From the Institut de Biologie Moléculaire et
Cellulaire, Unité Propre de Recherche 9022, CNRS,
"Réponse Immunitaire et Développement chez les
Insectes," 15 rue René Descartes, 67084 Strasbourg Cedex,
France and § Rhône-Poulenc Agro,
Research Triangle Park, North Carolina 27709-2014
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ABSTRACT |
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Lepidoptera have been reported to produce several antibacterial peptides in response to septic injury. However, in marked contrast to other insect groups, no inducible antifungal molecules had been described so far in this insect order. Surprisingly, also cysteine-rich antimicrobial peptides, which predominate in the antimicrobial defense of other insects, had not been discovered in Lepidoptera. Here we report the isolation from the hemolymph of immune induced larvae of the lepidopteran Heliothis virescens of a cysteine-rich molecule with exclusive antifungal activity. We have fully characterized this antifungal molecule, which has significant homology with the insect defensins, a large family of antibacterial peptides directed against Gram-positive strains. Interestingly, the novel peptide shows also similarities with the antifungal peptide drosomycin from Drosophila.
Thus, Lepidoptera appear to have built their humoral immune response
against bacteria on cecropins and attacins. In addition, we report that
Lepidoptera have conferred antifungal properties to the well conserved
structure of antibacterial insect defensins through amino acid replacements.
Cotton is grown in more than 60 countries and plays a major role
in the economy of these countries. The demand for cotton has not fallen
despite the rapid growth in synthetic fiber production since 1960. The
major biotic yield constraints are animal pests and weeds, although in
all growing regions there are also diseases causing losses of cotton
yields. Cotton plants appear to have little inherent defense against
attack by insect pests. In fact, many insect species have been reported
on cotton, and some of these species are regarded as major pests that
can destroy the plants in a few days. In most growing regions, the
principal pests are larvae of lepidopteran species that attack the
roots, leaves, flowers, and bolls. This is in particular the case for
the following species: Pectinophora gossypiella (pink
bollworm), Heliothis armigera (American bollworm), H. virescens (tobacco budworm), and Earias spp. (spiny
bollworm). Each year, these species cost the cotton industry millions
of dollars in yield loss and control measures (1). In this context, the
knowledge of the immune response mechanisms of the lepidopteran larvae
is of great interest. Indeed, these mechanisms can represent a
potential target for controlling these pests. In the present study, we
have chosen H. virescens as a biological model.
The host defense of insects relies on cellular and humoral mechanisms.
The cellular response consists of phagocytosis and encapsulation of
invading microorganisms by blood cells (for a review, see Ref. 2). The
humoral facet of the defense reactions of insects has been extensively
studied, and it is now clear that it is essentially based on the
synthesis of a battery of cationic antibacterial and antifungal
peptides/polypeptides exhibiting broad activity spectra. These
antimicrobial molecules are produced in the fat body (a functional
equivalent of the mammalian liver) and released into the hemolymph
(blood). Today, more than 150 antimicrobial peptides have been
characterized from various insect species. Although they exhibit great
structural diversities, certain common structural patterns are
apparent, and the peptides are often grouped into four families: (i)
cecropins, (ii) cysteine-rich peptides, (iii) proline-rich peptides,
and (iv) glycine-rich peptides/polypeptides (for reviews, see Refs.
2-4). Cecropins, which were the first inducible antibacterial peptides
to be characterized (5), are linear 4-kDa cationic peptides isolated
from Lepidoptera and Diptera. They are devoid of cysteine residues and
consist of two As regards the order of Lepidoptera, to which belongs H. virescens, a number of inducible antibacterial peptides had
already been isolated when we engaged in the present study. These
include, in addition to the cecropins and attacins mentioned above,
gloverin (17), moricin (18), and lebocins (19). Gloverin is a 14-kDa inducible cationic antibacterial polypeptide isolated from pupae of the
giant silk moth Hyalophora cecropia. It contains a large number of glycine residues but is devoid of cysteine residue. Moricin
is a 4-kDa antibacterial peptide isolated from Bombyx mori.
Neither moricin nor gloverin range into any of the four groups of
antimicrobial peptides/polypeptides defined above. Finally, lebocins
that were isolated from hemolymph of immunized B. mori are
structurally related to proline-rich peptides. Interestingly, the
unique threonine residue in each lebocin is
O-glycosylated.
From H. virescens, our experimental model, the presence of
cecropin-like and attacin-like molecules has been described, but no
amino acid sequences have been published (20, 21). Intriguingly, no
cysteine-rich peptides have been reported so far from Lepidoptera, and
no antifungal activities have been recorded from this order.
We present here the isolation of a novel inducible antifungal peptide,
the structure of which shares similarities with insect defensins, with
the Drosophila antifungal peptide drosomycin, and with
antifungal plant defensins.
Insect Immunization and Hemolymph Collection
Fifth instar larvae of the lepidopteran H. virescens
were individually pricked with a 30-gauge needle dipped into a combined bacterial pellet obtained after centrifugation of 37 °C overnight cultures of Micrococcus luteus and Escherichia
coli 1106. After 24 h, the insects were chilled on a bed of
ice, and several drops (30 µl/larva) of hemolymph were recovered by
sectioning an abdominal appendix and gently squeezing the abdomen. The
hemolymph was pooled in ice-cold polypropylene tubes containing
aprotinin as a protease inhibitor (20 µg/ml final concentration) and
phenylthiourea as a melanization inhibitor (20 µM final
concentration). After a centrifugation at 14,000 × g
for 1 min at 4 °C, the cell-free hemolymph was frozen at Purification Procedure
Sep-Pak Prepurification
Two ml of cell-free hemolymph, from untreated or
bacteria-challenged larvae, were acidified to pH 3 with 0.1% (v/v)
trifluoroacetic acid. The acidic extraction was performed for 30 min
under gentle shaking in an ice-cold water bath. After centrifugation
(10,000 × g for 30 min at 4 °C), the supernatant
was loaded onto Sep-Pak C18 cartridges (Waters)
equilibrated with acidified water (0.05% trifluoroacetic acid).
Elutions were performed with 10, 40, and 100% acetonitrile in
acidified water. All fractions were first concentrated in a vacuum
centrifuge (Speed-Vac, Savant) in order to remove the organic solvent
and trifluoroacetic acid and subsequently reconstituted with MilliQ water.
First Step of Purification
The two 40% Sep-Pak fractions from immunized and control
insects were purified in parallel by reversed-phase
HPLC1 on a semipreparative
Aquapore RP-300 C8 column (250 × 7.0 mm, Brownlee)
equilibrated with 2% acetonitrile in acidified water. Elution was
performed with a linear gradient of 2 to 60% acetonitrile in acidified
water over 120 min at a flow rate of 1.5 ml/min.
Second Step of Purification
The fraction with antifungal activity was applied on an
analytical Aquapore OD-300 C18 column (220 × 4.6 mm,
Brownlee) developed with a linear biphasic gradient of acetonitrile in
acidified water from 2 to 22% over 10 min and from 22 to 32% over 50 min at a flow rate of 0.8 ml/min.
Final Step of Purification
The fraction that contained the antifungal activity was further
applied on a narrow bore reversed-phase column (Delta Pak HPIC18, 2 × 150 mm, Waters) equilibrated with 2%
acetonitrile in acidified water and developed with a linear biphasic
gradient of acetonitrile in acidified water from 2 to 24% over 10 min
and from 24 to 44% over 100 min at a flow rate of 0.25 ml/min and at a
temperature of 30 °C.
The first and second steps of HPLC purification were performed with a
Beckman Gold HPLC system equipped with a Beckman 168 photodiode array
detector. The last step of purification was carried out at a
temperature of 30 °C using an all PEEK Waters HPLC system (Waters
model 626 pump) attached to a tunable absorbance detector (Waters model
486) and equipped with an oven. During the purification procedure, the
internal diameter of the column was chosen according to the amount of
peptide to be purified, while the modification of the gradient was
necessary to increase peptide separation.
In all HPLC purification steps, the column effluent was monitored by
absorbance at 225 nm to have the most appropriate signal/noise ratio.
Fractions were hand-collected in order to have one individual peak per
fraction. The presence of antimicrobial activity was detected using
liquid growth inhibition assays under the conditions described below.
Bioassays
Microorganisms
Bacterial Strains--
The following are the bacterial strains
used with their source (gifts from colleagues): M. luteus
A270 from the Pasteur Institut Collection (Paris) and E. coli SBS363 and E. coli 1106 from P. L. Boquet
(Center d'Etudes Nucléaires, Saclay).
Fungi and Yeast Strains--
The filamentous fungi and the yeast
strains used in this study were generous gifts from different
colleagues: Aspergillus fumigatus (H. Koenig, Laboratoire de
Mycologie, Faculté de Médecine, Strasbourg, France),
Fusarium culmorum (IMI 180420), Fusarium oxysporum (MUCL 909), Nectria hematococca (Collection
Van Etten 160-2-2), Neurospora crassa (CBS 327-54),
Trichoderma viride (MUCL 19724) (Université Catholique
de Leuven, Belgique), Candida albicans, Candida
glabrata, Cryptococcus neoformans (H. Koenig,
Laboratoire de Mycologie, Faculté de Médecine, Strasbourg,
France), and Saccharomyces cerevisiae (Société
Transgène, Strasbourg, France).
Antibacterial Assay
During the various steps of purification, antibacterial activity
was monitored by a liquid growth inhibition assay on the Gram-positive
strain M. luteus and on the Gram-negative strain E. coli 363. Ninety µl of a suspension of a midlogarithmic phase culture of bacteria at a starting A600 = 0.001 in Poor Broth nutrient medium (1% Bacto-Tryptone and 0.5% (w/v) NaCl,
pH 7.5) were added to 10 µl of fraction to analyze. Microbial growth
was assessed by an increase in A600 after a 24-h
incubation at 30 °C using a microtitration plate reader.
Antifungal Assays and Determination of the Minimal Inhibitory
Concentration
Fungal spores (final concentration 104 spores/ml)
were suspended in 1/2 Potato Dextrose Broth (Difco), and the
yeast strains were suspended at a starting A600 = 0.001 in the yeast complete medium YPG (1% yeast extract, 1%
peptone, 2% glucose). The medium was supplemented with tetracyclin (10 mg/ml) and cefotaxim (100 µg/ml), dispensed by aliquots of 80 µl
into wells of a microplate containing 20 µl of either water or the
fraction to be analyzed. Growth of fungi and yeast was evaluated after
24 h at 30 °C by optical microscopy and after 48 h by
measuring the culture absorbance at 595 nm using a microplate reader.
In the conditions where the antifungal assay was performed in the
presence of salt, the 1/2 Potato Dextrose Broth medium was prepared in phosphate-buffered saline, 137 mM NaCl.
The procedure used for the determination of the minimal inhibitory
concentration (MIC) was identical to that for the antifungal assay. The
MIC values are expressed as an interval (a-b), where a is the highest peptide concentration tested at which fungi
or yeast are still growing and b is the lowest concentration
that causes 100% growth inhibition.
Fungicidal Assay
Spores of N. crassa were cultured in the presence of
0.04-20 µM peptide. After 48 h, the 1/2 Potato Dextrose Broth medium containing the peptide was removed and
replaced by fresh medium. Two days later, the cultures were examined
microscopically and spectrometrically.
Structural Characterization
Capillary Zone Electrophoresis
Peptide purity was ascertained by capillary zone
electrophoresis. Analysis was performed on a model 270-HT capillary
electrophoresis system (PE Applied Biosystems). Two nl of the sample
were injected using vacuum to a 50 µm × 72-cm fused silica
capillary and run in 20 mM citrate buffer at pH 2.5 under
20 kV from anode to cathode at 30 °C for 20 min. Migration was
monitored at 200 nm.
Microsequence Analysis
Automated Edman degradation of the native and pyridylethylated
peptides and detection of phenylthiohydantoin derivatives were performed on a pulse liquid automatic sequenator (PE Applied
Biosystems, model 473A).
Reduction and S-Pyridylethylation
One nmol of purified peptide was submitted to reduction and
S-pyridylethylation. The procedure used was reported
previously (22).
Matrix-assisted Laser Desorption/Ionization Time of
Flight Mass Spectrometry (MALDI-TOF-MS)
MALDI-TOF-MS was performed on a Bruker BIFLEXTM
(Bremen, Germany) mass spectrometer operating in a positive linear mode
as described previously (23).
Enzymatic Digestions
Endoproteinase Lys-C Treatment
The pyridylethylated peptide (200 pmol) was treated with
endoproteinase Lys-C (Achromobacter protease I; Takara,
Otsu, Japan). Digestion was carried out at 37 °C for 6 h in 50 µl of 10 mM Tris-HCl (pH 9) in the presence of 0.01%
Tween 20 at a peptide/enzyme ratio of 1:40 (w/w). The reaction was
stopped by acidification with 1% trifluoroacetic acid, and the peptide
fragments were separated on a narrow bore reversed-phase column (Delta
Pak HPIC18, 2 × 150 mm, Waters). Elution was
performed with a linear gradient of acetonitrile in acidified water
(0.05% trifluoroacetic acid) from 2 to 60% over 80 min at 37 °C at
a flow rate of 0.2 ml/min. The fragments were analyzed by MALDI-TOF-MS,
and the enzymatically derived fragment corresponding to the C terminus
of the protein was sequenced by Edman degradation.
Determination of the Cysteine Arrangement by Thermolysin
Digestion
Eight µg of the native peptide were treated with thermolysin
at a peptide/enzyme ratio of 1:2 (w/w) for 1 h in 0.1 M MES buffer at pH 7.4 at 37 °C in presence of 2 mM CaCl2. The digestion was stopped by adding
50 µl of 70% formic acid. The peptide mixture resulting from the
enzymatic digestion was subjected to the same reversed-phase HPLC
column as above. Elution was performed with a linear gradient of
acetonitrile in acidified water from 2 to 50% over 100 min after an
isocratic period at 2% acetonitrile during 10 min. The peptides
generated by protease treatment were characterized by MALDI-TOF-MS.
Production of Recombinant Peptide
Strains and Media
The E. coli strain DH5 Construction of the Synthetic Gene
A synthetic gene encoding the 44-amino acid peptide was
constructed by ligating three double-stranded oligonucleotide
cassettes, preceded by the last five amino acids of the MF
Sites for restriction enzymes, which do not alter the coding sequence
were incorporated into the synthetic gene where possible. The
HindIII-SalI fragment of the resulting plasmid
containing the synthetic gene and the
SphI-HindIII fragment of M13JM132 containing the
MF Large Scale Production and Purification of Recombinant
Peptide
S. cerevisiae strain TGY 48-1 was transformed with
the expression plasmid pSEA2, and transformants were selected by growth at 29 °C on YNBG medium supplemented with casamino acids lacking uracil. For large scale peptide preparation, the culture medium was
inoculated at a dilution of 1:50 with a 48-h preculture of the selected
transformant, and an additional 48-h growth was performed at 29 °C.
Yeast were harvested by centrifugation at 4000 rpm for 30 min at
4 °C. The supernatant was carefully decanted, and the pellet was
discarded as the recombinant peptide was secreted from the cells. The
supernatant was subjected to solid-phase extraction on an open column
filled with C18 reversed phase (preparative C18, 125 Å, Waters; 48 g of phase/4 liters of
supernatant), solvated with methanol, and further equilibrated with
acidified water. Elution was performed with 40% acetonitrile in
acidified water and lyophilized under vacuum, reconstituted in MilliQ
water (Millipore Corp.), and applied on a cation column (Aquapore
cation, 20 µm, 250 × 10 mm, Brownlee) equilibrated in 25 mM ammonium acetate buffer at pH 3.6. Elution was performed
with a linear gradient of 0-100% 1 M NaCl in ammonium
acetate buffer over 30 min at a flow rate of 2 ml/min. During the
purification step, the column effluent was monitored by absorbance at
225 nm. The collected fraction containing the peptide was desalted on a
reversed-phase column (Aquapore preparative C18, 250 × 10 mm, Brownlee) equilibrated with 2% acetonitrile in acidified
water. Elution was performed with a linear biphasic gradient of
acetonitrile in acidified water from 2 to 20% over 10 min and from 20 to 40% over 30 min at a flow rate of 3 ml/min. The fraction containing
the peptide was lyophilized and kept as dry powder.
Isolation of the Antibacterial and Antifungal Peptides from
Bacteria-challenged Insects--
One hundred fifth instar larvae of
H. virescens were challenged by injection of bacteria, and
their hemolymph (2 ml) was collected after 24 h. In parallel, 2 ml
of hemolymph from unchallenged larvae were treated under the same
conditions. The hemocytes were removed by centrifugation, and the
antimicrobial peptides were extracted from the cell-free hemolymph in
acidic condition during 30 min in an ice-cold water bath under gentle
shaking. The acidic extracts were then submitted to prepurification by
solid phase extraction onto Sep-Pak C18 cartridges. Elution
was sequentially performed with 10, 40, and 100% acetonitrile in
acidified water. After concentration, the various fractions obtained
from both acidic extracts were tested for their antimicrobial activity
by liquid growth inhibition assays against M. luteus, E. coli 363, and N. crassa. Only the 40% Sep-Pak
fractions were found to contain antimicrobial activity and were further
applied on an Aquapore RP-300 C8 column. Elution was
performed with a linear gradient of acetonitrile, and aliquots of the
eluted fractions were tested for their antimicrobial activity by liquid
growth inhibition assays against the three test organisms. Several
fractions from immune larvae were found to contain strong antifungal
and/or antibacterial activity (Fig. 1);
in contrast, weak activities were detected in the control experiment
(data not shown). Here we have focused on the purification of the
compound present in fraction A eluted at 28% acetonitrile in acidified water, which exhibited the strongest antifungal activity. The active
molecule was further purified by a two-step purification procedure (see
"Materials and Methods"). Pure antifungal compound was obtained, as
judged by capillary zone electrophoresis (data not shown). MALDI-TOF-MS
gave a single molecular mass at 4784.8 Da, confirming the purity of the
antifungal molecule (data not shown).
Characterization of the Isolated Antifungal Peptide--
One nmol
of purified antifungal peptide was subjected to reduction and
S-pyridylethylation and subsequently to MALDI-TOF-MS measurement. The obtained molecular mass at 5422.3 Da, in excess of
637.5 Da to the mass measured on the native molecule, corresponds to
six pyridylethylated groups, suggesting the presence of six cysteine
residues. One hundred pmol of the S-pyridylethylated peptide
were then submitted to Edman degradation, and a partial 41-residue
NH2-terminal amino acid sequence was obtained (Fig. 2). The mass calculated for this
N-terminal sequence is lower by 333.4 Da than the measured molecular
mass of the native peptide at 5422.3 Da. This clearly establishes that
the sequence obtained by Edman degradation is only a partial sequence.
To obtain the full sequence, we used endoproteinase Lys-C to cleave the
pyridylethylated peptide after the Lys residues at positions 2, 23, and
28 under the conditions described under "Materials and Methods."
The digest was analyzed by reversed-phase HPLC (data not shown), and
the peptide fragments were measured for their respective molecular masses by MALDI-TOF-MS. Five major peaks (2661.7, 2001.2, 2779.3, 2535.7, and 5422.5 in MH+) were obtained. The peak at
5422.5 MH+ (mass/charge) corresponds to the native
molecule, while the peptide fragments yielding the molecular masses at
2778.3 and 2534.7 Da correspond to the predicted fragments from the
N-terminal sequence. Only the fragments at 2661.7 and 2001.2 MH+ did not fit with any possible cleavage product,
suggesting that both include the C-terminal end. The mass difference of
659.5 Da between these two fragments can be explained by the presence of the additional sequence Arg-Arg-Gly-Tyr-Lys (residues 24-28) in the
N-terminal part of the 2661.7 MH+ fragment obtained by
Edman degradation of the native peptide. To obtain the C-terminal
sequence information, the shortest fragment was subjected to Edman
degradation and the following sequence was obtained:
Gly-Gly-His-Cys*-Gly-Ser-Phe-Ala-Asn-Val-Asn-Cys*-Trp-Cys*-Glu-Thr, where Cys* represents a pyridylethylated cysteine residue. The calculated mass of the sequence is in perfect agreement with the molecular mass of 2000.2 Da measured by MALDI-TOF-MS, establishing that
there is no C-terminal amidation. The overlap observed with the
N-terminal sequence confirmed the identity of this fragment as the
C-terminal part of this 44-amino acid antifungal peptide. The full
sequence of the molecule is shown in Fig. 2. The comparison between the
calculated mass after Edman degradation (4791.3 Da) and the measured
mass by MALDI-TOF-MS (4784.8 Da) indicates that the six cysteine
residues are engaged in the formation of three intramolecular disulfide
bonds. The data bank analysis (BLAST program in the data base
Swiss-Prot; Ref. 26) showed that the peptide has significant sequence
similarities with the Drosophila antifungal peptide
drosomycin and insect defensins. Therefore, we assumed that the
cysteine arrangement is identical to that of insect defensins
(Cys(1)-Cys(4), Cys(2)-Cys(5), and Cys(3)-Cys(6)) (27), which is
also conserved in the core of drosomycin (Cys(2)-Cys(5), Cys(3)-Cys(6), and Cys(4)-Cys(7)) (25). The disulfide array of the
peptide was determined by thermolysin digestion, separation of the
peptidic fragments by HPLC, and measurement of their respective molecular mass by MALDI-TOF-MS. The major peak (peak
6 in Fig. 3) had a molecular
mass of 4784.4 Da, corresponding to the native molecule. Four minor
peaks (peaks 1-4 in Fig. 3) corresponding to the digest
products yielded single molecular masses at 1184.2, 2192.9, 2355.7, and
2337.7 Da. Peak 1 yielded a molecular mass at 1184.2 Da, which is in
agreement with the peptidic fragment Ile4-Cys7
linked to the fragment Tyr27-Ser34 through an
intramolecular disulfide bridge between Cys7 and
Cys32 (see inset in Fig. 3). The mass of peak 4 can be explained by admitting the connection of the fragment
Tyr14-Gly26 to the fragment
Val38-Thr44. This indicates that the disulfide
bonds are Cys18-Cys40 and
Cys22-Cys42. The mass difference between peaks
2 and 3 of 162.8 Da corresponds to a Tyr residue, and the masses
obtained by MALDI-TOF-MS are in perfect agreement with the digestion
products proposed in Fig. 3. Taken together, the data are compatible
with the following disulfide array:
Cys7(1)-Cys32(4),
Cys18(2)-Cys40(5), and
Cys22(3)-Cys42(6), which is identical to that
determined for insect defensins and to the three internal disulfide
bridges of drosomycin.
Recombinant Synthesis of the H. virescensAntifungal Peptide and Analysis of Its Activity Spectrum--
In order to produce sufficient amounts of the peptide for detailed
studies on its activity spectrum, we expressed a synthetic cDNA in
the yeast S. cerevisiae. The synthetic gene was constructed by annealing and ligating six individual oligonucleotides to form a
double-stranded oligonucleotide cassette. This cassette is preceded by
the last five amino acids of the yeast mating factor MF The data presented in this report establish that the larval
hemolymph of a lepidopteran species, H. virescens, contains
a strongly inducible cysteine-rich antifungal peptide. The molecule was
purified to homogeneity and fully characterized at the level of its
amino acid sequence by a combination of reversed-phase chromatography,
capillary zone electrophoresis, Edman degradation, protease digestion,
and MALDI-TOF-MS. The peptide, which was named heliomicin, consists of
44 residues and contains six cysteines engaged in three intramolecular
disulfide bridges. Cysteine-containing antimicrobial peptides had so
far not been reported from lepidopteran insects, although this order
served for the first successful identification of antimicrobial
peptides and namely the discovery of cecropins and attacins by Boman
and associates (5, 13). Similarly, inducible antifungal activities had
not yet been recorded in Lepidoptera.
Sequence comparison with the group of insect defensins shows that the
Heliothis antifungal molecule presents the same cysteine arrangement (Cys(1)-Cys(4), Cys(2)-Cys(5), and Cys(3)-Cys(6)) as
that present in these antibacterial peptides (22, 28) (Fig. 4). In addition to the six cysteines, the
residues strictly conserved between heliomicin and insect defensins are
three glycines at equivalent positions (numbered 26, 29, and 30 in
heliomicin). The sequence of heliomicin is also evocative of that of
antifungal peptides recently reported from Drosophila
(drosomycin; Ref. 25) and plants (e.g. Rs-AFP1
from Raphanus sativus; Ref. 29) (Fig. 4). In particular,
heliomicin shares the cysteine array with these molecules. Note that
drosomycin and Rs-AFP1 have in addition two external
cysteines absent from defensins. Drosomycin and heliomicin also present
a conserved cluster of four amino acids (Cys-Trp-Cys-Glu) located on
the third
INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-helices linked by a short hinge (for a review, see
Ref. 6). Cecropins are frequently C-terminally amidated. They kill
Gram-positive and Gram-negative bacteria. The group of cysteine-rich
peptides contains members with 1-4 disulfide bridges with molecular
masses ranging from 2 to 6 kDa. The most widespread of insect
antimicrobial compounds, the insect defensins, belong to this group.
They contain six cysteine residues involved in three disulfide bridges
(7). Defensins are active against Gram-positive bacteria. Their
presence has been reported from many insect orders and also from
scorpions and molluscs (8, 9). Hitherto, they had not been isolated from Lepidoptera. In addition to these antibacterial cysteine-rich peptides, two cysteine-containing peptides with antifungal activity have been characterized so far from insects: drosomycin from the dipteran Drosophila melanogaster (10) and thanatin from the hemipteran Podisus maculiventris (11). In response to septic injury, larvae and adults of Drosophila produce considerable
amounts of drosomycin, a 5-kDa peptide with eight cysteine residues
that are engaged in the formation of four intramolecular disulfide bridges. Drosomycin is inactive against bacteria. It shows significant homology with plant defensins, e.g. Rs-AFP2 from
Raphanus sativus (10). Thanatin is a 2-kDa peptide
exhibiting both fungicidal and bactericidal activities. The third group
of antimicrobial peptides from insects, the proline-rich molecules,
have been isolated from Hymenoptera, Diptera, Lepidoptera, and
Hemiptera (for a review, see Ref. 3). They are active against
Gram-negative bacteria, and for some members of this group activity
against Gram-positive bacteria and fungi was also reported. Some of the
proline-rich peptides are O-glycosylated (e.g.
drosocin (12)). Finally, the glycine-rich peptides/polypeptides, which
are mostly large (8-24 kDa), are active against Gram-negative cells.
They include attacins, characterized in Lepidoptera and Diptera (13);
sarcotoxins II (14); and diptericins from Diptera (15, 16).
MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
20 °C
until use. In a control experiment, a hemolymph sample from
unchallenged H. virescens larvae was processed in the same manner.
was used in the cloning
procedures, and the S. cerevisiae strain TGY 48-1 was used
for expression of recombinant peptide. Yeast cells were transformed
using the lithium acetate method (24), and transformed colonies were
selected on YNBG medium (0.67% yeast nitrogen base, 1% glucose)
supplemented with 0.5% casamino acids, lacking uracil.
1
proregion, into the HindIII and BamHI sites in
the polylinker of pBS/SK+ (Stratagene). For this purpose, the six
following oligonucleotides were used: SEA-5
(5'-AGCTTGGATAAAAGAGACAAGTTGATTGGCAGCTGTGTTTGGGGCGCCGTCA-3'), SEA-6 (5'-ACTACACTAGTGACTGCAACGGCGAGTGCAAGCGCCGCGGTTACAAGGGTGG-3'), SEA-7 (5'-CCATTGTGGATCCTTCGCTAACGTTAACTGTTGGTGTGAAACCTGATAGGTCGACA-3'), SEA-8 (5'-GATCTGTCGACCTATCAGGTTTCACACCAACAGTTAACGTTAGCGAAGGATC-3'), SEA-9 (5'-CACAATGGCCACCCTTGTAACCGCGGCGCTTGCACTCGCCGTTGCAGTCACT-3'), and
SEA-10
(5'-AGTGTAGTTGACGGCGCCCCAAACACAGCTGCCAATCAACTTGTCTCTTTTATCCA-3').
1 promoter and the preprosequence were subcloned into the SphI and SalI sites of JM138 to create the vector
pSEA1. The SphI-SalI fragment of pSEA1
containing the full expression cassette for peptide production in yeast
was then cloned into the yeast shuttle vector pTG4812 to give the
expression plasmid pSEA2 (for more details, see Ref. 25). The plasmids
were all controlled by sequencing.
RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
First reversed-phase HPLC separation of
immune hemolymph of H. virescens. The 40%
acetonitrile fraction obtained after prepurification by solid phase
extraction was analyzed on an Aquapore RP-300 C8 column.
Elution was performed with a linear gradient (dotted
line) of acetonitrile in acidified water. Absorbance was
monitored at 225 nm (solid line). Antimicrobial
activities were detected by liquid growth inhibition assays against
M. luteus, (white column), E. coli (dotted column), and N. crassa (black column). The inset
shows the final purification of fraction A, which contains the active
antifungal compound.
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Fig. 2.
Amino acid sequence of the H. virescens antifungal peptide. After sequencing of 100 pmol of S-pyridylethylated peptide by Edman degradation, a
partial sequence was obtained. To get full sequence information, an
endoproteinase Lys-C treatment was performed, and the masses of the
peptidic fragments were obtained by MALDI-TOF-MS are shown in
MH+. Sequencing of the 2001.2 MH+ fragment
allowed full sequence information.
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Fig. 3.
Reversed-phase HPLC of the
H. virescens antifungal peptide treated
with thermolysin. After digestion by thermolysin, the native
antifungal peptide was loaded onto a narrow bore reversed-phase column.
Elution was performed with a linear gradient of acetonitrile in
acidified water (dotted line), and absorbance was
monitored at 225 nm (solid line). MALDI-TOF-MS
was performed on peaks 1-6. The
m/z values obtained for peaks
1-4 and the deduced sequences are indicated as well as the
linkage between the cysteine residues.
1 proregion to generate a HindIII site necessary for an in-frame fusion
with the preprosequence, responsible for secretion of the peptide by the yeast strain. The MF
1 promoter directs the expression of the
gene, and Kex2 endopeptidase cleaves the fusion protein after the
dipeptide site Lys-Arg at the C terminus of the MF
1 promoter. The
full expression block for peptide production was cloned into pTG4812 to
give the expression plasmid pSEA2, which was used to transform yeast.
The 48-h culture was centrifuged, and the cell-free supernatant was
subjected to solid-phase extraction. The fraction eluted with 40%
acetonitrile was applied on a cation exchange column, and the fraction
containing the peptide was desalted by reversed-phase HPLC and
lyophilized. With this system, the average recovery of pure peptide was
2.5 mg/1 liter of culture medium. MALDI-TOF-MS showed that the
recombinant peptide exhibits the same molecular mass as the native
molecule, and capillary zone electrophoresis analysis confirmed its
purity. The antifungal activity of recombinant and native peptide were
compared by measuring their MIC on N. crassa and found to be
identical for both molecules. The purified recombinant peptide was
tested on a variety of bacterial and fungal strains. No antibacterial
activity against 15 Gram-positive and 10 Gram-negative bacterial
strains was detected even at concentrations of up to 50 µM. However, in marked contrast, the H. virescens antifungal peptide showed a strong antifungal activity
against six fungal strains tested (Table
I). In liquid growth inhibition assays,
the pure recombinant peptide had marked activity against N. crassa (MIC = 0.1-0.2 µM), F. culmorum (MIC = 0.2-0.4 µM), and N. hematococca (MIC = 0.4-0.8 µM). The peptide
was also found to be active against F. oxysporum (MIC = 1.5-3 µM), T. viride (MIC = 1.5-3
µM), and A. fumigatus (MIC = 6-12
µM). Furthermore, it showed a strong activity against two
of the four yeast strains tested: C. albicans and C. neoformans at a MIC of 2.5-5 µM. The peptide was
inactive against C. glabrata, up to 50 µM, and
against S. cerevisiae, even at a concentration as high as
100 µM. The antifungal activity was tested in a similar
assay on N. crassa at physiologic ionic strength (137 mM NaCl), and the MIC recorded for the peptide was
identical to that of the classical tests, suggesting that the activity
of the peptide is not affected by salts. In contrast, an activity
reduction (4-fold) was observed for drosomycin in the presence of salts
(data not shown). Finally, spores of the fungus N. crassa
were incubated in the presence of various concentrations of the peptide
(0.04-20 µM). After 48 h, the medium containing the
peptide was replaced by fresh medium, and 2 days later no growth
recovery had occurred when the peptide concentration during the initial
incubation was 0.15 µM or higher. This result
demonstrates that the peptide is fungicidal at this concentration. We
propose the name of heliomicin for this novel antifungal peptide from
H. virescens.
Antimicrobial activity spectrum of recombinant heliomicin compared to
Phormia defensin and drosomycin from Drosophila
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-strand at the C-terminal part of drosomycin (30). They
also have in common the hydrophobic dipeptide Ala-Val at positions 11 and 12; three glycine residues at positions 5, 26, and 30; and a lysine
at position 23 (numbering relative to heliomicin). As regards plant
defensins, the residues conserved between heliomicin and
Rs-AFP1 are restricted to the six cysteine residues and to
two glycines at positions 5 and 30 (relative to heliomicin, as shown in
Fig. 4).
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Fig. 4.
Sequence comparison of heliomicin with other
antimicrobial peptides from insects and plants.
Heliomicin was compared (i) to two antifungal peptides
(drosomycin from D. melanogaster and Rs-AFP1 from
the Brassicaceae R. sativus) and (ii) to some of the most
representative insect defensins. Identical amino acids and conservative
replacements are shown in gray boxes.
Bars indicate gaps to optimize the alignments.
Heliomicin presents the structural motif CS. In this scaffold, an
invariant Cys-(Xaa)3-Cys sequence of an
-helix is linked to an Cys-Xaa-Cys sequence of a
-sheet. A similar motif named CSH
(for cysteine-stabilized helix) was
initially described on the basis of NMR studies by Tamaoki et
al. in endothelin (31). This motif appears now to be commonly
shared by a variety of antibiotics and toxins like short- and
long-chain scorpion toxins (32), insect defensins (first report in
Phormia defensin; Ref. 33), the Drosophila
drosomycin (30), plant defensins (e.g. Rs-AFP1; Ref. 34), and
-thionins (35). The three-dimensional structure has
been established by NMR for some of these defense molecules, and we can
anticipate that the lepidopteran peptide will adopt a similar conformation.
In order to obtain sufficient amounts of heliomicin for
three-dimensional structure determination by two-dimensional NMR and for detailed studies on its activity spectrum, we have successfully expressed a synthetic gene in the eukaryote S. cerevisiae.
The purified recombinant molecule exhibits the same characteristics as
the native molecule and was used to establish the activity spectrum of
the peptide. Heliomicin was found to be active against various fungal
and yeast strains, but it had no activity against either Gram-positive
or Gram-negative bacterial strains even at concentration as high as 50 µM, which corresponds roughly to 10 times the estimated
concentration of heliomicin in immune challenged H. virescens larvae (Table I). We noted that heliomicin is active against most of the filamentous fungi tested at concentrations significantly lower than those of the Drosophila antifungal
peptide drosomycin. Furthermore, the latter exhibits no antifungal
activity against the yeast strains C. albicans and C. neoformans. Note that Phormia defensin exhibits also
some activity against several fungi, but at concentrations 4 times
higher than heliomicin. At physiological ionic strength, heliomicin
exhibits the same activity as in the classical tests against the
filamentous fungus N. crassa. This contrasts with the
reduction of antimicrobial activity observed in the presence of salts
for other antimicrobial peptides such as drosomycin, human
-defensins 1 and 2 (36), and lebocins (37). This indicates that the
antifungal activity of heliomicin is not based on charge differences
but may involve other mechanisms such as interactions with a receptor
in the target organism.
In conclusion, septic injury in H. virescens results in the
production of an antifungal peptide, heliomicin. Sequence comparison indicates that heliomicin is related to insect defensins and may represent the insect defensin prototype of Lepidoptera. However, the
antifungal activity observed for the Heliothis peptide
prompts us to classify heliomicin in the group of antifungal peptides such as plant defensins and drosomycin.
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ACKNOWLEDGEMENT |
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We are grateful to Martine Schneider for capillary electrophoresis analysis.
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FOOTNOTES |
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* This work was supported by CNRS, the University Louis Pasteur of Strasbourg, and Rhône-Poulenc Agro. The peptide was patented April 15, 1998 under the number FR9804933.
The protein sequence reported in this paper has been submitted to the Swiss-Prot protein data bank with the accession number P81544.
¶ To whom correspondence should be addressed. Tel.: 33388417062; Fax: 33388606922; E-mail: bulet{at}ibmc.u-strasbg.fr.
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ABBREVIATIONS |
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The abbreviations used are: HPLC, high pressure liquid chromatography; MIC, minimal inhibitory concentration; MALDI-TOF-MS, matrix-assisted laser/desorption time of flight mass spectrometry; MES, 4-morpholineethanesulfonic acid.
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REFERENCES |
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