B2, presents chemotactic and Gram-negative selectivebactericidal activities Constance Auvynet1,2,*, Pierre Joanne2,, Julie Bourdais3, Pierre Nicolas2,, Claire Lacombe2,4, and Yvonne Rose
Trang 1B2, presents chemotactic and Gram-negative selective
bactericidal activities
Constance Auvynet1,2,*, Pierre Joanne2,, Julie Bourdais3, Pierre Nicolas2,, Claire Lacombe2,4, and Yvonne Rosenstein1
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: yvonne@ibt.unam.mx
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: claire.lacombe@upmc.fr
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 memmem-branes 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; DiSC3(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.
Trang 2At 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 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
Laboratoire 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
Trang 3bacteria 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-colorskin exudates by a two-step protocol Specifically,
1.1 mL of skin secretions recovered by gently
squeez-ing the parotoid glands of a ssqueez-ingle livsqueez-ing 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
A
B
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
V0
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.
Trang 4A 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.
Trang 5distincta, 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
Gia-protein-coupled receptor such as FPR or FPRL-1
[19] Pretreatment with pertussis toxin (PTX) – a specific
inhibitor of Gia-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.
Trang 6the 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 [DiSC3(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 DiSC3(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 aureusstrain 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 ’
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 preincubmedi-ated with medium,
PTX, fMLP (1.14 n M ), or fMLP (100 n M ) prior to challenge with
DRS-DA4, fMLP (1.14 n M ) or fMLP (100 n M ) (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.
Trang 7observed 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 main 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 12.8C, arising from the conversion of
the lamellar phase to the rippled gel phase, and a
sec-ond, more energetic, main transition at 23.2C,
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 13.1C with an enthalpy of about 1.1 kcalÆmol)1, and a main transition at 23.9C 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 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 (l M ) E coli 363 ATCC 1175 or S epidermidis BM
3302 transmembrane potential changes were induced by DRS-DA4 and assessed with the DiSC3(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.
a
See ref [26].
Trang 8The 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
Time (s)
0 2000 4000 6000 8000 10 000 12 000
Time (s)
20 µ M
10 µ M
5 µ M 1.2 µ M 0.3 µ M Control
5 10 15 20 25 30 35 40
0
5
10
15
20
25
30
1/20 1/100 DMPG
Temperature (°C)
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
Wavelength (nm)
DMPG DMPC Buffer
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.
Trang 9DRS-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 Gia-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 Ab1–42 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)
Trang 10importance 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 selectivactiv-ity [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
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 projecinforma-tion 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-coccusstrains [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
(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 lm 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%