Báo cáo khoa học: Dermaseptin DA4, although closely related to dermaseptin B2, presents chemotactic and Gram-negative selective bactericidal activities doc

Dermaseptin DA4, although closely related to dermaseptin
B2, presents chemotactic and Gram-negative selective
bactericidal activities
Constance Auvynet
1,2,
*, Pierre Joanne
2,
, Julie Bourdais
3
, Pierre Nicolas
2,
, Claire Lacombe
2,4,
à
and Yvonne Rosenstein
1
1 Departamento de Medicina Molecular y Bioprocesos, Instituto de Biotecnologia, Universidad Nacional Auto
´
noma de Me
´
xico, Col Chamilpa,
Cuernavaca, Morelos, Mexico
2 FRE 2852, Peptidome de la peau des amphibiens, CNRS ⁄ Universite
´
Paris–Pierre et Marie Curie, Paris, France
3 Independent scholar, Cuernavaca, Morelos, Mexico
4 UFR Sciences et Technologie, Universite
´
Paris 12–Val de Marne, Cre
´
teil, France

Keywords
antimicrobial peptide; chemotaxis;
dermaseptin; frog skin; peptide–membrane
interactions
Correspondence
Y. Rosenstein, Departamento de Medicina
Molecular y Bioprocesos, Instituto de
Biotecnologia, Universidad Nacional
Auto
´
noma de Me
´
xico, Avenida Universidad
2001, Col Chamilpa, Cuernavaca, Morelos
62270, Mexico
Fax: +52 73172388
Tel: +52 5 55 66 22 76 63
E-mail:
C. Lacombe, Laboratoire des Biomole
´
cules,
Universite
´
Pierre et Marie Curie-CNRS-ENS,
4 Place Jussieu, 75252 Paris cedex 05,
France
Fax: +33 1 44 27 55 64
Tel: +33 1 44 27 51 59
E-mail:
Present addresses

*INSERM UMR-S 945 Immunite
´
et Infec-
tion, Universite
´
Pierre et Marie Curie, Paris,
France
Biogene
`
se des Signaux Peptidiques
(BIOSIPE), ER3-UPMC, Universite
´
Pierre
et Marie Curie, Paris, France
Antimicrobial peptides participate in innate host defense by directly elimi-
nating pathogens as a result of their ability to damage the microbial mem-
brane and by providing danger signals that will recruit innate immune cells
to the site of infection. Dermaseptin DA4 (DRS-DA4), a new antimicrobial
peptide of the dermaseptin superfamily, was identified based on its chemo-
tactic properties, contrasting with the currently used microbicidal proper-
ties assessment. The peptide was isolated and purified by size exclusion
HPLC and RP-HPLC from the skin of the Mexican frog, Pachymedu-
sa dacnicolor. MS and amino acid sequence analyses were consistent with
the structure GMWSKIKNAGKAAKAAAKAAGKAALGAVSEAM. CD
experiments showed that, unlike most antimicrobial peptides of the derm-
aseptin superfamily, DRS-DA4 is not structured in the presence of zwitteri-
onic lipids. DRS-DA4 is a potent chemoattractant for human leukocytes
and is devoid of hemolytic activity; in addition, bactericidal tests and mem-
brane perturbation assays on model membranes and on Escherichia coli and
Staphylococcus aureus strains have shown that the antibacterial effects of

DRS-DA4 and permeabilization of the inner membrane are exclusively
selective for Gram-negative bacteria. Interestingly, despite high sequence
homology with dermaseptin S4, dermaseptin B2 was not able to induce
directional migration of leukocytes, and displayed a broader bactericidal
spectrum. A detailed structure–function analysis of closely related peptides
with different capabilities, such as DRS-DA4 and dermaseptin B2, is criti-
cal for the design of new molecules with specific attributes to modulate
immunity and/or act as microbicidal agents.
Abbreviations
DDK, dermadistinctin K; DiSC
3
(5), 3,3¢-dipropylthiadicarbocyanine iodide; DMPC, 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine; DMPG,
1,2-dimyristoyl-sn-glycero-3-phosphatidylglycerol; DRS-B2, dermaseptin B2; DRS-DA3, dermaseptin DA3; DRS-DA4, dermaseptin DA4;
DRS-L1, dermaseptin L1; DRS-S1, dermaseptin S1; DRS-S9, dermaseptin S9; DSC, differential scanning calorimetry; ERK, extracellular
signal-regulated kinase; fMLP, formyl-methionyl-leucyl-phenylalanine; FPR, formyl peptide receptor; FPRL-1, formyl peptide receptor-like 1;
Gal-ONp, 2-nitrophenyl b-
D-galactopyranoside; GPCR, G-protein-coupled receptor; ITC, isothermal titration calorimetry; LUV, large unilamellar
vesicle; MAPK, mitogen-activated protein kinase; MIC, minimum inhibitory concentration; MLV, multilamellar liposome vesicle; PMN,
polymorphonuclear; PTX, pertussis toxin; SDF1-a, stromal cell-derived factor 1a; TFA, trifluoroacetic acid.
FEBS Journal 276 (2009) 6773–6786 ª 2009 The Authors Journal compilation ª 2009 FEBS 6773
Introduction
At the interface of innate and adaptive immunity,
antimicrobial peptides have been shown to enhance the
overall immune response [1]. The majority of these
peptides are cationic, with a net charge of +2 to +7,
and contain up to 50% hydrophobic amino acids. This
amphipathic design, consisting of spatially separated
hydrophobic and charged regions, is believed to allow
the insertion of the peptide into microbial membranes.
Until recently, direct antimicrobial activity against

bacteria, fungi, parasites and viruses was considered to
be the primary function of antimicrobial peptides.
However, there is now increasing evidence that anti-
microbial peptides are multifunctional molecules of
fundamental importance in host defense, modulating
the innate and adaptive immune systems. In addition
to microbicidal activity, a large number of antimicro-
bial peptides, such as the human cathelicidin LL-37
and the defensins, have been found to modulate the
immune response by directing the migration of immune
cells to the site of injury, as well as by activating leuko-
cytes and promoting cytokine release, wound repair,
angiogenesis, and neutralization of microbial products
[2,3]. In particular, the chemotactic activity of antimi-
crobial peptides is mostly mediated through G-protein-
coupled receptors (GPCRs) such as the CC-chemokine
receptor 6, the formyl peptide receptor (FPR), and the
formyl peptide receptor-like 1 (FPRL-1) [4–7].
Frog skin is a rich source of antimicrobial peptides,
with more than half of the peptides described to date
having been isolated from South American Hylidae or
European, Asian or North American Ranidae; the
peptides are involved in the defense of the frog against
predation or invading microorganisms. More than 80
antimicrobial peptides have been isolated from only 12
species of the Phyllomedusinae subfamily, belonging to
the genera Agalychnis, Hylomantis, Pachymedusa, and
Phyllomedusa. Among them, the dermaseptins, a super-
family of structurally and functionally related peptides
produced by the Hylidae family, have potent micro-

bicidal activity at micromolar concentrations against a
wide range of microorganisms (Gram-positive and
Gram-negative bacteria, fungi, yeasts, and protozoa),
but no or little hemolytic activity [8]. The microbicidal
activity of these lysine-rich linear polycationic peptides,
most of which are composed of 24–34 amino acids
structured as an amphipathic a-helix in polar solvents,
is thought to result from the interaction of the amphi-
pathic a-helical structure with the membrane bilayer of
target microorganisms.
Most peptides belonging to the dermaseptin super-
family have been identified primarily on the basis of
their antimicrobial activity. However, additional bio-
logical functions have been recognized that may, or may
not, be directly associated with pathogen clearance. For
instance, adenoregulin [dermaseptin B2 (DRS-B2)] was
first identified as a peptide able to stimulate binding of
agonists to A1-adenosine receptors [9], and was further
shown to enhance the binding potency of several GPCR
agonists [10]. Frog skin insulintropic peptide (FSIP),
also a member of this superfamily, significantly stimu-
lates insulin release in glucose-responsive BRIN-BD 11
cells [11], and dermaseptin S1 (DRS-S1) has been
reported to stimulate the microbicidal activity of poly-
morphonuclear (PMN) leukocytes [12] and dermaseptin
S9 (DRS-S9) to chemoattract PMN leukocytes [13].
We report herein the isolation and characterization
of a new dermaseptin-related peptide, GMWSKIKNA
GKAAKAAAKAAGKAALGAVSEAM, named derm-
aseptin DA4 (DRS-DA4), according to the new derm-

aseptin nomenclature [14]. DRS-DA4 was obtained by
fractioning the skin exudate of Pachymedusa dacnicol-
or, and was first identified on the basis of its chemo-
tactic properties rather than on the classic assessment
of its antimicrobial activity. Interestingly, although
DRS-DA4 was found to share strong sequence homol-
ogy with DRS-B2, it has distinct biological activities.
DRS-DA4, but not DRS-B2, induced human leukocyte
migration and activation mainly through a GPCR,
probably FPRL-1; moreover, it was devoid of hemo-
lytic activity. Unlike DRS-B2, which is active on
Gram-positive as well as on Gram-negative bacteria,
DRS-DA4 only exhibited direct antibacterial activity
on Gram-negative bacteria, together with perturbation
of the inner membrane against of Gram-negative
àLaboratoire des Biomole
´
cules, Universite
´
Pierre et Marie Curie-CNRS-ENS, Paris
cedex 05, France
(Received 12 August 2009, accepted 21
September 2009)
doi:10.1111/j.1742-4658.2009.07392.x
Chemotactic and antimicrobial DRS-DA4 C. Auvynet et al.
6774 FEBS Journal 276 (2009) 6773–6786 ª 2009 The Authors Journal compilation ª 2009 FEBS
bacteria. The identification of a novel antimicrobial
peptide on the basis of its immunomodulatory capacity
broadens the panel of activities of these peptides
within Hylidae frog genera. In addition, our analysis

of the biophysical characteristics and properties of
these peptides contributes to our current knowledge
regarding the different activities of antimicrobial
peptides, and opens the possibility of using them as
templates for the design of new molecules with specific
attributes to modulate immunity and ⁄ or act as micro-
bicidal agents.
Results
Isolation, purification and structure of DRS-DA4
DRS-DA4 was purified to homogeneity from P. dacni-
color skin exudates by a two-step protocol. Specifically,
1.1 mL of skin secretions recovered by gently squeez-
ing the parotoid glands of a single living frog was first
fractionated on a Sephadex G-50 column (Fig. 1A),
and each fraction was tested for chemotactic activity.
The chemotactic fraction III was further purified by
RP-HPLC on a semipreparative column (Fig. 1B). A
peak with a retention time of 38.7 min was found to
have strong chemotactic activity, inducing the direc-
tional migration of human leukocytes (Fig. 1B, insert).
The sequence of the purified fraction determined by
tandem MS (experimental monoisotopic mass of the
protonated peptide: 3063.26) and Edman sequencing
gave unequivocally the sequence GMWSKIKNAGKA
AKAAAKAAGKAALGAVSEAM. According to the
new dermaseptin nomenclature [14], this peptide was
named DRS-DA4.
To confirm the sequence and to demonstrate that
the biological activities of the purified natural peptide
reflected its intrinsic properties, DRS-DA4 was synthe-

sized by the solid-phase method. After HPLC purifica-
tion on a semipreparative column, synthetic DRS-DA4
was indistinguishable from the natural product, eluting
exactly at the same position (38.7 min) as the natural
corresponding product and giving the same monoi-
sotopic mass to charge ratio (3063.28) by MALDI-TOF
MS (data not shown). Further characterization of
the conformational and biological properties was
performed with the synthetic peptide.
Acetonitrile (%)
A
B
0 10203040506070
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
Fraction III
Fraction II
Fraction I
V
0
Absorbance (280 nm)
Fraction number

Fig. 1. (A) Profile of fractionation of the
P. dacnicolor skin exudates on a Sephadex
G-50 column. The absorbance at 280 nm is
represented as a solid line. (B) RP-HPLC
separation of the recovered fraction III using
a semipreparative column. Elution was
achieved with a 0–60% linear gradient of
solvent (dotted line). The arrow points to
the elution position of synthetic DRS-DA4
under the same conditions. The absorbance
at 220 nm is represented as a solid line.
Insert: neutrophil migration induced in
response to the peak indicated by the
arrow. The peak solution was diluted 1 ⁄ 10
from column 6 to column 1. RPMI medium
was used as negative control (column 0)
and fMLP (100 n
M) as positive control.
C. Auvynet et al. Chemotactic and antimicrobial DRS-DA4
FEBS Journal 276 (2009) 6773–6786 ª 2009 The Authors Journal compilation ª 2009 FEBS 6775
A protein database was screened for similar
sequences, using the embl-ebi fasta 3 program, and
this revealed that DRS-DA4 belonged to the derm-
aseptin superfamily. A sequence alignment showed
that the antimicrobial peptides dermaseptin DA3
[DRS-DA3 (PD-33)] [15], DRS-B2 [9], dermadistinctin
K (DDK) [16] and ARP-AC1 [17], isolated from
P. dacnicolor, Phyllomedusa bicolor, Phyllomedusa
A
B

Fig. 2. (A) Amino acid sequences of DRS-DA4 and DRS-B2, together with some antimicrobial peptides with nearest sequences. Identical
amino acids between these peptides are underlined. (B) Helical wheel projection of the DRS-DA4, DRS-B2, DDK, and DRS-L1. Hexagonal
and round backgrounds refer to basic and acid amino acids respectively, pentagonal backgrounds to hydrophilic residues, and squares to
hydrophobic residues.
Chemotactic and antimicrobial DRS-DA4 C. Auvynet et al.
6776 FEBS Journal 276 (2009) 6773–6786 ª 2009 The Authors Journal compilation ª 2009 FEBS
distincta, and Agalychnis callidryas, respectively,
presented the highest sequence homology with DRS-
DA4 (87.9% identity and 93.9% similarity for
DRS-DA3, 84.8% identity and 97% similarity
for DRS-B2, and 84.8% identity and 93.9% similarity
for DDK; Fig. 2A). Both, DRS-DA3 and DRS-B2
have six positive charges (Lys), and only one negative
charge (Glu). The resulting total overall net charge for
DRS-DA4 is +5, whereas DRS-B2 has an overall net
charge of +4, owing to the carboxyamidated end,
three negative charges (Glu) and the extra positive
charge of the N-terminus. Figure 2A shows dermaseptin
L1 (DRS-L1) [18] from the lemur leaf frog Hyloman-
tis lemur, which showed only 48.6% identity and
22.4% similarity but whose biological properties led us
to make a comparison. The helical wheel projection of
Edmundson showed the partial amphipathic character
of the helix obtained for DRS-DA4, with hydrophobic
residues on one face of the helix and polar or charged
residues on the opposite face, corresponding to a
hydrophobic sector that subtends a radial angle of
160° (Fig. 2B).
DRS-DA4 induces directional migration of human
leukocytes

Like natural DRS-DA4, synthetic DRS-DA4 induced
the migration of human neutrophils (Fig. 3A) and
monocytes (Fig. 3B), with a typical bell-shaped dose–
response curve. Maximum activity was observed at a
concentration of 10 lm for both cell types. Addition
of various concentrations of DRS-DA4 to the upper
wells of the Boyden chamber abolished the migration
of the cells, suggesting that DRS-DA4 induced che-
motactic movement rather than enhanced random
movement (Fig. 3A). As described previously [13],
DRS-B2 did not induce the migration of leukocytes
within the range of concentrations tested for DRS-
DA4.
Like formyl-methionyl-leucyl-phenylalanine (fMLP),
most chemotactic antimicrobial peptides induce cell
migration through a seven-helix transmembrane
G
ia
-protein-coupled receptor such as FPR or FPRL-1
[19]. Pretreatment with pertussis toxin (PTX) – a s pecific
inhibitor of G
ia
-protein-coupled receptors – prior to
the onset of the chemotactic assay partially inhibited
the motility of neutrophils induced by DRS-DA4. As
expected, neutrophils preincubated with PTX before
exposure to fMLP failed to migrate (Fig. 4A).
Preincubation of neutrophils for 30 min with 100 nm
fMLP, an FPRL-1 agonist, inhibited by up to 50% the
migration of the cells in response to a gradient of

DRS-DA4 or fMLP (100 nm). However, preincubation
of the cells with 1.14 nm fMLP, a concentration at
which fMLP is considered to be an FPR agonist, did
not inhibit the motility of the cells in response to
DRS-DA4 or fMLP (100 nm). As expected, preincuba-
tion of neutrophils with fMLP (1.14 nm) prior to the
chemotaxis assay inhibited the migration induced by
fMLP (1.14 nm) (Fig. 4A).
Stimulation of a GPCR by chemotactic agonist
ligands can lead to the activation of mitogen-activated
protein kinase (MAPK) pathways in target cells [20].
Incubation of neutrophils with DRS-DA4 (10 lm)or
fMLP (100 nm) for 5 or 10 min induced the phos-
phorylation of the extracellular signal-regulated kinase
(ERK)1 ⁄ 2 MAPK, but not that of the p38 MAPK
(Fig. 4B). Preincubation with PTX or PD98059 (a
specific inhibitor of MEK1 and MEK2) before the
addition of DRS-DA4 (10 lm) or fMLP (100 nm)
inhibited the phosphorylation of ERK1 ⁄ 2 but induced
A
B
Fig. 3. DRS-DA4 is a potent chemoattractant of leukocytes. (A)
Neutrophil migration induced in response to DRS-DA4 or fMLP
(100 n
M) as a positive control and medium as a negative control.
Addition of the same concentrations of DRS-DA4 to the upper and
lower wells of the chemotaxis chamber abolished the chemotactic
effect. (B) Monocyte migration in response to DRS-DA4, DRS-B2 or
SDF1-a (1 n
M) as positive control. The data shown represent the

average cell migration of triplicate wells. Similar results were
obtained from three different experiments.
C. Auvynet et al. Chemotactic and antimicrobial DRS-DA4
FEBS Journal 276 (2009) 6773–6786 ª 2009 The Authors Journal compilation ª 2009 FEBS 6777
the phosphorylation of p38. Moreover, pretreatment
of neutrophils with PD98059 led to decreased migra-
tion in response to DRS-DA4 or fMLP (100 nm),
whereas SB202190, a specific inhibitor of the p38
MAPK, did not (Fig. 4C). All together, these results
suggest that DRS-DA4 chemotactic activity is medi-
ated through a GPCR, probably FPRL-1, and that it
is coupled to the ERK1 ⁄ 2 MAPK pathway.
DRS-DA4, but not DRS-B2, is only microbicidal
for Gram-negative bacteria
DRS-DA4 exhibited good to moderate antimicrobial
activity against all of the Gram-negative strains tested,
as no colony was counted when the peptide– Escherichia
coli (both E. coli strains tested) or peptide–Pseudo-
monas aeruginosa mixtures were incubated on LB agar
plates overnight, indicative of a bactericidal effect. How-
ever, no microbicidal activity was detected against any
of the Gram-positive strains tested, at concentrations up
to 100 lm. In comparison, DRS-B2 presented strong
antimicrobial activities against both Gram-negative and
Gram-positive strains (Table 1). No hemolytic or apop-
totic activities were observed on rat blood cells or leuko-
cytes with DRS-DA4 or DRS-B2, respectively, for
concentrations up to 100 lm (data not shown).
DRS-DA4 interacts preferentially with
1,2-dimyristoyl-sn-glycero-3-phosphatidylglycerol

(DMPG) vesicles mimicking Gram-negative bacteria
When we used the membrane potential-sensitive dye
3,3¢-dipropylthiadicarbocyanine iodide [DiSC
3
(5)] to
assess the ability of the peptide to damage, and thus
depolarize, prokaryotic membranes [21,22], we found
that addition of DRS-DA4 resulted in concentration-
independent increases in DiSC
3
(5) fluorescence within
the range 10–50 lm, indicative of equivalent membrane
depolarization for both strains tested, i.e. E. coli and
Staphylococcus epidermidis (Table 1). Thus, in contrast
to its Gram-negative-specific microbicidal activity,
DRS-DA4 was able to depolarize the membranes of
Gram-positive and Gram-negative bacteria.
To further characterize the bactericidal activity
of DRS-DA4, we measured its ability to damage the
bacterial membrane. Permeabilization of the inner mem-
brane can be assessed by monitoring the b-galactosidase
substrate 2-nitrophenyl b-d-galactopyranoside (Gal-
ONp). In E. coli strain ML35p and in Staphylococ-
cus aureus strain ST1036, which lack lac permease, Gal-
ONp entry is blocked by the integrity of the inner mem-
brane; if Gal-ONp crosses this barrier, it can be cleaved
by the cytoplasmic b-galactosidase, resulting in a color
change from clear to yellow, reflecting membrane dam-
age. As shown in Fig. 5A, the time needed to obtain the
maximum Gal-ONp hydrolysis in E. coli increased as

the concentration of DRS-DA4 decreased. On the other
hand, no permeabilization of S. aureus ST1065 was
10 ’ 10 ’ 5 ’ 10 ’ 10 ’ 10 ’ 10 ’ 10 ’ 10 ’ 10 ’
PTX – – – + – + – + – +
PD98059 – – – – + + – – + +
pErk1/2
Erk1/2
pp38
p38
A

B
C
Fig. 4. DRS-DA4-induced neutrophil migration through a seven-
transmembrane GPCR. (A) DRS-DA4-induced chemotaxis is medi-
ated through a GPCR. Neutrophils were preincubated with medium,
PTX, fMLP (1.14 n
M), or fMLP (100 nM) prior to challenge with
DRS-DA4, fMLP (1.14 n
M) or fMLP (100 nM). (B) ERK1 ⁄ 2 phosphor-
ylation is induced in response to DRS-DA4. Human neutrophils
were treated with medium, DRS-DA4, or fMLP, with or without
preincubation with PTX and ⁄ or PD98059. The same membrane
was stripped and blotted with antibody against ERK1 ⁄ 2 or antibody
against p38. Similar results were obtained from three separate
experiments. (C) ERK1 ⁄ 2 phosphorylation is necessary for DRS-
DA4-induced. Human neutrophils were preincubated with PD98058
(ERK1 ⁄ 2 inhibitor) or SB202190 (p38 inhibitor) prior to the chemo-
taxis assay, as described in (A). The data shown are representative
of two independent experiments.

Chemotactic and antimicrobial DRS-DA4 C. Auvynet et al.
6778 FEBS Journal 276 (2009) 6773–6786 ª 2009 The Authors Journal compilation ª 2009 FEBS
observed for concentrations of DRS-DA4 up to 100 lm
(Fig. 5B and data not shown). These results indicate
that the bacterial membrane of Gram-negative strains is
one of the main targets of DRS-DA4.
Differential scanning calorimetry (DSC) is a power-
ful, nondisturbing thermodynamic technique that is
useful for the study of lipid–protein interactions in
model membranes as well as for the evaluation of anti-
microbial peptide interactions with lipid bilayer model
membranes [23]. We evaluated the effect of various
concentrations of DRS-DA4 on the pretransition and
the m ain transition of the zwitterionic 1,2-dimyristoyl-sn -
glycero-3-phosphatidylcholine (DMPC) and anionic
DMPG membranes used as models for eukaryotic and
prokaryotic plasma membranes, respectively. DSC
thermograms illustrating the effect of the incorporation
of increasing quantities of DRS-DA4 on the thermo-
tropic phase behavior of multilamellar liposome vesi-
cles (MLVs) of DMPG or DMPC are shown in
Fig. 5C,D. In the absence of the peptide, DMPG
exhibited two endothermic events: a less energetic pre-
transition near 12.8 °C, arising from the conversion of
the lamellar phase to the rippled gel phase, and a sec-
ond, more energetic, main transition at 23.2 °C, result-
ing from the conversion of the rippled gel phase to the
lamellar liquid-crystalline phase. These results, together
with the enthalpy values for the pretransition
( 1 kcalÆmol

)1
) and the main transition (8.8 kcalÆ
mol
)1
), are comparable with previous data [24,25]. The
incorporation of DRS-DA4 into DMPG MLVs signifi-
cantly altered their thermotropic phase behavior. The
presence of the peptide abolished the pretransition,
even at the lowest concentration tested (peptide/lipid
ratio 1 : 100), which is within the same range of order
as the minimum inhibitory concentration (MIC) for
Gram-negative strains. Increasing concentrations of
DRS-DA4 broadened the DMPG main phase transi-
tion peak, probably because of a loss of cooperativity
during the lipid fusion resulting from the insertion of
the peptide molecules. The main transition was totally
abolished at 1 : 20 peptide ⁄ lipid ratios, indicating total
disorganization of lipid DMPG bilayer (Fig. 5C).
Like those of DMPG, aqueous dispersions of
DMPC showed two endothermic transitions, a pretran-
sition occurring at 13.1 °C with an enthalpy of about
1.1 kcalÆmol
)1
, and a main transition at 23.9 °C with
an enthalpy of 11 kcalÆmol
)1
, which was again within
the range previously published [24,25]. At low con-
centrations of DRS-DA4 (1 : 100 peptide ⁄ lipid ratio),
no modifications of the thermogram were observed.

The pretransition was then gradually abolished as the
peptide/lipid ratio increased. At greater ratios only
(1 : 20 peptide ⁄ lipid ratio), DRS-DA4 induced some
changes to the DMPC melting profile, indicating an
interaction with this type of vesicle, whereas the main
transition peak was still present, without modification
of its temperature.
CD spectra of DRS-DA4 (Fig. 5E) revealed that
DRS-DA4 adopted an a-helix conformation in the
presence of DMPG vesicles, whereas it remained as a
random coil in aqueous solution and in the presence of
DMPC liposomes, whatever the peptide/lipid ratio
(data not shown). In the presence of DMPG vesicles,
the spectrum of DRS-DA4 showed a profile with
minima at 208 and 220 nm, suggesting a major contri-
bution of a-helix (  40%). The conformational
propensity of DRS-DA4 contrasts with that of DRS-
B2, which has been found to be structured as an
a-helix (55%), whatever the lipidic composition of the
vesicles [26].
Discussion
Hundreds of antimicrobial peptides have been isolated
from frogs, but very few studies dealing with their
immunomodulatory capacities have been published.
Table 1. Antimicrobial activities of DRS-DA4 and DRS-B2. Bacterial strains were considered to be resistant (R) when their growth was not
inhibited by peptide concentrations up to 100 l
M. The data shown correspond to the MIC (lM). E. coli 363 ATCC 1175 or S. epidermidis BM
3302 transmembrane potential changes were induced by DRS-DA4 and assessed with the DiSC
3
(5) probe: membrane depolarization was

monitored by an increase in fluorescence after the addition of peptide at the MIC. Triton X-100 was used to fully collapse the membrane
potential. B, bactericidal; ND, not determined.
Antimicrobial activity (MIC) DRS-DA4 DRS-B2 Membrane depolarization assay
Gram-negative E. coli ML35p 5, B ND ND
E. coli 363 ATCC 11775 5, B 0.39, B
a
55 ± 9%
Ps. aeruginosa ATCC 27853 40, B 3.1, B
a
ND
Gram-positive En. faecalis ATCC 29212 R ND ND
S. aureus ATCC 25923 R 0.7, B
a
ND
S. epidermidis BM 3302 R ND 50 ± 5%
a
See ref [26].
C. Auvynet et al. Chemotactic and antimicrobial DRS-DA4
FEBS Journal 276 (2009) 6773–6786 ª 2009 The Authors Journal compilation ª 2009 FEBS 6779
The first antimicrobial peptide shown to exhibit immu-
nological properties was DRS-S1, a 34 amino acid
cationic antimicrobial peptide, which stimulates the
microbicidal activity of PMN leukocytes [12]. A few
other frog peptides, temporin A, rana-6 and pLR-like
peptides and, recently, another dermaseptin-related
peptide, DRS-S9, have been shown to be microbicidal
as well as immunomodulatory [13,27–29]. Here, we
report the isolation, by screening for chemotactic activ-
ity, of a new member of the dermaseptin superfamily,
0.0

0.5
1.0
1.5
2.0
0 2000 4000 6000 8000 10 000 12 000 14 000
0.0
0.5
1.0
1.5
2.0
A B
C
E
D
Absorbance (405 nm)
Time (s)
0 2000 4000 6000 8000 10 000 12 000
Time (s)
20 µM
10 µM
5 µM
1.2 µM
0.3 µM
Control
5 10 15 20 25 30 35 40
0
5
10
15
20

25
30
1/20
1/100
DMPG
Temperature (°C)
5 10152025303540
0
5
10
15
20
25
30
1/20
1/100
DMPC
Temperature (°C)
190 200 210 220 230 240 250 260
–6
–4
–2
0
2
4
6
8
10
12
14

16
(M
–1
·cm
–1
)
Wavelength (nm)
DMPG
DMPC
Buffer
Cp (kcal·k·mol
–1
)
Cp (kcal·k·mol
–1
)
Fig. 5. The bactericidal capacity of DRS-DA4 is linked to its interaction with DMPG. (A, B) Kinetics of bacterial membrane leakage of E. coli
ML35p (A) and S. aureus ST1065 (B) after treatment with increasing concentrations of DRS-DA4. The membrane leakage was followed by
measuring Gal-ONp hydrolysis at 405 nm. (C, D) DSC heating thermograms illustrating the effects of DRS-DA4 on the thermotropic phase
behavior of DMPG (C) and DMPC (D) MLVs. The top scan corresponds to the lipid alone, and the peptide ⁄ lipid molar ratios of the lower
scans are indicated. Thermodynamic parameters are given in Table 2. (E) CD spectra of DRS-DA4 in buffer, DMPG and DMPC with a
peptide ⁄ lipid molar ratio of 1 : 50. The data shown are representative of three experiments.
Chemotactic and antimicrobial DRS-DA4 C. Auvynet et al.
6780 FEBS Journal 276 (2009) 6773–6786 ª 2009 The Authors Journal compilation ª 2009 FEBS
DRS-DA4, from the defensive skin secretion of the
Mexican leaf frog P. dacnicolor.
We showed that DRS-DA4 triggered the in vitro
directional migration of human neutrophils and mono-
cytes with a typical bell-shaped dose–response curve,
and a maximal response at a concentration of 10 lm.

Our results suggested that the chemotactic effect was
mediated by the low-affinity G
ia
-protein-coupled recep-
tor FPRL-1, recently reported to interact with many
different ligands, such as fMLP, the a-helical antimi-
crobial peptides LL-37 and temporin A, and the amy-
loidogenic peptides Ab
1–42
and, presumably, DRS-S9
[13,27,30,31] (Fig. 4B). Whether FPRL-1 interacts with
its various agonists through different functional
domains remains to be investigated. In addition, as
only human leukocytes were used in this study, it
remains to be discovered whether DRS-DA4 is also
capable of recruiting frog leukocytes. Indeed, frog pep-
tides are secreted at the outer surface of the skin, and
it is presently not known whether they also enter the
blood circulation or inner tissues to act on leukocytes
or to modulate other biological functions.
As described for other agonists [20], the interaction
of DRS-DA4, presumably with FPRL-1, led to the
activation of the ERK1 ⁄ 2 MAPK pathway, but not to
that of the p38 MAPK pathway. The chemotaxis data
suggested that ERK phosphorylation, but not that of
p38, was necessary for the chemotactic process. Inter-
estingly, inhibition of FPRL-1 by PTX has been
reported to activate the p38 pathway [32]. Consistent
with this, inhibition of ERK1 ⁄ 2 phosphorylation by a
specific inhibitor (PD98059) activated the p38 MAPK

pathway in response to DRS-DA4, suggesting that
DRS-DA4 may additionally bind to a non-GPCR
receptor and, through a p38-dependent pathway, con-
trol other cell functions such as degranulation or cyto-
kine ⁄ chemokine gene expression and release. This is
in accordance with studies indicating that inhibition
of one signaling pathway could activate another one
[27,33].
The 32 amino acid DRS-DA4 exhibits the typical
characteristics of the dermaseptin superfamily: it is a
linear, Lys-rich cationic peptide with the conserved
Trp at position 3. A blast search revealed high
sequence homology of DRS-DA4 with DRS-DA3
(PD-33), also isolated from P. dacnicolor, and, more
surprisingly, with DRS-B2 isolated from Ph. bicolor,
DDK from Ph. distincta, and ARP-AC1 from A. cal-
lidryas [17] (Fig. 2A). However, unlike these other pep-
tides, DRS-DA4 is not carboxyamidated. Modeling
antimicrobial peptides as idealized helices revealed
their highly amphipathic nature, with hydrophobic
residues on one face of the helix and polar or charged
residues on the opposite face, leading us to propose
that the amphipathic a-helix structure is an important
feature of these membrane-permeating peptides. Secon-
dary structure prediction methods and CD spectros-
copy have also shown that dermaseptins contain
45–90% helix in structure-promoting solvents [16,26,
34,39]. We illustrated here by CD (Fig. 5E) that DRS-
DA4 was randomly coiled in water and, unexpectedly,
also in the presence of DMPC vesicles. In contrast,

DRS-DA4 adopted an a-helical structure in the pres-
ence of DMPG vesicles, consistent with its cidal action
against prokaryotic cells. Thus, DRS-DA4 fits the
model proposed by Khandelia et al. [35], in which the
composition of the target membrane (zwitterionic or
anionic) modulates the extent of helical content
induced in antimicrobial peptides. CD results are con-
sistent with data from the calorimetric tests and iso-
thermal titration calorimetry (ITC) experiments with
DMPC large unilamellar vesicles (LUVs) (data not
shown), again suggesting very weak or no interaction
with this lipid. In contrast, strong perturbations of the
pretransition and the main phase transition (Fig. 5C
and Table 2) were recorded in the presence of DMPG,
suggesting that electrostatic interactions participate in
the peptide–lipid interaction, and that DRS-DA4 is
able to penetrate the acyl chain region.
A highly cationic charge such as that of DRS-DA4
favors the accumulation of peptides on negatively
charged DMPG bilayers via electrostatic interactions,
suggesting that the bactericidal activity of DRS-DA4
towards Gram-negative bacteria results from the pref-
erential binding of the peptide to the negatively
charged lipopolysaccharides of the outer membrane,
and that the subsequent membrane damage occurs
through hydrophobic interactions with the inner target
membrane, which is rich in neutral phosphatidyletha-
nolamine. This hypothesis is supported by a study
showing that helical amphipathicity prevails over
hydrophobicity in interfacial binding, underlining the

Table 2. Thermodynamic parameters obtained by DSC for the
interaction of DRS-DA4 with MLVs of either DMPG or DMPC. –, no
pretransition observed.
Lipid
Peptide ⁄
lipid ratio
Pretransition Transition
T (°C)
DH
(kcalÆmol
)1
) T (°C)
DH
(kcalÆmol
)1
)
DMPG 0 12.8 1 23.2 9.6
1 : 100 – – 23.3 10.6
1 : 20 – – 23.9 4.3
DMPC 0 13.0 1.1 23.9 11.8
1 : 100 14.1 1.2 24.0 10.2
1 : 20 13.5 0.1 23.9 5.0
C. Auvynet et al. Chemotactic and antimicrobial DRS-DA4
FEBS Journal 276 (2009) 6773–6786 ª 2009 The Authors Journal compilation ª 2009 FEBS 6781
importance of amphipathicity as a driving force
for cell lytic activity. In addition, conformational
constraints and appropriate positioning of aromatic
residues for the formation of hydrophobic clusters
have been shown to be critical for antimicrobial activ-
ity and selectivity [36]. Moreover, the presence of

regions with different order and polarity within the
membrane has shown the existence of domains
enriched in phosphatidylethanolamine or phosphatidyl-
glycerol, localized in highly curved regions of the
bacterial membrane [37]. Segregation of the membrane
lipid components, leading to clustering of anionic lip-
ids through an induced lateral-phase separation, and
the subsequent perturbation of existing domains of the
membrane has been proposed as a mechanism contrib-
uting to the antimicrobial activity of numerous antimi-
crobial peptides. In agreement with this, as they have
significant amounts of both anionic and zwitterionic
lipids, most Gram-negative bacteria are more suscepti-
ble to this membrane-disrupting mechanism. Consis-
tent with this, and as reported for oligo-acyl lysine
[38], we found that Gram-negative bacteria were killed
by DRS-DA4.
It has been suggested that an uninterrupted section
of five hydrophobic residues, as identified on the heli-
cal wheel, is sufficient for good antimicrobial activity,
with reduced hemolysis [39]. This is the case for
DRS-DA4 and DDK. However, despite strong
sequence homology with DRS-B2, DRS-DA4 exhibits
distinct biological activities. Contrasting with the wide
microbicidal spectrum of DRS-B2, DRS-DA4’s bacte-
ricidal capacity was selective for Gram-negative bacte-
ria (Table 1), and, whereas DRS-B2 did not induce
leukocyte motility, DRS-DA4, at equivalent concentra-
tions, was a potent chemotactic agent for PMN leuko-
cytes (Fig. 4B). A comparison of the helical wheel

projections of DRS-B2, DDK, ARP-AC1 and DRS-L1
(a dermaseptin with a similar selectivity for Gram-
negative bacteria [18]) revealed that, if the last three
residues – which are rarely involved in the a-helix
formation – are disregarded, these peptides exhibit an
amphipathic distribution (Fig. 2B). The main informa-
tion provided by the Edmundson projection is that
DRS-DA4 and DRS-L1, the two peptides that are
active only on Gram-negative strains, do not expose a
negative residue on the apolar face, as do DRS-B2 and
DDK, which have a Glu at position 31, or ARP-AC1,
with an Asp at position 27. In agreement with this, the
position of acidic residues seems to be a critical para-
meter for the antibacterial activity against Staphylo-
coccus strains [40]. However, the ability of a peptide to
depolarize the cytoplasmic membrane does not neces-
sarily correlate with bactericidal activity. Indeed, our
data show that, like plasticins, DRS-DA4 was able to
depolarize the membrane of Gram-positive (S. epider-
midis ST1065) as well as of Gram-negative (E. coli
ML35p) strains of bacteria, but it was toxic only for
Gram-negative strains (Table 1).
In contrast to the situation with clinically used anti-
biotics, resistance to natural antimicrobial peptides is
not frequent, raising interest in the use of antimicro-
bial peptides to fight antibiotic-resistant microbes.
Moreover, recent data have suggested that antimicro-
bial peptides participate actively in preventing the
appearance of resistant mutants, and are thus the last
line of defense dealing with persistent infections

[41]. Our data showing the antibacterial potency of
DRS-DA4, resulting from its ability to interact with
anionic model membranes and to induce directional
locomotion of mammalian cells through a receptor,
presumably FPLR-1, highlight the multifunctionality
of antimicrobial peptides as antibiotics and immuno-
modulatory molecules [3,42,43]. Modification of DRS-
DA4 to enhance its direct bactericidal effect and to
extend its antibacterial activity to Gram-positive
strains, without compromising its immunomodulatory
potency, could be achieved, as for dermaseptin S4,
through acylation [44], or, as for magainin 2 analogs,
amidation [45]. This new class of peptides will be use-
ful for therapeutic application purposes.
Experimental procedures
Frogs
Male and female specimens of P. dacnicolor were captured
in the state of Morelos (Mexico) and housed in a nonsterile
environment, in a large plastic container covered by a
fence. phyllodendron, potos and dracena were used as
perches, and a water bowl was provided for nocturnal
baths. Once a week, the frogs were fed with crickets.
Purification of the peptide
Fresh skin exudate was recovered by gently squeezing the
latero-dorsal portion of a frog skin, resuspended in de-ion-
ized water, and centrifuged for 15 min at 400 g. The super-
natant was first fractionated by size exclusion
chromatography on a Sephadex G-50 fine column
(60 · 0.75 cm) eluted with 10% acetic acid. Absorbance was
monitored at 280 nm. Three main fractions were obtained

and tested for their chemotactic activity. Fraction III was
further fractionated by RP-HPLC on a semipreparative col-
umn (Nucleosil 5 l m C18, 250 · 10 mm), using a solvent
system composed of water containing 0.1% trifluoroacetic
acid (TFA) as solvent A, and acetonitrile containing 0.07%
Chemotactic and antimicrobial DRS-DA4 C. Auvynet et al.
6782 FEBS Journal 276 (2009) 6773–6786 ª 2009 The Authors Journal compilation ª 2009 FEBS
TFA as solvent B. The column was eluted at 5 mLÆmin
)1
with a 0–60% linear gradient of solvent B for 65 min.
ESI-MS, and peptide sequencing by ESI-MS/MS
and Edman degradation
ESI-MS and tandem MS (MS ⁄ MS) experiments were per-
formed on a Q-TOF II mass spectrometer (Waters, Guyan-
court, France) equipped with its standard ESI source and
operated under the control of masslynx 3.5 software
(Waters) [46].
Solid-phase peptide synthesis and purification
Synthesis and purification of the peptides were performed
by the platform ‘Inge
´
nierie des Prote
´
ines et Synthe
`
se
Peptidique of IFR83’ (UPMC, Paris, France). The synthesis
of DRS-DA4 and DRS-B2 was carried out by a solid-phase
Fastmoc chemistry procedure on an Applied Biosystems
433A Automated Peptide Synthesizer (Applera, Courtab-

oeuf, France). Resins and Fmoc-protected amino acids were
purchased from Merck Chemicals (Novabiochem, Notting-
ham, UK) and solvents from Carlo Erba, SDS Val de
Reuil, France (Aix-en-Provence, France), as previously
described [47]. Fmoc-Met-NovasynTGA (100–200 mesh)
was used for DRS-DA4. Briefly, synthesis products were
cleaved from the resin with a mixture of TFA (94%), H
2
O
(2.5%), ethanedithiol (2.5%), and tri-isopropylsilan (1%),
precipitated in ether, centrifuged at 5000 g for 10 min, and
lyophilized. Peptides were purified by semipreparative
HPLC (C18 reverse-phase column, PrepLC 25 mm module,
250 · 100 mm, 15 mm particle; Waters) with a Waters 1252
Binary HPLC pump (flow rate 8 mLÆmin
)1
). Purity was
assessed by MALDI-TOF MS (Voyager DE P60; Applied
Biosystems, Courtaboeuf, France).
Membrane polarization assay
The cytoplasmic membrane depolarization was determined
with the membrane potential-sensitive dye DiSC
3
(5) (Molec-
ular Probes–Invitrogen, Cergy Pontoise, France) and E. coli
363 ATCC 11775 or S. epidermidis BM3302, as described by
Zhu et al. [48]. Briefly, bacteria were harvested in the mid-
logarithm phase by centrifugation at 1000 g 10 min at 4 °C,
washed twice in 5 mm glucose and 5 mm Hepes (pH 7.2),
resuspended to an absorbance of 0.2 at 630 nm in the same

buffer, and incubated with 1 lm DiSC
3
(5) at 37 °C until a
stable reduction of fluorescence was achieved; once the dye
uptake was maximal, as indicated by a stable reduction in
fluorescence due to the quenching of the accumulated dye in
the bacteria, 0.1 m KCl was added to maintain a high mem-
brane potential gradient. Membrane depolarization was
monitored by measuring the change in the intensity of
fluorescence emission of the membrane potential-sensitive
dye DiSC
3
(5) (k
ex
= 622 nm, k
em
= 670 nm) after addition
of the peptides, on a Perkin-Elmer LS 50B spectrophoto-
meter with spectro winlab software (Perkin-Elmer, Cour-
tabeouf, France). Full dissipation of the membrane potential
was obtained by adding Triton X-100 (final concentration,
0.1%). The membrane potential-dissipating activity of the
peptides was calculated as follows: percentage membrane
depolarization = 100 · [(F
p
) F
0
) ⁄ (F
g
) F

0
)], where F
0
is the
stable fluorescence value after addition of the KCl, F
p
is the
fluorescence value 10 min after addition of the peptide, and
F
g
is the fluorescence signal after addition of Triton X-100.
Peptide-induced permeabilization of the
cytoplasmic membrane of E. coli
Permeabilization of the cytoplasmic membrane of E. coli
ML35p (generously provided by S. Rebuffat, Laboratoire
Mole
´
cules de Communication et Adaptation des micro-
organismes, Museum National d’Histoire Naturelle, Paris)
or of S. aureus ST1065 by DRS-DA4 was assayed by mea-
suring the b-galactosidase activity with the chromogenic
substrate Gal-ONp. Bacteria were grown in 10% LB broth,
washed twice with NaCl ⁄ P
i
(0.15 m phosphate, 0.2 m NaCl,
pH 7.4), and diluted in 10% LB in NaCl ⁄ P
i
to an absor-
bance of 0.5 at 630 nm. The assay was carried out in a
96-well microtiter plate. Aliquots (15 lL) of bacterial sus-

pension were mixed with 2.5 mm Gal-ONp and incubated
with various concentrations of peptide. The hydrolysis of
Gal-ONp was monitored by measuring the absorbance at
405 nm of released o-nitrophenol with a Fluostar Galaxy
(BMG LabTech, Champigny-sur-Marne, France).
Antimicrobial assays and hemolytic activity
The Gram-negative bacterial strains E. coli ATCC ML35p,
E. coli ATCC 11775, Ps. aeruginosa ATCC 27853, S. aureus
ATCC 25923, S. aureus ST1065 (generously given by M.
Falord and T. Msadek, Biology of Gram Positive Patho-
gens, CNRS URA 2172, Institut Pasteur, Paris), S. epide-
rmidis BM3302 (generously given by O. Chesneau, Unite
´
Membranes Bacte
´
riennes CNRS URA 2172, Institut
Pasteur, Paris) and Enterococcus faecalis ATCC 29212
were used. Strains were cultured and antimicrobial assays
were performed as described previously [13]. The hemolytic
activity of the peptide was determined using fresh rat
erythrocytes, as previously described [47].
CD spectroscopy
The CD spectra of the peptide were recorded on a Jobin-
Yvon CD6 dichrograph linked to a PC microprocessor, as
previously described [13]. Spectra were scanned at room
temperature in a quartz-optical cell with a 1 mm path
length. Spectral measurements were obtained at wavelengths
C. Auvynet et al. Chemotactic and antimicrobial DRS-DA4
FEBS Journal 276 (2009) 6773–6786 ª 2009 The Authors Journal compilation ª 2009 FEBS 6783
of 190–260 nm with a bandwidth of 1 nm. Typically, five

scans were accumulated and averaged. The CD spectrum of
the peptide was measured in phosphate buffer (10 mm phos-
phate, pH 7.0), and in the presence of DMPC or DMPG
vesicles, at a peptide ⁄ lipid molar ratio of 1 : 50. The relative
a-helix content was estimated from De (m
)1
cm
)1
)at
220 nm, according to Zhong and Johnson [49].
DSC and ITC
One milligram of DMPC or DMPG (Avanti Polar Lipids)
was dissolved in 100 lL of chloroform or 100 lL of chloro-
form ⁄ methanol (1 : 1), respectively. Lipid films were
obtained by evaporating solvents under a nitrogen stream,
desiccated for several hours under vacuum at 40 °C, and
resuspended in NaCl ⁄ P
i
to obtain MLVs at a final concentra-
tion of 1 mgÆmL
)1
. The peptide was then added to this solu-
tion to achieve lipid ⁄ peptide ratios of 100 : 1, 50 : 1, or
20 : 1. NaCl ⁄ P
i
and this mixture were then degassed under
vacuum for 15 min before being loaded into the reference cell
of a Nano-Differential Scanning Calorimeter III (Calorime-
try Sciences Corp., Lindon, UT, USA) and in the sample cell,
respectively. Scans were recorded between 0 and 40 °Cat

scan rates of 0.5 °CÆmin
)1
for the heating and 1 ° CÆmin
)1
for
the cooling. MLVs were used rather than LUVs for DSC
experiments, because they exhibit much more cooperative
lipid phase transitions. As the thermotropic phase behavior
was not completely reproducible, we performed at least 10
scans. Thermogram analysis, using the last scans once equi-
librium had been reached, was performed with cpcalc, the
software provided with the calorimeter. Final figures were
plotted with origin 6.0 (Microcal, Inc., Northampton,
MA, USA).
Cell preparation and chemotaxis assays
Neutrophils and monocytes were obtained as previously
described [13]. The chemotactic activity was tested on calcein-
labeled human neutrophils or monocytes, using a 48-well
microchemotaxis chamber (Neuro Probe, Gaithersburg,
MD, USA) as described previously [13]. Briefly, different
concentrations of DRS-DA4 or DRS-B2, each diluted in
chemotaxis medium, were placed in the bottom wells of the
chamber; fMLP (1.14 nm), fMLP (100 nm) or stromal cell-
derived factor 1a (SDF1-a)(1nm) was used as positive
control, and chemotaxis medium as negative control. The
chamber was incubated for 45 min (neutrophils) or 4 h
(monocytes) at 37 °Cina5%CO
2
humidified atmosphere.
Cell migration was assessed by measuring the fluorescence

of the lower face of the membranes with an Alpha Innotech
image analyzer, using fluorchem 8800 software from
Alpha Innotech. To differentiate between chemotaxis and
chemokinesis, experiments were performed in which the
same concentrations of DRS-DA4 present in the lower
wells were added simultaneously to the upper wells. For
some experiments, neutrophils were preincubated with PTX
(200 ngÆmL
)1
) (Sigma-Aldrich, St Louis, MO, USA), fMLP
(1.14 nm), fMLP (100 nm), PD98059 (30 lm) (Calbiochem)
or SB202190 (10 lm) (Calbiochem, Los Angeles, CA, USA)
for 30 min at 37 °C, prior to evaluation of their migration
towards DRS-DA4.
Cell activation and immunoblot
Purified human neutrophils (2 · 10
6
per sample) resuspended
in RPMI-1640 were incubated with DRS-DA4 (10 lm),
fMLP (100 nm) or chemotaxis medium for the indicated peri-
ods of time at 37 °Cin5%CO
2
. In some cases, neutrophils
were pretreated for 30 min at 37 °C with PTX and ⁄ or with
PD98059. Phosphorylation of MAPK ERK1 ⁄ ERK2 and p38
were detected with an antibody against phospho-ERK1 ⁄ 2
(Santa Cruz Biotechnology, Santa Cruz, CA, USA) and an
antibody against phospho-p38 (Cell Signaling Technology,
Inc., Danvers, MA, USA) by chemiluminescence with ECL
(Amersham Biosciences Inc., Piscataway, NJ, USA).

Membranes were reprobed for ERK1 ⁄ 2 (Santa Cruz Bio-
technology) and p38 (Santa Cruz Biotechnology).
Acknowledgements
This work was supported by grants from the Ministe
`
re
des Affaires Etrange
`
res (France), the Centre National
de la Recherche Scientifique (CNRS), the Universite
´
Pierre et Marie Curie, the Secretaria de Relaciones
Exteriores (Mexico), and CONACyT (Mexico). The
authors are grateful to C. El Amri (Universite
´
Pierre et
Marie Curie, UR4-Enzymologie Mole
´
culaire et Fonc-
tionnelle, Paris, France) for help with ITC experiments
and fruitful discussions, R. Thai (Laboratoire de Bio-
chimie des Prote
´
ines, CEA, Saclay, France) for peptide
sequencing, F. Bruston (Universite
´
Denis Diderot,
Unite
´
EA4413, Paris, France) for helpful advice, espe-

cially in CD, A. Alagon, G. Corzo, H. Clement and E.
Melchy (Instituto de Biotecnologica, Universidad Nac-
ional Auto
´
noma de Me
´
xico, Me
´
xico) for suggestions
and technical help with HPLC purification, F. Abassi
(Laboratoire de Biochimie, Faculte
´
de Me
´
decine de
Sousse, Sousse, Tunisia) for help with antimicrobial
assays, I. Alves (Laboratoire des Biomole
´
cules, Univer-
site
´
Pierre et Marie Curie-CNRS-ENS, Paris, France)
for the LUV preparation facilities, and A. Robin and
S. Khedimi for technical assistance.
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