Whereas spherical oligomers of Drs S9 exhibit antimicrobial activity, the soluble, weakly self-associated forms of Drs S9 act on human leukocytes to promote chemotaxis and⁄ or immunologi
Trang 1chemoattractant activities for dermaseptin S9
Constance Auvynet1,2, Chahrazade El Amri1, Claire Lacombe1, Francine Bruston1, Julie Bourdais2, Pierre Nicolas1and Yvonne Rosenstein2
1 UPMC Universite´ de Paris 06, CNRS FRE 2852, Peptidome de la Peau des Amphibiens, Paris Cedex 5, France
2 Departamento de Medicina Molecular y Bioprocesos, Instituto de Biotecnologia, Universidad Nacional Auto´noma de Me´xico, Cuernavaca, Morelos, Mexico
Antimicrobial peptides that play a role in host defence
against competing or pathogenic microorganisms are
small proteins, typically 10–50 residues long, interact
with lipid bilayers to alter cell-membrane permeability,
which often leads to cell death Structural studies have
revealed that the peptide secondary structures include
a-helices, b-sheet structures stabilized by two or three
disulfide bonds, and extended structures with
over-representation of one or two amino acids (W, P, H or G) [1] However, all these peptides, regardless of the secondary structure or length, are cationic and exhibit amphipathic properties upon interaction with lipid bilayers, with apolar amino acid residues segregating from the hydrophilic residues on opposite sides of the 3D structure These structural elements are believed to play a crucial role in the binding of cationic host
Keywords
antimicrobial peptide; chemotaxis;
dermaseptin; frog skin; infrared
spectroscopy
Correspondence
P Nicolas, UPMC University Paris 06, CNRS
FRE 2852, Peptidome de la Peau des
Amphibiens, Tour 43, 2 place Jussieu,
75251 Paris cedex 5, France
Fax: +33 1 44 27 59 94
Tel: +33 1 44 27 95 36
E-mail: pnicolas@ccr.jussieu.fr
Y Rosenstein, Departamento de Medicina
Molecular y Bioprocesos, Instituto de
Biotecnologia, Universidad Nacional
Auto´noma de Me´xico, Avenida Universidad
2001, Col Chamilpa, Cuernavaca, Mor.
62270, Mexico
Fax: +52 73172388
Tel: +52 5 55 66 22 76 63
E-mail: yvonne@ibt.unam.mx
(Received 29 April 2008, revised 11 June
2008, accepted 16 June 2008)
doi:10.1111/j.1742-4658.2008.06554.x
Dermaseptin S9 (Drs S9), GLRSKIWLWVLLMIWQESNKFKKM, iso-lated from frog skin, does not resemble any of the cationic and amphipathic antimicrobial peptides identified to date, having a highly hydrophobic core sequence flanked at either side by cationic termini Previous studies [Lequin
O, Ladram A, Chabbert A, Bruston F, Convert O, Vanhoye D, Chassaing
G, Nicolas P & Amiche M (2006) Biochemistry 45, 468–480] demonstrated that this peptide adopted a non-amphipathic a-helical conformation in trifluoroethanol⁄ water mixtures, but was highly aggregated in aqueous solutions and in the presence of sodium dodecyl sulfate micelles Circular dichroism, FTIR and attenuated total reflectance FTIR spectroscopies, combined with a surface plasmon resonance study, show that Drs S9 forms stable and ordered b-sheet aggregates in aqueous buffers or when bound to anionic or zwitterionic phospholipid vesicles These structures slowly assem-bled into amyloid-like fibrils in aqueous environments via spherical inter-mediates, as revealed by electron microscopy and Congo red staining Drs S9 induced the directional migration of neutrophils, T lymphocytes and monocytes Interestingly, the antimicrobial and chemotactic activities
of Drs S9 are modulated by its amyloid-like properties Whereas spherical oligomers of Drs S9 exhibit antimicrobial activity, the soluble, weakly self-associated forms of Drs S9 act on human leukocytes to promote chemotaxis and⁄ or immunological response activation in the same range of concentra-tion as amyloidogenic peptides Ab(1–42), the most fibrillogenic isoform of amyloid beta peptides, and the prion peptide PrP(106–126)
Abbreviations
ATR, attenuated total reflectance; DMPC, phosphatidylcholine; DMPG,
1,2-dimyristoyl-sn-glycero-3-phosphatidylglycerol; Drs B2, dermaseptin B2; Drs B4, dermaseptin B4; Drs S9, dermaseptin S9; ERK, extracellular signal-regulated kinase; fMLP, formyl methionyl-leucyl-phenylalanine; FPRL-1, formyl peptide receptor like 1; PTX, pertussis toxin; SPR, surface plasmon resonance; TFA, trifluoroacetic acid.
Trang 2defence peptides to the negatively charged outer leaflet
of bacterial bilayers Once bound, the hydrophobic
face of the amphipathic peptide allows the peptide to
enter the membrane interior, thereby triggering local
fusion of the membrane leaflets, pore formation,
cracks and membrane disruption [2–7]
Frog skin is by far the most important source of
antimicrobial peptides, with more than half of the
pep-tides described to date isolated from South American
Hylidae or European, Asian or North American
Rani-dae [1] Among these peptides, dermaseptins S and B,
from the skin of the South American tree frogs
Phyllo-medusa bicolor and P sauvagei, form a family of
amphipathic, a-helical, closely related antimicrobial
peptides with broad-spectrum microbicidal activities at
micromolar concentrations against Gram-positive and
Gram-negative bacteria, fungi, yeasts and protozoa [8]
In previous work, we have used the conservation of
the preproregion sequences of the preprodermaseptin
transcripts to identify a new member of the
dermaseptin S family, dermaseptin S9 (Drs S9),
structure of this peptide does not resemble that of any
antimicrobial peptide identified to date, having a
hydrophobic central core flanked by positively charged
termini This structure is similar to that of synthetic
peptides originally designed as transmembrane mimetic
models that spontaneously become inserted into
mem-branes [10] We have shown that Drs S9 adopted a
non amphipathic a-helical conformation in the
pres-ence of trifluoroethanol⁄ water mixtures, which are
known to promote helical structures, but that it
aggre-gated in aqueous solutions and in the presence of SDS
micelles [9] Other antimicrobial peptides of the
derm-aseptin S family, particularly Drs S4 [11], have been
reported to form aggregates, leading to the proposal
that the state of aggregation of a peptide in solution
might be an important determinant for selective
cyto-toxicity as well as other biological events [12] In the
present study, we characterized the self-organization of
Drs S9 in aqueous solutions and determined how this
might affect its biological activity Drs S4 forms
amor-phous aggregates, but Drs S9 is a tryptophan-rich
peptide that forms ordered aggregates in aqueous
solutions, two characteristics that are reminiscent of
amyloid peptides We thus determined whether Drs S9
could have amyloidogenic properties Our data provide
evidence that, similar to amyloids, Drs S9 folds into a
b-sheet structure that assembles into amyloid-like
fibrils via spherical oligomeric intermediates in a
time-and temperature-dependent manner, time-and that, unlike
several antimicrobial peptides that form amyloid-like
fibers in the presence of acidic phospholipids [13–15],
Drs S9 forms amyloid fibrils in an aqueous envir-onment
Antimicrobial peptides are fundamental components
of the innate immune response; in addition to killing invading microorganisms, they modulate several immune functions such as chemotaxis of immune cells [16] through specific cell-surface receptors, such as for-myl peptide receptor-like 1 (FPRL-1) [17] Interest-ingly, amyloid peptides, which are associated with neuroinflammatory disorders such as Alzheimer’s, prion or Parkinson’s diseases, also interact with FPRL-1 and promote chemotaxis, favouring inflamma-tion [18–21] The neurotoxin prion peptide fragment PrP(106–126) is a chemotactic agonist for the G pro-tein-coupled receptor FPRL-1 [18] Moreover, the vari-ous structural states of the amyloidal peptide Ab(1–42) modulate its biological activities: the low-molecular-weight form of the peptide seems to be chemotactic, while the oligomeric form seems to be neurotoxic [21,22] With this in mind, we also tested the chemo-tactic potential of Drs S9 and determined whether the amyloidogenic properties of Drs S9 could modulate its biological activities Maximum antimicrobial activity
of Drs S9 was detected in its oligomeric form, while, similar to amyloid-like peptides, Drs S9 was chemotac-tic for human leukocytes in its soluble, low-molecular-weight, self-associated form Together, the data pre-sented here establish that dermaseptin S9 is the first antimicrobial frog skin peptide to exhibit amyloido-genic properties with potent chemotactic and antimi-crobial activities
Results
Dermaseptin S9 is highly aggregated and forms b-structures
Amide H⁄ D exchange kinetics monitored by attenu-ated total reflectance (ATR) FTIR provided informa-tion on the solvent accessibility of the peptide NH amide groups The solid state was taken as 100% NH content for the NH⁄ ND exchange analysis After
15 min, lower NH⁄ ND exchanges were observed for Drs S9 dissolved in NaCl⁄ Pi (18%) than for Drs S9 dissolved in D2O (50%) (Fig 1A), which is concomi-tant with an increase in b-sheet content (see below) that underlies the self-associative propensity of the peptide in the saline buffer
The kinetics of interactions between a peptide and
an adsorbing surface can be monitored in real time by surface plasmon resonance (SPR) A shift in the reso-nance signal during the adsorption step may provide information about the concentration of the peptide
Trang 3on the adsorbing surface For the hydrophobic HPA
biosensor, consisting of long chain alkanethiol
mole-cules attached directly to the gold film, the SPR signals
(resonance units) plotted as a function of peptide
con-centration (1–300 lm) further suggest a self-associative
propensity of Drs S9 (Fig 1B) The resulting curve
was biphasic, in contrast to monophasic curves
obtained with another well-known dermaseptin,
Drs B2 [23] The density of Drs S9 peptide adsorbed
onto HPA was quantified under continuous flow,
assuming the manufacturer’s characteristics for the
detection surface (1.2 mm2) [24] The surface area
occupied by a 23-residue peptide molecule differs
depending on its conformation When in a-helix
for-mation, its contact surface may be regarded as a
rect-angle measuring 3.3 nm long by 1.5 nm wide, giving a
surface area of approximately 5 nm2; in the b-structure
formation, the peptide has a contact area of 10–
12 nm2 [25] Consequently, the hydrophobic surface
could bind 0.9 ngÆmm)2of helical and 0.4 ngÆmm)2 of
b-sheet peptide, respectively The measured adsorption
densities for 30 lm peptide were thus compatible with
a b-structure for Drs S9 (Table 1) Figure 1B shows that, in the range of 5–30 lm, a monolayer of Drs S9
in b-sheet formation was adsorbed (480 resonance units, see Table 1) From 30–300 lm, the resonance unit values were 2- to 4-fold greater, corresponding to various types of peptide–peptide association
0
20
40
60
80
100
D 2 O PBS
Time (min)
0 250 500 750 1000 1250 1500 1750
PS9 concentration (µ M )
200 220 240
–40
–20
0
20
40
60
PBS PB
H 2 O
–1 ·cm
Wavelength (nm)
1500 1550 1600 1650 1700 1750 1800 0.00
0.01 0.02 0.03 0.04
Wavenumber (cm –1 )
0 10 20 30 40
1624 (β-sheet)
1636
1650 1665
1685
( β-hairpin)
(β-turn)
( β-turnandbent)
(disordered)
A
E
B
Fig 1 Dermaseptin S9 is highly aggregated and forms b-structures (A) NH content kinetics obtained by ATR FTIR (B) Absorp-tion density (resonance units) versus con-centration (1–300 l M ) obtained by SPR (C)
CD spectra of Drs S9 (30 l M ) freshly dis-solved in water, phosphate buffer (PB) or NaCl ⁄ P i (PBS) (D) ATR FTIR spectra in the 1500–1800 cm)1region indicating the adop-tion of b-sheet structure in NaCl ⁄ P i medium (E) Distribution of component band contents for Drs S9 in NaCl ⁄ P i (grey) or D 2 O (open).
Table 1 Adsorption potencies of Drs S9 on the hydrophobic HPA biosensor as function of peptide concentration.
50 l M HPA 100 l M HPA 300 l M HPA SPR response
(resonance units)
Adsorption density (ngÆmm)2)
Adsorbed molecule
no ⁄ mm 2 (· 10 12 )
Complex density (ngÆmm)2) a
a The complex density (ng per mm 2 ) is evaluated from values of the SPR response taken at 180 s in the desorption step.
Trang 4more, for peptide concentrations between 30 and
300 lm, the density of the complex evaluated from
values observed at 180 s during the desorption step
were identical to adsorption densities, suggesting that,
in contrast to some frog antimicrobial peptides [26],
the rate of interaction between Drs S9 and the
hydro-phobic support is not limited by the surface
Dichroic spectra typical of b-sheet conformation
(Fig 1C), with a negative band at 215–218 nm (amide
nfi p* transition) and a positive band at 190–200 nm
(amide pfi p* transition) were obtained from freshly
dissolved Drs S9 in phosphate buffer or NaCl⁄ Pi The
spectrum profile of Drs S9 dissolved in H2O exhibited
a blue shift that can be attributed to a distorted b-sheet
conformation, underlining the impact of the
environ-ment on peptide structure stability as well as on the
degree of peptide self-association In addition, the
neg-ative band became broader in phosphate buffer at
230 nm, probably due to contributions from aromatic
side chains [27] The effect of temperature on the
stabil-ity of the peptide structure was also tested by subjecting
a solution of Drs S9 in 50 mm phosphate buffer to
tem-peratures ranging from 5 to 70C No clear transition
from an ordered structure, i.e a b-sheet conformation,
to a random coil structure was observed, demonstrating
that Drs S9 was quite stable, with a multimeric
arrangement (supplementary Fig S1)
The 1500–1800 cm)1 region of the ATR FTIR
spec-trum of Drs S9 dissolved in deuterated NaCl⁄ Pi
showed a peak at 1624 ± 2 cm)1, corresponding to an
intermolecular b-sheet structure (Fig 1D) Comparison
between the distribution of the Drs S9 amide I¢
com-ponent bands in the two buffers (NaCl⁄ Pi or D2O)
(Fig 1E) confirmed a higher content of ordered
struc-ture in NaCl⁄ Pi medium than in aqueous medium as
evaluated by second-derivative analysis Moreover,
Drs S9 freshly dissolved in H2O or in NaCl⁄ Pi
migrated with apparent molecular masses of
approxi-mately 6 and 12 kDa under SDS–PAGE
(supplemen-tary Fig S2), suggesting that Drs S9 could already be
self-associated as dimers or tetramers
Dermaseptin S9 has b-amyloid-like properties
As, similar to amyloidogenic proteins, Drs S9
exhib-ited a b-sheet-rich conformation and a high propensity
to aggregate in an aqueous environment, we assessed
its ability to form amyloid fibrils using classical
approaches [28]: (a) detection of yellow–green
bi-refrin-gence under polarized light upon staining with Congo
red, (b) identification of fibrils obtained by negative
stain transmission electron microscopy (TEM), and (c)
amide I¢ analysis by infrared spectroscopy After
7 days of incubation at 37C, Congo red-treated Drs S9 exhibited a red colour under normal light (Fig 2Aa), and the yellow–green bi-refringence charac-teristic of amyloidogenic peptides under polarized light (Fig 2Ab) In contrast, at day 0 or after 3 days of incubation at 37C, no characteristic bi-refringence was observed under polarized light (data not shown) Dried phosphate buffer, or a 7-day-old solution of Drs B2, an a-helical linear antimicrobial peptide [23], stained with Congo red in phosphate buffer did not display the characteristic red staining under normal light (Fig 2Ac,Ae) nor yellow–green bi-refringence under polarized light (Fig 2Ad,Af) Neither Congo red nor Drs S9 alone exhibited the red colour or the yellow–green bi-refringence At day 3, aggregates with
a granular appearance that looked like spherical oligo-mers could be visualized (Fig 2Ba) Consistent with Congo red staining data, electron microscopy revealed that, at day 7, the same solutions of Drs S9 exhibited, like amyloid fibrils, a long filamentous structure of
8 nm diameter (Fig 2Bb) As expected, at day 0, nei-ther spherical oligomers nor fibrillar structures could
be observed (data not shown) Thus, three structural states can be defined for Drs S9: day 0 (D0), low molecular weight; day 3 (D3), oligomeric; day 7 (D7), fibrillar A kinetic study of the structure evolution Drs S9 was monitored using CD (supplementary Fig S1) The amyloid-like arrangement of the peptide was confirmed by submitting a 7-day-old solution of Drs S9 stained with Congo red and dried onto a glass slide to ATR FTIR spectroscopy Maximum absorp-tion of the spectrum was registered at 1624 cm)1, with shoulders at 1665 and 1685 cm)1 (Fig 3A) Second-derivative analysis of the spectrum (Fig 3B) showed a 15% gain in the proportion of b-structures (Fig 3C) compared to freshly dissolved Drs S9 with 75% b-structure (Fig 1E) In addition, 1624 cm)1 and
1685 cm)1 individual component bands were identified
as fingerprints of intermolecular b-sheet aggregates and b-amyloid structures [29] In summary, these data show that Drs S9 exhibits the biophysical and morpho-logical characteristics of amyloidogenic peptides
Dermaseptin S9 induces chemotaxis via a seven-transmembrane G protein-coupled cell surface receptor
Amyloidogenic peptides have been shown to partici-pate in inflammatory processes by inducing cell migration [30] We investigated whether, similar to amyloid-like peptides, Drs S9 was also chemotactic
We evaluated the capacity of Drs S9 to induce the migration of human peripheral blood neutrophils
Trang 5and T lymphocytes, as well as that of human acute
monocytic leukaemia cells, THP-1 Drs S9 triggered the
migration of human neutrophils (Fig 4A), T
lympho-cytes (Fig 4C) and THP-1 monolympho-cytes (data not shown)
with a typical bell-shaped dose–response curve at D0
For all cell types, maximal response was observed at a
concentration of 50 lm Equivalent concentrations of
Drs B2 had no effect on leukocyte motility (Fig 4C)
Drs S9-induced cell migration reflected a chemotactic
rather than a chemokinetic movement, as addition of
various concentrations of Drs S9 to the upper wells of
the chamber abrogated neutrophil migration towards
the Drs S9-loaded lower wells (Fig 4B), suggesting
that the cell locomotion we detected was the result of
chemotactic movement rather than enhanced random
movement
To determine whether Drs S9-induced neutrophil chemotaxis was mediated through a G protein-coupled receptor, as is the case for Ab(1–42) peptides [30], we examined whether this chemotactic activity could be inhibited by pertussis toxin (PTX), a toxin that specifi-cally inhibits Giaprotein-coupled receptor signalling [31] Incubation of neutrophils with PTX for 30 min at
37C prior to the start of the chemotaxis assay inhib-ited the migration of neutrophils by 50% compared with freshly dissolved Drs S9 (Fig 5A) The ability of neutrophils to migrate towards formyl methionyl-leucyl-phenylalanine (fMLP) (100 nm) was abrogated
by PTX Together, these data suggest that, in addition
to a Giprotein-coupled receptor, Drs S9-dependent chemotactic signals are mediated by a non-PTX-sensitive receptor
a
A
B
b
Fig 2 Dermaseptin S9 forms amyloid-like fibrils (A) Drs S9 binds Congo red and exhibits yellow–green bi-refringence; Drs S9 (500 l M ) (a, b) or Drs B2 (500 l M ) (c, d), both suspended in 50 m M phosphate buffer incubated for 7 days at 37 C, or phosphate buffer alone (e, f) were stained with Congo red and observed under normal (a, c, e) or polarized light (b, d, f) (B) Negative staining electron microscopy micrograph of Drs S9 (500 l M ) in 50 m M phosphate buffer incu-bated at 37 C for (a) 3 days (bar, 1.1 nm) or (b) 7 days (bar, 50 nm).
Trang 6A variety of agonistic ligands have been described
for the low-affinity FPRL-1 receptor, including
fMLP at high concentrations (approximately 100 nm)
and amyloid Ab(1–42) peptides [32–34] To assess
whether Drs S9-induced chemotaxis could be
medi-ated through this receptor, experiments were per-formed in which neutrophils were pre-incubated with Ab(1–42) (50 lm) or fMLP (100 nm) for 30 min before testing their ability to respond to a gradient
of Drs S9, Ab(1–42) or fMLP in a chemotaxis assay Preincubating the cells with fMLP reduced the che-motactic activity of Drs S9 by approximately 50% (Fig 5B), similar to what we had observed with PTX Comparable results were observed in response
to Ab(1–42) or fMLP when the cells were preincu-bated with Ab(1–42), Drs S9 or fMLP (Fig 5B) All these data suggest that the chemotactic activity of Drs S9 could be partially mediated through a recep-tor similar to the one used by high concentrations of fMLP or by Ab(1–42) (FPRL-1)
Activation of a G protein-coupled receptor by a chemoattractant can lead to activation of the MAPK pathway [35,36] Incubation of neutrophils with freshly
0 10 20 30 40
1624 (β-sheet)
1636
1650 1665
1685
C
1590 1620 1650 1680 1710
Wavenumber (cm –1 )
1624
1665
1685
1675 1665 1650
1624
1685
B
A
(β-hairpin)
(Amyloid
characteristic)
(disordered)
(Amyloid
characteristic)
2
1
0
–1
–2
–3
0.04
0.03
0.02
0.01
0.00
4 (a.u.)
1590 1620 1650 1680 1710
Fig 3 Drs S9 exhibits aggregated b-sheet structures with an
amy-loid-like arrangement as demonstrated by ATR FTIR (A) Amide I¢
band (1580–1720 cm)1) for Drs S9 (500 l M ) incubated for 7 days
and stained with Congo Red (B) Second-derivative analysis of the
spectrum in (A) indicating a strong individual band at 1685 cm)1
that is present in b-amyloid structures (C) Distribution of the
indi-vidual band content of amide I¢ from the spectrum in (A).
Drs S9 day 0 Drs S9 day 3 Drs S9 day 7
Drs S9 in the lower wells Drs S9 in the lower and upper wells
upper wells
Drs S9 day 0 Drs B 2day 0
Concentration (µ M )
0 0.05 0.5 5 15 50 150 350 fMLP (100 n M )
400
A
B
C
350 300 250 200 150 100 50 0 400 350 300 250 200 150 100 50 0 250
200
150
100
50
0
0 0.05 0.5 5 50 350 fMLP (100 n M )
0 0.05 0.5 5 15 30 150 250 350 500 700 fMLP (100 n M )
Fig 4 Dermaseptin S9 is a potent chemoattractant (A) Neutrophil migration induced by freshly prepared Drs S9, or Drs S9 incubated for 3 or 7 days at 37 C, or by fMLP as a positive control and med-ium as a negative control (B) Abolition of the chemotactic effect
by adding the same concentrations of Drs S9 or fMLP in the upper and the lower wells of the chemotaxis chamber compared with addition of Drs S9 or fMPL in the lower wells only (C) Induction of migration of T lymphocytes by Drs S9, Drs B2 or fMLP Similar results were obtained from three different experiments.
Trang 7dissolved Drs S9 (100 lm) for 5 or 10 min or fMLP
for 5 min induced the phosphorylation of the
extra-cellular signal-regulated kinase ERK1⁄ 2, compared to
neutrophils incubated for the same time period with
medium alone (Fig 5C) Preincubation with PTX,
PD98059 (a specific inhibitor of MEK1 and MEK2,
the ERK MAPK kinases) or both, before adding
Drs S9 or fMLP, prevented ERK1⁄ 2 phosphorylation
These data suggest that Drs S9 is sensed in part
through a seven-transmembrane G protein-coupled
receptor, probably FPRL-1, coupled to the ERK1⁄ 2
MAPK kinase pathway
The b-amyloid-like behaviour of dermaseptin S9 modulates its chemotactic and antimicrobial activities
Taking advantage of better knowledge of the aggre-gative and amyloid properties of Drs S9, we investi-gated whether the fibrillization process influenced the chemotactic and antibacterial activities of the pep-tide The activity of Drs S9 freshly dissolved in che-motaxis medium was compared to that of Drs S9 incubated for 3 or 7 days at 37C In all cases, Drs S9 induced cell migration with a peak response
at 50 lm, but with maximum efficiency for freshly dissolved Drs S9 (Fig 4A) Interestingly, the a-helical Drs B2 freshly dissolved in the same medium or incubated for 3 or 7 days at 37 C was not a chemoattractant for T lymphocytes (Fig 4C), neutro-phils or THP-1 monocytes (data not shown) in the same range of concentration (0.05–350 lm), indicat-ing the importance of the peptide structure for selected biological properties
Drs S9 dissolved in NaCl⁄ Pi or RPMI-1640 exhib-ited antimicrobial activity against all Gram-negative bacteria strains tested at day 0 The antibacterial activ-ity of 3-day-old Drs S9 in NaCl⁄ Pi (Table 2) or RPMI-1640⁄ 1% BSA (data not shown) was stronger than that of Drs S9 at day 0 After 7 days of incuba-tion in NaCl⁄ Pi at 37C, Drs S9 exhibited little or no antibacterial activity No significant differences were observed when Drs B2, a potent a-helical antibacterial peptide, was dissolved in NaCl⁄ Pi or RPMI-1640⁄ 1% BSA and tested, or incubated for 3 or 7 days at 37C prior to testing its antibacterial activity Moreover,
300
250
200
150
100
50
0
Peptides
Concentrations of Drs S9 0 day aged (µM)
Neutrophils pre-incubated with medium for 30 min
Neutrophils pre-incubated with PTX (200ng mL –1 ) for 30 min
250
200
150
100
50
0
Pre-incubation with Drs S9 Pre-incubation with fLMP Pre-incubation with medium
Pre-incubation with Aβ(1–42)
A
B
C RPMI Drs S9
5′ 10′
PTX
PD98059
– –
– – – – –
–
+ + + +
Drs S9
pERK1/2
ERK1/2
Fig 5 Dermaseptin S9 induces cell migration through a
seven-transmembrane G protein-coupled receptor, presumed to be the
FRLP-1 receptor (A) Pre-incubating neutrophils with PTX
(200 ng mL)1) partially inhibits Drs S9-induced migration fMLP was
used as a positive control (B) Pre-incubating neutrophils with
Ab(1–42), Drs S9 or fMLP reduces the migration induced by freshly
dissolved Drs S9, Ab(1–42) or fMLP (C) ERK1 ⁄ 2 phosphorylation in
response to Drs S9 or fMLP is abolished by pre-incubating human
neutrophils with PTX and ⁄ or PD98059 The same membrane was
stripped and blotted with anti-ERK1 ⁄ 2 Similar results were
obtained from three separate experiments.
Table 2 Effect of Drs S9 oligomerization state on the antimicrobial activity of freshly prepared Drs S9 and Drs B2 or after incubation for 3 or 7 days in NaCl ⁄ P i or H 2 O at 37 C.
Bacterial strains
Minimal inhibitory concentration a (l M )
Drs S9 (NaCl ⁄ P i or RPMI)
Drs B2 (NaCl ⁄ P i )
0 day 3 days 7 days
0, 3 and
7 days
a The antimicrobial activity is expressed as the minimal inhibitory concentration (l M ), which is the minimal peptide concentration required for total inhibition of cell growth in liquid medium Strains were considered resistant (R) when their growth was not inhibited
by peptide concentrations > 100 l M
Trang 8after removing the Drs S9 from the wells and replacing
it with fresh LB medium overnight, none of the
sensi-tive strains were capable of resuming growth (data not
shown), suggesting that Drs S9 is a bactericidal agent
Together, these data indicate that Drs S9 is a more
potent bactericidal agent in the oligomeric spherical
form detected at 3 days of incubation at 37C, and is
a potent chemoattractant in its low-molecular-weight
oligomeric form (day 0)
Dermaseptin S9 differently affects vesicle lipid
assemblies
Bacterial membranes contain substantial (up to 30%)
amounts of negatively charged lipids such as
phosphat-idylglycerol, cardiolipin and phosphatidylserine [37],
thus 1,2-dimyristoyl-sn-glycero-3-phosphatidylglycerol
(DMPG) was used as a model system for determination
of the relationships between antimicrobial activity,
structure and membrane disturbances in the FTIR
study Phosphatidylcholine is widely recognized
as representative for mammalian cell membranes,
thus 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine
(DMPC) was used as the lipid model to assess the
inter-actions of Drs S9 with neutral eukaryotic membranes
We examined the conformation of Drs S9 in the
presence of DMPG or DMPC vesicles, and evaluated
the impact of freshly dissolved Drs S9 on the lipid
bilayer assembly by transmission FTIR spectroscopy
Maximum absorbance of the amide I¢ bands was
observed at 1624 ± 1 cm)1 in NaCl⁄ Pi, DMPC and
DMPG vesicles (Fig 6A) Analysis of the amide I¢
bands using a decomposition procedure described
recently [25] allows band-to-band comparisons (Fig 6B)
The coil⁄ helix contents (25%) were the same in
aque-ous NaCl⁄ Pibuffer and in the presence of DMPG and
DMPC, but phospholipid vesicles promoted
intercon-version from b-hairpin to b-sheet structure with better
efficiency
The stretching vibration mode of the DMPG
carbonyl groups [m(CO)] was used to check the
hydra-tion and conformahydra-tional changes of the membrane
interface region (Fig 7A) As shown in Table 3, Drs S9
caused redistribution of the two [m(CO)] components
(hydrogen bond at 1726 cm)1 and dehydration at
1743 cm)1), characterized by a higher value (2.1) of the
1726⁄ 1743 ratio compared to pure DMPG (1.8) Thus,
the interaction of Drs S9 with DMPG vesicles decreased
the water content associated with phospholipid
head-groups (CO), probably due to hydrogen bonding
inter-actions between the lipid head groups and the peptide
[26,38,39] To evaluate the perturbations generated by
Drs S9 in the hydrocarbon core of the DMPG bilayer,
we analysed spectra in the 2800–3000 cm)1 region (Fig 7B) The symmetric mS(CH2) stretching band at
2852 cm)1 and the antisymmetric mAS(CH2) stretching band at 2923 cm)1 were shifted towards higher wave numbers These displacements and redistributions could
be due to a gel to liquid crystalline-phase transition [39] Drs S9 strongly affected the antisymmetric mAS(CH3) stretching modes at 2956 cm)1, indicating enhancement
of the alkyl chain flexibility, as confirmed by the deter-mination of the bilayer core disruption (Table 3)
In contrast, Drs S9 did not perturb the DMPC bilayer
as neither the m(CO) lipid carbonyl groups nor the
mS(CH2-CH3) or mAS(CH2-CH3) stretching vibration-mode were affected (Fig 7B) This suggests that no noticeable interactions were established between the zwitterionic vesicles and the peptide These data are corroborated by adsorption density measurements by SPR on hybrid bilayers of DMPC and HPA, the results
0 20 40 60 80 100
β-hairpin
β-hairpin
β-hairpin
coil/helix coil/helix
coil/helix
β-sheet
β-sheet
β-sheet
0.002
0.015
0.010
0.005
0.000
Buffer A
B
DMPC DMPG
1500 1550 1600 1650 1700
Wavenumber (cm –1 )
Fig 6 Conformations of dermaseptin S9 in the presence of anionic DMPG and zwitterionic DMPC vesicles (A) Transmission FTIR spectra in the 1500–1700 cm)1 region for Drs S9 in NaCl ⁄ P i or DMPC or DMPG vesicles (B) Individual band contents (± 1%) resolved in the amide I¢ domain (lipid : peptide ratio = 10).
Trang 9of which were found to be very low and inferior to those
obtained on hydrophobic surfaces (Table 1) The
polar head groups of DMPC prevent strong peptide
adsorption
Discussion
The dermaseptin superfamily includes peptides with very different structural characteristics: (a) the derm-aseptins stricto sensu (dermderm-aseptins S and B) from Phyllomedusa sauvagei and P bicolor, amphipathic a-helical peptides that all have a conserved tryptophan residue at position 3 and a positive net charge attribut-able to the presence of lysine residues between alter-nating hydrophobic and hydrophilic sequences [8], (b) the plasticins, which are rich in glycine residues arranged in regular pentamer motifs GXXXG (where
X is any amino acid residue) and are characterized by
a high structural malleability [40], and (c) dermaseptin S9, from Phyllomedusa sauvagei, a highly aggregated and non-amphipathic peptide that has a hydrophobic core sequence flanked at both termini by several posi-tively charged residues [9] Taking advantage of earlier observations by Lequin et al [9] regarding the aggre-gative properties of Drs S9, we evaluated the peptide self-organization properties in aqueous solutions We found that Drs S9 self-assembled via spherical interme-diates into amyloid-like fibrils as evidenced by elec-tronic microscopy and Congo red staining (Fig 2) Antimicrobial peptides and amyloids are known to play a role in chemotaxis and to ultimately promote inflammation [16] We therefore evaluated the antimi-crobial and chemotactic potential of the various struc-tural forms of Drs S9, i.e monomeric and⁄ or weakly self-associated, oligomeric or protofibrillar, and fibril-lar forms Interestingly, these structural intermediates resulted in various biological properties (Fig 8)
Amyloid-like properties of dermaseptin S9 Assembly of proteins or peptides into amyloid-like fibrils is a multistep process initiated by conforma-tional changes, during which intermediate aggregation states such as oligomers, protofibrils and filaments are seen [41] Here we have shown that Drs S9 possesses most amyloidogenic characteristics [28] Drs S9 has a b-sheet-rich structure as shown by ATR FTIR, shows
–0.01
0.00
0.01
0.02
0.03
0.04
0.05
0.06
A
B
DMPG + Drs S9 DMPG
DMPC
DMPD + Drs S9
DMPG
DMPG + Drs S9
Fig 7 Dermaseptin S9 interacts with anionic DMPG and
zwitter-ionic DMPC vesicles (A) DMPG CO ester spectra with or without
Drs S9 (B) Normalized FTIR spectra (2800–3000 cm)1) of Drs S9
with zwitterionic DMPC or anionic DMPG vesicles.
Table 3 Disturbance of anionic lipid assembly by Drs S9; assignment and distribution of component bands (± 1%) in lipid m(CO) and m(CH) stretching vibration modes in FTIR spectra obtained with 2 m M Drs S9 in the presence of 20 m M DMPG or DMPC vesicles suspended in NaCl ⁄ P i
Interface: peptide ⁄ DMPG lipid CO (%) Bilayer core: peptide ⁄ DMPG alkyl chain (%)
1726 cm)1 hydrogen bond
1743 cm)1 dehydrated
1726 ⁄ 1743 ratio
2852 cm)1
mS(CH2)
2875 cm)1
mS(CH3)
2923 cm)1
mAS(CH2)
2956 cm)1
mAS(CH3)
Trang 10green bi-refringence as observed by microscopy after
Congo red staining, and produces spherical oligomers
and fibrils visualized by electron microscopy after 3
and 7 days of incubation at 37C, respectively
(Fig 2) FTIR allowed clear distinction between
amy-loidogenic peptides and simple b-aggregated peptides
by the observation of specific 1665 and 1685 cm)1
bands as reported previously [29] Multiple b-structures
were observed, and were consistent with the formation
of oligomeric species (3 days) and long fibrils (7 days)
(Fig 3) Measurement of aggregation by
physicochem-ical methods is a difficult task We have demonstrated
that the SPR technique can provide information on
the bound peptide structure through determination of
its adsorption density on the sensor chip surface
[25,42] Calculated adsorption densities on the HPA
hydrophobic surface as a function of peptide
concen-tration were in agreement with a b-structure for
Drs S9 and suggested b-oligomer elongation (Table 1)
This is in contrast with neutral plasticins, which also
aggregate and form b-structures, but differ in their
adsorption mode on the HPA hydrophobic support,
which is governed by the surface-limited rate [25] Our
data are in agreement with previous observations
indi-cating that the rate of adsorption of amyloidogenic
Ab(1–42) peptide to lipid and sensor chip surfaces is a strictly diffusion-limited process [43] Furthermore, the self-associating properties of Drs S9 were also corrobo-rated by slow NH⁄ ND exchange kinetics monitored by ATR FTIR (Fig 1A)
Amyloidogenic peptides self-assemble, probably through specific molecular interaction patterns, gener-ating ordered mature fibrils It is thought that, due to the multiple aromatic residues present in amyloid-like peptides, p–p stacking plays a crucial role in the fibril-lization process, by reducing the energetic barrier [44,45] As Drs S9 has a hydrophobic core sequence (residues 8–15) that is rich in aromatic residues, the peptide can potentially establish p–p interactions [46] that could be at the origin of its amyloid-like behav-iour The structural properties of most of the amyloi-dogenic peptides are environment-dependent For example, Ab(1–42) is a-helical in the presence of triflu-oroethanol [47], but is poorly structured and aggre-gated in water and shows b-sheet formation in phosphate buffer solutions [28,48] Similarly, Drs S9 was shown to fold into a non-amphipathic a-helical conformation in trifluoroethanol⁄ water mixtures [9] Amyloid peptides and proteins can be divided into two main categories: those associated with pathological
D3
D7
Amyloid-like fibrils
R E C E P T O R
G-PROTEIN
Chemotactic activity on eukaryotes
D0
Antimicrobial activity on prokaryotes
Fig 8 Summary of the main
oligomeriza-tion states adopted by Drs S9 in relaoligomeriza-tion to
its biological activities D0, freshly dissolved
Drs S9 is mainly dimeric or tetrameric and
exhibits maximal chemotactic activity D3,
after 3 days, Drs S9 has formed
self-associ-ated oligomers with potent antimicrobial
activity D7, after 7 days, Drs S9 has formed
amyloid-like fibrils.