lag time), reaching a final value in about 15–20 min and in a concentration-dependent fashion However, membrane permeation caused by Esc(1–18) was not maximal at levels up to twice the MIC This was manifested by a further enhancement of fluorescence, following the addition of
a detergent for the complete solubilization of phospho-lipid bilayers (Fig 5, arrow at 20 min) Then, to inves-tigate the size of membrane lesions induced by the peptide, we assessed the release of intracellular com-pounds, such as the cytoplasmic b-galactosidase, whose Stokes radius is equal to 69 A˚ [33] As reported in Fig 6, the enzyme release was almost 40% of maxi-mum when the peptide concentration was equal to the MIC These results underscore a disturbance of the IM, although to a smaller extent than that of the OM, and
30
25
20
15
10
5
0
40
35
30
25
20
15
10
5
0
48 44 40 36 32 28 24 20 16 12 8 4
0
50
45
40
35
30
25
20
15
10
2400 2000 1600 1200 800 400
0
5
0
Time (s)
A
B
C
Fig 3 Insertion of Esc(1–18) into E coli O111:B4 LPS
monolay-ers (A) Increments of surface pressure of E coli LPS monolayers
due to the addition of Esc(1–18) to the subphase are illustrated as
a function of peptide concentration at an initial surface pressure
varying between 19.2 and 21.0 mNÆm)1, or (B) an initial surface
pressure, with 1.0 l M peptide (C) Typical kinetics of surface
pres-sure increase related to Esc(1–18) penetration into E coli LPS
monolayers (p0= 14.2, with 1.0 l M peptide; an arrow indicates
peptide injection into the subphase) Each data point represents
the mean of triplicate measurements The standard deviation
varied between 0.1 and 0.9 mNÆm)1and, for the sake of clarity, is
not shown.
Trang 6indicate the existence of a direct correlation between
the peptide dose and the extent of both microbial death
and membrane disturbance
Synergistic activities with conventional
antibiotics
Checkerboard titrations were carried out using Esc(1–
18) in combination with different classes of clinically
available antibiotics As illustrated in Table 2, a clear
synergism was noted when the peptide was mixed with
cephalosporin C, erythromycin, nalidixic acid,
netilmi-4000 5000
2000 3000 4000
0 1000
Time (min)
Fig 5 Effect of Esc(1–18) on the permeation of the E coli ATCC 25922 IM Cells (4 · 10 7 CFUsÆmL)1) were incubated with
1 l M SYTOX Green in NaCl ⁄ P i When basal fluorescence reached a constant value, the peptide was added (first arrow, t = 0), and changes in fluorescence were monitored (kexcitation= 485 nm, k emis-sion = 535 nm) and plotted as arbitrary units SDS (0.1% in chloro-form) was added for the maximal membrane permeation (second arrow, t = 20 min) Data points represent the mean of triplicate samples with standard deviation values not exceeding 2.5% from a single experiment, representative of three different experiments The peptide concentrations used were as follows: 2 l M (s); 4 l M (*); 8 l M (e); 16 l M (d); 32 l M ( ); and 64 l M ( ) Fluorescence values of control (bacteria without peptide) were subtracted from each sample.
100
60
80
0
20
40
0
Time (min)
0.05
0.06
0.07
A
B
Absorbance 0.02
0.03
0.04
0
0.01
detergent lysis
Peptide concentration (µ M )
Fig 4 Permeation of E coli OM by Esc(1–18) (A) Effects of
differ-ent concdiffer-entrations of Esc(1–18) on permeation of the OM of E coli
ATCC 25922 (4 · 10 7
CFUÆmL)1), were followed spectrophotometri-cally by measuring the activity of periplasmic b-lactamase The cells
were resuspended in sodium phosphate buffer (pH 7.4) + 100 m M
NaCl, and incubated with different concentrations of peptide at
37 C for 20 min The enzyme activity was measured in the culture
filtrate by following the hydrolysis of 80 l M CENTA at 405 nm The
absorbances of all peptide-treated samples, bacteria without peptide
(control) and bacteria after lysis with 0.1% SDS in chloroform are
reported on the y-axis The values are the means of three
inde-pendent measurements ± standard deviations (B) Kinetics of OM
permeabilization caused by 1 · MIC of Esc(1–18) (32 l M ) Bacteria
(4 · 10 7 CFUÆmL)1) were incubated with the peptide at different
time intervals, and b-lactamase activity was detected as described
above and expressed as percentage of the total obtained after cell
lysis Data are the means ± standard deviations of three
indepen-dent experiments.
60 70
40 50 60
10 20
30
0
Peptide concentration (µ M )
Fig 6 Bacterial viability and b-galactosidase activity of E coli ATCC 25922 culture after treatment with Esc(1–18) Bacterial cells (4 · 10 7 CFUsÆmL)1) were grown in Mueller–Hinton broth at 37 C, diluted in sodium phosphate buffer (pH 7.4), and incubated with the peptide at different concentrations for 20 min at 37 C The number of surviving cells ( ) is given as the percentage of the total b-Galactosidase activity was measured in the culture filtrate
by following the hydrolysis of 2 m M Gal-ONp at 420 nm Enzymatic activity detected in the control (bacteria without peptide) was sub-tracted from all values, which are expressed as percentage of the total (e) Complete enzyme activity was determined by treating bacteria with 0.1% SDS in chloroform The values are the means
of three independent measurements ± standard deviations.
Trang 7cin, and rifampicin [a fractional inhibitory
concentra-tion (FIC)£ 0.5 indicates synergy; see Experimental
procedures] To obtain insights into the mode of action
underlying the synergistic activity, we investigated the
bactericidal action of the combination of Esc(1–18)
and erythromycin, the antibiotic that gives the best
synergy with the peptide, as indicated by the lowest
FIC (Table 2) Erythromycin is a hydrophobic
mole-cule that inhibits protein synthesis by blocking either
the peptidyltransferase reaction or the translocation
step, but cannot easily traverse the OM of
Gram-negative bacteria [34]
As expected, erythromycin displayed a weak
bacte-ricidal effect, causing about 35% microbial death at a
very high concentration (256 lgÆmL)1) and within 3 h
of incubation (Fig 7) Interestingly, when sublethal
concentrations of Esc(1–18) and erythromycin were
combined, 8% and 90% killing were detected after
20 min and 3 h, respectively (Fig 7) These results
provide additional support for the
membrane-permea-bilizing properties of Esc(1–18) Indeed, as no
reduc-tion in the number of viable cells was observed
within the first 20 min [killing kinetics of Esc(1–18)],
but a reduction was observed after a longer time
(2–3 h) (Fig 7), corresponding to the time-kill kinetics
of erythromycin, we can assume that the synergistic
activity between the two compounds is the result of
increased access of erythromycin to its intracellular
target, because of increased peptide-induced
perme-ability of the cytoplasmic membrane and⁄ or the LPS
layer
Permeabilization of large unilamellar vesicles (LUVs)
The peptide’s ability to alter the structure of the plasma membrane of E coli cells by a nonstereo-specific process was also confirmed by employing calcein-loaded liposomes made of PE⁄ PG (7 : 3, w:w) Different concentrations of peptide were added to LUV suspensions, and membrane permeability was measured by following fluorescence recovery due to calcein leakage from the liposomes [35] Calcein leakage occurred immediately after peptide addition, and reached a plateau within the first 15 min (Fig 8A) Figure 8B shows the dose–response curve of peptide-induced calcein release from PE⁄ PG vesicles The data clearly show a membrane-perturbing activity of Esc(1– 18) Note that this activity increased in a dose-depen-dent manner and reached its maximum ( 65% calcein leakage) at a peptide⁄ lipid molar ratio of 1.5 These results are comparable with those found for other membrane-active antimicrobial peptides, such as cathe-licidin LL-37 [36] However, at a peptide⁄ lipid molar ratio as low as 0.04, Esc(1–18) was more active than cathelicidin LL-37 [36] As illustrated in Fig 8B, Esc(1–18) did not fully permeabilize the lipid vesicles, and the calcein leakage diminished when the peptide⁄ lipid molar ratio exceeded 1.5, probably because of the peptide’s aggregation at high concentrations Taken together, these observations are in line with those made above using intact cells (Figs 5 and 6), and are consistent with the suggestion that Esc(1–18) binds and destabilizes the bacterial membrane, but to a lesser
Table 2 Interaction of Esc(1–18) with conventional antibiotics
against E coli ATCC 25922 The ranges of concentrations tested
were as follows: 0.25–64 mgÆL)1 for Esc(1–18) and 0.25–
256 mgÆL)1 for the other antimicrobial agents FIC indices were
interpreted as follows: FIC £ 0.5, synergy; 0.5 < FIC <1, additivity;
1 £ FIC < 4, indifference; and FIC ‡ 4, antagonism.
80 100 120
CFU (%) 40 20 60 80
0
Incubation time (min)
Fig 7 Synergistic effect in the bactericidal activity of erythromycin and Esc(1–18) E coli cells (1 · 10 6
CFUsÆmL)1) were incubated in Mueller–Hinton broth (diluted 1 : 2 with distilled water) in the pres-ence of 256 lgÆmL)1erythromycin (white bars), a sublethal concen-tration of erythromycin (8 lgÆmL)1, gray bars), or Esc(1–18) (1 lgÆmL)1, squared bars), and with the combination of erythromy-cin and Esc(1–18) at their sublethal doses (black bars) Aliquots were withdrawn at the time intervals indicated, and plated for counting The percentages of viable cells with respect to the con-trol (bacteria not treated) are reported on the y-axis Data are the means ± standard deviations of three independent experiments.
Trang 8extent than temporin-1Tl [37] According to what has
been stated for other antimicrobial peptides [38], such
a discrepancy between the two frog skin peptides
might be related to a higher fraction of
membrane-bound active temporin-1Tl than of Esc(1–18)
The ability of Esc(1–18) to induce the leakage of
liposome-encapsulated markers of different sizes was
also monitored PE⁄ PG LUVs were preloaded with
fluorescein isothiocyanate–dextrans (FITC-Ds) of 4,
10, 40 or 70 kDa average molecular mass (FITC-D 4,
FITC-D 10, FITC-D 40, and FITC-D 70), and then
incubated with the peptide The data shown in Fig 9
reveal that Esc(1–18) is able to cause the release of the
four dextrans used in a dose-dependent manner, and with a dependence on the size of the liposome-entrapped probe This indicates that Esc(1–18) does not have a detergent-like effect on the membrane [39], and that membrane lesions produced by this peptide are larger than 58 A˚ (Stokes radius of FITC-D 70 [40]), which is in agreement with its ability to promote the release of b-galactosidase from E coli cells
Scanning electron microscopy (SEM) The effect(s) of Esc(1–18) on E coli morphology were visualized by SEM (Fig 10) The exposure of
4· 107cellsÆmL)1 at the corresponding MIC of Esc(1–18) resulted in an irregular rod form with a deep wrinkling of the cell surface (within 5 min) However, all of these changes became more pronounced after a longer incubation time (20 min) With reference to untreated cells, bacteria appeared flat, with a collapsed cell structure and surface corrugation similar to that induced by temporin-1Tl [41], but in a milder form
Transmission electron microscopy (TEM) TEM was then used to directly examine the damage to bacteria induced by the peptide A local disturbance to the membrane was noted after the first 5 min of pep-tide treatment, and this was followed by more damage and loss of cellular integrity, with a partial discharge
of the cellular contents, within 20 min (Fig 11) These results correlate with the killing kinetics of the peptide, and show that the antibacterial activity of Esc(1–18) is concomitant with its membrane-perturbing activity
A
50
60
70
B
10
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0
(Peptide) : (Lipid)
[Peptide]:[Lipid]
50
1.5
0.08
0.0012
20
30
40
0
10
0
Time (min)
Fig 8 Calcein leakage from PE ⁄ PG LUVs after Esc(1–18)
treat-ment (A) Time course of calcein release from PE ⁄ PG (7 : 3, w ⁄ w)
calcein-loaded LUVs (final lipid concentration 200 l M ) after addition
of Esc(1–18) (arrow at time zero) at different concentrations Control
(broken line) consisted of liposomes not treated with the peptide.
Calcein release was detected fluorimetrically (k excitation = 485 nm,
100(F1– F0) ⁄ (F t – F0), where F1 and Ft denote the fluorescence
before and after the addition of detergent (0.1% Triton X-100),
respectively, and F 0 represents the fluorescence of intact vesicles.
Data points are means with standard deviations not exceeding 4%
from a single experiment, representative of three independent
mea-surements (B) Esc(1–18) was added to PE ⁄ PG LUVs (7 : 3, w ⁄ w)
at concentrations of 0.125–660 l M Calcein release was detected as
described above, after 15 min of peptide treatment Data points are
means with standard deviations not exceeding 3% from a single
experiment, representative of four independent measurements.
30 40 50 60
0 10 20
(Peptide) : (Lipid)
Fig 9 Effect of Esc(1–18) on the release of FITC-D from PE ⁄ PG liposomes Liposomes containing FITC-D 4 (r), FITC-D 10 ( ), FITC-D 40 ( ) or FITC-D 70 (s) were prepared as described in Experimental procedures, and incubated in the presence of differ-ent concdiffer-entrations of the peptide for 15 min at 37 C Dextran release was detected fluorimetrically (kexcitation= 470 nm; kemission=
520 nm) Leakage was calculated as 100(F1– F0) ⁄ (F t – F0), where
F 0 represents the fluorescence of intact vesicles, and F 1 and F t denote the intensities of the fluorescence achieved by peptide and Triton X-100 treatment, respectively Values are means of three independent measurements ± standard deviations.
Trang 9Proteomic analysis
To determine whether Esc(1–18) could evoke a cellular
reaction by modifying, within 20 min, the expression
levels of proteins under conditions where the peptide
did not affect the viability of E coli or reduced it by
40% (2 and 16 lm peptide, respectively; data not
shown), the bacterial proteome was analyzed by means
of 2D-PAGE and MS This analysis revealed a similar
pattern of responses to both sublethal and lethal
pep-tide doses, consisting of only a few significant
varia-tions in protein expression (11 protein spots) as
compared with untreated cells The majority of these
spots (Fig 12) were identified by peptide mass
finger-printing, reported in Table 3 In particular, reductions
in the expression levels of a number of OM proteins
(OMPs), such as OMPc, nmpC, and OMP F, all of
which form passive diffusion pores allowing the
pas-sage of small molecular weight hydrophilic materials
[42], were detected in peptide-treated bacteria
(Table 3), with stronger reductions being seen at 16 lm
Esc(1–18) Otherwise, a slight increase in OMP W
expression was found at 16 lm Note that the function
of this protein is not completely understood; however,
recent data have suggested that it may be involved in
the protection of bacteria against various forms of environmental stresses [43] Overexpression of trigger factor (TF) was also caused by both peptide concen-trations TF in E coli is a ribosome-associated chaper-one that initiates folding of newly synthesized proteins [44] The enhanced production of TF might contribute
to more streamlined de novo protein folding, by shield-ing nascent polypeptides on the ribosome, and thereby shortening degradation or aggregation processes [45]
In addition, as shown in Table 3, exposure of bacteria
to Esc(1–18) gave rise to a drop in the level of the fol-lowing enzymes: (a) glucosamine-fructose-6-phosphate aminotransferase, which catalyzes the formation of glucosamine 6-phosphate, a precursor of cell wall peptidoglycan synthesis [46]; and (b) the dihydro-lipoyllysine-residue succinyltransferase component of 2-oxoglutarate dehydrogenase complex, which catalyzes the conversion of a-ketoglutarate into succinyl-CoA as part of the tricarboxylic acid cycle [47]
Discussion
The repertoire of antimicrobial peptides has dramati-cally increased during the past two decades, and
> 800 antimicrobial peptides have been isolated from
Control
MIC, 5 min
MIC, 20 min
Fig 10 Scanning electron micrographs of
Esc(1–18)-treated E coli ATCC 25922 cells
(4 · 10 7 CFUsÆmL)1) Upper panels: control
bacteria Middle panels: bacteria after 5 min
of treatment with Esc(1–18) at the MIC
(32 l M ) Lower panels: bacteria after 20 min
of treatment with Esc(1–18) at the MIC.
See Results for other experimental details
and descriptions of the images Each image
has been magnified · 10 000 or · 20 000.
Trang 10different plant and animal sources, with more than 400
isoforms being obtained from amphibian species This
article discusses the antibacterial activity and mode of
action of the N-terminal region of esculentin-1b, an antimicrobial peptide from the skin of P lessonae⁄ ridi-bundus As no activity against microorganisms had been previously observed with the 19–46 fragment of this peptide, possibly because of its low positive charge
at neutral pH (+1 versus +5 for the whole molecule) [48], we analyzed the antibacterial activity of the 1–18 N-terminal portion of esculentin-1b Surprisingly, this activity was found to be similar to that of the full-length natural peptide [48,49], whereas complementary insecticidal properties were ascribed to the 19–46 fragment [50] Recent experiments have underscored the fact that Esc(1–18) possesses a wide spectrum of antimicrobial activity against several species of Gram-positive bacteria, Gram-negative bacteria, Candida and multidrug-resistant nosocomial pathogens, without being hemolytic [20,48]
Regardless of the precise mode of action, the effect(s) of antimicrobial peptides in general depends upon their interaction with the microbial membrane [51,52] In particular, the first step in this process is the electrostatic attraction between the cationic peptide and the negatively charged components of the cell envelope, such as the phosphate groups within the LPS molecules of the OM in Gram-negative bacteria
or the lipoteichoic acids on the surface of Gram-posi-tive bacteria In the case of Gram-negaGram-posi-tive bacteria, antimicrobial peptides initially cross the LPS layer, in
a self-promoted uptake process driven by hydrophobic interactions, and subsequently reach the IM [51] Nev-ertheless, studies performed with intact bacteria have shown that antimicrobial peptides, e.g pleurocidin derivatives and buforin 2, do not disturb the membrane of E coli at their minimal antimicrobial concentrations, but rather traverse it, accumulate intracellularly, and damage a variety of essential vital processes to mediate the lethal event, which occurs only at multiples of the MICs [7,11,26]
In this study, we have shown that Esc(1–18) dis-plays rapid bactericidal activity, at the MIC, against
E coli (Fig 2), concomitant with alteration of its inner and outer membranes (Figs 4–6) As shown by the biophysical and biochemical assays, this peptide strongly bound LPS and completely permeated the LPS OM (Figs 3 and 4) In addition, the intracellular influx of SYTOX Green (Fig 5), the extracellular leakage of b-galactosidase (Fig 6), calcein and dex-tran release from liposomes mimicking the E coli IM (Figs 8 and 9) and electron microscopy images (Figs 10 and 11) suggest that Esc(1–18) is a mem-brane-active peptide which kills bacteria by, primarily, injuring their membranes This interpretation is fur-ther supported by the small changes in the proteomic
1 µm
A
1 µm
B
1 µm
C
Fig 11 Transmission electron micrographs of Esc(1–18)-treated
E coli ATCC 25922 cells (4 · 10 7
CFUsÆmL)1) (A) Representative control (B) Representative bacterium after 5 min of peptide
treat-ment at the MIC (32 l M ) (C) Representative bacterium after
20 min of peptide treatment at the MIC See Results for other
experimental details and descriptions of the images.